Abstract
Manfred Ogris Hildegard Büning Anja Ehrhardt Andrew Baker Wolfgang Hammerschmidt Ernst Wagner
Gold sponsors: Chemicell, Plasmid Factory, Bio-M Silver sponsor: Eurofins MWG/Operon
The German Society of Virology, Study-Group «Viral Vectors and Gene Therapy» The DFG Research Priority Program 1230 «Mechanisms of Gene Vector Entry and Persistence» The British Society for Gene Therapy Nanosystems Initiative Munich (NIM) HelmholtzZentrum München
Detailed information regarding registration and hotel booking can be found at the official DG-GT2010 homepage
DG-GT2010
Pharmaceutical Biotechnology
Department of Pharmacy, University of Munich
Butenandtstr. 5-13, D-81377 Munich, Germany
Tel: +49 89 2180 77740
FAX: +49 89 2180 77791
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DG-GT 2010 Invited Presentations*
Design of Recombinant Adenoviral Vectors for Gene Therapy: Improvements and Challenges
Over the past decade recombinant adenoviral vectors (rAdVs) became one of the most prominent gene therapeutic vector systems used in preclinical and clinical approaches. Adenoviral vectors exhibit several targets for modifications, thus they are useful for a variety of applications. Different generations of rAdV based on human adenovirus serotype 5 were generated starting with first and second generation adenoviruses, which lack one or two viral genes to more sophisticated technologies such as high capacity adenoviral vectors (HD-Ad) deleted for all viral coding sequences. Besides these types of vectors which will be discussed in detail, oncolytic adenoviruses exhibiting restricted replication in tumor tissues have to be mentioned but are not the major focus of this lecture. Common for all rAdV is their ability to efficiently transduce a broad range of dividing and non-dividing cells and that they can be produced at high titers. However, in contrast to first and second generation adenoviruses, HD-AdVs have a significantly larger packaging capacity for large trangene expression cassettes and they display long-term transgene expression in preclinical studies. Nevertheless, the biggest challenge in adenoviral gene therapy is to circumvent the immune response against the vector. As HD-AdV lacks all viral coding sequences, there is no response to de novo synthesized viral proteins but immune response directed against the incoming viral capsid components may impair the outcome of a gene therapeutic approach. Thus, improvements with respect to the administration route and methods to modify the surface of the virion are the major focus of ongoing research and will be discussed within this lecture.
Session: Educational Session
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Lentiviral Vectors in Gene and Cell Therapy Approaches
Retroviruses are evolutionally optimized for transfer of their genetic material into cells and integrating their genome into the host cell genome. They are thus well suited for long-term stable gene expression. Recombinant vectors based on gammaretroviruses (MLV) have been widely used in the past in preclinical research as well as clinical trials. However, during clinical trials of SCID, severe side effects occurred leading to induction of leukaemia in five cases. More recently researchers have tried to harness lentiviruses, another genus of the retroviridae and developed recombinant vectors based on Human Immunodeficiency Virus (HIV-1), Simian Immunodeficiency Virus (SIV), Feline (FIV) Immunodeficiency Virus, equine infectious anaemia virus (EIAV) and others. Whereas commonly used gammaretroviruses like MLV depend on cell division and breakdown of the nuclear membrane, lentiviruses and their vectors can also infect and transduce non-proliferating cells. The general concepts on how these pathogens can be converted into efficient and safe gene delivery tools for cell modification, the correction of inherited or acquired diseases will be introduced. Packaging concepts and targeting of lentiviral vectors (LV) will be discussed, as well as self inactivating (SIN) LV that are thought to be less likely to activate or disrupt neighbouring genes upon integration. Additionally important areas of research focus on how lentiviral vectors can be modified to avoid integration and thus reduce risk of insertional mutagenesis or to target integration to specific sites in the genome. An overview on construction and use of lentiviral vectors as tools in molecular biology, transcriptional targeting and de-targeting of LV, regulation of LV-mediated gene expression, pre-clinical as well as recent and future clinical applications will be presented.
