Abstract
Invited Speakers
General Overview of Viral/Non-viral Gene Therapy
In distinguishing gene therapy from genetically-modified cell therapy (the two main areas covered by the ESGCT and BSGT) it is easiest to regard gene therapy as where transfection/transduction happens within the living body, whereas in cell therapy it happens in vitro or ex vivo. Hence my talk will focus on the strengths and weaknesses of vectors for use in vivo, considering both areas of local application (eg. to treat known local tissue targets such as the retina) and systemic delivery (to access widespread and dispersed targets). Because gene therapy is increasingly becoming regarded as a regular pharmaceutical approach (albeit where the active component chances its molecular form following arrival at the target site) it is important to assess pharmacological aspects of the agents being developed. Accordingly pharmacokinetic aspects of delivery of the input particles (viral and non-viral) will be considered at the fore, and particularly the effects on ‘particle kinetics’ of their interaction with the innate and adaptive immune systems. The innate immune system includes natural antibodies (mainly IgMs), complement and non-specific phagocytosis, all of which can negatively impact particle kinetics, whereas the adaptive immune system can have a directly neutralising activity against the vector (if already primed by prior exposure) or can have a negative impact on the duration of transgene expression. Other aspects (such as the ratio of target/non-target cells) must also be considered in understanding the delivery challenges faced. The strengths and weakness of different vector approaches will be considered in the context of our major disease goals.
Achieving reproducible and stable transgene expression for long-term therapeutic benefit
Long-term persistence of therapeutic genes within a mitotic, especially stem cell population can at present only effectively be achieved by stable transgene integration. This brings with it problems associated with epigenetic-mediated integration site position effect variegation (PEV; highly variable levels of expression including silencing), which can result in gene therapy failure.
There are two classes of genetic regulatory elements have been used in an effort to overcome PEV; those with a boundary or blocking function [insulators, scaffold/matrix attachment regions (SMARs)] and those with a dominant chromatin opening and transcriptional activating function [locus control regions (LCRs), ubiquitous chromatin opening elements (UCOEs)]. This presentation will review the use of these two different types of elements within a gene therapy context. In particular, data showing the unprecedented ability of UCOEs to provide reproducible and stable transgene expression including under conditions that do not provide a selective survival and growth advantage to the targeted, corrected cell population will be discussed.
Episomal modification of cells using non-viral S/MAR vectors
The early potential of gene therapy is slowly becoming realised following the recent treatment of patients with severe combined immunodeficiency and ocular disease. However, the development of alternative vectors is still an active field which is vital for further progress to produce vectors which provide improved safety and more efficiency of gene expression. One such class of vectors are those based on scaffold matrix attachment regions (S/MARs) elements, which provide persistent transgene expression and are maintained in cells extra-chromosomally.
This presentation will cover the fundamentals of non-viral vector design and development and will discuss their application in gene therapy. In particular it will focus on the basic science and the most relevant studies utilising S/MAR elements to episomally modify mammalian cells and tissues.
Stem Cells for Studying Heart Disease
Stem Cells for Studying Heart Disease Human pluripotent stem cells are unique in that they can be grown in the lab for long periods of time but can also be coaxed into become many other cell types, including beating heart cells. This ability provides new opportunities in biomedicine, including tissue repair, drug screening and gaining more understanding of genetic disease. We will discuss the progress in these areas. We will include some of our own work, which has used stem cells to investigate disorders such as Sudden Cardiac Death and Duchenne muscular dystrophy, including how these conditions may be treated with drug or gene therapy. Finally, the ability to produce stem cells at large scale using fully automated robotic platforms will be considered, as this will be important for industry to gain interest in the technology.
An overview of the immune system, the challenges and opportunities it provides for gene therapy
The immune system is vital for protection from infectious diseases. This involves the co-ordinated interplay of the innate and adaptive immune systems, which have evolved a variety of strategies to identify and eliminate ‘foreign invaders’. Innate responses provide the first line of defence, triggering inflammatory reactions that directly impede the pathogen and initiate the more specific, but slower, adaptive responses. Key adaptive components are antibodies produced by B cells, that target extracellular antigens; and T cells, whose antigen-specific receptors recognize peptides sampling the entire cellular proteome, presented at the cell surface by MHC proteins.
Gene therapists have widely adopted viral vectors to enable efficient gene transfer to target cells. However with in vivo delivery, the innate inflammatory responses can cause immediate toxicities, while pre-existing or newly induced adaptive immune responses may reduce the efficiency of gene transfer, or lead to elimination of successfully targeted cells, via recognition of viral proteins or transgene products perceived as ‘foreign’.
The immune system also provides important therapeutic opportunities for gene therapy. Indeed, inherited disorders of the immune system provided the first and best examples of successful gene therapies. Subtle antigenic differences between cancer cells and their normal counterparts can provide a basis for specific T cell recognition, and several gene therapy strategies aim to enhance the activation of endogenous anti-tumour responses, or to provide T cells with surrogate tumour-specific receptors to re-direct their cytotoxicity. Finally, GM viruses, bacteria and parasites will increasingly be used as vaccines against infectious diseases.
Development of therapeutic vaccines for cancer and chronic infections: from AML to HBV
We have previously demonstrated that tumour cells engineered to express immune co-stimulatory molecules and appropriate Th1 cytokines can induce immune mediated rejection of previously established tumours. We are now assessing this strategy in a Phase-I clinical trial in relapsed poor prognosis acute myeloid leukaemia (AML), consisting of vaccination with autologous AML cells that are genetically modified to express B7.1 (CD80) and IL-2.
An attractive alternative to cell based vaccination strategies is the direct use of tumour-associated antigens. However, successful vaccination against chronically experienced antigens requires the induction of not only appropriate, but also robust immunological responses. We have recently developed a new vaccination strategy based on Combined Adjuvants for Synergistic Activation of Cellular immunity (CASAC). CASAC contains TLR agonists able to engage complementary TLR pathways (MyD88 and TRIF), in combination with other synergistic stimulators of cellular immunity. CASAC vaccination with a single peptide (OVA or Trp2), induces IL-12 secretion, stimulation of Th1-biased CD4 T-cells, and high levels of antigen specific CD8 T cells (routinely >20% by tetramer staining, and in vivo antigen specific cytolytic activity, resulting in 2–3 log reduction in the number of antigen positive target cells). The magnitude of CASAC mediated immunity is substantially greater than can be achieved by other adjuvants (e.g. 10–100 fold greater than complete Freund's adjuvant). This strategy, which allows the breakdown of tolerance to self-antigens is now being developed for therapeutic vaccinations against cancer (WT1 in myeloid leukaemias) and infectious disease (chronic HBV infection).
Department of Medicine, University of California, San Francisco, 505 Parnassus Ave., Box 1270, San Francisco, CA 94143
Current allogeneic preparative regimens and post-transplantation immunosuppression are associated with considerable morbidity. Our goals are to develop safer and less toxic protocols for hematopoietic stem cells (HSC) transplantation for genetic disorders using a “positive/negative” selection approach. Genetic modification of HSC enabling in vivo amplification with successive cycles of low-dose chemotherapy after engraftment, may reduce the intensity of preparative regimens and immunosuppression required, and thereby reduce the risks of allogeneic transplantation.
The feasibility of allogeneic transplantation, without myeloablation or post-transplant immunosuppression, was tested using in vivo chemoselection of allogeneic HSC after transduction with a novel tricistronic lentiviral vector (MAGIT) containing P140K-O6-methylguanine-methyltransferase (MGMTP140K), HSV-thymidine kinase (TKHSV), and eGFP. This enables a) in vivo chemoselection of HSC by conferring resistance to benzylguanine (BG), an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU and, b) depletion of proliferating cells such as malignant clones or donor T cells mediating GVHD, by expression of the suicide gene TKHSV and Ganciclovir administration.
Non-myeloablative transplantation of transduced, syngeneic, lineage depleted (Lin-) BM in neonates resulted in 0.7% GFP+ mononuclear cells in peripheral blood. BG/BCNU, administered 4 and 8 weeks post-transplant, produced 50-fold donor cell enrichment. Transplantation and chemoselection of MHC-mismatched MAGIT-transduced Lin- BM produced similar expansion for >40 weeks. This transplantation approach was then successfully applied in Hbbth3 heterozygous mice by correction of β-thalassemia intermedia, without toxicity or GVHD. Negative selection, by administration of Ganciclovir resulted in donor cell depletion. Multi-lioneage-expansion of donor cells could be achieved with additional BG/BCNU treatment. These studies show promise for developing non-ablative transplant approaches using in vivo positive/negative selection.
Immune responses to vector and transgenes
In vivo delivery of viral vectors is associated with complex host immune responses. The most proximal and possibly most important response is activation of innate immunity which can cause acute toxicity and also drives subsequent adaptive immunity. The field has generally focused on effector responses that lead to the generation of antibodies to the vector capsid and T and B cell responses to the transgene product. We have learned that the quality and quantity of these responses are very much context-dependent. The structure of the vector capsid can greatly influence the activation of innate immunity and the transduction of antigen presenting cells. The state of the recipient such as systemic effects on immunity and local inflammation at the site of vector delivery can also play a role. Being aware of these responses and factors which can influence them is critical in proceeding safely and effectively into the clinic.
Assessing and avoiding insertional mutagenesis
Retroviral vectors have been successfully used for the correction of inherited immune diseases in clinical trials. However, with increasing efficiency and success in clinical retroviral gene therapy, the occurrence of vector-induced severe side effects has dramatically increased. Five patients of an otherwise successful X-linked severe combined immunodeficiency (SCID-X1) gene therapy trial acquired leukaemia due to insertional activation of cellular proto-oncogenes.
To assess the gene modified hematopoiesis we have performed qualitative and quantitative in vivo monitoring in four clinical gene therapy trials as well as in 1 gene marking study and 3 preclinical trials revealing that vector induced side effects, ranging from subtle and not clinically overt effects to clonal dominance and even leukemogenesis, have to be considered to occur as ‘normal’. Our studies of vector integration site (IS) distribution (>7000 IS) before and after transplantation showed unequivocally clonal dominance in a trial of ADA-SCID and in successful CGD gene therapy. In two SCID-X1 trials with nearly identical clinical protocols, the outcome differed dramatically. In the French study, individual integrants in or near LMO2 were found five times, compared to one in the British trial. In both trials, the CD34+ cell gene expression status revealed preferred vector integration in expressed genes and gene ontology analysis showed a significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. In the first successful gene therapy trial of chronic granulomatous disease (CGD), we also have observed very intensive insertional side effects resulting in activation of MDS1/EVI1, PRDM16 or SETBP1 5 months after therapy.
Incorporation of new sophisticated technologies like next generation/deep sequencing will help to substantially improve in depth analyses and to monitor individual and global clone contribution in a hitherto unexpected resolution. Prospective monitoring of vector integration sites in clinical gene therapy studies is feasible, can detect possible side effects of gene therapy in real-time and may gain new insights in basic mechanisms leading to specific clonal behaviour in vivo.
Max Delbrück Center for Molecular Medicine, Berlin, Germany
The Sleeping Beauty (SB) transposon is a nonviral, integrating vector system that offers robust, stable delivery of genes-of-interest in human cells and sustained transgene expression in preclinical models, thereby representing a promising instrument for gene therapeutic applications. We mapped large numbers of SB insertions by a LAM-PCR protocol and Illumina sequencing. In comparison with γ-retroviral, lentiviral and piggyBac and Tol2 transposon insertion sites, our data show a close-to-random insertion profile of SB without a preference for inserting into transcription units or transcriptional regulatory regions of genes. Introducing a bias into SB's target site selection properties may facilitate improvements in the safety profile of the SB system by lowering or eliminating potential genotoxic effects. We evaluated molecular strategies based on engineering at least one component of the transposon system, either the transposon vector DNA or the transposase (or factors interacting with either of these components) to be physically linked or interact with a heterologous DNA binding domain (DBD), which is to tether the transposase/transposon complex to defined sites in the human genome, and to facilitate integration of the transposon into adjacent sites. As DBDs, we evaluated the tetracycline repressor, SAF-A, the Rep protein of adeno-associated virus and zinc finger proteins with novel specificites. The engineered components were incorporated into the transpositional machineries, and their effects on target site distribution in human cells examined by analyzing insertion sites. The obtained data provide proof-of-concept for manipulating insertion profiles, and assist in the design of target-selected gene insertion systems with enhanced efficiency and specificity.
Targeted genome engineering with designer nucleases
Designer nucleases have developed into powerful tools to edit the genomes of complex organisms at will. Typically, zinc-finger nucleases (ZFNs) and recombinant meganucleases have been employed for this purpose. More recently, the DNA binding domains of transcription activator-like effectors (TALEs) derived from the bacterial pathogen genus Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. Overall, designer nucleases have been employed to trigger the targeted editing of genomes at over 60 different gene loci in more than 10 model organisms, including the fruitfly, zebrafish, mouse, rat and pig, as well as human stem cells.
In this educational talk I will explain the basics of designer nuclease mediated genome engineering, including a number of specific genome engineering strategies and some nuclease assembly platforms. Furthermore, some examples of successful nuclease-mediated genome editing in human stem cells will be discussed.
Gene Therapy for the Treatment of Heart Failure
Recent advances in the molecular basis of myocardial dysfunction, and the evolution of increasingly efficient gene transfer technology have placed heart failure within reach of gene-based therapies. Abnormalities in both human and experimental HF are a defect in sarcoplasmic reticulum (SR) function, which is responsible for abnormal intracellular Ca2+ handling. Deficient SR Ca2+ uptake during relaxation has been identified in failing hearts from both humans and animal models and has been associated with a decrease in the activity of the SR Ca2+-ATPase (SERCA2a). Over the last ten years we have undertaken a program of targeting important calcium cycling proteins in experimental models of heart by somatic gene transfer. This has led to the completion of a first-in-man phase 1 clinical trial of gene therapy for heart failure using adeno-associated vector (AAV) type 1 carrying SERCA2a. In this Phase I trial, there was evidence of clinically meaningful improvements in functional status and/or cardiac function which were observed in the majority of patients at various time points. Safety profiles of AAV gene therapy along with the positive biological signals obtained from this phase 1 trial has led to the initiation and recent completion of a phase 2 trial of AAV1.SERCA2a in NYHA class III/IV patients. In the phase 2 trial, gene transfer of SERCA2a was found to be safe and associated with benefit in clinical outcomes, symptoms, functional status, NT-proBNP and cardiac structure. Recent successful and safe completion of the CUPID trial along with the start of more recent phase 1 trials usher a new era for gene therapy for the treatment of heart failure.
Dubowitz Neuromuscular Unit, UCL Institute of Child Health
Duchenne Muscular Dystrophy (DMD) is a chronic X-linked genetic disorder characterized by an early onset of muscle degeneration due to the absence of functional dystrophin protein. Damaged muscle fibres can initially be regenerated by a muscle-resident stem cell population called satellite cells (SCs), which underlie the myofibre basal lamina. Following activation, SCs proliferate extensively and fuse with the damaged fibre, however, some SCs return to quiescence to maintain the stem cell pool. As dystrophic muscle is progressively wasted, regeneration eventually fails resulting in premature death. Long-term correction of dystrophic muscle and SCs requires the insertion of a functional dystrophin gene copy into the mutated genome. Lentiviral vectors (LVs) represent suitable candidates for DMD gene therapy, as they stably integrate their genome into dividing and non-dividing cells, thereby mediating long lasting expression in both proliferating myoblasts and post-mitotic myofibres. Importantly, physiological protein levels and restriction of transgene expression to only muscle tissue is favored to avoid off-target transduction and to circumvent associated safety concerns.
This study compares LVs carrying either a strong viral, a muscle-specific or a housekeeping promoter, and assesses variances in expression levels, integrated copy numbers and persistence of transgene expression. Tissue-specificity of promoters will be discussed. Transgene expression mediated by randomly integrated viral copies did not alter the potential of SCs to differentiate and self-renew in vitro and in vivo. Notably, human myoblasts showed significantly enhanced transduction efficiencies compared to murine myoblasts, thus underpinning the great translational potential of LVs in the area of muscle degenerative diseases.
Gene therapy for immunodeficiency
At the start of the 1990s, the first clinical trials of gene therapy were attempted for an inherited severe combined immunodeficiency (SCID) caused by deficiency of the intracellular enzyme adenosine deaminase. In the absence of definitive treatment, SCID of any molecular type is usually fatal within the first year of life, although patients with ADA deficiency can be supported by administration of exogenous bovine enzyme. Even so, this is often only partially effective, and is extremely expensive. The rationale for the development of gene therapy for SCID therefore derives from the severity of the illness, the inadequacy of conventional therapy, and the considerable morbidity and mortality associated with stem-cell transplantation, particularly from a mismatched donor. Efficacy in these early studies was limited, but a decade further on, gene transfer technology and cell handling protocols had been refined sufficiently to produce real clinical benefit. Four recent studies have demonstrated highly effective gene therapy for the X-linked form of SCID (SCID-X1) and ADA deficiency, using retroviruses to deliver the therapeutic genes into haematopoietic stem cells ex vivo. Bearing in mind the outcome and adverse effects of conventional therapy, these are remarkable results and the first clear indication that gene therapy can offer a cure for some human diseases. In a few patients the treatment has failed, indicating that there is more to learn about the effective dose of corrected cells and the potential for host factors to influence immune cell development.
Many different types of vector have been tested in laboratory experiments to deliver therapeutic genes, and their effectiveness is largely determined by the host and tissue type. For stable gene transfer to dividing cells, such as haematopoietic cells, the new genetic material has to be retained through cell division and passed on to daughter cells. Although retroviruses are highly effective for this, their dependence on chromosomal integration brings with it the risk of inadvertent gene activation or inactivation. Having initially achieved successful immunological reconstitution, several patients with SCID-X1 (out of a total of 19 treated worldwide) developed T cell lymphoproliferative disease up to 6 years after the gene therapy procedure. In four of these patients, the enhancer sequences in the retroviral vector, which are responsible for effective transgene expression, had activated the LMO-2 proto-oncogene. There are likely to be other factors that contributed to cell transformation, but they have not yet been defined. It is therefore unclear whether all patients are at significant risk, or whether this is more pronounced in a few with SCID-X1.
Fortunately, it is likely that much can be done to improve efficiency and safety of current protocols, and these developments have recently entered clinical testing for SCID and other disorders. The design of vectors used for gene delivery is clearly important, and modifications are possible that limit the risks of mutagenesis, and direct more physiological gene expression profiles. Ultimately, the development of homologous recombination or gene repair to accurately correct genetic mutations, target safe havens within the genome, or the construction of mitotically stable extrachromosomal vectors, would obviate many of these problems, but current technologies are inefficient.
Gaspar H.B., Cooray, S., Gilmour, K.C., Parsley, K.L., Adams, S., Howe, S.j., Ghonaium, A.A., Bayford, J., Brown, L., Davies, E.G., Kinnon, C., Thrasher, A.J. Long term persistence of a polyclonal T cell repertoire after gene therapy for X-linked severe combined immunodeficiency. Science Translational Medicine 2011 Epub Aug 2011.
Gaspar, H.B. Cooray, S., Gilmour, K.C., Parsley, K.L., Zhang, F., Adams, S., Bjorkegren, E., Bayford, J., Brown, L., Davies, E.G., Veys, P., Fairbanks, L., Bordon, V., Petropolou, T., Kinnon, C., Thrasher, A.J. Hematopoietic stem cell gene therapy for adenosine deaminase-deficient severe combined immunodeficiency leads to long-term immunological recovery and metabolic correction. Science Translational Medicine 2011 Epub Aug 2011.
Moving forward with cystic fibrosis gene therapy
Although we and others have demonstrated proof-of-principle for correction of the cystic fibrosis (CF)-specific chloride transport defect in airways of CF patients after gene therapy, it is currently unclear whether gene therapy can improve clinically relevant features of CF lung disease. This question can only be addressed in trials that are designed to ensure prolonged expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and that are sufficiently powered to detect clinical benefit. Over the past decade the UK CF Gene Therapy Consortium has worked towards conducting a large multi-dose phase 2B gene therapy study to assess whether CFTR gene transfer ameliorated CF lung disease. This work has been carried out in several phases: (1) Pre-clinical selection of the most efficient gene transfer agents suitable for repeated administration into lungs of CF patients, (2) Development of a plasmid supporting prolonged gene expression in airways, (3) Assessment of safety and efficacy of the formulation in a single-dose dose-ranging phase 2A trial, (4) Selection of patients and outcome measures in a 2-year longitudinal Run-in study, (5) Conductance of a GLP-mouse multi-dose toxicology and biodistribution study and (6) conductance of a GLP-like multi-dose sheep study. Data illustrating key findings from the various phases of the programme will be presented and the design of the multi-dose phase 2B study due to start in 2012 will be discussed.
Cell Therapy Approaches for Lung Diseases
Many lung diseases remain without cure and result in substantial morbidity and mortality. New potential therapeutic approaches are suggested by recent developments in stem cell biology and in cell therapy strategies. However lung is a complex anatomic and physiologic organ and there are significant barriers to applying cell-based therapies. To be reviewed will be the role of endogenous lung progenitor cells in injury repair and recent attempts to affect repair with administration of exogenous stem and progenitor cells obtained from bone marrow and from cord blood. Novel approaches to grow functional lung tissue ex vivo will also be examined. Finally, the ability to modulate inflammatory and immune responses in the lung by administration of exogenous stem cells will be discussed. This is the most promising strategy to date and has already led to the first clinical trial in the US evaluating cell-based therapy for chronic obstructive pulmonary disease (emphysema).
Monitoring Gene Therapy by Non-Invasive Optical Imaging
Caliper Life Sciences (formerly Xenogen Corporation) is a leader in the field of small animal optical imaging. Our technology allows biological processes, including gene expression that is both temporally and spatially defined (i.e., occurring in defined tissues and organs within an animal) to be non-invasively monitored, both three dimensionally and in real-time. Genes encoding optical reporter proteins (luciferases or fluorescent proteins) can be delivered in vivo to mimic gene therapy techniques where successful delivery of a construct results in light emission that can be visualized through the tissues of a live animal using specialized imaging equipment and software designed and built by the company. Furthermore, this technique is equally applicable to imaging of fluorescent dyes and particles (e.g., Qdots), allowing fluorescently tagged biological events, such as tracking of proteins produced by gene therapy, to be three dimensionally visualized both independently and in combination with genetically tagged events. To date, Caliper's technology has been used to facilitate drug discovery and innovative biological research in areas such as oncology, infectious disease, neurology, inflammation, metabolic disease, toxicology and stem cell research.
Molecular imaging of gene expression: NIS as a reporter gene
Molecular imaging of gene expression in vivo requires a reporter gene, a tracer and a scanner capable of detecting the presence of the tracer in the live, anaesthetised animal. This methodology has been extensively used in gene therapy to determine the biodidstribution of gene expression allowed by viral and non-viral gene delivery vectors and to study the specificity of promoter fragments driving the expression of reporter genes. In term of methodology, both optical and isotopic imaging modalities are available. In this presentation, I will focus on in vivo molecular imaging using radiotracers as probes. More specifically, I will present examples of imaging using the Na/I symporter (NIS) as a reporter gene. This membrane protein induces the cellular uptake of iodide and, through the utilisation of relevant radio-iodide isotope, Positron emission tomography (PET) or Single Photon Emission Tomography (SPECT) scanner can be used to detect the biodistribution of iodide in the experimental subject. In addition to its potential as a reporter gene, NIS can also be used as a therapeutic transgene in cancer gene therapy, promoting targeted radiotherapy. Examples of the combination of imaging and therapy mediated by NIS will be presented in the context of oncolytic adenovirus.
San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy
Site-specific integration may overcome the major hurdles of current gene transfer approaches, namely insertional mutagenesis and uncontrolled transgene expression. However, suitable genomic sites for transgene integration and the dynamic interplay that occurs between the integrated transgene and the targeted locus have remained elusive. Here, we address these issues by studying the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc finger-nucleases, on two genomic loci of human cells, CCR5 and AAVS1. Analyses of the loci performed before and after integration allowed on site tailoring of the expression cassettes to achieve robust and uniform transgene expression without inducing detectable transcriptional perturbation of the targeted locus and its flanking genes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be exploited in novel experimental paradigms and safer therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.
Therapeutic strategies with integration-deficient lentiviral vectors
Insertional mutagenesis-related complications constitute a major risk factor in gene therapy. One of several possible approaches to reduce this hazard is to modify integrating vectors, minimising genomic insertion while other positive features remain unaffected. In the case of lentiviral vectors, this has been possible through the use of class I integrase mutants. These are affected at integrase residues critical for genomic integration but irrelevant for other functions of this pleiotropic enzyme, including reverse transcription, nuclear import and morphogenesis. Such integration-deficient lentiviral vectors (IDLVs) transduce cells efficiently and are converted into metabolically stable circular episomes. The episomal circles support transient gene expression in proliferating cells, stable and high-level gene expression in quiescent tissues, homologous and site-specific recombination, and transposition. We are currently exploiting IDLVs for stable transgene expression in the CNS and for gene repair by homologous recombination in the haematopoietic system. Direct injections of IDLVs into the striatum and the spinal cord have resulted in transgene expression efficiencies similar to those obtained with standard lentiviral vectors. An integration analysis in the injected striatum has confirmed highly reduced frequencies of genomic insertion by IDLVs, both in wild-type rats and in a drug-induced Parkinson disease model. Finally, we have used IDLVs to deliver a zinc-finger nuclease and gene repair template targeting the mouse Prkdc scid mutation. Gene correction and phenotypic rescue have been obtained in scid fibroblasts, and experiments on haematopoietic progenitors are on-going. This work has been supported by Genoma España and the EU (NEUGENE, PERSIST and CLINIGENE).
San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET)
Metachromatic Leukodystrophy (MLD) is a demyelinating lysosomal storage disorder due to deficiency of arylsulfatase A (ARSA) lacking effective therapies. According to relevant preclinical data, a Phase I/II clinical trial of hematopoietic stem cell (HSC) gene therapy employing lentiviral vectors (LVs) was recently approved for the treatment of MLD patients and the first patients were treated in our Institute since May 2010. Upon administration of a myeloablative dose of busulfan, the patients received autologous BM HSC freshly and efficiently transduced with ARSA-encoding LVs. No serious adverse events were reported in the peri- and post-transplant phase; a sustained engraftment of the transduced cells and a sustained, above-normal ARSA activity were observed in all blood lineages and in BM CD34+ cells up to the longest follow up. These marking levels far exceed those reported in previous trials using gamma-retroviral vectors or LV and, if maintained long-term, would suggest a substantial gain in gene marking efficacy. Thus far, the follow up of the first treated patients demonstrates the short-term safety and feasibility of the procedure, but only long-term evaluation will establish its safety and efficacy.
Immune responses to capsids and transgene products in pre-clinical and clinical trials
Immune responses to transgene products potentially complicate successful applications of gene therapy. A wealth of knowledge has been established in murine models regarding T cell responses to transgene products in the setting of adenoviral vectors – which activate T cells – and AAV vectors – which suppress T cells. We recently focused on T cell responses to AAV-encoded transgenes in several translational programs and have made some interesting observations. The apparent immune tolerance afforded to AAV in mice is not as robust in larger animal models and humans. T cell responses based on classic assays such as the ELISPOT are detected against capsid and transgene in human and nonhuman primates although their relevance to outcome is unclear. The notion of central tolerance which should lead to the deletion of self-reactive T cells in the thymus has guided experimental and clinical trial design in the context of immunotoxicity to non-self transgene products. Our studies in humans indicate that the assumptions on which these decisions are based may be flawed and should be reconsidered. A greater understanding of the biology and relevance of capsid and transgene T cells would facilitate more rational attempts to circumvent problems such as those based on vector engineering or immune modulation.
Preclinical assays to assess genotoxicity and “phenotoxicity”
Clinical studies indicate the potential of gene-modified stem cells to correct genetic disorders of hematopoiesis. Novel technologies for gene delivery have been introduced, but there is a paucity of approaches to assess the risk of potential adverse events. To develop platforms for “transgene toxicology”, we use a combination of murine models of bone marrow transplantation (BMT), cell-based assays, and transcriptome microarrays. Thereby, we have identified several risk factors contributing to the potential genotoxicity of integrating gene vectors: the “stemness” of the target cells, culture conditions, strong enhancer-promoter in the integrated transgene, and the vectors' insertion pattern. Our recent studies also underline the issue of gene disruption by vector splice sites, thus potentially causing transformation by haploinsufficiency of tumor suppressors. Based on these data, we develop integrating gene vectors that reduce the risk of insertional transformation by modifying the expression cassette and the insertion pattern of the transgene. A murine model of an inherited aplastic anemia caused by deficiency of the cytokine receptor Mpl showed the potential collaboration of ectopic transgene expression (“phenotoxicity”) and genotoxicity in promoting leukemia, and also demonstrated the importance of milieu conditions for aberrant cell proliferation. Furthermore, we introduced sensitive assays to uncover dose-dependent off-target effects of DNA-modifying enzymes such as transposases or recombinases. In conclusion, a proactive analysis of potential off-target effects, including the mechanisms underlying the creation, expansion and homeostatic control of transformed clones, is possible. Quantitative assay systems are required to assess the practical value of prevention strategies.
Advancing your research career
The talk will introduce Vitae, the research council funded organisation established to champion the personal, professional and career development of doctoral researchers and research staff in higher education institutions and research institutes. It will briefly cover the work Vitae does to promote researcher careers and focus on the Researcher Development Framework - a new tool designed to aid researchers in developing the skills that will help them progress in their career.
European Research Council: supporting independent research careers
The European Research Council (ERC) is a European funding initiative, designed to support the best scientists, engineers and scholars in Europe. The ERC's mandate is to encourage the highest quality research in Europe through competitive funding and to support investigator-initiated frontier research across all fields of research, on the basis of scientific excellence.
One type of grant that is currently available to support researchers is the ERC Starting Independent Researcher Grant. The objective of this funding scheme is to support the independent careers of outstanding researchers, who are at the stage of establishing or consolidating their first research teams or programmes. It is open to researchers of any nationality who have been awarded their first Ph.D. at least 2 and up to 12 years prior to the publication date of the call for proposals.
This presentation will provide information to potential applicants on: the eligibility criteria; assessing who would be a competitive candidate; preparing an application; and the evaluation process.
Science Communication
Sense About Science is a small charity that equips people to make sense of science and evidence. This session will discuss science related controversies in media reporting and what scientists can do to respond to misconceptions in public discussions. Do you think it is important for good science and evidence to be communicated to a wider audience? What happens when research announcements go wrong; statistics are manipulated; risk factors are distorted; or discussions become polarised? Not yet the leaders in the field what can early career researchers do to encourage good science and evidence in the public domain? Using examples of myth-busting and evidence hunting projects of the Voice of Young Science network of early career researchers, Julia will discuss the impact and importance of standing up for science.
Using adult tissues for generating new organs: autologous cell replacement therapy for diabetes
Pancreatic islets replacement therapy for treating insulin dependent diabetic patients is severely restricted by the shortage in tissue availability from cadavers and by the subsequent need to prevent the implanted cells immunological rejection.
Our working hypothesis is that adult cells retain a considerable level of plasticity which allows them to switch their original developmental fate and function in a process dictated by transcription factors functioning as master regulators of organogenesis. In this case diabetic patients can use their own adult organs as autologous pancreatic progenitors.
We present the efficacy of irreversibly activating functional endocrine pancreas in adult human liver cells by transient ectopic expression of pancreatic transcription factors, primarily Pdx-1. These developmentally shifted human liver cells produce insulin, process the hormone and secrete it in a glucose regulated manner. The cells ameliorate diabetes when implanted in immunodefficient SCID-NOD mice for long periods. Pdx-1 plays a dual role in the reprogramming process; while dictating the activation of the pancreatic lineage it also induces dedifferentiation of the adult precursor cells. The restricted developmental regression, but not the separate activation of pluripotency (IPS cells) is obligatory to the activation of the alternate repertoire. Using a lineage tracing approach we demonstrate the adult origin of the precursor cells, and the universality of the transdifferentiation process, as it occurs also between developmentally unrelated tissues.
Mesenchymal to Epithelial Transitioning Enhances the Reprogramming of Human Non Pancreatic Endocrine Cells towards Beta Cells
Islet transplantation is seen as a potential therapy for type 1 diabetes that would circumvent problems related to hypoglycaemia and the late complications of the disease that are associated with the current use of exogenous insulin injections. However, multiple donor pancreases are required to treat a single patient as the effectiveness of the initial transplant decreases with time and further transplants are required. The overall aim of this study was to generate a top-up supply of islet tissue from the exocrine part of the pancreas that is normally discarded during the human islet isolation procedure. The idea was that as the recipient's graft begins to fail the top-up supply would be readily available, and since they were derived from the original donor, would have many advantages over cells from additional donors. The non-endocrine pancreatic cells (NEPCs) were reprogrammed using four transcription factors (TFs), namely Pdx1, Ngn3, Pax4 and MafA that play a critical role in the development of the endocrine pancreas. This led to abundant expression of glucagon (alpha cell) but little expression of insulin, IAPP, somatostatin or other islet markers. However, increased insulin and reduced glucagon expression was observed when the four TFs were applied along with 3 growth factors in serum free media. The lack of serum caused the cells to undergo a mesenchymal to epithelial transition (MET) that was crucial for efficient reprogramming. Thus, as seen in other systems, MET plays an important role in the transdifferentiation (and reprogramming) of adult cell types.
Laboratory for Regenerative Medicine, Department of Surgery, West Forvie Building, Robinson Way, University of Cambridge, Cambridge, CB2 0SZ, UK
Induced pluripotent stem cells (iPSCs) offer the possibility of generating limitless quantities of cells for autologous cell based therapy aimed at treating a broad range of inherited and degenerative diseases. However, the use of iPSCs in the context of genetically inherited human disorders requires development of a method to correct the genetic abnormality in a manner fully compatible with clinical applications. Here, we show that a combination of Zinc finger nucleases (ZFN) and piggyBac technology can achieve simultaneous bi-allelic correction of a point mutation in the SERPINA1 gene responsible for α-1 antitrypsin deficiency (AAT) to restore normal AAT protein structure and function in iPSC derived liver cells. This approach appears to be quicker and more efficient that any gene targeting technology currently available and crucially prevents contamination of the host genome with residual prokaryotic sequences. Our results therefore provide proof of principle for the potential of combining hIPSCs and gene therapy in generating cells for disease treatment.
Development of Human Embryonic Stem Cells for Therapeutic Applications
HESC-based regenerative cell therapies require 1) evidence for reliable production and quality control of product manufacturing, 2) rigorous safety testing in preclinical models, and 3) the design of clinical trial protocols that assess the safety and benefit of the therapy in appropriate patient populations. GRNOPC1 is a population of allogeneic cells containing oligodendrocyte progenitors derived from characterized, dedicated, human embryonic stem cell banks. GRNOPC1 induces myelination of axons in rats with spinal cord injuries and in Shiverer mice, which lack compact myelin, and also produces numerous neurotrophic factors such as midkine, BDNF, and activin. Extensive preclinical studies were performed to determine the distribution of GRNOPC1 as well as any potential toxicities after injection near the thoracic injury epicenter. A Phase I clinical trial to assess the safety of GRNOPC1 in patients with subacute, complete ASIA A, thoracic injuries whose last fully preserved neurological level is T3 to T11 is currently enrolling subjects. The presentation will include a discussion on the design of the clinical studies to assess the safety of GRNOPC1 in patients with spinal cord injury and the risk mitigation strategies incorporated in the trial. Additional application of GRNOPC1 in other CNS disorders will be covered.
Cancer Stem Cells: a source of new therapeutic targets?
The existence of a small self-renewing cell population within cancers, which is responsible for tumour recurrence post-therapy, has been proposed for more than 100 years. In prostate cancer, this tumour initiating or cancer stem cell (CSC) population consists of primitive, undifferentiated basal cells, which are resistant to conventional treatments. However the CSCs can differentiate both in vivo and in vitro into the luminal cell populations characteristic of most untreated prostate cancers. The CSC populations also exhibit resistance to ionising radiation and the genotoxic effects of DNA damaging agents.
Successful treatments for CSCs have required the development of not only new agents, but also different assays for agent efficacy. The normal rules of rapid cancer cell proliferation do not apply in the SC compartment. Successful therapeutic molecules should target SC survival or promote the CSCs from their protected microenvironment, for example by differentiation.
On the basis of a microarray gene expression screen, we have identified a series of CSC therapeutic targets and signalling pathways associated not only with the stemness of prostate cancers, but particularly with properties which permit the cancer stem cell to survive outside of its normal niche e.g. at metastatic sites. Inhibition of these functions both prevents new colony formation in vitro (clonogenicity) and also tumour initiation in vivo.
While we have yet to learn the best means of applying these therapies in man, they represent the first steps in a more all-encompassing treatment which seeks to eliminate cancer rather than simply to palliatively reduce tumour volume.
Exploring and exploiting TALE nucleases (TALENs) for targeted genome engineering
Designer nucleases have developed into powerful tools to specifically edit the genome of various cell types, including multipotent and pluripotent human stem cells. Typically, zinc-finger nucleases (ZFNs) and recombinant meganucleases have been used for this purpose. More recently, the DNA binding domains of transcription activator-like effectors (TALEs) derived from the bacterial pathogen genus Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. This novel DNA binding domain is composed of an array of highly conserved 34 amino acids repeats, and solely two residues within each repeat specify DNA binding. Based on the modular nature of the repeats, the ‘one repeat to one base’ code enables the engineering of customized TALE repeat arrays that recognize a user-defined target sequence. We have characterized the cleavage parameters for efficient TALEN-mediated genome editing in human cells and designed TALENs that target an EGFP marker gene and the human loci AAVS1, CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated toxicities. Moreover, CCR5-specific TALENs revealed considerably lower off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform may enable the design of nucleases with higher specificity. Given both the ease with which TALENs can be engineered and their superior activity-to-toxicity profile, TALENs are likely to have a significant impact on targeted genome engineering in the context of human gene therapy.
Development of stem cell therapy for the treatment of retinal degeneration
Retinal degenerations leading to loss of photoreceptors are a major cause of untreatable blindness in the UK. Inherited retinal dystrophies affect 1 in 3,000 of the population, and age-related macular degeneration (AMD) affects 1 in 10 people over 60 yrs. Currently no treatments restore lost photoreceptor cells and visual function and thus there is a need for new therapeutic approaches. As new photoreceptors need only make short, single synaptic connections to the inner retinal circuitry to contribute to visual function, retinal repair by photoreceptor transplantation represents one of the most feasible types of CNS repair. We have previously discovered that transplantation of photoreceptor precursor cells at a specific stage of development results in their integration and subsequent differentiation into photoreceptors that form synaptic connections (Maclaren et al Nature 2006). We have now demonstrated improvements in vision following transplantation in mice with visual deficits. Here I will discuss this work and will outline the many challenges of developing stem-cell derived photoreceptor transplantation for the treatment of retinal disorders and how we are addressing some of these issues.
Utilization of iPS cells for retinal degenerative diseases
The induced pluripotent stem (iPS) cell is expected as unlimited sources for cell therapies, a tool for drug development and also an ideal material to understand diseases. We have established the culture method that induces directed differentiation of human ES cells into mature retinal cells. Applying this method to human iPS cells, we could obtain photoreceptors and retinal pigment epithelial (RPE) cells.
Photoreceptor cells derived from iPS cells of retinitis pigmentosa (RP) patients' are worth investigating the individual mechanism of photoreceptor cell death and evaluating drugs for cell rescue. We evaluated rod photoreceptors derived from iPS cells from 5 patients with different genetic subtypes of RP. Rod photoreceptors differentiated from patient iPS cells degenerate in vitro, while those of wild-type cells maintained the cell number for this period. The effects of vitamins were different from patient to patient, which suggests that iPS cells can be used for the personalized medicine in the future.Retinal pigment epithelial (RPE) cells derived from iPS cells of age-related macular degeneration patients' will be a good candidate for the cell source of transplantation, since they are supposed to avoid the immune rejection. We are now adapting all the culture processes to the clinical level and preparing SOP. Furthermore, by evaluating purity and tumorigenicity of the RPE cell sheet, attempts are made to secure safety of this cell therapy.
Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy
The eye is small, enclosed and immune-privileged and therefore an attractive target for gene therapy. Given their safety and versatility, vectors derived from the small adeno-associated virus (AAV) are the most promising for retinal gene transfer. AAV serotypes have been exploited to maximize the efficacy of gene transfer to specific retinal cell types, including challenging targets like the photoreceptors which are the site of many specific inherited retinal degenerations. Sophisticated strategies to tailor gene expression to specific retinal targets or to overcome AAV limited cargo capacity are being developed. This has allowed to successfully treat a variety of animal models of retinal diseases, inherited either as dominant or recessive. Evidence of the safety and efficacy of subretinal administrations of AAV in patients with a rare form of congenital blindness highlights the potential of this strategy for treating retinal diseases.
Cell therapy with genetically modified lymphocytes in onco-hematology
Adoptive cellular immunotherapy has produced important clinical results in the treatment of cancer both in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and in the autologous setting, in the treatment of metastatic melanoma. Major hurdles limiting adoptive T cell therapy relate to toxicity (ie: graft-versus-host disease-GvHD in allo-HSCT), feasibility and efficacy (ie: difficulty in expanding rare, high-avidity tumor-specific CTLs). Gene transfer technologies may allow to overcome these hurdles. In phase I-II clinical trials, we showed that the transfer of a suicide gene into donor lymphocytes, prior to infusion to transplanted patients, allows to control GvHD, thus taming the toxicity of allo-HSCT. When applied to genetically half-matched (haploidentical) HSCT, the suicide gene strategy resulted in rapid and effective immune reconstitution, leading to increased survival of transplanted patients of all ages. In the autologous setting, the infusion of tumor-specific T-cells has produced exciting clinical results in patients affected by metastatic melanoma. However, translation of this approach to other types of cancer proceeded at a low pace, possibly due to the difficulty in isolating and expanding tumor-reactive T cells from less immunogenic tumors. The significant advances in gene transfer technologies developed in the last decade, offer new tools to enforce natural T cells, enabling to generate high numbers of “supernatural” tumour-reactive T cells from virtually every cancer patient. Supernatural T cells may express clonal receptors providing new specificities, factors increasing T-cell performance or safety factors enabling their elimination in case of toxicity. Our group developed a novel genetic approach that allows for the first time the complete editing of T cell specificity, by combining the disruption of the endogenous alfa and beta TCR chain genes with the transfer of a tumor-specific TCR. We designed Zinc Finger Nucleases promoting the disruption of both endogenous TCR β and α chain genes. ZFN-treated lymphocytes lacked CD3/TCR surface expression and were stable in culture with IL-7 and IL-15. Upon lentiviral transfer of an engineered TCR for the WT1 tumor antigen, these TCR-edited cells expressed the new tumor-specific TCR at high levels, were easily expanded to near-purity and proved highly effective and specific in recognizing and killing leukemic cells.
Clinical results, challenges and advances recently made to improve the potency and specificity of adoptive immunotherapy with genetically modified lymphocytes to treat cancer will be discussed.
Stem Cell Models for Studying Cardiotoxicity and Cardiac Disease
The ability to maintain human pluripotent stem cells (hPSCs) for extended periods of time in culture and yet induce their differentiation to a wide variety of lineages offers new opportunities in biomedicine, including regenerative medicine, drug screening and disease modelling. We will discuss the progress our lab has made in producing human induced pluripotent stem cell (hiPSC)-based in vitro models of cardiac disease. Examples will include electrical (e.g. Long QT Syndrome) and structural (e.g. Duchenne muscular dystrophy) disorders. We will discuss how hiPSC lines carrying these mutations can be differentiated into functional cardiomyocytes that can be used to evaluate different drug- and gene-based therapies. Finally, the ability to scale hPSC culture and differentiation on fully automated robotic platforms will be considered.
Jennerex Inc., San Francisco, CA
JX-594 is a first-in-class targeted oncolytic poxvirus engineered to selectively replicate in and destroy cancer cells with cell cycle abnormalities and epidermal growth factor receptor (EGFR)/ras pathway activation. Direct oncolysis plus granulocyte macrophage – colony stimulating factor (GM-CSF) expression also stimulates tumor vascular shutdown and anti-tumoral immunity. Expression of the β-galactosidase marker transgene allows for monitoring of JX-594 replication in patients. Over 115 cancer patients have been treated by intravenous (IV) and/or intratumoral (IT) injections on one of eight Phase 1 or Phase 2 trials to date, including ongoing Phase 2 trials in patients with primary liver cancer. Treatment with JX-594 by either route of administration was well-tolerated at the 1 × 109 plaque forming units (pfu) dose level, representing the maximum tolerated dose (MTD) following IT JX-594 injection; the MTD was not reached following IV JX-594 infusion. The most common adverse events were dose-related flu-like symptoms generally resolving within 24 hours following JX-594 administration. JX-594 was detected acutely in the serum following either IT or IV treatment. Delayed JX-594 reappearance (day 3–15) in the bloodstream after initial clearance was observed in a subset of patients and is consistent with replication at the tumor sites and release into the blood. Expression of JX-594 transgenes was observed in patients treated by IT or IV administration; GM-CSF could be detected in post-treatment serum and correlated with induction of white blood cells. Furthermore, induction of antibodies to the β-galactosidase marker protein was observed in patients treated with JX-594 IV or IT, demonstrating gene expression in vivo. Objective radiographic tumor responses (RECIST and Choi criteria) and tumor vascular disruption have been observed in patients with metastatic cancers following JX-594 treatment IT (injected and non-injected tumors) or IV; Choi (necrotic) responses have been observed in approximately two-thirds of patients treated on Phase 2 trials. Finally, multiple long-term survivors (>/= 12 months in treatment-refractory patients) have been reported on JX-594 trials; in a randomized liver cancer study, patients treated with high dose JX-594 exhibited a survival advantage when compared to the low dose group.
Virotherapy in the UK
In the last 5 years, we have witnessed a huge increase in clinical trials involving oncolytic virotherapy (OV). As a result of this initial experience, the regulatory steps involved in activating new studies are relatively straightforward. Importantly, for a number of promising agents we have moved beyond initial safety and tolerability studies in to phase II/III efficacy studies. In keeping with a true translational approach, this has been driven by preclinical laboratory data demonstrating mechanism-based interactions between OV and standard anti-cancer agents (chemotherapy, radiotherapy, targeted drugs).
My presentation will focus on recently completed or ongoing studies from the UK involving oncolytic reovirus, vaccinia virus and herpes simplex virus. In particular, data that have informed the design of phase II/III studies will be discussed. In addition, analysis of mechanisms of systemic virus delivery in clinical studies will be highlighted. These data support previous preclinical studies that have shown that cell-based delivery of OV allows the virus to evade immune neutralization and enhances tumour delivery. In the UK, these studies have benefited from close collaboration between a network of clinician scientists who are able to support laboratory and clinical programmes. Work from a collaborative network of clinician scientists at The Institute of Cancer Research (Kevin Harrington), Leeds Institute of Molecular Medicine (Alan Melcher, Richard Vile) and University of Surrey (Hardev Pandha) will be presented.
Molecular design of improved adenoviruses
Among different viruses, adenovirus is still a promising candidate for cancer virotherapy, having a gene expression program amenable to tumor-specific control and a safe track of clinical experience. However, major limitations have been identified related to tumor targeting, intratumour spread, and immune neutralization: blood cells and proteins that interact with the virus capsid abrogate systemic delivery to tumors; cellular and non-cellular components of the stroma inhibit virus spread, immune responses clear the virus. Arming oncolytic adenoviruses with transgenes can be used to tackle spread and immune rejection issues but requires appropriate backbones to avoid oversizing the genome while keeping all virus genes. Examples based on minimal E2F-binding sites of universal application armed with fusogenic and matrix-degrading transgenes.
Fetal and neonatal gene therapy
Over the past ten years gene therapy has shown clear clinical benefit in several diseases for which conventional medicine offers no treatment. Several clinical trials of gene therapy for single gene disorders have recruited young patients since older subjects may have suffered irrevocable pathological changes or have not survived because of early onset and lethality of the condition. The concept of perinatal gene therapy is an extension of this principle: Diseases in which irreversible changes occur very early in life may be prevented by gene supplementation, or repair, in the fetus, in the associated maternal tissues or in the neonate. Several recent preclinical studies in small and large animal models lend weight to the feasibility and motivation for very early gene therapy intervention.
Zooming in on the target: transductional and transcriptional targeting
The therapeutic index of gene delivery vectors can be significantly improved by targeting gene delivery at the level of transduction, transcription or genomic integration. Transductional targeting can be achieved by modifying the vector capsid or envelope in order to deliver the therapeutic gene into the appropriate target cells. We have previously demonstrated that AAV9 vectors have an exquisite cardiac tropism that can be exploited to develop improved gene therapy approaches for cardiovascular disease. Moreover, this cardiotropism of AAV9 offers unprecedented opportunities to assess the function of genes or miRNAs in the heart without having to resort to the generation of transgenic mouse models. We have established proof-of-concept that AAV9-mediated delivery of TSP-2 protects against age-related or inflammatory cardiomyopathy. Transductional targeting to the liver could be achieved using either AAV8 or AAV9 serotypes yielding supra-physiologic clotting factor IX expression levels. To achieve transcriptional targeting we validated a novel data-mining algorithm, that allowed us to identify specific combinations of evolutionary conserved cardiac-specific or liver-specific transcription factor binding sites (TFBS). These TFBS combinations represent common denominators among highly expressed cardiac-specific or liver-specific genes, respectively. Consequently, robust cardiac or liver-specific gene expression could be achieved using AAV vectors containing these de novo designed tissue-specific expression cassettes. 1. VandenDriessche, et al. J Thromb Haemost. 5: 16–24 (2007) + Commentary: Arruda V.R. and Xiao, W. It's all about the clothing: capsid domination in the adeno-associated viral vector world. J. Thromb Haemost. 5: 1–4 (2007). 2. Swinnen, et al. The absence of thrombospondin-2 causes age-related dilated cardiomyopathy. Circulation, 120(16):1585-97 (2009). 3. Swinnen, et al. Thrombospondin-2 prevents cardiac injury and dysfunction in viral myocarditis through the activation of regulatory T-cells (submitted). 4. Petrus, et al. Combinations of tissue-specific transcription-factor binding sites identified de novo using a novel bioinformatics algorithm yield robust tissue-specific gene expression (submitted).
Targeting DNA damage repair and paclitaxel resistance in ovarian cancer using oncolytic adenoviruses
The sensitivity of ovarian cancers to adenovirus cytotoxicity varies greatly. We have recently investigated mechanisms for this variability.
We observed profound over-replication of genomic DNA in highly sensitive cancer cells (TOV21G, Hct116), associated with the presence of extensive genomic DNA damage, as detected by H2AX phosphorylation and pulsed-field gel electrophoresis. Similar changes were also seen in highly sensitive primary cells cultured from ovarian cancer ascites. Inhibition of ATR-Chk1, but not ATM, promoted genomic DNA damage and over-replication in resistant A2780CP and partially sensitive IGROV1 cells. This was accompanied by increased adenovirus cytotoxicity both in vitro and in vivo. We also demonstrated that Cdc25A is upregulated in highly sensitive cells following adenovirus infection and is stabilized following loss of Chk1 activity. Knockdown of Cdc25A inhibited virus-induced DNA damage in highly sensitive cells and blocked the effects of Chk1 inhibition in resistant cells. Finally, inhibition of Chk1 decreased homologous recombination repair of virus-induced genomic DNA double strand breaks. Thus, virus-induced host cell DNA damage signalling and repair are key determinants of oncolytic adenoviral activity.
Using matched ovarian cancer cell pairs, we have also observed increases in CAR expression, infectivity, and consequent sensitivity to adenovirus cytotoxicity, in paclitaxel-resistant cells (SKOV3-TR, A2780Tx1000) compared to their parental counterparts (SKOV3, A2780). Preliminary data also suggest that CAR signalling contributes to cell sensitivity to adenovirus cytotoxicity beyond simple infectivity. We are also investigating the role of CAR signalling in paclitaxel resistance.
Tocagen Inc. San Diego CA USA
Toca 511 is a retroviral replicating vector (RRV) expressing a modified cytosine deaminase (CD) gene. In animal models of brain cancer, Toca 511 can selectively infect tumor cells which then express the CD gene, converting administered 5-fluorocytosine (5-FC) into 5-fluorouracil. In animals, a single intratumoral injection of Toca 511, followed by cyclic treatment with 5-FC has resulted in dramatic prolongation of survival and clearance of tumors. Until now, RRV's have not been explored clinically as a potential cancer therapy. This first-in-man ascending dose study will evaluate the safety and tolerability of a single dose of Toca 511, injected transcranially into recurrent high grade gliomas and followed by monthly, 6 day courses of oral 5-FC. Subjects must be ≥18, have failed surgery and chemoradiation, and have a single, supratentorial recurrence that measures ≤9cm2 in its greatest cross-sectional area. This study uses a standard 3 + 3 dose escalation design. After a single injection, Toca 511 is allowed to spread through the tumor for 4 weeks, at which time 5-FC is administered. If tolerated, courses of 5-FC are repeated monthly for up to 6 cycles. Three subjects have been enrolled in the first dosing cohort. To date there have been no dose limiting toxicities (DLTs) identified, and no grade 3 or 4 AEs considered related to treatment. Intratumoral injection of Toca 511, followed by cyclic treatment with 5-FC was well-tolerated at the first dose of Toca 5111 studied. These data support enrollment of subjects at higher doses of Toca 511.
Embryonic stem cell lines: from biology to products
Pluripotent human embryonic stem cells (hESC) have the ability to proliferate indefinitely and to differentiate into virtually any cell type. These features can be harnessed to generate large quantities of partially differentiated progenitors or terminally differentiated specialised cells. Recent technical advancements have now made it possible to culture hESC under standardised feeder free conditions which allows for cost efficient large scale cell production. This is a requirement for future developments of in vitro and in vivo applications based on these cells. Such applications can include in vitro toxicity testing, compound screening, or cell therapy. This presentation will illustrate new achievements using human pluripotent stem cells, from basic biology and generation of new cell lines, to commercialisation of products. Differentiation of hESC towards hepatocytes and cardiomyocytes will be highlighted together with examples of current and future opportunities to use these cells for the assessment of adverse side effects of drugs. It is anticipated that the implementation of human stem cell based assays in the drug discovery process will lead to lower attrition rates and the development of safer new drugs.
Endosomal escape pathways for delivery of biologicals
Despite continuous improvements in delivery systems, the development of methods for efficient and specific delivery of targeted therapeutic agents still remains an issue in biological treatments such as protein and gene therapy. The endocytic pathway is the major uptake mechanism of cells and any biological agents, such as DNA, siRNA and proteins. These agents become entrapped in endosomes and are degraded by specific enzymes in the lysosome. Thus, a limiting step in achieving an effective biological based therapy is the endosomal escape to ensure cytosolic delivery of the therapeutics. Bacteria and viruses use different mechanisms to penetrate the membranes of their target cells and escape the endosomal pathway. Several processes such as pore formation in the endosomal membrane, pH-buffering effect of protonable groups and fusion into the lipid bilayer of endosomes have been proposed to facilitate the endosomal escape. Several viral and bacterial proteins have been identified that are involved in this process. In addition, chemical agents and photochemical methods to rupture the endosomal membrane have been described. New synthetic biomimetic peptides and polymers with high efficacy in facilitating the endosomal escape, low pathogenicity and toxicity have been developed. Each strategy has different characteristics and challenges for designing the best agents. New methods to facilitate the endosomal escape are under development. In this presentation, several mechanisms and agents which may facilitate in endosomal escape are reviewed.
Exosome-mediated RNAi delivery for neurological disease
Successful delivery of macromolecular therapeutic agents across the blood brain barrier (BBB) to the central nervous system is a major challenge in the treatment of neurological diseases of aging. Recently, the exploitation of natural nanoparticles known as exosomes has been pioneered and demonstrates their potential as therapeutic delivery vehicles. The natural macromolecular delivery and targeting properties of exosomes are well exemplified with siRNA delivery systemically to brain to target genes implicated in Alzherimer's disease. Moreover, delivery of exosomes appears to be well tolerated. This novel natural nanotechnology therefore has potential for systemic drug delivery across the BBB to treat a range of neurological and neurodegenerative conditions.
ZFN-edited CD4 T cells for HIV/AIDS Therapy: Phase 1 Trials to Evaluate the Safety and Tolerability of SB-728-T in HIV-infected Subjects
HIV requires the CD4 co-receptors CCR5 or CXCR4 to infect its target cells. Elimination of these co-receptors from the cell surface blocks viral entry thereby protecting the CD4 T cells lost in the course of HIV/AIDS. This represents an attractive therapy for HIV patients, yet no current methods enable therapeutically relevant disruption of a chosen human gene. To this end we have developed zinc-finger nucleases (ZFNs) targeting the CCR5 gene to create a double strand break (DSB) at a predetermined genomic site. Natural DNA repair pathways imperfectly repair the DSB resulting in the permanent disruption of the targeted CCR5 gene. Recently, we showed that ZFN-mediated disruption of the CCR5 gene could render CD4+ T cells resistant to infection by R5-tropic HIV-1 in a pre-clinical animal model of HIV infection (Nat. Biotech. 26:808 ′08). Based on this data we have initiated two Phase I clinical trials evaluating the infusion of CCR5-ZFN treated autologous CD4 T cells (SB-728-T) in HIV infected subjects with the goal of demonstrating the safety and tolerability of this novel cellular therapy. This presentation will review the clinical data obtained to date supporting the successful manufacturing, engraftment, expansion, and trafficking of these modified cells, as well as, evidence that SB-728-T can increase CD4 counts, normalize CD4:CD8 ratios and reduce viral load. Together these data support the further evaluation of SB-728-T as a potential treatment for HIV/AIDS.
Genetic Vaccines for Malaria
Over the last few years the prospects for widespread future use of genetic vaccines have increased considerably. A recombinant yellow fever virus vaccine against Japanese Encephalitis has reached licensure. An MVA-based vaccine against tuberculosis is in large phase IIb scale efficacy trials in Africa and poxvirus vaccines designed to treat prostate cancer have shown initial evidence of therapeutic efficacy. In malaria the most promising genetic vaccine approach entails the use of a recombinant chimpanzee adenovirus vector to prime immune responses and MVA as a booster. This approach has shown significant efficacy in phase II sporozoite challenge trials in the UK and is now in age de-escalation studies in children and infants in West Africa. Similar approaches are in clinical development for immunisation against HCV and HIV. I shall review recent progress and discuss the prospects and challenges for deployment of such genetic vaccines against malaria.
Restriction Factors and Gene Therapy for HIV
Restriction factors are professional antiviral proteins that can block specific stages of the viral life cycle and protect the host. They have been particularly well characterised in their antiviral activity against HIV-1. TRIM5alpha is a restriction factor that inhibits incoming retroviral particles by recruiting them to the proteasome before viral reverse transcription. It is strongly interferon induced but present in most cells at basal levels. Human TRIM5alpha cannot restrict HIV-1 due to an inability to recognise the HIV-1 capsid sequence. Simian TRIM5alpha variants, however restrict HIV-1 potently. Over millions of years of evolution TRIM5alpha genes have been under strong selective pressure, presumably from pathogenic viruses. Variants of TRIM5 exist called TRIMCyps in which the viral binding domain of TRIM5 has been substituted for a protein called cyclophilin A. TRIMCyps can restrict HIV-1 potently and humanised versions can be generated comprising human TRIM5 fused to human cyclophilin A. These proteins can be used as transgenes for human gene therapy and are attractive as similar chimeric proteins have been generated during primat evolution on at least two occasions underlining their potency and utility as protective factors. Preliminary data outlining the use of human TRIMCyps as antiviral factors in gene therapy for HIV infection will be presented.
microRNAs in Post-Ischemic Angiogenesis
MicroRNAs (miRNAs) are post-transcriptional inhibitory regulators of gene expression that bind to complementary messenger RNA transcripts. miRNAs have recently come into focus of cardiovascular research. Because each miRNA can repress many target mRNAs, it is possible that dysregulation of a single miRNA might play important roles in complex pathological situations.
Our laboratory is focussing on the identification of miRNAs which are involved in ischaemic disease, with particular interest on diabetes peripheral complications. We have reported for the first time the importance of miRNA-503 (miR-503) in diabetes mellitus–associated ischemic disease. miR-503 represses cell cycle–associated genes. In vitro, the combination of high glucose and starvation enhances the expression of miR-503 in human endothelial cells, and so does diabetes mellitus in endothelial cells extracted from murine ischemic limb muscles. Forced expression of miR-503 inhibits endothelial cell proliferation and endothelial network formation. In a diabetic mouse model of limb ischemia, local inhibition of miR-503 activity accelerated vascular healing and blood flow recovery. Importantly, miR-503 is up-regulated in muscular biopsies and peripheral blood–derived plasma of diabetic patients with critical limb ischemia. From a therapeutic perspective, manipulation of miR-503 may represent a novel molecular means to foster reparative angiogenesis in diabetic patients. I will also present unpublished data related to other miRNAs.
Gene therapy of familial hypercholesterolemia
Familial hypercholesterolemia (FH) is an inherited disease of lipid metabolism caused by a defect in the receptor for low density lipoprotein (LDLR) leading to increased circulating low-density lipoprotein (LDL) levels and premature cardiovascular disease. Liver-directed gene therapy is a potential new treatment for a variety of inherited liver disorders. Delivery of the functional gene can be achieved by the use of viral vectors which are able to efficiently transduce cells. Successful treatment of FH requires life-long correction of hepatic cells. We have focused on testing vector systems mediating long-term expression of the LDLR gene in the liver. Retro-, lenti-, and AAV vectors carrying the rabbit LDLR gene were injected into the livers of WHHL rabbits and animals were followed for up to three years. As a result significant lowering of serum cholesterol levels and a prolonged life-span were seen in rabbits receiving the LDLR gene with both retro-and lentiviral vectors whereas this was not seen with AAV mediated LDLR gene transfer. In the long-term follow-up after transduction with retroviral or lentiviral vectors no major histological changes were seen in the liver whereas 1 year after AAV-mediated gene transfer bile duct proliferation was noted in some of the animals. These results indicate that in vivo gene transfer of the LDLR results in a long-term therapeutic effect but the optimal vector needs to be assessed.
Purification and analysis of virus, VLP and pDNA vectors for gene therapy with CIM® Monolith Chromatography
Viral, VLP and plasmid vectors present a common challenge for chromatographic purification. They are typically excluded from traditional particulate media, being much larger than proteins.
Most chromatography media are packed beds of porous particles and depend on diffusion for efficient binding and desorption within the pores. Large bioparticles diffuse very slowly. This limits dynamic capacity.
Polymeric CIM monoliths are cast as a unit block of porous material designed to allow flow transport directly to and from binding sites. Kinetics of mass transfer becomes essentially independent of flow rate.
Fast, highly selective purifications can be achieved from complex protein and gDNA impurities. Structural pore connectivity promotes laminar flow with no dead-end pores. This morphology delivers high capacity for large particles whilst laminar flow promotes high yield. Capacity of up to 2 × 1014 particles or ∼6mg pDNA per ml column bed volume will be shown.
These same characteristics also pose a challenge for sample analysis. Analytical columns are typically fouled by gDNA, and selectivity is limited for crude sample materials.
CIMac® analytical monolith morphology permits repetitive runs of crude samples on 100ul columns with short run times.
A fast fingerprinting method will be shown to evaluate developing product quality and assist with optimised harvesting of bioproducts. The use of CIMac columns for resolution of supercoiled, open circular and linear plasmid forms will also be shown.
Manufacture of clinical-grade lentiviral vectors for ex vivo use
Genethon is a non-profit organization that develops gene-therapeutics to treat rare genetic diseases. Gene-modified hematopoietic stem cells (HSC) are among the products developed, and these advanced therapy medicinal products have encouraging prospects for the treatment of certain primary immune deficiencies (PID) or congenital hematopoietic diseases. Genethon is currently sponsoring PhI/II clinical trials for the treatment of Wiskott Aldrich syndrome (WAS), a rare X-linked PID. The therapeutic approach is based on ex vivo transduction of patients autologous HSC with a VSV-G pseudotyped rHIV1-lentiviral vector encoding the WAS protein under the control of the natural WAS gene promoter. To support this project and similar applications in other diseases, Genethon has developed a scaleable lentiviral manufacturing process that has been approved for the manufacture of clinical grade products to conduct clinical trials in the EU (UK, France) and in the US. The lentiviral vector upstream process involves a 4-plasmid transfection in HEK293T cells. Downstream process involves ion exchange chromatography coupled to ultrafiltration and gel filtration steps to purify, concentrate and formulate the bulk vector, which in turn is filter-sterilized to generate the product that is further aliquoted for clinical use. Five clinical grade preparations and more than 10 large-scale preclinical preparations of vectors with various transgenes have been manufactured to date in our facility. Although the process has not yet been validated, to a large extent analytical data generated show good reproducibility and robustness. In an effort to improve yield and residual contaminant profiles of the vector, a few modifications were recently introduced in the process. Process yield and analytical data will be discussed in the perspective of regulatory requirements.
m.barkats@institut-myologie.org ; Biothérapie des Maladies Neuromusculaires, Um76 UPMC - UMR 7215 CNRS -U974 Inserm, Institut de Myologie, Faculté de Médecine, 105 Bd de l'Hôpital, Paris 75013, France
We previously demonstrated that systemic injection of self-complementary AAV9 vectors (scAAV9) allowed neuronal gene transfer in both neonate and adult mice or cats despite the presence of the blood-brain-barrier. We next analyzed the therapeutic potential of this gene transfer methodology in a severe mouse model of spinal muscular atrophy (SMA), the “SMNdelta7” mice. The scAAV9 vectors were engineered to express a codon-optimized sequence of the “Survival of Motor Neuron” (SMN) gene under control of the PGK promoter (scAAV9-SMNopti). After injection of scAAV9-SMNopti into the temporal vein of PND1 SMNdelta7 mice, the survival analysis showed that this treatment rescued 100% of the mice, increasing life expectancy from 27 to over 355 days (median survival 199 days) in mice that normally survive about 13 days. The intravenous scAAV9 therapy also mediated complete correction of motor function, prevented motor neuron death and rescued the weight loss phenotype close to normal. This study reports one of the most efficient rescuing of SMA mice to date after a single intravenous injection of a SMN encoding AAV. We recently investigated whether alternative routes of scAAV9 injection, in particular the intramuscular route, could also provide therapeutic benefit in SMA mouse models. Our results showed that intramuscular injection of PND1 SMNdelta7 mice with scAAV9-SMNopti increased mouse rescue to a level never reached so far following intramuscular injection of gene vectors (median survival 163 days). Together, these studies highlight the considerable potential of systemic delivery of SMN encoding scAAV9 for the treatment of human SMA.
Genetic therapies for Duchenne muscular dystrophy
Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there is no effective treatment but new gene-based therapies are currently being developed with particular noted advances in using conventional gene replacement strategies, RNA-based approaches, or cell-based gene therapy with a main focus on Duchenne muscular dystrophy (DMD). DMD is the most common and severe form of muscular dystrophy and current treatments are far from adequate. However, genetic and cell-based therapies, in particular exon skipping induced by antisense strategies, and corrective gene therapy via functionally engineered dystrophin genes hold great promise, with several clinical trials ongoing. Proof-of-concept of exon skipping has been obtained in animal models, and most recently in clinical trials; this approach represents a promising therapy for a subset of patients. In addition, gene-delivery-based strategies exist both for antisense-induced reading frame restoration, and for highly efficient delivery of functional dystrophin mini- and micro-genes to muscle fibres in vivo and muscle stem cells ex-vivo. In particular, AAV-based vectors show efficient systemic gene delivery to skeletal muscle directly in vivo, and lentivirus-based vectors show promise of combining ex vivo gene modification strategies with cell-mediated therapies.
Genethon, Evry – France
In Duchenne Muscular Dystrophy (DMD) the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in in-frame mRNA transcripts that are translated into shorter but functionally active dystrophin.
The goal of our project was to determine in the dog model of DMD, the GRMD, the effective dose of our therapeutic product defined as a recombinant Adeno-Associated Virus serotype 8 (rAAV8) expressing a modified U7 snRNA specific for the skipping of exons 5 to 10 of the GRMD dystrophin transcript. The mode of delivery was the locoregional high-pressure intravenous (IV) injection of a forelimb.
Five groups of 2–3 GRMD dogs were exposed to different rAAV8-U7snRNA doses (from 2.5 1012 vg/kg to 2.5 1013 vg/kg diluted in two different volumes) + three dogs injected with same volume of ringer-lactate. Each dog was followed ≈3 months after injection until sacrifice and full autopsy. The primary outcomes were the restoration of dystrophin expression and the improvement of the tissue pathology in the injected limb and in muscles behind the tourniquet.
Results demonstrate high level (about 70–80%) of dystrophin expression in dogs injected with 2.5 1013 vg/kg, not only in the injected limb, but also in lower quantity in other muscles behind the tourniquet. Lower levels of dystrophin expression (∼30–40%) and (∼10%) were observed in dogs injected with a dose 5 or 10 times lower, respectively. An excellent correlation was found between the viral copy number/diploid genome and the dystrophin amount. Strength improvement was demonstrated in muscles with more than 40% dystrophin expression. In addition, we observed a dose dependant correction of various RMN pathological indexes.
This demonstrates the feasibility and the dose-dependant effect of locoregional rAAV8 U7snRNA approach in a large animal model of DMD and opens the way for a human trial in upper limb of non-ambulatory patients.
This project is supported by AFM (Association Française contre les Myopathies) and by ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by research and innovation aid from the French public agency, OSEO.
Cellular Recognition and Restriction of Viruses and Vectors
Viral vectors for gene therapy take advantage of the innate ability of viruses to transfer genetic material into target cells. It is important to understand how cells respond to DNA deposited in the nucleus by viruses and vectors, since this will impact the efficiency of the transduction process. Studying virus-host interactions we have uncovered ways in which the cellular DNA repair machinery recognizes and processes the genomes of three DNA viruses commonly used as vectors for gene delivery: adenovirus (Ad), adeno-associated virus (AAV) and herpes simplex virus (HSV). Both Ad and HSV can manipulate aspects of the cellular DNA damage response. AAV activates a unique type of damage response and transduction by vectors is limited by repair proteins. In addition to DNA repair, we found that APOBEC3A is a potent inhibitor of AAV replication and progeny production. Our work highlights how exploring the interactions between viruses and the host DNA repair machinery reveals novel host responses to virus infections. In addition, identifying the fate of vector genomes and the responses that they elicit will provide insights into the transduction process and how it can be optimized for gene therapy vectors.
Manipulating insertion profiles of transposon-based vectors
The Sleeping Beauty (SB) transposon is a nonviral, integrating vector system that offers stable delivery and expression of genes-of-interest in human cells and in preclinical models. We performed genome-wide integration site analyses evaluating SB and two other transposon systems, piggyBac (PB) and Tol2, for their propensities to integrate into genes, transcription start sites and histone modifications. SB had a random integration profile with no apparent bias for integrating into genes or near transcriptional regulatory regions. By contrast, PB showed a non-random integration profile with significant bias towards integrating into genes and transcription start sites. Tol2 showed an intermediate integration profile with no bias for inserting into genes, but preferential integration into transcriptional regulatory regions. Tol2 and PB integrations were particularly favoured near transcription-associated histone modifications, but disfavoured in regions rich in histone marks associated with heterochromatin. These data suggest that Tol2 and PB integration preferences resemble those of retroviral vectors. We also evaluated molecular strategies based on engineering transposon components with heterologous DNA-binding domains, which are to tether the transposase/transposon complex to defined sites in the human genome, and to facilitate integration of the transposon into adjacent sites. We evaluated the Rep protein of adeno-associated virus and zinc finger proteins with novel specificites. The engineered components were incorporated into the transpositional machineries, and their effects on target site distribution in human cells examined by analyzing insertion sites. Our data provide proof-of-concept for manipulating insertion profiles, and assist in the design of target-selected gene insertion systems with enhanced efficiency and specificity.
Via dei Castelli Romani 22, Pomezia, Italy
Replication defective Adenovirus vectors based on the human serotype 5 (Ad5) have been shown to induce protective immune responses against diverse pathogens and cancer in animal models, and to elicit robust and sustained cellular immunity in humans. However, most humans have anti-Ad5 neutralising antibodies that can impair the immunological potency of such vaccines. We have found that most other human Adenoviruses from rare serotypes are far less potent as vaccine vectors than Ad5 in mice and non-human primates, casting doubt on their potential efficacy in humans. Therefore, we have generated and characterized a large collection of novel Adenovirus vectors derived from chimpanzees (ChAd) that are not neutralised by antibodies present in humans, have high immunological potency and can be produced in human cell lines approved by regulatory agencies.
ChAd induce long-lasting memory responses that could be readily re-expanded in vivo by second encountering with the encoded pathogenic antigen. We also showed that ChAd can protect from infection by different pathogens (RSV, Influenza, Ebola) in relevant animal models.
Two of the most potent ChAd vectors were selected for clinical studies as carriers for Malaria and Hepatitis C virus genetic vaccines where they proved to be safe and immunologically potent. Heterologous prime/boost regimens with ChAd and MVA vectors were shown to achieve unprecedented levels of cellular immunity in humans.
ChAd represent a large collection of non cross-reactive and potent vectors that can be exploited for diverse vaccine strategies against infectious diseases and cancer.