Session: Educational Session
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AAV-Based Vectors for Cardiovascular Diseases: Therapeutic Potential in Chronic Ischemia
Therapeutic neovascularization of chronic ischemic muscle tissue is a treatment option for otherwise no-option patients, which are not suitable for interventional or surgical interventions. Protein application of pro-angiogenic factors, such as VEGF or bFGF provided vessel growth and perfusion gains in small and large animal studies. However, in patient studies no clearcut success was achieved by pro-angiogenic protein application. Similar discrepancies were idetified for plasmid therapy, e.g., by liposomal transfection: significant improvement in preclinical experiments was followed by mixed results in human. Even overexpression of pro-angiogenic genes via adenoviral vector, leading to a transgene overexpression for 5-7 days, failed to significantly improve perfusion of an ischemic myocardium (Yla-Herttuala, 2007, Lavu JMCC 2010) Since prolonged transgene expression currently appears as a critical variable for pro-angiogenic gene therapy, long-acting adeno associated viral vectors might no-option patients. Adeno-associated virus (AAV), a member of the parvovirus family, is a non-pathogenic DNA virus, which transduces dividing and non-dividing cells and leading to a long-term overexpression, displaying a favorable immunogenic profile compared to adenoviral vectors (Gruchala, 2004). Recently, we identified the pseudotyped virus strains AAV2/6 (carrying a partial genome of AAV2 and the envelope of AAV6) and AAV2/9 as highly efficient vectors in large animals (pigs, Raake et al., JACC 2008, Kupatt et al., JACC 2010), the latter sufficing to resolve hibernating myocardium after chronic coronary artery occlusion. Moreover, the long-lasting transgene expression after AAV transduction may require inducible transgene activity. We established the combination of AAV vectors and a Tet-off transgene system, where the pro-angiogenic factor Thymosin β4 was used to provide neovascularization being active only 2d a week. In a model of chronic hindlimb ischemia, pulsed pro-angiogenic activity appeared non-inferior to constitutive transgene expression up to 4 weeks after vector application. Taken together, the combination of of AAV vector and an efficient pro-angiogenic transgene may offer therapeutic potential in patients with ischemic cardiomyopathy or peripheral artery disease and exhausted conventional options.
Session: Educational Session
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Introduction into Nonviral Gene Delivery – Overview of Physical and Chemical Delivery Methods
Physicochemical methods for gene delivery have been intensively investigated during the last decades and first products have been approved for veterinary use. Although transfection rates achieved with nonviral delivery systems are frequently lower and gene expression is of only short duration when compared with viral vectors, they offer the advantage of being less immunogenic, less restricted to the size of the delivered transgene and being less expensive with respect to production. The currently used repertoire of physicochemical gene delivery methods have evolved from various basic concept using either naked DNA or complexed with gene transfer agents. Moreover, a variety of methods have been established which make use of different physical phenomena to introduce DNA into cells. In this lecture, basic concepts of various physicochemical gene delivery methods making use of either nanoparticle formation or physical forces for transfection will be discussed. In addition, advantages and restrictions of the physicochemical gene delivery methods will be discussed.
Session: Educational Session
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Imaging in Cancer Gene Therapy
The field of gene therapy has made considerable strides in the last decade by the development of new vectors and an increasing repertoire of therapeutic genes. Non-invasive monitoring of the in vivo distribution of viral and non-viral vectors as well as biodistribution, level and duration of transgene expression have been recognized as critical elements in the design of clinical gene therapy trials. Besides bioluminescence imaging using luciferase, reporter genes that have been evaluated for possible human studies are the herpes simplex thymidine kinase and the dopamine D2 receptor. Cloning of the sodium iodide symporter (NIS), that mediates the active transport of iodide in the thyroid gland and represents the molecular basis for radioiodine scintigraphy, has provided us with one of the most promising reporter genes available today. NIS represents a non-immunogenic protein with a well-defined body distribution that mediates the transport of readily available radionuclides such as 131I, 123I, 124I, 99mTc, 188Re or 211At, which can be used for gamma camera scintigraphic imaging, SPECT and PET imaging. Several research groups including our own have studied the potential of NIS as reporter gene in various applications, demonstrating that in vivo imaging of radioiodine accumulation correlates well with the results of ex vivo gamma counter measurements as well as NIS mRNA and protein analysis. NIS was successfully used to monitor in vivo biodistribution of synthetic vectors and mesenchymal stem cells as gene delivery vehicles after systemic application as well as replication-competent viral vectors using conventional 123I- or 99mTc-gamma camera imaging or 99mTc-SPECT/CT fusion imaging. In addition, PET imaging using 124I provides significant advantages for exact localization and quantitative analysis of NIS-mediated radioiodine accumulation due to enhanced resolution and sensitivity. Taken together, currently available data clearly demonstrate the enormous potential of NIS as a novel reporter gene, that in its dual function as reporter and therapy gene also allows therapeutic radionuclide application.
Session: Educational Session
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Gene Therapy Around the Globe
Gene therapy is a whole new approach to medicine. Instead of designing drugs to treat the symptoms of a disease, gene therapy seeks to identify the root genetic cause of a disorder and to treat it at that level. For diseases caused by mutations in ‘single genes’, this provides the possibility of treating the disease very effectively by providing healthy copies of the mutant gene, allowing production of normal proteins and restoring the healthy phenotype. Accordingly this approach has exciting potential for treatment of many gene-based diseases. Despite its promise, however, gene therapy is often limited by the difficulty of delivering therapeutic genes effectively into the diseased cells. Normally scientists try to do this using viruses as gene-vectors; however, even viruses find it challenging to access all diseased cells within a body. This is the central challenge of gene therapy. Where delivery has been successfully addressed, the results have been very encouraging. For example, children with bone marrow-based immune deficiencies can now be treated very effectively by introducing normal genes into their bone marrow, a procedure that is normally performed in the test tube before reintroducing the bone marrow to the patient. This approach has a success rate comparable to or higher than bone marrow transplantation, and can be applied to all patients (with no requirement to identify a matched bone marrow donor). Similarly a recent study has shown excellent treatment of adrenoleukodystrophy (the disease featured in the film ‘Lorenzo's Oil’) using this approach. However, the choice of viral vectors is important, since treatment of these disorders requires life-long expression of the therapeutic genes, and early generations of viruses used have been found to damage the DNA upon inserting into the genome, leading to leukaemia. Accordingly scientists are now seeking to identify safer viruses, to enable this approach to be used widely. Another approach to efficient delivery is in the field of retinal blindness, where viruses expressing therapeutic genes can be identified directly into the diseased area. Again, very encouraging results have been seen, with clear improvements in sight resulting. Gene therapy can also apply to the use of genetic vaccines, siRNA, and tumour-killing ‘oncolytic’ viruses, all areas where we are seeing rapid progress. As knowledge increases rapidly, so the day when genetic medicines are a routine part of life comes steadily closer.
Session: Educational Session
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Clinical Experience with Gene Therapy in Pediatric Patients
The division of infectious diseases at the Department of Pediatrics, University of Munich, takes care for three patients who were treated with gene therapy protocols. This presentation will highlight their clinical follow up after gene therapy. The first patient was diagnosed with SCID-X1 in early infancy. No HLA-identical sibling was avialable and no matched bone marrow donor was available. Therefore, the patient was transferred to Hospital Necker-Enfants Malades, Dr. Fischer, where an ex vivo retroviral mediated gene therapy was performed. The lymphocyte development after gene therapy showed a successful reconstitution of the immune system. After gene therapy the patient developed a clonal T-cell proliferation, but is now in good clinical condition. The second patient was diagnosed with ADA-SCID and was transferred to Hospital San Raffaele, Dr. Aiuti, Milano, where an ex vivo gene therapy protocol was performed. After gene therapy the patient showed reconstitution of the immune system and is now also in good clinical condition. A third patient was diagnosed of chronic granulomatous disease and at the age of 5 years was having severe infection with Aspergillus nidulans of the lung and of the thoracic vertebral column. He showed progressive tetraparesis. Antimycotic therapy showed only limited success. No HLA-identical sibling was avialable. No matched unrelated donor found. In this situatuion the patient was transferred to Zurich, where an ex vivo gene therapy protocol has been performed. After gene therapy the patient showed 20% gene corrected cells in the PBL for about 4 weeks, then less than 1%. He showed progressive recovery and plays no football and goes to school.