Psioxus Ltd, Cherwell Innovation Centre, Heyford Park, Oxfordshire OX25 5HD
Recombinant viral vaccines have demonstrated remarkable efficacy, both as prophylactic and therapeutically for a range of disease indications and animal models. Readily modified to express combinations of antigens and immune-modulators, viral vectors offer tremendous flexibility for vaccine design. Probably the greatest advantage of viral vectors is that they are self-adjuvanting, presenting the immune system with the illusion of a genuine infection. However the immunogenicity of viral vectors is also their greatest disadvantage with immuno-dominance issues and host anti-vector responses. Our research group is looking at how delivery routes and extracellular interactions with antibodies, serum proteins and matrix components impact on virus activity and behaviour in vivo. The main challenge here is to adapt laboratory models to reflect clinical reality because many host-virus interactions are species specific. Using passively immunised mice and luciferase imaging, we have measured the rate of virus infection and time course for antibody neutralisation. We have also looked at parameters of dose, volume and dose fraction on the immune response against vector and antigen. To overcome unwanted interactions and modify vector immuno-dominance we have developed a range of polymer coating strategies designed to improve virus performance in human tissue fluids and pre-immune animal models.
Institut de Génétique Moléculaire de Montpellier, Universités de Montpellier 1, Montpellier, France, Universités Montpellier 2, France
In the primate central nervous system (CNS), efficient, specific, widespread and long-term transduction of neurons with gene transfer vectors is challenging. In this presentation, we combined helper-dependent (HD) canine adenovirus type 2 (CAV-2) vectors and a novel nonhuman primate, Microcebus murinus, to address efficacy of gene transfer to the CNS. We found that injections of HD CAV-2 vector into the striatum resulted in neuron-specific expression at the injection site and in numerous regions and nuclei that harbour neurons that project into the striatum. Transgene expression was stable for at least 6 months, the longest duration assayed, without induction of an adaptive immune response as determined by the lack of anti-vector antibody production or the generation of anti-vector T cells. Transgene expression correlated with the quantification of vector genomes in each region, which is consistent with retrograde transport of vectors into afferent structures. Injection of CAV-2 vectors into the hindleg muscles also led to preferential and efficient (up to 75%) transduction of the innervating motor neurons in the anterior horn of the spinal chord. Transgene expression in the spinal chord was also long lasting (6 months, the longest time assayed). To address the molecular basis of CAV-2 tropism, we show that retrograde transport and preferential neuronal transduction is consistent with coxsackievirus adenovirus receptor expression at synapses in the CNS and neuromuscular junctions. Our model and results have notable fundamental and clinical implications for safety and efficacy of genetic modification of the CNS, in particular in the context of modelling, preventing and treating neurodegenerative diseases.
Department of Gene Therapy, Centre for Neurosciences, Division of Experimental Medicine, Faculty of Medicine, Imperial College London, Hammersmith Campus, Burlington Danes, Du Cane Road London W12 ONN
Rabies Virus glycoprotein (RVG) can pseudotype lentiviral vectors, albeit at a lower efficiency to that of Vesicular Stomatitis Virus glycoprotein (VSVG). Transduction with VSVG pseudotyped vectors of rodent CNS leads to local neurotropic gene transfer whilst with RVG pseudotyped vectors additional disperse transduction of neurons located at distal efferent sites occurs via axonal retrograde transport. We have constructed several chimeric RVG/VSVG glycoproteins and found that a construct bearing the external/transmembrane domain of RVG and the cytoplasmic domain of VSVG shows increased incorporation onto HIV-1 lentiviral particles and has increased infectivity in vitro in 293T cells and in differentiated neuronal cell lines of human, rat and murine origin. Stereotactic application of vector pseudotyped with this RVG/VSVG chimera in the rat striatum resulted in efficient gene transfer at the site of injection of cells showing both neuronal and glial tropism. Distal neuronal transduction in the substantia nigra, thalamus and olfactory bulb via retrograde axonal transport also occurs after intrastriatal administration of RVG/VSVG chimera pseudotyped vectors at similar levels to that observed with a RVG pseudotyped vector. The enhanced pseudotyping with this chimeric envelope should enable easier production of higher titer pseudotyped lentiviral vectors that exhibit efficient local and dispersed neuronal transduction in the CNS.
Current Advances in Gene Therapy for Spinal Muscular Atrophy
Spinal muscular atrophy (SMA), a recessive autosomal disorder, is one of the most common genetic causes of death in childhood. It is caused by mutations of the survival motor neuron (SMN) gene. We previously reported that Lentiviral vector expressing SMN was successfully used to increase the life expectancy by 5 days1. The marginal efficacy of this therapeutic approach, however, prompted us to explore different strategies for gene delivery to motor neurons to achieve a more clinically relevant result.
The first part of this presentation will describe the efficiency of scAAV9 mediated SMN gene replacement in SMA mouse model. We report that a single systemic injection of SMN-expressing scAAV9 vector reversed the phenotype of SMNΔ7mice2, a well established animal model of SMA. Most notably, SMN replacement led to substantial increase in the life expectancy, thereby achieving one of the highest therapeutic effects reported in the field to date3. The second part will discuss the clinical development of SMN strategy. Significant progress has been made in initiating regulatory pre-clinical studies required for clinical applications in SMA patients.
1. Azzouz et al. J. Clin. Inv.
2. Le et al. Hum Mol Genet
3. Valori et al. Sci. Transl. Med.
Engineering bacteria for simultaneous imaging and therapy of cancer
Use of microbial system has attributed to the recent advances in targeted molecular therapy application especially for its tumor-specific accumulation and proliferation. In an attempt to investigate tumor-specific targeting, we used an in vivo optical imaging system to monitor the spatial and temporal migration of light-generating E. coli or attenuated Salmonella typhimurium (carrying pLux) following injection into mice tumor models. In addition to optical imaging, we employed PET-based molecular imaging strategy to visualize bacteria through targeting of bacterial DNA synthesis. The phenomenon of selective targeting and proliferation of bacteria was observed in a diverse range of tumors with multimodal imaging technologies with optical imaging and PET, as it accumulated in every type of grafted tumor we tested.
Bacterial therapy possesses many unique applications for treating cancer that are unachievable with standard methods. Bacteria could controllably induce cytotoxic proteins. When using tissue specific or inducible system, bacteria could be engineered to express target genes specifically in the tumor area, rather than other organs, leading to maintenance of therapeutic efficacy and reduction of toxicity. The combination with engineered bacteria (BT) and radiotherapy (RT) had an synergistic effect to completely remove the subcutaneously grafted tumor.
In this research, we propose that synthetic biology techniques can be used to solve many key challenges that are associated with bacterial therapies, such as toxicity, stability and efficiency, and can be used to tune their beneficial features, allowing the engineering of ‘perfect’ bacteria for cancer treatment.
Tumour-specific bacterial growth: A vector toolbox for localising cancer therapeutics
Bacteria present an attractive class of gene vector for cancer, possessing a natural ability to grow specifically within tumours following systemic administration. We describe a range of strategies under investigation by our group, designed to exploit tumour-specific bacterial replication, utilising both non-pathogenic and pathogenic/invasive bacterial vectors. We have engineered a number of replication-competent non-pathogenic probiotic bacteria to express heterologous genes and mediate long-term production of agents within tumour masses (external to tumour cells) following systemic administration. Use of safety attenuated invasive strains permits intracellular DNA, RNA or protein delivery. Luminescent reporter gene (bacterial lux) tagging permits real-time tracking of vector without the need for substrate administration, with high-resolution detail of in vivo bacterial growth within deep tissue achieved using 3D BLI tomography. We have demonstrated that the various vectors can be exploited in a wide range of therapeutic strategies and tumour models, presenting a powerful and safe approach to specific gene/cell therapy of primary tumours and secondary metastases.
CEA-CNRS MirCen Fontenay aux Roses France
L-Dopa and dopamine agonists provide the primary standard of care for PD and are highly efficacious in the early stages of disease. However their long term use is associated with severe motor and non-motor side effects that seriously impact on the quality of life of the patients. These adverse events are believed to be caused by the fluctuating nature of dopaminergic stimulation that arises from oral drug administration and may be reduced by therapies that provide a continuous tonic supply of dopamine, locally to the striatum.
ProSavin® is a lentiviral vector derived from the equine infectious anaemia virus expressing the three key dopamine biosynthetic enzymes (tyrosine hydroxylase, aromatic L-amino acid decarboxylase and GTP cyclohydrolase-1). Transduction of striatal neurons with ProSavin® converts these cells to dopamine ‘factories’ and provides a continuous source of dopamine to the striatum. ProSavin® has been demonstrated to mediate dopamine production and cause behavioural correction in the rat (Azzouz et al., 2002) and in a severe MPTP-lesioned non human primate model (Jarraya et al., 2009). A phase I, open label clinical study has been initiated in which fourteen PD patients have received ProSavin® in four cohorts in a dose escalation phase. Nine patients have completed at least 12 months' follow up and the first cohort are now approaching their third year post treatment. ProSavin® has been demonstrated to be safe and well tolerated at all doses evaluated to date. There were no “OFF” state dyskinesias and no immune responses or serious adverse events related to Prosavin®. Furthermore, encouraging signs of efficacy have been observed on a number of endpoints including UPDRS Part IIII, patient diary and quality of life measures. An update on the trial and future plans for ProSavin® will be presented.
OncoVEX GM-CSF: From bench, to bedside; to approved drug?
Department of Haematology, University College London and
A distinct approach for haemophilia B (HB) gene transfer is being tested in the clinic. This Phase I/II clinical trial entails peripheral vein administration of a single dose of our novel self complementary AAV (scAAV2/8-LP1-hFIXco) vector encoding a codon optimised human FIX transgene into adult subjects with severe HB. Using a typical dose escalation design, scAAV2/8-LP1-hFIXco vector has been administered without immunosuppression at three dose levels consisting of the low (2x1011 vg/kg), intermediate (6 × 1011 vg/kg) and high (2 × 1012 vg/kg) dose, with two subjects treated at each dose level. The safety and efficacy of our approach in the subjects recruited will be discussed with a follow-up period of up to 18 months.
Oral Abstracts
San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, 20132, Italy
Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia, infections, autoimmunity and lymphomas. We previously demonstrated that a lentiviral vector (LVV) encoding for human WAS under the control of homologous 1.6 kb length promoter efficiently corrected human and mouse cells and is safe in the preclinical WAS-KO mouse. We validated the transduction protocol on patients' derived bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells using highly purified, clinical grade LVV. Patients' cells showed a robust transduction and restoration of WASp expression without evidence of toxicity. Patients will receive transduced CD34+ cells following preconditioning with anti-CD20 mAb and reduced intensity busulfan and fludarabin; ATG will be included in case of autoimmune manifestations. The first patient was treated with autologous BM and MPB derived transduced CD34+ cells, showing high gene transfer efficiency (1.4 and 1.9 vector copy number, VCN, respectively and 88–92% in clonogenic progenitors). The patient did not experience toxicity and is currently well, independent from platelet transfusions. One year after gene therapy engraftment analyses showed the presence of polyclonal vector transduced cells at substantial levels in different peripheral blood (PB) (VCN range: 0.43–1.47), and BM lineages (VCN range: 0.2–1.04), including CD34+ cells (0.39) and clonogenic progenitors (40.2% transduction). WASp expression was detected in PB platelets, monocytes and at higher levels in lymphocyte lineages, as expected from the selective advantage. Long-term studies will provide key information on the safety and efficacy of gene therapy for WAS patients using LVV transduced HSC in combination with reduced intensity conditioning.
3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan
Oncolytic virotherapy is an emerging and promising anticancer therapy. Here, through large-scale two-step screening of 28 wild-type enteroviruses, we demonstrate that coxsackievirus B3 (CVB3) had a specific oncolytic activity against various human non-small cell lung cancer (NSCLC) cell lines. Our subsequent surveys revealed that CVB3 infection caused substantial apoptosis which contributed to CVB3-triggered cytotoxicity and correlated with CAR receptor expression. Furthermore, in vitro inhibition assays demonstrated that both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) survival signaling pathways were responsible for regulation of CVB3 replication in human NSCLC cells. In vivo mouse studies showed that CVB3 administrations into pre-established tumors in the right flank elicited significant regression of untreated, contralateral pre-established tumors, in which replication competent CVB3 was detected. In regard to its safety, intratumoral CVB3 administrations elicited liver dysfunction and creatinine kinase elevation, but lung and kidney tissue remained unharmed. Notably, no mice died of side effects due to CVB3 treatment. Moreover, intratumoral CVB3 administration recruited robust numbers of NK cells, granulocytes co-expressing degranulation marker CD107a, macrophages, and mature dendritic cells (DCs) into the CVB3-treated xenografts, suggesting its immunostimulatory properties in tumor microenvironments. In conclusion, our findings indicate that CVB3 holds promise as a novel oncolytic virotherapy agent against NSCLC.
AAV9 gene transfer to the nose for pharmacologically-regulated systemic expression of therapeutic proteins
Adeno-associated virus (AAV)-based vectors have been shown to effectively transduce cells of the mouse nasal airway epithelium. The nose is easily accessible and topical application is accurate, noninvasive and painless. We evaluated the potential of using the nose for production of systemically secreted therapeutic proteins, such as erythropoietin (Epo). In addition, we evaluated the ARGENTTM system to pharmacologically regulate Epo expression. C57BL/6 mice were injected intranasally (IN; 10μl per nare) with AAV9-ARGENT vector expressing rhesus Epo (rhEpo) and AAV9-vector expressing the transcription factor. As controls, mice were injected with the constitutive AAV9-rhEpo IN or intravenously (IV). Expression of functional rhEpo results in a significant increase of hematocrit (HCT) levels over baseline (∼55%). Within 14 days, the HCT of mice treated with AAV9-rhEPO IN or IV were 68% and 77%, respectively. For mice treated with the AAV9-ARGENT-rhEpo vector, HCT levels prior to induction were in the normal range (∼45%). Expression of rhEpo was then induced by a topical (IN) administration of a small volume of rapamycin (5μl per nare) so as to effectively restrict induction of rhEpo expression from only the nasal airway epithelium. Levels of HCT increased within 24hrs to 60% and peaked at 48hrs to 65%, remained stable for 3 days and returned to baseline by day 7. Here we demonstrate the application of AAV-mediated gene transfer to the nose as a noninvasive way to achieve high levels of systemically secreted therapeutic proteins. Furthermore, expression of these therapeutic proteins can be regulated by a safe pharmacological approach.
Laboratory for Molecular Virology and Gene Therapy, K.U. Leuven, Flanders, Belgium
Research Dept. Cell and Gene Therapy, Clinic for SCT, UCCH, Hamburg-Eppendorf, Hamburg, 20246, Germany
We have recently introduced a new technique for clonality analyses in vitro and in vivo, named RGB marking (Nat Med 2011; 17:504). RGB marking is based on the physical principle that any spectral colour can be generated by mixing the three basic colours
Inst. of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany
The incorporation of heterologous proteins into the structural components of the retroviral particle facilitates transient modification of target cells. As we have shown, this strategy of Retroviral Protein Transfer (RPT) efficiently introduces fluorescent proteins and recombinases (Flp, Cre) into iPS and other cells (Voelkel et al., PNAS 2010). In order to expand our understanding of RPT and virus biology, we opted to characterize components of the pre-integration complex, namely integrase localization and capsid, as potential carriers for heterologous proteins.
Incorporation of Flp downstream of reverse transcriptase mediated recombination in a plasmid based transfection assay, but not in transduced cells. Western Blots of producer cell lysates indicated aberrant proteolytic processing and low levels of full-length gag-pol as the main limitations of this approach. Since gag is ∼10-fold more abundant than pol, we next focused on capsid modifications. A unique restriction site was thus introduced downstream of the natural p12–capsid protease cleavage site followed by a flexible linker. In a single round infection assay, mutant gag/pol constructs produced integration competent particles albeit at low titer. Copackaging of wild-type gag/pol with GFP labeled capsid mutants showed colocalization with fluorescently labeled integrase in packaging cells, increased titers, and allowed for “fluorescent painting” of target cells. Finally, viral particles harboring Flp fused to capsid, were infectious and mediated recombination of a reporter cell line.
Our study thus shows that targeted modifications of the gag/pol precursor will help to understand steps of the virus life cycle and facilitate the production of functionalized viral particles.
Department of Internal Medicine II, University Hospital of Munich - Campus Großhadern, Ludwig-Maximilians-University, Munich, Germany
The tumor-homing property of mesenchymal stem cells (MSC) has suggested their potential use as delivery vehicles for therapeutic genes such as the sodium iodide symporter (NIS). NIS used as either a diagnostic or therapeutic gene allows non-invasive imaging of functional NIS expression by 123I-scintigraphy, or therapeutic treatment by use of 131I, respectively. Upregulation of the chemokine CCL5 (RANTES) by MSCs within the tumor stroma is thought to play an important role in cancer growth and metastasis. We have therefore made use of the CCL5 promoter for a tumor-targeted expression of MSC-transgenes.
In the current study we stably transfected human MSCs with NIS driven by the CCL5 promoter (RANTES-NIS-MSC). Using a human HCC xenograft model (Huh7) we investigated distribution and tumor recruitment of RANTES-NIS-MSCs by 123I-scintigraphy, ex vivo gamma counting and immunofluorescence analysis after systemic tail vein injections of RANTES-NIS-MSCs in nude mice.
After systemic RANTES-NIS-MSC injection, 123I-scintigraphy revealed active MSC recruitment into the tumor stroma of Huh7 xenografts and CCL5 promoter activation as shown by tumor-selective iodide accumulation (7% ID/g 123I, biological half-life: 3.7 h). Immunofluorescence analysis using a NIS-specific antibody, and measurement of ex vivo 123I biodistribution verified active recruitment of RANTES-NIS-MSCs in the tumor stroma, whereas non-target organs showed no significant MSC recruitment/transgene expression.
Our results demonstrate selective recruitment of NIS-expressing MSCs driven by the RANTES promoter into HCC tumors resulting in induction of tumor-specific iodide accumulation, expanding the prospect of NIS-mediated radionuclide therapy of metastatic cancer after MSC mediated gene delivery.
Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Germany
Integrating lentiviral vectors (LV) are the preferred gene transfer vehicles enabling both efficient and stable gene transfer for the treatment of monogenetic liver diseases. However, the potential risk of insertional mutagenesis in postnatal liver gene therapy is insufficiently investigated. In this study we describe a serial hepatocyte transplantation model utilising C57BL/6-Fah(−/−) mice. Lentiviral Fah expression significantly improved survival (p=0.0311) compared to medication with the drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione).
Serial transplantation of gene corrected liver cells into four generations of mice forced approximately 88 hepatocyte doublings (total of 89 repopulated mice in four independent experiments). Liver tumours developed in all groups of mice in an observation time of one year after gene correction, as the Fah−/− mouse model is prone to develop hepatocellular carcinoma, but this was not associated with lentiviral vector integration. Reduced long term survival of 4th generation mice (p=0.0057) was associated with progressively shortened telomeres (18–29% reduction).
Insertion site analysis by LM PCR and high throughput 454 sequencing (2483 individual integration sites in 38 repopulated animals) revealed selective enrichment of insertion sites at genes linked to EGF signalling and other known tumour associated pathways. These identified genes such as Alcam and Adcy9 may be new candidates for liver regeneration or cancer research. Polyclonality of liver repopulation, however, was maintained in 4th generation livers. We conclude that insertional mutagenesis caused mild clonal imbalance within a polyclonal background in a model of forced proliferative stress, but this was not associated with tumour formation or reduced survival.
National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany
The adeno-associated virus (AAV) vector system AAV1-LPLS447X has been developed for the long-term correction of lipoprotein lipase deficiency in adult patients. As even low frequent integrating vectors as AAV may lead to oncogenesis, we were interested in analysing the persistence and integration frequency of AAV1-LPLS447X in mice. In the first setting, 1013 gc/kg were injected intramuscularly into C57BL/6 mice and adductors from the hind limb were collected 4 weeks post injection (p.i.). In a second approach, C57BL/6 mice received 6×1013 gc/kg intramuscularly and hind limb muscles and livers were collected 4 or 12 weeks p.i. LAM-PCR was performed on the 3′ end of the vector using the two different restriction endonucleases MseI and Tsp509I. >41000 AAV derived LAM-amplicons were 454 pyrosequenced, revealing >1500 unique integration sites (IS). The chromosomal distribution and the distribution in respect to gene coding regions showed a close to random integration of the AAV1-LPLS447X vector. Analysis of common integration sites (CIS) revealed a low level clustering. Despite a high number of IS was detected, the vast majority of identified sequences was related to concatemeric vector forms. Furthermore, the low relative sequence count of each exact mappable IS indicated the absence of dominating clones. Complete inverted terminal repeat (ITR) sequencing was shown and partially deleted ITRs with preferred breakpoints within the first loop of the ITR hairpin structure were detected. Taken together, our findings indicate safe AAV gene transfer and are promising for ongoing and upcoming AAV clinical trials.
San Raffaele-Telethon Institute for Gene Therapy, Milan, Italy
We generated a novel replication-defective Lentiviral Vector (LV) specifically tailored to achieve efficient insertional mutagenesis in hepatocytes and induced hepatocellular carcinoma (HCC) in three different mouse models of cancer or chronic liver injury. LV injection in newborn mice was able to induce HCC in 30% of Cdkn2a−/− mice (P=0.005), in 27% of liver-specific Pten-null mice (P=0.04) and in 75% of wild type mice coupled to CCl4 administration (P=0.002). From 30 LV-induced HCCs we could retrieve 172 unique integrations that allowed the identification of Braf, Fign, Sos1 and Dlk1-Dio3 region as candidate cancer loci. LV integration in Braf and Sos1 generated constitutively active oncogenic proteins, while integration in Fign and Dlk1-Dio3 region significantly deregulated these loci. We validated in vivo the causative role of these genes in hepatocarcinogenesis by forced expression in mouse liver. Moreover, we found that Braf and Fign are novel regulators of the Dlk1-Dio3 imprinted region in murine and human hepatocytes and that the Dlk1-Dio3 region also modulates energy metabolism in cancer cell, thus providing a new mechanistic framework to explain its role in the maintenance of stemness and induction of cancer.
The newly identified cancer genes are deregulated and/or amplified/deleted in human HCC, and their expression levels or the specific gene expression signatures consequent to their deregulation can distinguish different HCC stages and predict the overall survival and the disease-free survival of HCC patients.
By this approach we discovered new human liver cancer genes that may provide novel candidate therapeutic targets and diagnostic markers for human HCCs.
University of Cologne
Vectors based on the adeno-associated virus (AAV) have emerged as one of the leading delivery systems in gene therapy. One of the key features of AAV vectors is low immunogenicity. Nevertheless, induction of humoral as well as capsid-specific T cell responses has been reported that limits vector re-application and therapeutic efficacy. Since adaptive immune responses are a consequence of pathogen recognition by the innate immune system, we rigorously investigated innate pattern recognition of rAAV in the liver, which is one of the main targets in human gene therapy. Rodents and humans differ in their pathogen recognition receptor composition. We therefore decided to focus on primary human liver cells using mixed liver cell cultures and monocultures of hepatocytes, of Kupffer cells (KC) and of liver sinusoidal endothelial cells (LSEC) as model systems. Our study on primary human liver cells identified a new mechanism of innate immune recognition for AAV. Briefly, we could demonstrate that in contrast to Toll-like-receptor (TLR)-9-mediated recognition of AAV vector genomes in plasmacytoid dendritic cells (pDCs), AAV capsids are sensed at the cell surface of KC and LSEC. Sensing by KC and LSEC occurs through TLR-2 and results in up-regulation of inflammatory cytokines and chemokines, but - in contrast to pDCs – not in a type I interferon response. These novel insights on the mechanism and consequences of innate immune recognition of AAV are crucial for developing strategies to improve the efficacy of this vector system in human clinical trials.
Enhanced Mmyogenic potential of mesoangioblast-derived induced pluripotent stem cells
Mesoangioblasts (MABs) are a myogenic subset of pericytes, present in the adult skeletal muscles of mice, dogs and humans. MABs can migrate and efficiently engraft into chronically damaged fibres of dystrophic murine and canine muscles. However, MAB expansion is limited by senescence after 15–20 passages. Induced pluripotent stem cells (iPSCs) constitute a novel frontier in the regenerative medicine. iPSCs are obtained from somatic cells via overexpression of a combination of pluripotency-related transcription factors and/or microRNAs. iPSCs share many aspects with embryonic stem cells (ESCs), e.g. wide differentiation potency and vast proliferation capacity, and circumvent ESC-related ethical issues. Nevertheless, iPSCs should still carefully be investigated before a real clinical application, in order to avoid teratomas and to efficiently achieve in vivo regeneration. Recently, iPSCs were generated from murine MABs (MAB-iPSCs), through retroviral overexpression of Oct4, Sox2, Klf4 and cMyc. MAB-iPSCs were comparable to fibroblast-derived iPSCs (f-iPSCs) and to ESCs in morphology, pluripotency marker expression and teratoma formation ability. Strikingly, MAB-iPSC-derived teratomas showed 70% of striated muscle tissue (5% in f-iPSC teratomas). Moreover, flanking regions of myogenic markers were hypomethylated in MAB-iPSCs, as compared to f-iPSCs. In addition, following transient transfection with Pax3 and Pax7, MAB-iPSCs yielded in vitro, at a higher rate than f-iPSCs, CD56+ myogenic progenitors. MAB-iPSC-derived CD56+ and CD56- cells were intramuscularly injected into tibialis anterior muscles of αsg-null dystrophic mice. Only CD56+ progenitors did robustly engraft into severely damaged fibres, partially recovering αsg expression and reconstituting, even if rarely, the satellite cell pool.
Genetics, UCL Institute of Ophthalmology, University College London, London EC1V 9EL, UK
Gene Therapy Program, University of Pennsylvania, Philadelphia, PA 19104, USA
Age-related macular degeneration is the leading cause for visual disability among persons over the age of 60 and affects millions worldwide. Current treatments include vitreal injections of the VEGF antagonists bevacizumab (Avastin®) or ranibizumab (Lucentis®). These drugs are expensive, require repeated vitreal injections and while they are initially present at high concentrations, the levels are sub-therapeutic during the long trough periods before the next injection. In a nonhuman primate study we evaluated the levels and antiangiogenic activity of AAV-expressed bevacizumab or ranibizumab. Cynomolgus or rhesus macaques were injected intravitreally or subretinally with 1011 genome copies of AAV8 vector expressing bevacizumab or ranibizumab under the CMV promoter. No expression was observed in the anterior chamber (AC) fluid of macaques injected intravitreally with either vector (2 eyes/vector). Following subretinal vector injection stable expression of bevacizumab or ranibizumab was observed in the AC fluid (160 and 615 ng/ml, respectively). To pharmacologically regulate ranibizumab expression in the retina, we injected the AAV8-ARGENTTM vector subretinally followed by an intravenous injection of rapamycin to induce expression. Levels of ranibizumab were undetectable prior to induction and close to 200 ng/ml following induction. The antiangiogenic activity of ranibizumab or bevacizumab produced in the AC fluid by the constitutive or the ARGENTTM vector (for ranibizumab) was assayed in the HUVEC model. AAV2/8-expressed ranibizumab and bevacizumab inhibited VEGF-induced proliferation by ∼50% and ∼75%, respectively. In summary, AAV2/8 vectors can express clinically relevant levels of VEGF antagonists in the eye and their expression can be regulated by a safe pharmacological approach.
Sangamo BioSciences, Richmond, CA, USA
Allogeneic cell therapy has the potential to reconstitute cellular function in patients. However, due to the limited availability of donor cells, it is difficult to find an HLA-matched cell product. A solution to improving the availability of cells and tissue for transplantation would be to infuse donor-derived cells that avoid immune-mediated rejection caused by host T cells recognizing disparate major histocompatibility antigens on the infused product. To broaden the application of allogeneic cell-based therapies, we investigated whether HLA gene expression can be permanently disrupted by designer zinc-finger nucleases (ZFNs). ZFN pairs targeting exon 3 of the HLA-A locus resulted in 10% genetic modification of the HLA-A loci in the human kidney cell line HEK293. HLA-Anull HEK293 clones were isolated and shown to evade HLA-A-restricted lysis by T-cell clones, even after interferon-g and TNF-a treatment was used to upregulate HLA expression. Next, we tested these ZFNs in primary T cells and demonstrated that transient expression of in vitro transcribed mRNA generated 40% HLA-Anull primary T cells, which could be readily enriched by paramagnetic bead separation. An attractive clinical application for allogeneic HLAnull T cells is to redirect their specificity independent of HLA via expression of a chimeric antigen receptor (CAR) targeting CD19. Thus, we eliminated HLA-A expression from CD19-specific CAR+ T cells and demonstrated that they (i) evade HLA-A-restricted lysis by T-cell clones, and (ii) specifically lysed CD19+ tumor targets. These data support the concept of the development of “off-the-shelf” allogeneic cells that can be transplanted across HLA barriers on demand.
San Raffaele Scientific Institute, Division of Regenerative Medicine, Stem Cells, and Gene Therapy, Experimental Hematology Unit, Milan, Italy
The ultimate goal of leukemia treatment is the elimination of cells able to survive to radio-chemotherapy, causing disease relapse. Chemoresistance is strictly influenced by signals provided within the bone-marrow (BM) niche. CD44 and its isoforms are crucial players of this crosstalk. With the aim of targeting chemoresistant acute myeloid leukemia (AML) cells, we constructed a second-generation chimeric antigen receptor targeting CD44 variant 6 (CD44v6-CAR.28z). After stimulation with anti-CD3/anti-CD28 cell-sized beads and IL-7/IL-15, CD44v6-redirected T cells efficiently expanded, while preserving a central memory phenotype. In co-culture experiments, T cells were effective against AML cells isolated from patients, but not against healthy CD34+ stem cells. Importantly, chemoresistance induced by AML interaction with BM-derived stromal cells, did not translate in resistance to the immune attack mediated by CD44v6-redirected T cells. The efficacy of CD44v6-redirected T cells was also proved in the autologous setting. Interestingly, similar results were observed on multiple myeloma (MM) cells derived from patients, which also express CD44v6. CD44v6-redirected T cells proved to have major antileukemia activity, as ascertained in a xenograft model in NSG mice. Co-expression of CD44v6-CAR28z and the thymidine kinase suicide gene by a lentiviral vector carrying a bidirectional promoter enabled the elimination of CD44v6-CAR28z+ T cells upon prodrug administration, providing a safety switch in case of toxicity. The use of the inducible-caspase-9 as suicide gene allowed a more rapid elimination of modified T cells. These results warrant the clinical implementation of CD44v6-redirected suicide gene-modified T cells for safe and effective eradication of chemoresistant AML and MM cells.
San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy
The transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive strategy for cancer immunotherapy. However, TCR gene transfer into mature lymphocytes results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. To address these limitations, we developed a novel strategy based on zinc finger nucleases (ZFNs) that allows for the first time the editing of T-cell specificity at the DNA level. We established a protocol that sequentially disrupted the endogenous TCR chains with high efficiency (averages: 36% and 18%) and was followed by lentiviral transfer of the tumor-specific TCR chains (average efficiencies: 65% and 25%). This procedure resulted in a population of TCR-edited lymphocytes expressing only the tumor-specific TCR that, in the absence of competition, was expressed at high and physiological levels. TCR-edited cells were easily expanded to near-purity and proved superior to matched cells undergoing conventional TCR gene transfer in specific antigen recognition, including killing of primary leukemias. Moreover, in a humanized GvHD model, we showed that while 100% of mice infused with unmanipulated T cells, and 80% of mice receiving TCR-transferred lymphocytes develop lethal GvHD, no GvHD is observed upon infusion of matched TCR-edited cells, despite substantial and similar engraftment rates. Together, these data demonstrate complete editing of T-cell specificity at the genetic level to generate safe and high-avidity tumor-specific T cells.
Hematopoiesis and Gene Therapy Division. CIEMAT/CIBERER. 28040, Madrid, Spain
Conducting gene therapy in genetic aplasias has specific difficulties due to the reduced number of HSCs available for genetic correction. As recently shown, the lentiviral-mediated correction and reprogramming of skin cells offer the possibility of generating large numbers of disease-free hematopoietic progenitors from Fanconi anemia (FA) patients. In this study we aimed to generate genetically-corrected iPSCs using a safer approach in which the therapeutic FANCA gene was targeted into the “safe harbor” AAVS1 locus using ZFNs. A donor IDLV containing a promoterless EGFP gene and the PGK-FANCA flanked by AAVS1 homology arms was constructed. Upon targeted integration into the AAVS1 locus, GFP is expressed from the endogenous promoter of the PPP1R12C gene, located upstream of AAVS1. Fibroblasts from three FA-A patients were co-transduced with AAVS1-ZFNs AdVs and the donor FANCA-IDLV. Interestingly, the percentage of targeted FA-A fibroblasts progressively increased from 0.2–4.5% to up to 40%, showing the proliferation advantage of corrected cells. The integration of FANCA in the AAVS1 locus was confirmed by PCR. Additionally, the restored FA pathway in these cells was verified by the formation of nuclear FANCD2 foci after DNA damage. The transduction of corrected cells with the reprogramming STEMCCA LV allowed us to generate 19 iPS clones. In these clones a low number of FANCA copies (0.8–1.2 copies/cell) was detected, suggesting that only the targeted copy was integrated in the genome of FA-A iPS cells. Our data demonstrates the efficacy gene editing in FA cells, opening new possibilities of homologous recombination-mediated gene therapy in FA.
Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
Severe combined immunodeficiencies (SCID) are the most severe form of inherited blood disorders. Failure of the adaptive immune response is due to the absence of T and/or B/NK cells. The NOD.SCID mouse is a valuable model for human SCID in general and radiosensitive SCID (RS-SCID) in particular. A point mutation in exon 85 of the prkdc gene, which encodes for the DNA repair factor DNA-PKcs, leads to a T−/B− immune phenotype. Here, we generated zinc-finger nucleases (ZFNs) directed against intron 84 of the prkdc locus in order to establish a gene targeting approach for RS-SCID. We showed that ear fibroblasts from adult NOD.SCID mice are deficient for DNA-PKcs dependent signaling and sensitive to the DNA double strand break inducing agent bleomycin, hence reproducing the radiosensitive cellular phenotype. ZFN-mediated integration of cDNA encompassing exons 85/86 into these cells re-established expression of functional DNA-PKcs and restored DNA-PK dependent phosphorylation of RPA2. Moreover, gene targeted fibroblast clones showed a 4.5-fold reduced sensitivity to bleomycin. Using conditions that protected the cellular genome from free radicals, we successfully generated induced pluripotent stem cells (iPSCs) from NOD.SCID ear fibroblasts. These RS-SCID iPSCs revealed an intact karyotype and expression of pluripotency markers. When applying the same ZFN-based correction strategy, ∼90% of iPSC clones showed targeted integration of the exon 85/86 encoding cDNA into intron 84, and DNA-PK signaling was restored. Our findings demonstrate that a targeted, ZFN-based genome engineering approach can be applied to correct an RS-SCID phenotype in primary cells.
Harelbekestraat 72, Gent, Belgium
The groundbreaking work of Yamanaka's group (2006) proved the possibility to reprogram fibroblasts into induced pluripotent stem cells or iPS by viral introduction of 4 types of plasmid DNA, each encoding a defined factor (Oct4, Sox2, Klf4, cMyc). Since then, laboratories all over the world tried to improve the efficiency or safety, among others by using other (Nanog and Lin28) or less factors, other transfection methods (non-integrating vectors, protein-based reprogramming). Recently, it became clear that mRNA, however always believed to be too unstable, can transfect cells efficiently. mRNA not only has the potential to introduce the 4 above-mentioned factors in somatic cells and thereby induce their reprogramming, it has several advantages, such as no risk of insertional mutagenesis and lower immunogenicity.
Our group has elaborate experience in mRNA transfection by non-viral means. Because mRNA induced protein expression is in situ transient, it is imperative that expression of the 4 factors lasts long enough to induce reprogramming. Therefore, we introduced mRNA encoding luciferase (by lipofection) in two cell lines (Hela and MSC) and ensured detectable luciferase levels up to 9 days post transfection. Then, we subjected fibroblasts to multiple transfections with mRNAs encoding the reprogramming factors (Oct4, Sox2, Klf4, cMyc). Flow cytometry following immunostaining proved successful transfection of all introduced factors, moreover, expression of pluripotency markers (e.g. Nanog an SSEA-1) were significantly increased. Preliminary results, based on morphology and immunohistochemistry, indicate that iPS colonies have indeed been formed. The final proof by means of embroid body and teratoma formation is currently being performed.
Experimental Hematology Unit, Division of Regenerative Medicine, San Raffaele Scientific Institute, Milan, 20132, Italy
Duchenne muscular dystrophy (DMD) is the most severe form of genetic muscular dystrophies and no effective therapies are available at present. Mesoangioblasts (MAB) are myogenic stem cells that produce dramatic functional improvement in animal models of muscular dystrophies. Following this preclinical evidence, a phase I-II clinical trial based on the systemic infusion of MAB isolated from HLA-identical familiar donors started at our Institute. We characterized the immunological properties of MAB to investigate their ability to induce alloreactive immune responses that could hinder cell therapy. In vitro isolated and expanded MAB expressed HLA-I molecules but displayed very low levels of expression of HLA-II, immune adhesion molecule ICAM-1 and costimulatory molecules. To mimic inflammation, we exposed MAB to IFN-g (g-MAB), and observed significant up-regulation of HLA-II and ICAM-1 but not of costimulatory molecules. When MAB were used for direct sensitization of allogeneic T cells, a mixed Th1/Th2 polarization was observed, while g-MAB induced a preferential Th1 response. Interferon-g-pretreatment induced the generation of cytotoxic T cells (CTL) which recognized g-MAB but not untreated MAB. Furthermore, MAB induced the expansion of Th17 but not of Treg/Tr1 cells, independently from IFN-g treatment. Finally, g-MAB and myotubes, but not MAB, harvested from male donors, were recognized by a CTL clone specific for the minor histocompatibility alloantigen H-Y, ubiquitously expressed on male cells. Altogether these results suggest that, although immunologically inert, MAB may become targets of the alloreactive responses upon inflammation, justifying and recommending the use of immunosuppressive and/or anti-inflammatory drugs in allogeneic cell therapy of DMD.
Dep. of Pediatrics, Children's Hospital Bambino Gesù and University of Rome Tor Vergata School of Medicine, Rome
Chronic Granulomatous Disease (CGD) is caused by defective NADPH oxidase function in patients' phagocytes leading to increased susceptibility to fungal and bacterial infections. Our goal was to develop a novel gene transfer approach into hematopoietic stem cells (HSC) for safe and efficacious therapy of X-CGD using 3rd generation SIN lentiviral vectors (LVVs) encoding gp91phox by a cellular promoter (LVV-PGK.gp91). To reduce the risk of ectopic gp91phox expression in HSC, we introduced miR-126 target sequences (miR-126T) into a gp91phox expression cassette (LVV-PGKgp91_126T), to allow post-transcriptional downregulation of gp91phox expression by miR-126 which is found to high levels in HSC but not in the myeloid progeny (Gentner et al., Sci Transl Med 2010).Transduced myeloid (PLB985) and EBV-B cell lines defective for gp91phox showed a restored protein expression and NADPH oxidase activity. Furthermore, in the presence of miR-126 expressing LVV, only LVV-PGKgp91_126T-transduced cells showed dramatically reduced gp91phox expression.
We then transduced healthy donors or X-CGD CD34+ cells to investigate the effect of miR126 mediated regulation system. LVV.PGK.gp91- transduced cells displayed an ectopic expression of gp91phox in CD34+ cells, which was substantially reduced in cells transduced with LVV-PGKgp91_126T. Following in vitro differentiation into granulocytes, human X-CGD CD34+ cells and X-CGD mouse Lin- transduced with the different vectors showed restored gp91phox expression and NADPH function. In vivo studies in the murine model are ongoing to test the safety and the efficacy of our system. Our strategy based on the use of SIN-LVVs with miRNA target sequences could represent an efficient approach for X-CGD gene therapy.
Department of Haematology, University College London, London, WC1E 6BT
Recombinant adeno-associated virus (AAV) vector based gene therapy for Haemophilia A (HA, factor VIII deficiency) has been hampered by the relatively large size of the hFVIII cDNA, (∼7.0 kb) which far exceeds the normal packaging capacity of this vector. We have begun to address some of these limitations through the development of a 5.7kb AAV expression cassette (rAAV-HLP-codop-hFVIII-N6) which consists of a novel more potent codon-optimised hFVIII (codop-hFVIII-N6) including a short 226 amino-acid B-domain spacer which has 6 N-linked glycosylation sites. This variant is under the control of a small ∼200bp liver specific promoter. Tail vein injection of AAV serotype 5 or 8 rAAV-HLP-codop-hFVIII-N6 in C57Bl/6 mice resulted in FVIII levels of 23±6 IU/ml and 54±12 IU/ml respectively. This level of expression is 400-fold greater than required for therapeutic efficacy (0.05IU/ml) and at least 10 fold higher than achieved in mice transduced with a comparable dose of rAAV encoding either the BDD or N6 variant of hFVIII. To confirm correction of the bleeding phenotype, either 4x1011 or 4x1012 rAAV5-HLP-codop-hFVIII-N6 vector genomes were injected into the tail vein of HA knockout mice. Peak hFVIII levels, as determined by a one-stage clotting assay, were 137±27% and 374±18% of normal levels in the low and high-dose cohorts of F8−/− mice respectively. These levels were sufficient to arrest bleeding in a modified tail clip assay. Therefore, the higher potency of our novel codop-hFVIII-N6 construct and the ability to package this FVIII variant within AAV virions has substantially improved the prospects of effective gene transfer for Haemophilia A.
Cancer Gene Therapy Group, University of Helsinki, Finland
We created a fully serotype 3 oncolytic adenovirus Ad3-hTERT-E1A. Ad3 uses receptor(s) which are suggested to be abundant in tumors. Recently it was shown that desmoglein 2 is a major attachment receptor for Ad3. Clinical data with serotype 5 oncolytic adenoviruses show rapid induction of neutralizing antibodies. We hypothesize that in a repeated dosing regimen switching the serotype might restore effective tumor transduction and give an alternative immunological stimulus. Our preclinical in vitro and in vivo data give a proof of principle.
So far 25 patients with advanced solid tumors, refractory to standard therapies, have been treated with Ad3-hTERT-E1A. The safety profile was good with doses up to 4x1012 VP. Overall, all patients experienced grade 1–2 flu-like adverse events. No life-threatening adverse events were seen.
Objective signs of efficacy (RECIST/PERCIST/tumor marker) were seen in 71% of patients treated with Ad3-hTERT-E1A. Also few complete responses were noted. Median survival of patients was 210 days. Neutralizing antibodies against serotype 3 increased in all patients (median 12 fold, N=13). However, serotype 5 neutralizing antibodies with the same patients were stable (increase 3, decrease 3, stable 7). Treatment with Ad3-hTERT-E1A cleared lymphocytes from the blood for several weeks. In blood a varying numbers of interferon gamma secreting lymphocytes were observed, after stimulation with survivin or cancer specific peptide mixes, before and after treatment.
All together, these findings suggest that Ad3-hTERT-E1A is an effective oncolytic virus that can be used in humans and that the alternative immunological response might be beneficial.
Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London
Adenoviruses have broad applications in medicine, and one such example is cancer virotherapy. Given the fact that E3 is not necessary for adenovirus replication in vitro and E3 deletions provide more space to arm adenovirus vectors, the large majority of oncolytic adenoviruses in clinical trials have deletions within the E3B region. We previously have demonstrated that the anti-tumour potency of the E3B-deleted mutant (dl309) was inferior to wild type virus (Ad5). Tumours treated with dl309 showed a significant infiltration of macrophages compared to Ad5-treated tumours. However, the functional mechanisms for this were not known. Here we demonstrated that E3B gene expression inhibited secretion of chemokines in macrophage after adenovirus infection and in turn reduced macrophage migration. Further stepwise investigations revealed that E3B proteins inhibit Stat 1 function by preventing its expression and nuclear translocation; the E3B-14.7k protein physically interacted with Stat 1. Stat 1 inhibitor, fludarabine, rescued the effect of E3B-14.7k by down-regulating target chemokine gene expression in macrophage and significantly enhanced the antitumor efficacy of E3B-deleted oncolytic adenovirus in vivo. These findings have important implications for clinical use of E3B-deleted oncolytic adenovirus and other E3B-deleted adenovirus vector-based therapy.
Uppsala University
Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surface of tumor cells. Furthermore, infected cells overproduce adenovirus fiber proteins, which are released prior to cell lysis. The released fibers block CAR on non-infected neighboring cells thereby preventing progeny virus entry. Our aim was to add a fiber-independent/CAR-independent infection route to Ad5 to increase the infectivity of tumor cells with low CAR expression and prevent the fiber-masking problem. We constructed Ad5 viruses that encode the protein transduction domain (PTD) of the HIV-1 Tat protein (Tat-PTD) in hyper variable region 5 (HVR5) of the hexon protein. Tat-PTD functions as a cell-penetrating peptide and Tat-PTD-modified Ad5 showed a dramatic increased transduction of CAR-negative cell lines compared to unmodified vector. Moreover, while tumor cell infectivity was severely reduced for Ad5 in the presence of fiber proteins it was only marginally reduced for Tat-PTD-modified Ad5. Furthermore, because of the sequence alteration in the hexon HVR, coagulation factor X (FX)-mediated virus uptake was significantly reduced. Mice harboring human neuroblastoma and neuroendocrine tumors show suppressed tumor growths and prolonged survival when treated with Tat-PTD-modified oncolytic viruses. Our data suggests that modification of Ad5 with Tat-PTD in HVR5 expands its utility as an oncolytic agent.
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA research center, Austrian Cluster for Tissue Regeneration, Vienna, Austria
Sonoporative gene transfer, a minimal invasive non-viral gene transfer method, has been shown to trigger expression of plasmid vectors in vivo. The combination of Bone Morphogenetic Protein 2 (BMP2) and Bone Morphogenetic Protein 7 (BMP7) in a co-expression strategy has been proven highly osteoinductive.
Therefore, the aim of this study was to design and test a BMP2/BMP7 co-expression plasmid for its regenerative potential in in vivo models.
BMP2 and BMP7 were cloned into a modified pVAX1 constitutive expression plasmid and an inducible TetON system for co-expression. Induction of osteogeneic differentiation was tested in vitro in C2C12 cells with an osteocalcin specific reporter system and standard methods. Subsequently, the plasmids were administered in an ectopic mouse model and 3 days post fracture in a rat femur non-union model using sonoporation. Animals received 5 transcutaneous treatments on 5 subsequent days. Controls received luciferase plasmid, which allowed monitoring of gene transfer efficacy through bioluminescence imaging. All animals were imaged using an in vivo μCT on days 28 and 56.
Bioluminescence showed strong luciferase expression after sonoporation. μCT images showed ectopic bone formation in muscle in mice and significantly enhanced bone volume as well as unions in critical size rat femur defects after treatment with BMP2/BMP7 sonoporation. Therefore, we conclude, that sonoporative gene transfer is an effective non-viral method to mediate transient transgene expression in ectopic and orthotopic settings and to improve bone regeneration in orthotopic settings.
Facharzt für Kinder- und Jugendmedizin Moorenstr. 5 40225 Düsseldorf
Correction of genetic mutation by homologous recombination (HR) is a very promising new approach for gene therapy. Meganucleases (MNs) are highly specific enzymes that can induce HR in different types of cells. Consequently, these enzymes can be used as tools to induced HR with a DNA repair matrix in murine hematopoietic stem cells (mHSC).
To develop this approach, we chose the Artemis deficiency mouse model which was created by substitution of exon 12 of the Artemis gene by a restriction site for the I-Sce1 MN.
To deliver I-Sce 1 and to provide the Artemis repair matrix we designed two SIN-integrase-defective lentiviral vectors (SIN-IDLV): IDLV-CMVI-Sce1 and IDLV-Art in order to avoid non-specific integration and transgene over-expression.
In this work, we showed that transduction of Art−/− mHSC with both vectors, resulted in a reversion of the Artemis deficient phenotype indicating successful gene targeting in 2 out 10 experiments (2,4 and 1,3% CD8+/CD4+ DP cells respectively). Site specific insertion of Artemis exon 12 by HR was confirmed by nested-PCR and genomic DNA sequence analysis.
Even though the efficiency of genomic reparation needs to be further improved and its potential genotoxicity remains to be evaluated, this new approach of gene therapies is very promising for the future.
MHH Inst. Experiment. Haematol. D-30625 Hannover
Until recently usage of replicating episomes has been restricted to viral systems. The EBV-paradigm shows that replication relies on (i) chromosome association, (ii) initiator-of-replication- (IR) and (iii) DNA strand separation (SIDD-) potential. Functions (i) and (iii) could be provided by an S/MAR element placed downstream from an active transcription unit. While this combination meets fundamental requirements, present improvements concern the IR-functions derived from lamin B. Besides a limited cloning capacity silencing actions remained the shortcomings of first-generation episomes. These restrictions could be overcome by Flp-catalyzed recombination, i.e. conversion to minicircles (MCs). Other than conventional MCs that persist only in non-dividing tissues, these present the first examples suitable for the modification of dividing cells due to their authentic replication and segregation. Supported by bioinformatic routines effective S/MAR modules were identified and assembled yielding a ∼3 kb MC “M18” with just the active transcription unit in addition to a 730 bp S/MAR-fragment. M18 derivatives prove a virtually unlimited replication capacity in dividing cells. Their potential to modify tissues, stem cells and zygotes is under investigation. The scope of applications is expanded as different MCs can be established side-by side allowing the immediate expression of multi-subunit proteins and the design of regulated systems encoding the transactivator and a tet-regulated GOI, respectively. While the minicircle production process could continuously be refined, MC generation is also feasible in the recipient mammalian cell. This “all-in-one” principle serves mainly exploratory purposes, but it can be linked to existing MC production routines enabling entities with authentic ccc-status (coop.: PlasmidFactory).
Development of minicircle-DNA vectors as non-viral liver-directed gene therapy for phenylketonuria (PKU)
We have previously reported long-term correction of hyperphenylalaninemia in the PKU mouse model, C57Bl/6-Pahenu2, after liver-directed gene transfer with recombinant adeno-associated viral (AAV) vectors (Ding et al 2006, Ding et al 2008, Rebuffat et al 2010). However, safety requirements for targeting newborn and paediatric patients for potential life-long treatment remain a challenge for virus-dependent approaches. Specifically, questions of high vector load with pure AAV preparations and its potential for insertional mutagenesis and subsequent toxicity remain to be solved. Currently, we are developing and evaluating highly efficient non-viral gene transfer methods by targeting the murine liver as a potential alternative gene-therapeutic approach. In our study, we report the use of the minicircle (MC) technology for gene therapy of PKU the mouse model. Our MC-DNA vectors contain a liver-specific promoter, the Pah-minigene, a luciferase reporter plus a downstream mammalian scaffold/matrix attachment region (S/MAR) element for episomal maintenance and/or extra-chromosomal stability. Delivery was mediated by hydrodynamic tail vein (HTV) injection as a liver-targeted approach. Luciferase expression was monitored by quantitative bioluminescence in a non-invasive imaging technology in living mice (IVIS screening). We compared longitudinal luciferase expression between MC-DNA vector and conventional plasmid DNA (pDNA). Our data showed that vectors were exclusively delivered to the liver and the luciferase expression in mice injected with MC was more than 20-fold higher than mice injected with pDNA for at least 9 months. MC gene delivery for maximizing safety and sustained gene expression is a potential new approach for the hepatic treatment.
Orally administered chitosan nanoparticles for gene delivery of FIX to treat hemophilia B
Non-viral vectors show excellent safety profiles and allow repeated administration over long periods of time. Hemophilia B, a bleeding disorder caused by deficiency of coagulation factor IX (FIX), is especially well suited for a gene therapy approach since plasma levels above 1% can already prevent major long-term complications. In this study we are developing an oral delivery system for the treatment of hemophilia B based on the cationic polymer chitosan known to protect DNA from degradation. In cell culture experiments transfection with nanoparticles resulted in expression of up to 100 ng/ml (2% of normal plasma concentration). Next, we treated groups of mice (n=4–5/group) with naked DNA or nanoparticle complexes containing FIX or mock DNA. Circulating plasma levels in all groups remained below the therapeutic range (<1%). To improve therapeutic efficacy, different amino acid substitutions known to increase FIX clotting activity were combined and introduced into the expression cassette by site-directed mutagenesis. One protein variant was identified with 1500% FIX specific clotting activity compared to the wild-type protein. Following oral gene transfer in mice, FIX antigen expression levels remained low but shortened clotting time with a circulating FIX activity of 1–4% compared to plasma activity of normal controls. Immune histochemical staining confirmed FIX accumulation in the extracellular matrix of the gut. In conclusion, gene transfer strategies based on non-viral vectors are still hampered by their low efficacy but in combination with engineered proteins might provide a tool to overcome this shortcoming.
Hypoxia-regulated HO-1 expression improves post-ischemic blood flow and skeletal muscle regeneration
Heme oxygenase-1 (HO-1) is a heme-degrading enzyme that generates carbon monoxide (CO), bilirubin and iron ions. Through these compounds HO-1 mitigates cellular injury by exerting antioxidant, anti-apoptotic and anti-inflammatory effects. Uncontrolled overexpression of HO-1 can, however, cause some detrimental effects. Here, we report the successful construction and use of plasmid vector expressing human HO-1 under the regulation of three hypoxia response elements and a minimal cytomegalovirus promoter (pHRE-HO-1). Human microvascular endothelial cells (HMEC-1) transfected with pHRE-HO-1 and cultured in hypoxic chamber with 0.5% oxygen had significantly increased expression of HO-1. In contrast, the basal level of HO-1 in these cells cultured n normoxic conditions was very low, demonstrating that our construct is functional. Additionally, such hypoxia-upregulated HO-1 was effective in conferring protection against H2O2-induced cell death and in promoting the proangiogenic phenotype of HMEC-1 cells. More importantly, when delivered in vivo, pHRE-HO-1 significantly improved the post-ischemic foot blood flow and decreased the incidence of toe necrosis in C57BL/6 mice subjected to femoral artery ligation. These protective in vivo effects were associated with reduced levels of pro-inflammatory cytokines (IL-6 and CXCL1) and decreased expression of caspase-3. Moreover, HO-1 delivered into mouse skeletal muscles seems to influence the regenerative potential of myocytes as it significantly changed the expression of transcriptional (myogenin) and post-transcriptional (miR-146a and miR-206) regulators of genes involved in satellite cell differentiation and skeletal muscle regeneration. Our results demonstrate that pHRE-HO-1 vector can be further investigated as a tool to provide therapeutic effects in critical limb ischemia.
Reducing LDL cholesterol with exon-skipping antisense oligonucleotides
Familial hypercholesterolaemia (FH) causes extremely high levels of plasma low-density lipoprotein (LDL) due to defective hepatic LDL-receptors. Patients with homozygous FH have severe cardiovascular disease and often childhood mortality. Current hypocholesterolemic drugs, such as statins, are inadequate and alternative therapies to reduce LDL are needed.
The structural apolipoprotein in LDL particles is APOB100. We have developed 'skip27' antisense oligonucleotides (ASOs) that induce the skipping of APOB100 exon 27 to generate a truncated form, APOB87(SKIP27). This mimics the truncated APOB isoforms found in families with hypobetalipoproteinaemia who have very low LDL and cholesterol levels.
Here, we screened >30 ASOs in HepG2 cultures to increase skip27 efficiency up to 15-fold over a previously published ASO. By using two quantitative real-time PCR assays to detect the skip27 (sk) and full-length (fl) APOB isoforms, we optimized ASO sequence and chemistry to achieve a mean skipping efficiency of 58.7% (sk/(fl+sk)*100; n=3). Western blotting confirmed expression and secretion of truncated APOB87.
Complexation of skip27 ASO with Invivofectamine 2.0 and intravenous injection into transgenic mice expressing human APOB100 resulted in abundant ASO delivery to hepatocytes and high skipping activity (25 mg/kg dose; 29.1% skipping 24 h post-injection; n=5), which persisted for >6 d (10.8% skipping; n=2). This was significantly higher than achieved with ASO hydrodynamic injection (25 mg/kg; 1.13–1.54% skipping; 24 h post-injection; n=9).
Preliminary in vivo data show that LDL-cholesterol falls in mice injected weekly with skip27-ASO complexes. Our findings suggest that ASO-mediated exon skipping of hepatic APOB100 is a promising therapeutic approach to treat FH.
Role of antioxidants genes in angiogenesis: significance for endothelial progenitor cells
Hypoxia and oxidative stress stimulate revascularization but they could also affect bone marrow-derived endothelial progenitor cells (EPC). Several antioxidant enzymes are involved in EPCs protection. Among them, heme oxygenase-1 (HO-1) has been demonstrated to be crucial the processes of angiogenesis and post-natal vasculogenesis (Dulak et al., Circulation 2008, 117: 231–241). Here we examined the role of HO-1 and Nrf2, the major transcription factor regulating HO-1 and other anti-oxidant genes expression, in angiogenic potential of EPCs and revascularization after hind-limb ischemia. Bone marrow-derived EPCs (Sca-1+, VEGFR-2+, CD45-) isolated from Nrf2−/− mice were more sensitive to oxidative stress and produced more reactive oxygen species in comparison to Nrf2+/+ EPCs. Lack of Nrf2 inhibited proliferation and migration of EPCs upon VEGF or SDF-1α stimulation, respectively. Accordingly, Nrf2−/− EPCs have decreased angiogenic potential as evidenced by tube formation on Matrigel and spheroid angiogenic assay. Similar effect was observed in EPCs derived from HO-1 −/− mice. Accordingly, revascularization after hind-limb ischemia is attenuated by HO-1 deficiency both in normoglycemic and streptozotocin-induced diabetic conditions and it is related to impaired angiogenesis in HO-1−/− mice. Interestingly, although the lack of Nrf2 impairs EPC functions, and EPC mobilization after hind-limb ischemia is attenuated in Nrf2−/− mice, surprisingly the revascularization is augmented in Nrf2−/− mice in comparison to wild-type animals. Thus, although both Nrf2 and HO-1 significantly affect EPCs functions, their effect on revascularization appears to be different. It may be dependent on different extent of inflammatory response during ischemia in mice of different genotype of antioxidant genes.
Viral Vector Production Laboratory, Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota 55905 USA
Measles viruses (MVs) have shown potent oncolytic activity as a therapeutic against a variety of human cancers in animal models and are currently being tested in clinical trials in patients. MV oncolytic activity depends on high concentrations of infectious virus. However, MV is extremely shear-sensitive and pleiomorphic in size necessitating a gentle aseptic production process. Previously, our Good Manufacturing Practices (GMP) MV production process limited the maximum MV dose in patients to levels estimated to be sub-therapeutic based on preclinical animal studies. We have made process improvements that increased volumetric MV productivity by 10-fold and infectious titer of the final product 100-fold that will allow clinical trials to reach doses predicted to be in the therapeutic range. Changing the process from using adherent Vero cells to suspension HeLaS3 cells enabled the development of standard conditions that yield >10-fold higher levels of infectious MV. Cell culture supernatant containing MV is clarified by filtration, and then treated with Benzonase® to digest contaminating nucleic acid. The MV in the treated supernatant is then concentrated and purified using hollow-fiber tangential flow filtration (TFF) and diafiltration. We updated our TFF system from one using autoclavable TFF cartridges to a flexible system using disposable, pre-sterilized components and a TFF module with an increased pore size. After a final clarifying filtration, GMP preparations of MVs now reach titers of >3×109 TCID50/mL and yields of >7×1011 TCID50 units from 75 L MV infected cell supernatant. The transition to suspension cell culture coupled with the improved TFF process will facilitate further product scale-up.
Tau reprogramming by RNA trans-splicing: a gene therapy strategy for neurodegenerative diseases
The microtubule-associated protein Tau (MAPT) is predominantly expressed in neurons where it promotes microtubule polymerization and stabilization. Exon 10 (E10) of the MAPT gene encodes the second of four imperfect microtubule-binding repeats. Exclusion or inclusion of E10 gives rise to Tau isoforms with three (3R) or four (4R) microtubule-binding repeats, respectively. In the normal adult human brain the ratio 4R/3R is 1. Recent genetic evidence demonstrated that some neurodegenerative diseases characterized by the intracellular aggregation of tau (tauopathies) are associated with abnormal alternative splicing of E10, which affects the normal ratio of 4R/3R Tau isoforms.
We developed a strategy to modulate Tau 4R/3R ratio by reprogramming the inclusion of E10 in endogenous Tau via spliceosome-mediated RNA trans-splicing (SMaRT). SMaRT creates a chimeric mRNA through a trans-splicing reaction between an endogenous mRNA and an exogenously delivered RNA pre-trans-splicing molecule (PTM). To test the potential therapeutic utility of this strategy for tauopathies we used a mouse model that carries the human Tau gene in a Tau knock-out background (hTau mice). hTau mice present an imbalance in 3R/4R Tau isoforms in the adult brain and develop Tau aggregation and cognitive deficit from 9–12 months old.
We delivered Tau-PTMs into differentiated primary neurons of hTau mice by lentiviral vectors. Trans-spliced product was detected both at the RNA and protein level, demonstrating efficient translation of the chimeric RNA as well as isoform conversion between 3R and 4R Tau. Together, our results provide promising perspectives of a plausible gene therapy approach for human tauopathies.
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany
Retroviral vectors are important tools for human gene therapy. However, due to their semi-random integration in the genome, they bear the risk of deregulating gene expression and subsequently transforming cells. Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies have been conducted with alpharetroviral vectors harboring viral coding sequences and intact LTRs.
We have developed a self-inactivating (SIN) alpharetroviral vector system with an advanced split-packaging design, lacking viral coding sequences in the vector genome and enabling high-titer vector production in human cells. In a mouse model of serial transplantation we compared alpharetroviral, gammaretroviral and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs) at low MOI. Importantly, alpharetroviral SIN vector integrations were decreased in the vicinities of transcription start sites, CpG islands, and cancer genes compared to gammaretroviral integrations, and decreased in intragenic regions compared to lentiviral integrations. Hence, the favorable alpharetroviral integration pattern also applied to the newly developed SIN vectors in serially transplanted HSCs. Analyzing the correlation of the transcriptome and intragenic integrations in engrafting cells, we observed coincidences to be strong for lentiviral vectors, lower for gammaretroviral, and inverse for alpharetroviral vectors. Furthermore, sensitive genotoxicity studies using the In Vitro Immortalization Assay revealed a decreased incidence of immortalization for alpharetroviral vectors.
These experiments suggest that alpharetroviral SIN vectors are suitable for genetic modification of HSCs and that their favorable integration spectrum is reflected in a decreased risk of insertional cell transformation.
Genetic modification of cancer cells using non-viral, episomal S/MAR vectors for in vivo tumour modeling
We are developing non-viral vectors for the prophylactic gene therapy of Birt–Hogg–Dubé (BHD) syndrome (caused by mutations in the tumour-suppressor protein folliculin) whose principle life-threatening manifestation is an increased risk of renal neoplasia.
We hypothesise that if a protective copy of folliculin is introduced into the renal cells of BHD patients these would become resistant to BHD-associated neoplasia.
We have generated a novel class of S/MAR vectors which are designed to be maintained episomally in cells and provide safe, sustained and regulated gene therapy without limitations of toxicity or insertional mutagenesis.
We have generated a range of vectors, which express folliculin and luciferase/eGFP and describe their application for BHD gene therapy in vitro, ex vivo and in vivo.
We describe methods we have developed for generating folliculin-Luciferase/eGFP expressing cells in knockout cell-lines using these episomal constructs. We show restored folliculin expression using RT-PCR and Western analysis and show clear differences between the viability of corrected cells and controls and the restoration of FLCN-mediated regulation in their TGF-β pathway. Additionally, we have xenografted these corrected cells into immunodeficient mice and will describe the differences between folliculin supplemented cells and their knockout controls.
The safe and persistent restoration of folliculin to BHD cells and the new methodology we have developed for the effective delivery of these vectors to renal cells in vivo demonstrate the potential of S/MAR vectors for the genetic correction and prophylactic treatment of BHD.
Institute of Cardiovascular and Medical Science, College of Medical, Veterinary and Life Sciences, University of Glasgow, UK
Stroke remains the 3rd leading cause of death in the UK with <5% of patients receiving therapeutic thrombolysis. We propose by combined neuroglobin (Ngb) overexpression and JNK inhibition, using gene- and drug-delivery, a greater beneficial effect than either agent alone will be seen in an in vitro proof-of-concept study and subsequently in a robust preclinical experimental stroke model. Lentivirus-mediated Ngb overexpression (5vp/cell) combined with JNK inhibition (SP600125, 20μM) significantly reduced oxidative stress following 9 hrs hypoxia/24 hrs reoxygenation in rat neuronal (B50) cells determined by electron paramagnetic resonance (normoxic 551±6, hypoxic 838±30, combined treatment 571±11* counts/min/ng protein, *p=0.001 vs. hypoxic) and malondialdehyde assay (normoxic 0.365±0.03, hypoxic 1.369±0.12, combined 0.359±0.03* MDA μmol/μg protein, *p=0.0002 vs. hypoxic). Treatment also significantly reduced apoptosis assessed by cell death ELISA (normoxic 0.103±0.004, hypoxic 0.302±0.006, combined 0.108±0.006* absorbance/μg protein, *p<0.005 vs. hypoxic) and immunostaining for nuclear-localised caspase 3. Male hypertensive rats (5 groups, n=9) subjected to 45 minutes transient middle cerebral artery occlusion (tMCAO) (tMCAO; control green fluorescent protein-expressing canine adenovirus, CAV-GFP; Ngb-expressing CAV, CAV-Ngb; SP600125, 1mg/kg i.v.; CAV-Ngb+SP600125) or sham procedure were longitudinally assessed for neurological outcome (+14days) before infarct determination. CAV (3x109vp) was administered stereotactically into the cortex 5 days pre-tMCAO. Neurological outcome and survival were improved in CAV-Ngb and CAV-Ngb+SP600125 groups. Ongoing studies will determine if this is reflected in reduced infarct volumes. Our translational study demonstrates combined Ngb overexpression with JNK inhibition improves neurological outcome greater than either therapy alone in vitro following hypoxia/reoxygenation and in vivo following transient cerebral ischaemia.
BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, G12 8TA, UK
Hepatocyte transduction following intravenous administration of adenovirus 5 (Ad5) is mediated by interaction between coagulation factor X (FX) and the hexon. The FX serine protease (SP) domain tethers the Ad5/FX complex to the hepatocyte surface through binding heparan sulfate proteoglycans (HSPGs). Pharmacological blockade of the heparin-binding proexosite (HBPE) in the SP domain prevents FX-mediated cell binding. The aim of this study was to identify the specific residues of FX which mediate Ad5 attachment to HSPGs. Charge to alanine site directed mutagenesis was performed at the seven residues R93, K96, R125, R165, K169, K236 and R240 previously shown to bind heparin. Stable cell lines were generated to constitutively produce the protein in the presence of vitamin K. The conditioned media was affinity purified using 4G3 mouse monoclonal antibody coupled to sepharose. The rFX proteins were quantified by ELISA, had the predicted molecular weight and could be converted to FXa in the presence of tissue factor and FVIIa. Surface plasmon resonance analysis demonstrated the SP mutations had no effect on FX-specific binding to the Ad5 hexon. However the proexosite mutations ablated FX-mediated Ad5 cell surface binding, internalisation, cytosolic transport and gene transfer as shown by confocal microscopy, qPCR and quantification of transgene expression. The SP mutations also inhibited FX-mediated Ad5 binding to mouse liver sections ex vivo. Taken together, this study uncovers the basic residues (R93, K96, R125, R165, K169, K236 and R240) of FX have a fundamental role in Ad5/FX complex engagement with HSPGs at the surface of target cells.
ICH/UCL, 30 Guilford Street, London WC1N 1EH
Department of Immunology, UCL
TCR gene transfer can generate tumour antigen-specific T cells for adoptive immunotherapy. Following TCR gene transfer, transduced T cells usually display the same functional avidity as the parental clone from which the TCR was isolated. However, tumour-antigen specific T cells typically recognise over-expressed self antigen and are often of low/moderate avidity. It is known that optimal recognition of target cells by CTL requires binding of the cognate peptide MHC class I complex by both TCR and the CD8 co-receptor. Some CD8b chain mutations have been shown to increase CD8 binding affinity with pMHC and enhance T cell effector function.
We have previously generated murine CD8b chain mutants, affecting both MHC binding sites (L58R, S53L, S54V and L58R/I25A) and glycosylation sites (T120A, T121A T124A, T121A/T124A). The mutated CD8b molecules were introduced into murine splenocytes using retroviral vectors together with tumour antigen-specific TCRs.
Using CD8αα T cells obtained from CD8β knockout mice, we have introduced CD8β mutants or control CD8β wild type (WT) chains. All T cells were co-transduced to express the murine F5 TCR (which recognizes the model tumour antigen, influenza A nucleoprotein (NP366) presented by H2Db. The L58R MHC binding CD8 co-receptor mutant (L58R) demonstrated better IFN-γ and IL2 production in response to relevant peptide. We have generated a retroviral vector encoding the human CD8 co-receptor with a homologous mutation (I59R) which has been used to transduce human T cells, together with a CMV-specific TCR. The I59R CD8+ T cells demonstrated reduced antigen-specific proliferation but higher IL-2 production and cytoxicity.
Antigen-expressing immunostimulatory liposomes: a novel genetically-programmable vaccine platform
We propose a novel DNA vaccination platform that is based on the production of protein antigens from DNA templates inside liposomes. This approach circumvents the need for plasmid delivery into the nucleus of endogenous cells of the vaccine recipient, a process which is often very inefficient. Instead, antigens are produced inside the aqueous core of liposomes that can subsequently be taken up by professional APCs. To show proof-of-concept, a bacterial in vitro transcription and translation system together with a gene construct encoding the model antigen β-galactosidase were entrapped inside multilamellar liposomes. Production of β-galactosidase inside liposomes was confirmed by fluorescence microscopy and flow cytometry. Vaccination studies in mice showed that such antigen-expressing immunostimulatory liposomes (AnExILs) elicited higher specific humoral immune responses against the produced antigen (β-galactosidase) than control vaccines (i.e. AnExILs without genetic input, liposomal β-galactosidase or pDNA encoding β-galactosidase). Further studies show a strong adjuvant effect of the AnExIL formulation with induction of chemotaxis of dendritic cells in vitro.
Improved systems for generating and evaluating adenovirus vaccine vectors reveal differences in immunogenicity between vectors of different adenoviral species
Recombinant adenoviruses have emerged as promising vectors for delivery of vaccine antigens. We, and others, have shown that the potential of vectors to elicit transgene product specific immune responses is partly dependent on the serotype of virus on which the vector is based. It is therefore important that we understand the mechanisms behind these differences in immunogenicity.
We have developed a system for generating, modifying and titering novel adenovirus vectors to enable a reliable comparative assessment of immunogenicity. The use of bacterial artificial chromosomes in vector construction has enabled seamless and flexible modification of virus genomes through
Our approach has enabled us to clearly demonstrate superior immunogenicity of AdHu5 (species C) based vectors over a selection of species E chimpanzee adenovirus vectors in mice. The mechanism behind this difference is as yet unclear, but through recombineering we have been able to exchange the virus associated RNA (VA-RNA) between serotypes to investigate whether elements other than the capsid proteins influence vector immunogenicity.
CNC, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. The CAG expansion translates into a toxic polyglutamine tract within the mutant protein, leading to the pathology. No treatment able to modify the disease progression is available.
We previously showed that lentiviral-mediated RNA interference robustly reduces striatal neuropathology in a lentiviral rat model of MJD (Alves et al 2008, 2010). Here, our goal was to investigate if this silencing strategy would decrease: a) the neuropathological features of the disease in the cerebellum, one of the most attained regions in MJD, b) the motor behavior deficits. Therefore, we injected lentiviral vectors encoding the silencing sequences (shAtx3), specifically targeting mutant Atx3, into the cerebellum of a transgenic mouse model expressing a truncated form of mutant ataxin-3 (Torashima et al 2008), and already presenting a dramatic ataxic phenotype by the time of injection at 3–4 weeks of age.
Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant alleviation of gait, balance, locomotor and exploratory activity impairments in shAtx3 injected mice (n=8), as compared to control ones injected with shGFP (n=8) as soon as 4 weeks post-injection. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin immunoreactivity and molecular and granular layers thickness.
These data demonstrate the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities even after the onset of the disease, supporting the use of this strategy for therapy of MJD.
University of Modena and Reggio Emilia, Modena
Retroviral integration is a non-random process, whereby pre-integration complexes of different viruses recognize components or features of the host cell chromatin in a specific fashion. By using deep sequencing technology, we mapped >60,000 MLV and HIV integration sites in the genome of human CD34+ hematopoietic stem/progenitor cells and defined genome-wide integration maps in this cell type. MLV integrations cluster around regulatory elements of genes involved in hematopoietic functions, and to chromatin regions bearing epigenetic marks of active or poised transcription. On the contrary, HIV integrations are clustered in regions marked by histone modifications associated to the body of transcribed genes, and are under-represented in regulatory regions. Although >80% of the genes targeted by HIV integration are expressed by Affymetrix analysis, expressed genes are not equally targeted. By a statistical analysis, we define a set of <500 genes that are targeted by HIV at significantly higher frequency than matched random controls after normalization for gene length, and a smaller set of genes that are targeted at significantly lower frequency. Functional clustering analysis shows that highly targeted genes are involved in chromatin remodelling and transcription, and are enriched in housekeeping functions. This analysis identifies a set of “high-risk” genes in hematopoietic cells, the function of which is more likely to be influenced by lentiviral vector integration in clinical gene therapy. Many of these genes are over-represented in collections of lentiviral vector integrations from patients treated by gene therapy, indicating that lentiviral “common integration sites” are determined by the HIV target site selection rather than clonal dominance in vivo.
A new lentiviral vector pseudotype outperforms by far VSV-G-LVs for gene transfer into hematopoietic stem cells
HSC based gene therapy holds promise for the cure of many inherited and acquired diseases. The field is now moving towards the use of lentiviral vectors (LVs) evidenced by two recent clinical trials for adrenoleukodystrophy and Beta-thalassemia. In these trials, VSV-G-LVs were applied at high doses to obtain therapeutically relevant transduction levels. Moreover, high gene transfer was only achieved with very strong cytokine-cocktails compromising the 'HSC' character. Adding to this the huge vector-production costs, we can summarize that current LVs are unsatisfactory for gene therapy applications.