Session: Educational Session
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Gene Transfer Approaches for Gene Addition, Knockdown and Cellular Reprogramming In Vivo
Vectors that allow for DNA directed-RNA transcription can be used to treat a broad number of diseases. Our laboratory has been developing both minicircle plasmid based vectors and recombinant adenoassociated viral (rAAV) vectors for the purpose of developing platforms for gene addition, knockdown, and cellular reprogramming in vivo. These platform technologies are being tested in mice, dogs, and humans with hemophilia B (Factor IX deficiency), mouse models of Hepatitis C Virus Infection, and mouse models of juvenile onset insulin dependent diabetes mellitus, as examples of gene addition, knockdown, and cellular reprogramming strategies, respectively. Minicircle DNA plasmid vectors are devoid of all bacterial plasmid backbone DNA providing 20 to 1000 times more persistent transgene expression compared to routine plasmids when transfected into quiescent cells in vivo. The mechanistic differences compared to routine plasmid DNA vectors are beginning to be elucidated. From a practical standpoint, we have developed a simplified method for minicircle vector preparation that is nearly equivalent to a routine plasmid preparation making it feasible for these DNAs to replace routine plasmids for all mammalian expression studies. Novel recombinant AAV vectors with altered transduction properties and/or site-specific integration into the ribosomal DNA locus are being derived. These expanded vector properties increase their utility for treating serious diseases in people. We will present our current studies using these improved vectors for treating the three platform diseases stated above.
Session: Keynote Lecture
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Delivery Issues for Oncolytic Viruses
While there are several examples where gene therapy approaches are looking promising, they all reflect situations where the gene vectors can be introduced efficiently into target cells – either by direct injection or by transduction ex vivo. Indeed, cancer gene therapy can be very effective if tumour-killing viruses are injected directly into tumours. Unfortunately three quarters of people who develop cancer in the West go on to die from metastatic disease. In this situation it is not possible to inject gene therapy vectors into all the tumour nodules, and intravenous ‘systemic’ therapy is required. However, the human bloodstream represents a very aggressive environment for most gene therapy vectors. Therapeutic microbes delivered via the bloodstream encounter many host defences and anatomical barriers that must be surmounted to enable their access to disseminated cancers. This is particularly true for adenovirus type 5 in humans, where most recipients have powerful pre-existing adenovirus-neutralising activity. We have recently shown that human (but not murine) erythrocytes provide an additional barrier by sequestering adenovirus onto the Coxsackie and Adenovirus Receptor and (via antibodies) complement receptor 1. Coating adenovirus with a layer of hydrophilic polymer can prevent this interaction and allow virus to circulate free in the plasma, showing passive targeting to disseminated tumours and mediating good anticancer efficacy. Entry of polymer-coated virus particles into the tumour mass is a product of fluid transfer, and is directly proportional to the area under the plasma concentration-time curve. Increasing extravasation of fluid through tumour-associated endothelium using permeability-enhancers such as Tumour Necrosis Factor alpha can improve virus particle entry into tumours over 100-fold, reaching as high as 10% injected dose (virus particles) per tumour. This provides the possibility for highly efficient targeting to tumours and good anticancer efficacy. An alternative approach is to target agents to infect tumour-associated vasculature. However while this provides a vulnerable target to traditional gene therapy approaches, endothelial cells do not normally support ‘oncolytic’ viruses. One approach to overcoming this problem is to encode syncytium-forming proteins within the endothelial, to enable trans-complementation of virus replication by tumour-associated factors.
Session: Vector Development
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Adenovirus Vector Engineering for Gene Therapy
Adenoviral vectors are used frequently for gene therapy but much of their potential is limited by their tropism for liver and spleen, leading to effects on virion, sequestration, limited gene transfer to alternate tissue and toxicity. Recent studies have elucidated the mechanism underlying much of this tropism and the consequence of this infectivity profile.1–4 We have shown the precise role of coagulation factor X (FX) in mediating liver uptake of adenovirus in rodent models in recent years. This occurs through a nM interaction between the FX Gla domain and the hypervariable regions of the Ad5 hexon trimer. We modeled our cryoelectron microscopy data from the Ad5:FX interaction. We observed specific contact points within the hexon in hypervariable regions 5 and 7. Creation of novel vectors with deletions and amino acid mutations in these regions had dramatic effects on FX-mediated gene delivery in vitro and in vivo. We have also assessed the impact of HSPG structure on adenovirus uptake. These findings highlight the fundamental importance of the hexon:FX interaction dictating in vivo tropism as well as novel avenues for vector retargeting to alternate sites in vivo. 1Parker AL et al., Blood (2006); 2Waddington SN et al., Cell, (2008); 3Kalyuzhniy, O et al., PNAS (2008); 4Di Paola, N et al., Immunity (2009).
Session: Vector Development
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Laboratory of Molecular Biology, Medical Research Council, Cambridge, UK
Duchenne muscular dystrophy (DMD) is an X-linked genetic disease that affects 1 in 3500 male births. Mutations in the dystrophin gene result in aberrant splicing of the pre-mRNA and hence a truncated and inactive protein. Dystrophin connects actin to the dystrophin-associated complex at the sarcolemma membrane and the lack of dystrophin leads to progressive muscle degeneration and a significantly shorter life span. There is no effective treatment currently available. A number of potential gene therapy approaches are in development. Meanwhile a promising treatment has reached clinical trials, which involves use of antisense oligonucleotides (ON). ONs are targeted to bind to dystrophin pre-mRNA and redirect splicing, resulting in “exon skipping” that restores the correct protein reading frame. The resultant expressed protein is shorter than natural dystrophin but can substitute very effectively. This phenotype is similar to Becker muscular dystrophy that shows generally only mild symptoms in patients. Two chemistry types for an exon 51 targeting ON are currently in clinical trials, 2′-O-methyl phosphorothioates in Holland and Belgium and phosphoramidate morpholino oligonucleotides (PMO) in the UK. Early results in both cases have shown some dystrophin production in patients following systemic delivery. However, levels of dystrophin restored are variable and it is unclear yet whether a therapeutically useful effect will be reached in all muscle types at moderate dosage levels. To enhance activity, Arginine-rich peptides have been developed as conjugates of PMO targeting exon 23 that showed in an mdx mouse model of DMD substantially improved dystrophin production compared to naked PMO. We have been developing novel Arginine-rich Pip peptides that when attached to exon 23-targeted PMO have shown very high dystrophin generation in mdx mice, including in hard-to-reach heart muscle. These and other Arginine-rich peptides involving muscle-specific targeting domains are currently being evaluated as candidates for possible use as a peptide-PMO conjugate in a future clinical trial for DMD.
Session: Vector Development
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Vesalius Research Centrum KUL-VIB, Leuven, Belgium
Adult stem cells and induced pluripotent stem cells (iPS) hold great promise for regenerative medicine. The development of robust non-viral approaches for stem cell gene transfer would facilitate functional studies and potential clinical applications. We therefore generated hyperactive transposases derived from Sleeping Beauty, using an in vitro molecular evolution and selection paradigm. These hyperactive transposases resulted in superior gene transfer efficiencies and expression in mesenchymal and muscle stem/progenitor cells, consistent with higher expression levels of therapeutically relevant proteins including coagulation factor IX. Their differentiation potential and karyotype was not affected. Most importantly, relatively efficient stable gene transfer could be obtained in bona fide hematopoietic stem cells that are capable of hematopoietic reconstitution and multi-lineage gene marking (Mates, Chuah et al., Nature Genetics, 41(6):753–761, 2009). Moreover, stable transposition could also be achieved in iPS which retained their ability to differentiate along neuronal, cardiac and hepatic lineages without causing cytogenetic abnormalities. Most importantly, transposon-mediated delivery of the myogenic PAX3 transcription factor into iPS coaxed their differentiation into MYOD + myogenic progenitors and multinucleated myofibers, suggesting that PAX3 may serve as a myogenic “molecular switch” in iPS. Hence, this hyperactive transposon system represents an attractive non-viral gene transfer platform with broad implications for regenerative medicine, cell and gene therapy.