We generated here for the first time a LV pseudotyped with a BaEVgp derived from a baboon endogenous retrovirus. We evaluated these vectors for hCD34+-cell transduction under mild cytokine prestimulation (rSCF+rTPO) that allow to preserve better the 'HSC' characteristics. After a single application at a low vector doses, BaEV-LVs stably transduced up to 80–90% of hCD34+-cells, where VSV-G-LVs reached 5–10% transduction at the same vector doses. Even more striking was that these new BaEVgp-LVs allowed at low vector doses efficient transduction of up to 25% of quiescent hCD34+-cells where VSV-G-LVs only allowed up to 5% quiescent cell transduction. Importantly, reconstitution of rag2−/−,gammaC−/−,BalbC mice with BaEV-LV transduced hCD34+-cells (MOI=10) resulted in 60–70% transduction of all analyzed myeloid and lymphoid engrafted lineages. In contrast, VSV-G-LVs (MOI=100) did not result in equivalent transduction levels of all hematopoietic lineages with a strong bias toward B-cell lineage. Together, these results strongly suggest that the novel BaEV-LVs efficiently transduce true HSCs.
Dept Clin Pharm, University of Oxford, Oxford, OX3 7DQ
The anti-tumour efficacy of intravenously delivered oncolytic adenoviruses (OA) is limited by poor circulation kinetics and inefficient extravasation at target sites. We have addressed these limitations with the use of polymer coating, to raise levels of OA in the bloodstream, combined with the application of focussed ultrasound which provides a targeted stimulus for the movement of these raised levels into the tumour.
A new generation of hydroxypropylmethacrylamide polymer containing pH sensitive hydrazone groups was synthesised and its ability to coat OA in a manner which was reversible upon exposure to low pH, was demonstrated. Following polymer coating, the binding of OA to human blood cells, complement proteins and clotting factors was reduced. In intravenously injected murine models, hepatic infection was reduced >8,000-fold (p<0.001) and the dose remaining in the bloodstream at 30 min was enhanced 49-fold (p<0.005). Focussed ultrasound parameters were then optimised (0.05 mL SonoVue, 50 frequency: 0.5 MHz, peak rarefractional focal pressure:1.2 MPa, pulse duration: 50,000 cycles, pulse repetition frequency: 0.5 Hz for 4 min) to provide a powerful but safe extravasation stimulus, which could be passively and remotely detected in real time. In mice bearing ZR75.1 xenograft tumours, exposure of polymer coated-OA to focussed ultrasound was found to enhance infection levels by up to 30-fold (p<0.005). In ongoing studies this led to a retardation of tumour growth and an extension of survival.
Tracking hematopoietic stem cell fate in humans by retroviral tagging
Upon retroviral mediated gene correction of haemopoetic stem cells (HSC) each transduced progenitor is univocally marked by an integration site and can be tracked in vivo by retroviral tagging. We previously showed that ADA-SCID gene therapy (GT) with CD34+ cells resulted in multilineage engraftment, in the absence of aberrant expansions. We performed a comprehensive longitudinal insertion profile of distinct bone marrow (BM) and peripheral blood (PB) cell types in 4 patients 3–6 years after GT, retrieving to date 2350 unique insertion sites by LAM-PCR and high-throughput sequencing. We could uncover in each lineage the frequency of identical integrants among different haematopoietic compartments. BM cells and PB granulocytes displayed the highest proportion of shared integrants (up to 58.1%), reflecting the real-time repopulating activity of gene-corrected progenitors. Strikingly, we detected “core integrants”, shared between CD34+ cells and both lymphoid and myeloid lineages at multiple time points, stably tagging long-term multipotent progenitors overtime. Tracking two of these integrants (proximal to the MLLT3 and LRRC30 genes) by specific PCR we confirmed the multilineage contribution to haematopoiesis of these clones, showing fluctuating lineage outputs over a period of 5 years. We also retrieved 170 integrations in T cell subtypes from patients who received infusions of transduced lymphocytes, showing evidence that naive T cell clones may survive for up to 10 years after infusion. In conclusion, through retroviral tagging, we can track single transduced haemopoietic stem cells directly in humans. This study could provide information of reference for ongoing and future GT approaches for hematological diseases.
San Raffaele Telethon Institute for Gene Therapy (Milan, Italy)
Lentiviral vector (LV) integration site analysis in patients' derived cells from a Hematopoietic Stem Cell (HSC)-based clinical trial in for X-linked adrenoleukodystrophy (ALD) showed that a relevant number of Common Insertion Sites (CIS) were present. We generated our own profile of LV integrations from transduced human HSC after transplant in immunodeficient mice and compared it to LV the integrations of the ALD clinical trial, and other gene therapy trials that reported insertional mutagenesis, as well as in retroviral and transposon-mediated oncogene tagging studies in mice. LV CIS clustered in megabase-wide chromosomal regions of high LV integration density. Conversely, CIS found in malignant cells from clinical trials and mice, do not form clusters, and target always a single gene, the culprit of oncogenesis. Further statistical analysis show that genotoxic CIS, but not LV CIS, have a higher targeting frequency with respect neighboring genes.
Moreover, integration site analysis was also performed on a patient enrolled in a recent clinical trial of HSC gene therapy for metachromatic leukodystrophy (MLD) in our Institute (6-months follow-up). About 5000 unique LV integration sites were obtained from transduced in vitro cultured CD34 cells and in blood at 30, 90 and 120 days after transplant. CIS genes are largely overlapping with those reported in the other LV-based studies above, are also clustered in the same megabase-wide chromosomal regions.
These findings imply that LV CIS in our experimental model, the ALD and MLD clinical trials are produced by an integration bias towards specific genomic regions rather than by oncogenic selection.
San Raffaele Telethon Institute for Gene Therapy, Milan, Italy
We recently developed and validated a new in-vivo assay based on systemic vector injection into newborn Cdkn2a−/− mice to address the genotoxic potential of different lentiviral-vectors (LV). Treatment with a SINLV harboring the SF enhancer/promoter sequences in internal position (SINLV.SF.GFP) caused a significant tumor onset acceleration with respect to mock-controls and demonstrates the high sensitivity to vector genotoxicity of this in-vivo platform. Injection of an LV containing the SF sequences in the LTR (LV.SF.LTR) caused an even earlier acceleration of tumor onset with respect to SINLV.SF.GFP-treated mice. To understand the reasons for the different genotoxicity of these vectors, we deleted the GFP-ORF from the SINLV.SF.GFP-vector and tested this modification (LV.SF) in-vivo. Strikingly, LV.SF induced tumor onset acceleration similar to the one of LV.SF.LTR. Hence, the presence of an ORF downstream the SF and not the position or the number of enhancers per se has a major role in increasing the safety profile of the SIN design. Analysis of >5000 LV integrations from tumors revealed the presence of several CIS in the different vector-treated groups (i.e.Braf) and demonstrate that tumor onset acceleration was consequent to vector driven insertional mutagenesis. Moreover, LV.SF.LTR and the LV.SF frequently produced chimeric LV/Braf transcripts by a read-through/splicing-capture mechanism using available splice donor signals present in the vector or in the surrounding cellular genome. No LV/Braf chimeric transcripts were detected in SINLV.SF.GFP induced-tumor, indicating that the presence of an ORF has a major role in decreasing the genotoxicity of SINLV-vector by reducing read-though transcription and splicing-capture events.
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany
Generating patient-specific pluripotent stem cells by reprogramming of somatic cells offers great potential for regenerative therapies and disease studies. Since these “induced” pluripotent stem cells (iPSC) can self-renew and differentiate into virtually all cell types of the human body, they represent an important source for cell replacement therapies and for the study of disease-specific phenotypes. This is especially important when patient material is limited, as for rare immunodeficiencies (e.g. congenital neutropenia).
To generate iPSC we constructed a self-silencing lentiviral vector expressing all four human codon-optimized reprogramming factors, flanked by Flp recombinase target sites. By optimizing culture conditions we could successfully generate low vector-copy iPSC clones from different disease models (e.g. Hax1 −/−, p14 −/−). This allowed us to excise the reprogramming vector using a newly developed method of retroviral particle-mediated Flp recombinase protein transduction. Generated “factor-free” iPSC from knockout mice are being tested for pluripotency and differentiation potential to allow disease studies in gene-corrected and uncorrected hematopoietic cells. Mapping the remaining enhancer-deprived lentiviral “footprint” in clones of different genetic backgrounds using ligation-mediated-PCR, we identified clones with intergenic insertions, representing potential safe harbors. This genetic mark is useful to detect iPS cell progeny in an autologous transplantation scenario or minimal residual disease of teratomas elicited by iPSC.
In summary, we have established an efficient platform for generating “factor-free” iPSC with a unique clone-specific genetic identifier. In future, we want to combine retro-/lentiviral gene transfer and directed differentiation of disease-specific iPSC to study phenotypic correction of defects in vitro and in vivo.
Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany
Zinc finger nucleases (ZFN) enable gene editing in living cells by introducing a targeted DNA double-strand break (DSB) at a preselected genomic locus. Due to the rapid repair of DSB in living cells, strategies to analyze the genome-wide activity of synthetic nucleases remain limited and mostly rely on in vitro binding assays and bioinformatics predictions. Here we show that non-homologous end-joining (NHEJ) incorporates integrase-defective lentiviral vectors (IDLV) at DSB, thus stably tagging these otherwise transient events. Genome-wide mapping of >1500 captured IDLV vector genomes by high-throughput integration site analysis identified the sites of actual ZFN cleavage of a panel of ZFNs targeting the human CCR5 and IL2RG genes in K562 cells. ZFN activity was detected at a few off-target positions in the genome outside of the target locus. By ranking nuclease activity at these sites we could monitor the degree of ZFN specificity reached in vivo. In vivo DNA cleavage required binding of the ZFN monomers in specific spatial arrangements on DNA bearing homology to the intended target site and tolerated mismatches only at select individual nucleotides within the ZFN binding sites. Importantly, despite that the in vivo derived consensus binding sequence of the ZFN closely matched that obtained biochemically, the actual ZFN cleavage sites could not be predicted in silico solely based on the degree of their sequence identity to the intended target site. Comprehensive mapping of the genome-wide ZFN activity in vivo will facilitate their broad application in translational research.
Developing Safer Gene Therapy Approaches by Zinc Finger Nucleases-Mediated Gene Editing
Gene targeting by homologous recombination holds great promise for gene therapy as it may overcome the risks of insertional mutagenesis and uncontrolled transgene expression associated with the use of conventional gene transfer vectors. The development of Zinc Finger Nucleases (ZFN) has brought this long-sought objective within the reach of gene therapy. A ZFN-induced DNA double strand break at a predetermined site of the genome can trigger homology-directed repair (HDR), a pathway that can be exploited to insert new sequences with high efficiency into the ZFN target site. Yet, the full potential of this technology requires solving the challenge of delivering the required machinery to clinically relevant cell types. We exploited the infectivity of integrase-defective lentiviral vectors to express ZFNs and provide template DNAs for HDR in a panel of human cell lines, achieving high rates (up to 50%) of gene editing at three endogenous loci. We then extended and optimized the application of ZFN-mediated gene editing to several primary human cell types, including T lymphocytes, fibroblasts, keratinocytes, neural stem cells, induced Pluripotent Stem cells, and hematopoietic stem/progenitor cells, and achieved up to 10% targeting efficiency with an overall 94% specificity measured on bulk-treated cell populations and in a large panel of single cell-derived clones. Finally, we assessed the dynamic interplay that occurs between the integrated transgene and the targeted locus by studying the transcriptional and epigenetic impact of different transgene expression cassettes targeted by ZFNs into two human loci. Analyses of these loci before and after mono- or bi-allelic integration allowed on site tailoring of the expression cassettes to achieve robust and uniform transgene expression without inducing detectable transcriptional perturbation of the targeted locus and its flanking genes. These studies provide a framework for sustainable gene transfer that can be exploited in novel experimental paradigms and safer therapeutic applications.
Young Investigator Award
Poster Abstracts
University of Southampton
Peptide major histocompatibility complex (pMHC) arrays can improve our understanding of the specific T cell populations involved in immune responses during conventional treatment and immunotherapy clinical trials. In the longer term this could guide clinical decisions towards more individualised patient treatments. One major advantage of the technique is its ability to simultaneously analyse multiple T cell populations using small number of CD8+ T cells (∼1.2×106 cells/array). In addition pMHC arrays use very small amounts of pMHC per spot (1ng, 1/1,000th of that used in flow cytometry) and can analyse a range of T cell populations without haplotype restriction. CD8+ T cells were negatively isolated from leukaemia patients, lipophillically dyed with DiD and incubated with pMHC arrays printed with more than 40 tumour-associated antigen and viral epitopes (including CMV and Flu). Positive scoring of T cells bound to pMHC were only made when T cells were consistently bound to spots of the pMHC specificity in two distinct regions on the array. We have analysed 20 leukaemia patients to date and found that four of the six HLA-A2+ acute myeloid leukaemia patients had specific-T cell populations recognising epitopes within the tumour antigens PASD1, WT1, MelanA and/or Tyrosinase. We are now investigating whether these responses correlate with treatment. We have developed a robust method for the simultaneous analysis of T cell populations in leukaemia patients, which can indicate a short-list of T cell populations for further investigation of T cell function minimising reagent and sample use.
Efficient regulation of GDNF expression in the brain using targeted proteasomal degradation
A new system to regulate gene expression acting at a post-translational level has recently been developed. The system uses destabilized protein domains (DD) to target proteins for destruction. If the antibiotic trymehoprim (TMP) is present, the protein of interest will be protected, becoming stable and expressed.
DD were fused to Glial-cell Derived Neurotrophic Factor (GDNF), in a N-terminal (DD-GDNF) or a C-terminal position (GDNF-DD). Lentiviral vectors expressing wildtype GDNF, DD-GDNF and GDNF-DD were used to transduce 293T cells. Treatment of the cells with TMP resulted in a 3.8 fold induction of DD-GDNF, reaching a concentration of 19 ng/ml. GDNF-DD had a 21 fold induction, resulting in a concentration of 10.8 ng/ml upon TMP induction. Furthermore, both DD were able to activate GDNF signalling pathways to levels comparable to the wildtype GDNF. However, uninduced levels of DD-GDNF were also sufficient to activate GDNF signalling pathways.
Due to the in vitro performance, GDNF-DD was chosen for in vivo studies and lentiviral vectors expressing GDNF-DD were delivered to the striatum of rats. The animals were then treated with TMP in their drinking water. Treatment with TMP resulted in a broad GDNF distribution throughout the striatum, reaching 124 pg/mg of tissue. In contrast, when the animals were not given TMP, the GDNF-DD concentration reached a maximum of 13 pg/mg tissue. Furthermore, only the induced GDNF-DD was able to activate GDNF signalling pathways in SNpc neurons. Thus, DD system was able to efficiently regulate GDNF expression in vivo.
Institute of Neuroscience and Psychology, School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8QQ, UK
Mutations in MECP2 cause the neurological disorder, Rett syndrome (RTT). The protein product of this gene, MeCP2, is expressed at high levels in the nuclei of postnatal brain neurons. Studies in knockout mice suggest that restoration of MeCP2 to neurons can both reverse and prevent RTT-like neurological deficits. We tested the effectiveness of lentiviral and AAV9 vectors in delivering a transgene encoding MeCP2 to neurons in vivo.
Murine Mecp2 was cloned as a FLAG-RFP fusion construct under neuronal or ubiquitous promoters in a series of lentiviral vectors. Human MECP2 was cloned as a fusion with a Myc epitope tag under the endogenous Mecp2 core promoter in an AAV9-encapsidated AAV2 vector. After testing in primary neuronal culture, viral particles were injected into wild-type and knockout (Mecp2stop/y) mice.
Our results showed that vector-derived, nuclear MeCP2 could easily be visualized by immunohistochemistry (IHC). The per-cell level of lentiviral vector-derived protein expression was typically less than double (177±6%) the endogenous level. Changes in neuronal morphology consistent with phenotypic rescue at the molecular/cellular level were evident. MeCP2 regulation (S421 phosphorylation) was also demonstrated. AAV-mediated delivery of Mecp2 resulted in high-efficiency transfection of brain neurons (12% for intravenous and 70% for intracranial delivery) across the CNS. There was no improvement in RTT-like disease phenotype. Moreover, the MECP2-vector-injected mice developed a novel, severe hindlimb dysfunction.
These findings suggest that viral delivery of Mecp2 can efficiently induce functional, correctly-targeted MeCP2 expression at potentially useful cellular levels, while highlighting the risk of potentially adverse physiological effects in vivo.
Dept. of Biotherapy, Hôpital Necker Enfants-Malades, AP-HP, Paris 75015, France
The long-term follow-up of the first gene therapy trial for X-linked severe combined immunodeficiency (SCID-X1) demonstrates that gene therapy provided a clear therapeutic benefit in eight of the nine treated patients despite the occurrence of vector-related leukemia in four patients. The sustained presence of transduced T cells is observed for up to 12 years following gene therapy. Seven patients (including the three leukemia survivors) exhibited sustained immune reconstitution, with the presence of various T cell subsets and functions. Remarkably, most patients (including the 3 who received chemotherapy) have naive T cells, strongly suggesting the presence of ongoing, long-term thymopoiesis that could originate from transduced progenitor cells with persistent self-renewal capacity. Significant advances in viral vector technology have enabled the design of safer vectors with the development of enhancer-deleted LTR-SIN vectors containing an internal promoter. It will be critical to see if the SIN vectors are as safe as expected in the current SCID-X1 No.2 gene therapy trial. So far two patients have been enrolled aged 8 (P1) and 5.5 (P2) months at the time of treatment. Both were severely affected with disseminated BCG, pneumopathy and several viral opportunistic infections at the time of admission. Furthermore P2 presented with a massive enlargement of secondary lymphoid organs and of liver due to EBV infection. Despite their high-risk presentations, both patients restored the T-cell compartment with the same kinetics as that described in trial No.1 and are simultaneously clearing their infections. The follow-up data will be described in our presentation.
Centre for Immunodeficiency, Molecular Immunology Unit, Institute of Child Health, University College London, UK
Genetic defects in the purine salvage enzyme adenosine deaminase (ADA) lead to severe combined immunodeficiency (ADA-SCID) with profound depletion of T, B, and NK cell lineages. HLA-matched allogeneic haematopoietic stem transplantation (HSCT) offers a successful treatment option. However, individuals that lack a matched donor must receive mismatched transplants, which are associated with significant morbidity and mortality. Enzyme replacement therapy (ERT) for ADA-SCID is available, but the associated suboptimal correction of immunological defects leaves patients susceptible to infection. Here, six children were treated with autologous CD34-positive haematopoietic bone marrow stem and progenitor cells transduced with a conventional gammaretroviral vector encoding the human ADA gene. All patients stopped ERT and received mild chemotherapy prior to infusion of gene-modified cells. All patients survived, with a median follow up of 43 months (range: 24–84). Four of the six patients recovered immune function as a result of engraftment of gene-corrected cells. In two patients, treatment failed due to disease-specific and technical reasons: Both restarted ERT and remain well. Of the four reconstituted patients, three remained off enzyme replacement. Moreover, three of these four patients discontinued immunoglobulin replacement, and all showed effective metabolic detoxification. All patients remained free of infection, and two cleared problematic persistent CMV infection. There were no adverse leukemic side effects. Thus, gene therapy for ADA-SCID is safe, with effective immunological and metabolic correction, and may offer a viable alternative to conventional unrelated donor HSCT.
Erasmus University Medical Center, Department of Hematology, Rotterdam, the Netherlands
Pompe disease is a lysosomal storage disease, characterized by systemic storage of glycogen leading to progressive muscle weakness and death within the first year of life in its most severe, infantile form. Pompe disease is caused by mutations in the gene encoding acid alpha-glucosidase (GAA). Enzyme replacement therapy is the current option for treatment, but is not beneficial to all patients, may result in antibodies against the recombinant enzyme and the costs are high. We used a Gaa-/- mouse model to develop an ex vivo lentiviral vector mediated gene therapy strategy to modify syngeneic hematopoietic stem cells. Using this strategy with an optimized human therapeutic GAA gene, life-long high levels of (human) alpha-glucosidase were achieved in leukocytes and all affected tissues of Pompe mice using relatively mild conditioning. Supranormal levels of GAA activity were obtained (including skeletal muscle) as well as a complete reduction of glycogen storage in most affected tissues. Heart morphology and function as well as skeletal muscle function were normalized, and glycogen levels were highly reduced in cartilage and brain tissue.
Thus, ex vivo hematopoietic stem cells lentiviral vector mediated gene therapy corrects the Pompe phenotype in the Gaa-/- mouse model, warranting preparation of clinical implementation in CRIM-negative patients.
Molecular Immunology Unit, Institute of Child Health, UCL
X-linked Severe Combined Immunodeficiency (SCID-X1) is an immunopathy caused by a mutation of the common gamma chain (γc) gene which results in a lack of T cells, NK cells and with dysfunctional B cells. Current gene therapy methods involve the addition of a correct γc gene via integrating viral vectors. However, these current non-targeting gene addition strategies can result in transformation of the cell. A novel solution to this problem is met by targeted gene correction via homologous recombination stimulated by a specific cleavage event caused by zinc finger nucleases (ZFN) within the disease gene.
A γc deficient mouse has been created by replacing the murine γc gene locus with a mutated human γc gene containing a point mutation frequently seen in SCID-X1 patients. The mutant human γc gene is transcribed and initial analysis of this new SCID-X1 model has revealed a phenotype mirroring γc gene knockout mice. This murine model of SCID-X1 is an appropriate platform to assess the efficiency of various gene targeting and correction strategies for the human mutation including ZFN induced homologous recombination.
We have successfully achieved targeted homologous recombination in both a human T cell SCID-X1 cell line model and the humanised mouse embryonic stem cells with γc gene specific ZFN.
Department of Genetics, University College London Institute of Ophthalmology, 11–43 Bath Street, London, EC1V 9EL
Cell transplantation is a potential therapeutic strategy for the irreversible loss of photoreceptors occurring in retinal degeneration. In the degenerating retina, Müller glia undergo gliosis, a process that includes increased production of the intermediate filaments GFAP and vimentin and hypertrophy. Glial scarring is likely to have a major impact on regenerative strategies, including cell transplantation, by acting as a physical barrier and/or as a reservoir of inhibitory molecules. We examined the role of gliosis in impeding photoreceptor integration success in the rhodopsin knockout (rho-/-) model of retinal degeneration. Gliosis was examined by western blot and immunostaining. GFAP and vimentin expression were manipulated using viral shGFAP (and/or shVimentin) vectors. Photoreceptor transplantation was assessed by transplanting FACS-sorted rod precursors into rho-/- recipients at early (4 week), mid (6 week) or late (10 week) degenerative stages. GFAP expression was higher at 4 wks compared to wildtype, and increased significantly between 4 and 10 weeks. Increasing numbers of GFAP+ve processes were observed extending into the outer nuclear layer (ONL), in addition to the appearance of lateral glial scars at the outer edge of the ONL. Transplanted photoreceptor integration was significantly lower in the rho-/- mouse than in age-matched wildtype controls and declined markedly with degeneration. To determine whether or not gliosis is responsible for this decrease, we used shGFAP (and/or shVimentin) to impair GFAP upregulation in the degenerating rho-/- retina in conjunction with photoreceptor transplantation. Gliosis represents a significant barrier to transplanted photoreceptor integration in the degenerating retina.
LBPA/ CLINIGENE, Ecole Normale Superieure (ENSC), 94235 Cachan Cedex, France
We have used new short synthetic genetic insulator elements with view to reducing enhancer mediated insertional mutagenesis. We could identify a specific combination of four CTF-repeats, which translates into optimal functional activity, high titers, boundary effect in both gammaretro and lentivectors (4xDCaro4). In comparison, constructs which comprise 3 repeats of CTCF1-CTCF2 cannot be produced at same high titers and show a strong silencing effect in transduced cells. With 4xDCaro4, a more homogenous level of expression is monitored which strictly depends on the promoter. 4xDCaro harbours boundary properties, which translate into enhanced promoter specificity, e.g.: by two orders of magnitude in CD4 cells using a CD4 specific promotor. Genetic stability has been monitored ex-vivo both in HeLa cells over three months and primary human cord blood HSCs for two months. In comparison, control vectors mediate heterogeneous expression which depends on the MOI and prove unstable; these features also hold when a 7xCTF repeat is used instead of 4x, which indicates that genetic stability is repeat-number dependent. In vivo, 4xDCaro-transduced CD34+ cells wholly reconstitute NOG-mice including secondary hosts. Genotoxicity potential has first been assessed in vitro, in comparing integration patterns ingenuity in HeLa sampled over three months: a reduction in clonality is monitored using pyro-sequencing. In cancer-prone mice, the animals' half-life is prolonged as compared to non-insulated lentivector. We have developed new versatile tools toward improvement of integrating vectors safety (a combination approach is presented in Artus et al).
Centre for Immunodeficiency, Molecular Immunology Unit, Institute of Child Health, University College London, London, UK
X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine-receptor gamma chain. These mutations classically lead to a complete absence of functional T and NK cell lineages as well as intrinsically compromised B cell function. Although HLA-matched hematopoietic stem cell transplantation (HSCT) is highly successful in SCID-X1 patients, HLA-mismatched procedures can be associated with prolonged immunodeficiency, graft versus host disease, and increased mortality. Here we present a follow-up of ten children treated with autologous CD34+ hematopoietic stem cells transduced with a conventional gammaretroviral vector. The patients did not receive myelosuppressive conditioning and were monitored for immunological recovery after cell infusion. All patients were alive after a median follow up of 80 months (range 54–107 months), and a functional polyclonal T cell repertoire was restored in all patients. Humoral immunity only partially recovered, but was sufficient in some patients to allow for withdrawal of immunoglobulin replacement; however, three patients developed antibiotic-responsive acute pulmonary infection after discontinuation of antibiotic prophylaxis and/or immunoglobulin replacement. One patient developed acute T cell acute lymphoblastic leukaemia due to upregulated expression of the proto-oncogene LMO-2 from insertional mutagenesis, but maintained a polyclonal T cell repertoire through chemotherapy and entered remission. Therefore, gene therapy for SCID-X1 without myelosuppressive conditioning effectively restored T cell immunity and was associated with high survival rates for up to 9 years. Further studies using vectors designed to limit mutagenesis and strategies to enhance B cell reconstitution are warranted to define the role of this treatment modality alongside conventional HSCT for SCID-X1.
Delivery of siRNA to the brain by systemic injection of targeted exosomes
Despite recent advances in delivering siRNA, targeting specific tissues or cell types while avoiding nonspecific delivery, especially to the liver, remains challenging. Another major barrier is immunogenicity of the siRNA or delivery vehicle, especially if repeated dosing is needed to treat chronic or degenerative diseases. Naturally occurring RNA carriers, such as exosomes, might provide an untapped source of effective delivery strategies.
Here we show that exosomes, endogenous nanovesicles that transport RNAs and proteins, can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.
RVG exosomes are especially capable of delivering siRNA specifically and safely after systemic administration and therefore represent a promising vehicle for gene therapies targeting chronic neurodegenerative disorders. Moreover, lack of nonspecific knockdown in the liver and other organs marks a major improvement over most current siRNA delivery strategies (Alvarez-Erviti et al. Nature Biotechnology, 2011).
UCL Cancer Institute, 72 Huntley street, London, WC1E 6BT
The generation of patient-specific induced pluripotent stem cell (iPS) lines and subsequent derivation of in-vitro disease models provides a unique opportunity to investigate cellular underpinnings of genetic disease. Potential benefits are especially pronounced for neurological disorders affecting midbrain structures, where there is a real dearth of alternative model systems. To establish the potential for iPS cell-derived midbrain we have focused on rapid-onset dystonia parkinsonism (RDP), an incurable autosomal-dominant motor disorder predominantly affecting young adults and characterised by the sudden onset of combined dystonia and parkinsonism with rapid deterioration of symptoms. It is caused by mutations in the ATP1A3 gene, which lead to dysfunction rather than death of midbrain neurons by an as-yet unknown cellular mechanism. Skin fibroblasts from two subjects with RDP were transduced with onco-retroviral vectors. A large number of human-embryonic stem cell like colonies were visible within two weeks. Pluropotency was confirmed by expression of Tra 1–60, Nanog and Rex1. Next, these iPS cells were differentiated into dopaminergic Tyrosine Hydroxylase (TH)+ neurons by coculture with PA6 feeder cells. A large number of TH+-neurons were obtained and shown to express DAT-DOPA-Decarboxylase and midbrain markers Ptx3 and Lmx1b, as well as the disease gene ATP1A3. These dopaminergic neurons provide a unique opportunity to study the effects of aberrant Na+/K+ ATPase function on neuronal function in a context relevant to humans, thus raising the prospects of developing novel treatment opportunities.
CABIMER, Americo Vespucio S/N, Seville, Spain
Human embryonic stem cells (hESC) hold great promise for the treatment of patients with many neurodegenerative diseases particularly those arising from cell loss or neural dysfunction including spinal cord injury. This study evaluates the therapeutic effects of transplanted hESC-derived oligodendrocyte progenitors (OPC) and/or motoneuron progenitors (MP) on axonal remyelination and functional recovery of adult rats after complete spinal cord transection. OPC and/or MP were grafted into the site of injury in the acute phase. Based on Basso-Beattie-Bresnahan scores recovery of locomotor function was significantly enhanced in rats treated with OPC and/or MP when compared with control animals. When transplanted into the spinal cord immediately after complete transection, OPC and MP survived, migrated, and differentiated into mature oligodendrocytes and neurons showing in vivo electrophysiological activity. Taken together, these results indicate that OPC and MP derived from hESC could be a useful therapeutic strategy to repair injured spinal cord.
851 S. Morgan St. SEO 218, MC 063, Chicago, IL, 60607, United States
Gene therapy offers completely new avenues for treating disorders of the central nervous system such as chronic pain or traumatic cord injury. Without modifying the genome, gene silencing with short interfering RNA (siRNA) enables down-regulation of specific proteins in a target region. Previous clinical studies show siRNA administered intrathecally successfully reduces the pain response in rodents. The engineering of human gene silencing therapies requires the coupling of quantitative data on biodistribution with intracellular reaction kinetics.
After intrathecal infusion, the therapeutic response not only depends on biodistribution and half-life of siRNA in the cerebrospinal fluid and tissue, but also the intracellular mechanisms that follow siRNA uptake. A non-invasive method is employed to determine unknown transport and kinetic parameters of radio-labeled siRNA from distributed imaging data such as positron emission tomography (PET). The match between experimental and simulated concentration profiles of siRNA allows for the estimation of kinetic parameters that govern in vivo reaction pathways. Using the optimal parameter set, accurate predictions of biodistribution and therapeutic response for intrathecally infused unprotected siRNA and vehicle-delivered siRNA are made. To maximize siRNA delivery to the dorsal horn of the spinal cord, a novel co-infusion therapy design with a decoy molecule effectively decreases siRNA endocytosis in untargeted cells. Results show that the co-infusion technique increases siRNA concentration in target cells. In summary, the discovery of unknown kinetic parameters from imaging data and the prediction of siRNA biodistribution as well as intracellular kinetics will facilitate the systematic development of gene therapy for humans.
Gene-cell therapy for multiple sclerosis in MOG-induced murine experimental autoimmune encephalomyelitis model
Multiple sclerosis (MS) is a severe debilitating disorder characterised by progressive demyelization and axonal damage of the central nervous system (CNS). MS usually begins as a relapsing-remitting (RRMS) disease which, in 65% of the cases, develops into secondary-progressive MS (SPMS). Although current therapies are able to reduce inflammation, they are ineffective both in blocking the autoimmune response and repairing the damaged CNS. Gene and cell therapy strategies for MS treatments aim to increase the efficiency of the therapeutic proteins and to favour CNS regeneration and/or neuroprotection. In this study we have compared the therapeutic efficiency of direct injection of a lentiviral vector expressing VIP cDNA (LentiVIP) versus administration of MSCs expressing VIP for the treatment of a mouse model of chronic MS (MOG-induced EAE in C57Bl/6 mice). While neither MSCs nor LentiVIP administration were able to achieve any therapeutic benefits at this stage of disease, MSC/LentiVIP treatment increased survival and reduced EAE average score from 3.3 to 2.3 (n=10). MSC/LentiVIP was more efficient compared to non-transduced MSC in decreasing astrogliosis and protecting neuron degeneration. We also found higher inhibition of autoimmune T cell responses in MSC/LentiVIP-treated mice. However, the reduction in proinflammatory cytokines important in the pathogenesis of EAE (IL-17, TNF-alpha, IL-6 and iNOS) were similar in MSC and MSC/LentiVIP-treated mice. In summary, the increased therapeutic effects of MSC/LentiVIP require the immunoregulatory and neuroprotective roles of both VIP and MSCs and the ability of the MSCs to migrate to peripheral lymph organs and the inflamed CNS.
INSERM U745, Paris, France and Department of Pediatric Neurology, Hôpital Bicêtre, Le Kremlin Bicêtre, France
Metachromatic Leukodystrophy (MLD) is an untreatable demyelinating disease due to deficiency of Arylsulfatase A (ARSA). The most severe form starts around 1–2 years, leading to death within a few years.
We have demonstrated efficiency and safety of intracerebral delivery of adeno-associated-vector serotype 10 (AAVrh.10) encoding ARSA in MLD mice. Importantly, we observed rapid and significant expression and overactivity of ARSA, not only in neurons but also specific correction of oligodendrocytes.
Towards clinical application submission, the neurosurgical procedure was optimized in non-human primate (NHP) based on MRI selection of the injection sites followed by simultaneous infusion of the vector at 12 different brain locations, and MRI-based evaluation of tolerance post-surgery. Feasibility and safety of this clinical protocol were demonstrated in 4 NHP, using the vector dose (in respect to brain size) that will be used in children. Intracerebral injection of AAVrh.10/ARSA vector (1.1.1011 vg/hemisphere) resulted in significant diffusion of the vector (66 to 90% of the injected hemisphere) and increased ARSA activity (up to 31%), rapidly reaching foreseeable therapeutic levels (a crucial requirement in this very rapidly progressing disease). Results of the toxicological studies in NHP and submitted clinical protocol will be presented. This phase I/II tolerance and efficiency study includes five patients with rapidly progressing MLD. AAVrh.10/ARSA vector (total dose 4.1012 vg) is administrated to 12 locations in the CNS. Safety and efficiency parameters will be evaluated up to 2 years, a period that will allow to assess the potential therapeutic efficiency of this strategy.
School of Biological Sciences, Royal Holloway-University of London, Egham, Surrey TW20 0EX, UK
Gene addition strategies to correct inherited diseases may hold promise for treatment in the clinic. An improved approach to treat both dominant and recessive genetic disorders would be to repair the mutant gene by homologous recombination-mediated gene targeting. Recently, gene targeting frequencies have dramatically increased through the development of (i) efficient DNA delivery tools, including integration-deficient lentiviral vectors (IDLVs); and (ii) improved designer zinc finger nucleases (ZFN), able to induce specific double-strand breaks at their target locus. To demonstrate therapeutic gene repair in a pre-clinical model of immunodeficiency we have developed an ex vivo system to correct the classical scid mouse, a model of human DNA-dependent protein kinase catalytic subunit (PRKDC) deficiency. We have produced donor templates to correct the mutation, obtained and optimised a ZFN pair that targets mouse Prkdc close to the scid mutation, and incorporated ZFN genes and donor templates into IDLVs. We have demonstrated specific ZFN activity in scid fibroblasts and haematopoietic progenitors using a Cel-I assay, which detects modifications introduced at the target site upon repair by nonhomologous end-joining. We have observed ZFN-mediated gene targeting of the scid mutation via the incorporation of a diagnostic restriction site from the donor template into the targeted locus. In fibroblasts, we have shown rescue of DNA-PKcs activity and increased resistance to DNA damage upon gene correction. In haematopoietic progenitors we have detected gene correction when the ZFN genes are delivered by integrating lentivectors, and we are currently optimising IDLV-mediated gene targeting.
Molecular Immunology Unit, Institute of Child Health, University College London, London, WC1N 1EH, UK
Type I Gaucher Disease (GD) is a lysosomal storage disorder which is currently treated with bimonthly infusions of an enzyme replacement therapy. The goal of this work is to develop a gene therapy for GD using lentiviral vectors carrying a functional copy of the glucocerebrosidase gene. We have created a variety of lentiviral vectors and are currently testing the expression and efficiency levels of the enzyme they encode. We are also attempting to increase cross-correction of cells by fusing the glucocerebrosidase gene to a modified version of the protein transduction domain of the HIV-1 TAT protein. We expect this to enable cells to take up glucocerebrosidase independently of the endogenous uptake pathway. We are looking to compare two methods of vector delivery, ex vivo delivery to haematopoietic stem cells and direct intravenous delivery. We are also looking to work with osteoclasts in order to develop a therapy which can treat the bone manifestations of GD which are not remedied by the enzyme replacement regimen but which could be targeted by a genetic approach.
Gene therapy for lysosomal storage diseases using an enhanced lentiviral vector delivery system
LSDs arise from specific enzyme defects which lead to the accumulation of toxic storage compounds that affect normal cellular function in different organ systems both peripherally and in the brain. The clinical consequences result in severe disability and/or death in early childhood or in progressive disease with age. The key to effective disease correction is dependent on successful delivery of enzyme to both the CNS and peripheral organs. Therapeutic strategies are limited to ERT, which cannot cross the BBB and has limited effect on CNS, or allogeneic HSCT, which carries considerable toxicity and does not provide sufficiently high enough enzyme levels for disease correction. Gene therapy of autologous HSCs has been shown in recent pre-clinical studies to provide supra-physiological levels of enzyme and CNS correction through gene modified myeloid cell microglial differentiation, but current vector designs are limited by their ability to correct both CNS and peripheral abnormalities.
We have developed lentiviral vectors expressing transgenes under the transcriptional control of the EFS promoter, which drives expression in all hematopoietic lineages, and which also encodes the essential elements of the locus control region of the β-globin gene thereby upregulating transgene expression specifically in erythroid cells. With the aim of demonstrating the effectiveness of this design in LSDs such as mucopolysaccharoidosis type I, we carried out in vitro assays using these vectors expressing α-L-iduronidase. Our experiments have shown that these vectors are able to restore the enzymatic activity in patient cell lines and also provide enhanced levels of expression in erythoid lineage.
German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany
Monogenetic diseases have successfully been treated by gene therapy using retroviral vectors, but it was also shown that the insertion of functional retrovirus LTRs can lead to severe side effects. A reduced genotoxic risk of lentiviral vectors with self-inactivating (SIN) was reported in preclinical testing's and already led to promising clinical trials. In the present study, we report on the first clinical trial using a HIV-1 based lentiviral SIN-vector to treat the genetic defect in cerebral adrenoleukodystrophy (X-ALD) which showed a stable and sustained restoration, and is so far not accompanied by signs of clonal dominance or even premalignant disproportional distribution of cellular contributions. We performed integration site (IS) analysis by LAM-PCR and subsequent 454 pyrosequencing on samples from the first three patients treated. Our analyses showed a polyclonal repopulation of the hematopoietic system (unique mappable IS: P1: 4040; P2: 5929, P3: 5119) two to four years after transplantation, displaying the characteristic integration profile of lentiviral vectors with a preferred integration within genes (P1: 74%; P2: 74%; P3: 72%). We observed common integration sites in the same genomic regions in all three patients, likely reflecting an integration preference of the vector for particular genomic regions. Our IS data further indicate a successful ex vivo transduction of early hematopoietic progenitors underlined by the presence of identical IS identified in myeloid and lymphoid lineages in all three patients. High-throughput integrome analyses suggest that lentivirus vectors offer great promise for safe, effective and sustained correction of human stem and progenitor cells.
UCL Cancer Institute, 72 Huntley Street, London WC1E 6DD
We explored adeno-associated viral vector (AAV) mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease that is associated with early postnatal life-threatening hemorrhage. In new-born mice with plasma FVII<1% of normal, a single temporal vein administration of AAV (1×1013vector genomes [vg]/kg) resulted in expression of murine FVII at 266%±34% of normal for ≥100 days, which mediated protection against fatal hemorrhage and significantly improved survival in the post natal period. Codon optimization of human FVII (hFVIIcoop) improved AAV mediated transgene expression by 37-fold when compared with the wild-type hFVII cDNA, likely through improved post transcriptional processing. In adolescent macaques, a single peripheral vein injection of 2×1011vg/kg of the hFVIIcodop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7%±3.1%) and females (12.3%±0.8%). In-utero delivery of this vector in macaques conferred expression of hFVII at birth of 20.4%±3.7%, which remained detectable for 7 weeks. Re-administration of an alternative serotype at 12 months of age increased hFVII levels to 165%±6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus perinatal gene transfer with AAV vectors shows promise for monogenetic disorders with onset of pathology early after birth.
Telethon Institute of Genetics and Medicine, Naples, Italy
Crigler-Najjar syndrome is an autosomal recessive disorder presenting with severe unconjugated hyperbilirubinemia due to deficiency of liver-specific uridine diphospho-glucuronosyl transferase 1 A1 (UGT1A1). Current treatment relies on phototherapy to prevent life-threatening elevations of serum bilirubin levels but liver transplantation is the only permanent treatment. Although correction of the deficient enzymatic activity in the affected organ, i.e. the liver, would be most straightforward, therapeutic gene expression within an ectopic tissue to clear toxic metabolites from the circulation is very attractive. The muscle is the preferred tissue for this goal because of its simple and safe access through intramuscular (IM) injections. We have investigated the efficacy of muscle-directed gene therapy for Crigler-Najjar syndrome type 1 using adeno associated viral (AAV) vectors. Serotype 1 and 8 AAV vectors expressing the UGT1A1 under the control of the muscle-specific promoter were injected at the dose of 3×1012 genome copies/kg into the muscles of one month-old Gunn rats, the animal model of the disease. AAV injections resulted in long term expression of functionally active UGT1A1 enzyme in the muscle. AAV-injected Gunn rats showed an approximately 50% reduction of baseline serum bilirubin levels by 3 weeks post-injection which were sustained for at least 1 year post-injection. Serum bilirubin reduction was associated with increased urinary bilirubin excretion. Taken together, these data show that clinically relevant and sustained reduction of serum bilirubin levels can be achieved by simple and safe IM injections in Gunn rats. AAV-mediated muscle directed gene therapy has potential for treatment Crigler-Najjar syndrome type 1 patients.
Telethon Institute of Genetics and Medicine and Department of Pediatrics of Federico II University of Naples, Naples, Italy
Crigler-Najjar syndrome type 1 is a severe inborn error of bilirubin metabolism due to mutations in the uridine diphosphoglucuronate glucuronosyltransferase (UGT1A1) gene. Affected patients have elevated serum bilirubin levels which may be life-threatening and are often treated with liver transplantation. Several lines of evidence indicate that even a small number of corrected hepatocyte (1–5%) can provide a significant phenotypic improvement. Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high level transgene expression from transduced hepatocytes with no chronic toxicity and are attractive for treatment of Crigler-Najjar syndrome. However, due to a steep threshold effect, systemic high dose administration required for efficient hepatic transduction results in the activation of an acute inflammatory response which is potentially lethal. Novel strategies to achieve efficient hepatic transduction with reduced toxicity are desirable. To overcome this steep threshold effect, we have investigated the safety and efficacy of direct injections in the liver parenchyma of an HDAd vector expressing UGT1A1 under the control of a liver-specific promoter in the Gunn rats, the animal model of Crigler-Najjar syndrome. We found that intraparenchymal injections resulted in reduction of hyperbilirubinemia at lower doses as compared to systemic intravenous injections. Moreover, intraparenchymal injections caused a reduction of the systemic inflammatory response as compared to the intravenous injections of the same vector doses. In addition, biodistribution studies showed that intraparenchymal injections resulted in reduced vector dissemination. In conclusion, direct hepatic injections improve the therapeutic index of HDAd and may be a safe and efficient approach for liver-directed gene therapy of Crigler-Najjar syndrome.
Telethon Institute of Genetics and Medicine and Second University of Naples, Italy
Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate-aminotransferase (AGT), which catalyzes the conversion of glyoxylate to glycine. Organ transplantation as either preemptive liver transplantation or combined liver/kidney transplantation is the only therapeutic strategy available to prevent kidney failure. Gene therapy is an attractive option to provide a definitive cure for PH1. Towards this goal, we are investigating helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. We have injected PH1 mice with an HDAd encoding the AGT under the control of a liver-specific promoter and observed a sustained reduction of oxalate urinary excretion and reduction in kidney stone formation. Recently, we have developed a minimally invasive method to improve the therapeutic index of HDAd (Brunetti-Pierri et al., 2009). This approach based on balloon occlusion catheter to achieve preferential delivery of the vector to the liver, results in higher efficiency of hepatocyte transduction using clinically relevant low vector doses. Therefore, this method may permit correction of PH1 using clinically relevant doses of HDAd and may thus pave the way to clinical application of HDAd for PH1 gene therapy.
Molecular Immunology Unit, Institute of Child Health, UCL, London
Haemophagocytic lymphohistiocytosis (HLH) is a devastating disorder of early childhood arising from defects of T and NK cell cytotoxicity. The most common form of this disease is due to mutations in the perforin gene. Current treatment options of HLH are limited, thus the development of gene therapy may have great benefit for such patients.
Two lentiviral vector constructs were designed; with perforin expression being driven by the PGK promoter or by the Perforin promoter (specific for expression in NK and CD8+ T cells). Both vectors were able to reconstitute cytotoxicity activity in vitro in NK and CD8+ T cells derived from perforin deficient mice.
These two vectors were used to reconstitute perforin deficient mice through transplant of transduced lineage negative cells into lethally irradiated mice. Two month after transplant the number of T, NK, B cells and granulocytes were shown to be completely reconstituted, but the levels of expression of GFP and perforin were only around 5–8%. NK and CD8+ T cells were isolated from the spleens of these animals and cytotoxicity assays were performed. The results show that the cytotoxicity of NK cells was recovered up to 60% and in CD8+ T cells was recovered up to 80% of the cytotoxicity from normal animals.
The recovery of cytotoxicity in transplanted perforin deficient mice represents evidence that lentiviral vectors expressing perforin can correct this gene defect. The next step will be to verify if this correction is sufficient to protect the mouse model against development of HLH through infection with LCMV.
Demonstration of pre-clinical efficacy of AAV gene therapy for methylmalonic acidemia with a transgene suitable for human clinical trials
Methylmalonic acidemia (MMA) can still be a lethal disease even when patients are being properly managed, underscoring the need for new therapies for this disease. We have previous shown efficacy for AAV gene therapy with the delivery of a murine methylmalonyl-CoA mutase cDNA to an established murine model of MMA. However, amino acid sequence difference between the human and murine methylmalonyl-CoA mutase make the murine ortholog a potential antigen in humans. Because immunoresponses to transgene products in gene therapy have been well established, our lab has developed and tested an AAV vector carrying the human methylmalonyl-CoA mutase cDNA. This new AAV vector is capable of rescuing the murine model of MMA from neonatal lethality and significantly reducing toxic metabolites for greater than 18 months. Since patients with MMA typical carry at least one missense mutation, the human methylmalonyl-CoA mutase is less likely to elicit an immune response in this patient population, than in a patient population that does not express or expresses a truncated form of the protein being delivered. The AAV vector we have developed is the first that could potential be used in a human clinical trial.
LBPA/ CLINIGENE, Ecole Normale Superieure (ENSC), 94235 Cachan Cedex, France
Fanconi anemia (FA) is a rare inherited genomic instability syndrome, with severe bone-marrow deficiency developing in children. In FA, the single therapy available is allogeneic haematopoietic cells (HSC) transplantation. We have recently achieved long-term reconstitution both in vitro and in vivo with retroviral-mediated gene transfer of FANCA (main complementation group) and protecting cells from oxidative stress. In order to help reduce the risk for induced malignancies in this cancer-prone disease, we have designed and developed a new generation of genetically stable insulated vectors (see Duros et al, and Artus et al, ibid). Insulated lentivectors have been cloned to encode for either FANCA cDNA or eGFP as control. Three different internal have been tested: the strong viral Fr-MuLV-U3, the housekeeping hPGK or the FANCA endogenous promotor. Transduction of FANCA-/- HSC72 lymphoblastoids cells with all lentivirus constructs expressing FANCA results in phenotypic correction of FA cells which is maintained over 12 weeks of sustained culture and functionally assessed by both: (i) overcoming the cell-cycle G2M block with high doses of Mitomycin C and (ii) restoring FancD2 mono-ubiquitination. FANCA-/- human primary mesenchymal cells have further been transduced and similarly assayed: gene-corrected cells slowly recover a wild type pattern, the earliest being monitored with the U3 promotor. Experiments are underway which address the feasibility of generating diseased induced pluripotent stem cells, as a model towards establishing the best conditions for FA- gene therapy, making use of these safety improved vectors.
Gene Therapy Research Unit, The Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, NSW 2145, Australia
A major challenge when using gene therapy for treatment of metabolic liver disease is maintaining persistent levels of therapeutic gene expression in a growing liver. We have addressed this in the context of ornithine transcarbamylase (OTC) deficiency, a urea cycle disorder resulting in hyperammonemia. Using recombinant adeno-associated viral vector (rAAV)-mediated gene delivery we have successfully treated adult spfash OTC deficient mice based on normalization of urinary orotic acid. Following neonatal vector delivery, however, only ∼5% stable gene-modified cells remain at adulthood. While this was not sufficient to prevent orotic aciduria, it may be sufficient to prevent hyperammonemia. However, the spfash mouse has only a mild phenotype, with ∼5% endogenous OTC activity which is sufficient to maintain normal ammonia levels. We have therefore developed a model with a severe phenotype, using a short-hairpin RNA to knock down endogenous OTC activity (shRNA-OTC). Neonatal spfash mice were injected with rAAV2/8.OTC (2.5×1011 vg/mouse) and as adults (8–12 weeks) with rAAV2/8.shRNA-OTC (1×1012 vg/mouse). However, knockdown was inefficient as transduction was inhibited by an immune response to the initial injection. To overcome this, the shRNA was pseudo-serotyped with an alternate AAV capsid. Knockdown was successful but despite achieving a level of 5–13% OTC activity, this was not sufficient for protection against hyperammonemia. Given that endogenous OTC activity of ∼5% protects against hyperammonemia under normal conditions, our experiments indicate that the proportion and distribution of gene-modified cells across the hepatic lobule is a critical factor in determining therapeutic success.
Amsterdam Molecular Therapeutics (AMT) B.V., 1105 BA Amsterdam, NL
Department of Physiology, BioSciences Institute, University College Cork, Ireland
Cystic fibrosis is most commonly caused by the ΔF508 mutation, a 3 bp CTT deletion, in CFTR.
Mutations can be repaired by creating a zinc finger nuclease (ZFN) site-specific double-stranded break (DSB) to stimulate homology-directed repair (HDR) using a wild-type (WT) donor DNA molecule in >10% of treated cells1.
We have previously described ZFNs that create a DSB ∼200 bp upstream of the DF508 mutation2. Here, we use these ZFNs to repair the DF508 mutation in a DF508 homozygous cell line (CFTE) using a 4.3 kb WT genomic CFTR sequence cloned between adeno-associated virus ITR sequences (pCFTR-Donor). This WT CFTR sequence should repair the CTT deletion and change an A/G SNP (rs213950) in exon 10. pCFTR-Donor-XC contains XhoI and ClaI restriction sites to help quantify HDR efficiency.
CFTEs were transfected as follows: pZFNs, pCFTR-Donor, pCFTR-Donor-XC, pZFNs and pCFTR-Donor, or pZFNs and pCFTR-Donor-XC. Seventy-two hours post-transfection, RNA was extracted and RT-PCR performed using a WT-specific primer. A 675 bp band was seen only when cells were transfected with pZFNs and pCFTR-Donor, or with pZFNs and pCFTR-Donor-XC. Sequence analysis confirmed gene repair had occurred.
Frequency of gene repair will be established by genomic DNA sequence analysis, and the ability of ZFN-HDR to restore normal airway surface liquid levels will be assessed in an established cell based system3.
[1] Lee CM et al., (2008). Irish Journal of Medical Science
[2] Urnov FD et al., (2005). Nature;
[3] Worthington EN & Tarran R (2011). Methods Mol Biology;
Laboratory of Gene Pharmacotherapy, Department of Pharmacological and Toxicological Chemistry, Faculty of Chemical and Pharmaceutical Sciences, Universidad de Chile, Santiago, Chile
Genetic vulnerability to alcoholism is mainly ascribed to genes which encode enzymes of alcohol metabolism. In the liver, ethanol is oxidized to acetaldehyde by alcohol dehydrogenase and, subsequently, acetaldehyde is oxidized to acetate by mitochondrial aldehyde dehydrogenase (ALDH2).
An inactivating polymorphism in ALDH2 protects from alcoholism: upon alcohol intake, blood acetaldehyde levels increase, resulting in dysphoric effects and aversion to ethanol. Treatment for alcoholism with disulfiram mimics this phenotype by inactivation of ALDH2. However, drug efficacy is offset by individual variability, toxicity, and short half-life. Ribozymes recognize and cleave an mRNA in a specific manner and may thus provide a therapeutic alternative having greater specificity and longer lasting effects.
Adenoviral vectors encoding a hairpin ribozyme, a control ribozyme (incapable of cleavage) or a control RNA were injected into the tail vein of alcohol dependent UChB rats (n=5). A single administration (2×1012 vp/kg) of the adenoviral vectors encoding either ribozyme resulted in ∼50% decrease in alcohol intake (34 days), while the control adenovirus and the vehicle did not diminish consumption (p<0.0001). Blood acetaldehyde (gas chromatography) increased to more than twice (p<0.001) that of the control animals after an intraperitoneal administration of alcohol (1 g/kg). The ALDH2 activity in liver homogenates (spectrophotometry) decreased ∼24% (p<0.02), a specific effect, since the activity of other ALDHs was unaffected.
In conclusion, hairpin ribozyme genes reduce the rat ALDH2 activity in vivo, curtailing voluntary alcohol intake for a month, thus providing a basis for developing a gene therapy for alcoholism.
FONDECYT 1040555, VOLO 2008-02.
Department of Physiology, Department of Microbiology, BioSciences Institute, University College Cork, Ireland
Cystinosis is a multi-system autosomal recessive disorder caused by mutations and/or deletions in both alleles of ctns, encoding for the low pH dependent lysosomal cystine exporter cystinosin. Cystinosis is a rare disease with one case per 200,000 live births resulting from accumulation of cystine in the lysosomes by an, as yet, incompletely characterised mechanism.
Use of virus vector modified autologous hematopoietic stem cells is being explored as a potential therapeutic approach in a mouse model of cystinosis.
Zinc finger nuclease (ZFN) homology directed repair (HDR) can correct gene sequences in vitro and in vivo with high efficiency. We have corrected mutations in cftr using ZFNs and have made ZFNs that can target ctns.
Here we report an assay to provide functional evidence for restoration of CTNS via ZFN HDR. In addition to apoptosis, autophagy is also a key regulator of tissue injury in cystinotic cells. Ctns specific ZFNs will be used to test the hypothesis that ZFN HDR will accurately restore cystinosin expression and change the expression of an autophagy marker by immunostaining.
Department of Research and Development, Amsterdam Molecular Therapeutics BV, Amsterdam
The development of RNAi-based gene therapy of genetic diseases has been investigated extensively with adeno-associated viral vector (AAV) being the delivery method of choice. However, several safety concerns have been raised including short hairpin RNA (shRNA) and microRNA (miRNA) toxicity due to oversaturation of the RNAi machinery and unanticipated gene off-targeting effects. To circumvent the toxicity and specificity problems, the use of regulated miRNA expression is an important requirement because it allows tight control on the effector molecule concentrations upon administration of an inducer drug. Here, we used the Gene Switch (GS) mifepristone-inducible system to express artificial miRNA targeting Apolipoprotein B (ApoB) for therapy of hypercholesterolemia. ApoB gene expression was silenced in murine livers transduced with AAV-H1shApoB or AAV-LP1miApoB resulting in a strong decrease of plasma cholesterol. Further, miApoB was expressed from the GS system (GS-miApoB) and its efficacy and inducibility was demonstrated in vitro. Ongoing research aims to determine if tight and controlled long-term GS-miApoB expression can be obtained in murine liver upon induction with mifepristone, without signs of toxicity.
In parallel to the gene therapy approach for hypercholesterolemia, we are using RNAi to treat Huntington's disease. Approximately 100 artificial miRNAs (miH) targeting the wild-type and/or mutant Huntingtin (Htt) gene were designed and verified for their knock-down efficacy in vitro. We have identified the highly potent miH5, miH10 and miH12 targeting exon 1 or miS7 and miS15 targeting a HD-associated SNP in exon 67 of Htt. As a next step, those miRNAs will be expressed from the GS-inducible promoter and tested in rodent brains.
University of Zurich, Div Clin Chem Biochem, Univ Child's Hosp, Zurich, Switzerland
Tetrahydrobiopterin (BH4) is an essential cofactor for phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH). BH4 is synthesized de novo from guanosine triphosphate (GTP) by three enzymes: GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase (SR). Conventional treatment of patients with BH4 cofactor deficiency due to autosomal recessive mutations in the PTS gene, encoding PTPS, requires control of elevated blood-phenylalanine (Phe) levels by BH4 supplementation and oral replacement therapy with L-dopa and 5-OH-tryptophan, precursors of dopamine (catecholamines) and serotonin, respectively. We have recently developed a viable mouse model for PTPS deficiency, the compound heterozygous Pts-ki/ko mouse, which exhibit low PTPS activity, hyperphenylalaninemia and low brain dopamine and serotonin levels. Currently we are testing a triple-gene transfer to target liver using AAV2 pseudotype 8 vectors (rAAV2/8) in combination with luciferase co-expressed for in vivo hepatic imaging. Coordinate expression of PTPS, TH and TPH is thought to lead to hepatic synthesis of BH4, L-dopa plus 5-OH-tryptophan, respectively, and eventually to restoration of systemic phenylalanine clearance and endogenous supply of the essential precursors for monoamine neurotransmitter biosynthesis in the CNS.
Genetic Therapeutics, Jichi Medical University, Shimotsuke 329-0498, Japan
Phenylketonuria (PKU) is caused by a deficiency of the liver enzyme phenylalanine hydroxylase (PAH) that converts phenylalanine to tyrosine. We showed that liver-targeted gene transfer with adeno-associated virus (AAV) vectors completely corrected hyperphenylalaninemia in Pahenu2 mouse, a murine ortholog of human PKU (Mochozuki et al, Gene Ther 2004; Yagi et al, J Gene Med 2011). Among these, a self-complementary AAV (scAAV) vector was particularly effective, presumably by immediate formation of double-stranded DNA upon transduction. Here, we addressed whether intramuscularly administered scAAV vectors would impact on PKU phenotype. Skeletal muscle is an attractive target for PKU gene therapy, because of its large mass and easy accessibility. This strategy, however, raises another challenge that one must provide muscle with tetrahydrobiopterin (BH4), a necessary cofactor for PAH, since the extrahepatic tissues do not synthesize BH4. In addition to one encoding PAH, we constructed scAAV vectors for the expression of GTP cyclohydrolase I (GCH) and 6-pyruvoil tetrahydrobiopterine synthase (PTS), two key enzymes in BH4 synthesis (Ding et al, Mol Ther 2008). Intramuscular injection of 2×1011 vector genomes (vg) of PAH vector did not show a significant effect in Pahenu2, despite that BH4 was supplied intraperitoneally. Simultaneous injection of three scAAV vectors encoding PAH, GCH and PTS (4×1011 vg each) resulted in a significant, still moderate decrease of blood phenylalanine in Pahenu2. These results suggested that higher dosing of vectors or further vector development is required to fully correct PKU phenotype by muscle-directed gene transfer.
Centre d'Investigation Clinique intégré en Biothérapies, Hôpital Necker-Enfants Malades, 149 rue de Sèvres 75015 Paris
Blnk deficiency is an autosomal recessive immune disorder characterized by the absence of B cells in periphery and the absence of any serum immunoglobulins due to an early blockage at the pro-B cell stage in the bone marrow. The very rare patients affected by blnk deficiency develop severe infections. In the murine model of the disease, a similar blockage in B cell development is described as well as susceptibility to infections and to pre-B lymphomas.
A homozygote stop mutation in blnk gene was identified in a 8-yr old boy. Bone marrow cells analysis revealed that CD34+ CD10+CD24-CD19- lymphoid progenitors were present as well as CD34+CD10+CD24+CD19- early B cells and CD34+CD19+ pro B cells. However, no surface IgM or serum Ig were detected in this patient. To demonstrate the implication of Blnk in the B-cell differentiation process, we transduced CD34+ sorted bone marrow cells from this patient with a lentiviral construct containing human wild type Blnk cDNA. The transduced cells were intravenously injected into irradiated NOD/SCID/IL2rg knock-out mice. Twelve weeks after transplantation, recipients were analysed. Human engraftment was detected in bone marrow and spleen. Among bone marrow human CD45+ cells, more than 80% were CD19+ and 6 to 8% express surface IgM. In the spleen, between 14 and 30% of CD19+ cells were detected. Eight to 42% of CD19+ cells expressed surface IgM.This is the first demonstration that Blnk is absolutely required for the differentiation of pro-B cells toward mature B cells.
Department of Physiology, University College Cork, Cork, Ireland
Zinc finger nucleases (ZFNs) are chimeric enzymes, composed of a zinc finger protein (ZFP) DNA binding domain and FokI nuclease domain, designed to create targeted genomic double-stranded breaks (DSBs). DSBs are repaired by either non-homologous end joining or homology directed repair allowing ZFNs to mediate targeted gene KO or repair. Despite successes with ZFNs targeted to several different genes, there is no standard protocol for efficient ZFN design in publicly available literature. Modular assembly (MA), which is based on linking zinc fingers together, has a high failure rate1. A refinement of this system, context dependent assembly (CoDA), has a higher success rate but reduces the number of targetable sites2. The OPEN screening method, although more labour intensive, has been shown to yield ZFNs with high efficiency and specificity; the original study doubled the number of targeted genes in available literature3.
Cystinosis is a rare multi-system autosomal recessive disorder caused by mutations in CTNS 4. This work describes the application of the OPEN screening protocol to CTNS after both the MA and CoDA approaches failed to yield active CTNS-specific ZFNs. Candidate ZFN sites were selected using the online tool ZiFit with a bias towards exonic sites. ZFP libraries were tested in a bacteria 2 hybrid assay to isolate ZFPs with high specificity for candidate target sites. Further studies are required to assess ZFN function in cell based assays.
1. Ramirez et al. 2008 PMID:18446154
2. Sander et al. 2011 PMID:21151135
3. Maeder et al. 2008 PMID:18657511
4. Town et al., 1998 PMID:9537412
Institute of Experimental Hematology, Hannover Medical School
Congenital amegakaryocytic thrombocytopenia (CAMT) is a genetic disorder caused by inactivating mutations in the thrombopoietin receptor MPL, which causes severe thrombocytopenia and aplastic anemia. To develop a gene therapy for CAMT we retrovirally expressed Mpl in a bone marrow transplantation model (C57Bl/6 mice). Expression of Mpl by conventional gammaretroviral vectors induced uncontrolled expansion with subsequent exhaustion of hematopoietic stem cells (HSC), and increased frequency of insertional leukemias. The HSC loss, but not myeloproliferation and leukemogenesis, could also be induced by expressing an intracellular truncated, signalling deficient Mpl, which induced HSC transition into G1/S phase of the cell cycle and loss of the HSC transcriptome signature. To avoid “phenotoxicity” of ectopic Mpl expression we developed self-inactivating lentiviral vectors that express Mpl from different lineage-specific promoters (human or murine Mpl or human GPIba). In a murine model for CAMT we observed long-term survival of Mpl-/- mice after gene therapy. Recipients of gene corrected cells had increased numbers of lineage negative, Sca1 and cKit positive (LSK) cells with a normal contribution of CD34-LSK cells. However, only mice that expressed Mpl from the GPIba promoter showed correction of platelet counts (>300×106/ml, 11/13 mice). Mpl expression from the PGK promoter in Mpl-/- mice caused death 6 days post transplantation (6/7 mice) due to splenic rupture with erythroid hyperproliferation highlighting the extreme sensitivity of Mpl-negative hematopoiesis to ectopic Mpl expression. In conclusion, severe complications of ectopic Mpl expression could be avoided dependent on the vector's promoter, and transcriptionally targeted lentiviral vectors regenerated HSC and platelets.
Molecular Medicine and Gene Therapy, Lund University, Sweden
Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by nonfunctional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. Gene therapy could be an alternative treatment to allogeneic stem cell transplantation for IMO patients.
The aim of this study was to rescue the phenotype of IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA.
CD34+ cells from peripheral blood of 3 IMO patients were cultured with M-CSF, GM-CSF, IL-6, SCF and Flt3L for 2 weeks. During culture the cells expanded 500 fold and gradually lost CD34 expression while 50% became CD14+. The cells were transduced with SIN lentiviral vectors expressing either endogenous or codon optimized TCIRG1 under a SFFV promoter. The transduction efficiency was approximately 40% at 2 weeks. Cells were then differentiated for 10 days on bone slices with M-CSF and RANKL to mature GFP+ osteoclasts.
qPCR analysis and western blot revealed increased mRNA and protein levels of Tcirg1 compared to controls. Vector corrected IMO osteoclasts generated increased Ca2+ release and bone degradation products such as CTX-1 into the media, while non-corrected IMO osteoclasts failed to resorb bone. Resorption per osteoclast (CTX-1/TRAP ratio) was 20–50% of that of osteoclasts derived from normal CD34+ cord blood cells.
In conclusion we provide the first in vitro evidence of lentiviral-mediated correction of a genetic disease involving the osteoclast lineage, supporting further development of gene therapy of IMO and other diseases affecting these cells.
Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany
Nine out of ten patients were treated successfully in the German WAS clinical gene therapy trial. Linear amplification-mediated PCR (LAM-PCR) in combination with high throughput sequencing revealed between 2.000 and 13.000 unique integration sites (IS) so far. Eight treated patients showed a polyclonal reconstitution of the hematopoietic system until the latest time point analyzed. However, for each of the first two patients we observed a predominant repopulating clone with an IS upstream of CCND2 (patient 1) and within the MDS1 locus (patient 2), respectively. The contribution of these clones increased over time to more than 10%, but declined again at later time points. Furthermore, we could show an expression of MDS1-EVI1 in patient 2 at later time points with a high MDS1 contribution, whereas in early time points no expression was detectable. Strikingly, one patient (patient 6) developed a T-lineage acute lymphoblastic leukemia (T-ALL) 489 days after gene therapy. The analysis of the leukemic time point revealed an IS 33 kb upstream of the LMO2 locus. The contribution of this clone increased from less than 1% at day 405 to more than 70% at day 489 after gene therapy. After successful chemotherapy, the LMO2 contribution again decreased to less than 1%. Reverse Transcriptase (RT)-PCR analysis showed an overexpression of LMO2 at the leukemic time point, whereas at earlier time points and after chemotherapy no or only a weak expression was detectable. Clinical and molecular follow-up is ongoing and will allow clarifying the risk/benefit ratio in these patients.
Hematopoiesis and Gene Therapy Division CIEMAT/CIBERER 28040, Madrid, Spain
The engraftment of immunodeficient mice with human hematopoietic stem cells (HSC) constitute one of the best models for assessing the efficacy of new therapies for diseases affecting the hematopoietic system. In the case of Fanconi anemia (FA), however, current studies have not being able to reproducibly engraft immunodeficient mice with FA-HSCs. The aim of our work was to develop a new humanized mouse model based on the transplantation of immunodeficient mice with FANCA-interfered human CD34+ cells obtained from healthy donors. To this aim, LVs carrying different anti-FANCA shRNAs and the marker EGFP gene (shFA-LVs) were initially constructed. Cord blood (CB) CD34+ cells transduced with sh7FA-LV showed a spontaneous reduction in the clonogenic potential of these cells, particularly when secondary colonies were generated. Additionally, a marked hypersensitivity to both mitomycin C (MMC) and TNF. was observed, resembling the phenotype of CD34+ cells from FA patients. The transplantation of sh7FA-transduced CB CD34+ cells into immunodeficient mice showed an evident engraftment with EGFP+ human hematopoietic cells at 30 days post-transplantation. Significantly, the engraftment of these animals progressively decreased, indeed due to the compromised repopulating ability of FA-like stem cells. To test the efficiency of gene therapy in this humanized FA mouse model, FANCA-interfered samples were transduced with a LV expressing FANCA. Interestingly, this therapeutic LV corrected the phenotype of FA-like CD34+ cells, validating the efficacy of this humanized model for the development of new therapies of FA, including gene therapy.
ICH, 30 Guilford street, London WC1N 1EH
X-linked Chronic Granulomatous Disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte NADPH-oxidase catalytic subunit gp91phox. Due to the lack of NADPH oxidase activity patients suffer from life threatening bacterial and fungal infections and have an impaired inflammatory response. A recent clinical trial for X-CGD using a Spleen Focus-Forming virus (SFFV)-based gammaretroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to methylation and silencing of the viral LTR. To improve safety and efficacy, we have designed a lentiviral vector (pCCLCHIMGp91WPRE4) that directs transgene expression primarily in myeloid cells. The lentiviral vector has been tested in a murine model of stem cell gene therapy for its ability to restore a normal NADPH oxidase activity. Transplanted mice were challenged with an inflammatory stimulus using an air pouch model and showed increased ability to resolve inflammation when compared with XCGD mice.
The same vector has recently been used to treat a patient in extremis. Two weeks after gene therapy, peripheral blood neutrophils showed 25% of NADPH oxidase activity. The patient is routinely tested for oxidase activity and gene marking.
Molecular Immunology Unit, UCL Institute of Child Health, London, WC1N 1EH, United Kingdom
Sin-lentiviral vectors can mediate stable gene transfer to keratinocyte stem cells (KSC). Gene modified KCS can be used to generate epidermal skin sheets suitable for grafting. The production of gene modified skin grafts may provide therapeutic benefit for inherited skin diseases as well as for systemic diseases. We have previously reported pre-clinical gene correction for the inherited skin disease, Netherton Syndrome (NS), a defect of the serine protease inhibitor LEKTI, which arises due to mutations in SPINK5. Lentiviral vectors incorporating an internal SFFV retroviral promoter, in combination with a codon optimised SPINK5 (SPINKco) transgene supported high levels of LEKTI reconstitution and robust correction of skin architecture. However, subsequent experiments have uncovered unanticipated silencing phenomena, with loss of LEKTI expression from the SPINKco transgene over time. We report how the inadvertent introduction of CpG sites during the codon optimisation of SPINK5 rendered the vector susceptible to silencing due to DNA methylation within the promoter/transgene boundary. In contrast, substitution of the methylation-prone retroviral promoter with a minimal human involucrin promoter (INVOp) (which encodes very few CpG sites) supported durable, methylation-resistant gene expression. Using three dimensional skin equivalent organotypic cultures in vitro, and a human:murine skin graft model in vivo, we demonstrate that INVOp mediates highly compartment specific reconstitution of LEKTI expression and supports architectural correction in NS. The findings highlight unexpected consequences relating to the use of synthetic gene constructs optimised to support increased protein production through increased GC content designed to enhance mRNA stability.
Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK
MPSIIIA is caused by a mutation in the SGSH gene, leading to cellular accumulation of 16etard16 sulphate and progressive neurodegeneration in patients. Our aim was to compare promoters in a clinically applicable lentiviral vector using a stem cell gene therapy approach in WT and MPSIIIA mice.
GFP lentiviral vectors under the ubiquitous (PGK) or monocyte specific (CD11b or CD18) human promoters were used to transduce lineage depleted cells and then transplanted into WT mice. Flow cytometry showed full chimerism and over 80% transduction with an average of 5 copies per blood cell. Mice were sacrificed 6 months post-transplant, and cells from blood, bone marrow, spleen and brain were stained with CD45, CD3, CD19 and CD11b antibodies. Gene expression was not lineage specific, but expression from PGK and CD11b was similar, with lower expression from CD18. Around 25% of CD11b positive cells in the brain were transduced.
HSCs transduced with GFP lentivirus had 3 fold more copies than cells transduced with SGSH-long (cDNA+3′ and 5′ UTRs). Copy number was increased when using a codon-optimised SGSH (SGSH-CO) (cDNA only) and remained high when a stop mutated SGSH-CO-X was used, suggesting that vector size, not SGSH toxicity is important in HSC transduction. We are currently assessing PGK-SGSH-CO and CD11b-SGSH-CO in MPSIIIA mice.
We have achieved excellent HSC transduction, shown that vector size is important in HSC transduction, and that PGK and CD11b are equally good promoters in blood cells. This is an important step towards clinical application of this approach.
Optimization of 3′ trans-splicing in the type VII Collagen gene
Dystrophic epidermolysis bullosa (DEB) is an inherited mechano-bullous skin disease caused by mutations in the type VII collagen gene COL7A1. Gene-therapeutic approaches in DEB are hindered in terms of the size of the COL7A1 transcript (9 kb). The size of the therapeutic insertions can be reduced by spliceosome-mediated RNA trans-splicing (SMaRT).
The sequence of COL7A1 was analyzed to determine an intron located in the middle of the gene. We decided to target Intron 46 in order to repair all possible mutations within the COL7A1 gene with just two repair molecules.
An RTM library consisting of RTMs with random BDs specific for COL7A1 Intron46/Exon47 was produced. Double transfection of single RTMs and a target molecule into HEK293AD cells showed different trans-splicing efficiencies among the various RTMs. The most efficient RTMs analyzed via fluorescence activated cell sorting (FACS) induce specific trans-splicing in up to 95% of RTM and target expressing cells.
The target constructs will be stably integrated into the genome of a HEK293AD cell line to mimic an endogenous target pre-mRNA at a constant level. This should exclude RTM efficiency variations due to varying target concentrations in the cells.
The methodology of RNA trans-splicing may provide a promising tool for gene therapy in DEB patients.
Gene Therapy for Metabolic Diseases, Gene Therapy and Hepatology/CIMA, Universidad de Navarra, Pamplona, 31008, Spain
Adeno-associated vectors (rAAV) have been engineered to attain long-term liver gene expression. Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria) benefits from liver gene transfer in mouse models and clinical trials are about to begin. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Macaques given a rAAV5 vector encoding human PBGD develop cellular and humoral immunity against viral capsids but not towards the transgene. Mycophenolate mofetil (MMF), anti-thymocyte immunoglobulin, tacrolimus and rituximab were given in combination in such a way that anti-AAV immune responses were abolished during 12 weeks but rebounded following cessation of the immunosuppressants. Viral antigen persistence in circulation due to poor clearance under reduced antibody production explains the rebound. Subsequent gene transfer with a rAAV5 vector encoding enhanced green fluorescent protein was not possible, even in a macaque in which the intensive immunosuppression regimen was instigated again. Twenty five weeks after the rAAV5-pbgd administration, enhanced transgene expression was only observed in the macaques which received immunosuppression only circa the first rAAV exposure. Results in mice indicate that MMF decreases rAAV5-encoded transgene expression in the liver via a mechanism that involves a functional adaptive immune system.
Dr Lin Zhang
Gene delivery into hematopoietic stem cells by gammaretroviral vector is an effective treatment for Adenosine deaminase (ADA) deficient-SCID. However the current gammaretroviral vectors carry long terminal repeat (LTR) enhancers with insertional mutagenic potential as shown in animal work and clinical trials. This has necessitated the need for safer vector development. To improve the safety for ADA-SCID gene therapy and achieve greater efficacy, we have generated a vector using a third-generation vesicular stomatitis virus G pseudotyped self-inactivating lentivirus, in which ADA gene transcription is under the control of the elongation factor- 1α mammalian promoter.
We compared the lentiviral vector with the clinical gammaretroviral vector in an in vitro immortalization assay combined with proliferation assay, which selects for viral insertional transformants with robust replating phenotypes in limiting dilution. The lentiviral vector showed less transforming potential and therefore significantly reduced genotoxicity, compared to the gammaretroviral vector. In vitro studies with wildtype and ADA deficient murine lineage negative progenitor cells show that, compared to the gammaretrovirus, the lentiviral vector has higher transduction efficiency in quiescent cells but with similar levels of protein expression and enzymatic activity. Data from in vivo studies in which human CD34+ cells from ADA-SCID patients were transduced with the lentiviral vector encoding ADA and engrafted into humanized NOD-SCID/γc-/- mice suggests that the presence of intracellular ADA delivered by the lentiviral vector promotes successful engraftment CD34+ cell engraftment, as well as lymphoid lineage differentiation. These studies indicate that the self-inactivation lentiviral vector has both efficacy and improved safety for ADA-SCID gene therapy.
Generation of X-CGD CD34+ cells from primary human hematopoietic cells via lentivirally delivered shRNAs against gp91phox
Chronic granulomatous disease is a severe inherited immunodeficiency often with a fatal outcome. The only curative treatment currently available is matched hematopoietic stem cell transplantation. For patients lacking a matched donor, gene therapy of autologous hematopoietic stem cells could be an option. The retroviral vectors used for gene transfer need to be evaluated for their safety and capability to correct the disease phenotype. The predictive values of these assays depend on the test systems used. For X-CGD, only minute amounts of patient derived primary material are available for research and thus murine models of the disease are mostly used for vector testing.
In an approach to overcome the acute paucity of primary human X-CGD cells, we developed a lentiviral vector for the stable knockdown of gp91phox in normal human hematopoietic stem cells. The knockdown is mediated via two distinct Pol III-driven shRNAs which are expressed along with a fluorescence marker. Efficient knockdown of gp91phox was achieved in a myelomonocytic cell line and in primary CD34+ cells as assessed by FACS and Western blot, resulting in complete loss of NADPH-oxidase activity. Reconstitution of NADPH-oxidase function in these cells was feasible after introduction of a second lentiviral vector expressing a recombinant codon-optimized gp91phox cDNA along with novel far-red fluorescent marker. This approach presents a valuable system for the testing of viral vectors in human primary X-CGD cells as well as for research focusing on the basic biology of gp91phox.
University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Division of Woman and Child, Leuven, Belgium
LBPA/ CLINIGENE, Ecole Normale Superieure (ENSC), 94235 Cachan Cedex, France
We have investigated potential ways of integrating vectors safety improvement in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing integration to heterochromatin. Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, the insulated lentivector which shows efficient and stable expression into human primary cells: 4xIns2-DCaro4, has been further tested and compared to its non-insulated counterpart, with chimeric HIV-1 derived integrases targeting heterochromatin through either histone H3 or methylated CpG islands (ML6 & ML10 chimeras, respectively). With DCaro4 and ML6 chimeras, a homogeneous expression is sustained over time. With the control, GFP expression is just over background double-mutant in catalytic and ledgf binding-sites while expression can be induced with HDAC. In CD34+ cells from cord-blood, these data are partially recapitulated with the ML6 chimera and entirely with the ML14, which includes an additional mutation translating in an enhanced targeting specificity by one order of magnitude. Preliminary high throughput integration sites analysis on cell-lines reveals a distinct profile with histone H3 targeting chimeras; experiments are being performed on human primary cells: fibroblasts and CD34+ cells from cord blood. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent risk for enhancer-activation mediated insertional mutagenesis, in combination with a potent and stable genetic insulation, regardless of the integration site inside heterochromatin.
Mesenchymal Stem Cell and Gene Therapy Ameliorate Ketamine Induced Toxicity on Leydig Cells
HTGSCs (Human Tooth Germ Stem Cells) isolated from human tooth germs have characteristics of MSCs (mesenchymal stem cells). Leydig cells produce testosterone in mammalian testis maintaining steroidogenic functions under control of Luteinizing Hormone (LH). Ketamine, an anesthetic drug used for human and veterinary medicine has been shown to be toxic for nervous system and reproductive system. Vascular endothelial growth factor (VEGF) gene, key regulator of angiogenesis, was shown to be induced by hypoxia and stimulates testosterone production in Leydig cells. In this study we tested effect of MSCs and VEGF in reducing ketamine induced toxicity of TM3 leydig cells.
MSCs were isolated from human impacted third molars and analyzed for their surface antigens by flow cytometry followed by co-culture with leydig cells which were exposed to 200μg/ml of ketamine for 24hrs. After 24 hours, the cell viability was measured by the MTS-assay checking the expression of apoptosis related genes, P53 and Caspase3 genes by using Real Time PCR. It was also tested that whether VEGF recombinant protein and VEGF gene delivery reduce toxicity of Ketamine on leydig cells by both checking cell viability and apoptosis related genes. Our results revealed that both MSCs therapy and VEGF have protective effects on TM3 cells exposed to ketamine. MCSs naturally secrete VEGF into cell culture medium which might be the main cause of the protection against Ketamine stress on cells. This study also showed that direct VEGF gene delivery might be an alternative treatment in severe Ketamine induced toxicity on reproduction system.
Medicinal Virology, Department of Oncology and Institute of Biomedical Engineering, Department of Engineering Science, University of Oxford, Oxford, OX3 7DQ, UK
The use of three-dimensional hydrogel in stem cell cultures can provide a microenvironment that mimics the stem cell niche. The niche is crucial in promoting cell to cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization. This study aims to culture and characterise human embryonic stem cells (hESCs) in a 3D microenvironment using alginate hydrogel. Two types of scaffold e.g. alginate (Al) and poly-D-lysine coated alginate (Al-PDL) are prepared in 1.2% (wt) calcium alginate and cell seeding density was 1×106cells/ml. Cell viability was assessed by using trypan blue and live-dead cell staining. The results showed that Al-PDL supported cell growth, higher than Al in day-1 and day-2 culture however both hydrogels possessed the same number of viable cells at day-3 (2.875×105). Cell viability was then decreased parallel with the degradation of hydrogels. Hydrogels that sustained their integrity (1.8% wt calcium alginate) up to 28 days promoted cell growth and formed cell aggregates which were pluripotent. Live-dead cell staining demonstrated positive expression from live cells and a number of dead cells encapsulated in both AL and AL-PDL. Quantification of live-dead cells is on-going to compare the feasibility of the two different microenvironments. In conclusion, the preliminary results showed that AL-PDL provides higher mechanical integrity and maintains cell viability better than Al hydrogel.
MCTT, Seoul, Korea
Department of Genetics and Bioengineering, College of Engineering and Architecture, Yeditepe University, 26 Ağustos Campus, Kayisdagi cad., Kayisdagi, TR-34755 Istanbul, Turkey
Mesencyhmal stem cells (MSCs), important components of the tumor microenvironment, could modulate tumor growth and development in many ways. MSCs have been used as targeted gene delivery vehicles against cancer because of their potential of tumor tropism. In this study, we investigated the effects of Human Tooth Germ (HTG) derived MSCs on MCF-7 (breast cancer cells) and SH-SY5Y (neuroblastoma cells) cells treated with doxorubicine and paclitaxel, anticancer drugs. We also tried to determine in vitro effect of genetically modified MSCs secreting apoptosis inducers (Tumor necrosis factors related apoptosis inducing ligand (TRAIL), Dickkopf-related protein-1(Dkk-1)) on SHSY5Y cells.
TRAIL and Dkk-1 genes were transferred into MSCs by electroporation. A GFP gene transfected group was used as a control. Normal and genetically modified MSCs were co-cultured with SHSY5Y cells for 24 hours. After 24 hours cell viability and expression of apoptosis related genes Bcl-2, Bax, FADD, caspase3 were analyzed by MTS test and Real time PCR respectively.
The results showed HTGSCs increased the survival of cancer cells treated with doxorubicin and paclitaxel by around 30% reducing the doxorubicin and paclitaxel induced apoptosis. However, genetically modified MSCs secreting TRAIL and Dkk-1 reduced cell survival of cancer cells by inducing apoptosis and inhibiting the proliferation. Our findings demonstrated that MSCs reduced the efficiency of anti-cancer drugs on cancer cells which might be attributed to the secreted cytokines or chemokines by MSCs. On the other hand genetically modified MSCs might be used as gene delivery vehicles in cell and gene therapy of cancer.
Inst. of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Germany
Stem cell therapy is applied for a wide field. For some applications it is necessary to encapsulate and differentiate the cells. Therefore biocompatible matrices are required which supports the differentiation in the desired cell type. In this work we analyzed potential matrices for the regeneration of the nucleus pulposus, part of the intervertebral disc. hMSC-TERT (human mesenchymale stem cells- telomerase reverse transcriptase) have been embedded in gelatin and a “gelatin-porcine nucleus pulposus” matrix. Also we could compare this with work previously done on agarose matrix.
The immobilized cells were cultivated for 21 days in media, containing growth and differentiation factors. Viability of the cells in the different matrices was proofed by using a tetrazolium salt (WST-1). At certain times RNA isolation was done following the phenol/guanidine isothiocyanate protocol. RNA was transformed into cDNA followed by a RT-PCR. Different primers were used to analyze the differentiation of hMSC-TERT.
Via WST1-test the survival of hMSC-TERT in gelatine was proofed. The differentiation status of hMSC-TERT in the two different matrices (gelatine, NP extract) was analyzed. The PCR analysis showed a differentiation in a chondrogenic type, less with the NP extract. In agarose a differentiation in a nucleus pulposus cell type could be obtained. This leads to the conclusion that the influence of matrices on cell differentiation is greater than recently surmised. Therefore it is necessary to find a “close to nature”- matrix for the need in cell therapy.
ITQB-UNL/IBET
The successful transfer of human pluripotent stem cell (hESC) technology and cellular products into clinical and industrial applications needs to address issues of automation, standardization and the generation of relevant cell numbers of high quality. Another major challenge is the establishment of integrated bioprocesses capable to guarantee efficient cell banking and distribution after large scale-expansion.
In this study, we combined microcarrier technology and environmentally controlled stirred tank bioreactors, to develop an efficient and scalable system for expansion of pluripotent hESCs (SCEDTM461, Cellartis AB). We demonstrate the importance of controlling pO2 at 30% air saturation to improve hESCs growth. This concentration allowed for a higher energetic cell metabolism, increased growth rate and maximum cell concentration in contrast to 5% pO2. Importantly, the incorporation of an automated perfusion system in the bioreactor enhanced culture performance and allowed the continuous addition of small molecules assuring higher cell concentrations for a longer time period. The expanded hESCs retained their undifferentiated phenotype and pluripotency.
In addition, cell microencapsulation technology was investigated not only to improve cell expansion yields but also as a main strategy to facilitate bioprocess integration with cryopreservation protocols.
Our results show that the use of environmentally controlled bioreactors, microcarriers and cell microencapsulation technology are critical to ensure the production and cryopreservation of hESCs in high quality and relevant quantities. When compared to the standard 2D colony culture, our 3D strategy improves the final yield of hESCs by 15-fold, providing a potential bioprocess to be transferred to clinical and industrial applications.
Quality assessment of umbilical cord blood units before freezing and at the time of transplantation
Quality of umbilical cord blood (UCB) unit was usually assessed by total nucleated cell (TNC) count, CD34+ cell count, content of colony-forming unit-granulocyte-macrophage (CFU-GM), and cell viability. Data of Unit quality have been usually provided by cord blood banks (CBBs), however, it is unclear whether pre-freezing tests or pre-transplant release tests performed by CBBs are reproducible. The aim of this study was to find reliable in vitro tests which consistently reflected the number of hematopoietic stem cells in UCB units for transplantation. Samples were taken from 54 UCB units for assessment of post-thaw characteristics. TNC counts and CD34+ cell contents measured at our hospital before infusion showed good correlations with values assessed in pre-freezing tests (r=0.900 and 0.943,respectively) and pre-transplant release tests (r=0.829 and 0.930, respectively). Our data reveal that the TNC counts and CD34+ cell contents determined by pre-freezing and pre-transplant release tests, which are the most important UCB unit selection criteria, accurately related the quality of infused UCB units. However, CFU-GM content was poorly correlated (r=0.560 and 0.606). Correlation of post-thaw cell viabilities measured before infusion and during the pre-transplant release tests was also poor (r=0.308). In conclusion, our study suggested that the TNC count and CD34+ cell content calculated prior to cryopreservation and at the time of transplantation accurately reflected the quality of infused UCB units. These two tests can assist the transplant physicians in selection of appropriate UCB units.
Versatile Extensions of the Flp-Recombinase-Mediated Cassette Exchange Technology: multiplexing RMCE meets the needs for predictable genome engineering
Recombinase mediated cassette exchange is used for the specific modification of defined genomic loci. It was successfully applied for the elaboration of various cell lines and embryonic stem cells. Flp-RMCE has proven to have a reliable efficiency and its outcome avoids the genomic co-introduction of prokaryotic vector parts or a selection marker. An extension of this system is called Multiplexing RMCE, which enables the simultaneous or sequential exchange of two genomic loci. Multiplexing RMCE relies on seven novel heterospecific FRT-sites. These mutated Flp recognition sites were evaluated for function by self-recognition and no cross-interaction among heterospecific entities in vitro. After characterisation several pairs of these novel FRTs were chosen for episomal and genomic RMCE in NIH3T3 cells. In a final approach we were able to target two previously established loci simultaneously in a single experiment. We conclude that the novel FRTs work equally in terms of efficiency and safety as the established pair F3-F and are ready for use.
Applications for the system could be the defined and independent expression of protein subunits, T-cell receptors or the stepwise elaboration of chromosomal domains in cell lines, primary cells, embryonic stem cells and induced pluripotent cells.
Targeted inactivation of Hepatitis B virus cccDNA using engineered Zinc Finger Nucleases
Over 350 million people are chronically infected with Hepatitis B virus (HBV), which results in approximately in 600,000 liver cancer-related deaths annually. Despite the availability of therapeutic immunomodulators and nucleoside/nucleotide analogues, as well as advances in RNAi-based gene therapies, a cure for chronic HBV infection does not yet seem realistic. Treatment failure has been attributed to the persistence of episomal cccDNA, the fundamental viral replication intermediate that is required for HBV transcription. As cccDNA forms minichromosomes, it is capable of establishing a dormant reservoir of viral DNA. Currently, anti-HBV therapeutics target viral replication post-transcriptionally without affecting cccDNA. We are developing Zinc finger nucleases (ZFNs) to inactivate cccDNA. Three sets of ZFNs against the HBV polymerase, core and surface genes are being generated using the bacterial two- hybrid (B2H) Oligomerized Pool Engineering (OPEN) methodology. As the viral genome comprises overlapping reading frames, ZFNs targeting core and surface, will additionally target the polymerase gene. ZFN half-sites were generated from ZF DNA pools, and the B2H system was used to select efficient cccDNA-binding proteins from a library of approximately 8.5×105 different context-dependant arrays. This approach significantly increases the likelihood of successfully generating ZFNs, unlike modular assembly methods. Investigations are currently characterising the efficiency of ZFNs using reporter gene assays, and targeted mutagenesis of cccDNA. Successfully using ZFNs to disable cccDNA in hepatocytes of HBV carriers should contribute significantly to advancing the treatment of this serious infection.
Characterizing the key determinants for ZFN-mediated gene disruption
Simple methods to increase zinc-finger nuclease (ZFN) activity are highly attractive. Recent studies reported that transient hypothermia can increase the level of ZFN-induced gene disruption, but the underlying mechanism has remained undetermined. Here we aimed at characterizing the parameters for efficient ZFN-mediated gene knockout by comparing the activities of six different ZFN pairs (directed against EGFP, human AAVS1, CCR5, IL2RG, CFTR and HOXB13) in five human cell types (primary fibroblasts, HeLa, HEK293, K562, U2OS) under different conditions. The ZFN-expression plasmids were delivered either by nucleofection, lipofection or polyethylenimine-based transfection. Subsequently, cells were incubated at 30°C or 37°C and the extent of ZFN-induced insertions/deletions (indels) was quantified. On average ∼20% of alleles showed indels. The main determinants of the mutation frequency were DNA delivery method and temperature, although a beneficial effect of hypothermia was only observed when ZFN expression vectors were nucleofected. In accordance, in vitro cleavage assays did not reveal a difference in ZFN activity at 30°C vs. 37°C, implying that the temperature shift does neither affect DNA binding nor the catalytic activity of ZFNs. Assessment of ZFN expression levels revealed that hypothermia increased and prolonged transgene expression by effectively inhibiting cell division, as determined by proliferation assays and cell cycle analysis. As opposed to ZFN-stimulated homology-directed repair, however, the cell type had no significant influence on gene disruption rates. In summary, our results show that hypothermia can partially rescue suboptimal transfection efficiencies by inducing cell cycle arrest that leads to an increase in intracellular ZFN concentration.
Constraint-free Common Sites Definition in Clinical Gene Therapy
Viral vector integration is a process exploited in gene therapy (GT) to correct defective cells of an individual and driving the health status from the disease to a normal long-term condition. As consequence of this perturbation some of the affected cells could step to a secondary state when the vector is integrated in certain genomic positions leading to a deregulated expression of an important cellular gene.
Insertional mutagenesis can be used to discover new disease candidate genes and help to design safer GT protocols.
Clusters of integration sites, termed Common Insertion Sites (CIS), may be used as an indicator to evaluate and predict the safety in clinical gene therapy. However, the definition of CIS is not standardized and is based on the number of insertions in a specific region. Usually the number of IS required and/or the dimension of the region to define a CIS are defined before the analysis. We call this approach constrained.
We developed a fast graph based method to detect CIS independently from the IS number and using a constraint-free approach. This approach has numerous advantages over other methodologies: 1) thousands of IS are analysed in few seconds without any prior constraint, 2) resulting CIS are represented by graphs that are easy to display and understand, 3) graph theory could be used to infer characteristics and properties of the resulting networks and 4) CIS are exported in standard graph format for further downstream analyses via common software already used in system biology studies (i.e. Cytoscape).
Cellectis Therapeutics, 13 Rue Watt, 75013, Paris, France
Targeted approaches constitute an alternative to current random insertion strategies for gene therapy, by circumventing the potential risk of insertional mutagenesis. Such approaches include the repair of deleterious mutations at their endogenous locus as well as the insertion of a functional coding sequence at a chosen locus. These strategies require the use of specific endonucleases inducing high frequencies of homologous gene targeting at their cleavage site. Natural meganucleases, also called Homing Endonucleases, are the most specific endonucleases in nature, thus providing ideal scaffolds for the creation of genome engineering tools. By using a two-step combinatorial method to redesign the DNA-binding interface of the I-CreI LAGLIDADG meganuclease, we have created many endonucleases cleaving human genes. We show that natural meganucleases as well as engineered derivatives can be used to induce up to 15% of gene insertion into chosen sequences in immortalized cell lines.
Therapeutic applications are among the most challenging ones and their effective use will depend on several criteria. Intrinsic properties of engineered meganucleases, such as a favorable activity/specificity ratio, as well as the use of appropriate vectorization methods are essential for optimizing the efficacy of targeted recombination. We have tested several meganucleases for their ability to induce targeted modifications in primary hematopoietic cells. Depending on vectorization, frequencies could reach more than 1%. We will present data concerning our strategy for the development of therapeutic meganucleases including the selection and screening of potential candidates, the development of appropriate methods of vectorization and the use potential therapeutic meganucleases for the engineering of primary cells.
Division of Molecular Medicine, KU Leuven, Kapucijnenvoer 33, VCTB+5, Leuven, B-3000, Belgium
Retroviruses are commonly used as delivery vehicles to correct genetic diseases because of their precise integration mechanism. However, adverse events in which vector integration activated proto-oncogenes, leading to clonal expansion and leukemogenesis complicate their application. During lentiviral infection, the host cell–encoded lens epithelium-derived growth factor/p75 (LEDGF/p75) binds the lentiviral integrase and targets integration to active transcription units. We demonstrated that vector integration can be targeted away from genes by replacing the LEDGF/p75 chromatin interaction-binding domain with another DNA-binding protein, such as CBX1. CBX1 binds histone H3 di- or trimethylated on K9, which is associated with pericentric heterochromatin and intergenic regions, whereas the LEDGF/p75 C-terminal end still binds the viral integrase. Here, we show that transient expression following RNA-electroporation of the chimeric protein supports efficient transduction of lentiviral vectors and directs the integration of lentiviral vectors near CBX1 binding sites, outside of genes. In wild-type cells these regions are generally disfavored for integration. Despite integration in regions rich in epigenetic marks associated with gene silencing, lentiviral vector expression remained efficient. Thus, these results demonstrate that transient expression of engineered LEDGF/p75 chimera provide technology for controlling lentiviral vector integration site selection.
Institute of Experimental Hematology, Hannover Medical School, 30625 - Hannover, Germany
The use of self inactivating (SIN) retroviral or lentiviral vectors (LV) can minimize the risk of enhancer mediated oncogene activation. However, the recently reported clonal dominance in a clinical trial for gene therapy of beta-thalassemia mediated by deregulated gene expression due to alternative splicing highlights potential risks of SIN-LV mediated insertional mutagenesis.
Here we report a case of B-cell leukemia in a murine bone marrow (BM) transplantation model after integration of a SIN-LV vector expressing eGFP from an intron-containing glycoprotein-I-b-alpha promoter. The leukemia was clonal and transplantable. Two integrations were identified, one in the first intron of the Nance-Horan-Syndrome gene, the other in the intron 8 of the Early B-cell factor 1 (Ebf1) gene, a major regulator of B-cell development. No further genetic alterations were identified by spectral karyotyping and comparative genome hybridisation. Detailed PCR analyses revealed splicing between Ebf1 and the integrated SIN-LV leading to strong downregulation of Ebf1 mRNA and protein in leukemic cells compared to stage-matched BM B-cell progenitors. Genome wide expression analysis of leukemic cells showed downregulation of Ebf1 target genes and definite signs of transdifferentiation to the myeloid lineage, as described for loss of Ebf1 or its downstream target Pax5. In addition we observed strong upregulation of Flt3 and high levels of STAT5 activation, in line with recent reports of B-cell leukemia development in Ebf1+/- mice overexpressing constitutively active STAT5. Taken together our results highlight the risk of intragenic SIN-LV vector insertions that can induce alternative splicing with resulting inactivation of haploinsufficient tumour suppressor genes.
Hematopoiesis and Gene Therapy Division. CIEMAT/CIBERER. 28040, Madrid, Spain.
Pre-clinical studies and clinical gene therapy trials have provided evidence of genotoxicity associated to certain retroviral integrations, vector designs and expressed transgenes. In this work, we investigated the potential genotoxic effects of a PGK-FANCA-Wpre* lentiviral vector (LV), recently proposed for the gene therapy of Fanconi Anemia-A patients (Gonzalez-Murillo et al. Human Gene Therapy 2010, 21: 623–630). Briefly, lineage depleted bone marrow cells from male FA-A mice were transduced at high MOIs (above 20 T.U./cell) with VSV-G packaged PGK-FANCA-Wpre* LVs. After overnight transduction, cells were transplanted into lethally irradiated FA-A female recipients. Six months after transplantation, recipients were killed and BM cells re-transplanted into secondary FA-A recipients. Periodic peripheral blood samples and end-of-analysis bone marrow were obtained to perform 12etard12logical analyses. Besides, analyses of proviral copy number, functional correction of FA phenotype and stem cell marking were performed. As deduced from the analysis of gender specific sequences (quantitative PCR), high VCN in peripheral blood cells, and reversion of MMC sensitivity to DNA cross-linking drugs (mitomicin C), it was concluded that almost all tested recipients (currently, 40 primary and 20 secondary recipients) were highly engrafted with transduced cells. Significantly, no signs of myeloid or lymphoid leukemia were noted either in primary or secondary FA-A recipients. LAM-PCR studies are in progress to characterize the clonal gene marking and the insertional pattern of the therapeutic vector in the genome of repopulating cells.
Research Group Reprogramming REBIRTH, Hannover Medical School, Hannover 30625. Germany
Severe adverse events due to insertional mutagenesis by integrating vectors present a major safety concern for human gene therapy. However, most of the preclinical risk assessment is done in mice. Due to inherent differences between murine and human cells this may be problematic, and in contrast to the murine system we did not observe empty vector-induced transformation of human hematopoietic stem cells (HSC) in the In-vitro-immortalization-assay. Thorough assessment of genotoxic risks appears particularly relevant in the context of drug resistance gene-mediated in-vivo-selection and expansion of transduced human HSC as here the genotoxic risk may be augmented by the accumulation of cytotoxic agent-induced genotoxic lesions. In this respect, the DNA repair protein MGMT, and especially the O6-benzylguanine (BG) resistant MGMTP140K point mutant allows for efficient enrichment of transduced hematopoiesis by the combined application of BG and 1,3-Bis-(2-chlorethyl)-1-nitroso-urea (BCNU) or Temozolomide. Human CD34+ cells transduced with an MGMTP140K expressing lentiviral vector conferred 30% and 9% colony survival in methylcellulose assays at 20uM and 40uM BCNU and prior BG treatment while no colonies survived at concentrations higher than 5uM BCNU in control cells. Therefore, we transplanted MGMTP140K-transduced human CD34+ cells into NOD.SCID.IL2Rgc-/- mice. At 6 weeks post transplant 22–39% of human CD45+ with 1–4.5% MGMTP140K -transduced cells were detected in the peripheral blood. BG/BCNU-mediated in vivo selection has been initiated and the safety of this selection approach will be investigated post chemotherapy by clonality analysis of engrafted cells before and after drug application utilizing LAM-PCR technology.
Institute for Biomedical Research Georg-Speyer-Haus, Frankfurt, Germany
The analysis of retroviral insertion sites in our X-CGD gene therapy trial revealed an unprecedented clustering of integrations in or around the MDS1/EVI1 and PRDM16 genes, with more than 60 insertions found to be squeezed within a DNA stretch of only 3 kB within the first intron of the PRDM16 gene. Although retroviral target site selection is still poorly understood, recent data has shown a remarkable association between epigenetic marks linked to active or poised transcription and integration site selection. Such open chromatin regions are usually associated with promoter/enhancer activity and define S/MARs or DNAse I hypersensitive DNA regions. In silico analysis of the PRDM16 integration cluster predicted the existence of a S/MAR element within this region. Plasmid vectors containing this region were maintained as episomes in CHOK1 cells, confirming the S/MAR function of this element. Furthermore preliminary data from ChIP experiments showed that the acetylation level of histone H3K9 is increased and the overall histone H3 load is decreased at the PRDM16 cluster region suggesting a highly accessible chromatin structure. In luciferase assays we showed that a 1.5 kb DNA fragment derived from the integration cluster region was able to enhance luciferase activity in PRDM16 expressing cells, while DNA regions flanking the integration site cluster lacked enhancer activity. Thus the PRDM16 integration cluster defines a transcriptional regulatory region within the first intron of PRDM16 which may control PRDM16 gene expression in hematopoietic progenitor cells.
San Raffaele-Telethon Institute for Gene Therapy, Milan, Italy
We devised and validated sensitive genotoxicity assays based on Cdkn2a or wild type (WT) mouse strains to assess the biosafety of lentiviral vector (LV) developed for liver gene transfer. As a positive control of insertional mutagenesis in hepatocytes, a LV carrying Enhanced Transthyretin enhancer/promoter (ET) in the Long Terminal Repeats (LV.ET.LTR) was generated and used to induce hepatocellular carcinoma (HCC) in mice. Systemic LV.ET.LTR injection induced HCCs in 30% of Cdkn2a-/- mice (p<0.01) and 75% of WT mice in combination with CCl4 treatment (p<0.01). We also administered a vector with SIN LTRs carrying only the mutated Posttranscriptional Regulatory Element (LV.mPRE) to Cdkn2a-/- mice. The mPRE sequence, as recently described, contains a hepatospecific enhancer/promoter. LV.mPRE injection induced 30% of grade 1 HCCs in Cdkn2a-/- mice (p<0.05), thus showing that our in vivo genotoxicty assays are sensitive to SINLV insertional mutagenesis.
We exploited these mouse models to test the safety of self-inactivating (SIN) LVs developed for the therapy of hemophilia B. A SINLV that express factor IX transcript under the control of ET was injected in Cdkn2a-/- (N=39) or WT mice (N=24). The HCC incidence was monitored till 1 year after treatment. The therapeutic vector did not induce HCCs as assessed by histopathological analysis neither in tumor prone nor WT mice +CCl4. Conversely, the mice injected with matched doses of LV.ET.LTR developed HCCs at frequency similar to previous experiments. Analysis of integration is currently ongoing. This study provides encouraging results on the biosafety profile of SINLVs-based liver gene therapy.
Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy
Transplantation of autologous, genetically corrected Epidermal Stem Cells (EpSC) has been successfully used to treat junctional epidermolysis bullosa, a genetic skin adhesion disorder. Targeted transgene integration overcomes the issue of potential random insertional mutagenesis associated with retroviral vectors, and thus may provide a safer alternative. We developed a gene-targeting platform based on the use of Zinc-finger nucleases (ZFN) and integrase-defective lentiviral vectors (IDLV) to insert a transgene by homologous recombination (HR) into the AAVS1 locus on chromosome 19. We evaluated the targeting efficiency in a keratinocyte cell line (HaCaT) by IDLV-mediated delivery of an AAVS1-specific ZFN pair together with an HR construct driving the insertion of a GFP expression cassette into the site of cleavage. We achieved high efficiency of targeted insertion of single copies or concatamers of the GFP cassette into the AAVS1 locus (up to 30% of the infected cells), as analyzed by PCR, Southern blotting and sequencing on individual HaCaT clones. Evidence of HR-mediated targeted integration was also obtained at a lower but significant frequency in primary human keratinocyte cultures, where the efficiency of gene delivery by IDLVs appears to be a limiting factor. Overall, these experiments provide proof of principle for the feasibility of ZFN-mediated transgene integration in safe and pre-determined genomic locations in human keratinocytes, opening new possibilities for the treatment of genetic skin diseases. Moreover, our studies reveal the need to develop improved methods for delivering both ZFN and donor DNA to keratinocytes for high efficient gene transfer.
A Zinc Finger Nuclease specific for the human beta-globin gene
Beta-thalassemia major and severe sickle cell disease are, together, the most common monogenic diseases worldwide; both are caused by mutations in the beta-globin gene. Current treatments are blood transfusions or bone marrow transplants, but these are limited, respectively, by iron overload and donor availability. Gene therapy by gene replacement in autologous haematopoietic stem cells (HSCs) is subject to the dangers of uncontrolled vector integration and inappropriate and/or unstable expression levels. Gene correction, by homologous recombination (HR) with a delivered DNA repair template, would circumvent all these issues and is stimulated to practical frequencies by cleavage of the chromosomal target gene. The development of zinc finger nucleases (ZFNs), and other customised endonuclease technologies, has therefore raised hopes that gene correction will soon become a viable approach for the treatment of many monogenic diseases. At present, however, no β-globin gene-specific ZFN has been described. We have used public and commercial approaches to develop a β-globin specific ZFN and will describe our results. We have detected specific ZFN-mediated cleavage of the β-globin gene in two cell lines. In the absence of a delivered repair template, cleavage is indicated by the formation of small deletions/insertions due to inaccurate repair by non-homologous end-joining. Specificity is indicated by analysis of the closely related delta-globin gene which suffers no such cleavage/repair events under identical conditions. We have also demonstrated ZFN-stimulated HR that depends on the presence of the β-globin target sequence. Development of vectors for delivering the ZFN to human HSCs is in progress.
San Raffaele Telethon Institute for Gene Therapy
We developed and validated a new in-vivo genotoxicity assay able to assess the oncogenic potential of integrative vectors based on their systemic injection into newborn tumor prone Cdkn2a-/- mice. With this platform we addressed the genotoxic potential of a VSVG-pseudotyped Lentiviral Vector (LV) harboring Self-Inactivating (SIN) LTRs, the Spleen Focus Forming Virus (SF) enhancer-promoter in internal position and driving GFP expression. With respect to mock-controls, the injected mice developed significantly earlier hematopoietic tumors that were marked by genomic integrations clustered at Common Insertion Sites (CIS). Gene expression analysis from these vector-induced tumors demonstrated that the targeted CIS genes were activated by different insertional mutagenesis mechanisms including enhancer-mediated overexpression. The high sensitivity to vector genotoxicity of our platform is ideal to test and validate new vector designs harboring additional safety features. Therefore, we introduced a validated 4xIns2 insulator element into the SIN.LTR of our oncogenic vector and tested its genotoxic potential in-vivo. Our results show that Cdkn2a-/- mice injected with 2.5×10e8TU of the insulated LV display a slightly improved median survival with respect to mice injected with 1×10e8TU of its un-insulated counterpart (204 vs 189 days). Hence, insulator elements are able to slightly reduce the genotoxicity of SINLV with strong enhancer-promoter. However, this appears to be not sufficient to fully prevent the oncogenic potential of this vector in our mouse model. Integration analysis and gene expression studies will dissect if the residual genotoxicity of the insulated vector is consequent to either enhancer-mediated activation of nearby oncogenes or by different mechanisms.
INSERM UMR948, Biothérapies Hépatiques, Université de Nantes, CHU Hôtel-Dieu, Nantes, France
Delivery of AAV vectors to the newborn liver results in a rapid loss of episomal vector due to hepatocytes division. Integration of rAAV genome can lead to a sustained expression of the transgene in selected hepatocytes. The safety of in vivo gene therapy with rAAV has been questioned by a study reporting a high incidence of hepatocellular carcinoma (HCC) in mice that received an AAV injection at birth, associated with provirus integration events.
In order to reveal the tumorigenic potential of AAV transduction in the liver, groups of newborn rats received intravenous injection of either self-complementary AAV vectors encoding GFP with or without target sequences for miR142.3 (scAAVGFP, scAAVGFPmirT), or PBS, or DiethylNitrosamine (DEN), a well-known tumor-initiator as a control. All the rats were then fed on a diet containing 2-acetylaminofluorene (2-AAF), a potent liver tumor-promoting agent. After 2 months, animals were sacrificed and their liver analyzed. Preneoplastic nodules were identified by GSTp staining, and GFP expression detected by immunohistochemistry. To complete this study, vector genome integration events analysis was undertaken.
The number of GSTp positive clones was comparable in the PBS and the scAAVGFP groups and was significantly lower than in the DEN group. However, the overall GSTp positive surface was significantly bigger in the scAAVGFP compared to the scAAVGFPmirT group. A very low frequency of GFP clones in GSTp positive areas was observed in both groups. In conclusion AAV injection to newborn rat does not appear to induce an increased risk of liver tumorigenesis.
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA research center, Austrian Cluster for Tissue Regeneration, Vienna, Austria
Axonal injury close to cell bodies of motoneurons induces the death of the vast majority of affected cells. Neurotrophic factors, such as brain derived neurotrophic factor BDNF and glial cell derived neurotrophic factor (GDNF), therapeutically produced in situ in a non-regulated manner typically induce good survival of damaged motoneurons and sprouting of axons without functional reinnervation.
Therefore, we devised an ex vivo gene therapy to restrict expression of BDNF to approx. two weeks using in vitro transfected rat adipose tissue-derived stem cells (rADSCs) applied in vivo periradicularly, i.e. around the reimplanted ventral root, embedded in collagen gel.
rADSCs were transfected with plasmids for BDNF or GDNF expression using lipofection. Expression kinetics were evaluated by ELISA. An optimized transfection protocol was established and verified using FACS and cell viability assays. After strong induction of BDNF/GDNF expression in the first days after transfection, a significant decline in expression has been observed after 10–14 days at a transfection yield of approx. 25%. In vivo results suggest, that axons of injured motoneurons were able to enter the reimplanted root following reimplantation and BDNF treatment and produce morphological and functional reinnervation. The axons of surviving motoneurons showed elongative growth typical of regenerative axons, without the presence of aberrant or sprouting axonal growth.
These findings provide evidence that damaged motoneurons require limited and spatially directed amounts of BDNF to support their survival and regeneration. Moreover, neurotrophic support appears to be needed only for a critical period of time not longer than for two weeks after injury.
Assessment of the variability between human AC133+ stem cells of different donors in delivery to skeletal muscle and subsequent contribution to muscle regeneration
Duchenne muscular Dystrophy, in which the protein dystrophin is missing, is a severe muscle wasting disease leading to wheelchair dependency around the age of twelve and ultimately death around thirty years of age. To counteract the loss of muscle tissue, creating new muscle by means of stem cells is an interesting option. One of the stem cell types that has recently showed promising results are muscle derived AC133+ cells, that can contribute to muscle regeneration after intra-arterial or intra-muscular grafting in immunodeficient host mice. Using a human specific qPCR, complemented with standard microscopy techniques, we have been able to make a comparison between AC133+ stem cells derived from different human donors. We have compared their ability to home to the skeletal muscle after intra-arterial delivery into immunodeficient, dystrophin-deficient host mice and subsequently their contribution to muscle regeneration at different time points after grafting. The results give an indication of the level of variability that should be taken into account in experiments with stem cells. Further, detailed examination of the genetic expression of the cells of the different donors could reveal characteristics that are beneficial for stem cell efficiency. Since currently most stem cells do not home to skeletal muscle and contribute to regeneration after systemic injection, discovering such characteristics is urgently needed.
Impact of PTEN modulation on motor neuron survival in experimental models of motor neuron disease
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by loss of motor neurons in the brainstem, spinal cord and cerebral cortex. There is growing evidence for the role of phosphatidylinositol-3 kinase (PI3K)/Akt signalling pathway in regulating motor neuron survival. PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a key negative regulator of this pathway. Recent reports demonstrate that PTEN plays curial roles in brain development, axonal growth and neuroprotection against neuronal apoptosis. The aim of this study is to investigate the role of PTEN and its downstream signalling pathways on motor neuron survival in experimental models of ALS.
NSC34 cells carrying the SOD1G93A mutation and purified motor neuron (MN) cultures from (E13) transgenic SOD1G93A mice were transduced with lentiviral vector expressing PTEN siRNA to mediate PTEN silencing. Protein lysate was harvested at day 6 for western blot analysis. MNs were fixed at day 6 for cell counting and immunocytochemistry. PTEN silencing increased phosphorylation of Akt, and p70S6 (a downstream effecter of the mTOR pathway) and prolonged motor neuron survival in wild type and transgenic SOD1 G93A MNs. PTEN depletion in NSC34 SOD1G93A and wild type cells also activates the PI3K/Akt pathway. Lentiviral vector mediated PTEN silencing increases motor neuron survival through activation of the PI3K/Akt pathway. We are currently exploring the impact of Adeno-associated virus (AAV)-mediated PTEN modulation in ALS SOD1G93A mouse model.
SWAN Institute of Biomedical and Life Sciences, School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, UK
Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterised by ptosis, dysphagia and proximal limb weakness. Autosomal dominant OPMD is caused by a short (GCG)8–13 expansions within the first exon of the poly(A) binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres.
In the transgenic mouse model of OPMD (A17.1) a severe and progressive muscular atrophy is associated with a reduction in muscle strength from the expression of the mutated PABPN1 protein and its nuclear inclusions. The A10 transgenic mouse model expresses the ‘wild-type’ PABPN1 protein but at higher levels than normal.
Myostatin is a member of the transforming growth factor-β (TGF-β) superfamily and is produced almost exclusively in skeletal muscle tissue where it is secreted and acts as a negative regulator of muscle mass. It is naturally inhibited by its own propeptide and naturally occurring Myostatin mutants exhibit a ‘double muscling’ phenotype.
Adeno-associated virus-8 (AAV8) myostatin propeptide vector (AAV8-ProMyoFc) was systemically introduced in 8 week old and 6 month old A17.1 and A10. After 10 weeks, ProMyoFc expression was stable and a significant increase in muscle mass was seen. The use of Myostatin inhibition in gene therapeutic strategies for age-related or disease-related muscle loss is been evaluated through this model.
Dubowitz Neuromuscular Unit, UCL Institute of Child Health
Duchenne Muscular Dystrophy (DMD) is a chronic X-linked genetic disorder characterized by an early onset of muscle degeneration due to the absence of functional dystrophin protein. Damaged muscle fibres can initially be regenerated by a muscle-resident stem cell population called satellite cells (SCs), which underlie the myofibre basal lamina. Following activation, SCs proliferate extensively and fuse with the damaged fibre, however, some SCs return to quiescence to maintain the stem cell pool. As dystrophic muscle is progressively wasted, regeneration eventually fails resulting in premature death. Long-term correction of dystrophic muscle and SCs requires the insertion of a functional dystrophin gene copy into the mutated genome. Lentiviral vectors (LVs) represent suitable candidates for DMD gene therapy, as they stably integrate their genome into dividing and non-dividing cells, thereby mediating long lasting expression in both proliferating myoblasts and post-mitotic myofibres. Importantly, physiological protein levels and restriction of transgene expression to only muscle tissue is favored to avoid off-target transduction and to circumvent associated safety concerns.
This study compares LVs carrying either a strong viral, a muscle-specific or a housekeeping promoter, and assesses variances in expression levels, integrated copy numbers and persistence of transgene expression. Tissue-specificity of promoters will be discussed. Transgene expression mediated by randomly integrated viral copies did not alter the potential of SCs to differentiate and self-renew in vitro and in vivo. Notably, human myoblasts showed significantly enhanced transduction efficiencies compared to murine myoblasts, thus underpinning the great translational potential of LVs in the area of muscle degenerative diseases.
Antisense-induced myostatin exon skipping leads to muscle hypertrophy in mice following treatment with octaguanidine-conjugated oligonucleotides
Patients suffering from cancer, chronic obstructive pulmonary disease (COPMD), AIDS etc, tend to lose body weight, a condition called cachexia, responsible for increased risk of death due to the underlying condition in such patients. Degenerative muscular like muscular dystrophies could actually be fatal at an early age of late twenties. Muscle mass loss resulting from ageing and obesity also has a very high impact on public health. Myostatin is a negative regulator of muscle mass, and natural mutations in myostatin in whippets, Belgian blue bull, myostatin mull mice and even in humans have resulted in significantly increased muscle mass relative to their normal counterparts.
In this study the use of antisense oligonucleotides (AOs) of three different chemistries: 2′O-methyl RNA (2′OMePS-with a phosphorothioate backbone), phosphorodiamidate morpholino (PMO) and octa-guanidine conjugated PMO (vivo-PMO) to induce myostatin exon skipping by modulating myostatin mRNA splicing has been demonstrated. Systemic administration of vivo-PMO led to a significant increase in the mass and cross sectional area of soleus muscle of treated mice. In order to examine the longevity of PMO action in treated muscles, exon skipping and muscle mass was evaluated in mice across an 8 week period in response to a single intramuscular administration and exon skipping in the myostatin mRNA was recorded from week 1 through to week 8. In addition, a significant increase in muscle mass was observed after 4 and 8 weeks. Thus, myostatin knock-down by exon skipping exhibits a potential for therapeutic strategy to counter muscle wasting conditions.
Efficient Conversion of Embryonic Stem Cells into Myogenic Lineage Using an Inducible Gene Expression System in vivo
We established genetically engineered ES (ZHTc6-MyoD) cells that harbor a tetracycline-regulated expression vector encoding myogenic transcriptional factor MyoD, for the therapy of muscle diseases, especially Duchenne muscular dystrophy (DMD). Almost all the ZHTc6-MyoD cells were induced into muscle lineage after removal of tetracycline. The undifferentiated ZHTc6-MyoD cells are Sca-1+ and c-kit+, but CD34−, all well-known markers for mouse hematopoietic stem cells. In addition, they are able to maintain themselves in the undifferentiated state, even after one month of culture. Therefore, it is possible to obtain a large quantity of ZHTc6-MyoD cells in the undifferentiated state that maintain the potential to differentiate only into muscle lineage. Additionally, the cells were infected with lentiviral vector carrying GFP gene as maker. We injected the infected cells into vein of mdx mice, a model mouse of DMD, for systemic delivery. A few GFP positive cells were detected in the muscle of the mice. Therefore, ES cells have considerable therapeutic potential for treating muscle diseases.
SWAN Institute of Biomedical and Life Sciences, School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, UK
Repetitive allotransplantation of bone marrow stromal cells to muscular dystrophy model mice drastically improves the body build and life span
Room B26, Sheffield Institute for Translational Neuroscience, University of Sheffield, 385a Glossop Road, Sheffield S10 2HQ, UK
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons with no effective treatment to date. Although it has a multi-factorial aetiology, oxidative stress is hypothesized to be one of the key pathogenic mechanisms. It is proposed that manipulation of the expression of anti-oxidant genes may serve as a therapeutic strategy for the protection of motor neurons. It has been previously found that two specific anti-oxidant genes (Prdx3 and NRF2) are down-regulated in the presence of mutant SOD1 (mutated in approximately 5% of ALS cases). The main aim of this project is to alter the expression of one or more target genes using viral vectors and study the effect in vitro and in vivo. The lentiviral vectors expressing each of the two target genes were tested in NSC34 cells (mouse motor neuron and neuroblastoma hybrid cell line), and the oxidative stress levels as well as cell survival was measured in cells stably transfected with human SOD1 gene incorporating the ALS-causing G93A mutation. The target genes were selected for in vivo studies in the G93A mouse model. In the cellular model, stable over-expression was achieved, and they showed neuroprotective properties. Cells over-expressing Prdx3 showed a 40% decrease in oxidative stress, whereas cells over-expressing NRF2 a 60% decrease. These two genes were taken forward to the animal model in which intramuscular AAV6 injections were used as the administration route. Preliminary data from the in vivo studies suggest that there is no significant difference between treated and control groups.
Department of Neuroscience, Sheffield Institute of Translational Neuroscience, University of Sheffield, 385a Glossop Road, Sheffield, S10 2HQ United Kingdom
Ribeirão Preto School of Medicine-University of São Paulo Av Bandeirantes, 3900 Ribeirão Preto-SP, Brazil
Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder affecting the skeletal muscle compartment, which is caused by mutation(s) in the dystrophin gene. Gene and cell therapy using different approaches have been used as therapeutic strategy for DMD. In this field, the use of mesenchymal stem cells is promising, given its regenerative and anti-inflammatory effects. In this way we compared two different routes of mesenchymal stem cells administration in a murine model (mdx mice). We evaluated the migration capacity of these cells and the cytokines production in the gastrocnemius muscle and diaphragm. Using GFP+ mesenchymal stem cells, we analyzed by flow cytometry the localization of the cells in several organs seven days after the injection. The intramuscular route showed to be interesting since we found these cells in diaphragm and gastrocnemius muscle of the mdx mice and fewer cells were found in the liver when compared with the intravenous route. After mesenchymal cells intravenous injection we found diminished IL-17 and IL-6 production and more Foxp3 in diaphragm. In the gastrocnemius muscle these cytokines levels had no change but TNF-alfa production increased. When we tested the intramuscular route, we found diminished production of TNF-alfa, IL-6, IL-17 and Foxp3 both in gastrocnemius muscle and diaphragm. Taken together, these results indicate that, after the intramuscular injection, the mesenchymal stem cells can migrate for other sites and better modulate the immune response in the dystrophic muscle, when compared with the intravenous route.
Department of Physiology, Anatomy and Genetics, University of Oxford, OX1 3QX, UK
Myostatin is a secreted growth factor that acts to negatively regulate muscle mass. Myostatin null mutations result in increased musculature in cattle, mice and humans and thus myostatin blockade is being considered as a putative therapy for muscle wasting disorders such as Duchenne muscular dystrophy (DMD). Various myostatin blockade strategies have been shown to induce functional improvement in the mdx mouse model of DMD. Similarly, the combination of myostatin blockade with dystrophin splice correction has results in greater functional improvement than either strategy alone. Transcriptional Gene Silencing (TGS) can be induced by small RNA molecules complementary to target gene promoter sequences. TGS effectors target chromatin modifying activities to low copy number transpromoter RNAs which induce silent state chromatin structure and, in some cases, promoter DNA methylation. Consequently, TGS may be permanent and heritable.
In order to investigate the possibility of silencing myostatin by TGS we designed small interfering RNAs (siRNAs) targeting the myostatin promoter. These were transfected in differentiated C2C12 mouse myotubes. Knock-down was observed in multiple transfection experiments, and with both conventional and nuclear-targeting transfection reagents. The results were replicated in the disease relevant H2K-mdx cell line indicating that the effect is not unique to C2C12 myoblasts Silencing was sensitive to the histone deactelyase inhibitor Trichostatin A suggesting that epigenetic remodelling at the myostatin promoter is responsible for the observed effect. Similarly, enrichment of the silent state histone mark H3K9me2 was observed at the myostatin promoter following treatment with promoter-targeting siRNA. We also report progress towards achieving myostatin transcriptional silencing in vivo.
School of Clinical Sciences, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol, BS1 3NY, United Kingdom
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that results in a progressive loss of motor neurons in the central nervous system. ALS patients suffer from symptoms of muscle weakness and slurred speech, as motor neurons in the spinal cord and brain stem gradually deteriorate. Typically, the debilitating condition causes paralysis and death within 3–5 years of diagnosis. A known cause of familial ALS is a toxic gain of function mutation in superoxide dismutase (SOD1). Transgenic mice expressing a mutated form of human SOD1 (SOD1G93A) experience age-dependent motor neuron degeneration comparable to humans.
Here we show that efficient transduction of the ependyma cells in the ventricles of mice can be achieved following a single intracerebroventricular (ICV) injection of a GFP-expressing lentiviral vector based on EIAV. In addition vectors encoding either the human or mouse genes of the neuroprotective protein, vascular endothelial growth factor (VEGF-A), have been generated. Transduction of ependymal cells with these vectors will support the long term production and release of the VEGF-A protein into the CSF. The circulating VEGF will be able to access the motor neurons of the brain and spinal cord via the perivascular spaces in the parenchyma. We have performed an efficacy study to determine the therapeutic benefits of ICV delivery of EIAV vectors expressing the VEGF-A gene to SOD1G93A mice. VEGF-A mediated improvement in motor performance using the rotarod test and extension of survival has been assessed.
UCL Cancer Institute, University College London, London WC1E 6BT, UK
Synthetic antisense oligoribonucleotides (AONs) can be used to modulate gene splicing by masking motifs on the pre-mRNA required for spliceosome assembly. Transfected AONs display a transient activity whereas stable expression is obtained using chimeric small RNAs containing the antisense sequences, which are delivered by gene transfer vectors. Here we try to maximize the amount of active antisense sequences delivered by creating new and tissue-specific expression cassettes for these small RNA shuttles.
In the murine model of Duchenne Muscular Dystrophy a chimeric U7 snRNA (U7Dtex23) induces skipping the mutated exon 23 and restores the Dystrophin mRNA reading frame. To optimize this system we used the muscle-specific enhancer MHCK7 to drive the expression of the U7Dtex23 cassette. AAV2/5 vectors were tested in myoblast cultures and our results show that the enhancer improves chimeric U7snRNA expression in myotubes and increases exon 23 skipping.
We have also explored the possibility of using small nucleolar (sno) RNAs as new shuttles for AONs. The MBII-52 snoRNA regulates the 5-HT2C mRNA alternative splicing through its 18nt antisense, suggesting that pre-mRNA processing can be modulated with snoRNAs. We have replaced the original antisense of MBII-52 with the Dtex23 sequence and observed skipping of exon 23. While our observation validates the approach, the efficiency of skipping is still considerably lower than with the U7snRNA cassette and requires further improvements.
Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College
MicroRNAs (miRNAs) are thought to play important roles in the pathogenesis of autoimmune diseases. MiRNA-223 (miR-223) can promote osteoclast differentiation and its expression is upregulated in rheumatoid arthritis (RA) patients. Here we tested the feasibility of using lentiviral vectors expressing the miR-223 target sequence (MiR-223T) to suppress miR-223 activity as a therapeutic strategy in a mouse model of collagen-induced arthritis (CIA). Levels of miR-223 expression in the synovial tissue of RA and osteoarthritis (OA) patients as well as ankle joints of CIA mice were determined by real-time PCR. Lentiviral vectors expressing miR-223T (LVmiR-223T) or luciferase shRNA (LVshLuc) serving as a control vector were injected intraperitoneally into CIA mice. The treatment responses and disease-related bone mineral density were monitored. Expression of NFI-A, a direct target of miR-223, was detected by immunohistochemistry. Osteoclasts were assessed by TRAP staining. The levels of miR-223 expression were significantly increased in the synovium of RA patients and the ankle joints of CIA mice as compared with OA patients and normal mice, respectively. LVmiR-223T treatment significantly reduced the arthritis score, histologic score, miR-223 expression, osteoclastogenesis and bone erosion in CIA mice, as compared with LVshLuc treatment. Increased NFI-A expression was detected in the synovial tissue from LVmiR-223T-treated mice. This report is the first to demonstrate the amelioration of clinical symptoms of CIA mice by a lentiviral vector carrying the miR-223 target sequence. Furthermore, our results implicate that suppression of miR-223 activity may be further explored as a therapeutic strategy for patients with RA.
Expression kinetics of non-invasive intramuscular sonoporative gene transfer in vivo
Non-viral ultrasound mediated gene therapy is a minimally invasive method for transient gene expression in vivo for broad ranging applications.
In order to define the kinetics of transient transgene expression in the muscle, the aim of this study was to optimize sonoporation using different doses of DNA and to compare expression patterns to naked intramuscular DNA transfer. Expression was quantified using non-invasive bioluminescence imaging employing a luciferase plasmid in mouse hindlimb muscles.
Plasmids were intramuscularly injected and transcutaneously sonoporated. Transfection efficacy and expression kinetics using different amounts of plasmid were investigated. Sonoporation without microbubble reagent or simple naked DNA injection served as controls. Bioluminescence signals were determined to obtain optimized DNA amounts at different time points for up to 28 days. Anti-luciferase immunohistology stainings were carried out to investigate tissue distribution of the expressed transgene.
Sonoporation with different doses showed similar results, indicating that there is no dose dependency in the applied range. However, differences were obtained comparing sonoporation versus controls. We were able to obtain characteristic expression kinetics for both, naked DNA transfer (low expression within the first 3 days) and sonoporation (high expression for up to 28 days with a peak at day 7), which enables to distinguish the contribution of naked DNA transfer to observed expression levels/kinetics from DNA transfer mediated exclusively by sonoporation.
We conclude from our in vivo findings, that intramuscular sonoporative gene transfer induces higher and more sustained transgene expression than simple naked DNA transfer and that successful sonoporation follows a distinct expression pattern.
Investigation of systemic siRNA biodistribution using near-infrared imaging of PEI-siRNA complexes
RNA interference (RNAi) has great therapeutic potential for treatment of numerous conditions. Treatment strategies that rely on downregulation of potentially ubiquitously expressed targets require reliable delivery and transfection protocols for successful systemic knockdown.
For successful therapies it is vital to obtain information on the spatio-temporal distribution of siRNA after systemic delivery. Therefore, it was the aim of this study to follow polyethyleneimine (PEI) siRNA biodistribution in mice after tail vein injection using near-infrared imaging.
Female, CD-1 mice received 80μg of Alexa Fluor 680 (AF680)-labelled or unlabelled scrambled (SCR) siRNA complexed to PEI and were sacrificed 6h later for blood and organ sampling. Unstained paraffin sections were made and analyzed on a Licor Odyssey Imaging station and by confocal laser scanning microscopy (CLSM). Furthermore, blood parameters were analyzed to investigate potential PEI toxicity.
Imaging revealed increased accumulation of AF680 labelled siRNAs in liver, lung, spleen, and kidney but not in brain samples. CLSM confirmed this observation and enabled to image cellular localization of siRNA-PEI complexes. Imaging at the chosen wavelength has been shown superior to other fluorophores because of almost absent tissue autofluorescence at 680nm. Blood-parameters as well as microscopic evaluation indicate liver toxicity at the employed dose of 80μg and the N/P-ratio of 10.
We conclude from our in vivo findings, that siRNA delivery has been achieved for certain organs using tail vein injection of PEI complexed siRNAs. Furthermore this study again confirms that liver toxicity might be an inherent drawback of cationic polymers such as PEI used for delivery.
Department of Gene and Cellular Theraphy/GENYO, Fundación Progreso y Salud, Granada, 18007, Spain
Biological Sciences, Royal Holloway University of London, United Kingdom
Development, evaluation and optimisation of HIV-1/SIV genetic vaccine components to generate multiple T cell CTL epitopes, is currently one of the primary strategies in HIV-1/SIV vaccination protocols.We have identified sugar mixtures that give optimal preservation of infectivity whilst maintaining the strength and rigidity of a micro-needle array. Vectors retained substantial infectivity (i.e.>55% eGFP+ cells) for up to 3 months through a temperature range (−20°C to +40°C). Transcutaneous injections of desiccated Ad-OVA-GFP vectors (4×10E9 vg) in C57BL/6 mice and footpad-mediated migration of Ad5-CMV-eGFP to lymph nodes were successfully performed. We originally aimed to induce immunity to HIV-1 infection by developing a cohort of adenoviral [human (Ad5/Ad11)/simian (Ad30/37)] vectors in which HIV-1 genetic components (gag/pol/env/nef) have been genetically engineered to stimulate broader CTL responses. As an example, full size HIVCN54 or SIVmac251 gag genes including an across clade conserved epitope were engineered, fused to mono-(±N-end rule) or tetra-ubiquitin (Ub) sequences and further tested on DC and non-DC cell lines by using plasmid, in vitro synthesised mRNA, or human adenoviral vectors. We subsequently constructed a cohort (n=34) of rAd vectors carrying genetically fragmented ubiquitinated (4, 1, 0 Ub) fusions for either HIVCN54 (n=10) or SIVmac251 (n=7) gag genes to reduce antigenic competition and/or alter epitope dominance. We are currently extending our studies using an in vitro CTL epitope-mapping system employing human monocyte-derived DCs from healthy and HIV-1+ individuals alongside mouse in vivo studies impacting the antigen ubiquitination and genetic fragmentation on the quality of the generated immune responses. SIV constructs are currently forward tested in non-human primate (NHP) models (cynomolgus macaques).
Antigen-Inducible Self-Amplifying Therapeutic B cells Producing Anti-HIV Monoclonal Antibodies
For many disease indications a neutralising antibody response would provide significant therapeutic benefit. However, for conditions such as Human Immuno-deficiency virus (HIV) infections, the humoral immune response fails to effectively control the pathogen. In order to correct this failing, we have designed non-replicating Epstein Barr virus amplicon vectors that express the genes encoding the broadly neutralising anti-HIV antibody, 2F5. We show that these vectors can be used to express significant quantities of anti-HIV antibodies and that the addition of the endogenous plasma membrane tag for IgG1 can sequester the antibody to the cell surface to act as a B cell receptor mimic. We anticipate that this alteration could allow antigen specific B cell expansion in vivo, thereby amplifying the therapeutic construct with B cell division. We are currently investigating if these vectors are faithfully maintained during extended B cell division and confirming the anti-viral activity of the antibody produced.
The genetic modification of B cells to express new therapeutic antibodies could represent a new paradigm for genetic vaccination. We propose that this approach could be used for many disease conditions and circumvents the problem of poor gene delivery by allowing gene amplification within the host following targeted vaccination post gene transfer.
Molecular Immunology Unit, Institute of Child Health, University College London, London, WC1N 1EH
Alternative therapies against HIV based on lentiviral mediated transfer of anti-HIV elements offer the possibility of long term protection against the virus. Certain non-human primate species are protected against HIV by naturally occurring anti-HIV factors such as TRIM5α-Cyclophilin A (TRIM5CypA) fusion proteins. These innate antiviral factors have arisen on at least two separate occasions following retrotransposition of CypA cDNA into the TRIM5 gene during primate evolution and are potent inhibitors of lentivirus without any evidence of resistance. We have investigated using human TRIMCypA variants in a gene therapy approach against HIV-1 by generating lentiviral vectors to express these factors in susceptible cell populations. We found that human TRIM5CypA mediates robust protection against HIV-1, and confers a survival advantage to T cells in the presence of replication competent virus. However, we found that TRIM5CypA expression interferes with endogenous TRIM5α activity, and renders cells permissive to infection by murine retrovirus. Interestingly, substitution of TRIM5 with the corresponding RING. B-Box and coiled coil domains of another TRIM protein, human TRIM21, produces a TRIMCypA variant protein which mediates equally effective restriction of HIV-1, without impacting on endogenous TRIM5α antiviral activity. Furthermore, endogenous TRIM21 function, quantified by restriction of secondary Adenovirus infection, was not compromised in cells modified to express either TRIM5CypA or TRIM21CypA. In conclusion, lentiviral vectors expressing TRIMCypA proteins could form the basis of an intracellular vaccination against strategy against HIV-1, and TRIM21CypA offers highly effective protection without disruption of endogenous TRIM activity.
San Raffaele Scientific Institute, Milan
T cell engineering against tumor antigens aims at ameliorating current immunotherapeutic approaches. To date, however, the suboptimal persistence of the transferred cells represents a serious limitation of this approach. The most appropriate T cell subsets to be infused should ensure optimal in vivo persistence and yet appropriate anti-tumor activity. Here we report that culturing highly purified naïve T (TN) cells with anti-CD3 and anti-CD28 beads, allows the retrieval of a novel post-mitotic CD45RA+CD62L+CCR7+ T cell population, which requires IL-7 and IL-15 for expansion and maintenance. This population is highly proliferative and sensitive to RV and LV transduction, expresses low levels of IFNg and cytotoxic molecules and is best defined as IL-7Ra+CXCR4+c-kit+ CCR5-HLADR-PD1-. The gene expression signature is typical of antigen-experienced lymphocytes and classifies these cells between TN and central memory (TCM) lymphocytes. Because of this, we refer to it as precursor to TCM (TpreCM). We identified and functionally validated the natural counterpart of this population in healthy donors as a rare fraction of CD45RA+CD62L+ cells expressing CD95 and CD45ROdim. When infused in immunodeficient mice, genetically manipulated and in vitro generated TpreCM proved superior engraftment and longer persistence than transduced TCM cells, and xenoreactivity comparable to that of unmanipulated lymphocytes. TpreCM, but not engineered TCM, maintained engraftment and xenoreactivity in serial transplantation experiments, indicating unique self-renewal abilities. We believe that this novel T cell subset, traceable in nature, and generated in vitro in selected culture conditions from TN cells, might recapitulate protective immunity in vivo and improve cancer adoptive immunotherapy.
Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, 30625 Germany
Integration deficient lentiviral vectors (IDLV) have been shown to be effective vaccines in mouse models, but studies in humanized systems are lacking. We generated integration deficient bicistronic lentiviral vectors expressing combinations of cytokines capable to induce self-differentiated dendritic cells for stimulation of multivalent immune responses. Overnight transduction of human monocytes with high titer IDLV was followed by in vitro and in vivo analyses. Highly viable and immunophenotypically stable DCs generated with IDLV-GM-CSF/IL-4 were called “SmartDCs”, whereas DCs induced with IDLV-GM-CSF/IFNα were called “SmyleDCs”. In vitro cultured of both DCs demonstrated stable secretion of transgenic and several endogenously produced inflammatory cytokines (IL-8,TNF-a,MCP) and potent stimulation of T cell proliferation in allogeneic and autologous mixed lymphocyte reactions. SmyleDCs promoted stimulation of NK cells in vitro. Expansion of multi-antigenic pp65-reactive effector and memory CTLs were obtained after in vitro stimulation with SmartDcs and SmyleDCs pulsed with a CMV-pp65 peptide pool or co-transduced with LV-pp65 as evaluated by tetramer analyses and IFN-γ ELISPOT. In order to evaluate the effects of SmartDCs and SmyleDCs in the expansion of anti-pp65 CTLs in vivo, NOD.Rag1−/−.IL2rγ−/− (NRG) mice were pre-immunized with DCs co-expressing pp65, and 7 days later, mice were infused with autologous CD8+ T cells from CMV seropositive donors. Both types of ID-LV-induced DCs promoted in the expansion of multi-antigenic pp65 reactive CTLs in peripheral blood and spleen. Due to their potential capacity to stimulate stronger Th1-immune responses and multivalent responses, SmyleDCs are promising candidates for prophylaxis or immunotherapy of chronic CMV infection in the transplantation setting.
Dep. Biochemistry and Immunology – College of Medicine of Ribeirão Preto – University of São Paulo, Ribeirão Preto, SP, Brazil, 14049-900
Paracoccidioides brasiliensis is a dimorphic fungus that causes paracoccidioidomycosis (PCM), a chronic, granulomatous and progressive disease, which is associated with various degrees of suppressed cell-mediated immunity. Preliminary data from our group shown that P. brasiliensis has a fetuin-binding protein fraction (FBP) that induces a worsening of the infection. Initially, this study aims to purify and identify which protein was responsible for immunosuppressive activity. Three proteins, with MW of 60, 58 and 50 kDa, were purified and submitted to sequencing by mass spectrometry and all were identified as heat shock protein of 60 kDa from strain 18 of P. brasiliensis (Hsp60_Pb18). Through the predicted protein sequence obtained from GenBank was possible to perform molecular modeling of protein Hsp60_Pb18 based in 1WE3A (PDB). These results suggest that the protein Hsp60_Pb18 can be responsible for immunosuppressive activity in experimental PCM. Because the immunosuppressive property of Hsp60, it becomes a vaccine target. Therefore gene subcloning in vaccine vectors is undertaken to develop DNA vaccine and therapy to PCM.
Financial Support: FAPESP (09/14777-1 and 09/03235-3)
Inserm, U 951, Molecular Immunology and Innovative Biotherapies, Genethon, Evry, France
The possibility of targeting gene transfer to MHC-II+-antigen-presenting cells (APC) in vivo provides an opportunity to specifically immunize and to study the effects of various types of APC in gene-specific cellular immune responses. We herein describe and validate a model for such studies. A novel immunogenic transgene was generated by fusing GFP to antigenic epitopes from the murine HY male gene. Cells expressing GFP-HY are detectable by FACS with reduced (about a log) fluorescence levels compared to native GFP. To target APCs for gene transfer in vivo, we used lentiviral vectors pseudotyped with engineered measles envelope glycoproteins targeting MHC class II with a ScFV (Ageichik et al., 2011). The vector is produced by co-transfecting 5 plasmids in 293T cells and concentrated by ultracentrifugation. Under optimal conditions (2500g, 24h, 4°C), concentrated viral stock containing about 2000 ng RT/ml; 3×1010 viral RNA/ml is produced i.e. increasing viral RNA titer 170 fold, from the harvested supernatant. In vitro, the vector specifically transduced MHC class II+ spleen cells following infection of a whole spleen cell population. In vivo, intravenous injection of concentrated vector (10 to 25 ng of RT per female mouse) induced male-specific cellular Th1 immune response. Fourteen days after injection, peptide re-stimulated spleen cells generated 150–400 IFNγ SFU/millions lymphocytes whereas PBS and Vector-Like Particules (lacking envelope glycoproteins) injected animals did not respond. These results provide an initial validation of this new experimental model. Further experiments are ongoing to challenge immunized mice and to target various types of APCs.
Biotechnology and Genetic Engineering Group, Department of Molecular Diagnostics and Epidemiology, Central Research Institute of Epidemiology, Moscow, 111123, Russia
Department of Nursing, Chung Hwa University of Medical Technology, Tainan Hsien, Taiwan
Pro-opiomelanocortin (POMC) is a precursor of various neuropeptides. POMC-derived neuropeptides are potent inflammation inhibitors and immunosuppressants. Evidence that osteoarthritis (OA) is an inflammatory disease is accumulating. We assessed whether intraarticular gene delivery of POMC ameliorates experimentally induced OA in a rat model. OA was induced in Wistar rats by anterior cruciate ligament-transection (ACLT) in the knee of one hind limb. Adenoviral vector encoding human POMC (AdPOMC) was injected intraarticularly into the knee joints after ACLT. The transgene expression and the inflammatory responses were evaluated using immunoblotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). The treated joints were assessed histologically for manifestations of the disease. Human POMC was expressed in the chondrocytes and synovial membrane after the intraarticular injection. POMC gene transfer reduced nuclear factor-kappa B activity and the levels of interleukin-1 beta in HTB-94 chondrosarcoma cells and Raw 264.7 macrophages; it also reduced microvessel density in the synovium. Histological examination showed that symptoms of OA in AdPOMC-treated rats were less severe than in rats treated with either empty adenoviral vector (AdNull) or normal saline. Intraarticular injection of adenoviral vectors expressing POMC significantly suppressed the progression and severity of OA, and reduced inflammatory responses and angiogenesis. POMC gene delivery may offer novel therapeutic approach for treating OA.
Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK
Toll-like receptor ligands are potent immune response modifiers that can enhance both the quality and magnitude of immune responses when used as adjuvants in vaccine formulations. The adoption of many promising toll-like receptor ligands has been limited by systemic toxicity and poor efficacy when formulated with protein and peptide antigens. These shortcomings are largely due to the failure of current formulation strategies to address the need for toll-like receptor ligands and antigens to be co-delivered to phagosomal compartments of antigen presenting cells. To overcome these limitations, we have developed chemical strategies to conjugate multiple toll-like receptor-2, or −7/8 agonists to protein and peptide antigens through polymer linkers, thereby permitting co-delivery of antigen and toll-like receptor ligands. We have recently completed the synthesis of a first-generation combinatorial chemical library of TLR-2 and −7/8 agonist-polymer conjugates and have screened these materials for biological activity in vitro using human reporter and primary blood cells. Preliminary results have revealed how chemical properties (spacer length, ligand density, etc.) influence the magnitude and profile of the innate immune responses elicited. The synthetic schemes and results of our biological screens will be presented.
Efficient lentiviral transduction and transgene expression in activated primary human B cells
Primary human B cells are an attractive target for gene-therapeutic applications. Up to now B cells have been found to be relatively resistant towards transduction with lentiviral vectors (LVV), even though a number of different envelope pseudotypes were tested. Moreover, low transgene expression in primary human B cells has impeded the use of LVV for this target cell.
We investigated the transduction potential of gibbon-ape-leukaemia-virus (GALV)-env pseudotyped LVV for primary human B cells CD40L-activated in vitro. Using optimised conditions (including transduction kinetics and multiplicities-of-infection) we were able to regularly obtain transduction efficiencies of more than 65%. Noteworthy, with the utilisation of GALV-pseudotyped LVV we could achieve an up to tenfold higher yield of transduced activated B cells in direct comparison to measles-GP-pseudotyped LVV. Besides, phenotyping of transduced primary B cells revealed a majority of memory B cells, a long-lived phenotype, presumed to be well suited for enduring therapeutic interventions.
Finally we show that combining the enhancer (Eμ) and the matrix/scaffold-attachment regions (MAR) of the human immunoglobulin heavy-chain with the promoter of spleen focus forming virus (SFFV) facilitates significantly higher transgene expression compared to the SFFV-promoter alone in various B-cell lines. Indeed, in all primary human B-cell samples tested transgene expression was strongly increased (mean factor of three). In summary, we have established a novel protocol for the efficient lentiviral B cells transduction and introduced vector modification to ensure better transgene expression.
Different MicroRNA array expression in Dendritic Cells of mice with prokaryotic DNA or mRNA stimuli
The microRNAs have been identified as important regulators of gene expression like suppress the expression of specific target genes at posttranscriptional level. The mechanism is involved in a fine control of physiological responses, including cell development and homeostasis. In the immune response are able to induce differentiation and cell activation, and signaling pathways acting by transcription factors. In the immune response the gene therapy is a very important tool for induced treatment of several diseases. Dendritic cells (DC) are powerfully activated by a prokaryotic DNA and mRNA molecules, but the regulation of miRNA in the cells are unknown yet. In this way, we decided to compare the stimulus between the mRNA and DNA molecules to identify different miRNAs induced by these stimuli.