Session: On the Route to Clinical Application
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University College London, London, UK
A number of inherited neurological diseases are characterised by neurodegenerative changes at or around birth. Prognosis remains dismal and for several of these diseases, including Acute Neuronopathic Gaucher Disease there is no treatment and palliative care remains the only option. Owing to the aggressive nature of this and related diseases fetal or neonatal gene therapy may be the only potential means of treatment. Recently, AAV9 has been shown to cross the blood brain barrier following intravenous injection into neonatal mice, cats and macaques. We have investigated this further by studying vector tropism after intravenous injection into fetal mice. In utero intravenous injection of single-stranded (ss) and self-complimentary (sc) AAV9 expressing green fluorescent protein (GFP) into mice (embryonic day 16) resulted in efficient global transduction of neurons in the CNS. Furthermore, there was a stark contrast in cell type transduction when compared to neonatal administration, confirmed by scanning confocal microscopy. The efficiency varied depending upon ss or sc configuration of the vector. Examination of injected mice by fluorescent microscopy, immunohistology, and GFP ELISA revealed extensive and efficient transduction in the visceral organs in addition to muscle, bone, eye, and skin. Gene expression was also seen in the peripheral nervous system. Similarly, extensive transduction was also observed following intravenous administration of AAV9 to the fetal macaque. The combination of CNS and visceral organ transduction is well suited to the study, and potential treatment for diseases such as acute neuronopathic Gaucher disease where both CNS and visceral pathology require targeting and a suitable mouse model is available.
Session: On the Route to Clinical Application
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Division of Molecular Therapy, UCL Institute of Ophthalmology and UCL/Moorfields Eye Hospital Biomedical Research Centre for Ophthalmology, London, UK
Early-onset severe retinal dystrophy caused by defects in the gene encoding the retinal isomerase RPE65 is associated with poor vision at birth and complete loss of vision in early adulthood. In a phase I/II dose-escalation trial, we have delivered subretinally recombinant adeno-associated virus (rAAV) vector expressing RPE65 under the control of an RPE65 promoter in 9 human subjects with early onset severe retinal dystrophy associated with mutations in RPE65. We have examined systemic vector dissemination and immune responses following vector delivery, assessed visual function pre- and post-vector delivery using a range of psychophysical techniques, and performed detailed electrophysiology and retinal imaging studies. There have been no serious adverse effects of surgical delivery of vector in the subjects enrolled to date. We have detected no systemic dissemination of vector genome. Although we have detected an increase in systemic neutralising antibodies to AAV capsid in two subjects, there have been no evidence of immune responses to RPE65 protein. We have measured significant improvements in retinal sensitivity by microperimetry and dark-adapted perimetry, and improved performance in a test of visually-guided mobility. The outcomes in the first 9 subjects to date suggest that subretinal delivery of rAAV vector can be safe in humans in the short term and can improve retinal sensitivity. These findings support further clinical studies in subjects with RPE65 deficiency and the development of gene therapy for other inherited retinal disorders.
Session: Gene Therapy
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Amsterdam Molecular Therapeutics (AMT), Amsterdam, The Netherlands
Gene therapy is coming of age. Correction or supplementation of genetic defects through gene therapy is amenable to both monogenic disorders as well as to diseases with a key protein playing a key role in the pathology. Alipogene tiparvovec (Glybera®, AMT-011) is the first gene therapy product for a metabolic disease in late stage clinical development. The product contains a gain-of-function variant of the human lipoprotein lipase gene in an AAV1 based vector (AAV1-LPLS447X). Alipogene tiparvovec has been developed for the long term correction of lipoprotein lipase deficiency, to control or abolish symptoms and prevent complications in adult patients clinically diagnosed with lipoprotein lipase deficiency (LPLD). LPLD is a rare, seriously debilitating autosomal inherited monogenic disorder of lipid metabolism. Deficiency of LPL function results in chylomicronaemia. Recurrent pancreatitis is known as the most frequent, potentially lethal, complication; other severe sequelae include diabetes and increased tendency for atherosclerosis. During the presentation the various steps to develop a gene therapy medicinal product will be discussed. Alipogene tiparvovec in LPLD will be used to demonstrate that persistent gene transduction after one-time intramuscular administration is feasible, and results in long-term clinical improvements in the ‘chylomicronaemia syndrome’ caused by LPLD.