For this approach, we used DC from spleen of Balb/c mice. They are incubated for 30 minutes with doses of 10mg to both molecules: mRNAhsp65 or pcDNA3Ahsp65. The miRNA were obtained after the stimulus and a pool of each stimulus were performed. The pools were used in triplicate in microarray analysis using the Agilent slides and the results get using the GeneSpring and TargetScan programs. After bioinformatics analysis we observed that the treatment with mRNA or DNA showed two principles microRNA regulation. The miRNA mmu-miR-92b was down expressed after stimulus with DNA compared with RNA, and the mmu-miR-139-3p was up regulated, after stimulus. These results showed that each molecules can induces alterations in the expression of different miRNAs, and are important to improved the knowledge in gene therapy.
Dep. Biochemistry and Immunology. School of Medicine of Ribeirao Preto, University of Sao Paulo. Av. Bandeirantes. 3900. 14049-900, Ribeirao Preto, SP. Brazil
Paracoccidioides brasiliensis is a dimorphic fungus, which is the causative agent of paracoccidioidomycosis (PCM). PCM is a chronic and progressive disease that mainly attacks lungs and mucosa of the mouth and nose. Often PCM treatment lasts for up to 2 years and patients experience toxic drug side effects and relapses. These problems have motivated us to develop strategies to improve the host immune reactions. Here, we investigated the protection of BALB/c mice against P. brasiliensis infection through transfer of murine bone marrow dendritic cells, which were electroporated with total RNA of P. brasiliensis, into the tail veins. The animals were infected with P. brasiliensis either 30 days before (therapy) or 30 days after (prophylaxis) cell transfer. The therapeutic treatment led to an increase in the pulmonary fungal burden, mainly because of hyperactivation of the immune system, determined through the dosage of cytokines. In contrast, when the dendritic cells were used as a vaccine, there was a significant decrease in the pulmonary fungal burden. Taken together these data indicate that our strategy is good to prime the immune system when used prophylactically but not as a therapeutic agent.
Supported by FAPESP (Procs. No. 2009/00087-3 and 2009/14777-1).
Cancer Gene Therapy Group, Molecular Cancer Biology Program, University of Helsinki, Helsinki, P. O. Box 63, 00014, Finland
Induction of an antitumor immune response is usually not sufficient to eradicate cancer because of the immune suppressive nature of tumor. A promising approach to reduce the tumor immune evasion mechanisms is the blockade of cytotoxic T lymphocyte antigen 4 (CTLA-4). CTLA-4 is an inhibitory receptor for the costimulatory molecules B7.1 (CD80) and B7.2 (CD86), and functions as a negative regulator of T-cell activation.
The use of monoclonal antibodies (mAbs) such as ipilimumab and tremelimumab to block CTLA-4 has shown promising clinical results. Systemic administration of immunostimulatory agents may cause severe immune-related side effects, however. Thus, local production of anti-CTLA-4 would be desirable.
We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-delta24-aCTLA4 coding for anti-CTLA-4. We tested Ad5/3-delta24-aCTLA4 in vitro, in vivo and in peripheral blood mononuclear cells (PBMC) of normal donors and patients with advanced solid tumors. Anti-CTLA-4 mAb produced by Ad5/3-delta24-aCTLA4 was biologically functional and caused T-cell activation in PBMCs of patients, but not with healthy donors. Further, we saw a direct anti-CTLA-4 mediated pro-apoptotic effect in cancer cells. The replicating virus produced locally 43-fold higher (p<0.05) anti-CTLA-4 mAb concentration in tumor versus plasma. Plasma levels in mice remained below what has been reported safe in humans. The replication competent Ad5/3-delta24-aCTLA4 produced 81-fold higher (p<0.05) tumor mAb levels than its replication deficient control Ad5/3-aCTLA4.
Genetically modified T-cells for prostate cancer
Prostate cancer is one of the most frequent cancer types in the Western world. Initial treatment is surgery (prostatectomy) or irradiation, followed by hormonal treatment, but there are no good available treatments for advanced hormone refractory cancer. Adoptive T-cell transfer is an emerging promising new therapy for cancer. T-cells specific for a tumor associated antigen can be isolated from the tumors, expanded and given back to the patient. TARP is a small protein specifically expressed in the prostate, and several epitopes have been predicted to be immunogenic. We were able to raise specific T-cells against HLA-A2-restricted TARP4–13 epitope, which was assessed by specific dextramer binding in flow cytometry. The T-cells produced high amount of IFN gamma in response to stimulation with the relevant peptide. We sorted the specific T-cell population and generated single T-cell clones by limiting dilution. The growing clones secreted IFN gamma and were able to bind dextramers. The TCR alpha and beta sequences were isolated and cloned into a lentiviral vector. To ensure equimolar expression of TCR alpha and beta chain, a T2A self-cleaving peptide sequence was introduced in between them. To improve pairing of the introduced alpha and beta chain, the constant domain from the murine T-cell receptor was used to replace the human constant domain. The lentivirally transduced T-cells bound to the dextramer and produced IFN gamma in response to peptide pulsed target cells or HLA-A2 positive cell lines in an antigen-specific manner.
Adenoviral Transduction of Mesenchymal Stem Cells: In Vitro Responses and In Vivo Immune Responses after Cell Transplantation
Investigating the T Cell Receptor Repertoire at the Molecular Level
T cell receptor (TCR) is a heterodimeric glycoprotein composed of α-β (or γ-Δ) chains. The antigen recognition is determined by the hypervariable complementarity determining region 3 (CDR3) that originates by the somatic rearrangement of noncontiguous variable (V), diversity (D, for β and Δ chains), joining (J) and constant (C) gene segments and the insertion/deletion of non-template nucleotides at the V-D-J junction. We have adopted a LAM-PCR approach combined with deep sequencing for the comprehensive characterization of TCR clonality. We have processed peripheral blood cells from 2 different healthy donors and, with only 48000 (Donor 1) and 49000 sequences (Donor 2), respectively, we have detected the majority of V, D, J and C genes of the beta chain. More in detail, we have identified 239 (Donor 1) and 185 (Donor 2) different V-J pairings. Furthermore, from the deep analysis of the CDR3 region we have characterized 7022 and 5373 clonotypes at the nucleotide and amino acid level respectively for Donor 1, and 5967 and 4398 clonotypes at the nucleotide and amino acid level respectively for Donor 2. Our results show the occurrence of the convergent recombination, i.e. different nucleotide sequences coding for the same amino acid sequence. In addition, we have detected 57 nucleotide sequences shared between the 2 Donors (“public clones”). Sequencing of the alpha chain rearrangements as well as mouse TCRs is ongoing. These results demonstrate the feasibility of our technology to monitor the dynamics over time of immunogenetic reactions in vivo.
Co-expression of 4-1BBL+IL-12 by carcinoma cells induces strong proliferation of functional human NK cells, and establishment of ‘memory’: potential for cancer immuno-gene therapy
4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells can also express 4-1BB, and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however the response of human NK cells to 4-1BB ligand (4-1BBL) is not clearly defined. We show human NK cells proliferate in response to OVCAR-3 cells expressing 4-1BBL or IL-12, while the combination 4-1BBL+IL-12 is superior for activation, proliferation and function of NK cells from healthy donors and patients with ovarian or renal cell carcinoma. Up to 1000-fold increase in NK cell numbers could be obtained in 21 days, with repeated 4-1BBL+IL-12 stimulation. The expanded cells secreted IFN-g and were active in cytotoxicity assays. While about 90% of NK cells in blood are CD56dimCD16+, the expanded NK cells are predominantly CD56brightCD16-; we show that isolated CD56dimCD16+ NK cells can switch to a CD56brightCD16- phenotype in response to 4-1BBL+IL-12. Resting NK cells do not express 4-1BB; its upregulation required multiple stimuli — either target OVCAR-3 cells plus IL-12, or 4-1BBL-expressing targets plus additional “help” from other lymphocytes. Following primary stimulation with OVCAR-3 cells expressing 4-1BBL+IL-12 and subsequent resting, NK cell cultures subsequently demonstrated enhanced secondary response to targets. Expansion and activation of NK cells by 4-1BBL+IL12 has significant potential for in vivo or ex vivo immuno-gene therapy of cancer.
Comparative study of DNA-based immunization vectors against botulinum, anthrax and smallpox
Botulinum, anthrax and smallpox have been considered the most probable bioweapon-induced disease. Based on the hypothesis that immune outcome can be influenced by the form of antigen and its efficient targeting to antigen-presenting cells, in the current study, we evaluated the efficiency of genetic vaccination with botulinum, anthrax, and smallpox. The eukaryotic expression vectors were designed with HCR, D4, and L1R which contains codon-optimized genes. The plasmid IgM-D4, IgM-HCR, and IgM-L1R, encoding the target protein, have IgM sequence for secretion of the expressed protein and SV40 enhancer for over expression of target proteins. We transfected 293T cell with constructed plasmids and performed Western blotting and ELISA assay. Transfection studies indicated that HCR, D4 and L1R proteins were produced. The antibody against HCR, D4 and L1R were produced in Balb/c mice vaccinated with 200 μg IgM-HCR, IgM-D4, IgM-L1R via intramuscular routes. These findings suggest that these HCR, D4 and L1R encoding plasmids-vectored vaccines may be suitable as candidate vaccines and we suggest alternative strategy to conventional protein vaccines against botulinum, anthrax and smallpox.
Department of Immunology, University College London Hampstead Campus, Royal Free Hospital, Rowland Hill Street, London NW3 2PF, U.K.
Alloreactive immune responses directed against malignant cells in recipients of allogeneic haematopoietic stem cell transplants are able to cure patients with haematological cancers. However, such immune responses may cause severe morbidity when directed against healthy recipient tissue, resulting in graft-versus-host disease (GvHD).
Naturally occurring regulatory T cells are CD4+ T cells characterised by their expression of the transcription factor Foxp3. Whilst adoptively transferred polyclonal Tregs suppress GvHD in several murine models, their lack of specificity may compromise beneficial immunity against malignancy or infection.
We have generated antigen-specific ‘converted’ Tregs through retroviral transfer of genes encoding Foxp3 and a specific T cell receptor (TCR) into conventional CD4+ T cells. We used the 2C-TCR, which recognises the MHC class I molecule H2-Ld, expressed in Balb/c and other H-2d mice, in complex with the ubiquitously expressed peptide p2Ca.
Consistent with the acquisition of regulatory function in vitro, Foxp3 2C-TCR-transduced B6 CD4+ T cells are hyporesponsive to stimulation and are able to suppress the alloreactive proliferative response of B6 CD4+ and CD8+ T cells to Balb/c splenocytes.
When given to lethally irradiated Balb/c recipients of MHC-mismatched B6 bone marrow, CD4+ and CD8+ T cells, Foxp3 2C-TCR transduced B6 CD4+ T cells reduce early proliferation of donor T cells, weight loss and GvHD score in the recipients.
Our preliminary data have shown that Foxp3 2C-TCR-transduced CD4+ T cells can suppress allogeneic responses in vitro and in vivo. Confirmation of antigen specificity would strongly support the use of class I-restricted TCRs alongside Foxp3 in CD4+ T cells to direct regulatory activity, with important implications for translation into clinical practice.
Generation of MHC-I restricted CD4+ CMV and EBV specific T cells for the management of leukaemia patients
EBV and CMV reactivation after allogeneic stem cell transplantation remains a significant cause of mortality and morbidity. Adoptive transfer of donor-derived virus-specific CD8+ CTLs has been successfully used in controlling of reactivation and viral infection. But this approach has two major limitations. Firstly, when the donor is sero-negative, it is very difficult to generate virus specific T cells; and secondly, without CD4 help, these CD8+ CTLs are often less effective. As most tumours do not express MHC class II molecules, provision of conventional CD4 help in a tumour immunotherapy setting is challenging.
In this study, we report the generation of CMV and EBV specific CD8+ CTLs and CD4+ Th cells via TCR gene transfer. TCR engineered both CD8+ CTLs and CD4+ Th cells produced cytokines, and displayed antigen-specific killing activity towards tumour cells endogenously expressing the target antigens. In vivo, we showed that both CMV and EBV TCR engineered CD4+ Th cells can reject tumors. When a CD8 molecule together with these TCRs was introduced into CD4+ T cells, the antigen-specific functionality of the TCR modified CD4+ T cells was improved dramatically, they can produce more INFg than the same TCR engineered CD8+ CTLs, and rejected tumor in vivo as effective as the TCR engineered CD8+ CTLs. Therefore, the results described here will open a new way of generating antigen-specific CD4+ Th cells for adoptive immunotherapy of virally associated malignancies in the immunocompromised bone marrow transplant patients. This will also have implications for immunotherapy of other forms of cancers.
IBET/ITQB-UNL, Apartado 12, 2781-901 Oeiras, Portugal
Process development for production of pharmaceutical grade plasmid DNA to be used in gene therapy and DNA vaccination
The number of plasmids used in clinical trials dealing with gene therapy or DNA vaccination is increasing. Therefore the demand for pharmaceutical grade plasmid DNA is growing continuously. To be able to provide sufficient material a manufacturing platform for GMP compliant plasmid DNA is being established.
During process development one focus is on the widely-used method of alkaline lysis, particularly in the separation of the precipitate. The impact of different neutralizing buffers and the separation method (e.g. centrifugation or flotation) on yield and the ratio between open circular and supercoiled plasmid is evaluated.
Another focus is on a better understanding of plasmid formation in E. coli. As a result a model is proposed which clearly demonstrates the relationship between specific growth rate and plasmid formation.
Cell Therapy Unit, Cardiocentro Ticino, Lugano, CH-6900, Switzerland
The Cell Therapy Unit of Cardiocentro Ticino is authorized for the production of experimental advanced therapy medicinal products (ATMP).
Among other clinical trials, the METHOD study (“Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease”, Swissmedic reference TrP_I_2010_001) is currently ongoing (initial feasibility phase: 3/10 patients treated). The ATMP for this trial consists of fresh mononuclear cells isolated from autologous bone marrow (BM) (100ml aspirate from iliac crest) through density gradient centrifugation; cells are formulated in saline/HSA and tested for safety (sterility, endotoxin), identity/potency (CD45/CD34/CD133, viability) and purity (contaminant granulocytes and platelets).
The aim of the present work was to further develop the manufacturing process in order to reduce contaminants (current specifications: granulocytes ≤75%, platelets ≤2E9/dose). Moreover, additional potency assays (CFC, CFU-F, invasion) were introduced in view of the upcoming more advanced phases of the METHOD study and of a new trial (CIRCULATE), in which the same ATMP will be administered to patients with peripheral artery disease.
The current manufacturing protocol (A. std Ficoll-PaqueTM/719gx20min) was compared with two modified protocols (B. std Ficoll-PaqueTM/400gx30min; C. low-density Ficoll-PaqueTM/400gx30min), using sternal BM. Preliminary results suggest that protocol B does not differ from A in terms of product purity and potency, while protocol C may improve both purity (% granulocyte removal: B. 97.88±0.94, C. 98.85±0.69; p<0.05/T-test) and potency (CFC: B. 4967±1713/1E6MNC, C. 7417±1835/1E6MNC, p<0.05/T-test; CFU-F and invasion ongoing).
After confirmatory experiments, GMP validation of the optimized manufacturing and testing methods will be performed and regulatory approvals will be requested.
Molecular immunology Unit, Institute of Child Health (ICH), 30 Guilford Street, London, WC1N 1EH, United Kingdom
In the UK, clinical trials of gene modified, cell based, investigational medicinal products or advanced therapy medicinal products (ATMP) are regulated by the MHRA. Clinical application of requires vector production capacity, cell transduction facilities and infrastructure to support regulatory and ethical approvals. Here we describe a pathway from the production of a master cell bank for the production of a therapeutic retroviral vector, to clinical batch production and ex-vivo transduction of T cells for a phase 1 study of suicide gene therapy. Vector manufacturing was performed as an academic collaboration, with safety testing outsourced to specialist subcontractors. Therapeutic scale manipulations were undertaken at the trial site using modified protocols and reagents to ensure GMP compliance. Peripheral blood lymphocytes isolated from blood or by leukapheresis, were activated with CD3/CD28 conjugated beads and cultured in X-Vivo 10 supplemented with human serum and therapeutic grade interleukin 2. Transductions were facilitated by Retronection and all manipulations performed in a closed bag system. After magnetic removal of activation beads, transduced cells were enriched to a high degree of purity by CliniMacs selection, and then cryopreserved to provide multiple dose aliquots of the IMP. A phase 1 study of suicide gene modified T cells after haploidentical stem cell transplantation in children has recently opened and the technological expertise gained is now being applied to additional cell therapy studies.
Department of Biomedicine, Aarhus University, Denmark
VEGF plays an essential role in ocular angiogenic diseases including the late-stage form of AMD, the primary cause of vision loss in the western world. Over-expression of VEGF leads to development of vasculature emanating from the choroid, invading the subretinal space through breaks in Bruch's membrane. Strategies leading to long-term suppression of inappropriate ocular angiogenesis are required. A panel of 10 shRNAs targeting the coding region of human VEGF165 was tested in HEK293 cells and in the human retinal pigment epithelial cell line, ARPE-19. VEGF knock-down up to 92% was achieved by co-transfecting shRNA-expressing constructs with plasmid encoding the Renilla luciferase gene fused to the VEGF165 sequence. For in vivo delivery of the most potent shRNA cassette, both single-stranded and self-complementary rAAV vectors were packaged in serotype 8 capsids. Intramuscular administration in mice led to localized expression and 96% knock-down of endogenous VEGF. Using eGFP as a marker, efficient gene transfer of retinal pigment epithelial cells, the cells thought to be responsible for the abnormal VEGF production, was obtained by subretinal delivery of rAAV2.8 vectors. The capacity of rAAV-encoded shRNAs to silence endogenous VEGF gene expression was evaluated in the laser-induced murine model of choroidal neovascularization (CNV). In this mouse model of AMD, sizes of the CNV were found to be significantly reduced following rAAV-shRNA subretinal delivery. Thus, our results indicate that gene transfer combining AAV-mediated delivery with triggering of the endogenous RNAi pathway can be used for anti-VEGF therapy and holds great promise for the treatment of AMD.
UM80 UPMC, UMR_S968 Inserm, UMR7210 CNRS, Centre de recherche Institut de la Vision, 75012 Paris, France
Retinitis pigmentosa (RP) are retinal degenerative diseases which lead to peripheral vision loss evolving toward blindness. Rhodopsin is the most frequently mutated gene in RP. Rhodopsin, the photopigment of the rod photoreceptors, is a G-protein coupled receptor that is activated when hit by a photon, leading to initiation of the phototransduction cascade. Most of the rhodopsin mutations involved in autosomal dominant RP are gain-of-function or dominant-negative mutations. Moreover, degeneration of photoreceptors is also observed in heterozygous knockout mice, emphasizing the importance of rhodopsin expression level regulation. Spliceosome-mediated RNA trans-splicing, the therapeutic strategy selected, will allow to respect this constraint. This technique consists to bring, in the target cell, an exogenous pre-mRNA able to bind the endogenous rhodopsin pre-mRNA, in order to promote a splicing event in trans between the two pre-mRNA molecules. Targeting of the first intron should thus allow to repair any mutation present in exons 2 and following. A major advantage of this approach is that the expression level of the “repaired” protein depends only of the endogenous gene expression regulation.
To model rhodopsin mutations in cell culture and in rodents, we have constructed lentiviral vectors encoding wild-type and mutated alleles of the human rhodopsin genes. After screening of different trans-splicing molecules in the cellular model of rhodopsin mutation, the most efficient will be introduced in an AAV vector, and tested in “humanized” mouse model of RP. In this case, the therapeutic tool should be directly applicable to humans.
Non- clinical studies for the treatment of Stargardt disease for clinical trial using direct administration of an EIAV Lentiviral vector (StarGenTM)
StarGen™ is a lentiviral vector-based gene therapy in development by Oxford BioMedica (OBM) for the treatment of Stargardt disease. Stargardt disease is the most common inherited juvenile degenerative retinal disease, resulting from degeneration of retinal pigment epithelium (RPE) and photoreceptor cells in the retina. StarGen™ which expresses a functional ABCA4 gene will be administered by subretinal administration; this should result in the long-term expression of a functional ABCA4 protein, thus suppressing further RPE and photoreceptor degeneration. StarGen™ is an equine infectious anaemia virus (EIAV)-based Lentiviral vector and is currently under clinical evaluation in the US.
GLP non-clinical combined toxicology, shedding and biodistribution studies were conducted using subretinal administration of StarGen™in two species to support a regulatory submission for StarGen™,. A wide range of biological samples, including a variety of target and non-target sample types, were taken both in-life and at necropsy to evaluate the bio-distribution of vector, vector shedding and vector persistence. Samples were assessed for the presence of vector by quantitative real-time reverse transcriptase PCR (qRT-PCR) for vector genomic RNA. Persistence of vector was assessed by quantitative real-time PCR (qPCR) for vector associated DNA.
The most striking general observation from these studies was the fact that vector and vector associated sequences were highly restricted to target tissue (eye). An overview of the data from these studies will be presented.
1Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA 2Center of Ophthalmology / Institute of Anatomy, Essen University Hospital, Germany
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA
Center of Ophthalmology / Institute of Anatomy, Essen University Hospital, Germany
Non-clinical studies for the treatment of Usher syndrome 1B for a first in man clinical trial using direct administration of an EIAV Lentiviral vector (UshStat®)
UshStat
GLP non-clinical combined toxicology, bio-distribution, vector persistence and shedding studies have been conducted in non-human primates (NHPs) to support the regulatory submission. A range of biological non-target sample types distal to the administration site, were taken both in-life and at necropsy. Vector bio-distribution was assessed by extracting DNA using a real-time PCR-based (qPCR) assay. Vector persistence and shedding were assessed by analysing extracted RNA using a reverse-transcriptase real-time PCR-based (qRT-PCR) assay. EIAV DNA or RNA sequences were not detected at quantifiable levels during this study, indicating that, following the administration of UshStat
LentiVector® platform, a highly effective Equine Infectious Anaemia Virus-based lentiviral gene therapy platform for ocular disease
Oxford BioMedica is developing Ocular gene therapies based on its proprietary recombinant Equine Infectious Anaemia Virus (EIAV) LentiVector® platform. Currently, two Ocular therapies are undergoing evaluation in Phase I/II clinical trials, namely RetinoStat® and StarGen™ for the treatment of age-related macular degeneration and Stargardt macula dystrophy respectively. A first clinical trial with UshStat®, for the treatment of Usher Syndrome 1B, is due to start by the end of the year.
The development of lentiviral vector-based gene therapies to treat eye diseases is an attractive option due to the vector's innate ability to express therapeutic genes for extended periods, either to correct inherited disease, or to interfere with disease aetiology. The accessibility of the eye both for administration and evaluating therapeutic benefit, combined with the anatomical separation from the rest of the body preventing spread of the vector, are also significant advantages.
RetinoStat®, StarGen™ and UshStat® have been extensively characterized in GLP safety and biodistribution studies following subretinal administration. All three products exhibited a transient inflammation in the absence of prophylactic anti-inflammatory medication. Subretinal administration of vector caused no long-term detrimental changes within the eye. Biodistribution studies of vector demonstrated that the lentiviral vector products did not escape the ocular compartment, and as a result little or no antibody responses were observed in these studies.
In conclusion, the EIAV-based lentiviral gene therapy platform has been shown to be an effective and safe system for the delivery of relatively large genes into target retinal cells resulting in stable and long-term therapeutic expression.
AAV9 gene therapy rescues a murine model of Menkes disease
Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Consistently effective treatments for this devastating disorder of copper metabolism are limited. In an animal model, the mottled-brindled (mo-br) mouse, that recapitulates the Menkes disease phenotype, we recently reported improved biochemical and neurobehavioral parameters, and substantially increased average life span using combined brain-directed AAV5 gene transfer plus copper (Donsante et al., Molecular Therapy, advance online publication August 30, 2011).
Systemic gene therapy for Menkes disease would enable safer, less invasive delivery. To refine prospects for clinical translation of gene therapy for this disorder, we evaluated transduction efficiency and therapeutic effectiveness of AAV serotype 9, which crosses the blood-brain and blood-CSF barriers. In a cohort of mo-br mice, we administered 5 × 1011 viral genome particles of AAV9 harboring a human ATP7A cDNA by intraperitoneal (i.p.) injection on day 2 of life, followed by 50 ng copper chloride into the brain lateral ventricles on day 3. Western blot analysis indicated high ATP7A transgene expression in skeletal and cardiac muscle. AAV9-ATP7A transduction of liver, kidney, heart, lung, intestine, cerebral cortex and choroid plexus epithelia was confirmed by quantitative PCR. Median life span doubled in comparison to untreated affected mo-br mice, demonstrating the efficacy of systemic AAV9. Comparison of various treatment modes suggested that brain-directed AAV9 may offer even more effective long-term outcomes than AAV5, based on broader neuronal tropism. Our findings provide the first evidence that AAV9 gene therapy may have clinical utility for treatment of Menkes disease.
Department of Genetics, UCL Institute of Ophthalmology, London, UK
The retinal pigment epithelium (RPE) interacts closely with photoreceptors and is important for maintaining visual function. Primary or secondary loss of RPE in age-related macular degeneration or some forms of inherited retinal disease leads to loss of photoreceptors and contributes to vision loss. Under normal conditions RPE cells are growth-arrested but retain their proliferative capacity. We here test the hypothesis that gene transfer of E2F2, a potent transcriptional regulator of cell proliferation, to RPE cells might induce mitosis and increase RPE cell density. Non-integrating lentiviral vectors were used to deliver E2F2 (LNT-E2F2) or hrGFP (LNT-hrGFP) as a control to either confluent, serum starved ARPE19 cells (MOI 5) or by subretinal injection to the RPE of C57Bl/6 mice. Quantitative RT-PCR and Western Blot analyses were used to verify overexpression of the transgenes and showed that LNT-E2F2 infection in vitro leads to a 100-fold increase of E2F2 mRNA and to increased protein levels. Co-localization of E2F2 with Ki67, a marker for cell cycle activation, showed increased numbers of Ki67 positive ARPE19 cells after LNT-E2F2 infection. Eyes injected with LNT-E2F2, LNT-hrGFP or buffer alone were assessed for RPE cell proliferation on RPE flat mounts by Bromodeoxyuridine (BrdU) labelling and showed a 90-fold increase in EF2F positive RPE cells and a 50-fold increase in BrdU positive cells after LNT-E2F2 infection compared to both controls. This data suggests that E2F2 can induce RPE cell proliferation in vitro and in vivo and may lead to a new treatment concept for retinal degenerations with RPE loss.
Role of lentivirus-mediated overexpression of Programmed Death Ligand 1 on corneal allograft survival
Oxford BioMedica (UK) Ltd, Oxford Science Park, Oxford, OX4 4GA, UK
Over 100,000 corneal transplants are carried out each year, and as more national eye banking systems are initiated this figure is likely to increase substantially in the near future as more of the 10 million people stricken with corneal blindness worldwide are treated. Corneal transplantation has a high success rate, with only 14% of grafts failing in the first year. This is due to the immune-privilege status of the cornea, which is underpinned by the avascular nature of the tissue depriving the immune system access to the cornea. As the most common reason for graft failure in patients is irreversible immunological rejection, it is unsurprising that neovascularisation (both pre- and post-grafting) is a significant risk factor for subsequent graft failure. It is logical therefore to target neovascularisation to prevent corneal graft rejection.
EncorStat® is an ex vivo gene therapy product which enables the genetic modification of donor human corneas to be inherently angiostatic. The modified corneas are engineered by the ex vivo delivery of the genes encoding secretable forms of the angiostatic human proteins endostatin and angiostatin. The ex vivo gene therapy product used to modify the cornea is a non-replicating, recombinant lentiviral vector product derived from the Equine Infectious Anaemia Virus (EIAV), and shares the same active ingredient with RetinoStat®, itself an Oxford BioMedica gene therapy product currently in clinical development to treat wet age-related Macular Degeneration (AMD).
Here we present early preclinical and the first proof-of-concept data for the EncorStat® programme.
Heme oxygenase-1 affects myoblasts differentiation by targeting microRNAs
Heme oxygenase-1 (HO-1) is an anti-oxidative and anti-apoptotic enzyme, which degrades heme to biliverdin, iron ions and carbon monoxide (CO). HO-1 improves cell survival and hence could be beneficial in cell-based therapies. Here we show that HO-1 overexpression impairs the differentiation of murine myoblasts. HO-1 downregulated miRNA processing proteins, Lin28 and DGCR8, affecting the expression of 11.4% of microRNAs out of 695 investigated. Importantly, impaired formation of myotubes by HO-1 overexpressing satellite cells is accompanied by decreased expression of MyoD, myogenin, or myosin, and reduced production of miR-1, miR-133a, miR-133b, and miR-206 myomirs. In turn, HO-1 overexpressing cells generate more SDF-1 and display increased levels of miR-146a. Inhibition of myogenic development is independent of anti-oxidative properties of HO-1 but relies on decreased expression of miR-133b and miR-206. The effects of HO-1 were fully reversed by pharmacological (tin protoporphyrin) or genetic (siRNA) HO-1 inhibition and lack of HO-1 in primary satellite cells from HO-1−/− mice accelerated myoblast differentiation. Accordingly, effects of HO-1 overexpression were mimicked by HO-1 products (iron ions or CO) and were mocked by treatment of cells with SDF-1 protein while reversed by overexpression of myomirs. Surprisingly, HO-1 overexpressing cells injected to gastrocnemius muscles form fast growing, hyperplastic tumors, infiltrating the surrounding tissues, and metastasizing to the lungs, the effect not observed for control cells, which formed myoblasts. In conclusion, HO-1 inhibits differentiation of myoblasts in a SDF-1 and myomir-dependent manner. Thus, although HO-1 increases the cell survival, its overexpression, affecting numerous microRNAs, can induce cell transformation.
Center for Stem Cell Biology and Regenerative Medicine, IMSUT, University of Tokyo, 4-6-1 Shirokanedai Minato-ku, 108-8639, Tokyo, Japan.
Embryonic stem cell-derived retinal cells transplanted to the adult retina
Embryonic stem (ES) cells provide a promising source of cells for retinal cell transplantation, a novel strategy to repair the degenerate retina. We previously demonstrated that post-mitotic photoreceptor precursors can functionally integrate into the adult wild type and degenerating retina. We sought to investigate the feasibility of ES cell-derived retinal cell transplantation to the adult murine retina.
Using a mouse EB5 ES cell line containing a GFP reporter driven by the early retinal transcription factor Rx, we assessed the efficacy of differentiation to retinal cell types by a serum-free floating culture method (SFEB/DLFA culture, Osakada, et al, 2008) with or without selection of Rx.GFP positive cells. Following AAV9.CMV.GFP labelling, differentiated retinal cells were transplanted subretinally into adult mice. Photoreceptor cell integration and retinal cell survival was evaluated 2 weeks post transplantation.
Further differentiation of Rx.GFP+ retinal progenitors generated significantly greater numbers of Crx+ and Rhodopsin+ photoreceptors than mixed populations of unselected cells. The peak of rod photoreceptor birth was at day 20 of culture and transcriptional analysis demonstrated an increase in Crx and Nrl expression, photoreceptor precursor markers, until day 28. We therefore dissociated cells at this stage for subretinal transplantation. However, following transplantation into adult mice no ES cell-derived integrated photoreceptors were observed.
Despite the presence of post-mitotic photoreceptor precursors in the differentiated cell population these cells failed to integrate when transplanted to the adult retina. These results suggest the selection of photoreceptor precursor cells prior to transplantation may be required to enable their integration into the adult retina.
Optimizing Adenoviral Transduction of Endothelial Cells under Flow Conditions
Major challenges while targeting adenoviral vectors (AdV) to the vascular endothelial system are accumulation of therapeutically relevant doses of vectors to the site of interest and protection of vectors from inactivation by the immune system. The aim of this study was to target AdV to endothelial cells and analyze transduction efficiency under flow conditions in vitro.
Complexes of reporter gene expressing AdV with positively charged magnetic nanoparticles were formed by means of electrostatic interaction with or without subsequent shielding with negatively charged polyethylene glycol based polymer. Transduction of HUVEC was analyzed in vitro under flow conditions. Protection from inactivation by the immune system was analyzed by pre-incubation of AdV and complexes with serum and subsequent reporter protein analysis of infected cells.
Physical association of AdV with MNP and polymers was demonstrated by radioactive labelling of components and co-sedimentation in a magnetic field. Conditions resulting in net zero surface charge of complexes were found.
Ad-MNP+/- polymer resulted in efficient transduction of HUVEC, depending on MOI and flow rate upon application of magnetic field, whereas no transduction was observed, in the absence of complex formation with MNP or without magnetic field. Association with MNP did result in protection from neutralizing antibodies, with slightly increased protection provided by polymer.
Complex formation of AdV with MNP with or without polymer is a viable means for targeting vectors by magnetic fields to areas of interest. Efficient gene transfer under flow conditions and protection from inactivation by components of the immune system was achieved.
Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laboratory, University College Cork, Ireland
Bacteria can be exploited as gene/cell therapy vectors due to their inherent tumour-specific growth. One strategy involves delivery of non-invasive bacteria to the tumour site with bacterial expression of genes locally (external to the tumour cell). By transfection with plasmids suitable for bacterial expression of heterologous genes, these bacteria can home to tumours, replicate within them and locally express therapeutic proteins. E.coli nissle and Bifidobacterium breve are native, harmless residents of the human gut (commensal bacteria), and are on the market as probiotic preparations.
We report the novel finding that oral administration of E.coli nissle or B. breve is equally as effective as IV administration in achieving specific targeting of distal tumours in mice. Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal tract (GIT) to extra-intestinal sites and is traditionally associated only with pathogenic bacteria and/or sick hosts. Lux-labelled B.breve or E.coli nissle fed to mice bearing subcutaneous tumours were detected by whole body imaging specifically in tumours, at similar levels to IV administration. Findings were confirmed by bacterial culture and mice remained healthy throughout experiments. With regards the mechanism of this bifidobacterial translocation, cytokine analyses indicated a significant up-regulation of IFN( in the GIT of bifidobacteria-fed mice, which is associated with increases in epithelial permeability. However, B.breve feeding did not increase systemic levels of other commensal bacteria.
These findings indicate potential for safe and efficient gene based treatment and/or detection of tumours via ingestion of non-pathogenic bacteria expressing therapeutic or reporter genes.
Oxford BioMedica Ltd, The Medawar Centre, Oxford Science Park, Oxford, OX4 4GA, UK
ProSavin® is a gene therapy product that is currently being evaluated in a clinical trial for mid-stage Parkinson's disease. ProSavin® consists of a tri-cistronic EIAV based vector encoding three enzymes for dopamine synthesis. The clinical administration protocol involves intra-striatal injection using multiple needle tracts and multiple deposits of vector along each tract. Although this approach was designed to maximise vector distribution throughout the striatum it necessitates a relatively long surgical procedure that may limit the capacity for dose escalation.
Previous studies have demonstrated that continuous infusion of small molecules and AAV vectors mediates efficient distribution in the brain in a process termed convection enhanced delivery. It has not been previously studied whether larger lentiviral vectors can be effectively distributed using continuous infusion. We have investigated different delivery parameters for optimising gene delivery of lentiviral vectors into the macaque striatum using an EIAV-LacZ vector. Delivery parameters investigated were volume of administration, rate of delivery and injection device diameter. Analysis of gene transfer indicated that reducing the injection device diameter and increasing administration rate resulted in improved distribution of vector. This was achieved using a single injection of vector compared with five injection tracts in the previous multi-tract approach. There were no indications of increased tissue damage or inflammatory responses with these optimised parameters.
The refined delivery approach has now been successfully translated to the clinical trial resulting in a simpler and shorter surgical procedure. The method has also allowed dose escalation that may not have been feasible with the previous approach.
Self-assembled siRNA lipoplexes of LinOS-cholesterol mixtures: enhanced siRNA uptake and gene silencing in HeLa cells
Cholesterol is used as a helper lipid in many DNA-lipid complexes (for gene therapy) and it plays an important role in membrane fusion. We synthesized N 4-linoleoyl-N 9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS - a spermine diacyl fatty acid derivative) and prepared self-assembled siRNA lipoplexes with mixtures of LinOS/cholesterol. Self-assembled siRNA lipoplexes were prepared by adding ethanolic LinOS/cholesterol mixtures to siRNA in OptiMEM. These lipoplexes were then evaluated for their efficiency to deliver siRNA and to mediate gene-silencing, and for their effect on cell viability (alamarBlue® assay). Transfection was on HeLa cells stably expressing EGFP in DMEM (10% FCS). Uptake of Alexa Fluor 647-tagged siRNA against EGFP and EGFP expression were evaluated by FACS 48 h post-transfection.
The lipoplexes prepared with LinOS/cholesterol resulted in nanometer sized particles (113–118 nm). These are of significantly smaller particle size than the lipoplexes prepared with LinOS only (194–294 nm). The LinOS/cholesterol (1:2) lipoplexes resulted in a 40-fold increase of siRNA delivery compared to LinOS only. These LinOS/cholesterol (1:2) lipoplexes also resulted in better gene silencing than the lipoplexes of LinOS without a helper lipid (reduced EGFP expression to 20% and 32% respectively) and comparably efficient to the commercial siRNA delivery reagent TransIT TKO. The prepared lipoplexes resulted in cell viability of over 85% in HeLa cells. Our results show that LinOS/cholesterol mixtures can form self-assembled lipoplexes with siRNA. Cholesterol enhances the siRNA uptake and the siRNA mediated EGFP silencing. Lipoplexes of LinOS/cholesterol are promising non-viral vectors for siRNA. (Supported by a fully-funded PhD studentship from the Egyptian Government to AAM).
Investigating the potential of Herpesvirus-saimiri as a novel gene-delivery vector for reprogramming somatic cells
The potential for embryonic stem cells for therapeutic uses is widely recognised. However, ethical issues surrounding their derivation leads to tight regulations on their use for therapy and research purposes. Induced Pluripotent Stem Cell technology involves reprogramming somatic cells into a pluripotent, stem cell-like state using defined factors, thereby avoiding these ethical issues. Moreover, this technology can also be used to reprogram cancer cells to their pre-cancerous, precursor state, which may help in the study of exogenous factors involved in cancer formation. Currently, the most widely used method of iPS gene delivery is using retroviral vectors. These provide long-term expression of transgenes, but integrate into the host cell genome, which may cause insertional mutagenesis. Herpesvirus saimiri has several advantages as a gene-delivery vector, such as a large capacity for heterogeneous DNA (∼150kb) and it is capable of forming persistent infections without integrating into the host genome by means of episomal maintenance elements. Furthermore, deletion of the STP and Tip genes in all HVS based vectors removes any transforming ability, thus generating a safe viral vector. Interestingly, HVS has been shown to maintain its genome in differentiating cell populations. Therefore, we are currently exploring the potential of HVS as a gene delivery system for iPSC generation. Here we describe the engineering of HVS-based vectors containing the Thompson reprogramming genes, Oct4, Sox2, Lin28 and Nanog. Data will be presented on which reprogramming genes are required to efficiently reprogram three Ewing's Sarcoma cell lines and one neuronal stem cell line.
The direct, real-time and multi-parameter visualisation and analysis of individual virus and gene delivery nanoparticles by laser microscopy and Nanoparticle Tracking Analysis
Using a specially configured laser illumination system, it is possible to directly visualise and analyse biological nanoparticles as small as 20–30nm (e.g. viruses, VLPs, drug delivery particles, nanovesicles and exosomes, etc..) on an individual basis in suspension and in real time using conventional microscopical instrumentation which is commercially available and becoming widely established in a number of sectors in the biosciences (
Each particle can be individually but simultaneously analysed for i) size (through measuring its Brownian motion); ii) high resolution particle size distribution profile (model independent); iii) light scattering properties (refractive index); iv) fluorescence (e.g. through presence of Ab-mediated fluorescent label) allowing phenotyping or speciation; v) zeta potential through the application of electric fields and, significantly, vi) concentration (i.e. numbers of particles per millilitre) of any given size class of nanoparticle detected given the detection volume is fixed and known. Plotting particle size against light scattering intensity allows high resolution discrimination of particles of different refractive index but similar size. Suitable concentration ranges lie between 107–109 particles/ml.
Examples will be shown of the successful enumeration and characterisation of a range of virus and phage types as well as examples of numerous forms of drug and gene delivery systems. Similarly, we will discuss the recent application of multi-parameter nanoparticle tracking analysis of the detection of exosomes and microvesicles which have been recently proposed as showing significant potential as diagnostic structures and as therapeutic agents.
benoit.chatin@univ-nantes.fr
Induced pluripotent stem cells (iPSc) technology offers the possibility to generate patient-derived cells applicable for disease modelling, drug screening and ultimately, autologous cell replacement therapy. However, despite several efforts invested in this field, these cells are difficult to obtain, due to very low reprogrammation efficiency. Moreover, iPSc are usually obtained by viral transduction of the reprogramming transcription factors, leading to genetic modifications and to an increase of the oncogenicity due to vector integration, making these cells unsuitable for iPSc-based therapies. Non integrative strategies, including the direct intracellular delivery of reprogramming proteins, are consequently a promising approach. We showed that liposomes based on aminoglycoside-derived cationic lipids are an efficient and versatile system for intracellular protein delivery. These vectors are capable of delivering reporter proteins in various cell lines, including primary human cells in vitro. Using this system, we intend to deliver purified reprogramming transcription factors under their native form into somatic cells, to modulate gene expression and bring them to a pluripotent state. This approach could lead to an efficient generation of iPSc without genomic integrations, suitable for many applications including cell-based therapy.
Welsh School of Pharmacy, Cardiff University, Cardiff, CF10 3NB, United Kingdom
The delivery of nucleic acid therapies to their intracellular target site is one of the greatest challenges in gene therapy. In human skin this challenge is further complicated by the presence of the outermost barrier layer, the stratum corneum. We aim to optimise a method for delivery of nucleic acid therapies into the skin using microneedle device (MN) technology, with a view to influencing the pathogenesis of inflamed dermatopathogenic conditions such as pyoderma gangrenosum.
The ability to dry coat a siRNA formulation onto MNs was investigated using Lamin A/C as a model gene for knockdown studies. Immortalised human keratinocytes (HaCaT cells) and primary keratinocytes, isolated and cultured from excised human breast skin, were used to evaluate the functionality of MN loaded siRNA. RNA interference (RNAi) activity was evaluated through polymerase chain reaction (PCR) for gene expression, western blotting for protein synthesis and confocal microscopy for visualisation of cell protein immunofluorescence.
Up to 30mg siRNA was successfully dry-coated onto the surface of MNs. The coating and drying process did not appear to reduce the biological functionality of siRNA, demonstrated by the significant reduction in gene expression/protein synthesis of Lamin A/C in both HaCaT and primary keratinocyte cells. Current studies are using MNs as a minimally invasive method to facilitate effective cutaneous delivery of siRNA to human skin explants and in vivo models and we are encouraged by our preliminary experiments where dry-coated pDNA (100mg; pCMVβ) has been successfully delivered, and subsequently expressed, in human skin explants using MNs.
The use of mRNA-loaded microbubbles for ultrasound-assisted transfection of dendritic cells
Dendritic cells (DCs) play a crucial role in stimulating immune responses. Therefore, they have emerged as outstanding candidates to induce immune responses against cancer cells. In this study we evaluated the possibility to transfect DCs with mRNA-lipoplex-loaded microbubbles in combination with ultrasound. Upon exposure to ultrasound, the gas-filled, lipid microbubbles implode and create transient pores in membranes of nearby cells. Simultaneously release of the attached lipoplexes allows their entry directly into the cytosol.
The binding of mRNA complexes to microbubbles was confirmed by confocal microscopy. Intracellular localisation of mRNA-lipoplexes in DCs was demonstrated by flow cytometry. Cell viability after transfection amounted up to 84%. To evaluate the ability of mRNA-loaded microbubbles to transfect DCs, an mRNA encoding luciferase was employed. Significant protein expression occurred already as early as 30 min after ultrasound application. Maximal levels of luciferase were detected after 8 hours. Transfection with mRNA encoding green fluorescent protein showed more than 24% of positive DCs as evaluated by flow cytometry. In contrast, no protein expression was detected when only mRNA lipoplexes were added to the cells. The transfection protocol only induced a minor activation of the DCs, as revealed by the expression pattern of co-stimulatory markers and transfected DCs were still able to respond to maturation stimuli. Our data demonstrate a proof-of-concept that mRNA-lipoplex loaded microbubbles in combination with ultrasound can transfect DCs. In contrast to current ex vivo transfection methods applied in the field of DC-based vaccines, this technique offers perspectives towards in vivo transfection of DCs.
BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, G12 8TA, UK
The safety and efficacy of adenovirus 5 (Ad5) as an intravenously delivered clinical gene therapy vector is hampered by substantial liver gene transfer. Hepatic transduction is mediated by a high affinity interaction between coagulation factor X (FX) and the adenovirus hexon1. The aim of this study was to use a high throughput approach to screen a compound library to identify an inhibitor of Ad5:FX gene transfer. We developed a fluorescence based high throughput assay using SKOV3 cells (CARlow) seeded in a 384-well microplate format to analyse FX-mediated Ad5GFP infection. This was used to screen 10,240 low molecular weight compounds from the “Pharmacological Diversity Drug-Like Set” library. Using an integrated liquid handling platform cells were infected with Ad5GFP in the presence of physiological concentrations of FX (10μg/ml) and 10μM of each compound. 48 hours post-infection GFP expression was analysed using high-content cellular screening microscopy. Employing a 75% decrease in FX-mediated Ad5GFP expression as the criterion for further evaluation 288 compounds were identified (∼2.8 % hit rate). All potential hits were rescreened to re-evaluate the compound activity both manually and using screening robotics. Any compounds causing toxicity as assessed by propidium iodide staining and MTS assay were disqualified. Upon completion of these assays 10 compounds were identified as causing over 75% inhibition of FX-mediated gene transfer without causing any cytotoxicity. These preliminary candidate compounds will be further analysed to determine IC50 values, structure-activity relationships, effects on Ad5 binding to FX and potential lead compounds will undergo in vivo study.
1. Waddington 2008 Cell
Dpto. Farmacología, Fac. Medicina, Universidad de Valencia, Spain
Dpto. Farmacología, Fac. Medicina, Universidad de Valencia, Spain and Hospital Universitario y Politécnico La Fe, Valencia, Spain
Sheffield Institute for Translational Neurosciences, University of Sheffield, 385a Glossop Road, Sheffield, S10 2HQ
Polymersomes are nanometer sized spheres that self assemble in water from amphiphilic block co-polymers. We have been using PMPC25-PDPA70 co-polymers to make polymersomes which can be used to encapsulate DNA in their aqueous core, and can deliver their cargo to the cell cytosol. We aim to use these polymersomes to deliver therapeutic genes for motor neuron diseases including spinal muscular atrophy (SMA). SMA is autosomal recessive disease affecting young children due to mutations or deletions in the survival of motor neuron (SMN) gene. This disease causes weakness and progressive paralysis due to the death of motor neurons in the spinal cord. SMA has no cure and in its most severe form children only live to about 2 years of age. We hypothesise that polymersomes can be used to encapsulate SMN gene for delivery to the central nervous system (CNS) to enhance expression of SMN.
Rhodamine-labelled polymersomes were produced and toxicity was assessed by MTT assay and uptake was assessed by FACS analysis using NSC34 cells. Transfection efficiency of polymersomes in HEK cells was determined using E2-Crimson fluorescent plasmid. We have shown that PMPC25-PDPA70 polymersomes are not toxic in cell lines and the majority of polymersomes are uptaken by NSC34 cells by 6 hours. We demonstrated that polymersomes can encapsulate plasmid DNA by electroporation and can deliver the plasmid to cells. Delivery of polymersomes encapsulating SMN plasmid can increase SMN levels in SMA type 1 patient fibroblasts.
Delivery of Interleukin-4 gene using a novel amphipathic peptide for the treatment of psoriasis
Psoriasis is a chronic and relapsing autoimmune disease affecting the skin, joints, tendons and ligaments. Although the cause of psoriasis is not completely understood, a combination of genetic predisposition and environmental factors are thought to contribute to the disease process. To date treatments for severe forms of the disease involve general immunosuppression. However, the lack of specificity and side-effects caused by these drugs means there is an urgent need to find more effective and specific treatments.
Previous studies have shown that topical delivery of the interleukin-4 (IL-4) gene has a significant anti-psoriatic effect. However, the delivery systems chosen for this gene therapy are highly inefficient and do not actively transport the DNA to the nucleus. We propose the concept of delivering IL-4 using a synthetic pH-responsive amphipathic peptide linked to a nuclear localisation signal (NLS) that is designed to overcome biological barriers at a cellular level. The peptide/DNA nanoparticles are small enough to penetrate the cellular membrane and are designed to disrupt endosomes and actively translocate to the nucleus.
This work involves comparisons between the efficacy of the RALA peptide with either the SV40 (NLS) or the REV (NLS) incorporated into the sequence. To date the peptide/DNA nanoparticles have been characterised via size and zeta potential, stability in the extracellular matrix and in vitro transfection efficacy.
Cancer Gene Therapy Group, University of Helsinki, Helsinki, 00290,Finland
Oncolytic viruses represent a novel tool for cancer treatment. In addition to killing cancer cells, these agents provide danger signals useful for stimulation of an anti-tumor immune response. As a consequence of oncolysis, the innate and the adaptive arms of the immune system gain access to tumor antigens resulting in cross-priming and vaccination effects. Wild type adenoviruses feature reduced CpG content to reduce stimulation of toll like receptor 9, a key anti-adenoviral defense circuit, resulting in suboptimal danger signal. Here the aim was to study whether we could enhance this adjuvant capacity by incorporating immunostimulatory CpG motifs into the adenoviral genome.
We found that a combination of oncolytic adenovirus with CpG oligonucleotides had increased anti-tumor activity compared with virus or oligonucleotides alone in lung cancer xenografts. Consequently we engineered the genome of a p16-Rb pathway selective oncolytic adenovirus (Ad5Δ24) by inserting 20 immunostimulatory CpG islands (Ad5Δ24-CpG). This virus showed increased anti-tumor efficacy compared not only to Ad5Δ24 but also to the combination of Ad5Δ24 with CpG oligonucleotides. Splenocytes from mice treated with Ad5Δ24-CpG showed an enhanced capability to kill human tumor cell lines.
When Ad5Δ24-CpG was used in immune competent mice with B16-OVA tumors, we observed increased anti-tumor efficacy and an enhancement of the tumor-specific CD8+ T cells response. Moreover we demonstrated that stimulation of TLR9 at the tumor site by Ad5Δ24-CpG reduced inhibition by myeloid-derived suppressor cells (MDSCs).
In conclusion we report an oncolytic adenovirus backbone capable of stimulating TLR9 for an anti-specific immune response while suppressing MDSCs.
Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK
We have designed and synthesized seven new N 4,N 9-diacylated asymmetrical spermine conjugates having two different fatty acids varying in length and/or saturation state, and evaluated them as siRNA delivery vectors. We compared transfection with asymmetrical conjugates to transfection with mixtures of their counterpart symmetrical derivatives. Cationic lipids were synthesised by stepwise coupling of fatty acids to N 1,N 12 protected spermine. Transfection was of HeLa cells stably expressing EGFP in DMEM (10% FCS). Uptake of Alexa Fluor 647-labelled siRNA silencing EGFP and EGFP expression were measured by FACS 48 h post-transfection. Cell viability was evaluated using the alamarBlue® assay.
In these cationic lipids, one chain was kept constant (18:1). Increasing chain length (22:1 and 24:1) caused an increase in siRNA delivery compared to 18:1 and 20:1. EGFP expression reduction (3 μg/well lipid) was down to 24% EGFP (18:2) and to 38% EGFP (18:0). Increasing chain length (from 18 to 24) resulted in lower EGFP reduction (26%, 38%, 51%, and 54% at 6 μg/well of 18:1, 20:1, 22:1, and 24:1 respectively). Cell viabilities 48 h post-transfection were above 74%. Asymmetrical 18:2 showed better EGFP reduction compared to diacyl conjugate mixtures of 18:1/18:2. Diacyl conjugate mixtures of 18:1/22:1 showed better EGFP reduction compared to asymmetrical 22:1. Unsaturated spermine conjugates resulted in better EGFP reduction, while saturated conjugates showed better delivery. Mixtures of symmetrical conjugates resulted in different EGFP reduction compared to asymmetrical conjugates. Asymmetrical 18:2 conjugate (LinOS) is an efficient siRNA delivery vector. (Supported by a fully-funded PhD studentship from the Egyptian Government to AAM).
Synthesis and evaluation of N4,N9-fatty acid conjugates of N1,N12-diamidino-spermine as non-viral siRNA delivery vectors
Guanidine functional groups, with their high pK a values (∼12.5), are fully protonated at physiological pH and attractive for incorporation in novel cationic lipids. Four N 1,N 12-diamidino-spermine N 4,N 9-fatty acid derivatives were designed, synthesized, purified, characterized, and evaluated as non-viral vectors for siRNA delivery. The N 4,N 9-fatty acid spermine conjugates were prepared by DCC coupling of the corresponding fatty acids after di-N-phthalimido N 1,N 12 primary amine protection, followed by deprotection and guanidinylation with 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine/triethylamine and a final TFA deprotection yielded the di-TFA salts of the cationic lipids. Transfection of HeLa cells stably expressing EGFP, in DMEM (10% FCS), using Alexa Fluor 647-labelled siRNA silencing EGFP was measured by FACS 48 h post-transfection. Cell viability was evaluated by the alamarBlue® assay.
The particle sizes of the lipoplexes were 132–575 nm with zeta-potential +28 to +50 mV. N1,N12-Diamidino-N 4-linoleoyl-N 9-oleoylspermine showed both the best siRNA uptake compared to the other vectors and the best EGFP knock-down (EGFP reduced to 26%). N1,N12-Diamidino-N 4,N 9-dioleoylspermine was the next most efficient with EGFP reduction to 43%. Although N1,N12-diamidino-N 4,N 9-dierucoylspermine showed comparable siRNA uptake to the dioleoyl, the EGFP fluorescence was reduced only to 85% at the highest N/P=18. The cell viability assay showed that HeLa cells 48 h post transfection had viability of 64%-83% compared to non-transfected cells, except the C12 conjugate which resulted in very low viability at different N/P values. N1,N12 -Diamidino-spermine with unsaturated fatty acids of chain length C18 conjugated to N 4,N 9 are good non-viral vectors for siRNA delivery. (Supported by a fully-funded PhD studentship from the Egyptian Government to AAM).
Safer and improved Sleeping Beauty DNA transposition from DNA minicircles
With the reconstructed Sleeping Beauty (SB) DNA transposable element as a driving force, DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T-cells, and clinical trials with transposon-engineered cells are on the way. To further optimize SB-based vectors for clinical use, we have examined SB DNA transposition from DNA minicircles devoid of the bacterial backbone of the plasmid. Using a genetically engineered E. coli strain with inducible expression of minicircle-assembly enzymes, we produced pure SB minicircles harboring a selection cassette flanked by SB inverted repeats that were separated by a short backbone linker. We demonstrate potent DNA transposition, directed by the hyperactive SB100X transposase, from the minicircle transposon donor. With low doses of SB100X, the efficiency of transposition from the minicircle donor in HeLa cells was significantly enhanced (up to 8-fold) relative to the level of transposition obtained with the same molar amount of parental plasmid DNA. Flow cytometry analysis of transiently transfected HeLa cells showed a 2.5 fold increase in positive cells with minicircle DNA compared to plasmid DNA, suggesting enhanced nuclear uptake of minicircle molecules. DNA transposon minicircles are easily produced, are less likely to trigger an innate response in transfected cells, and are robust substrates for DNA transposition. Based on our findings, DNA minicircles may become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system.
Department of Biomedical Sciences and Medicine, University of Algarve, Faro, 8005-139, Portugal
The aim of this work is to test the efficiency of enhanced plasmids, based on pEPI-1 and pEPito backbones, in retinal gene transfer. These plasmids contain a number of modifications to enhance the length of gene expression [1]. These plasmids have 1) a matrix attachment region sequence to permit replication as episomal particle, 2) lower CpG content, to avoid silencing and 3) different promoters, including a tissue-specific promoter for targeted expression.
The transfection efficiency of pEPI-1 and pEPito-based plasmids was evaluated both in vitro and in vivo. In vitro transfection efficiency was quantified by flow citometry using two RPE cell lines. In vivo studies were performed in C57Bl6 mice by intravitreous injection of these plasmids and 10 μm sections of the eyes analysed for GFP expression at different time points up to 32 days post-injection (dpi).
Flow citometry analysis shows that plasmids with the pEPito backbone have higher transfection efficiencies than those of plasmids with the pEPI backbone. Plasmids with the tissue-specific RPE65 promoter have lower transfection efficiencies compared with plasmids containing either CMV or hCMV promoter. Moreover, the cell line markedly influences the transfection efficiency. GFP expression in injected eyes of C57Bl6 mice was sustained for at least 32 dpi for plasmids containing the RPE65 promoter.
Plasmids containing the RPE65 promoter display lower transfection efficiencies in vitro than those with CMV or hCMV promoter. However, this is reversed in vivo, where the former were shown to have higher and lengthier GFP expression.
[1] Haase et al., BMC, 2010.
Department of Biomedicine, Aarhus University, DK- 8000 Aarhus C, Denmark
Previously we observed sustained levels of circulating human growth hormone (hGH) after electrotransfer of hGH plasmids into the muscle of immunodeficient dwarf mice (lit/scid) and these treatment led to a significant weight increase of 33.1% (Oliveira et al., 2010, J. Gene Medicine 12, 580–5). In order to develop a clinical relevant gene therapy protocol we have started to compare a panel of growth hormone plasmids with various promoters (UbiC or CMV promoters) transferred to skin and muscle using different electro-transfer equipment (Cliniporator/IGEA vs ECM830/BTX). Transgenes encoding human or mouse growth hormone and as controls erythropoietin plasmids were tested in NMRI mice. Initially we performed electrotransfer to muscle and followed the mice for 1 month. Here the UbiC promoter linked to a genomic hGH gene lead to highest weight increase (17,25%±13,02%), the CMV promoter linked to the genomic hGH gene somewhat lower effect (13,85%±5,29%), the CMV promoter linked to hGH cDNA caused some effect (12,85%±7,47%) while the weight increase of the control group treated with saline was lower (5,18%±4,03%). In mouse skin the UbiC promoter linked to an erythropoietin gene seems less efficient compared to the CMV promoter and the effects were less compared to muscle expression. Our results indicate that hGH expressed in muscle causes weight increase in normal mice 30 days after a single treatment.
The effects of antioxidative additives on electroporation efficacy
Electroporation is a widely used method for gene transfer. The efficacy is limited by several factors including the oxidative damage caused by electric pulses to the cells. Antioxidants could potentially improve cell survival, but their effect on gene transfer is unknown. The aim of our experiment was to test the effects of antioxidative additives on gene transfer and cell survival after electroporation.
Melatonin and a combination of vitamin C and E were added directly to the electroporation buffer or the cells were pretreated by growing in a medium containing these antioxidants. Cell survival was measured spectrophotometrically and by cell cultivation under selective conditions.
Interestingly, the antioxidants slightly decreased cell survival and growth, if no electroporation was applied. No effects was found when the antioxidants were applied directly to the electroporation buffer. On the other hand, the number of transgenic cells after electroporation increased by an order of magnitude, if the cells were treated with antioxidants during 3 hours.
The results indicate that the idea of antioxidative protection during electroporation pulses is of interest and should be followed in vivo. It might be hypothesized that the effect is tissue-specific, non-linearly dose-dependent and associated with the ability of the antioxidant to protect the cell membrane from oxidative damage.
LUNAM Université – Ingénierie de la Vectorisation Particulaire, Angers, 49933, France;
Two types of promising non-viral nanocarriers have been developed in our laboratories, DNA lipid nanocapsules (DNA LNCs) on the one hand and DNA multimodular systems (DNA MMS) on the other hand. Both systems are based on the formation of lipoplexes with different cationic lipids and are then either coated by a corona of steric stabilizers (DNA MMS) or encapsulated in lipid nanocapsules (DNA LNCs). The different DNA nanocarriers, encapsulating a luciferase coding plasmid, were characterized by physico-chemical methods. Encapsulation of a fluorescent probe, DiD, allowed us to follow their biodistribution with in vivo biofluorescence imaging (BFI) after systemic administration on healthy animals. Afterwards, the biodistribution of intravenously injected PEG DNA LNCs (DNA LNCs coated with PEG2000), encapsulating pHSV-tk, was analysed by in vivo imaging on an orthotopic melanoma mouse model. In this model, luminescent melanoma cells, implanted subcutaneously in the right flank of the mice, allowed us to follow tumour growth and tumour localisation with in vivo bioluminescence imaging (BLI). The BF-images confirmed a prolonged circulation-time for PEG DNA LNCs as was previously observed on an ectotopic model of glioma; comparison with BL-images evidenced the colocalisation of PEG DNA LNCs and melanoma cells. After these promising results, treatment with PEG DNA LNCs and GCV, using the gene-directed enzyme prodrug therapy (GDEPT) approach, was performed and the treatment efficacy measured by BLI. The first results showed tumour growth reduction tendency and so, once optimised, this therapeutic strategy could become a new option for melanoma treatment.
Bio-inspired delivery of RUNX2 siRNA as an adjuvant therapy for metastatic prostate cancer
RUNX2 belongs to the family of runt-related transcription factors shown to be upregulated in tumour cells that have a propensity to metastasize to the bone. RUNX2 activates survivin expression which has correlated with relapse and chemoresistance. RUNX2 has also been shown to protect against apoptosis through survivin, BCL2 and BMP7. No drug is currently available to inhibit RUNX2, so using siRNA technology to inhibit expression of RUNX2 in prostate tumours is a viable concept.
However, RNAi therapies have been significantly limited by the lack of a suitable delivery system, especially in vivo. Only by understanding the biological barriers to be overcome can sequences be designed to perform discrete functions that can aid nucleic acid delivery. To that end we have designed a novel 30mer peptide, RALA, that can deliver nucleic acids in vitro and in vivo. We now aim to characterise this delivery system with RUNX2 siRNA.
RALA neutralised the negatively charged RUNX2 siRNA at N:P ratios ≥4 with an average nanoparticle size <150 nm from N:P ratios ≥6. Transmission electron microscopy also revealed spherical RALA/RUNX2 siRNA nanoparticles <100nm. The RALA/RUNX2 siRNA nanoparticles remained stable in 10% serum for up to 6 hours. Western blotting confirmed knockdown of the RUNX2 gene. Finally, we found that RALA is superior to the commercially available oligofectamine transfection agent for delivery of fluorescently labelled siRNA to the prostate cancer PC-3 cell line. Taken together these results indicate that RALA is a highly effective delivery system with a wide range of applications for siRNA therapies.
Bacterial gene therapy of colitis by reprogramming of target cells
The etiopathogenesis of inflammatory bowel diseases is not yet fully understood. Current therapy is mainly based on suppression of the inflammation. Induced pluripotent stem (iPS) cells rise from reprogramming and dedifferentiation of adult cells and are the starting point for differentiation into specialized cell types. However, iPS cells have never been generated in vivo using gene delivery strategies. The aim of our work was to test the gene therapy of DSS-induced colitis in mice by delivery of plasmid carrying the genes encoding transcription factors Oct-4, Klf-4 and Sox-2. Bacterial strain Salmonella Typhimurium SL7207 was used as a vector for oral delivery of the plasmid. Bacteria were administered via gastric gavage every other day either concurrently with the course of colitis or preventively. There were no differences found between the therapeutic group and the control groups in weight loss, stool consistency and colon length at the end of the therapeutic experiment. In the preventive treatment experiment we found some improvement in weight loss and stool consistency in the therapeutic group at the end of the experiment. The expression of the transgenes in colon of the therapeutic groups was increased. However, despite the improvement of some clinical outcomes seen in the preventive experiment and increased expression of the transgenes, we cannot definitely conclude the effectiveness of this approach for therapy of colitis.
Dpto. Farmacología, Fac. Medicina, Universidad de Valencia, Spain;
Waking Up the Sleeping Beauty for T Cell Receptor Gene Transfer in Adoptive Cell Therapy
To date, the gold standard for the stable integration of T cell receptor (TCR) genes for adoptive cell therapy into lymphocytes has been the use of retroviral vectors. However, for therapeutic applications in the clinic this gene transfer method requires a laborious and costly infrastructure to generate GMP grade retroviral particles. Furthermore, retroviral gene transfer possesses a potential safety issue due to the propensity of viral vectors to integrate within actively transcribed regions. A promising alternative to virus-mediated gene transfer systems may be formed by transposon-based systems, in particular, with the very recent description of a highly optimized ‘Sleeping Beauty’ transposase. This system is solely based on nucleic acids (DNA or RNA) which can be obtained in GMP grade by expending acceptable efforts. Additionally, the integration pattern of this transposon-system has been recurrently reported to be invariably random, this way reducing potential genotoxicity of genomic integration.
In this study we investigated the potential of this novel approach for mediating TCR gene delivery into human lymphocytes. We developed and optimized a nucleofection based protocol for delivering the required vectors efficiently in our target cells. As a result, we achieved high and stable cell surface expression of a TCR reactive against the MART-1 melanoma antigen. In subsequent work we have compared and are comparing the in vitro functionality of these transposon-based engineered T cells versus retrovirally modified T cells.
From our studies we conclude that the transposon-based system is a valid and promising alternative to the classic retroviral system for future applications in the field of adoptive cell therapy with TCR engineered lymphocytes.
Thermo Fisher Scientific, Rochester, NY, USA
Multi-potent human mesenchymal stromal cells (hMSC) are a potential source for autologous cells for tissue repair, either differentiated into cells targeted for a specific tissue or as un-differentiated hMSC. Whether the requirements are for clinical or research use, obtaining a substantial number of cells can constitute a bottle neck for the investigator. We here present a protocol enabling the clinician or researcher to rapidly expand a population of hMSCs utilizing the potential of Thermo Fischer Scientific HyClone AdvanceSTEM media, developed specifically for the optimal expansion and maintenance of undifferentiated hMSCs and Nunc brand cell factories. Several different seeding densities from 350 – 4000 cells/cm2 was investigated in flask format and a relatively low seeding density of 350 cells/cm2 was chosen for use in the cell factory hMSC expansion protocol. In the cell factories the hMSC population was rapidly expanded more than 800 fold in 12 days. The easy to use protocol provides the investigator with several decision points regarding growth format, cell density and culture period while maintaining the multi-potency of the hMSCs. Subsequent to expansion the differentiation of the expanded hMSCs into adipocytes and osteoblast lineages was demonstrated in cell factories and chondrocytes differentiation in polypropylene tubes. The differentiation was documented using commercial kits.
Biocompatible polyplex nanomicelle for safe and effective gene introduction minimizing inflammatory responses
Non-viral techniques of gene introduction using nanocarrier are attracting much attention for clinical uses. The high-level safety is essential not to induce undesirable responses at the targeted site. PEG Shielding of nanocarriers is a promising strategy to reduce toxicity by preventing non-specific interactions with surrounding molecules. However, PEG tends to reduce the transgene expressions by hampering the intracellular processing of carriers.
In this study, we evaluated a new methodology to solve the dilemma of PEG. Based on our original polycation, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), which possesses high capacity of endosomal escape and safety due to its biodegradable nature, showing effective therapeutic effects on lung disease models [Itaka et al. 2010, Harada-Shiba, 2009], we prepared polyplex nanomicelles as DNA nanocarriers, by the combinatorial use of two forms, PEG-block-PAsp(DET) (B) and homo PAsp(DET) (H), with changing their mixing ratios. In in vitro transfection, the transgene expressions were increased in parallel with increased ratios of H. In contrast, by in vivo intratracheal administration toward lung, the nanocarriers with the ratios of B/H=50/50 – 75/25 showed highest expressions. With more proportion of H, the nanomicelles showed aggregation in lung tissue, activating the macrophages and highly inducing inflammatory cytokines. Interestingly, this phenomenon was not observed after administration of free polymers (without DNA). Thus, controlling the behavior of gene nanocarriers in lung played a key role to achieve good expressions, by reducing the inflammatory responses that are induced by aggregation of the nanocarriers. The optimized conditions of B/H formulation provide a highly practical system for gene therapy.
Characterization of hydrophobic drug conjugated polyamidoamine dendrimer as gene carriers
Polyamidoamine (PAMAM) dendrimers have been developed as polymeric gene carriers with relatively high gene transfection efficiency and low cytotoxicity. In this research, hydrophobic drug, dexamethasone, was conjugated to PAMAM G1 or G2 dendrimer and dexamethasone conjugated PAMAM (PAMAM-Dexa) was characterized as a gene carrier. In gel retardation assay, plasmid DNA (pDNA) was retarded by the addition of PAMAM-Dexa. In addition, dynamic light scattering (DLS) showed that PAMAM-Dexa formed small particles with pDNA. PAMAM-Dexa protected pDNA from DNaseI attack for more than 20 min. In vitro transfection assay showed that PAMAM-Dexa had the highest transfection efficiency at a 10:1 weight ratio (carrier:pDNA). Furthermore, PAMAM-Dexa had higher transfection efficiency to HEK293 cells than polyethylenimine (PEI25k, 25 kDa). The MTT assay suggested that PAMAM-Dexa was not toxic to HEK293 cells. Therefore, PAMAM-Dexa may be useful for therapeutic gene delivery.
E-mail address:
Development of dexamethasone conjugated low molecular polyethylenimineas a gene delivery carrier to brain cancer cell
Dexamethasone conjugated low molecular polyethylenimine (PEI-Dexa) was synthesized and evaluated as a gene delivery carrier to brain cancer cells. Dexamethasone translocated into nucleus by binding to the glucocorticoid receptor in the cytoplasm. During this process, PEI-Dexa was co-translocated into the nucleus with plasmid DNA, facilitating nucleic trafficking of plasmid DNA. Complex formation between PEI-Dexa and DNA was confirmed by a gel retardation assay. The complete retardation of DNA was observed at and above a 1:2(DNA: carrier) weight ratio. The average mean diameter of complexes was approximately 100 nm. Zeta potential study showed that DNA/PEI-Dexa complex had positive surface charges at various ratios. To evaluate the DNA binding affinity and DNA protection ability of PEI-Dexa, a heparin competition assay and a serum stability assay were performed. In heparin competition assay, plasmid DNA was released from DNA/PEI-Dexa complexes with aless amount heparin, compared with DNA/PEI25k complexes. A serum stability assay showed that PEI-Dexa protected plasmid DNA for more than 60 min. In vitro transfection assay was performed to evaluate the transfection efficiency of PEI-Dexa. To optimize the transfection conditions, pSV-Luc/PEI-Dexa complexes were prepared at various weight ratios and transfected into brain cancer cells. Luciferase assays showed that DNA/PEI-Dexa complexes had higher transfection efficiency than lipofectamine and the efficiency of DNA/PEI-Dexa was comparable to PEI25k. PEI-Dexa induced cell death and inhibited proliferation of brain cancer cells more efficiently thanlipofectamine and PEI25k. However, in normal cells, PEI-Dexa had lower toxicity than lipofectamine and PEI25k. The results suggested that PEI-Dexa is an efficient gene carrier for brain cancer cells.
Department of Internal Medicine II, University Hospital of Munich-Campus Großhadern, Ludwig-Maximilians-University
We have recently demonstrated the high potential of various polyplexes for tumor-specific delivery of the sodium iodide symporter (NIS) after systemic application. In the current study we used novel polymers based on linear polyethylenimine (LPEI), shielded by polyethylene glycol (PEG), and coupled with the synthetic peptide B6 as a transferrin (Tf)-receptor-specific ligand to achieve active tumor targeting to human HCC cells after systemic delivery of NIS DNA.
We complexed LPEI-PEG-B6 with a NIS-expressing plasmid and analyzed levels of functional NIS expression after transfection of HuH7 (high Tf-receptor expression level) as compared to RKO cells (low Tf-receptor expression level) in vitro and in vivo.
In vitro incubation of HuH7 cells with LPEI-PEG-B6/NIS resulted in a 9-fold increase in iodide uptake activity as compared to RKO cells. After establishment of subcutaneous HuH7 and RKO tumors in nude mice, NIS-conjugated nanoparticles or control vectors were injected i.v. followed by analysis of radioiodine biodistribution using 123I-scintigraphy. After injection of LPEI-PEG-B6/NIS, a significant perchlorate-sensitive iodide accumulation (8.5–10.9 % ID/g 123I; eff. half-life of 5 h) was observed in HuH7 tumors. Tumoral iodide uptake activity and NIS mRNA expression were significantly lower in RKO cells confirming the specificity of Tf-receptor-targeted nanoparticle vectors.
These results clearly demonstrate that systemic in vivo NIS gene transfer using nanoparticle vectors coupled with a Tf-receptor-targeting ligand is capable of inducing tumor-specific radioiodide uptake, which represents a promising innovative strategy for the NIS gene therapy approach in metastatic cancer.
Surgery/Surgical Oncology, Max-Delbrueck-Center for Molecular Medicine, Robert-Roessle-Str. 10, Berlin, 13125, Germany
In a clinical phase I gene transfer study we have shown, that the nonviral jet-injection transfer technology leads to safe and efficient transgene expression using conventional plasmids as vectors. To meet the stringent safety requirements and to improve gene transfer and expression efficiency, we compared minimalistic nonviral vectors in vitro with their respective parental plasmids. These studies have shown, that the MIDGE system (Mologen AG, Berlin, Germany) is the most efficient vector for transient gene expression, especially when physical transfer methods are used. We therefore used the MIDGE vector for the expression of human tumor necrosis factor alpha (TNF-a) to chemosensitize human melanoma cells. To approach this, first we compared the MIDGE vector with its parental plasmid in vitro and in vivo regarding expression efficiency, vector biodistribution and therapeutic potential in combination with chemotherapy. Furthermore, we analyzed the underlying mechanism of improved cell killing and antitumor activity of the MIDGE-mediated TNF-alpha expression in combination with vindesine chemotherapy in melanoma models.
We have shown in vitro and in vivo, that the MIDGE vector is superior compared to its parental plasmid regarding transfer efficiency and transgene expression. Due to its reduced size MIDGE vector offers a great therapeutic potential for chemosensitization strategies, since MIDGE-mediated TNF-alpha expression leads to effective tumor cell killing in vitro and reduced tumor growth in vivo, if combined with vindesine chemotherapy.
Pharmacy Department, King's College London, 150 Stamford Street, London SE1 9NH
The zwitterionic helper lipid Dioleoylphospatidylethanolamine (DOPE) is usually incorporated with cationic lipid gene delivery vectors to improve their transfection efficiency by facilitating escape of the DNA from the endosomal compartment. In this study the effect of DOPE on the integrity of model early and late endosomal monolayers was investigated through Langmuir trough and neutron reflectivity experiments.
Mixtures of either neutral or neutral and anionic lipids representing early or late endosomal membranes respectively were dissolved in chloroform and spread on the surface of a Langmuir trough in MES buffer at pH 5 then compressed to 30mN/m. Lipoplexes composed of the cationic lipid Dimethyldioctadecylammonium bromide (DDAB) or equimolar mixtures of DDAB:DOPE at 2:1 NP charge ratio were injected in the subphase and changes in surface pressure and neutron reflectivity were measured with time while holding constant area. Interestingly, injections of DDAB:DOPE lipoplexes resulted in a marked increase in surface pressure by up to 15mN/m to 45mN/m in both early and late endosomal models whereas injections of the same concentration of DDAB lipoplexes in the absence of DOPE did not result in a change in surface pressure. Simultaneous neutron reflectivity measurements, however, showed that DDAB in the presence or absence of DOPE has been incorporated in both early and late endosomal membrane models within several minutes of injection. This difference in the two experimental techniques is thought to be due to DOPE's ability to form non-lamellar hexagonal phases when the cationic lipid undergoes charge neutralisation upon interaction with the endosomal lipids resulting in an increase in surface pressure.
Construction and characterisation of a designer biomimetic vector for iNOS gene therapy
The potential for gene therapy to treat a vast range of diseases is impeded by the lack of an effective delivery system. For systemic administration of therapeutic genes, a suitable vector for clinical applications should have low cytotoxicity/immunogenicity, high transfection efficiency, tissue specificity, be cost effective and remain stable in vivo. Potentially designer biomimetic vectors (DBV) could provide a solution.
DBVs are based upon fusing several beneficial viral sequences to form a biomacromolecule. The DBV in this project, known as RMGT, consists of genetically bio-engineered discrete motifs, each one with a single function. It contains: i) a DNA condensing motif (DCM) to form nanoparticles with plasmid DNA (pDNA), ii) a TMTP cancer cycling targeting peptide (TP) for specificity, iii) an endosomal disruption motif (EDM) to aid cellular uptake and iv) a nuclear localisation signal motif (NLS).
Thus far it has been demonstrated that RMGT condenses plasmid DNA forming nanoparticles less than 150nm in diameter at a N:P ratio of 8. The nanoparticles are stable in serum for up to 6 hours and current work is showing promise of delivering DNA into prostate cancer cell nuclei (PC3) in vitro. Future work will focus on optimisation, characterisation and in vitro studies using the therapeutic gene inducible nitric oxide synthase (iNOS).
School of Pharmacy, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL Northern Ireland
This study examines the use of a designer biomimetic vector (DBV) that is essentially a recombinant fusion protein to deliver the therapeutic inducible nitric oxide synthase (iNOS) gene to breast cancer cells in vitro and in vivo. The DBV is composed of several discrete motifs each designed with single function architecture including a) a DNA condensing motif (DCM) obtained from the adenovirus mu peptide, b) a ZR-75-1 breast cancer cyclic targeting peptide (TP) for specific delivery of the nanoparticles, c) an endosomal disruption motif (EDM) that mimics the influenza virus fusogenic peptide and d) a nuclear localization signal (NLS), rev, obtained from the human immune-deficiency virus type-1.
The DBV was able to condense piNOS into spherical nanoparticles between N:P ratios of 4–10. At a N:P ratio of 9, piNOS was fully condensed with an average size of 75.1nm. Transfection with the DBV/piNOS nanoparticles resulted in a maximum of 62% cell kill. INOS overexpression was confirmed and total nitrite levels were in the range of 18μM. Furthermore 48 h after i.v. injection of the DBV/pEGFP-N1 nanoparticles GFP protein was detected in all the organs. The addition of chloroquine did not enhance the expression of GFP indicating functionality of the EDM. Furthermore the addition of nocodazole resulted in a reduction in GFP expression again indicating NLS functionality in vivo. Finally when the DBV/iNOS nanoparticles were administered i.v. there was a significant tumour growth delay in ZR-75-1 xenografts.
Chemical Biology Section, Department of Chemistry, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK
Linear poly(ethylenimine) (l-PEI) is the “gold standard” polymeric gene delivery vector. It is hypothesised that molecular weight (MW) relates to toxicity and efficacy. Synthesis of l-PEI is typically carried out by the polymerisation of 2-ethyl-2-oxazoline followed by hydrolysis leading to broad polydispersity. Improved methodology can produce monodisperse polymers, but only up to a MW of ∼10 kDa (∼4 kDa l-PEI).1 Chromatographic separation suggests the optimal balance between toxicity and efficacy is at ∼15 kDa.2
The commercial synthesis is sub-optimal. Exploration of reaction conditions suppressed chain transfer-coupling to the point where ∼15 kDa l-PEI with PDI values of 1.03 instead of ∼1.50 were synthesised. This allowed us to synthesise higher MW monodisperse l-PEI than previously possible, upto what may be the “ideal” l-PEI.
A range of monodisperse l-PEIs are currently being used to probe disruption of cellular membranes and to test alternate theories of internalization and endosomal escape. LDH release shows a linear dependence on molecular weight with a low MW threshold that produces no statistical increase in cell membrane disruption over 4 hours, even at 0.1 mg/ml doses. 22 kDa broad l-PEI lysed the cells at this dose. This correlates with the lack of endocytosis of low MW l-PEI and can be used to calculate the ideal MW for macromonomers for triggerable release.
1. Park, J.-S. & Kataoka, K., Macromolecules
2. Falco, A., Encinas, P., Carbajosa, S., Cuesta, A., Chaves-Pozo, E., Tafalla, C., Estepa, A. and Coll, J. M., Fish & Shellfish Immunology
Gene linker 2A, naturally occurring and active in cellular organism: Amphioxus
Linking two or more genes in one single open reading frame (ORF) would be highly desirable for co-expression of multiple proteins. Various strategies have been developed: alternative RNA splicing, IRESes, several promoters and the use of 2A or 2A-like elements.
Originally 2A was characterised in Foot-and-Mouth Disease Virus. Site directed mutagenesis identified a C terminus consensus DV/IExNPGP and it is proposed that 2A interacts with the exit tunnel of the ribosome in a way that a specific peptide bond is skipped between the last glycine of 2A and the first proline of 2B, thus providing a discontinuity in translation, resulting in release of discrete proteins from one single ORF.
Previously several other viruses have been identified with active 2A-like elements, as well as one cellular organism: trypanosome. Here we report another cellular organism Branchiostoma floridae (Amphioxus), where five 2A-like elements were recruited with the conserved consensus DV/IExNPGP, cloned in the synthetic reporter system GFP-2A-GUS, proved to be active. Out of these five, four were located in genes, one noticeably in front of a CATERPILLER gene (innate immunity) and finally the last one at the N terminus of a retrotransposon.
Further bioinformatic analysis of retrotransposons in Amphioxus revealed other potential 2A-like elements similar in sequence to the motif DV/IExNPGP in the CR1 clade, suggesting a natural “genetic linker” role for these elements as well as potential new 2A-like motifs.
Universidade do Algarve Campus de Gambelas Dept. Ciências Biomédicas e Medicina, Ed.8, Faro 8005-139, Portugal
Our goal is to develop chitosan-based non-viral vectors optimized for ocular gene therapy. For this, chitosan-DNA nanoparticles were prepared using two NH3:PO4 ratios. These nanoparticles were characterized regarding their morphology, stability, and transfection efficiency on retinal pigment epithelial cells.
The method to produce the nanoparticles proved to be reproducible, consistently yielding particles with a spherical morphology and sizes below 500 nm. Both types of nanoparticles were stable at least for 15 days and capable of efficiently protecting DNA from the action of DNase. Transfection efficiency, evaluated by fluorescence microscopy and flow cytometry was low compared with commercial transfection reagents.
In order to elucidate why the nanoparticles have low transfection efficiency we have chemically coupled rhodamine to chitosan, which will allow us to track the pathway undertaken by the nanoparticles within the cell based on rhodamine fluorescence. Chitosan-rhodamine B isothiocyanate nanoparticles were shown to efficiently enter the cell and travel towards the nucleus, which means these nanoparticles are able to escape endosomal degradation, unlike many other non-viral vectors. This suggests that the low transfection efficiency observed for these nanoparticles may be due to reduced plasmid release once inside the nucleus. The next step will be to devise a release trigger to allow timely DNA release.
The properties of chitosan nanoparticles, such as adequate size, stability, efficiency in penetrating the cell, and ability to escape endosomal degradation support and their potential use in gene therapy.
Unité de Pharmacologie Chimique et Génétique et d'Imagerie, Inserm U1022, CNRS UMR8151, Université Paris Descartes, Faculté de Pharmacie, Chimie-Paris Tech, Paris, France, F-75006
Institut de Chimie et des Matériaux Paris Est, UMR 7182, Thiais, France
Laboratoire de Pharmacologie Chimique et Génétique UMR 8151 CNRS-U1022 INSERM, Université René Descartes, Chimie Paristech, Paris, France
The delivery of nucleic acids into cells represents a promising technology for the treatment of a variety of genetic and acquired diseases. However, in order to reach this goal, efficient and safe delivery systems have to be developed.
We previously reported that cationic amphipathic histidine-rich peptide antibiotics can efficiently deliver plasmid DNA into eukaryotic cells. Our lead compound, LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA), demonstrated transfection efficiencies comparable to those of commercially available reagents such as PEI. Synthesis and evaluation of LAH mutants provided evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide. In fact, our results indicate a dual role of the peptide during the transfection process, namely DNA complexation and membrane permeabilization.
Using a human cell line stably transfected with a luciferase-encoding expression vector, we have now evaluated the ability of LAH4-L1 and 5 derivatives thereof to deliver siRNAs and silence gene expression. Our results show that the six peptides are all efficient siRNA delivery vehicles whose efficiency in mediating gene silencing in 911-Luc cells was greater than that of commercially available compounds including Lipofectamine, DOTAP and polyethylenimine. Further, we present evidence that inclusion of 2,3-diaminopropionic acid in place of the histidine residues produces peptides which have very similar transfection properties than LAH4-L1.
Experimental Biophysics, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, Germany
Here, we give an overview on our technologies for production and purification of minicircle DNA, a safe and efficient vector system for future gene therapy and genetic vaccination approaches.
Our minicircle technology is removing needless sequences like marker genes, the bacterial origin of replication etc., only used for stable maintenance and amplification of plasmids in bacteria. The resulting minicircle DNA consists almost only of the gene of interest, leading to significant size reduction and improved performance.
However, the first challenging step is to construct the so called parental plasmid containing the gene cassette of interest, in the example shown here the reporter gene lacZ under control of a CMV promoter, as well as specific motifs needed for the subsequent two main steps in the minicircle production process: 1) An inducible, sequence specific and very efficient in vivo recombination of the parental plasmid, resulting in the minicircle and a miniplasmid containing the residual sequence motifs. 2) A chromatographic purification technology for the isolation of minicircle DNA from the mixture of minicircle and miniplasmid.
Here, in addition to their improved safety profile, minicircles have shown their potential to increase the efficiency of transgene expression. Hence, PlasmidFactory's minicircle production technology facilitates the production of highly pure minicircle DNA for applications in gene therapy and vaccination as well as e.g. virus production or transient gene expression in any producer cells.
Brain-derived neurotrophic factor for gene therapy of peripheral nerve trauma
Traumatic injuries of peripheral nerves are the major cause of temporary or permanent patient's disability. Regeneration of nerve axons requires prolonged and stable expression of neurotrophins, including brain-derived neurotrophic factor (BDNF), which ensures the survival and regeneration of neurons. Therefore most promising way to stimulate the axonal growth and nerve recovery is using of plasmid constructs that encode BDNF.
Human BDNF cDNA (NCBI ACC#DQ893966) was cloned to pVax1 vector (Invitrogen). To increase BDNF expression efficiency, rare triplets were replaced by ones that encode the same amino acid, but found in human genes most frequently (BDNFopt). HEK293 cells transfected with pVax1-BDNFopt produced 325±16 pg/106 of BDNF, whereas cells transfected with empty pVax1 vector secreted no more than 13±6 pg/106 cells. To study the effect of pVax1-BDNFopt on nerve regeneration, 60 μg of each vectors were electroporated into the anterior tibial muscle immediately after damaging of common peroneal nerve in C57/Black mice. Nerve recovery was analyzed using electrophysiological parameters on the superficial branch of common peroneal nerve. The length of latency period of total nerve action potential in mice electroporated with pVax- BDNFopt was 1.5 fold shorter compared to control pVax1 group (n=11, p≤0.05). We also observed significant recovery of the amplitude of total nerve action potential action potential in animals electroporated with pVax1- BDNFopt compared to control pVax1 group (0.52±0.03mV and 0.220.06±mV, n=11, p≤0.05, respectively).
These data indicate that electroporation of pVax1-BDNFopt into muscle innervated by injured nerve stimulates its post-traumatic recovery.
PDMAEMA as a delivery vector for gene therapy on retina cells
Gene therapy is a potentially great therapeutic for a wide range of diseases. Consequently, it is critical to develop adequate delivery vectors. In this study, we evaluated if poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), which proved to be an efficient delivery vector for OVCAR-3 cells [1], is also suitable to transport nucleic acids to the retina. Reversible addition-fragmentation chain transfer polymerization (RAFT) was used to synthesize PDMAEMA with an average molecular weight of 200000g/mol. H1 NMR spectroscopy confirmed the chemical structure of the polymer and potentiometric titration showed that PDMAEMA has a pKa value around 7. The cytotoxity of PDMAEMA was evaluated in retinal pigment epithelium (RPE) cell lines ARPE-19 and D407 and HEK293 cells and found to be proportional to polymer concentration. However, for the concentration to be used in vivo, the cytotoxicity was negligible. Poliplexes of polymer:DNA (nitrogen:phosphorus, N:P) ratios of 5, 10 and 20 were able to encapsulate and protect DNA from DNase action. Dynamic light scattering and zeta potential measurements revealed nanosized particles with sizes around 213 nm, with sizes decreasing with increasing polymer amount. Fluorescence microscopy showed all three cell lines efficiently transfected with these poliplexes, although to a lesser extent in RPE cells. Preliminary results for intravitreal administration of these poliplexes in the eyes of C57Bl6 mice have shown no inflammation. Altogether these results suggest that PDMAEMA is a good candidate as a non-viral delivery vector for gene therapy of the retina.
Non-viral Gene Delivery Laboratory, Discipline of Biochemistry, University of KwaZulu-Natal, Privat Bag X54001, Durban 4000, South Africa
Gene therapy using small interfering RNA (siRNA) has attracted attention as a new therapeutic approach for the treatment of various diseases. The delivery of siRNA still remains a major obstacle for their therapeutic use. Cationic liposomes are the most widely studied non-viral vectors and have shown potential to efficiently bind and deliver siRNA.
We have formulated cationic liposomes containing either cytofectin, 3β[N-(N′,N′-dimethylaminopropane carbamoyl] cholesterol (Chol-T), or N,N-dimethylaminopropylaminylsuccinylcholesterylformylhydrazide (MS09) respectively, together with the neutral helper lipid, dioleoylphosphatidyl-ethanolamine (DOPE) and varying amounts of DSPE-PEG2000 (0, 2 and 5 mole percent). Liposome:siRNA interactions were determined using band shift, dye displacement and nuclease digestion analyses. Cytoxicity and gene silencing were determined in the human cervical carcinoma cells stably expressing the luciferase gene (HeLa-Tat-Luc).
All liposomes showed effective siRNA binding and protection against nuclease digestion. Significant luciferase gene knockdown (60–80%) was observed using 20 nM, 30 nM and 50 nM of anti-luciferase siRNA bound to the respective cationic liposome, compared to that of the control naked anti-luciferase siRNA. Furthermore it was seen that an increase in the degree of liposome pegylation reduced the gene knockdown activity. The results obtained thus far suggest that these cationic liposome formulations show great promise in siRNA therapeutics. This liposomal-mediated strategy could be useful in reducing expression of target genes in model biological processes or to provide therapy for dominant human diseases.
Arginine-based short peptide micelles delivery anti-microRNA21 antagomir into glioblastoma cells
Antagomirs are single strand RNAs or DNAs, which have complimentary sequences to microRNAs. MicroRNA is composed of 18 to 26 nucleotides single strand RNA and it inhibits target gene expressions. MicroRNA-21(miR-21) is one of the anti-apoptosis factors in glioblastoma, suggesting that effective inhibition of miR-21 may be useful for the treatment of glioblastoma. In this study, the R3V6 peptide(R3V6)was evaluated as a carrier of the anti-miR21 antagomir into C6 glioblastoma cells. R3V6 is composed of a positive region containing 3 arginines and a hydrophobic region containing 6 valines. Antagomir/R3V6 complex was analyzed by a gel retardation assay. The complex was completely retarded at a 1:3 weight ratio (antagomir:R3V6). Heparin competition assay determined that R3V6 formed tighter complexes with antagomir than polyethylenimine(25 kDa, PEI25K). FACS analysis demonstrated that antagomir/R3V6 complex was taken up by cells more efficiently than the antagomir/PEI25K complex and naked antagomir. RT-PCR results suggested that R3V6 delivered the antagomir and inhibited miR-21 more efficiently than antagomir/PEI25K complex. In MTT assay, R3V6 was less toxic than PEI25K. Anti-miR-21 antagomir inhibited the action of miR-21 and reduced the tumor growth in C6 glioblastoma xenograft mouse model. Therefore, miR-21 antagomier/R3V6 peptide complexes may be useful for gene therapy of glioblastoma.
E-mail:
Centre for Experimental Drug and Gene Electrotransfer, Department of Oncology, Copenhagen University Hospital Herlev, Herlev 2730, Denmark
Gene electrotransfer is an effective non-viral technique for delivery of naked DNA into muscle tissues. As long term high-level transgenic expression is obtainable, DNA electrotransfer to muscle has clinical potential with systemic delivery of therapeutic proteins. Using a porcine model and a reporter gene (firefly luciferase pCMV-Luc) we investigated the optimum dose of DNA for gene electrotransfer to muscle tissue in larger animals.
Yorkshire cross bred pigs were subjected to general anesthesia and a small area of the gluteal muscles were exposed. Intramuscular injection of increasing doses of DNA (1 – 1500 μg pCMV-Luc) was performed with electrotransfer using a combination of high and low voltage pulses. Forty-eight hours after treatment animals were sacrificed and biopsies were collected from and around each transfection-site. Transfection efficacy was evaluated by luciferase activity in muscle lysate, as well as PCR detection of the plasmid DNA.
Interestingly, it was possible to obtain high gene expression using as low as 10 μg of plasmid DNA. At 50 μg of plasmid DNA, luciferase activity reached a plateau in the range of 1–11 μg Luc/gram muscle whereas GFP transfected controls showed no Luc activity. The level of measured luciferase activity correlated well with the amount of Luc plasmid DNA in the muscle lysates as tested by semi-quantitative PCR.
In conclusion, it is highly encouraging that even low amounts of DNA led to high expression levels in a large animal model. This points to muscle DNA electrotransfer being a clinically relevant method for administration in clinical gene therapy trials.
Department of Academic Surgery, Cardiff University School of Medicine, Cardiff, CF14 4XN, UK
Pyoderma gangrenosum (PG) is a rare neutrophilic dermatosis, characterised by painful cutaneous ulcers. Treatment is empirical; first-line therapy is corticosteroids and/or cyclosporine, with topical wound management and analgesia. PG is a good target for gene therapy since the skin is easily accessible. Liposomes are particularly suited for this use: they readily penetrate the lipid bilayers, and have generally localised effects. However, gene therapy strategies alone are rarely successful clinically, thus gene therapy as an adjunct to other treatments is an interesting area for study.
This study aimed to investigate the effect of drugs commonly used to treat PG on the efficiency of in vitro transfection of keratinocytes with the reporter gene β-galactosidase, using the FDA-approved vector, Lipofectamine.
Cells of the HaCaT line were cultured in growth medium containing either no drug, or one drug used for treatment of PG: hydrocortisone, methotrexate, or infliximab. After 24 hours, the keratinocytes were transfected with β-gal. Expression was measured at 4 hours, and on days 1, 3, 5, 7, 9, and 11 post-transfection.
Transgene expression was greatest using culture medium containing hydrocortisone. Expression was greatest on day 1 post-transfection in hydrocortisone-treated cells, and day 3 in other treatment groups.
To conclude, transfection efficiency of the HaCaT cell line was enhanced by the presence of hydrocortisone. Optimum duration of exposure of the cells to transfection reagents was 1 to 3 days. This study is a step towards development of topical gene therapy-based treatments for PG, a potential strategy in the management of this debilitating condition.
Center for Regenerative Medicine, University of Modena and Reggio Emilia, Via Gottardi 100, 41125 Modena, Italy
Transplantation of autologous, genetically corrected epidermal stem cells was successfully used to treat junctional epidermolysis bullosa (EB), a genetic skin adhesion disorder. The dystrophic form of EB is caused by mutations in the type-VII collagen gene (COL7A1). Delivering the >9 kb COL7A1 cDNA by a retroviral vector is problematic, due to the large size and highly repeated nature of its sequence, which induce genetic rearrangements during reverse transcription and integration. We tested the feasibility of using a non-viral vector system based on Sleeping Beauty (SB)-derived transposons, taking advantages of the recently developed, high-capacity “sandwich” version of the SB transposon and the “hyperactive” SB 100X transposase, which showed high transposition efficiency in human cells. We tested the system in HeLa cells and in a keratinocyte cell line (HaCaT), which were co-transfected with the SB 100X transposase and either the normal or the sandwich version of the SB transposon containing a reporter gene. In both cell lines, transposition was obtained in up to 80% of the transfected cells with the sandwich transposon, compared to ∼50% obtained with the older version. These data were confirmed in type XVII collagen-deficient patient cells, and in human primary keratinocytes isolated from foreskin biopsy. The sandwich transposon integrates at a much higher copy number than a conventional transposon (7 and 1.5 copies, respectively). Clonal analysis demonstrated that the integrated copies were accompanied by a minimal risk of rearrangements. Integration profile of sandwich transposon is investigated by deep sequencing of large number of insertion sites.
Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, 141 86 Huddinge, Stockholm, Sweden
Huntingtin (HTT) is a ubiquitous protein that has a crucial function in embryonic development and other biological processes. Huntington disease (HD) is a severe neurodegenerative disorder caused by the increased number of CAG repeats leading to formation of a mutant HTT. Zorro-LNA is a newly developed, oligonucleotide (ON)-based, Z-shaped construct with the ability of strand invasion and specific binding to its target sequence in duplex DNA. We designed 4 Zorro ONs, two of them targeting adjacent sequences in the 5′-Untranslated region (5′UTR) and the other two targeting a stretch in the polyA1 region of the HTT gene. Each pair of Zorros was transfected using a cationic lipid-based transfection reagent into Human Embryonic Kidney 293 cells. Post transfection, RNA was prepared and multiplex RT-PCR was run to determine the remaining amount of HTT mRNA versus that of an endogenous gene. Interestingly, we have found that Zorros, in a final concentration of 50 nM, caused down-regulation of HTT gene ranging from 40–60%. Moreover, in time-course experiments, we have noticed that the onset of the blocking effect started already one day after transfection and lasted for four days (last time-point analyzed). Combination of Zorros targeting the two different sequences was markedly more efficient; 75% blocking of HTT was obtained. In this study, we show for the first time, that Zorro-LNA delivered by a cationic lipid could down-regulate HTT gene on the DNA level in mammalian cells. This finding represents a novel treatment strategy for HD.
Perinatal Brain Repair Group, Institute for Women's Health, UCL, London, WC1E 6HX
To test the effect of microglial activation on the neuronal regenerative response after spinal cord injury, we created a non-integrating lentiviral vector (NILV) expressing the potent microglia mitogen and activator GM-CSF and the reporter protein eGFP.
In vitro, supernatant from HEK-293T cells infected with GMCSF/eGFP NILV caused a dose dependent increase in cell density of the murine microglial BV-2 cell line. In vivo, stereotactic injections of GMCSF/eGFP NILV to outbred Sprague Dawley (SD) rat CNS revealed a strong neuroinflammatory response. To determine a viral concentration that results in moderate microglial activation with little CNS damage, rats were injected with GMCSF/eGFP NILV titre of 103-107 (PFU/ml). A dose dependent increase in microglia density and number of phagocytic microglia/macrophages, correlated with an increase in TUNEL labelling and a decrease in eGFP+ cells. The titre 103-104 PFU/ml of GMCSF/eGFP NILV produced moderate microglial activation but little cell death and phagocytosis. We are currently testing rats for possible trophic effects of GM-CSF on spinal cord regeneration.
Injections in the outbred CD1 mouse strain in the majority displayed no tissue damage and little microglial activation. To see whether this low susceptibility is general in mice, we have screened the response in six inbred mouse strains- C3HN, Balb/C, FVB, SJL, SVJ, and C57Bl6. 14 days after viral injection to the striatum, all strains demonstrated efficient transfection and microglial activation. However, C57Bl/6 showed avid neutrophil recruitment, Balb/C demonstrated strong microglial activation but no phagocytosis and the C3HN strain revealed many activated brain macrophages as those seen in SD rat after GMCSF treatment.
GE2.4, Bavarian Health and Food Safety Authority, Oberschleissheim, 85764, Germany
Viral vectors based on different adeno-associated virus (AAV) serotypes belong to the most promising tools in human gene therapy. For production of recombinant AAV vectors, researchers are focusing predominantly on cross-packaging an artificial AAV genome based on serotype 2 (AAV2) into the capsids derived from other serotypes. Within the packaged genome the inverted terminal repeats (ITRs) are the only cis-acting viral elements required for AAV vector generation and depict the lowest common denominator of all AAV2 derived vector genomes. Up to now, no quantitative PCR (qPCR) for the detection and quantification of AAV2-ITRs could be established due to their extensive secondary hairpin structure formation. Current qPCR-based methods are therefore targeting vector-encoded transgenes or regulatory elements. Herein we established a robust molecular biological method that allows accurate and reproducible quantification of AAV2 genomes based on an AAV2-ITR sequence-specific qPCR. Primers and labelled probe are located within the ITR sequence and have been designed to detect both, wild type AAV2 and AAV2-based vectors. This method is suitable for detecting single-stranded DNA derived from AAV2 vector particles and double-stranded DNA derived from vector plasmids even in the presence of high concentrations of spiked genomic DNA. The limit of detection has been determined at 50 ITR sequence copies per reaction by using a plasmid standard. In conclusion, this method describes the first qPCR system facilitating the detection and quantification of AAV2-ITR sequences. Since this method can be used universally for all AAV2-genome based vectors, it will significantly simplify AAV2 vector titrations in the future.
MolMed SpA, via Olgettina 58, 20132 Milano
Despite different technologies disclosed to generate packaging cells for lentiviral vectors (LV) manufacturing, as of today none corners the market yet. Hence new large scale production systems meeting safety, quality, cost containment and process feasibility demands are needed. As MolMed SpA is a leader on cellular and gene therapy product development counting on a cGMP facility authorized for production and release of medicinal products for humans, we generated two packaging cell lines for semi-stable (PK-7 clone) and stable (RD2-MolPack-Chim3.14 clone) manufacturing of HIV-based LV. We conceived a new strategy based on the use of integrating vectors for viral genes insertion into packaging cells. To obtain the PK-7 clone, we transiently transfected HEK-293T cells with a plasmid encoding the adeno-associated virus (AAV) Rep-78 protein and then infected them with a hybrid baculovirus-AAV vector, in which the baculovirus backbone contains an integration cassette expressing the HIV-1 gag, pol, rev genes and the hygro-resistance gene flanked by the AAV inverted terminal repeats (ITR) sequences. This system allows the Rep78-mediated integration of the ITR-flanked cassette into HEK-293T genome. From PK-7 we obtained the RD2-MolPack-Chim3.14 clone through the sequential integration of SIN-LV expressing either tat or RD114-TR envelope, and the LTR-LV expressing the anti-HIV Vif dominant negative transgene called Chim3. We provide evidence that PK-7 and RD2-MolPack-Chim3.14 clones, as an example of a prototype PK-7-based stable packaging cells, are suitable for large-scale production of LV for clinical application. It is envisageable that their employment will increase safety, cut the costs and reduce manufacturing time.
Characterising Receptor Usage and Trafficking of FX Binding-Ablated Adenoviruses in Vascular Cells
Although type 5 adenoviral vectors (Ad5) are excellent candidates for gene therapy, their efficacy and safety is severely limited by the sequestration of virus particles in the liver after intravascular delivery. Hepatocyte transduction by Ad5 is mediated by coagulation factor X (FX), which ‘bridges’ hexon proteins in the adenovirus capsid to the cell surface. We recently showed that a FX-binding ablated Ad5 vector (Ad5-HVR5*HVR7*E451Q/AdT*) could be retargeted to the lung by pseudotyping with an Ad35 fiber containing two amino acid modifications conferring increased affinity to CD46 (AdT*F35++). AdT*F35++ may also have potential for vascular gene therapy as CD46 is highly expressed in smooth muscle and endothelial cells. Here we characterise the receptor usage and endocytosis of AdT*F35++ in primary human vascular smooth muscle and endothelial cells.
FX significantly increased Ad5-mediated transduction of human saphenous vein endothelial cells (HSVECs) but not smooth muscle cells (HSVSMCs). No FX-mediated increase in transduction was observed using AdT* or AdT*F35++, however AdT*F35++ showed high levels of transduction in both cell types. The CD46 antibody MEM258 significantly reduced transduction by AdT*F35++ but had no effect on Ad5 or AdT*, while enzymatic removal of heparan sulphate (HS) ablated FX-mediated transduction by Ad5 only. FACS analysis of HSVECs and HSVSMCs demonstrated that while CAR expression was uniformly low, CD46, HS and αv integrins were expressed at high levels. Finally, although efficient endocytosis of fluorescently-labelled Ad5, AdT* and AdT*F35++ was observed, trafficking of AdT*F35++ to the nucleus was comparatively slower. These data indicate that AdT*F35++ is a promising candidate vector for vascular gene therapy.
San Raffaele Telethon Institute for Gene Therapy, Milan, Italy
Oncogenesis induced by insertional mutagenesis with integrating vectors occurs mainly by deregulation of proto-oncogenes found at or nearby the insertion site. Proto-oncogene activation occurs by an enhancer-mediated mechanism or by a process of splicing-capture generating chimeric transcripts comprising portions of vector and cellular mRNAs. Although oncogene activation may be reduced by the use of self-inactivating design and moderate cellular promoters, how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear. We developed a new PCR technique named Complementary-Linear-Amplification-Mediated-PCR (cLAM-PCR) which allows the retrieval of aberrantly spliced mRNAs that contain LV sequences fused with cellular transcripts from LV-transduced cell lines and primary human HSCs. We identified 70 different fusion transcripts, 84% of which were fused to known splice sites of genomic exons, 6% were fused to uncharacterized cryptic splice sites located in introns and the remaining 10% were fused to genomic sequences not corresponding to any annotated gene. Quantitative-PCR on different portions of the LV-backbone allows measuring the relative contribution to the aberrant splicing process of each LV splice site identified. The amount of transcription occurring in vector regions outside the expression cassette reached up to the 3% of that measured for the transgene. The cLAM-PCR technique, coupled to high-throughput sequencing allows gaining insights into the biology of vector-mediated splicing alteration. Since this process could induce neoplastic transformation by the generation of aberrant oncogenic protein, its in-depth characterization is instrumental in the development of next-generation LV with a higher safety profile.
Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, NSW 2145, Australia
There are many genetic metabolic disorders of the liver that are difficult to treat using conventional therapies. Liver-directed gene therapy presents a feasible therapeutic approach in these cases. Adeno-associated virus (rAAV) vectors have shown exceptional promise for liver-directed gene therapy, with numerous reports of successful phenotype correction in small and large animal models. One challenge that exists in moving research to the clinic is evaluating vector performance across a species boundary. The humanised FRG mouse model opens up new opportunities to test therapeutics in human hepatocytes in an in vivo setting.
This study set out to pseudoserotype AAV vectors with different capsids and exploit the humanised FRG mouse to study vector tropism and transduction efficiency in primary mouse, human and primate hepatocytes. After successfully engrafting hepatocytes into the mice they were transduced with rAAV2/2 and 2/8 vectors which expressed the reporter gene, eGFP, under the control of a liver specific promoter. Despite equivalent levels of vector genomes present in mouse and human cells after 2/8 delivery, transgene expression in human hepatocytes was approximately 20-fold less. This observation held for both single stranded and self complementary vectors. Interestingly, rAAV2/2 vectors more readily transduced human compared to mouse hepatocytes, with at least 8 times more vector present in human cells, although this did not translate to significantly higher expression levels. This study has come to highlight the importance of testing gene therapy vectors in a species-specific manner as well as understanding intracellular fate of vectors.
Novel disease-specific promoters for use in gene therapy for Parkinson's disease
Gene therapy is becoming one of the most promising future therapeutic tools for the treatment of Parkinson's disease (PD). The promoter used in the vector is important to make the therapy as safe and beneficial as possible.
In this study we show two approaches, one biased and one unbiased, to find novel promoters that are relevant for PD. In the biased approach, Drd1a, Drd2 and Dyn were chosen since they are involved in pathways affected by PD. In the unbiased approach, RNF25, ACE, DNAJC3, GALNS and MAP1a were chosen since they are highly expressed in patients with PD.
The promoters were evaluated in rat striatum using lentiviral vectors and eGFP as a transgene. RNF25, DNAJC3 and MAP1a showed the highest efficiency (6.1, 6.5 and 10%). All candidates had a neuronal specificity of more than 90%; Drd1a, MAP1a and GALNS showing an exclusive or nearly exclusive phenotype. Some candidates were also evaluated in the 6-OHDA model of PD to study the effect of dopamine depletion on transgene expression. DNAJC3 showed a non-significant tendency of a higher efficiency on the lesioned side.
This study shows that these approaches can be used to find novel promoters with high efficiency and specificity for use in gene therapy for PD.
Généthon, Evry, F-91002 France;
Retroviral vectors, and in particular HIV-1-based lentiviral vectors (LVs) are promising delivery systems for stable gene transfer into stem cells. Currently, the efficiency of gene transfer into CD34+ hematopoietic stem cells (HSC) is limited by incomplete transduction of the cell population. Transduction efficiency is dependent upon several factors such as vector infectious titers, the vector pseudotype or cell types. To potentiate lentiviral transduction of hCD34+ cells, protocols include the addition of fibronectin fragments or cationic compounds that enhance vector entry into target cells. The cationic amphipathic peptide LAH4-L1, an antibiotic and DNA transfection agent, was investigated for its capacity to promote lentiviral transductions. Our results show that LAH4-L1 strongly improves the transduction of human cord blood CD34+ cells with various lentiviral pseudotypes (i.e. GALVTR-LVs) but not with VSV-G-LVs. Using structure-function approaches, we designed LAH4-L1 derivatives. Two of them, LAH2-A6 (renamed Vectofusin1) and LAH4-A4 (Vectofusin2), strongly enhance the transduction of hCD34+ cells, ranging from a four-fold increase for VSV-G-LVs to two orders of magnitude for RD114TR-LVs. The efficiency of Vectofusins is comparable to RetronectinTM. Interestingly, viral particles lacking an envelope glycoprotein are unable to transduce target cells in the presence of Vectofusins, indicating that Vectofusins action is dependent on the establishment of a receptor-mediated entry pathway. Finally, using an HIV-1 fusion assay adapted to LVs, we show that Vectofusins are acting at the entry step by promoting the fusion between viral and cellular membranes. Vectofusins are therefore promising additives that could significantly enhance the efficacy of CD34+ cell-based gene therapy.
Université de Lyon, F69000; EVIR, Inserm, U758, Human Virology Department, F-69007; Ecole Normale Supérieure de Lyon, F-69007; Université Lyon 1, F-69007, Lyon, France.
Lentiviral transduction of quiescent lymphocytes is key for gene therapy. However, transduction with classical VSVG-pseudotyped LVs needs at least cell cycle entry to occur. We generated lentiviral vectors pseudotyped with measles virus glycoproteins (MV-LVs) that showed to be independent of the cell cycle status therefore allowing efficient and stable transduction of G0/G1a T and B cells with the bonus of maintaining their quiescent state. Since MV-LVs recognizes CD46 and SLAM molecules on the target cells' membrane we looked into the roles of each of these receptors on the transduction process. LVs that recognized only SLAM or CD46 receptors did not result in stable transduction of resting lymphocytes. Looking in depth, CD46-tropic vectors accomplished vector-cell binding, fusion, and reverse transcription but no provirus integration while SLAM-tropic vectors were blocked already at the level of fusion. Importantly, efficient and stable transduction of quiescent T and B cells only occurred when both CD46 and SLAM binding sites were present in cis in the MV-hemagglutinin envelope glycoprotein. In addition, the way of entry of MV-LVs appears to be crucial for the efficient transduction observed and strongly resembles macropinocytosis. Taken together we propose that vector entry and reverse transcription can already occur through CD46 receptor but SLAM binding and signalling is absolutely required to ultimately achieve successful integration and stable transduction of quiescent lymphocytes.
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany
The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. We compared retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, and found that SB expression may trigger premitotic cell cycle arrest followed by apoptosis. However, an improved approach of retrovirus particle-mediated mRNA transfer (RMT) prevented cytotoxicity by expressing SB in a transient and dose-controlled manner. Cytotoxic effects induced by continued SB overexpression were strictly dose-dependent and occurred even in the absence of a co-transfected transposable element. High and prolonged levels of SB were self-limiting due to the induction of caspase-mediated apoptosis within a few days after transduction. We observed substantial induction of p53 and c-Jun and increased numbers of γH2AX foci, suggesting genotoxicity as the underlying mechanism. However, inactivating mutations of the SB transposase DDE catalytic triad or variants impaired in nuclear entry reduced but could not abrogate cytotoxicity. Ongoing studies address the underlying mechanisms with the aim to generate a SB variant deprived from cytotoxic side effects. When using conventional SB or SB100X, we recommend reducing the level and duration of transposase expression via RMT or other transient expression methods to avoid the previously described overproduction inhibition and the newly discovered cytotoxicity.
Department of Genetics, UCL Institute of Ophthalmology, London
Optimization of miRNA-based knockdown lentiviral vectors
RNA interference is a post-transcriptional process that leads to specific gene silencing by targeting mRNAs. In combination with viral vectors this technique has emerged as a powerful tool for the elucidation of gene function. Despite the current advances, the development of more robust and versatile RNA-based silencing strategies remains a field of intense research. We combined the use of integrating HIV-based lentiviral vectors with the potency of artificial miRNA-based shRNAs (miR-shRNA).
In a first step, we evaluated whether the potency of miRNA-based knockdown improved by introducing multiple hairpin sequences (1, 2, 4 and 6 miR-hairpins) targeting the same seed-sequence. Second, we also combined miR-shRNAs that were aimed at different positions within the same target mRNA of the same reporter gene. Knockdown efficiency was monitored by qPCR analysis and functional assays.
We generated miR30-based constructs against a reporter gene (monomeric Red Fluorescent Protein) and 2 endogenous genes (human PSIP1 and PINK1). Lentiviral transduction of laboratory cell lines showed that increasing number of repeats improved knockdown efficiency only slightly. Introduction of more than 4 repeats even had a negative effect on the knockdown. In addition, introduction of two different target sequences against the same mRNA did not improve the degree of knockdown.
Goethe University Hospital, Department of Medicine II, Hematology/Oncology, Frankfurt, Germany
The potential of gene therapy as a curative treatment for monogenetic disorders has been clearly demonstrated in a series of recent phase I/II clinical trials. CD34+ cells used in gene therapy trials consist of a heterogeneous cell population including a large amount of cells with limited long-term repopulating capabilities. This poses a problem for gene therapy of Chronic Granulomatous Disease (CGD), in which low long-term engraftment was observed in most patients treated in clinical gene therapy trials.
The aim of this project is the improvement of HSC based gene therapy by specifically targeting cells capable of long-term engraftment. For this purpose we constructed lentiviral gene transfer vectors (LV) specific for CD133, a marker for long-term repopulating HSC. Targeting is achieved by incorporating the measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein into LVs. The hemagglutinin protein is blinded for the MV receptors and displays a single-chain antibody specific for CD133 (CD133-LV).
We are now investigating whether CD133-LV is superior to the commonly used VSV-G pseudotyped particles in transducing long-term repopulating CD133+/CD34+ HSCs. In in vitro cultures we detected an expansion of gene marked CD34+ cells after incubation with CD133-LV, while VSV-G-LV mediated transduction resulted in a decrease of transduced cells over time. Furthermore, CD133-LV transduced HSCs show a significantly improved engraftment capability in vivo in NOD-Scid/gc-mice. The power of this targeting approach is currently evaluated in a competitive repopulation assay. In summary, this new strategy may be promising to overcome the engraftment dilemma in CGD-gene therapy.
Department of Internal Medicine II – Campus Grosshadern, University Hospital of Munich, Germany
We have demonstrated the high potential of the sodium iodide symporter (NIS) in its dual function as reporter and therapy gene. Recombinant adenovirus-mediated gene therapy is a powerful strategy in cancer treatment. However, high promiscuity due to widespread expression of the coxsackie-adenovirus-receptor (CAR) and pooling in the liver are major limitations for its clinical application. Polymer coating of adenoviral vectors represents a promising tool to detarget adenoviral vectors away from CAR and liver towards tumor-specific targets.
We therefore coated a replication-deficient adenovirus carrying the hNIS gene under the control of the CMV-promoter (Ad5-CMV/NIS) with poly(amidoamine) dendrimers (PAMAM-G5). In this study we analyzed transduction efficacy and altered tropism of these coated virus particles using NIS as reporter gene.
In vitro experiments with PAMAM-G5 coated adenoviruses demonstrated enhanced transduction efficiency in CAR-negative cells (SKOV-3), which showed a 22-fold increase in iodide uptake. In vivo experiments showed high levels of radioiodine accumulation in the liver of mice four days after i.v. injection of 1×109 PFU Ad5-CMV/NIS as shown by123I γ-camera imaging, which were reduced by 70% after systemic injection of coated virus particles. Inhibition of liver pooling resulted in increased tumoral transduction efficiency as demonstrated by increased levels of iodide uptake in liver cancer xenografts. Liver detargeting and enhanced tumoral NIS expression of mice treated with polymer-coated virus were confirmed by qPCR and immunohistochemical analysis.
In conclusion adenovirus coating with synthetic polymers represents an efficient way to overcome adenoviral pooling in the liver, one of the most limiting barriers for systemic adenovirus-mediated gene delivery.
A novel replication-selective vaccinia virus vector to express diagnostic or therapeutic genes in hypoxic environments
Oncolytic viral therapy is a promising approach to treat cancers resistant to conventional therapies. However, hypoxic cells represent an aggressive and treatment resistant fraction of most tumours and even the efficacy of many viral vectors is attenuated in hypoxia.
Vaccinia virus is a DNA virus used as a vector for oncolytic viral therapy. We have previously reported that vaccinia virus replication and therapeutic gene expression are maintained in hypoxia. We have subsequently shown that Vaccinia virus replication is augmented by vascular endothelial growth factor (VEGF) in vitro and in vivo. Many hypoxic tumours express high levels of VEGF indicating that vaccinia virus is an ideal agent for targeting hypoxia.
A conventional Hypoxia Response Element (HRE) promoter based strategy cannot be used with vaccinia virus to target therapeutic or diagnostic gene expression to the hypoxic tumour fraction as virus replication occurs exclusively in the cytoplasm using viral transcription factors. As a proof of concept we developed a hypoxia targeting replicating vaccinia virus using a 5′ internal ribosomal entry site, the hypoxia inducible factor-1α oxygen degradation domain (ODD) and the VEGF 3′ untranslated region. This allows post-transcriptional, translational and post-translational regulation of reporter gene expression. This novel system minimised luciferase expression in normoxia, while allowing robust expression in hypoxia. This finding was reproducible in multiple cell lines, activity was sensitive to the degree of hypoxia and could be replicated using hypoxia mimetics. This novel vector may have potential application for development of new generations of oncolytic vaccinia viruses for diagnosis and treatment.
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
Recombinant human adenoviruses have great potential in anticancer therapy. One strategy to improve their tumor-cell specificity and anti-tumor efficacy is to include tumor-specific targeting ligands in the viral capsid. This can be achieved by fusion of polypeptide targeting ligands with the minor capsid protein IX. Previous research suggested that protein IX-mediated targeting is limited by inefficient release of protein IX-fused ligands from their cognate receptors in the endosome. This may thwart endosomal escape of the virus particles. Here we show that the targeted transduction of tumor cells is augmented by a cathepsin-cleavage site between the protein IX anchor and the HER2/neu-binding ZH affibody™ molecule as ligand. The cathepsin-cleavage site did not interfere with virus production and incorporation of the ZH Affibody™ molecules in the virus capsid. Virus particles harboring the cleavable pIX-ligand fusion in their capsid transduced the HER2/neu positive SKOV-3 ovarian carcinoma cells with increased efficiency in monolayer cultures, 3-dimensional spheroid cultures, as well as in SKOV-3 tumors grown on the chorioallantoic membrane of embryonated chicken eggs. These data show that inclusion of a cathepsin-cleavage sequence between protein IX and a high-affinity targeting ligand enhances targeted transduction. This modification further augments the applicability of protein IX as a natural anchor for genetic inclusion of tumor-targeting ligands into the adenovirus capsid.
Evaluation of a regulatable lentiviral vector overexpressing RARβ2 in the central nervous system
Damage to the adult central nervous system (CNS) can produce debilitating consequences. Previously, we have shown that lentiviral vector-mediated expression of the transcription factor retinoic acid receptor b2 (RARb2) promotes functional recovery in rodent models of corticospinal tract injury. We propose to express RARb2 in a regulatable manner, allowing us to finely tune the timing and dosage of RARb2, thereby optimising the therapeutic window of opportunity.
We used a novel regulatory system that tags an unstable protein domain (destabilizing domain - DD) to a protein of interest and targets it for degradation (Iwamoto et al. 2010). However in the presence of the ligand trimethoprim (TMP), the fusion protein can accumulate in the cell. The ability of TMP to cross the blood-brain barrier enables the tunable regulation of proteins in the mammalian CNS.
We first tested this system in human immunodeficiency virus (HIV)-based lentiviral vectors in vitro and in vivo using the reporter gene green fluorescent protein (GFP). In cell lines and primary neuronal cultures, the DD-GFP fusion protein showed dose-dependent expression over a range of TMP concentrations, with very low basal levels in the absence of TMP. Regulation was similarly tight in vivo, where GFP levels could be modulated by dietary TMP. Based on these findings, we constructed a DD-RARb2 fusion construct and tested its regulation in vitro and in vivo models of axonal regeneration. The development of a regulatable lentiviral vector overexpressing RARb2 will allow us maximise its therapeutic effects after nerve injury.
Généthon, Evry, F-91002 France;
Lentiviral vectors (LVs) are used for various gene transfer application, notably for hematopoietic gene therapy. Various envelope pseudotypes can be used although it is not entirely understood how cellular entry mechanisms contribute to cellular transduction. This prompted us to study the fusion step of LVs with hematopoietic target cells to determine whether it affects transduction efficiency. For this purpose, we adapted a fluorescence resonance energy transfer (FRET)-based fusion assay, initially designed to study replicative HIV-1 virions (Cavrois et al., Nat.Biotech., 2002), to study non replicative LVs. The assay utilizes recombinant LVs containing b-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) to measure fusion with target cells via delivery of BLAM-Vpr into the cytoplasm, which is detected by flow cytometry using the CCF2-AM substrate. We successfully generated high-titer, infectious recombinant BLAM-LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) and encoding the surface marker DLNGFR (truncated form of low affinity nerve growth factor receptor) using 6 plasmids co-transfection. The adapted FRET fusion assay using this VSV-G BLAM-LV is specific, sensitive (detection of fusion with less than 5% transduced cells), vector dose-dependent, and shows that transduction correlates with fusion in HCT116 cells and CD34+ cells. This fusion assay was used to characterize the effect of new transduction enhancers called Vectofusins. Data show that Vectofusins enhance VSV-G fusion with target cell membranes. In conclusion, this LV-based fusion assay is a powerful and versatile tool to concomitantly study LVs fusion and transduction in primary cells. Further investigation is ongoing to characterize Vectofusin peptides with different target cells.
Antiviral Gene Therapy Research Unit, School of Pathology, University of the Witwatersrand Medical School, Johannesburg, South Africa
Chronic infection with hepatitis B virus (HBV) remains a global public health problem of particular importance to sub Saharan Africa and parts of Asia. Licensed therapies for HBV infection do not effectively suppress viral replication. Consequently, life-threatening complications of the infection cannot be averted. Development of novel and effective therapies against this virus is therefore a priority.
Exploiting RNA interference (RNAi) enables sequence-specific target RNA degradation and has enormous potential for the development of novel antivirals. Lentiviral vectors have the useful property of stable integration into host cells enabling sustained expression of RNAi-activating sequences.
The recombinant lentiviruses were capable of transducing human liver-derived Huh7 and HepaRG cell lines in culture. Northern blot analysis verified that the anti-HBV primiRNA sequences were processed to form RNAi-activating guide sequences. When stably transduced cells were transfected with a HBV replication-competent plasmid, potent inhibition of markers of viral replication was achieved. Sustained, hepatotropic HBV silencing was effected with limited disruption of endogenous micro RNA function. Current evaluation of preclinical proof of principle in SCID mice that have been xenografted with lentivirus transduced human hepatocytes will provide insights into the potential utility of ex vivo gene therapy for serious chronic viral infections of the liver.
TetR-regulated all-in-one Lentiviral vector system for the easy generation of Tet-ON primary cell lines
Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and/or gene therapy approaches. However the controlled delivery of therapeutic genes represents one of the limiting factor of LV. The inducible systems based on antibiotics or hormones, represent an excellent alternative to this limitation. Among the existing inducible transcriptional gene regulatory systems, those, based on the Tet transactivator (tTA), a TetR-VP16 chimera, are the most widely used. However, several side effects has been recently described in some animal models due to the expression of transactivator domains. To avoid these secondary effect, a doxycycline-regulated system based on the original TetR repressor was developed as an alternative to the TetR-VP16 chimeras. In the present study, we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) or the EF1a promoter (ETetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the rtTA transactivator, can be expressed in excess to bind and modulate a high number of TetO operons. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells.
Georg-Speyer-Haus, Frankfurt, 60596, Germany
The alpharetrovirus family comprises simple retroviruses, such as the avian sarcoma and leukosis viruses (ASLV), with characteristics that make them highly attractive for gene therapy. ASLV are apathogenic in humans, ASLV-derived vector can be produced at high titers and even transduction of non-dividing cells has been reported. Moreover, studies on the integration pattern of ASLV have shown no preferences for integration close to transcription start sites or into active genes as it has been reported for gammaretro- and lentiviral vectors, respectively. Consequently, the genotoxicity profile of ASLV vectors is expected to be lower compared to other types of integrating vectors, thus reducing the risk of insertional mutagenesis. For our studies we have constructed and tested replication-deficient ASLV vectors with a self-inactivating LTR for the treatment of X-linked Chronic Granulomatous Disease (X-CGD). The construct under evaluation encodes a codon-optimized gp91phox gene driven by the constitutively active elongation-factor 1α short (EFS) promoter. Transduction of the human myelomonocytic cell line X-CGD PLB985 resulted in long-term stable expression of gp91phox and functional rescue of the X-CGD phenotype as determined by superoxide production, even at low vector copy numbers. Furthermore, primary murine (BM-Lin-) and human (CD34+) hematopoietic stem cells can be transduced efficiently with ASLV-derived vectors pseudotyped with a variety of envelopes. Studies on transplanted animals models of X-CGD are ongoing as are studies on optimal transduction of human CD34+ cells including transplantation in a humanized mouse model.
National Laboratory for Lentivirus Vector, Macrogen
Caprine arthritis encephalitis virus (CAEV), like all lentiviruses, can infect and replicate in dividing cells as well as in terminally differentiated and non-dividing cells. However, efforts to develop CAEV-based delivery systems have not been successful yet.
In order to develop an efficient CAEV-based lentiviral vector system we constructed CAEV transfer vectors carrying a high-level gene expression cassette and a hybrid 5′LTR sequences (hCMV enhancer/R/U5) and lacking the major splicing donor and acceptor sequences. However, although lentiviral vectors, as opposed to gammaretroviral vector systems, has been known not to show promoter interference, the CAEV transfer vectors produce abundant transcripts from internal promoter rather than full-length genomic RNA in transfected packaging cells. The promoter interference could not be overcome neither by replacement with other heterologous promoters nor by inserting with an insulator sequences.
When introducing splice sites in front of the internal promoter to make a coupling transcription and splicing, we obtained a strong enhancement in production of transcripts expressed from the 5′ hCMV enhancer/R/U5 hybrid promoter. However, most of the full-length transcripts were efficiently spliced and processed into non-pakagable, sub-genomic RNA, a major limitation for obtaining high titer of CAEV vectors. In order to enhance export of unspliced genomic RNA, Rev/RRE systems of CAEV and HIV-1 were introduced into the CAEV transfer vector system. As expected, the expression of CAEV or HIV-1 Rev proteins increased the amount of unspliced genomic RNA transcribed from the CAEV transfer vectors carrying CAEV RRE or HIV-1 RRE sequences, respectively.
Measles virus glycoprotein based lentiviral targeting vectors are partially protected against neutralizing antibodies
We have recently developed a targeting method for lentiviral vectors (LVs). To generate such LVs the measles virus (MV) glycoproteins hemagglutinin (H), responsible for receptor recognition, and fusion protein are incorporated into LVs. The H protein displays a single-chain antibody (scAb) specific for the target receptor and is blinded for the MV receptors by mutations in its ectodomain. Targeting vectors directed against e.g. CD20+ B-lymphocytes, CD105+ endothelial cells or CD133+ hematopoietic stem cells show high specificity. A theoretical impediment to systemic application of the vectors in humans is pre-existing antiviral immunity as almost all individuals have circulating anti-measles antibodies due to immunization or natural infection.
We compared transduction efficiencies of targeting vectors and non-targeting vectors pseudotyped with unmodified MV glycoproteins (MV-LV) in presence of MV antibody-positive human plasma. Using same MOIs and serial plasma dilutions, MV-LV was completely neutralized at dilution 1:160, whereas CD105-LV, CD133-LV and CD20-LV showed relative transduction efficiencies of 60%, 70% and 90%, respectively. At plasma dilution 1:80 an at least 4-times higher MOI of MV-LV had to be applied to obtain similar transduction efficiencies as with targeting vectors. Targeting vectors also exhibited less sensitivity to neutralization when normalized to p24 levels. In contrast, in MV antibody-free serum, targeting vectors and MV-LV did not behave differently and were not neutralized.
The reduced sensitivity to neutralization may be due to the modifications in the H protein ectodomain and the displayed scAb, which might hamper recognition by MV antibodies. These results are promising for in vivo applications in humans.
RHUL
The ideal vector for gene addition therapy needs to deliver DNA into the cell and promote transgene expression at the desired level without the need for re-administration and without interfering with host DNA. Non-integrating vectors have an improved safety profile compared to integrating vectors, but transgene retention is problematic as episomes usually get lost during cell division. To this end, we have developed an integration-deficient lentiviral vector (IDLV) system generating mitotically stable episomes capable of long-term transgene expression.
The potential of different DNA elements and cell culture conditions for assisting in episome establishment was explored. Structural elements of genomic DNA called the Scaffold/Matrix Attachment Regions (S/MARs) have been implicated in episome stability, and we further examined whether manipulating cell culture conditions following vector transduction affected the establishment of episomes.
In our system, IDLV transduction of CHO cells resulted in 15–50% of cells expressing the transgene for over 100 cell generations. Investigations into the episomal status of the vector genome using Fluorescent In Situ Hybridisation, Southern blotting and Linear Amplification –Mediated PCR followed by deep sequencing of vector-genome junctions strongly suggest that the vector is not integrated in the vast majority of cells. We are currently testing whether the system is similarly effective in a variety of cell lines. If maintained, such an approach could significantly improve the safety profile of a range of stem and progenitor cell gene therapies.
NEWVECTYS, Hôpital Pitié Salpêtrière, PARIS, France
In spinal cord injury (SCI), reactive astrocytes build-up a dense cellular structure, the glial scar, that forms both physical and chemical barriers considered to be detrimental for axonal regeneration. Reactive astrocytes present hypertrophic processes resulting mainly from increased expression of glial fibrillary acidic protein (GFAP) and vimentin, two astrocyte cytoskeletal intermediate filament proteins. It had been shown, in mice knockout for both GFAP and vimentin, that due to the absence of glial reactivity, reinnervation and partial motor recovery occurred after lateral spinal cord hemisection.
In order to translate this proof of principle into a therapeutic strategy, we have developed an experimental approach based on the use of siRNA carried by lentiviral vectors. We have developed lentiviral vectors Lv-siGFAP and Lv-siVIM which inhibit the in vitro formation of a glial scar in a Scratch model of lesion. Here we report that in a mouse model of SCI, intraspinal application of lentiviral vectors Lv-shGFAP and Lv-shVIM efficiently reduces glial reactivity by silencing GFAP alone or associated with vimentin, permits axonal regrowth and improves functional motor recovery.
With the objective of translation to clinics, we are adapting the work performed on mice to a large animal model (domestic swine), in order to mimic the clinical settings. We thus first designed and generated lentiviral vectors Lv-siGFAPsw able to inhibit GFAP expression in swine astrocytes. In parallel, we started to define the experimental surgical procedures of lentiviral transduction in the swine injured spinal cord by hemisection.
INSERM UMR649, Nantes, France
In nonhuman primate (NHP) skeletal muscle, recombinant Adeno-Associated Virus (AAV) genomes are stabilized as episomes with a chromatin-like structure. Epigenetic modifications targeting chromatin play a key role in mammalian transcriptional regulation. To assess the possible involvement of such modulation on the expression of a therapeutic protein, we injected mice and NHP via the intramuscular (IM) or the intravenous (IV) route with rAAV2/1 or rAAV2/8 vectors carrying a reporter transgene under the control of the constitutive Rous Sarcoma Virus promoter (RSVp). We followed the transgene mRNA expression level by RT-Q-PCR in skeletal muscle and liver samples as well as CpG methylation and histone post-translational modifications (PTMs) associated with the rAAV genomes. These two major epigenetic marks were investigated using respectively bisulfite high-throughput sequencing and chromatin immunoprecipitation (ChIP). We observed that the RSV promoter was much more efficient to drive expression in skeletal muscle than in the liver. DNA methylation analyses indicate no significant de novo CpG methylation along the RSV promoter short and long-term in transduced murine and nonhuman primate tissues. As direct DNA methylation is not responsible for this tissue-differential/specific expression, we looked for evidence of other regulatory mechanisms. Interestingly we showed that rAAV genomes are rapidly marked with either repressive or active histone marks after vector administration. This preliminary work suggests a negative impact on transcription due to particular histone PTMs.
Université de Lyon, F69000; EVIR, Inserm, U758, Human Virology Department, F-69007; Ecole Normale Supérieure de Lyon, F-69007; Université Lyon 1, F-69007, Lyon, France;
Gene transfer into quiescent T and B cells is of importance for gene therapy and immunotherapy approaches to correct hematopoietic disorders. Previously, we generated lentiviral vectors (LVs) pseudotyped with Edmonston measles virus (MV) hemagglutinin (H) and fusion glycoproteins (H/F-LVs), which represent the first tool allowing efficient transduction of quiescent human T and B cells. However, a major obstacle in the use of H/F-LVs in vivo for transduction of T and B cells is the fact that most of the population is vaccinated against measles. Importantly, the human humoral immune response appears to be almost exclusively directed against the H protein of MV. In a first approach, we therefore mutated the 2 immunodominant epitopes in H, Noose and NE. LVs pseudotyped with these mutated glycoproteins (HNoNE/F-LVs) escape inactivation by several monoclonal antibodies but were still neutralised by human serum. Thus, in a second approach, we took advantage of new naturally emerging MV genotypes that seems to be less sensitive to MV vaccination due to different glycosylation pattern. Introduction of these corresponding mutations into HNoNE/F-LVs reduced neutralisation by 50% as compared to the original H/F-LVs. Of importance, the modified H/F-LVs still allow high-level transduction of quiescent T- and B-cells, the in vivo gene therapy targets. Finally, in a “in vivo like setting”, we demonstrated that these modified H/F-LVs transduced efficiently quiescent T and B cells in the presence of high concentration of human serum, where the original H/F-LVs failed.
Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP
Baculoviridae comprises a unique group of arthropod-specific DNA viruses and in the last decade the use of these viruses as vectors for gene delivery into mammalian cells has increased. Their non-replicative nature, low cytotoxicity and ability to accommodate multiple genes or large DNA inserts make them an attractive alternative to mammalian viruses. Ischaemia reperfusion injury (IRI) commonly occurs during organ transplantation and is associated with hypoxia and free radical formation. Evidence suggests that the mitochondrial isoform of manganese superoxide dismutase (sod-2) is involved in cellular protection against free radical damage and mammalian viruses carrying sod-2 have previously been used in gene therapy to ameliorate ischaemic damage. In this study, the ability of baculovirus as a delivery vector to improve the “health” status of injured human embryonic kidney cells (HEK 293T) was tested. An in vitro ischaemia model causing intracellular ATP depletion was established using antimycin A and 2-deoxyglucose. A recombinant baculovirus carrying sod-2 under the control of the CMV/IE enhancer/promoter was constructed and the effect of recombinant gene expression on IRI was investigated. Furthermore, although baculoviruses infect arthropods, in this study the effect of the virus vector and the recombinant gene on the innate and adaptive immune response of HEK 293T cells was evaluated. RT2 Profiler PCR arrays showed that the presence of sod-2 activated the innate immune response pathway to a higher degree than the presence of the virus vector itself. These findings may have important safety implications in the future development of baculovirus as a gene therapy vector.
Adenovirus E1A binding to p300 is essential for synergistic cell killing in combination with chemotherapeutics in prostate cancer cells
Oncolytic adenoviruses and chemotherapeutics interact synergistically to enhance tumour cell killing with clinical efficacy demonstrated in several trials in combination with chemotherapeutics. It has previously been demonstrated that the early expressed viral E1A gene is essential for the sensitization to chemotherapeutics, while the underlying mechanisms have not been identified. We generated non-replicating adenoviral mutants expressing the small E1A12S protein with specific deletions in cell cycle regulatory protein-binding sites. We found that mutants with intact E1A-p300 binding (AdE1A12S, AdE1A1102, AdE1A1108) potently sensitized all tested prostate cancer cells including PC3 (androgen receptor (AR-) and p53-) and 22Rv1 (AR+, p53wt) to mitoxantrone and docetaxel while mutants with the p300-binding site deleted (AdE1A1104) could not enhance drug-induced cell killing. Deletion of the p400-binding (AdE1A1102) or the pRb-binding (AdE1A1108) did not compromise the sensitization to drugs. Comparing the non-replicating E1A mutants with the corresponding replicating deletion-mutants verified that both AdE1A1102 and dl1102 caused synergistic cell killing with mitoxantrone in 11 and 14 out of 16 conditions in PC3 cells and in 13 and 11 out of 16 in 22Rv1 cells respectively. The AdE1A1104 and dl1104 in combination with mitoxantrone either caused antagonism or low levels of synergy in both cell lines. These results indicate that the E1A-p300-binding region has an important role in sensitization of prostate cancer cells to chemotherapeutics. To identify the mechanism of enhanced cell killing and synergy, miRNA and mRNA array screening was performed and data from these studies will be reported.
Institute of Virology, Department of Pathobiology, University of Veterinary Medicine, 1210 Vienna, Austria
Vectors based on enveloped viral particles are widely used for gene delivery purposes. One way of modifying such vectors and thus adapting them to specific purposes is to change the protein composition of the viral envelope. Proteins modified with a post-translational glycosylphospatidylinositol (GPI) modification can be re-inserted to lipid bi-layer membranes such as viral envelopes after purification. This process, called molecular painting, can be used to modify the surface of retroviral or lentiviral (RV/LV) vectors as well as herpes virus particles. Modification is quick and flexible, since a broad range of proteins can possibly be deposited on different viral species. The method was originally described in 2008, demonstrating the deposition of the human complement regulatory factor CD59 on murine leukemia virus (MLV) and human immunodeficiency (HIV)-based viral vectors. Progress has been made regarding technical characterization and possible applications of the technique. Gene therapy approaches can benefit from viral painting in a number of ways: from simple viral particle labeling to modular transduction targeting systems. The most recent developments including dual painting, fluorescence and growth factor painting have further underlined the potential of molecular painting as an emerging technique and a valuable addition to the gene therapy toolbox.
Optimization of efficacy in AAV-mediated siRNA expression
Adeno-associated virus (AAV) vector is useful in applications to gene expression. Approach to gene silencing by encoding siRNA molecules is also promising. In search of effective vector structure for knockdown experiments, we made a series of AAV vectors and compared their capability. For monitoring of gene transfer efficiency, GFP sequence driven by CMV promoter was incorporated as a downstream expression cassette. The vector was designed to allow exchange siRNA sequence easily using H1 or U6 promoters. For each promoter, two different vector structures were made; the same or the reverse orientation to GFP cassette. These four structures were compared in the following aspects. At first, we tested the production rate of vectors based on AAV2 capsid and no differences were found. Secondly, we transduced 293 and HeLa cells using these vectors. Again, no differences were found in GFP expression levels. To determine the efficacy of siRNA expression quantitatively, siRNA sequence against GAPDH, a housekeeping gene, was incorporated. In both 293 and HeLa cells, GAPDH expression was significantly suppressed at lower vector dose. In both cells, vector construct using U6 promoter in the same orientation of GFP cassette worked best, suppressing more than 95% of endogenous GAPDH mRNA at a regular vector dose (30,000 vg per cell). These results indicate that AAV-mediated efficient knockdown of gene expression is feasible, and the vector structure affects efficacy of mRNA knockdown.
Molecular Immunology Unit, Institute of Child Health UCL, London WC1N 1EH, UK
The autologous transplant of genetically modified hematopoietic stem cells, or stem cell gene therapy (HSC-GT), is a novel strategy that has been successfully used to treat disorders of the immune system, such as the severe combined immunodeficiency due to lack of adenosine deaminase (ADA-SCID), or degenerative disorders such as the X-linked Adrenoleukodystrophy.
In cases where the lack of a functional protein affects more than one system, it would be advantageous to provide a constant, systemic supply of the protein. We have developed a gene expression system in a lentiviral vector whereby use of the b-globin LCR (b-LCR) upstream of the EFS-1a promoter allows for high transgene expression in the erythrocyte lineage, whilst maintaining expression in other hematopoietic lineages.
We have tested our lentiviral vector in cell lines and in vitro models of erythroid differentiation, and shown that the b-LCR vector upregulates transgene expression in erythroid lineages. In a murine transplant model, the b-LCR vector showed equivalent levels of expression to a control vector in lymphoid and myeloid lineages but >20x over expression of a reporter (GFP) or therapeutic transgene (ADA) in erythrocytes even up to a year after transplant. Experiments to assess the safety of our lentiviral vector are underway but in a standard in vitro immortalisation assay there is no evidence of transformation above control values.
This vector system has great potential for correction of a number of diseases including ADA SCID and other metabolic conditions where disease correction is dependent upon high level systemic enzyme delivery.
Georg-Speyer-Haus, Frankfurt, Germany
During the follow-up of our X-CGD gene therapy trial we observed clonal expansion and downregulation of transgene expression due to CpG methylation at the promoter region. To counteract epigenetic effects on transgene expression we used ubiquitous chromatin opening elements (UCOEs), which are methylation-free CpG islands spanning divergently transcribed promoters of housekeeping genes with chromatin opening capability. For our studies we used a 1.5 kb DNA fragment derived from the core region of the UCOE from the human HNRPA2B1-CBX3 gene locus. This element includes the promoter region and the first exon of the HNRPA2B1 and CBX3 genes. Introduction of this element in combination with the myeloid specific promoter MRP8 into a lentiviral vector resulted in high, persistent, specific and copy number dependent expression of gp91phox. However, initial safety assays in growth factor dependent cell lines revealed a potential for insertional mutagenesis caused by aberrant splicing of transcripts initiated from HNRPA2B1 promoter. This observation was confirmed by the analysis of aberrant transcripts at the site of vector integration. In all cases analyzed, fusion transcripts between the first HNRPA2B1 exon and cellular exons were found. To reduce the probability of insertional mutagenesis, we modified the HNRPA2B1 splice donor site, added the polyadenylation signal downstream of the HNRPA2B1 promoter, and included the target sequence of miRNA142 within the HNRPA2B1 transcript. The effect of these modifications on expression specificity from the MRP8 promoter was confirmed in vivo. Currently the genotoxicity potential of this construct is being tested by qRT-PCR and in cell culture assays.
Suppression of miRNA activity by vector-encoded inhibitors
miRNAs have emerged as important regulators of translation and are believed to play a role in almost any cellular process and as potential triggers of disease. Whereas emerging therapeutic strategies aim at mimicking the action of miRNAs to knock-down gene activity, a new generation of small RNA drugs may target miRNAs rather than mimicking them. Suppression of miRNAs can be achieved by administration of synthetic chemical miRNA inhibitors, but this strategy may suffer from the need for repeated administration and poor delivery in hard-to-transfect cell types. Here, we investigate the suppression of miRNA activity by DNA-encoded RNA inhibitors as an alternative to synthetic agents. The efficiency of suppression of seven different types of vector-encoded miRNA inhibitors (including five miRNA decoys, a premiRNA-directed shRNA, and a miRNA target site mask) was compared by using a luciferase reporter assay in which the luciferase expression was made responsive to the target miRNA. In studies of miR-16 targeting in HEK-293 cells, neither the shRNA nor the mask relieved miR16-mediated suppression of the reporter, whereas three of the five decoy strategies exhibited potent suppression of miR-16. We are currently analyzing the seven inhibition strategies for targeting of other miRNAs, and studies have been initiated that will compare the miRNA suppression capacities of the seven types of inhibitors when expressed from lentiviral vectors.
Development of a HEK293T clonal suspension cell line for the production of high titre EIAV lentiviral vector
Equine Infectious Anaemia Virus (EIAV) derived lentiviral vectors (LVs) are being developed by Oxford BioMedica for a number of gene therapy applications. Currently, EIAV LV material is manufactured by the transient co-transfection of adherent HEK293T cells with three plasmids that encode the necessary components for LV production. However, this method is cumbersome and not easily scalable as it is restricted by the adherent growth mode. One solution is to develop a suspension HEK293T cell line that could be transiently co-transfected with three plasmids that encode the necessary components for vector production. This would be advantageous over the current adherent process as EIAV LV production could be performed at large scale (bioreactors) and in a serum-free (SF) environment. Like all transformed cell types HEK293T cells show karyotypic abnormalities with published karyotypes varying greatly. It could be that isolation of clones from the current HEK293T cell bank could lead to the identification of a clone whose karyotype is advantageous for EIAV LV production.
Here we describe the isolation of one-hundred HEK293T clones capable of producing high titre EIAV LVs, and the adaptation of the best clones to SF suspension culture. The suspension clones were then screened for their ability to produce EIAV LV and compared to EIAV LV produced using the current adherent HEK293T transient co-transfection process.
Laboratoire de Thérapie Génique, INSERM UMR649, Nantes, 44007, France
We have developed A549-based packaging cell lines, which contain the AAV2 rep gene and the cap gene from various AAV serotypes. These cell lines efficiently assemble rAAV particles upon dual adenovirus infection (wild-type Ad5 followed by hybrid Ad/AAV), which is correlated with amplification and high-level expression of the integrated rep-cap genes. The cell lines are fully documented and their stability has been extensively tested. In addition, they are adapted to low serum conditions but they still remain in an adherent state.
For production scale-up, we chose to use a fixed-bed bioreactor system: the high-cell density iCELLis™ system developed by ATMI. This technology consists of a disposable bioreactor system with a fixed-bed design operating in perfusion mode, which is available at different scales and has a modular design: in function of the single cell volume, the target cell density and the required cell quantity, several fixed-bed volumes and compactions can be provided.
For cell culture process development, we used our A549/rep2-cap8 cell line maintained in medium supplemented with 1% FBS. The cells were adapted into the iCELLis™ nano system for optimization of culture parameters, then rAAV2/8 production was tested following dual adenovirus infection. Several production assays were performed, each giving comparable yield of rAAV2/8 per cm2 in the bioreactor as compared to a production conducted in CellStack vessel.
Thus, the iCELLis™ system provides a scalable cell culture support by which large-scale, GMP-compliant AAV production can be considered without the need for our cell lines to be adapted to suspension culture.
Gene Transfer Technology Group, Institute for Women's Health, University College London, 86–96 Chenies Mews, London, WC1E 6HX, UK
Many genetic diseases present pathology in a range of tissues and organs throughout the body. One example is Gaucher disease where a variety of tissues are affected including the liver, spleen, nervous system, bones and a range of haematological abnormalities. Although enzyme replacement therapy can effectively treat the visceral manifestations, some tissues, such as the bones, remain refractive to therapies and lifelong treatment of the disease is expensive. Furthermore, enzyme replacement therapy is not available for rarer diseases.
We evaluated the tropism of single-stranded (ss) and self-complimentary (sc) AAV2/9-GFP following intravenous administration to fetal (E15) and neonatal (P1) mice. The animals were culled and the visceral tissues were analysed for GFP expression using fluorescence stereoscopic microscopy, immunohistochemistry, immunofluorescence, scanning confocal microscopy and qPCR analysis. We report highly efficient gene delivery to a range of tissues, organs and cell types using both ss- and sc-AAV2/9 e.g. liver, lung, heart, spleen, skin, kidney, intestine, bladder, muscle and vasculature. Interestingly, bone and cells within the bone marrow were also transduced. To further investigate vector tropism scAAV2/9 was intravenously administered to late gestation fetal macaques. We observed widespread gene delivery and confirmed our observations in mice within a non-human primate model.
We, and others, have previously demonstrated that systemic administration of AAV2/9 mediates efficient gene delivery to the nervous system. The data presented here support the potential that this vector has for treating perinatal lethal systemic diseases that affect both the brain and the viscera for which conventional medicine currently offers no treatment.
Department of Haematology, UCL Cancer Institute, 72 Huntley St, London, WC1E 6BT, UK
Our on-going haemophilia B (HB) clinical trial show therapeutic levels of human FIX following a single bolus administration of recombinant adeno-associated virus (rAAV) vector in all six subjects recruited. However, immune-mediated, AAV8 capsid-specific clearance of transduced hepatocytes remains a concern. As this phenomenon is vector dose dependent, there is an urgent need to develop more potent vectors so that therapeutic gene transfer can be achieved using lower doses of rAAV. To this end, we have developed a novel single-stranded rAAV that contains a liver-specific regulatory element (LP1), codon optimised human FIX (hFIX) cDNA and an pigenetic modifier (ssAAV-LP1-hFIXco-EM). Tail vein injection of 4×1012vg/kg of AAV8 pseudotyped ssAAV-LP1-hFIXco-EM in male C57Bl/6 mice resulted in hFIX expression at 188% of normal, which was at least 10 fold higher than in mice transduced with the same dose of ssAAV-LP1-hFIXco (17±1% of normal), which does not contain the EM. Administration of a log lower dose (4×1011vg/kg) of ssAAV-LP1-hFIXco-EM resulted in human FIX expression at 7±2.2% of normal, sufficient to ameliorate the bleeding phenotype in patients with severe HB. In contrast, subtherapeutic (0.3%) levels of hFIX were observed in mice transduced with a comparable dose of ssAAV-LP1-hFIXco. Increased hFIX expression following gene transfer with ssAAV-LP1-hFIXco-EM was associated with higher hFIX RNA levels and a 3 fold decrease in the AAV associated H3K9me2 heterochromatin marker. Thus EM improves the potency of rAAV by preventing epigenetic silencing of the provirus. These data are promising as they enhance the prospects of safe and efficient gene transfer in humans.
Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany
Primary murine hepatocytes are partially refractory to lentiviral (LV) vector transduction. The inefficient transgene delivery to the target cells poses a problem for the ex vivo gene transfer to mouse models of monogenetic liver diseases. In our attempts to optimize the transduction protocols we found that culture media supplemented with 10 ng epidermal growth factor (EGF) significantly improved transduction efficacy of mouse hepatocytes at various multiplicities of infection (MOI 1–128, VSVg pseudotype). Transduction in absence of EGF resulted in a maximum of 30.3% (±15.1%, MOI of 128) transgene positive cells, whereas the LV gene transfer in presence of the growth factor enabled us to achieve levels of up to 88.4% (±1.6%, MOI of 128). The increased number of transgene expressing cells correlated with increased nuclear import of pre-integration complexes (1.8 fold) as determined by 2-LTR circles, upregulation of genes active in the priming phase of hepatocytes (c-fos, cyclin D1) and elevated global metabolic activity. EdU incorporation assays, the absence of S- and M-phase specific cyclins and the unsuccessful attempts of gammaretroviral transduction ruled out the possibility of proliferation as the driving force for enhanced transgene delivery to hepatocytes. EGF had a significant positive effect on long term (up to day 6 in culture), short-term (24 hours in adherence) as well as ultra-short term (4 hours in adherence or suspension) transduction. After over-night transduction in adherence and subsequent transplantation hepatocytes still showed liver repopulation capacities similar to freshly isolated cells in alb-uPA mouse recipients.
Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany
The effective entry of retroviruses into target cells depends on the presence of viral envelope (Env) proteins and cognate cellular receptors, such as the murine cationic amino acid transporter mCAT-1 for the ecotropic murine leukemia virus (MLV-E). We examined whether human cells internalize MLV-E as well as other retroviral and lentiviral particles with different pseudotypes irrespective of the presence of a specific receptor. Using fluorescently tagged Gag to monitor viral internalization and treating cells with the lysosomotropic substances chloroquine or bafilomycin A1, we show that viral uptake did not depend on mCAT-1 or Env. Confocal microscopy revealed colocalization with late endosomes and lysosomes. Further studies testing the Gag-mediated transfer of recombinant protein or mRNA revealed that non-specific endocytosis prevented the release of intact particle-associated proteins and nucleic acids into the cytosol. Similar results were obtained for MLV particles pseudotyped with measles virus H and F proteins as well as for ecotropic or non-enveloped, “bald” HIV-1 particles: non-specific and thus, non-productive, endocytosis occurred independent of the pseudotype, and led to a surprisingly long persistence of transduced protein in exposed cells (several days for primary T lymphocytes). We conclude that retroviral receptor-targeting strategies are unlikely to escape unspecific uptake unless appropriate protective principles will be discovered, thus potentially trapping viral particles in endosomes and contributing to antigen presentation. Conversely, as lysosomal degradation was found to inactivate functional mRNA and proteins embedded into retroviral particles, receptor-targeting is a useful strategy for the targeted delivery of biomacromolecules by retrovirus-like particles.
German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany
Severe side effects caused by therapeutic gammaretroviral vectors have been observed in gene therapy trials for the treatment of various immunodeficiencies. We have previously reported on a clinical trial for the treatment of X-linked chronic granulomatous disease (X-CGD) where insertional activation of MDS1-EVI1 has led to clonal expansion of gene marked myeloid progenitor cells. Since the gammaretroviral vector has also proven to be an efficient tool for the transduction and correction of hematopoietic stem cells, an optimized self-inactivating vector was developed (SINfes.gp91s) aiming to improve the safety and efficacy of X-CGD gene therapy. High throughput integration site (IS) analysis allows us to follow the fate of transduced cells in vitro and in vivo. Here, we present the comparative analysis of a murine serial transplantation model with SINfes.gp91s- versus SF91eGFP-transduced hematopoietic stem cells using LAM- and LM-PCR and subsequent next generation sequencing (454/Roche).
In primary as well as in secondary mice we observed a more polyclonal IS pattern for SINfes.gp91s mice with an average of 754 and 170 unique IS compared to 530 and 83 unique IS in SF91eGFP mice. While the overall chromosomal and genomic distribution of IS was similar with both vectors, we detected significantly fewer clones with IS in or close to the MDS1-EVI1 locus in SINfes.gp91s mice and with no signs of clonal dominance. Furthermore, we observed significantly more common integration sites of higher order in secondary mice of SF91eGFP mice than of SINfes.gp91s mice. Overall, we found that SINfes.gp91s displays a more favorable safety profile than the SF91eGFP vector.
Brain tumor selective gene expression and therapy mediated by recombinant retroviruses containing the SSX4 promoter
Despite many efforts to develop effective therapy, the outcome of malignant glioma remains poor. Gene therapy for this disease using retroviral vector is attractive, because the virus can infect only mitotic cells. Previously, we reported the eradication of mouse glioma by retroviral-mediated gene therapy. In this study, a tumor-specific targeting system was studied to develop the effective and safe gene therapy. We searched for genes expressing at high frequency in brain tumors but not in normal human astrocyte (NHA) among cancer testis antigen (CTA) genes. MAGEA3 and SSX4 were identified as tumor-specific genes. The promoter of both genes was cloned into luciferase reporter vector and the activity was measured in glioma, telomerase-immortalized fibroblast and normal human astrocyte cells. The SSX4 promoter but not MAGEA3 showed the tumor-specific activity. The minimal promoter of SSX4 was defined as a 256 bp fragment upstream of transcriptional start site. In order to define a useful tumor-specific promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to restrict the expression of suicide gene HSVtk in retroviral vector. The glioma cell lines transduced with the retroviral vector were killed efficiently by addition of ganciclovir (GCV), but telomerase-immortalized fibroblast BJ-5ta cells were not. Mouse glioma RSV-M cells transduced with the retroviral vector were transplanted into syngeneic mice subcutaneously. The administration of GCV suppressed the tumor growth completely. These results support identification of a tumor-specific promoter capable of providing safe and effective retroviral-mediated gene therapy for malignant glioma.
Investigation into the Entry and Intracellular Trafficking of a Bacteriophage-based Gene Therapy Vector
Bacteriophage-based gene therapy vectors have shown great promise for systemic delivery of cytotoxic transgenes to cancer. RGD-Phage vectors, which are targeted to alpha-v integrins overexpressed on tumour vascular endothelium and tumour cells, have successfully demonstrated targeting and tumour regression in several in vivo cancer models. Bacteriophage vectors have several advantages over their traditional eukaryotic counterparts, in that they are cost-effective and considered safe as they have no intrinsic tropism for mammalian cells unless engineered for this purpose. However, one major disadvantage of bacteriophage-based vectors is low transduction efficiency as, unlike eukaryotic viruses, they have not evolved strategies for escaping the cell's natural defences to viral pathogens. This has led us to investigate the entry and intracellular trafficking events involved in transduction by RGD-Phage, in order to identify barriers to transduction in an attempt to engineer more efficient gene therapy vectors.
We have used a combination of microscopy techniques, biochemical analysis and reporter gene assays to elucidate RGD-Phage uptake in a number of in vitro cancer models. We have shown that targeted Phage entry is alpha-v integrin dependent and that virus entry appears to occur independently of clathrin-mediated endocytosis. Following entry, our data suggests that transgene expression is limited by sequestration of the majority of RGD-phage particles by the endosomal/lysosomal degradative pathway. These findings may have important implications for the development of the next generation of bacteriophage vectors.
Centre for Neurosciences, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Burlington Danes Building, Du Cane Road - London W12 0NN
Lentiviruses have been effectively used as vector systems for the delivery of genes to cell populations in vitro and in vivo. The use of heterologous glycoproteins in place of the lentiviral native envelope, a process known as pseudotyping, can greatly expand the host range and tropism of the vector, often resulting in tropism similar to that of the virus from which the glycoproteins were derived. Venezuelan Equine Encephalytis Virus (VEEV) pseudotyped lentiviruses have not been tested to date in vivo, but successful use of other alphaviral pseudotypes has been reported. High titers of eGFP HIV-1 lentiviral vectors pseudotyped with strain 3908 of VEEV-G were generated and concentrated 2000-fold prior to injecting stereotaxically in the striatum of adult rats. Brains were harvested 5 and 8 weeks post injection and analysed by immunohistochemistry and confocal microscopy. At the injection site, VEEV pseudotype exhibited distinct neuronal tropism (NeuN), that besides medium spiny neurons also involved cholinergic (ChAT) neurons. The neuronal transduction efficiency increased over time. No microglial (ED1) and only minor astroglial (GFAP) tropism was observed, interestingly decreasing over the two time points examined. Anterograde transport was detected in cortical areas of the frontal lobe, in the olfactory bulb and in substantia nigra (SN) pars reticulata, but no transduction of SN pars compacta dopaminergic neurons was observed, implying absence of retrograde vector transport. In this study we demonstrate sustainability of expression and minimal host immune response in vivo, making this pseudotyped vector, potentially useful for therapy of neurodegenerative diseases.
Gene Transfer Technology Group, Institute for Women's Health, University College, London, 86–96 Chenies Mews, London, WC1E 6HX
Haemophilia A is a compelling candidate for treatment with gene therapy as therapeutic benefit only requires a modest increase in the endogenous coagulation factor level, response to treatment can be easily monitored, and FVIII expression can be mediated by many cell types in vivo. B-domain deleted (BDD) FVIII protein retains full procoagulant function and is expressed at higher levels than wild type FVIII. However a partial deletion of the B-domain leaving an N-terminal 226 amino acid stretch (N6) has been previously reported to increase in vitro secretion of FVIII tenfold compared to BDD-FVIII.
Previously, our group has shown that expression of FVIII protein from a codon optimised cDNA sequence increases expression in comparison to the wild type sequence over 30-fold in vivo yielding over 200% normal human FVIII levels in neonatal mice after injection of a SIN lentiviral vector. A longstanding goal for treatment of haemophilia A using gene therapy is to maintain sustained production of FVIII in the absence of an immune response in adult mice. To achieve this we have produced an optimised vector system including our codon-optimised FVIII N6 cDNA sequence under control of the liver specific promoter LP1 to prevent off target expression and maximise expression in hepatocytes. Further to this, we have also included target sequences for the hematopoietic specific microRNA, miR-142-3p, to eliminate off target expression in antigen presenting cells. With this approach we hope to achieve stable long term expression of FVIII in adult FVIII knock-out mice.
Gene therapy of Fabry disease - using foamy virus vectors
Foamy virus (FV) vectors have several characteristics, which make them perfect candidates for gene therapy, such as a large packaging capacity, self-inactivation, integration in the genome, lack of tumorigenesis and high rates of hematopoietic stem cells (HSC) transduction. An additional advantage of these retroviruses is their apathogenicity in their natural host. Fabry disease is a X-linked recessive lysosomal storage disorder caused by the lack of alpha galactosidase A (a-gla A) which results in a disability to catabolize alpha-D-galactosyl glycolipid moieties. This leads to an accumulation of globotriaosylceramide (Gb3) in various tissues and organs, causing a wide range of systemic symptoms including renal failure and cardiac symptoms.
In this study, we generated novel FV vectors which express the a-gla A gene under the control of various promoters. To test the therapeutic potential of this vector, primary cells derived from a Fabry knock out mouse were used. We measured the galactosidase activity in HSCs and embryonic fibroblasts of wild type and knockout mice which confirmed efficient gene transfer and restoration of a-gla A enzymatic activity. To achieve a whole body expression of the enzyme, HSC from a knockout mouse were isolated and transduced overnight with a FV vector, these cells were then injected in lethally irradiated sister mice. The mice were analyzed for a-gla A activity in the plasma and different organs at several time points post injection. This in vivo model provided the first experimental prove of principle and evaluated the efficacy of FV vector system for gene therapy of Fabry disease.
Group of Gene Therapy, Department of Biotherapeutics, National Institute for Biological Standards and Control, Blanche Lane, Potters Bar, EN6 3QG, UK
One problem during transient in vitro production of viral vectors is the formation of incomplete particles that lack vector genome, hence reduced the amount of functional particles and can cause an immune response or be toxic when used in gene therapy trial.
To characterise vector preparations vector particles need to be purified from the cell culture supernatant. In current purification methods for lentiviral vectors a compromise has to be made between high vector yield and high purity of vector particles.
We applied Size Exclusion Chromatography (SEC) to purify two types of lentiviral vectors: cat endogenous retrovirus RD114 pseudotypes produced by stable packaging cells and transiently produced VSV-G pseudotypes. We have also compared them without addition of the viral genome to find possible difference between complete and empty particles. The majority of vector particles of all four preparations eluted in the void volume, but problems in recovery of infectivity have been identified.
Advantages and limitations of this purification method will be outlined.
Gene Expression and Therapy Group, King's College London School of Medicine Department of Medical and Molecular Genetics, Guy's Hospital, London, SE1 9RT, UK
The Ubiquitous Chromatin Opening Element from the human HNRPA2B1-CBX3 locus (A2UCOE) provides reproducible and stable expression within lentiviral vectors independent of the site of integration in haematopoietic cells. Furthermore, an A2UCOE-IL2RG lentiviral vector efficiently rescues the SCID-X1 phenotype in mice at a low (<1) vector copy number (VCN) per haematopoietic stem cell (HSC). Here we tested A2UCOE transgene expression in vivo under circumstances where unlike the SCID-X1 condition, selection of corrected cells would not occur. Human blood clotting factor IX (hF.IX) cDNA-A2UCOE (A2UCOE-hF.IX) lentiviral vector was functionally analysed by prenatal and ex vivo HSC approaches in mice.
Prenatal delivery of A2UCOE-hF.IX via the vitelline vessel at embryonic day 14 resulted in efficient transduction of the fetal liver. A constant plasma level of hF.IX (1.74mg/ml) was detected up to 7 months post-natally. The average concentration of hF.IX per 200mg total liver protein was 1.26mg/ml with average VCN of 0.2 per liver genome detected. No vector copies or hF.IX protein were found to be present in either peripheral blood or bone marrow cells.
Ex vivo HSC delivery gave on average 0.25mg/ml plasma hF.IX with a VCN of 0.47 and 0.44 per peripheral blood and bone marrow cell respectively at 2 months post-transplantation.
These results show that the A2UCOE-hF.IX cassette is highly effective as low VCN in peripheral blood and bone marrow cells (0.44 copies per cell) and especially liver (0.2 copies per cell) readily provides stable, curative hF.IX levels for the treatment of Haemophilia B under non-selective conditions.
Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guilford Street, London, WC1N 1EH, UK
Loss of gene expression due to epigenetic-mediated silencing following gene transfer has been an obstacle in achieving efficacy in gene therapy. We have previously shown that the human HNRPA2B1-CBX3 housekeeping gene locus (UCOE) can give rise to stable transgene expression in haematopoietic stem cells (HSCs) in vivo (zhang et al. Blood, 2007). Furthermore, we have also shown that, unlike the viral promoter, the stability of UCOE in driving transgene expression is due to its resistance to DNA methylation-mediated silencing (Zhang et al. Mol. Therapy, 2010). In our recently study, we have assessed a long term efficiency of UCOE in driving interleukin-2 receptor gene (IL2-RG), in the mouse model SCID-X1. It is shown from our study that the lentiviral vector containing IL2RG driven by UCOE can fully rescue the phenotype of SCID-X1 mouse model following the engraftment of transduced HSCs. The reconstituted T and B cells remained at a stable level at the different time points (5, 8 and 12 months post-transduction). Splenocytes from the transduced mice showed a similar level of proliferation index compared to the wild-type mice in response to cytokine and mitogen stimulation. Moreover, the efficiency of UCOE is achieved at a low vector copy number (VCN) (the average VCN of 0.18/cell in bone marrow, and 0.29/cell in spleen). There was no observed genotoxicity among the experimental mice (n=32). These data provide convincing evidence that the UCOE indeed confers a reliable, long-term transcriptional activity in HSCs In vitro immortalization assay for assessing genotoxicity of UOCE vectors is underway and will be discussed.
Division of Medical Biotechnology, Paul-Ehrlich-Institut, Paul-Ehrlich-Str. 51-59, 63225 Langen, Germany
T cell receptor (TCR) gene transfer which introduces tumor-specific TCRs into T cells is an attractive strategy in cancer immunotherapy. Currently used gene-transfer tools, like VSV-G pseudotyped lentiviral vectors (VSV-LV), do not distinguish between different human cell types and equally transduce CD8-positive and −negative T cells. Here we present CD8-targeted LVs and their applicability for TCR gene transfer.
CD8-LV mediated selective and stable reporter-gene transfer into CD8 T cells, both, in vitro in human peripheral blood mononuclear cells (PBMC), and in vivo in NOD-scid-IL2rγnull mice engrafted with human PBMC. This vector also efficiently delivered the coding information for a high and a low affinity tyrosinase specific TCR (T58 and D115) into CD8 T cells as revealed by tyrosinase tetramer staining. T58 expressing T cells specifically lysed HLA-A2+/tyrosinase+ melanoma cells via granzyme B and perforin. Strikingly, the genetically modified T cells killed tumor cells reproducibly more efficiently when transduced with CD8-LV than with VSVG-LV. This effect became even more pronounced, when the low affinity TCR D115 was applied. TCR surface density, expression levels of IFNγ, IL-2, and TNFα, as well as the cell proliferative activity were excluded as potential causes for this difference between targeted and non-targeted vector. However, the CD8-surface-expression level in CD8-LV transduced T cells was significantly higher than that of VSV-LV transduced cells.
We hypothesize that CD8 targeting results in an increased CD8 density which enhances antigen recognition and tumor cell killing. These promising results suggest that CD8 targeting can improve the efficiency of TCR gene therapy.
Department of Gene Therapy, University of Ulm, Ulm, 89081, Germany
Plasmodium falciparum is the causative agent of tropical malaria. One of the most promising candidate antigens for vaccination is MSP-1, a protein expressed on the surface of P. falciparum merozoites. However, due to the high AT content of its gene, virus-based vectors expressing full-length MSP-1 frequently have unstable genomes and are difficult to produce. Here we demonstrate that using codon-optimized full-length MSP-1 allows for the high-titer generation of ΔE1 and high-capacity adenovirus vectors and we provide a comparison of MSP-1-specific B- and T-cell responses induced by these vectors in mice.
After a single intramuscular immunization with HC-Ad or ΔE1-Ad expressing full-length MSP-1 broad antibody responses against the MSP-1 subunits p83, p38, p42 were induced. The titers of the antibodies were equivalent for both vector types (HC-Ad or ΔE1-Ad) and independent of the subcellular localization of MSP-1 (GPI-anchored or secreted). Importantly, independent of vector format, antibody titers were three-fold higher when MSP-1 expression was driven by the chicken ß-actin/CMV hybrid promoter (CAG) instead of a CMV promoter.
To analyze MSP-1-specific T cell responses, we identified 3 Ld-restricted epitopes on MSP-1. After immunization of Balb/c mice with ΔE1-Ad, CD8 T cells specific for all three epitopes were induced. Surprisingly, after immunization of mice with HC-Ad, the frequencies of specific IFN-γ-secreting CD8 T cells were significantly higher compared to immunization with ΔE1-Ad.
Overall we demonstrate that expressing full-length MSP-1 by adenovirus vectors is a promising strategy and our data suggest that HC-Ad may have advantages for this purpose.
Clinic for Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
Polysialic acid (pSia) is a cell surface antigen that plays an important role in neuronal development but is not expressed in most adult tissues. However, expression of pSia is reactivated during oncogenesis of several cancers including glioblastoma, rhabdomyosarcoma and small cell lung cancer (SCLC). pSia therefore represents an excellent tumor selective target for oncolytic adenovirus based therapies.
In this study, we developed a strategy to retarget oncolytic Adenoviruses (OAV) to pSia-expressing tumors using a recombinant bispecific adapter protein. For the adapter CARex-pSia, the soluble CAR ectodomain was fused to a pSia-binding domain linked via a trimerization motif. By infection experiments in CHO and pSia-negative CHO mutant cell, we could show that CARex-pSia treatment of Ad5 efficiently allowed viral cell entry in a pSia-selective manner. Furthermore, CARex-pSia treatment allowed efficient adenoviral infection of normally refractory pSia-expressing human tumor cells. After intravenous injection of CARex-pSia-treated Luciferase-Ad we could observe a significant reduction of adenoviral hepatotropism in mice and could show improved adenoviral infection of s.c. grown pSia-expressing human tumor xenografts in mice. Therapeutic investigations were performed in a syngeneic, murine SCLC model induced by intravenous delivery of transgenic, pSia-expressing CMT64 cells in C57/Bl6 mice. Systemic administration of CARex-pSia pretreated OAV allowed for successful transduction of lung colonies as shown by in vivo imaging and qPCR of virus DNA, and finally led to significantly improved survival of mice.
Together, we could demonstrate that retargeting OAVs to cell surface pSia represents a promising strategy for the oncolytic treatment of clinically relevant pSia-expressing tumors species.
Dev Infection and Immunity, Royal Free Hospital, London, UK. NW3 2PF
The function of T cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR a/b heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognised lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared to T cells modified by TCR only. Following tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when re-challenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.
Inhibition of Immune-suppressive TGFβ in Tumour Models
Despite expressing antigens that can induce immune-surveillance and eradication, tumours are able to overcome anti-tumour immunity. Recently, this has been attributed to what is described as ''locally acquired immune privilege'' which is currently a major obstacle for tumour immunotherapy, and vaccination. Key mediators of this effect are soluble pleiotropic cytokines including tumour growth factor beta (TGFβ) and Interleukin-10, which can be released by tumour and stromal cells, and are thought to play a significant role in producing this local immune-suppression. To understand this mechanism in greater detail we developed a system to allow the selective inactivation of soluble immunosuppressive molecules within the tumour microenvironment, to allow their effects on tumour growth, and tumour immunity status to be assessed. To antagonize TGFβ we established a stable cell line of mouse Colon Carcinoma CT26 cells expressing the soluble extracellular domain of TGF beta receptor II (TGFβRII) under a tetracycline inducible expression system (tet-on). The ability of the system to allow tight regulation and the induction of high levels of gene expression was demonstrated both in vitro and in vivo using a CT26 cell line expressing luciferase. This study shows that the use of the tet-on inducible expression system can allow the temporal control of genes which may have important roles in tumour development and immune control. This system should allow better understanding of the influence of individual cytokines on the development of local immune-suppression in tumours and help to develop new strategies to overcome it.
Targeting of CD133-specific tumor stem cells with oncolytic measles virus
Tumor stem cells (TSCs) are regarded as attractive target for novel anti-tumoral strategies as they have been shown to be responsible for metastases formation and to be resistant towards radio- and chemotherapy. A prominent marker for TSCs of glioblastoma is CD133 also known as prominin-1. Here we propose to develop a novel anti-tumoral strategy based on oncolytic Measles viruses (MV) with high selectivity for the infection and killing of TSCs. We fused the reading frame of a CD133-specific single-chain antibody to that of mutated H protein unable to enter cells via the natural MV receptors CD46 and SLAM. The resulting virus (MV-CD133) was absolutely specific for CD133-positive cells such as the hepatocellular cancer derived HuH7 cell line. MV-CD133 infection resulted in a substantial and dose dependent killing of HuH7 cells, which was more efficient than that of the unmodified parental virus MV-Nse. Upon intravenous injection into mice carrying a subcutaneous HuH7-derived tumor, MV-CD133 infected larger areas of the tumor tissue than MV-Nse. Intratumoral injection of MV-CD133 into subcutaneously growing HuH7 tumors resulted in a substantial reduction of tumor growth and a prolonged survival as compared to control treated animals. Remarkably, survival was even significantly longer than that of mice treated with MV-Nse, which is currently used in clinical trials. Our data suggest that MV-CD133 will serve as a novel tool to improve our understanding about the role of tumor stem cells in oncology and as innovative strategy in tumor therapy.
Department of Research & Development, Amsterdam Molecular Therapeutics; Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
ATP-binding cassette (ABC) transporters are drug efflux pumps responsible for the multidrug resistance phenotype causing hepatocellular carcinoma (HCC) treatment failure. Here we studied the expression of 15 ABC transporters relevant for multidrug resistance in 19 paired HCC patient samples (15 untreated, 4 treated by chemotherapeutics). Twelve ABC transporters showed up-regulation in HCC compared to adjacent healthy liver. These include ABCA2, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, ABCC10, ABCC11, ABCC12 and ABCE1. The expression profile and function of some of these transporters have not been associated with HCC so far. Since cellular microRNAs (miRNAs) are involved in post-transcriptional gene silencing, we hypothesized that regulation of ABC expression in HCC might be mediated by miRNAs. To study this, miRNAs were profiled and dysregulation of 90 miRNAs was shown in HCC compared to healthy liver, including up-regulation of 11 and down-regulation of 79. miRNA target sites in ABC genes were bioinformatically predicted and experimentally verified in vitro using luciferase reporter assays. In total 13 cellular miRNAs were confirmed that target ABCA1, ABCC1, ABCC5, ABCC10 and ABCE1 genes and mediate changes in gene expression.
CAR Mechanics Group Research Oncology 3rd Floor Bermondsey Wing Guy's Hospital St. Thomas Street SE1 9RT London
In patients with squamous cell carcinoma of the head and neck (SCCHN), treatment-associated morbidity is common and 5 year survival rate remains at 50%. Therefore, new therapies with greater efficacy are urgently required. To address this, we have developed a GMP compliant process for the expansion of genetically enhanced tumour-specific T-cells. Tumour specificity is imparted by engineering T-cells to express a chimeric antigen receptor (CAR) named T1E28z. T1E28z recognises an array of homo- and heterodimers of the four members of the ErbB family, which are over-expressed in over 90% of SCCHN and are implicated in the pathogenesis of SCCHN. T1E28z is co-expressed with a chimeric cytokine receptor named 4ab, using a T2A-containing retroviral vector. The 4ab provides a strong IL-2-like growth signal upon binding of IL-4, facilitating selective expansion of transduced T-cells using this cytokine.
Blood samples were obtained from patients with SCCHN. T-cells were activated using CD3+/CD28+ paramagnetic beads and transduced 72 hours later. Selective expansion of T4+ was facilitated by growth in IL-4. After 14 days, the functionality was assessed through coculture with ErbB expressing tumour monolayers. Despite similar initial transduction rates, the two donors showed very different enrichment and expansion patterns over time. Cells from both patients were able to destroy tumour monolayers in a T4-ErbB interaction dependent manner. The efficiency of tumour destruction and cytokine production correlates with the rate of T4 expression. These results indicate that this protocol can be applied in a forthcoming phase I clinical trial involving patients with loco-regional recurrent SCCHN.
Lucy Cavendish College, Cambridge, CB3 0BJ
Oncolytic adenoviruses replicate selectively within cancer cells, causing lysis and cell death. However, sensitivity to virus cytotoxicity varies greatly, even within cells of similar infectivity. Our data show that virus-induced genomic DNA damage and checkpoint signalling are critical determinants of oncolytic cell death. In highly sensitive TOV21G cells, E1A-induced expression of cdc25A leads to over-replication of genomic DNA, accumulation of replication-associated DNA breaks and activation of the ATR-Chk1 signalling pathway. In contrast, cells resistant to adenovirus oncolysis undergo minimal over-replication following infection, thereby evading lethal genomic damage.
Inhibition of ATR-Chk1 signalling using both siRNA and small molecule inhibitors (UCN-01, PF) promotes virus-induced genomic over-replication via two mechanisms: (i) suppression of proteasome-dependent degradation of cdc25A to facilitate overreplication and associated genomic DSB formation; and (ii) blockade of Chk1-mediated recruitment of Rad51 to sites of DNA damage, thereby augmenting the accumulation of DSB by impeding homologous recombination (HR) repair. ATR-Chk1 pathway inhibition sensitises resistant (A2780CP) and partially sensitive (IGROV1) ovarian cancer cells in vitro to both Ad5 WT and the E1A CR2 deletion mutant dl922–947 and increases dl922–947 activity in mice bearing IGROV1 intraperitoneal xenografts.
Agents that promote the generation of virus-induced genomic DNA damage, either through aberrant initiation of genomic DNA replication or by inhibition of HR repair, may be used to potentiate virus oncolysis.
Institute of Hepatology, Foundation for Liver Research, London, WC1E 6HX, UK
Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death, with a 5-years survival rate for advanced HCC of only 7%. Recombinant adeno-associated virus (rAAV) vectors may be ideally suited for gene transfer-based therapeutic approaches for HCC because of their excellent safety profile and remarkable tropism for the liver. Tail vein injection of rAAV8 in tumour-bearing mice resulted in selective transduction of the liver as well as HCC but not neuroblastoma xenografts. The specificity of rAAV-mediated transgene expression for HCC was further improved by cloning the nucleotide sequences with perfect complementarity to microRNA-122a into an expression cassette that included a novel liver-specific promoter (HLP), thus taking advantage of the differential expression of miR-122a, which is abundant in normal liver tissue but is down regulated in most HCC. Transduction of HCC cell lines with this vector showed that normalized transgene expression was at least 26.5 fold higher in cells negative for 122a when compared to those that were positive. Tail vein injection of rAAV8-HLP-LUC resulted in high transgene expression in liver but mice transduced with an equivalent dose of rAAV8-HLP-Luc-122aT had little or no luciferase expression in the liver despite the presence of the proviral DNA. In contrast, subcutaneous HCC xenografts in mice showed strong luciferase signal following tail vein injection of both of these vectors. Our novel vector, therefore, has the potential to deliver therapeutic transgene to HCC in preference to normal hepatocytes, thus limiting toxicity.
Department of Oncology, ORCRB, University of Oxford, Oxford, OX3 7DQ, UK
Coload1, a chimeric adenovirus based on group B adenoviruses Ad11p and Ad3 was recently developed by using a bioselection methodology. However, the oncolytic behaviours of both Ad11p and Coload1 have not yet been systematically investigated.
In this study, we assessed the oncolytic abilities of Coload1 compared with Ad11 and Ad5, including the potency and kinetics of oncolysis, virus infectivity and cellular uptake, selectivity of cell killing and replication and neutralisation by blood factors. The results have shown that Coload1 and Ad11p share similar properties of high cellular uptake and resistance to human serum neutralization; in contrast, Ad5 has a much poorer performance in most of the analyses. Coload1 also shows a strict selectivity in replication and killing of tumour cells rather than normal cells including primary hepatocytes, fibroblasts and endothelial cells; moreover, Coload1 is the most potent oncolytic agent among the three viruses measured both by the end point IC50 value and by killing kinetics. In addition, the virus is found capable of circumventing cancer drug resistance in studies using drug resistant HCT116 (human colorectal cancer) and Capan1 (human pancreatic cancer) cell lines. The virus also induces efficient bystander killing in a fibroblast/A549 co-culture system. Overall, these data suggest Coload1 is a potent oncolytic virus that could have good activities in clinical trials in the future.
Clinical Data of Recombinant Adeno-viral Human p53 Gene (rAd-p53), Gendicine®, in Treatment of Human Solid Tumors
P53 protein has multiple anti-tumor functions and p53 gene mutation is found in about 50% of human malignant tumors. Recombinant adenoviral human p53 gene (rAd-p53) was approved by SFDA as the first anti-tumor gene product in the world. Since it's approval in 2004, rAd-p53 has been used for treatment of over fifty thousands of tumor patients and over 50 types of solid malignant tumors. It is also used for pre-cancerous lesions such as oral leukoplakia and cervical displasia. Over 100 scientific publications demonstrated that rAd-p53 is effective and safe in treatment of solid malignant tumors and a synergic effect when combined with chemotherapy, radiotherapy, or other anti-cancer therapies. The pre-registration phase II clinical trial was a multi-center, open-labeled, active control, randomized study, and enrolled 135 subjects with advanced nasopharyngeal cancer. The overall response rate (RR) for experimental group (EG: rAd-p53+ radiotherapy) was 93%, significantly higher than that of 79% from control group (CG: radiotherapy alone). The overall survival (OS) rate and progress free survival (PFS) rate of 5 years in EG were 66.7% and 58.7%, respectively, which were both higher than those of CG (59.2% and 47.0). With a similar study design, a phase III study enrolled 400 cases with advanced nasopharyngeal cancer, 300 subjects in EG and 100 in CG. The RR of the EG was 92.69%, significantly higher than that of CG (79%). The 3-years OS and PFS of the EG are significantly higher than those from CG.The safety results from various clinical studies indicate rAd-p53 is safe. The main adverse effect is self-limited fever.
Department of Internal Medicine II – Campus Grosshadern, University Hospital of Munich, Germany,
We recently reported therapeutic efficacy of 131I in hepatocellular carcinoma (HCC) after adenoviral NIS gene delivery under control of the tumor-specific α-fetoprotein promoter (AFP). In the current study we investigated the efficacy of a replication-selective adenovirus, in which E1A is driven by the AFP promoter and hNIS is inserted in the E3 region driven by the E3 promoter (Ad5-E1/AFP-E3/NIS) using NIS as reporter gene.
In an earlier study in vitro experiments in HCC and control cancer cells demonstrated high transduction efficiency and tumor-specific replication of Ad5-E1/AFP-E3/NIS. In the current study in vivo experiments showed functional NIS expression in HCC xenograft tumors after adenoviral NIS gene transfer as shown by 123I gamma camera-imaging as well as qPCR. HCC xenografts accumulated a maximum of approx. 25% ID/g (percentage injected dose/gram tumor tissue) 72h after intratumoral injection of 5×108 PFU Ad5-E1/AFP-E3/NIS with an average effective half-life of 5h, while control tumors (thyroid cancer and melanoma xenografts) revealed no significant iodide uptake. A single intratumoral injection of Ad5-E1/AFP-E3/NIS resulted in a significant reduction of tumor growth with prolonged survival of virus-injected mice, as compared to control groups.
These studies demonstrate high transduction efficiency, tumor-selectivity and strong oncolytic activity of a replication-selective adenovirus that expresses NIS in a replication-dependent manner, which opens the prospect of image-guided oncolytic adenovirus therapy of liver cancer with the possibility of further enhancing therapeutic potency by additional radionuclide therapy.
Department of Internal Medicine II – Campus Grosshadern, University Hospital of Munich, Germany
Recombinant adenovirus-mediated oncolytic and gene therapy are powerful strategies often combined in cancer treatment. Polymer coating of adenoviruses represents a promising tool to increase the load of viable virus reaching peripheral tumors after systemic application by detargeting adenoviral vectors away from the coxsackie-adenovirus receptor (CAR) and liver. We therefore coated a replication-selective oncolytic adenovirus, in which E1A is driven by the α-fetoprotein (AFP) promoter and hNIS is inserted in the E3 region driven by the E3 promoter (Ad5-E1/AFP-E3/NIS), with poly(amidoamine) dendrimers (PAMAM-G5). In this study we analyzed transduction efficacy and oncolytic potential of these coated virus particles in vivo in a liver cancer xenograft mouse model using NIS as reporter gene.
After a single intravenous injection of 1×109 PFU of Ad5-E1/AFP-E3/NIS, a significant delay in tumor growth was observed when polymer-coated adenovirus was used as compared to uncoated adenovirus, which showed no therapeutic effect, thereby indicating higher levels of viable virus reaching the tumor after polymer coating. Serial 123I γ-camera-imaging confirmed significantly higher levels of tumor-specific, NIS-mediated iodide accumulation after application of coated adenovirus as compared to uncoated virus. Pronounced tumoral iodide uptake was detected up to 15–18 days after infection with a maximum at 3 days, demonstrating high transduction efficiency and efficient replication of Ad5-E1/AFP-E3/NIS in the tumor.
Our data demonstrate that polymer coating is able to increase the load of viable virus reaching peripheral tumors after systemic application thereby increasing transduction efficiency and therapeutic efficacy of an oncolytic adenovirus.
Oncolytic Adenovirus Driven by the SCG3 Promoter and ASH1 Enhancer for Neuroblastoma Therapy
Replication-selective oncolytic adenoviruses are widely used in cancer therapy. Here, we evaluated an oncolytic adenovirus Ad5PTD(ASH1-SCG3-E1A) in which the E1A gene is controlled by the human secretogranin III (SCG3) promoter and the murine achaete-scute complex homolog 1 (ASH1) enhancer. The virus is further modified with a cell penetrating peptide (Tat-PTD) in the hexon hyper variable region 5 (HVR5), which we have previously shown increased transduction efficiency for many neuroendocrine tumor cell lines.
Secretogranin III belongs to the chromogranin/secretogranin family of neuroendocrine secretary proteins and it is widespread in endocrine and neural tissues. The promoter of SCG3 has previously not been characterized. We identified a 0.5kb region upstream of the SCG3 gene that can selectively drive luciferase expression in neuroblastoma cell lines. In addition, we found the specificity and luciferase expression level to be increased by adding the ASH1 enhancer in front of the SCG3 promoter.
Based on these finding, the oncolytic adenovirus Ad5PTD(ASH1-SCG3-E1A) was constructed. It shows selective and efficient killing of neuroblastoma cell line in vitro. The therapeutic effect will next be evaluated on tumor bearing nude mice.
Cancer Gene Therapy Group, University of Helsinki, Finland and Department of Obstetrics and Gynecology, Helsinki University Central Hospital, Finland
Redirecting and augmenting the immunological antitumor response is a promising way to enhance the efficacy of oncolytic adenoviral therapy. We constructed Ad5/3-D24-GMCSF (CGTG-102), a 5/3 capsid modified virus coding for GMCSF. CGTG-102 was used to treat patients with advanced solid tumors refractory to standard therapies.
Evidence of biological activity of the virus was seen in 62% of patients receiving a single injection. 67% showed objective disease control (CT scans). Virus replication was detected in 80% of patients for up to 58 days.
Since it is well established that immune response can be boosted by multiple dosing, we compared a single injection to administration of three doses 3–4 weeks apart (n=109). Serial treatment did not increase frequency or grade of adverse events. All patients experienced grade 1–2 adverse events including flu-like symptoms. Grade 3–4 nonhematological adverse events were seen in 11% of single treated versus 12% or 7% (after 2nd and 3rd treatment) of serial treated patients. Median overall survival increased from 112 days to 277 days with serial treatment, while 300 day survival increased from 33% to 48%. Although the inclusion and exclusion criteria are identical, it should be noted that randomization was not performed. Both patient series seem to compare favorably to a historical controls. Induction of antitumor T-cells was more frequent in serial treatment.
In conclusion, more immunological effects and longer survival are seen in serial treated patients, while adverse events are not increased. Thus, we plan to use multiple injections in an upcoming phase 1–2 clinical trial.
vincent.kao@kcl.ac.uk
Chimeric antigen receptors (CARs) are fusion molecules that can re-target T-cell specificity against a chosen tumour-associated cell surface antigen. In breast cancer, aberrant expression of the MUC1 mucin and members of the extended ErbB family are commonly found in different molecular sub-types. We have previously engineered CARs directed against MUC1 (HOX) and the extended ErbB family (T1E28z). T-cells engrafted with HOX or T1E28z exert anti-tumour activity in-vivo when administered to SCID Beige mice bearing established breast cancer xenografts. In each case however, therapeutic effect is transient and T-cell longevity is a limiting factor. To improve this, we have engineered a chimeric cytokine receptor named 4αβ. In 4αβ, the ectodomain of the IL-4 receptor α subunit has been fused to the endodomain of the shared β receptor used by IL-2 and IL-15. Addition of IL-4 to 4αβ+ T-cells elicits a potent growth and survival signal, comparable to that delivered by IL-2 or IL-15. Here, we have developed a system to achieve targeted IL-4 delivery to the tumour microenvironment, in order to support immunotherapy using 4αβ-HOX or 4αβ-T1E28z-engineered T-cells. To achieve this, we have engineered a plasmid vector that encodes for a latent form of IL-4 (LAP-IL-4) in which the cytokine is surrounded by a LAP-TGFβ shell. This shell is cleaved by matrix metalloproteinase (MMPs), which are found at high levels in breast tumours. Tumour models to test this approach have been established and proof of principle for the ability of this system to achieve sustained IL-4 release in-vivo has been obtained.
Dept. Hematology, Erasmus MC, 3015 GE Rotterdam, The Netherlands
The approval in China of the cancer gene therapy drugs Gendicine (in 2003) and Oncorine (2005) startled the research field; the issue of medical tourism to China for gene therapy treatment received wide attention and concern. However, in recent years special attention to gene therapy in China has faded. The present study was initiated by The Netherlands Commission on Genetic Modification to retrieve information from Chinese sources, esp. Chinese literature databases, Chinese scientists and scientific meetings in China related to gene therapy development and risk assessment. Even though no special gene therapy hallmarks have been reported in China recently, the number of publications on gene therapy increased nearly 10-fold between 2000 and 2009, esp. in the last 5 years, whereas these numbers were more or less stable in other countries. The total number of gene therapy clinical studies approved in China that could be retrieved for this study was 44. Research activities are similarly diverse as in Europe and the United States. More than 10 companies in China are focused on gene therapy. A prominent focus is apparent for disease areas that have a relative high prevalence in China, e.g., liver related disorders. Although predictions on the further development of the gene therapy field in China cannot be made from the study, potentially it may become the country that contributes most data from gene therapy clinical trials. Recording and publication of these data should be further improved to consolidate clinical developments in the gene therapy field and foster collaborative development.
A YB-1-dependent, armed oncolytic adenovirus as a candidate for nuclear reporter gene imaging and suicide tumour gene therapy
Oncolytic adenoviruses (AdV) are promising tools in gene therapy, as they have the ability to eradicate tumour cells. Hence, we investigated whether a YB-1-dependent oncolytic AdV armed with the reporter and suicide gene herpes simplex virus thymidine kinase (HSV1-sr39tk) displays an enhanced tumour cell killing through the combination of oncolysis and HSV1-sr39tk-Ganciclovir (GCV) mediated cytotoxicity in human glioma cells, and if this AdV is suitable for in vivo imaging studies. YB-1-dependent oncolytic AdV have so far not been combined with suicide genes. Previous data indicate that the oncolytic activity of YB-1-dependent AdV can be promoted by cytostatics via triggering the accumulation of YB-1 in the nucleus. We tested if oncolysis can be enhanced with Temozolomid (TMZ), a commonly used cytostatic in glioma therapy.
An enhanced overlapping oncolytic and HSV1-sr39tk-GCV-mediated cytotoxic effect (>95%) was observed in infected glioma cells, even at low viral doses and GCV concentrations <0.1 μg/ml. The strongest cell killing was achieved if GCV was applied 2–3 days after infection. We also observed an increased bystander effect in co-cultured infected and uninfected cells. Furthermore, the enzymatic function of HSV1-sr39tk was measured by uptake of [18F]-FHBG, a radio-labelled prodrug-analogue. As expected, HSV1-sr39TK displayed an enhanced accumulation of [18F]-FHBG within infected cells. Even at 2.5 μM TMZ and co-administrating TMZ with GCV, oncolysis increased drastically (>80%).
The results of this study indicate that the HSV1-sr39TK expressing oncolytic adenovirus effectively induces tumour cell killing and radiotracer accumulation ([18F]-FHBG) and might allow for in vivo PET imaging within a limited time frame.
Effects of Producer Cell Type on the Properties of Oncolytic Sindbis Virus
A major obstacle in the field of virotherapy is the production of a vector that is capable of systemic intravenous delivery to treat tumour metastases that are too small to resect with surgery. In vivo half-life is limited primarily by clearance from the bloodstream by the liver and the stability of the viral vector following exposure to blood components.
Sindbis virus displays natural tumour selectivity and has demonstrated efficacy in mouse models against several tumour types. During transmission of Sindbis from infected mosquitoes to humans, virus particles are exposed to blood. As such, we hypothesised that the virus may have significant stability in human blood, making it compatible with intravenous delivery. Furthermore, we postulated that Sindbis virus produced from insect and mammalian cells may have significantly different blood stability and tissue tropism due to differing envelope lipid composition and glycosylation patterns on surface proteins.
We utilized mammalian and insect producer cells to produce two separate preparations of Sindbis virus. In vitro we compared their infectivity on a range of cell lines, ability to activate complement and their inhibition of infection by human blood components. In vivo we analysed their respective pharmacokinetics and biodistribution in mice. We found a 3.62 fold higher activation of complement in vitro by insect derived compared to mammalian derived Sindbis virus. No significant differences, however, were observed in in vitro cell line tropism, in vitro inhibition of infection by human blood components, or pharmacokinetics and biodistribution in mice.
Uppsala University; Dept Immunology, Genetics, Pathology; Rudbeck Laboratory; Uppsala, Sweden
Gastrointestinal (GI) neuroendocrine tumors (NET) have generally metastasized to the liver at the time of diagnosis, and surgery is seldom curative at that point. It is our belief that oncolytic viruses could be beneficial for this group of patients.
We have systematically developed an oncolytic virus for GI-NET liver metastases. The virus is based on human adenovirus serotype 5 (Ad5) and the E1A gene, which controls the viral gene expression cascade, is under transcriptional control of the human chromogranin A (CgA) promoter. This leads to selective virus replication in neuroendocrine cells. Furthermore, in order to selectively detarget virus activity from normal liver cells, six copies of the target sequence for the liver-specific microRNA, miR122, was incorporated in the 3′UTR of E1A. This led to specific degradation of E1A mRNA and blockage of virus replication in hepatocytes.
Some GI-NET cells express low levels of CAR, the native receptor for Ad5 infection. However, they often express high levels of somatostatin receptors (SSTRs). Therefore, we introduced cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site for SSTRs in the virus fiber knob. We show that FWKT-modified Ad5 binds to SSTR2 on NET cells by using proximity ligation. Furthermore, it transduces ileal NET cells from liver metastases about 3–4 times better than non-modified Ad5. We conclude that a replication-selective oncolytic adenovirus modified with the FWKT motif may be beneficial for therapy of neuroendocrine tumors.
NO.14 3rd Part Renmin South Road, Chengdu 610041, China
zhe.liu@kcl.ac.uk
The aim of this project is to develop prostate cancer immunotherapy using T-cells that express the P28z chimeric antigen receptor, targeted against prostate-specific membrane antigen (PSMA). The first step in this process is to develop an animal model of metastatic PSMA+ prostate cancer. Bone is the most common site for prostate cancer spread in patients. However, animal models that spontaneously recapitulate this are not available. The PC3LN3 prostate cancer cell line undergoes metastatic spread to draining lymph nodes following orthotopic implantation. When engineered to express PSMA, the resultant PC3LN3-PSMA cells are highly metastatic, but do not spread predominantly to bone. Increasing evidence suggests that bone metastasis requires (i) CXCR4 expression by tumour cells and (ii) interaction between E-selectin on bone marrow endothelium with sialyl Lewis antigen-containing E-selectin ligands, expressed on cancer cells. We found that both PC3LN3 and PC3LN3-PSMA cell lines express CXCR4. However, neither cell line expresses E-selectin binding ligands or sialyl Lewis antigens. To investigate the mechanism underlying this, we quantified expression of glycosyltransferases required for sialyl Lewis antigen expression. Using real-time PCR, low fucosyltransferase (FT) 3 expression was consistently found. In preliminary studies, we have observed that delivery of a FT3-encoding retroviral vector to PC3LN3 and PC3LN3-PSMA enables them to express sialyl Lewis X and to acquire E-selectin binding activity. Bioluminescence imaging will be used to study the effect of this upon their pattern of metastatic spread in SCID Beige mice. This will serve as a platform to test immunotherapy using P28z+ T-cells.
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden
T-cells genetically engineered with chimeric antigen receptors (CAR) are showing promise in clinical trials for patients with B-cell malignancy. However, increased levels of anti-apoptotic proteins, a common trait among B-cell tumors, may hamper treatment efficacy. ABT-737 is a small molecule inhibitor of anti-apoptotic proteins. ABT-737 has been shown to efficiently promote apoptosis in B-cell tumors as exemplified in models of pre-B-ALL. Recently, ABT-737 was shown to synergize with TRAIL to induce apoptosis. This prompted us to investigate if ABT-737 could be combined with T-cell therapy to enhance tumor cell death. PBMCs from healthy donors and pre-B-ALL patients were genetically engineered with a second generation CAR targeting CD19 on B-cells. The T-cells and ABT-737 were tested individually and in combination for their cytotoxic capacity. The expression of anti-apoptosis molecules in the cell lines was investigated by PCR. PCR confirmed expression of BCL family proteins in the cell lines tested. CD19-targeting T-cells specifically induced apoptosis in CD19+ tumor cells. Similarly, but less efficiently, ABT-737 as single agent increased apoptosis in the cell line panel. When combining T-cell and ABT-737 therapy, the tumor cell death was significantly increased to that of single agent treatment. The tumor cell lines did not change the level of antigen presenting molecules (MHC I and II), death receptors (Fas) or adhesion or costimulatory molecules (ICAM-I, CD80, CD86) upon ABT-737 treatment. Hence, the effect did not represent increased killing by enhanced physical interaction between T-cells and tumors. In conclusion, the apoptosis inducer ABT-737 is potently enhancing CD19-targeting T-cell therapy.
Dept of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Sweden
Immunotherapy such as adoptive transfer of T cells is hampered by the accumulation of suppressor cells such as myeloid-derived suppressor cells (MDSCs) in the tumor milieu. Preconditioning the patients with cyclophosphamide, and/or irradiation reduces the number of suppressor cells which is beneficial for treatment outcome. However, preconditioning is harsh and limits the number of patients. New strategies to overcome immune escape mechanisms that terminate T cell responses are needed. CD40L is a potent stimulator of tumor immunity. Adenovector CD40L (AdCD40L) has shown promising results in experimental models and in man (phase I-II trials). We hypothesize that AdCD40L may affect MDSC because of its broad range as an immune activator. Herein, we determined the level of GR1+ CD11b+MDSCs in tumors and spleens from mice treated with AdCD40L gene therapy and found that MDSCs were significantly reduced in AdCD40L-treated tumors. To determine the effect of CD40L on MDSCs they were cocultured with tumors +/- AdCD40L. Tumors promoted survival of MDSCs but the presence of CD40L did not alter the viability or phenotype of MDSCs. However, MDSCs were increased in the cocultures with splenocytes and tumors, suggesting that the tumor promotes generation of MDSCs. The generation of MDSC was blocked if the tumor expressed CD40L. Instead, DCs were detected and the lymphocytes expanded vigorously. In a migration assay, CD40L-expressing tumors had less attractive capacity on MDSCs. In conclusion, AdCD40L blocks the generation and migration of MDSCs to the tumor. CD40L gene therapy may act as a sensitizer to T cell therapy.
CAR Mechanics Group, Department of Research Oncology, King's College London, London SE1 9RT, UK
The ErbB family consists of EGFR, HER-2, ErbB-3 and ErbB-4, which undergo ligand-induced homo- and heterodimerisation. Over-expression of individual ErbB receptors has been linked with the development of several solid tumours. Importantly however, the family operates as a dynamic and integrated network within which heterodimers (notably EGFR/HER-2 and HER-2/ErbB-3) elicit the greatest transforming activity. To exploit this, we have developed an immunotherapeutic approach that targets these and other tumour-associated ErbB dimers. A chimeric antigen receptor (T1E28z) was engineered in which the promiscuous ErbB ligand, T1E, is fused to a CD28+CD3z endodomain. When expressed in human T-cells, T1E28z mediates the recognition of 32D cells engineered to express EGFR homodimers, all EGFR-based heterodimers and the HER-2/ErbB-3 heterodimer. However, neither HER-2 nor ErbB-3 alone is targeted. Equipped with this targeting specificity, human T1E28z+ T-cells destroy several ErbB+ tumour cell lines of diverse tumour origin. Therapeutic efficacy in-vivo was evaluated in mice bearing established tumour xenografts, driven either by EGFR/HER-2 or HER-2/ErbB-3 respectively. Potent anti-tumour activity was observed using bioluminescence imaging in three distinct models, representing head and neck, breast and ovarian cancer. Human T1E28z+ T-cells also engage murine ErbB receptors efficiently. Crucially however, no toxicity was observed in SCID Beige mice following IV or IP administration of doses that extrapolate beyond the clinical range. These findings support the clinical development of a platform cancer immunotherapy in which ErbB dysregulation is exploited safely for therapeutic gain. Phase 1 testing in patients with head and neck cancer will begin in 2012 at King's College London.
Effective DARPin-targeted oncolytic measles viruses directed against HER2/neu, EGFR, or EpCAM expressing tumor cells
In order to improve safety while conserving tumor-killing efficiency of recombinant oncolytic viruses (OVs), we intended to exploit the binding specificity of
We utilize DARPins directed against HER2/neu, EGFR, or EpCAM as binding domains of receptor-blind MV hemagglutinin (H). DARPins directed against either target revealed the capacity for functional retargeting of MV glycoprotein activity. Insertion of the DARPin-H attachment protein gene into the MV genome allowed the generation of replicating MVs carrying either HER2/neu-, EGFR-, or EpCAM-specific DARPins as targeting domain. With HER2/neu as the specific target, the respective MV variants revealed specific targeting, genomic stability, efficient viral replication and oncolytic activity in receptor-positive cells in vitro. We found evidence that the target cell killing efficiency of the targeted viruses was dependent on the receptor density on target cells and the affinity of the DARPins to the target receptors. In receptor-high target cells, DARPin-targeted MVs displayed the cytotoxicity of parental, non-targeted oncolytic MV, killing up to 90% of target cells in three days. Side-by-side comparison of the DARPin-MV with published scFv-MV targeted against HER2/neu in a cytolytic assay indicated an enhanced oncolytic efficiency of the analysed DARPin-MV. In vivo experiments confirmed the anti-tumoral efficacy and revealed first evidence for in vivo targeting.
In conclusion, DARPins are an effective targeting module for oncolytic MVs.
Enhancing the efficacy of T cell receptor (TCR) gene therapy by co-transfer of TCR and additional CD3 molecules into CD4+ T cells
The majority of TCR gene therapy studies have focused on the transfer of TCR genes into CD8+ T cells. However the transfer of antigen specific CD8+ T cells in the absence of antigen specific CD4+ T cells leads to impaired anti-tumour responses and impaired memory development in vivo. We previously transduced class I restricted F5-TCR (specific for influenza peptide NP presented by H2-Db class I molecules) into murine CD4+ T cells. F5 TCR-transduced CD4+ cells produced cytokines in response to specific antigen and provided help for tumour protection in murine models.
In order to improve the function of class I restricted TCR expressing CD4+ T cells we co-transduced additional CD3 molecules as TCR-CD3 complex assembly is rate limiting for surface expression of introduced TCR. Using retroviral transduction, CD4+ T cells were transduced with F5-TCR only or with F5-TCR and CD3. CD4+ T cells transduced with F5 and CD3 have increased surface expression of TCR compared to cells transduced with F5 alone. In vitro, F5-CD3 CD4+ T cells show increased proliferation and production of IL-2 and IFN-γ in response to specific antigen when compared to F5 CD4+ T cells and have increased functional avidity. F5-CD3 CD4+ T cells can mediate tumour protection following adoptive transfer but transfer of equivalent doses of F5 CD4+ T cells did not lead to tumour regression. F5-CD3 CD4+ T cells were also shown to have increased levels of persistence/expansion in vivo in response to specific antigen than F5 CD4+ T cells.
San Raffaele Scientific Institute, Division of Regenerative Medicine, Stem Cells and Gene Therapy, Experimental Hematology Unit, Milan, Italy
Leukemia is highly sensitive to chemotherapy, but after initial responses, often relapses. This is thought to depend on the survival of chemoresistant leukemia cells, whose persistence is imprinted by signals provided by the bone-marrow niche. Targeting chemoresistant cells is therefore advocated for the eradication of leukemia. The variant 6 isoform of CD44 (CD44v6) is expressed by primary acute myeloid leukemia (AML, 36%) and it correlated with poor prognosis. CD44v6 expression is regulated by the cross-talk whit the microenvironment. Accordingly, co-culturing AML cells isolated from patients with bone marrow-derived mesenchymal stromal cells (MSCs) caused the up-regulation of CD44v6. Interestingly, the interaction with MSCs also induced significant resistance to chemotherapy. This protection is mediated by a combination of soluble factors as evinced by culturing AML cells with MSC supernatant. MSC-induced chemoresistance was also evident in multiple myeloma (MM) cells, which as well express CD44v6. Strikingly, the level of CD44v6 expression directly correlated with the ability to resist to chemotherapy upon stroma interaction. To further investigate this issue, we downregulated CD44v6 expression with short hairpin RNA delivered by a third generation lentiviral vector. Importantly, CD44v6-silenced AML and MM cell lines lost MSC-induced chemoresistance. Albeit resistant to chemotherapy, AML and MM cells co-cultured with MSCs were efficiently cleared in vitro by T cells genetically redirected by means of a CD44v6-specific chimeric antigen receptor (CAR). These results validate CD44v6 as a suitable target for disease eradication in patients suffering from AML and MM.
Barts Cancer Institute
Pancreatic cancer is a rapidly progressing disease with poor prognosis and a high fatality rate. The lack of curative treatments for advanced disease necessitates the development of new therapies. Replication-selective oncolytic adenoviruses represent a novel anticancer strategy with demonstrated efficacy, tumor-selectivity and overall safety for several mutants, both in preclinical and clinical studies. We previously demonstrated that an adenovirus type 5 mutant, lacking the anti-apoptotic protein E1B19K (AdΔ19K), had high efficacy in pancreatic cancer cells, and further enhanced cell killing when combined with the chemotherapeutic drug gemcitabine, the standard first-line treatment for pancreatic cancer. Here we investigate the molecular mechanisms leading to enhanced cell death when AdΔ19K is combined with DNA-damaging drugs, such as gemcitabine and irrinotecan. We show that AdΔ19K enhances gemcitabine-induced apoptosis and that gemcitabine-induced DNA damage responses, could not be completely prevented by virus, despite the potent inhibition of DNA-repair mechanisms by adenovirus alone. In addition, AdΔ19K induces sustained expression of proteins involved in mitosis and a dose-dependent appearance of chromosomal and mitotic aberrations, which are further increased in the presence of gemcitabine or irrinotecan. In particular, AdΔ19K induces the formation of large multinucleated aneuploid cells, partly as a result of re-replication, in a time- and dose-dependent manner and this aneuploid population is reduced in the presence of gemcitabine. Further investigations are aimed at determining the cellular pathways responsible for the synergistic antitumor efficacy to enable refinement and future translation into clinical trials.
CAR Mechanics, Research Oncology/Kings College London, London, SE1 9RT, UK
T-cells that express the Vg9Vd2 T-cell receptor possess innate anti-tumour activity and are activated by phosphoantigen intermediates of the mevalonate pathway, notably IPP. Tumour cells have a greater requirement for farnesylated and geranylated signalling molecules than their normal counterparts. Consequently, they generate increased levels of phosphoantigens thereby rendering them susceptible to immune surveillance by gd T-cells. The nitrogen-containing bisphosphonate drug zoledronic acid can further enhance phosphoantigen production owing to inhibition of a key enzyme in the mevalonate pathway, farnesyl-pyrophosphate-synthetase. Exploitation of this property provides an important opportunity to render tumours susceptible to immunotherapy using gd T-cells. Here, we have tested the applicability of this system to develop adoptive immunotherapy for epithelial ovarian cancer (EOC). Peripheral blood mononuclear cells from healthy donors and from patients with newly diagnosed EOC were activated in-vitro using zoledronic acid, IL-2 and IL-15. As a result, expansion and marked enrichment of gd T-cells ensued over 15 days. We obtained 100 to 1000 fold increase of Vg9Vd2 T-cells from EOC patients, over the 15 days expansion period. To test anti-tumour activity, ovarian cancer cell lines and autologous tumour cells were pulsed in-vitro for 24 hours with zoledronic acid and then co-cultured with gd T-cells. We observed that SKOV-3, IGROV-I and autologous EOC tumour cells were highly susceptible to killing by gd T-cells after pre-treatment with 1mg/mL zoledronate. In each case, gd T-cell activation was accompanied by production of nanogram quantities of interferon-g. Taken together, these data indicate the promise afforded by targeted gd T-cell immunotherapy of EOC.
Cancer Gene Therapy Group, University of Helsinki, Helsinki, 00290, Finland
The immune suppressive environment of advanced tumors is recognized as one of the major obstacles for immunostimulatory and cancer vaccine approaches. Oncolytic adenovirus is an attractive platform for immunotherapy because virus replication is highly immunogenic. Arming the virus with immune stimulatory molecules, such as CD40 ligand (CD40L), can improve efficacy further. Therefore, a double targeted chimeric oncolytic adenovirus Ad5/3-hTERT-E1A-CD40L was constructed. In a syngeneic mouse model, murine CD40L induced recruitment and activation of antigen presenting cells which increased IL-12 production in splenocytes. Furthermore, we noticed the enhanced presence of macrophages and cytotoxic CD8+ T cells in tumors treated with Ad5/3-CMV-mCD40L.
Next, 9 patients with advanced solid tumors were treated intratumorally with Ad5/3-hTERT-E1A-hCD40L. Concomitant low-lose cyclophosphamide was given to reduce regulatory T-cells. Two patients experienced transient grade 3 liver enzyme increases and one patient showed grade 3 lymphocytopenia. No measurable increases in systemic levels of CD40L were recorded suggesting local production at tumors. Peripheral blood mononuclear cells were analyzed by interferon gamma ELISPOT and induction of both tumor specific and adenovirus specific T-cells were recognized after treatment. Patient serum samples were analyzed for cytokines and 4 out of 7 evaluable patients showed dominance of a Th1 type immune response after treatment. For antitumor efficacy, 6 patients were evaluable with RECIST criteria and 3 showed stable disease and 3 had progressive disease. Furthermore, 3 patients were evaluated with PET-CT and all three showed stable disease by PERCIST criteria. These data suggest that Ad5/3-hTERT-E1A-CD40L is a promising agent for further clinical evaluation.
Adoptive immunotherapy for patients with chronic lymphocytic leukaemia using a chimeric antigen receptor targeting CD23
We have developed a distinct gene transfer based adoptive immunotherapy for patients with chronic lymphocytic leukaemia (CLL), which involves the generation of bi-specific gene modified T cells that recognise CMV-infected cells via their native T-cell receptor (TCR) and CLL cells via a CD23-specific chimeric antigen receptor (CD23CAR). CD23 is expressed at high levels on CLL cells from patients with a poor prognosis. Expression on normal B cells is low or undetectable, thus reducing potential for off-target toxicity observed with CD19CAR approaches currently being evaluated. High titres (3X108 transducing units/ml) of a self-inactivating lentiviral vector consisting of a humanised CD23 single chain antibody linked to a tripartite cytoplasmic signalling domain were generated. A protocol was developed for optimal transduction of CMV-specific cytotoxic T-cells (CMV-CTLs) isolated from human peripheral blood using Miltenyi IFNg capture technology. After an 8–10 day rest period, CMV-CTLs were effectively transduced (30–50%) with CD23CAR-lentivirus (MOI of 10) without alteration of phenotype or growth kinetics. Importantly, these transgenic cells were capable of potent CD23-specific but MHC-unrestricted killing of tumour cell lines (SKW6.4, Raji) and primary CLL cells derived from patients with poor prognosis CLL, with secretion of high levels of IFNg (30–300ng/ml) in this context. Thus CMV-CTLS can be efficiently transduced without phenotypic alteration and redirected to killing tumour cells, which supports further preclinical evaluation of this approach.
Human prostate transglutaminase, a highly prostate-specific gene with a non-canonical regulation and the implications for prostate-targeted gene therapy
Department of Microbiology and Immunology, National Cheng Kung University Medical College
For achieving sufficient tumor transduction and selective tumor killing, replication-competent oncolytic viruses hold promise as cancer therapeutic agents. E1B55K-deleted adenovirus can replicate in tumor cells with intrinsic ability to shut down host protein synthesis and export late viral RNA in the absence of E1B55K. However, it is still unclear how cells that are permissive for E1B55K-deleted adenovirus replication shut down host protein synthesis. Here we report a novel mechanism underlying the tumor selectivity of E1B55K-deleted adenovirus replication. YB-1 (Y-box-binding protein 1), a transcription factor and translational inhibitor, has been shown to be associated with tumor progression and adenovirus replication. Defects in the expression or nuclear translocation of YB-1 have been documented to restrict viral replication in cancer cells. We showed that E1A could shut down host protein synthesis through upregulation of YB-1 expression, which in turn activated Akt to trigger nuclear localization of YB-1. Moreover, we constructed an E1B55K-deleted adenovirus driven by the multidrug resistance 1 (MDR1) promoter which is regulated by YB-1 and demonstrated its high oncolytic activity particularly in combination with chemotherapy. This is attributed to a positive feedback regulation that involves E1A, YB-1 and the MDR1 promoter. Therefore, exploiting the positive feedback loop of E1A and YB-1 for designing E1B55K-deleted adenovirus driven by YB-1-responsive promoters may be a better strategy for virotherapy of drug-resistant tumors.
Chmielna 12/9 20-075 Lublin, Poland
Multidrug resistance of tumor cells to cytostatic activity is one of main factors limiting the effectiveness of cancer chemotherapy. Currently, many scientists try to understand the mechanisms of this phenomenon, identifying the genes responsible for the appearance of insensitivity of cells to cytostatic drugs. It seems that SHH is a potent inducer of multidrug resistance. The aim of this study was to test Sonic Hedgehog (SHH) influence on sensitivity of paclitaxel on cells of studied prostate cancer cell lines: Du145, LNCaP and SHH gene stable transfected cell lines: DU145/SHH, LNCaP/SHH with particular emphasis on defining the role of ABC transporters in the determination of the resistance.
We made the stable transfection Du145/SHH cell line with plasmid DNA. To assess the sensitivity of control cells and transfected cells, exposed to paclitaxel, we used two colorimetric assays and flow cytometry. We examined level of transcripts of the following genes: SHH, MDR1, MPR1, BCRP.
We showed that the increased expression of SHH gene in cancer cells affects their resistance to paclitaxel -Du145/SHH cells are less sensitive to drug than Du145. Additionally, in Du145/SHH cells we found elevated levels of MDR1 pump, the protein responsible for reducing drug accumulation in cells, and often involved in the development of multidrug resistance of tumor cells to cytostatics. Preliminary studies indicate that the mechanism of resistance dependent on SHH signaling pathway may be related to the influence of SHH on the activity of ABC protein pumps. The obtained results underline the clinical importance of the Sonic Hedgehog pathways.
Dep. Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, 30625, Germany
“SmartDCs” consist of highly viable multivalent DCs induced to differentiate with lentiviral vectors after an overnight ex vivo transduction. Tricistronic vectors co-expressing cytokines (GM-CSF, IL-4) and a melanoma antigen (TRP2) were used to transduce mouse bone marrow cells and human monocytes. 16 hours after transduction, the cells were dispensed in aliquots and cryopreserved for batch release analyses, potency and safety tests. Thawed SmartDCs readily differentiated into cells with DC characteristics. Repeat dose administration of 10e6 thawed murine SmartDCs s.c. into C57BL/6 mice resulted into TRP2-specific CD8+ T cell responses, protection against melanoma challenge, and low degree of vitiligo/ alopecia (TRP2 is expressed in melanoblasts which accumulate in the hair follicles). Thus, prime/boost with SmartDCs abrogated tolerance against TRP2. Human SmartDCs were effectively generated with transduced monocytes obtained from healthy donors or melanoma patients and induced T cell proliferation in allogeneic and autologous MLR assays. Thawed human SmartDCs injected s.c. in NOD.Rag1−/− IL2rγ−/− mice were viable for a month in vivo and did not cause any signs of pathologies for over 6 months. For GMP development, leukapheresis was followed by CD14+ selection (CliniMACs), lentiviral transduction was performed in a closed bag system and the cells were cryopreserved. Thawed SmartDCs produced in two independent runs expressed GM-CSF/IL-4 and several cytokines (TNF-a, MCP, IL-8). Seven days after thawing, approximately 2.5 copies of integrated LV per cell were detected in the homogeneous DC populations. Genotoxicity analyses by vector integration studies and IVIM assays are ongoing. Multivalent SmartDCs will be evaluated in a melanoma immunotherapy trial.
A non-replicating adenovirus expressing Hey1 is cytotoxic to prostate cancer cells
Androgen receptor (AR) cell signalling is active in most hormone-refractory prostate cancer (PCa) tumours and suppression is hypothesized to impede cell proliferation. Hey1, a co-repressor of AR is being investigated as a therapeutic transgene for treatment of late-stage PCa. A replication-defective recombinant adenovirus was constructed (Ad5Hey1). Ad5Hey1 was cytotoxic in AR-positive cells, 22Rv1 and LNCaP and AR-negative DU145 cells. The dose required to kill 50% of cells (EC50) were comparable to cytotoxic E1A-12S gene expressed from an identical vector (AdE1A-12S). A luciferase assay showed Ad5Hey1 repressed AR activity in a dose dependent manner in 22Rv1 cells. Knock down of AR in 22Rv1 cells by siRNA decreased Ad5Hey1-mediated but not AdE1A-12S-mediated cytotoxicity. These data demonstrate Ad5Hey1 cytotoxicity is partially mediated by repression of AR-activity. p53 expression levels increased with increasing doses of Ad5Hey1 whereas p21 levels increased at low doses but decreased at higher doses in 22Rv1 and LNCaP cells (both p53 wt) suggesting cells are being driven towards either cycle arrest and/or apoptosis. In 22Rv1 cells, the population in G2/M-phase increased in an Ad5Hey1 but not AdE1A-12S dependent manner. An increase in cyclin B1 expression reflects the Ad5Hey1-dependent increase in G2/M phase. At >100ppc, a larger proportion of cells were in sub-G1 indicating that both viruses induce apoptotic death. Detection of caspase-3 activation by flow cytometry in both Ad5Hey1 and AdE1A-12S-infected cells further indicated apoptotic death. Data is presented to suggest Hey1 can be expressed from a replication selective adenovirus to improve on oncolysis anti-tumour efficacy in vitro and in prostate cancer xenograft models.
Harelbekestraat 72, B-9000 Ghent
Viral and non-viral carriers have been under investigation for their ability to deliver nucleic acids. Although non-viral systems present advantages, such as being less toxic and immunogenic than viral vectors, they are less efficient in overcoming intracellular barriers. Polycationic amphiphilic cyclodextrins (CDs) are promising nucleic acids carriers. In this study we aimed at specific targeting of hepatocytes (HepG2 cells) via the asialoglycoprotein receptor. To that end we evaluated transfection efficiencies of several CDs modified with galactose moieties.
Transfecting HepG2 cells with pDNA complexed with different CDs resulted in hardly any transfection. To determine the cause of this failure, we performed a set of experiments. Our results showed that pDNA complexes are efficiently taken up by the cells, implying that internalization is not the problem. To verify whether endosomal release of CDplexes limits their efficiency, we applied a technique called photochemical internalization. It did not improve transfection by pDNA CDplexes. Competition experiments showed that pDNA can be released from the complexes, indicating that the lack of transfection can neither be attributed to release of free pDNA from the CDplexes. Taken together, these results suggest that the nuclear envelope represents the main obstacle for pDNA CDplexes to become efficient. To verify that, we transfected HepG2 cells with mRNA, which is translated in the cytosol. The fraction of GFP positive cells obtained with differently composed mRNA complexes reached 26±2%. These results strongly indicate that modified CDs complexed with mRNA represent a promising system to transfect hepatocytes.
Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan
The majority of malignant mesothelioma possesses the wild-type p53 gene with defective INK4A/ARF locus that includes the p14ARF and the p16INK4A genes. Loss of p14ARF and p16INK4A subsequently decreased p53 expression levels through enhanced Mdm2 and induced pRb phosphorylation through up-regulated CDK 4/6, respectively, which presumably contributes to the resistance to conventional chemotherapy and radiotherapy. We examined then whether forced expression of p53 inhibited the growth of mesothelioma cells and produced anti-tumor effects by a combination of cisplatin (CDDP) or pemetrexed (PEM), the current first-line drugs for mesothelioma treatments. Transduction of mesothelioma with adenoviruses bearing the p53 gene (Ad-p53) induced phosphorylation of p53 at Ser 15 and 46, up-regulated cleaved Mdm2 and p21 expression and decreased phosphorylation of pRb, demonstrating that both p53 and pRb pathways were functionally intact and could be suppressed. The transduction generated cleavage of caspase-8 and -3 but not -9, suggesting that intrinsic mitochondrial-mediated apoptosis was less likely to be involved. Cell cycle analysis showed increased G0/G1- or G2/M-phase populations and subsequently sub-G1 fractions, depending on cell types and Ad-p53 doses. Transduction with Ad-p53 suppressed viability of mesothelioma cells and augmented the growth inhibition by CDDP or PEM mostly in a synergistic manner (less than CI<1 with CalcuSyn software) and cell cycle analysis supported the synergism. Intrapleural injection of Ad-p53 and systemic administration of CDDP produced anti-tumor effects in an orthotopic animal model. These data collectively suggest that Ad-p53 is a possible agent for mesothelioma in combination of the first-line chemotherapeutics.
Medicinal Virology, Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK
Improvement of virus spread through tumour nodules is expected to lead to a greater overall anti-tumour efficacy. The inhibitory effects of the extracellular matrix as well as cellular debris, as a result of virus-induced cell lysis, are thought to be major factors restricting the spread of oncolytic viruses. Therefore, our initial objective was to decipher whether these individual components have a direct inhibitory effect on adenovirus infection and if these effects can be reversed with the addition of key enzymes.
Initially, we demonstrated in vitro that the addition of exogenous hyaluronic acid, collagen, elastin and actin in the presence of a replication incompetent reporter type 5 adenovirus had varying activity on viral infection. To address whether these factors are responsible for sequestering the virus and thereby inhibiting infection, assays were performed in which cells were infected in the presence of cell and tumour lysates treated with a variety of enzymes. Whilst the presence of the lysate significantly inhibits infection, treatment with hyaluronidase, proteinase K and to a lesser extent dispase I, substantially reversed this effect and significantly enhanced adenovirus infection.
These data combined suggest the involvement of both intracellular factors and components of the extracellular matrix in preventing infection and spread of the virus and are therefore considered important targets in virotherapy.
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, UK
Stealthing of Adenovirus serotype 5 (Ad5) vectors with multivalent hydrophilic polymers can provide protection against the binding of antibodies, complement proteins and erythrocytes, making such formulations more active and less immunogenic. Here we report the characterisation of the new n-hydroxypropyl methacrylamide (HPMA) stealthing polymer EC236. In addition to coating Ad5 with very high efficiency, EC236 also appears to impart a new cell entry mechanism as infection activity was not CAR, HSPG or factor X mediated. In monolayer infections the activity of EC236 modified Ad5 surpassed that of non-modified virus by between 5 and 10-fold in 6 out of 10 cancer cell lines and showed resistance to neutralising human serum. In primary ascites samples from ovarian cancer patients EC236 coated wild-type Ad5 could achieve efficient killing even in anti-adenovirus antibody positive autologous ascitic fluid. Hence, EC236 modified Ad5 might present a major breakthrough in overcoming in vivo barriers to efficient gene and oncolytic therapy.
CAR Mechanics Group, Research Oncology, King's College London
Head and Neck squamous cell carcinoma (HNSCC) is the sixth most common cancer. However, 5-year survival rate remains at only 50%. ErbB1 is over-expressed in 90% of HNSCC, however therapeutic targeting of ErbB1 can lead to resistance. We have developed an immunotherapeutic approach to target the extended ErbB family using retrovirus-transduced T-cells. T-cells are engineered to express ‘T4’, a combination two transgenes: an ErbB-targeted chimeric antigen receptor (T1E28z) and a chimeric cytokine receptor (4ab). Expression of 4ab allows for selective expansion and enrichment of transduced T-cells using IL-4.
Using a murine HNSCC tumour cell line (B7E3), we have shown that T1E28z can bind murine ErbBs and induce tumour cell destruction by T4+ T-cells. This cross-species recognition allows us to use a murine model to assess the potential toxicity of treatment with human T4+ T-cells. SCID Beige mice were infused with up to 2×107 T4+ T-cells, administered IP or IV. No alteration in weight or behaviour was seen in any of the mice over the following month. For a more detailed toxicity assessment, T-cells were engineered to co-express T4 with ffLuciferase (T4luc), enabling us to monitor T-cell migration and longevity in vivo. Following IV administration, T4luc+ T-cells migrated throughout the whole body, with focal accumulation in the lungs and head. Subcutaneously injected T4luc+ T-cells remained at the injection area with minimal systemic trafficking. Despite expression of ErbB receptors by the majority of major organs, and demonstrable trafficking of T4luc+ T-cells to these sites, no toxic effects were apparent.
CAR Mechanics Group Research Oncology 3rd Floor Bermondsey Wing Guy's Hospital St. Thomas Street SE1 9RT London
Chimeric antigen receptors (CAR) are fusion molecules that couple the binding of a cell surface target to delivery of a tailored T-cell activating signal. Most commonly, CAR contain a modular endodomain in which co-stimulatory elements (eg CD28) are incorporated upstream of the T-cell receptor signalling subunit, CD3z. Consequently, potent T-cell activation is elicited in response to engagement of target antigen. Recently however, this has led to profound toxicity owing to T-cell activation within tissues where target is expressed at low levels. To circumvent this, we sought to render the activation of CAR+ T-cells contingent upon the binding of two tumour-associated antigens. We selected MUC1 and ErbB2 as targets to test this concept since they are commonly co-expressed in breast cancer. An ErbB2-specific CAR was engineered in which an ICR12-derived scFv was coupled to CD3z (Iz1). Using a T2A-containing retroviral vector, Iz1 was co-expressed with a MUC1-specific CD28 CAR. Comparison was made with several controls, including CARs that contain CD3z-, CD28- or a fused CD28+CD3z endodomain. We observed that dual targeted T-cells could kill ErbB2+ tumour cells efficiently. They also proliferated in a manner that required co-expression of MUC1 and ErbB2 by target cells. However, dual targeted T-cells produced much less interleukin-2 or interferon-g upon activation, when compared to Iz1+ T-cells or those that express a CD28+ CD3z-containing CAR. Together, these data indicate that dual targeting is feasible but that the achievement of synergistic T-cell activation is challenging. This may be the result of steric inhibition or disparate affinity of targeting moieties.
Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London
Oncolytic viruses (OV) have been developed as a new approach for the treatment of cancer that is resistant to standard therapies. However, the outcome of clinical trials of virotherapy as monotherapy has been disappointing. Exploiting our knowledge of cancer cell biology, immunology and virology, we now have developed a novel therapeutic regime by sequential combination of oncolytic adenovirus and vaccinia virus. We demonstrated that three low doses of oncolytic adenovirus followed by three low doses of vaccinia virus resulted in a superior antitumor efficacy to the reverse combination, or six doses of either virus alone, against pancreatic and kidney tumour models in immune-competent Syrian hamsters. A complete response was seen in 62.5% of animals bearing either tumor type treated with the sequential combination in vivo, accompanied with an effective induction of tumour-specific immunity.
The functional mechanisms of this novel regime have been further investigated. We found that Humoral immunity against the two viruses did not contribute to the enhanced efficacy of the sequential use of oncolytic adenovirus and vaccinia virus. Strikingly, depletion of T cells using our recently developed monoclonal antibody against Syrian hamster CD3 completely demolished the antitumor efficacy by the combination of oncolytic adenovirus and vaccinia virus. These findings demonstrate that sequential use of oncolytic adenovirus and vaccinia virus, both of which have been used individually in clinical trials, could be a promising approach for curing cancer in humans. These results have significance for the design of new regimens for cancer viro-immunotherapy and vaccines.
Inst. of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Giessen/Germany
Measles virus which is adapted to tissue culture showed selective tumor cell killing abilities with attenuated pathogenicity. According to the dose needed for measles vaccination in cancer therapy a higher amount of the virus is needed. In this work, the production of large quantities of measles virus particles in a standardized manufacturing process should be established. First results of the work are presented here.
Measles virus production was carried out in spinner system at 40 rpm, 5% CO2, 37°C and 32°C. The offline tracking of cell growth was carried out by fluorescence-based and activity-based assays. The measles virus concentration was estimated by the TCID50 method.
Vero cells have been adapted to a serum-free medium. Additionally, all products of animal origin have been replaced. The growth surface is provided by microcarriers for the cultivation of adherent cells in stirred systems. Kinetic studies were performed for cell growth, metabolite concentration and virus titers. Virus production rates showed similar results for cell associated and released virus. Research on the temperature stability of the virus was carried out, too. It has been shown that released virus is very unstable though production rates remained stable.
This data showed new results by getting much larger amount of virus by harvesting the virus continuously from the supernatant. Commercial and regulatory requirements are met by establishing a cell culture process employing stirred system and serum-free cell culture medium. The first results are promising for a successful scale up in the bioreactor.
Microenvironmental influence on gene therapy after gene transfer - The effects of VEGFs on monocytes
It has been recently recognized that chronic inflammation plays a major role in the pathogenesis of many cancers and that macrophages are central players in this inflammatory process. Accumulating evidence demonstrates a strict correlation between macrophage infiltration and progression of disease with poor prognosis. Paradoxically, these immune cells, which are suppose to function as guardians of the body, have been associated with tumor growth, angiogenesis, immunosuppression and metastasis. At least two different subpopulations of macrophages have been described; the M1 (classically activated) or M2 (alternatively activated) phenotypes.
The aim of this study was to evaluate the role of VEGFs on macrophages and to put this into context to Lodavin gene therapy, a biotin binding targeting construct used for targeted therapies of inoperable tumors. For that, the protein expression of THP-1 cells after hVEGF-A and D stimulation were evaluated under normoxic and hypoxic conditions, by using protein expression arrays. The results show that hVEGF-A and D have distinct effects on protein expression of human THP-1 cells. Furthermore, the results revealed that the proteins induced by VEGFs potentially could support tumor growth and metastasis by stimulating angiogenesis and the recruitment of inflammatory cells. Additionally, it could be seen that the effects were dependent on the status quo of the environmental conditions (i.e. normoxia and hypoxia).
In summary, the results show that in addition to the well known angiogenic effects of VEGFs on endothelial cells, VEGFs may play an important role in immunemodulation of macrophages and that the effects may support the growth and metastasis of tumors.
A phase II study of recombinant adeno-viral human p53 gene combined with radiotherapy and radio- and chemo-therapy in treatment of patients with advanced nasopharyngeal carcinoma
University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Division of Woman and Child, Leuven, Belgium,
Development of Tissue-Specific Expression Cassettes Using miRNA Targeting Sequences for Cardiac Gene Therapy
The heart is a target for gene therapy in disorders including heart failure and myocardial infarction. Regulated gene delivery would facilitate improved efficacy and safety. This could be mediated by microRNAs (miRNAs) which post-transcriptionally control gene expression.
Tissue-specific regulation using miRNA targeting sequences (TS) was investigated for selective cardiomyocyte expression. MiRNA_TS constructs were generated with either a mixture of tissue-specific miRNAs; miR-122 (liver), miR-126 (endothelial) and miR-206 (skeletal muscle) or miR-206 alone, each with either ×3, ×6 or ×9 copies. Constructs were transfected into cell lines representative of specific miRNAs: A549 (endothelial miR-126), HepG2 (liver miR-122), L6 & C2C12 (skeletal muscle miR-206), HeLa's (negative control) and H9c2 cardiomyocytes. In HeLa cells luciferase expression was unaffected. In L6 cells, miRNA-206_TS ×9 significantly decreased luciferase expression by 81% (P<0.005) while miRNA-mix ×9 produced a 67% (P<0.05) reduction compared to control vector, probably due to high endogenous miR-206 expression. The highest degree of repression was with miRNA-mix_TS ×9 in C2C12 cells (94%, P<0.001) due to high miRNA-206 and miRNA-122 levels. In H9c2 cardiomyocytes all constructs reduced luciferase expression again probably due to high endogenous miR-206 levels.
In summary, insertion of tissue-specific miRNA targeting sequences into an expression cassette regulated luciferase expression in different cell types with the degree of repression associated with increased number of miRNA target sequences. Incorporating miRNA target sequences into expression cassettes may enable the development of tissue-specific gene therapy in cardiovascular disease.
Department of Cardiology and Pneumology Charité - Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin 12200, Germany
Division of Cardiovascular Surgery, Department of Surgery, University of Verona, ITALY
Therapeutic angiogenesis in ischemic muscle: gene and cell therapy for gene delivery
Gene transfer is important for therapeutic angiogenesis. Current results demonstrate that clinical efficacy of existing approaches is not high enough thus indicating the need for development of new methods.
We tried to imply combined transfer of several therapeutic genes to enhance angiogenic response. Plasmid vectors with genes of human VEGF165, HGF, angiopoietin-1 and mouse urokinase were tested in murine hind limb ischemia to assess efficacy of sole genes or their pair combinations. We found that gene transfer of VEGF+HGF, VEGF+ANG1 or ANG1+HGF amplified limb perfusion compared to sole vectors yet uPA combinations failed to show synergistic interaction. Our findings were confirmed by histological studies and showed reduced necrosis, higher CD31+ and SMA+ density in combined gene transfer groups.
Next widely used approach is cell therapy using adipose-derived stromal cells. In our study we tried to use gene-modified ADSC overexpressing human VEGF165.
In vivo studies revealed that VEGF-expressing ADSC induce higher vascularization of Matrigel in nude mice compared to control cells. Vessel density was higher in Matrigel plugs from VEGF-ADSC animals indicating VEGF-driven formation of mature arterioles.
Hind limb ischemia test showed increased perfusion after VEGF-ADSC injection compared to controls and we found that VEGF-ADSC mice had higher perfusion than animals that received VEGF-carrying plasmid.
We can conclude that combined gene transfer and gene-modified cells can be used to enhance angiogenesis in ischemic tissue and present an attractive target for further translation.
Department of Physiology/Regenerative Institute of Medicine, National University of Ireland Galway, Ireland
Atherosclerosis is associated with initial endothelial dysfunction followed by accumulation of smooth muscle cells in the sub-intimal layer of the arterial wall. Endothelial nitric oxide synthase (eNOS) is the major source of nitric oxide (NO) production in blood vessels and is well known to inhibit the intimal hyperplasia and promote re-endothelialization (Sharif et al., 2011). In this study there was an interest in examining the gene profile of eNOS adenoviral transduced human coronary arterial smooth muscle (HCASMC) cells to further understand the mechanism for the eNOS inhibitory effect on smooth muscle cell proliferation. To this aim we performed a whole genome wide analysis on adenoviral eNOS transduced HCASMC after 48 hours. Hsp70 genes were identified as regulated by eNOS and a novel Hsp70B' gene family member was explored as a potential mediator of eNOS 14etard14log in smooth muscle cells. The identification of an eNOS smooth muscle specific target could aid in further developing gene therapeutic approaches to vascular repair.
Sharif F, Hynes SO, McCullagh KJ, Ganley S, Greiser U, McHugh P, Crowley J, Barry F, O'Brien T. Gene-eluting stents: non-viral, liposome-based gene delivery of eNOS to the blood vessel wall in vivo results in enhanced 14etard14logical14ion but does not reduce restenosis in a hypercholesterolemic model. Gene Therapy. 2011 Jun 30.
Engineering attenuated Salmonella typhimurium for selective targeting and gene expression in myocardial infarction
Optimization of the specific affinity of cardiac delivery vector could significantly improve the efficiency of gene/protein delivery, yet no cardiac vectors to date have sufficient target specificity for myocardial infarction (MI). In this study, we have developed engineered Salmonella typhimurium that specifically colonize infarcted myocardium and were engineered to express a variant Renilla luciferase gene (RLuc8) under the control of the hypoxia-inducible promoter systems.
We isolated promoters such as pepT, pflE and ansB which regulate gene expression under the hypoxic condition. Those promoters were designed to drive for expression of RLuc8 inside attenuated S. typhimurium defective in the ppGpp (SLΔppGpp). Murine MI models received an optimal dose of SLΔppGpp via tail-vein injection. Bacterial tropism for MI and gene expression was assessed in vivo by luminescence in vivo imaging system (IVIS).
Bioluminescence signal was selectively observed in myocardial infarction that corresponded well with histological staining, indicating that the administered bacteria accumulated in the hypoxic region of left ventricle. No signal of RLuc8 expression was detected in liver, spleen and normoxic myocardium on day 1 in spite of the presence of high number of bacteria, indicating that gene expression was strictly controlled under the function of hypoxia-inducible promoters.
The engineered Salmonellae processes a specific tropism for infarcted myocardium. The hypoxia-related promoters could drive gene expression from the bacteria selectively in the infarcted myocardium. This current proof-of principle study demonstrates the capacity of Salmonellae to target infarcted myocardium and to serve as a vehicle for the selective delivery of therapeutic agents in MI.
BHF GCRC, University of Glasgow, MVLS, 126 University Place, Glasgow, UK.
Human adenovirus type-5 (Ad5) is a commonly used vector for gene therapy but its clinical utility is often limited by high seroprevalence rates and off-target liver sequestration. Therefore, the use of rare species adenoviruses (eg. species D; here Ad48) is appealing. Targeted viral delivery to defined tissues (eg. tumours) can be achieved through the incorporation of peptide ligands within virus structural proteins and such strategies have been reported extensively for Ad5.
We used predictive structural modelling (SWISS-MODEL) to assess the CD, DG, HI and IJ loop domains of the Ad48 fiber knob, for the incorporation of a 20aa peptide, A20FMDV2 (A20). This peptide targets ανβ6 integrin, which is overexpressed in human carcinomas. Recombinant knob proteins (Knob48, Knob48-CD-A20, Knob48-DG-A20, Knob48-HI-A20 and Knob48-IJ-A20) were purified following expression in E.coli. Trimerisation of the fiber knob domain is critical for viral assembly and we confirmed that Knob48, Knob48-CD-A20 and Knob48-IJ-A20 formed stable homotrimers. However, Knob48-DG-A20 failed to form a trimer and we were unable to validate Knob48-HI-A20 due to low protein yield and the formation of insoluble inclusion bodies in E.coli. All A20-modified knob proteins, but not Knob48, were capable of blocking the transduction of Ad5-EGFPA20 via ανβ6, demonstrating that the inserted A20 peptide was functional. In conclusion, we have found that the CD and IJ loops of Ad48 represent suitable sites for targeting peptide incorporation. These data support the future generation of the corresponding Ad48 viral vectors, Ad48-CD-A20 and Ad48-IJ-A20, which may offer improvements in targeted delivery in vivo.
Prenatal Cell and Gene Therapy Group, UCL Institute for Women's Health, University College London, London, WC1E 6HX, UK
ESGCT 2011 Abstract Author Index
Abe, M, P 58
Abel, T, P 239
Abhay, KP, Inv 62
Abriss, DA, Or 31
Acosta-Saltos, A, P 185
Acosta-Saltos, C, P 185
Adams, S, P 10
Adamsen, D, P 35
Adjaye, J, Or 20
Aghi, MK, Inv 36
Aghighi, S, Inv 64
Ahmad, F, P 281
Ahmadi, M, P 114, P 237, P 262
Ahouansou, O, P 17
Aitchison, KL, P 19
Aitken, RA, P 291
Aiuti, A, Or 1, Or 22, Or 50
Ak, P, P 68
Al Ghonaium, A, P 10
Alamoudi, A, P 238
Alba, R, P 188
Albuquerque, D, Or 45
Alemany, R, Inv 32
Alexander, IE, P 29, P 190
Alfaro, C, P 47
Ali, RR, Inv 23, Or 13, P 8, P 128, P 195, P 134
Aliño, SF, P 145, P 146, P 159
Allay, J, Inv 64
Almeida, LP, P 85, P 104, P 105
Alonso-Ferrero, ME, P 20, P 216
Altemir, D, P 209
Alvarez, L, Or 18, P 42
Alvarez-Erviti, L, P 11
Alves, PM, P 57, P 115
Ambrosi, A, Or 50
Amidi, M, Or 43
Amiel, C, P 174
Amirache, F, Or 47, P 193, P 211
Ammar, I, Ed 11, Inv 54
Amofah, E, Ed 8a
Amsüss, S, Or 27
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Andre-Schmutz, IAS, P 37
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Antoniou, M, Ed 2, P 216, P 217, P 232, P 233
Appelt, JU, Or 9, Or 37
Appelt, U, P 208
Appukuttan, B, P 130
Arbuthnot, P, P 60, P 203
Archibald, KM, P 242
Arens, A, Or 54, P 41, P 109
Argyros, O, Or 38
Ariatti, M, P 179
Arsenijevic, Y, P 119
Artus, A, P 9, P 28, P 51
Arzberger, S, Or 11
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Asfahani, RI, Inv 58
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Askou, AL, P 119
Astord, S, Inv 49
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Athanassiadou, A, Or 29
Aubourg, P, Or 51, P 17, P 21
Aumann, J, P 166
Auricchio, A, Inv 25, P 23
Aurnhammer, C, P 186
Avale, ME, Or 36
Azzouz, M, Inv 59, P 76, P 83, P 84, P 147
Bach, P, P 234, P 239
Bachy, V, P 93
Baconnais, S, P 174
Baekelandt, V, P 196
Bahrami, S, P 91
Baiker, A, P 186
Bailey, MES, P 3
Bainbridge, JWB, Or 13
Bak, RO, P 152, P 218
Baker, AH, Or 40, P 144, P 188, P 290
Ball, C, P 41
Ballmaier, M, P 39
Baños, R, Or 18, P 42
Barbagalo, R, P 93
Barber, AC, P 8
Barber, L, Ed 8a
Barc, C, P 209
Baricordi, C, Or 1, Or 50
Barkats, M, Inv 49
Barker, R, P 167
Barnes, L, P 87, P 137
Bartholomae, CC, Or 10, Or 50, Or 52, Or 54, P 21, P 62, P 69, P 72
Bartolini, P, P 154
Bartolomae, CC, Or 51
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Bassi, G, P 286
Battaglia, G, P 147
Bauer, JW, P 46
Baum, C, Inv 12, Or 6, Or 8, Or 37, Or 53, P 4, P 26, P 39, P 59, P 65, P 194, P 205, P 223, P 224, P 271
Bayart, E, P 28, P 51
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Bazan-Peregrino, M, Or 48
Beattie, SG, Or 9, P 47
Bechinger, B, P 175
Becker, P, P 93
Bednarski, C, P 61
Bek, T, P 119
Bell, JC, Inv 30
Beltran, WA, Or 14
Bemelmans, A-P, P 120
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Benito, A, P 47
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Benten, D, Or 5
Berardi, E, Or 12
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Bernardi, M, P 263
Betts, C, P 11
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Bhalla, J, P 118
Bhatt, R, P 111
Bhattacharya, SS, P 13
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Bitoque, D, P 172
Bizário, JCS, P 85
Bjorkegren, E, P 5
Björquist, Inv 38, P 57
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Blanche, S, P 4
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Bloom, K, P 60
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Bock, M, Or 8, P 223
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Bogoslovskaya, E, P 100
Bohle, K, P 116
Boldrin, L, Inv 2, P 78
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Bonetti, A, P 106
Bonini, C, Inv 8, Inv 27, Or 16, Or 17, Or 21, P 96, P 263
Bonney, S, P 1
Borchers, S, P 97
Bordignon, C, Or 16, Or 17, P 187, P 263
Bordon, V, P 5
Borel, F, P 34, P 240
Borzone, R, P 25
Bosshard-Carter, L, P 241
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Bosticardo, M, Or 1
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Bouma, G, P 43
Bouquet, C, P 17
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Bradley, A, Inv 19
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Brigida, I, Or 50
Brisson, D, P 30
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Brochet, JB Julien, Or 28
Broll, S, Or 29
Brooks, S, P 1
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Brown, L, P 5, P 10
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Brynza, J, P 207
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Büning, H, Or 11
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Burke, D, P 20
Burke, J, Inv 30
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Bushman, F, P 4, P 64
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Canton, I, P 147
Cantore, A, P 69
Cantore, C, Inv 8
Cantz, T, Or 53
Capo, V, Or 22
Capotondo, A, Or 51
Carcenac, R, Inv 49
Carinhas, N, P 115
Carlier, P, Inv 51
Carlisle, R, Inv 56, Or 48, P 238, P 276
Carlon, M, Or 4, P 50, P 283
Carlucci, M, P 121, P 125, P 126
Carmo, M, P 26, P 216
Carmoy, N, P 156
Carpenter, B, P 113, P 262
Carpentier, DCJ, Inv 58
Carr, R, P 140
Carr, DJ, P 291
Carrondo, MJT, P 57
Cartier, N, Or 51, P 17, P 21
Carvalho, LS, Or 13
Casado, JA, P 42
Casiraghi, M, Or 1
Casorati, G, Or 17
Casper, CJ, P 12
Castello, R, P 25
Castiello, MC, Or 1
Casucci, M, Or 16, P 263
Cathomen, T, Ed 11, Ed 12, Inv 22, Inv 54, Or 19, P 7, P 61
Cattaneo, R, P 261
Cattoglio, C, Or 46
Cavazzana-Calvo, M, Or 28, P 4, P 21, P 37
Cawood, R, P 94, P 238, P 253
Cecchi, CR, P 154
Celec, P, P 155, P 158
Cerullo, V, Or 24, P 106, P 149, P 249, P 266
Cesana, D, Inv 8, Or 10, Or 51, Or 52, P 9, P 69, P 72, P 189
Cesani, M, Or 51
Chakraverty, R, P 113
Champlin, RE, Or 15
Chan, E, P 95
Chan, JKY, P 221
Chan, L, Ed 8a, P 118
Chandler, RJ, P 27
Chang, SM, Inv 36
Chard, L, P 199
Charrier, S, P 43
Chatin, B, P 141
Chavda, S, P 185
Chen, D, P 279
Chen, Q, P 162
Chen, SY, P 89
Chen, W, Ed 11, Inv 54
Cheong, SC, P 272
Cherel, Y, Inv 51, P 17
Chernajovsky, Y, P 250
Chernichovsky, E, Inv 17
Cherubini, G, P 264
Chevé, C, P 220
Chiapero-Stanke, L, P 241
Chiriaco, M, Or 22
Choi, J, Or 48
Chokshi, S, P 243
Chong, RHE, P 142
Chowdary, P, Inv 64
Chu, V, Or 17
Chua, I, Or 42
Chuah, M, Inv 34, Or 12
Chun, S, P 75
Cicalese, MP, Or 1
Ciceri, F, Or 1, Or 16, Or 17, P 263
Cicero, V Lo, P 117
Cichutek, K, P 261
Cieri, N, P 96
Ciesla, M, P 131
Ciré, S, P 99
Clarke, C, P 284
Cloughsey, TF, Inv 36
Cobb, SR, P 3
Cobo, M, P 16, P 204
Cocchiarella, F, P 96
Coelho-Castelo, AAM, P 85, P 98, P 104, P 105
Coffin, R, Inv 63
Cohen-Haguenauer, O, P 9, P 28, P 72, P 51
Coleman, J, Inv 64
Colle, MA, P 17
Collins, MK, P 99, P 216, P 217, P 231
Colombo, DF, Inv 8
Coluccio, A, P 70
Conchon, S, P 73
Connell, CM, Inv 35, P 242
Cooney, R, P 288
Cooper, JD, P 221
Cooper, LJN, Or 15
Cooray, S, P 5, P 10, P 43
Cornils, K, Or 5
Coroadinha, AS, P 115
Correia, C, P 57
Corti, G, Or 46
Corydon, TJ, P 119, P 181
Cosset, FL, P 193, P 211
Cosset, F-L, Or 47
Cossu, G, Or 21
Costa, A, P 178
Costa, C, Or 47, P 193, P 211
Cottingham, MG, Or 44
Coughlan, L, P 290
Coulman, SA, P 142
Coussios, C, Or 48
Creedy, JH, Inv 47
Cregg, M, P 108, P 129
Cronin, MC, P 136
Crowe, J, P 228
Crystal, RG, P 17
Cubizolle, A, Inv 57
Cunningham, SC, P 29, P 190
Custers, J, P 290
Czauderna, S, Or 34
Czermak, P, P 56, P 280
da Costa, A, P 172
da Cunha, BM, Or 4
da Cunha, MM, P 50
Dabkowska, A, P 167
Daenthanasanmak, A, P 97
Dagnaes-Hansen, F, P 119, P 154, P 181
Dal-Cortivo, L, P 4
Damo, M, Inv 8
Dana, R, P 122, P 123
Dane, AP, P 190
Dangerfield, JA, P 214
Daniels, AN, P 179
Daniels, R, P 93
Dannemann, N, P 7, P 61
Dannhauser, PN, P 224
Danos, O, Inv 46, P 88
Dautzenberg, I, P 200
David, AL, Or 41, P 291
David, S, P 156
Davidoff, AM, Inv 64, Or 23, P 22, P 222, P 243
Davies, A, P 208
Davies, DM, P 241, P 259, P 277
Davies, EG, P 5, P 10
Davies, M, P 267
Davison, A, P 157
de Almeida, LP, Or 45
de Belin, J, P 126
De Bellis, G, Or 46
de Boer, H, P 6
De Coppi, P, Or 41
de la Fuente, J, P 71
de Raad, M, Or 43
De Rijck, J, P 64
de Saint-Basile, G, P 4
De Smedt, SC, Or 20, P 143, P 273
De Temmerman, ML, P 143
de Villartay, JPV Jean-Pierre, Or 28
de Visser, TP, P 6
De Vrij, J, P 200
de Wal, J, P 30
Debras, F, P 220
Debré, M, P 4
Debyser, Z, Or 4, P 50, P 64, P 196, P 283
Degg, B, P 290
Déglon, N, Or 45
Deichmann, A, P 62
DeKoninck, P, P 50
Delgado, M, P 92
Delgado-Maroto, V, P 92
Demeester, J, P 143
Denis, C, P 156
Denning, C, Ed 4, Inv 29
Deprest, J, Or 4, P 50, P 283
Derlacz, R, P 131
Déry, S, P 30
Deschamps, JY, P 17
Desclaux, M, P 209
Desrosiers, M, P 120
Desseaux, C, P 63
Dev, KK, P 3
Dewitte, H, P 143
Di Cesare, S, Or 22
Di Matteo, G, Or 22
di Robilant, BN, Or 16, P 263
Di Serio, C, Or 46, Or 50
Di, WL, P 44
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Dias, J, P 106
Diaz, M, P 225
Dicks, MDJ, Or 44
Dickson, G, Inv 50, P 77, P 79, P 81, P 93, P 208
Dietrich, L, P 258
Dimberg, A, P 258
Dionisio, F, Or 50
Disterer, P, Or 33
Docherty, K, Inv 18
Doğan, A, P 52
Doglioni, C, Or 10, P 69
Dolp, PD, Or 7
Dolphin, P, Inv 62
Dominguez, E, Inv 49
Donnelly, RF, P 148
Donsante, A, P 127
Dorda, M, P 41
Dotti, G, Or 16
Dowell, A, P 111
Draganovici, DD, Or 7
Drepper, C, P 84
Dreyer, AK, P 61
Driessche, TV, Inv 34
Dubiel, M, P 131
Duffy, MR, Or 40, P 144
Dulak, JJ, Or 32, Or 34, P 131
Dumler, K, P 252
Duncker, D, P 6
Duqué, S, Inv 49
Duran, Y, P 134
Duros, C, P 9, P 28, P 51, P 72
Dyduch, G, P 131
Eckenfelder, A, P 88
Edwards, N, Or 44
Ehrhardt, A, P 186
Eis-Vesper, B, P 97
EL Andaloussi, S, P 86
Elgstrand-Wettergren, E, P 2
Elliott, T, P 1
Ellis, S, P 126, P 130
Ellison, SM, Inv 58, P 228
El-Sherbini, Y, P 53
Ely, A, P 203
Emanueli, C, Inv 44
Endo, H, P 132
Engler, T, P 235
English, R, Inv 13
Enriquez de Salamanca, R, P 47
Epp, C, P 235
Epple, M, P 124
Equihua, CM, P 232
Erceg, S, P 13
Erguen, S, P 124
Eriksson, FE, Or 26, P 107, P 258
Escutenaire, S, Or 24, P 106, P 149, P 266
Essand, ME, Or 26, P 107, P 248, P 254
Evangelio, C, Or 1
Fairbanks, LD, P 5, P 216
Falahati, R, Ed 8b
Falcone, L, Or 16, P 263
Falk, CSF, P 194
Farinelli, G, Or 22
Farzaneh, F, Ed 8a, P 118, P 271
Fasth, A, P 40
Fattah, C, P 284
Fechner, H, P 285
Federspiel, MJ, Or 35
Fehse, B, Or 5, P 103, P 118
Feichtinger, GA, Or 27, P 74, P 90, P 91
Fenard, D, P 192, P 202
Fenelon, G, Inv 62
Ferber, S, Ed 6, Inv 17
Fernandes, FF, P 98, P 105
Ferrige, G, P 121, P 125, P 126
Ferrua, F, Or 1
Ferry, N, P 73, P 203, P 210
Fichtner, I, P 166
Figueiredo, C, P 97, P 271
Filippis, C, P 261
Finocchi, A, Or 1, Or 22
Fischer, AFA, Or 28, P 4, P 21
Fisher, K, Inv 56, P 93, P 102, P 238, P 244, P 275, P 276
Flak, MB, P 242
Flebbe-Rehwaldt, L, Ed 8b
Florczyk, U, Or 34
Flutter, B, P 113
Flynn, R, P 31
Fontanellas, A, P 47
Fontoura, IC, P 85, P 104, P 105
Foster, H, P 77, P 81
Foster, JM, P 250, P 259, P 277
Foster, K, P 77, P 79
Frances, K, Inv 6
François, A, P 220
Frange, P, P 4
Frecha, C, Or 47, P 193, P 211
Freidig, A, P 30
Friedman, G, Or 54
Friedrich, K, P 261
Frisk, P, P 257
Fromes, Y, Inv 51
Fronza, R, Or 50, P 62
Frystyk, J, P 154
Fuchsbauer, HL, P 56
Fuchsluger, T, P 122, P 123, P 124
Fujimoto, Y, P 226
Fukamachi, T, P 274
Gabriel, R, Or 54
Gadalla, KKE, P 3
Gaensler, KML, Ed 8b
Galetto, R, P 63
Galla, MG, Inv 12, Or 53, P 194, P 224
Gallina, P, Or 10, P 69
Gallo, JM, Or 36
Galy, A, Inv 48, P 43, P 99, P 192, P 202
Gangestwaran, R, Or 25
Gansbacher, B, P 135, P 252
Ganser, A, P 97, P 271
Gao, D, Or 25, P 279
Gao, GG, P 245
Gao, L, P 114
Garate, Z, Or 18
García Fernández, JM, P 273
Garcia, L, Inv 51, P 120
Gardlik, R, P 158
Garritsen, H, P 271
Gasmi, M, Inv 48, Inv 51
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Gaudet, D, P 30
Gauthier, D, P 209
Gauttier, V, P 73
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Geisler, A, P 285
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Genries, S, P 192
Gentner, B, Or 22, Or 56, P 263
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Gerli, M, Or 21
Gerson, SL, Ed 8b
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Ghani, S, P 44
Ghorashian, S, P 113, P 114
Giandomenico, V, P 254
Giannelli, S, Or 1
Gijsbers, R, Or 4, P 64, P 196
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Gillen, S, Or 11
Gilmour, KC, P 5, P 10, P 118
Giordano, F, P 208
Girard, A, Or 47
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Glader, B, Inv 64
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Glazkova, D, P 100
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Goebel, B, P 197
Gogol-Döring, A, Ed 11, Inv 54
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Göke, BG, Or 7, P 165, P 198, P 246, P 247
Gonitel, R, P 185
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Gonzalez-Rey, E, P 92
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Gowers, K, Or 38
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Gray, SJ, P 3
Greenberg, PD, Or 17
Greentree, S, P 30
Gregory, LC, Or 35
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Griesenbach, U, Inv 4
Griesmann, GE, Or 35
Griffioen, A, Inv 64
Gritti, A, Inv 8
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Größl, T, P 285
Gruber, C, P 46
Gruber, HE, Inv 36
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Güenechea, G, P 18, P 66
Guiner, CL, Inv 51, P 210
Guinn, B, P 1
Günzburg, W, P 214
Gürlevik, E, P 236
Gurruchaga, J-M, Inv 62
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Gussing, F, P 191
Gutzmer, R, P 271
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Hackett, NR, P 17
Haddad, M-R, P 127
Haemmerle, R, P 65, P 67
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Hajjar, R, Inv 1, Ed 5
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Hallek, M, Or 11
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Hanauer, J, P 261
Hanley, J, P 222
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Hansen, B H, P 154
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Harigae, H, P 58
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Harrison, P, Ed 8a
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Hemmi, S, Or 24
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Hemminki, O, Or 24, P 106
Hennink, W, Or 43
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Herrmann, T, P 84
Hervás-Stubbs, S, P 47
Hervouet, C, P 93
Hettiarachchi, HDM, P 14
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Hiley, CT, P 199
Hill, AVS, Inv 42
Hill, S, Or 44
Hintner, H, P 46
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Hirai, H, Or 45
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Hitchman, RB, P 212
Hobbs, S, P 278
Hoeben, R, P 200
Hoefer, K, P 221
Hofierkova, D, P 155
Hofmann, AT, Or 27, P 90
Hogrel, JY, Inv 51
Hojman, P, P 181
Hollenstein, AP, P 160
Holler, A, Or 42, P 114, P 237, P 262
Hollywood, JA, P 31, P 33
Holm, PS, P 198, P 246, P 247, P 252
Holm, S-L, P 149
Holmes, MC, Inv 8, Or 15, Or 17, Or 18, Or 54, P 18, P 70
Honegger, A, P 234
Horn, S, P 103
Hösel, M, Or 11
Houghton, BC, P 201
Howe, SJ, Or 41, P 7, P 10, P 18, P 19
Hsieh, J, P 101
Hsu, Y, P 14
Hu, J, P 124
Hu, MH, P 255
Hu, WS, P 115
Huber, I, P 186
Hughes, DA, P 19
Huls, H, Or 15
Hurst, F, P 125
Huseinovic, A, P 34
Hwang, TH, Inv 30
Ibanez, CE, Inv 36
Iken, M, Or 8, P 223
Illingworth, S, Inv 56, P 244, P 275, P 276
Ingemarsdotter, K, Inv 35
Ingrao, D, P 192, P 202
Inoue, H, Or 2
Iqball, S, P 126
Irrazábal, T, P 32
Isaksson, C, P 2
Isgrò, A, Or 22
Ishida, E, P 226
Ishii, T, P 162
Ishioka, N, P 58
Ismail, A, P 76, P 84
Itaka, K, P 162
Ittrich, H, Or 5
Ivacik, D, P 203
Ivics, ZI, Ed 11, Inv 54, P 183, P 194
Iwamuro, H, Inv 62
Izsvack, Z, P 183
Izsvák, Z, Ed 11, Inv 54
Izsvak, ZI, P 194
Jacobs, R, P 97
Jahr, H, P 6
Jakobsen, M, P 154
James-Mason, A, P 175
Jansen, P, P 240
Jansson, E, P 191
Jarmin, S, P 77
Jarraya, B, Inv 62
Jazwa, A, Or 32, Or 34, P 131
Jeannon, JP, P 241
Jensen, J, P 57
Jensen, TG, P 119, P 181, P 154
Jeong, JH, Inv 60, P 289
Jiang, Y, P 274
Jin, CJ, Or 26, P 248
Joensuu, T, Or 24, P 249, P 266
Jolly, DJ, Inv 36
Jones, S, P 45
Jonuschies, J, Inv 2, P 78
Jost, C, P 261
Joung, JK, Or 19
Jozkowicz, A, Or 32, Or 34, P 131
Jurkunas, U, P 123
Kalatzis, V, Inv 57
Kaler, SG, P 127
Kalinina, N, P 177
Kalkman, E, P 144
Kampik, D, P 128
Kanerva, A, Or 24, P 106, P 149, P 249, P 266
Kang, J K, P 79
Kangasniemi, L, Or 24, P 249, P 266
Kao, VYC, P 232
Kao, YV, P 250
Käppel, C, P 73
Kaptein, L, P 251
Karim, A, P 14
Karlsson, H, P 257
Karyagyaur, M, P 177
Kas, A, Inv 62
Kasahara, N, Inv 36
Kaschig, K, P 31, P 33
Kataoka, K, P 162
Kaufmann, KB, P 49, P 205
Kawamura, K, P 274
Kawanishi, Y, P 226
Kay, M, Inv 64
Kazlauskas, A, P 123
Kehl, E, P 43
Kelleher, M, P 126, P 137
Keller, D, Or 15
Kernan, F, Inv 15
Khan, K, P 61
Khoo, B, Or 33
Kichler, A, P 174, P 175, P 192
Kim, DY, Inv 60
Kim, HA, P 164
KIm, JYK, P 163
Kim, NY, P 112
Kim, YJ, Or 10
Kim, YS, P 206
Kimura, E, P 80
Kimura, S, P 80
King, J, P 237
King, LA, P 212
Kingsman, S, Inv 62
Kinnon, C, P 5, P 7, P 10, P 19, P 43
Kirn, DH, Inv 30
Kirsten, R, Or 9
Klatzmann, D, Inv 26, P 9
Klausegger, A, P 46
Klavinskis, L, P 93
Kleeff, J, Or 11
Klein, C, Or 53, P 41
Kloos, A, P 236
Klutz, KK, P 165, P 198, P 246, P 247
Kneissl, S, P 197, P 207, P 234
Knight, SB, P 216, P 217
Knolle, P, Or 11
Knoop, KK, Or 7, P 165
Ko, YM, P 206
Kobayashi, H, P 274
Kobelt, D, P 166
Koch, C, P 135
Kochan, F, P 214
Kochanek, S, P 235
Koide, T, P 80
Kok, CY, P 29
Koller, U, P 46
Komaromy, AM, Or 14
Kondratenko, I, P 41
Konstantinova, P, P 34, P 240
Koo, T, P 81
Koornneef, A, P 34
Koppers-Lalic, D, P 200
Korn, M, P 235
Korner, G, P 35
Koski, A, P 249
Kostic, C, P 119
Kostka, L, Or 48
Kostova, Y, P 252
Kotlinowski, J, Or 34, P 131
Kovtun, A, P 124
Kozakowska, M, P 131
Krampera, M, P 286
Krattenmacher, A, P 49
Kremer, EJ, Inv 57, Or 39
Kreppel, F, P 235
Krishnamurthy, P, Ed 8a
Kuball, J, Or 17
Kubicka, S, P 236
Kudo, Y, P 58
Kudsiova, L, P 167
Kueberuwa, G, P 253
Kuehle, J, Or 53, P 59, P 194
Kues, W, Or 29
Kühlcke, K, P 41
Kühle, J, Or 19
Kühnel, F, P 236
Kume, A, P 36, P 215
Kurata, H, P 71
Kurreck, J, P 285
Kustikova, O, Inv 12
Kutschera, I, P 21
Kweder, H, P 211
Kwon, SY, P 93
Kymäläinen, HE, P 22, P 208
Lachmann, N, P 67
Laga, R, P 102
Lagresle-Peyrou, CLP, P 37
Laible, G, Or 29
Lakhal, S, P 11
Landgraf, TN, P 98
Lange, K, Or 8
Lange, KL, P 194
Langfield, KL, Or 35
Langford-Smith, A, P 45
Langford-Smith, K, P 45
Lanzi, A, Or 43
Larcher, F, P 44
Larkin, DFP, P 128
Latella, MC, P 183
Lavigne, M, P 51
Lavillette, D, Or 47
Lavisse, S, Inv 62
Lawrence, MJ, P 167
Lebkowski, JS, Inv 20
Leborgne, C, P 174
Lecomte, MJ, P 209
Lee, C, P 1
Lee, CM, P 31, P 33, P 38
Lee, DA, Or 15
Lee, HI, P 206
Lee, J, P 54
Lee, M, P 164
Lee, S, P 111
Lee, SY, P 53
Lee, SK, P 206
Leger, A, P 210
Leja, J, P 254
Leja, JL, Or 26, P 248
Lemke, K, P 131
Lemoine, NR, Or 25, P 199, P 213, P 264, P 272, P 279
Lentacker, I, P 143
Lenting, P, Or 23
Lenz, D, P 271
Leo, S, P 189
Lepetit, H, Inv 62
Lévy, C, P 193, P 211
Li, F, P 93
Li, LL, P 255
Li, Q, P 274
Li, X, P 279
Li, Y, P 251
Li, YT, P 89
Liau, LM, Inv 36
Liljenfeldt, L, P 258
Lim, A, P 4
Lima, M João, Inv 18
Limberis, MP, Or 3, Or 14
Lin, HT, P 132
Lin, J, Or 41
Linch, D, P 267
Lindemann, D, Or 6
Lindholm, L, P 200
Lindqvist, C, P 257
Linninger, AA, P 14
Lipinski, D, P 228
Litichever, N, Inv 17
Little, D, P 147
Liu, GL, P 282
Liu, PQ, Or 17
Liu, Z, P 256
Loader, J, P 121, P 125, P 126
Loboda, A, Or 34, P 131
Locanto, E, P 212
Lockley, M, P 242
Logan, K, P 93
Lomas, DA, Inv 19
Lombardo, A, Inv 8, Or 17, Or 18, Or 54, Or 55, P 70
Lonjon, N, P 209
Lopes, E, P 178
Lorain, S, P 120
Loskog, A, P 257, P 258
Lozano, ML, P 66
Lucia, T, P 117
Lüer, C, P 116
Luhmann, UFO, P 128
Lulay, C, P 41
Lundberg, C, P 2, P 191
Lundin, KE, P 184
Luo, H, P 286
Lynn, G, P 102
Macintosh, J, Inv 64
Macrae, IM, Or 39
Maczuga, P, P 34
Maeda, Y, P 82
Maeder, M, Or 19
Maetzig, TM, Inv 12, Or 6, Or 37, Or 53, P 194, P 224
Maghsoudlou, P, Or 41
Magnani, Z, Inv 8, Or 17, P 96
Magnusson, M, P 200
Maguire, AM, Or 14
Maher, J, P 241, P 250, P 256, P 259, P 265, P 277, P 278
Maitalnd, N, Inv 21, P 268
Majdalani, NM, Or 26
Majka, M, P 131
Makarevich, PI, P 287
Malardo, T, P 85, P 104, P 105
Małecki, M, P 270
Malerba, A, P 79
Maleszewska, M, Or 34
Malik, P, P 4
Mallet, J, P 209
Malric, A, P 28
Mancheño, U, P 47
Manns, MP, Or 8, P 223, P 236
Mantwill, K, P 252
Marais, T, Inv 49
Marcatti, M, P 263
Marie, C, P 173
Markelov, M, P 100
Maródi, L, P 41
Martí-Bonmatí, L, P 145
Martín, F, P 16, P 92
Martin, F, P 204
Martin, S, P 192
Martin, JF, P 291
Márton, G, P 74
Mastrobattista, E, Or 43
Mather, SJ, P 250, P 259, P 277
Mattar, CN, P 221
Mauleón, I, P 47
Mavilio, F, Or 46, P 70, P 96, P 183
Maya-Pineda, H, P 213
Mayr, E, P 46
Mazarakis, ND, Inv 58, P 228
Mazzucco, A, P 286
McBride, JW, P 168
McCabe, C, Or 39
McCaffrey, J, P 148
McCarthy, HO, P 148, P 157, P 168, P 169
McClorey, G, P 86
McCullagh, K, P 288
McFarland, T, P 130
McHale, AP, Or 27
McIntosh, J, Or 23, P 22, P 222, P 243
McNeish, IA, Inv 35, P 242
McVey, JH, Or 40, P 229
Meinl, AT, P 91
Meiser, A, P 93
Meivar-Levy, I, Inv 17
Melder, DC, Or 35
Melero, I, P 47
Melo, RVG, P 104
Méndez-Ardoy, A, P 273
Meng, J, P 75
Mennechet, F, Inv 57
Merella, S, Or 10, Or 50, Or 51, Or 52, P 189
Mestre-Francés, N, Inv 57
Méthot, J, P 30
Metwally, AA, P 138, P 150, P 151
Metzner, C, P 214
Mével, M, P 141
Meyer, J, P 39, P 65
Mezzadra, RE, P 160
Miguel, A, P 145, P 146, P 159
Mihelec, M, Or 13
Mikkelsen, JG, P 119, P 152, P 218
Milanov, PM, Or 31
Milili, MM, P 37
Miller, JC, Or 54
Milne, JS, P 291
Min, JJ, Inv 60, P 289
Minegishi, M, P 58
Mingozzi, F, Inv 64
Miranda, E, P 213
Mischak-Weissinger, E, P 97
Miselli, F, P 70
Miskey, C, Ed 11, Inv 54
Miskin, J, Inv 62, P 121, P 125, P 126
Mitrophanous, KA, Inv 62, P 87, P 126, P 130, P 137, P 219
Mittermayr, R, P 90
Miyamoto, S, Or 2
Mizukami, H, P 36
Moccetti, T, P 117
Mock, U, P 103
Modlich, U, Inv 12, Or 8, Or 37, P 39, P 65, P 67, P 223, P 271
Mohamed, AJ, P 184
Mojidra, C, P 118
Molakandov, K, Inv 17
Molina-Estevez, FJ, P 18, P 66
Mondino, A, P 96
Monkeviciute, A, P 216
Monnery, B, P 170
Montiel-Equihua, CA, P 26, P 48, P 216
Montier, T, P 156
Montilla, AI, P 145, P 146, P 159
Montini, E, Inv 10, Or 1, Or 10, Or 50, Or 51, Or 52, P 9, P 69, P 72, P 189
Montus, M, Inv 51
Moon, A, Inv 30
Morcos, M, P 129
Moreira, BP, P 85, P 104, P 105
Moreno, PM, P 184
Moreno-Manzano, V, P 13
Morgan, JE, Inv 2, P 75, P 78
Mori da Cunha, M, P 283
Moritz, T, P 67
Morris, EC, Or 42, P 113, P 114, P 237, P 262
Morris, J, P 256
Morris, K, P 86
Morrison, J, P 276
Morton, C, P 22, P 222
Moscatelli, I, P 40
Moullier, P, Inv 51, P 17, P 210, P 220
Mufti, GJ, Ed 8a, P 1
Mühlebach, MD, P 239, P 261, P 280
Muir, K, Inv 18
Müller-Kuller, U, P 217
Münch, R, P 261
Muñoz, P, P 16, P 204
Muntoni, F, Inv 2, P 75, P 78
Murauer, EM, P 46
Mykhaylyk, O, P 135
Mzukami, H, P 215
Nakanishi, Y, Or 2
Nakauchi, H, P 132
Naldini, L, Inv 8, Inv 10, Inv 28, Or 1, Or 10, Or 16, Or 17, Or 18, Or 22, Or 51, Or 52, Or 54, P 7, P 69, P 70, P 72, P 189, P 263
Nanou, A, P 83, P 147
Narita, A, P 58
Nascimento-Ferreira, I, Or 45
Nathwani, AC, Inv 64, Or 23, P 12, P 22, P 222, P 243, P 267
Nathwani, D, P 243
Naundorf, S, P 41
Navarro, S, Or 18
Navarro, V, P 145
Nawathe, S, Or 23
Naylor, S, Inv 62, P 126, P 130
Nègre, D, Or 47, P 193
Nehlsen, K, Or 29
Nelson, PJN, Or 7
Neri, M, Inv 8
Ng, C Y C, Inv 64
Ng, P, P 24, P 25
Nguyen, VH, Inv 60
Nicholson, E, P 262
Nickerson, ML, P 210
Nicklin, SA, P 188, P 284, P 290
Nicosia, A, Inv 55
Nienhuis, AW, Inv 64
Nilsson, BN, Or 26, P 107, P 248, P 254
Ning, K, P 76, P 83, P 84, P 147
Noble, A, Ed 8a
Nobrega, C, Or 45
Nógrádi, A, P 74
Noguera, I, P 146
Nokisalmi, P, P 249
Nosov, M, P 108, P 129
Noviello, M, Or 21
Nowrouzi, A, Or 9, Or 54, P 41, P 66, P 109
Nusco, E, P 23, P 24
O'Flynn, L, P 129
O'Hanlon, DOH, P 136
O'Sullivan, GOS, P 136
ÓValle, F, P 92
Öberg, K, P 254
Ocwieja, K, P 64
Odoardi, F, P 108
Odon, VMN, P 171
Ogilvie, C, P 208
Ogris, MO, P 165, P 198, P 247
Oh, S, P 93
Okada, T, P 81
Okamoto, S, P 274
Okanobo, A, P 122
Oldham, K, P 111
Oliveira, A, P 153, P 172, P 178
Oliveira, NAJ, P 154
Onofre, I, Or 45
Orchard, K, P 1
Ord, ENJ, Or 39
Ormán, I, P 42
Osada, K, P 162
Osorio, L, P 196
Osuchowski, MF, P 91
Otsu, M, P 132
Ott, M, Or 8, P 223
Oumard, A, P 68
Owen, J, Or 33
Ozasa, S, P 80
Ozawa, K, P 36, P 215
Padilha, E, P 85, P 104, P 105
Page, M, P 93
Pahujani, S, P 49
Pajenda, G, P 74
Palfi, S, Inv 62
Pala, M, Or 10
Palazzolo, G, Or 12
Pallant, C, P 7
Palmer, D, P 24, P 25
Pamukcu, C, P 55
Pantelidou, C, P 264
Panunto-Castelo, A, P 98, P 105
Papagatsias, T, P 93
Pâques, FP, Or 28, P 63
Parente-Pereira, AC, P 241, P 259
Parfyonova, YEV, P 287
Park, SH, Inv 60
Parker, AL, Or 40, P 290
Parker, CV, P 219
Parker, M, P 130
Parsley, KL, P 5, P 10, P 43, P 118
Paruzynski, A, P 41
Paschon, DE, Or 17
Passirani, C, P 156
Pastore, N, P 23, P 24
Patterson, S, P 93
Paul Wetterberg, G, P 257
Pearson, RA, Or 13, P 134, P 8
Pearton, M, P 142
Peccate, C, P 120
Peebles, DM, P 291
Peggs, K, P 267
Pellin, D, Or 46, Or 50
Penaud-Budloo, M, P 210
Peng, K-W, P 261
Pereira, ACP, P 265, P 277
Peretti, G, Or 21
Pérez, D, P 145
Perez, OD, Inv 36
Perini, I, Or 12
Peroni, CN, P 154
Perreau, M, Inv 57
Perrin, F, P 209
Pertschuk, D, Inv 36
Peruta, MD, P 22, P 243
Pesonen, S, Or 24, P 106, P 149, P 249, P 266
Petropolou, T, P 5
Petry, H, Or 9, P 34, P 47, P 240
Pezant, J, P 209
Phaltane, R, P 67
Picard, CP, P 37
Piccolo, P, P 25
Pie, J, Inv 64
Piguet, F, P 17
Pihlmann, M, P 119
Pilet, H, P 209
Pincha, M, P 271
Piovani, B, P 187
Pipino, C, Or 41
Pirlo, KJ, P 242
Pitard, B, P 141, P 156
Plank, C, P 135
Plati, T, Or 51
Plückthun, A, P 261
Poirot, LP, Or 28, P 63
Popplewell, L, P 79
Porada, CD, Or 41
Porras, O, P 40
Porter, A, P 71
Pörtner, R, P 280
Pospori, C, P 114
Pottage, K, P 140
Pournaras, JC, P 119
Pourzand, C, P 150
Préat, V, P 173
Preiss, C, P 68
Price, G, Inv 62
Prieto, J, P 47
Primo, MN, P 218
Privat, A, P 209
Protzer, U, Or 11
Provasi, E, Inv 8, Or 17, P 96
Prüfer, S, P 261
Pujol, R.M, P 42
Pule, M, P 267
Quade-Lyssy, PQL, Or 31
Quattrocelli, M, Or 12
Quintana, O, Or 18
Quintanilla, ME, P 32
Quintino, L, P 2, P 191
Quiviger, M, P 173
Raeven, P, P 91
Ragon, I, P 9
Rahim, A, P 185
Rahim, AA, P 19, P 221, P 229
Rahman, SH, Or 19
Raivich, G, P 185
Raj, D, P 267
Ralph, L, Inv 62, P 87, P 137
Ramelli, AL, Inv 62
Rammensee, H, P 1
Ranki, T, P 106, P 266
Ranzani, M, Or 10, Or 51, Or 52, P 69
Raoul, S, P 17
Rashid, ST, Inv 19
Rashkova, VR, P 107
Rauschhuber, C, P 186
Rebar, EJ, Or 15
Recchia, A, Or 46, P 70, P 96, P 183
Redl, H, Or 27, P 74, P 90, P 91
Reelfs, O, P 150
Reik, A, Or 15, Or 17
Reiss, U M, Inv 64
Rejman, J, Or 20, P 143, P 273
Remy, P, Inv 62
Resnier, P, P 156
Rethwilm, A, P 230
Reuser, AJ, P 6
Richard, M, P 173
Richter, J, P 40
Riddell, A, Inv 64
Riedel, H, Or 19
Rio, P, Or 18, P 42
Riou, M, P 209
Rischmueller, A, P 176
Riso, PL, Inv 8
Rittelmeyer, I, Or 8, P 223
Ritter, T, P 108, P 129
Rivera, G, P 268
Rivière, JRJ, Or 28
Rizvanov, AA, P 55
Rizzardi, GP, P 187
Rizzi, E, Or 46
Robbins, JM, Inv 36
Roberts, I, P 71
Roberts, T, P 86
Robson, T, P 157, P 168, P 169
Rocca, C, P 18
Roda, M, Inv 49
Rödl, WR, P 165
Rodrigues, AF, P 115
Rodríguez-Martín, T, Or 36
Roesen, N, P 93, P 244
Roeske-Nielsen, A, P 161
Romano, V, P 149
Ron, K, Inv 17
Roncarolo, MG, Or 1
Rosales, C, Inv 64, P 222, P 243
Ross, A, P 116
Rossetti, RAM, P 85, P 98, P 104, P 105
Rossi, P, Or 22
Rothe, M, Or 8, P 223
Rouiller, JR, P 37
Roujeau, T, P 17
Rouvinen-Lagerstrom, N, P 149
Rubina, K, P 177
Rudolph, C, P 65
Rüdrich, U, P 223
Ruggiero, E, P 109, P 271
Russell, SJ, P 193
Ryan, A, P 108
Ryan, M, P 171
Saban, D, P 122
Sabater, L, P 145
Sahel, J-A, P 120
Sahin, F, P 55
Saleun, S, P 220
Salguero, G, P 97, P 271
Salinas, S, Inv 57
Saller, V, P 142
Salmons, B, P 214
Salomoni, M, Or 1
Salvatori, F, P 187
Salzig, D, P 56, P 280
Sampaolesi, M, Or 12
Sampedro, A, P 47
Sánchez, M, P 146, P 159
Sandra, P, P 285
Santilli, G, P 43
Sanvito, F, Or 10, P 69
Sapag, A, P 32
Sapir, T, Inv 17
Sarkis, C, P 209
Sarradin, P, P 209
Sato, Y, P 58
Sauer, JA, Or 35
Sauss, H, P 237
Savoldo, B, Or 16
Scallan, MF, P 31, P 33, P 38
Scaramuzza, S, Or 1, Or 22, Or 50
Şahin, F, P 52
Schaller, T, P 95
Schambach, AS, Inv 12, Or 6, Or 8, Or 19, Or 37, Or 53, P 4, P 26, P 39, P 59, P 194, P 205, P 223, P 224, P 225
Scherman, D, P 173, P 174, P 175
Schiff, CS, P 37
Schiffer-Mannioui, C, P 63
Schlag, PM, P 166
Schlager, P, P 46
Schleef, M, Or 29, P 176
Schlegelberger, B, P 65
Schlichtiger, JS, Or 7
Schmeer, M, P 176
Schmidt, M, Or 9, Or 10, Or 37, Or 50, Or 51, Or 52, Or 54, P 9, P 21, P 41, P 51, P 62, P 66, P 69, P 72, P 73, P 109, P166, P 208, P 216, P 225, P 271
Schmiermund, A, P 56
Schneider, I, P 234
Scholtz, S, P 9, P 51
Scholz, S, P 216, P 225
Schrøder, LD, P 152
Schroff, M, P 166
Schultz, A, P 40
Schulz, R, P 216
Schumacher, TNM, P 160
Schüttrumpf, JS Jörg, Or 31
Schützenberger, S, Or 27
Schwarzer, A, Inv 12, P 65
Schwenk, NS, Or 7
Scorer, S, Inv 62
Scripps, V, P 130
Scupoli, M, P 286
Searle, PF, Ed 7
Searle, P, P 111
Seder, R, P 102
Segovia, JC, Or 18, P 42
Seifried, ES, Or 31
Seki, O, P 58
Self, S, P 93
Semenova, E, P 44
Semina, E, P 177
Sendra, L, P 145, P 146, P 159
Sendtner, M, P 84
Senekowitsch-Schmidtke, RSS, P 165, P 198, P 246, P 247
seng, EC, P 209
Seow, Y, P 11
Sepe, RM, P 23
Sergi, L Sergi, Or 22
Sergi, L, Or 10, Or 52, P 69, P 72
Sergijenko, A, P 45
Serra, M, P 57
Serratrice, N, Inv 57
Servais, L, Inv 51
Sessa, M, Inv 10
Sevin, C, P 17
Seye, A, P 192
Seymour, L, Ed 1, Inv 56, Or 48, P 53, P 93, P 94, P 102, P 238, P 244, P 253, P 275, P 276
Sgualdino, J, Or 51, P 189
Shafa, R, Inv 36
Shah, A, P 142
Sharma, N, P 152
Sharrack, B, P 76
Shaunak, S, P 170
Shaw, PJ, P 83, P 84
Shaw, SW, Or 41
Shen, P, P 101
Shevchenko, EK, P 287
Shevelev, AYA, P 287
Shi, Y, Ed 8b
Shiau, AL, P 89, P 101, P 269
Shibata, A, P 242
Shimada, H, P 274
Shimizu, K, P 226
Shin, JH, P 81
Shin, S, P 112
Shipulin, G, P 100
Shirley, R, Or 39
Siclari, F, P 117
Sierpniowska, A, P 131
Sigurdardottir, D, P 1
Silva, A, P 153, P 172, P 178
Silva, G, P 153, P 172, P 178
Simons, JP, Or 33
Sing, A, P 186
Singer, B, P 124
Singh, M, P 179
Siupa, A, P 140
Skawran, B, P 65
Skrzypek, K, P 131
Sleep, S, Inv 64
Smith, A, P 195
Smith, AJ, Or 13, P 8, P 128
Smith, CI, P 184
Smith, J, P 63
Smits, E, P 1
Snapper, J, Or 9
Snyder, RO, P 210
Sodeik, B, P 224
Soldati, G, P 117
Son, Y, P 54
Soncin, S, P 117
Sondhi, D, P 17
Song, H, P 180
Sousa, MFQ, P 57
Souza-Moreira, L, P 92
Sowden, JC, P 134
Spanggaard, I, P 181
Spence, Y, P 22
Spencer, A, Or 44
Spicer, J, P 241
Spinoulas, A, P 29, P 190
Spitzweg, C, P 198, P 246, P 247
Spitzweg, CS, Or 7, P 165
Spurrel, E, Or 25
Stabenow, D, Or 11
Stambolsky, D, P 177
Statkiewicz, M, P 270
Stauss, HJ, Or 42, P 114, P 262
Stefanska, A, P 131
Stein, S, P 225
Steinemann, D, P 65
Steinke, JHG, P 170
Stelzer, T, P 161
Stephan, SA, Or 35
Stepniewski, J, Or 32
Steuhl, K, P 124
Stewart, HJ, P 219
Stojkovic, M, P 13
Stok, M, P 6
Stoneham, C, P 227
Stornaiuolo, A, P 187
Stout, T, P 130
Strickland, IT, P 87, P 201
Strick-Marchand, H, Inv 19
Stripecke, R, P 97, P 271
Strüver, N, P 236
Su, CH, P 269
Subr, V, P 276
Suerth, JD, Inv 12, Or 37, Or 53
Sullivan, J, P 140
Sürder, D, P 117
Surralles, J, P 42
Sürth, JD, P 205
Suzuki, A, P 58
Sweeney, K, P 272
Swift, S, P 118
Swoden, JC, P 8
Symens, N, P 273
Sysoeva, VYU, P 177, P 287
Szymula, A, P 131
Tada, Y, P 274
Tagawa, M, P 274
Takahashi, H, P 58
Takahashi, M, Inv 24
Takayama, K, Or 2
Takayama, N, P 132
Takeda, S, P 81
Takeuchi, Y, P 231
Takiguchi, Y, P 274
Tampier, L, P 32
Tang, TT, P 282
Tangney, MT, Inv 61, P 136
Tani, N, Inv 62
Tani, K, Or 2
Tarantal, A, P 22
Tatsumi, K, P 274
Tavernier, G, Or 20
Teagle, A, P 182
Tedcastle, A, P 253, P 275
Tedesco, FS, Or 21
Tessari, M, P 286
Thanou, M, P 170
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Thiele, R, P 103
Thoma, C, P 276
Thomas, S, P 114, P 262
Thomaschewski, M, Or 5
Thöny, B, Or 30, P 35
Thorpe, R, P 231
Thorrez, L, Or 12
Thrasher, AJ, Inv 2, Inv 3, Or 41, P 4, P 5, P 7, P 10, P 18, P 20, P 22, P 26, P 43, P 44, P 48, P 78, P 95, P 118, P 185, P 216, P 229, P 232, P 233
Thudium, CS, P 40
Tkachuk, V, P 177
Tkachuk, VA, P 287
Tockner, B, P 46
Toelen, J, Or 4, P 50, P 283
Tollefson, A, Or 25
Tomaszewski, A, P 124
Tonlorenzi, R, Or 21
Tonon, G, Or 10
Tookman, L, Inv 35, P 242
Tordo, J, P 88
Torikai, H, Or 15
Torre, T, P 117
Toscano, MG, P 16, P 204
Tostoes, RM, P 57
Tothova, L, P 155
Toublanc, E, P 220
Touzot, F, P 4
Towers, G, Inv 43, P 95
Trabalza, A, Inv 58, P 228
Treacy, O, P 108, P 129
Trollet, C, P 77
Tsokolaeva, ZI, P 287
Tsuchiya, S, P 58
Tsukahara, T, P 36, P 215
Tsyganova, G, P 100
Tuddenham, EGD, Inv 64, Or 23, P 22
Turan, S, Or 53, P 59
Turchiano, G, P 183
Turko, P, P 3
Tye, G-J, Ed 8a
Tykodi, SS, Or 15
Tysome, J, P 279
Uchida, S, P 162
Uckert, W, P 234
Ugolini, M, P 149
Uhde, A, P 103
Uil, T, P 200
Ulbrich, K, Or 48, P 276
Uney, JB, P 201
Ungerer, CU, Or 31
Unzu, C, P 47
Urabe, M, P 36, P 215
Urata, Y, Or 2
Ursulet, S, P 209
Uttenthal, BJ, P 113
Uusi-Kerttula, H, P 290
Vaha-Koskela, M, P 149
Vakova, J, P 23
Valeri, A, P 42
Vallier, L, Inv 19
Valori, CF, P 83, P 84
van den Bulk, N, P 30
Van den Haute, C, Or 4, P 196
Van den Hengel, S, P 200
Van der Loo, H, P 4
van der Stegen, SJC, P 241, P 259, P 277
van Deventer, S, P 34, P 240
van Griensven, M, Or 27, P 90, P 91
van Schalkwyk, MCI, P 241, P 278
van Til, NP, P 6
Vandenberghe, LH, Or 14
Vandendriessche, T, Or 12
Vandermeulen, G, P 173
Vanier, MT, P 17
Vannocci, T, P 71
Van-Tendeloo, V, P 1
Vassaux, G, Inv 7
Venditti, CP, P 27
Verdier, JM, Inv 57
Verhoeyen, E, Or 47, P 193, P 211
Vessillier, S, P 250
Vetchinova, A, P 100
Vetrini, F, P 23, P 24
Vets, S, P 64
Vetter, AV, P 165, P 198, P 247
Vetter, R, P 285
Vevis, K, Inv 58
Veys, P, P 5, P 118
Vidovic, D, Or 4
Viecelli, HM, Or 30, P 35
Vile, R, P 149
Villa, A, Or 1
Vitek, L, P 23
Vlasik, TN, P 287
Voelkel, C, Or 6, Or 53, P 224
Vogelbaum, MA, Inv 36
Voigt, K, Ed 11, Inv 54
Voisine, C, P 237
Voit, T, Inv 49, Inv 51, P 120
Völker, I, P 239
Volpin, M, Or 52, P 69, P 72
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Waddington, SN, Inv 33, Or 23, P 19, P 22, P 221, P 229, P 232
Wagemaker, G, P 6, P 251
Wagner, EW, Or 7, P 165, P 198, P 247
Wagner, S, Or 29
Walker, HJ, Or 35
Wallace, JM, P 291
Wally, V, P 46
Walther, W, P 166
Wang, CR, P 89
Wang J, Or 25
Wang J, Or 54
Wang, JW, P 282
Wang, L, P 147
Wang, P, Or 25, P 279
Wang, S, P 279
Wang, Y, Or 25, P 199, P 279
Ward, NJ, P 229
Warlich, E, Or 53
Warner, TT, P 12
Warren, EH, Or 15
Was, H, P 131
Wassermann, K, Or 27
Waxman, J, P 237
Weber, K, Or 5
Wege, H, Or 5
Weiss, D, Inv 5
Weiss, K, P 280
Weitzman, M D, Inv 52
Wengel, J, P 184
West, EL, P 134
Wettergren, EE, P 191
Wheatley, SP, P 242
Whitcomb, LS, Or 35
White, K, P 188
Whitehouse, A, P 139
Wicke, DC, P 39
Widdowson, P, P 126, P 130, P 137
Wiktorowicz, T, P 230
Wilk, M, P 108, P 129
Wilkie, S, P 259, P 277, P 278
Wilkinson, F, P 45
Willassen, Y, P 191
Willhauck, MJW, Or 7, P 165, P 246
Williams, D, P 4
Williams, R, P 243
Wilson, JM, Ed 9, Inv 11, Inv 14, Inv 16, Or 3, Or 14
Winkler, S, P 231
Wintgens, V, P 174
Wintterle, S, P 39
Wirth, T, P 281
Wold, W, Or 25
Wolf, A, P 135, P 252
Wolfrum, K, Or 20
Woller, N, P 236
Wong, AMS, P 221
Wong, LF, P 87, P 201
Wong, SP, Or 38
Wood, MJ, Inv 40, P 11, P 86
Work, LM, Or 39
Wraith, E, P 45
Wright, G, P 237
Wright, GP, P 113
Wu, CL, P 89, P 101, P 269
Wunderlich, N, P 198, P 246
Wunderlich, NW, P 165
Wyles, M, P 83, P 84
Wynn, R, P 45
Xin, YX, P 282
Xu, S, P 195
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Yalvaç, ME, P 52, P 55
Yamada, M, Or 2
Yamauchi, S, P 274
Yáñez-Muñoz, RJ, Inv 9, P 18, P 208
Yañez, R, Or 18
Yang, JM, P 112
Yang, MY, P 255
Yang, S, P 274
Yawata, T, P 226
Ye, H, P 53
Yin, H, P 11
Ylä-Herttuala, S, Inv 45, P 281
Young, N, P 231
Yu, DY, Or 26, P 248, P 254
Yuen, C, Or 15
Yusa, K, Inv 19
Zachary, IC, P 291
Zaghloul, EM, P 184
Zamoyska, R, Or 42
Zamykal, M, Or 32
Zanetti, G, Or 51, P 189
Zanoni, G, Or 27
Zebzda, A, P 131
Zerah, M, P 17
Zhan, H, P 118
Zhang, F, P 5, P 233
Zhang, J, Ed 8b
Zhang, JZ, P 255
Zhang, L, Or 17, P 48, P 216
Zhang, LZ, P 282
Zhao, Y, P 231
Zheng, JZ, P 282
Zhogina, Y, P 100
Zholobenko, A, P 169
Zhou, J, Inv 64
Zhou, Q, P 234
Zhou, Y, Or 15
Zia, S, P 50, P 283
Ziehr, H, P 116
Zimmermann, H, Inv 37
Zischek, CZ, Or 7
Zong, S, P 235
Zu, MZ, P 282
Zucchelli, E, P 187
Zussy, C, Inv 57
Zychlinski, DZ, Or 53, P 194
Footnotes
Invited Speakers (Inv) are found on pp 2–20; Oral abstracts (Or) on pp 21–38; Poster abstracts (P) on pp 39–128.