Session: Gene Therapy
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Georg-Speyer-Haus, Frankfurt am Main, Germany
In our recent gene therapy trial for X-CGD we demonstrated reconstitution of superoxide activity in phagocytic cells and elimination of preexisting infections in two treated patients. However, an unexpected expansion of myeloid progenitors occurred five months after transplantation. Both patients developed a myelodysplastic syndrome (MDS) caused by insertional activation of MDS1/EVI1 followed by clonal progression and the gradual loss of chromosome 7. P1 died 27 months after gene therapy of MDS in combination with severe septicemia, the latter resulting from loss of bacterial killing activity in transduced cells. P2 underwent allogeneic stem cell transplantation. Forced overexpression of MDS1/EVI1 or EVI1 in human cells disrupted normal centrosome duplication, linking MDS1/EVI1 activation to the development of genomic instability. Optimized SIN gammaretroviral vectors have been shown to have an enhanced safety profile. From several SIN-gammaretroviral vectors expressing gp91phox, we selected a construct containing the promoter of the human c-fes gene for detailed preclinical studies. We used X-CGD mice for monitoring safety and functional reconstitution of NADPH oxidase activity in primary and secondary animals. The SINfes vector was safe, was resistant to CpG methylation and reconstituted superoxide activity to clinical relevant levels. We plan to use this vector for the next Phase I gene therapy trial.
Session: Gene Therapy
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Reprogrammed Measles Viruses as Cancer Therapeutics Three Points of Attack
Soon after viruses were recognized more than 100 years ago, tumor regressions occasionally documented after accidental infections suggested the idea of using them to fight cancer. Early virotherapy clinical trials based on wild type viruses were unsuccessful, but recent ones based on genetically modified viruses are built on much stronger foundations. They foresee extensive monitoring of viral replication, gene expression, and host immunity. Therapeutic efficacy is being assessed by well-defined biological end points, and can be improved. For future clinical trials more specific and potent oncolytic viruses are developed based on three types of modification: targeting, arming, and shielding. Targeting introduces multiple layers of cancer specificity; arming amplifies locally the effects of approved cancer therapeutics; and shielding provides temporary relief from the immune response. We have shown that the envelope of measles (MV) and related paramyxoviruses can be targeted to many designated receptors. Retargeted MV envelopes have become preferred tools to pseudotype lentiviral and other gene transfer vectors. We have developed protease activation targeting, and innate immunity control targeting. We have armed MV with a prodrug convertase, and shown that this virus synergizes with the chemotherapeutic fludarabine to eliminate mantle cell lymphoma xenografts. MV-based therapeutics developed by the Russell and Peng groups at Mayo are being administered to patients who, having exhausted other therapeutic options, have enrolled in clinical trials of ovarian carcinoma, glioma, and myeloma. Survival and quality of life data are being collected and will be critical for the approval of new therapeutic products. Cattaneo, R. (2010) Paramyxovirus entry and targeted vectors for cancer therapy. PLoS Pathogens 6: e1000973.
Session: Cancer Gene Therapy
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Jennerex Inc, San Francisco, USA
Engineered viruses have been developed for cancer therapy both as non-replicating gene therapy agents and as cancer vaccines. Oncolytic viruses, in contrast, were developed to replicate within and subsequently lyse, cancer cells. Clinical efficacy to date with each of these approaches has been limited by multiple factors including the inability to infect enough tumor cells in vivo locally within a tumor or systemically, and resistance of complex advanced tumors to a single mechanism-of-action (MOA). Over the last several years, however, a novel therapeutic class has emerged that combines the best features of all three approaches: targeted and armed oncolytic poxviruses. Recent preclinical and clinical results demonstrate convincingly that products from this therapeutic class can achieve highly selective and potent cancer destruction systemically through a multi-pronged MOA. Given recent clinical validation, we expect this therapeutic class to expand rapidly.
Session: Cancer Gene Therapy
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Oncolytic Vaccinia Virus for the Treatment of Cancer
The creation of novel “cancer lysing” or “oncolytic” viruses over the last 10-15 years has led to the development of a number of innovative anti-cancer therapeutic products many of which have entered into clinical testing. We are examining a number of poxvirus backbones for their ability to selectively target and kill tumour cells. A derivative of the Wyeth Strain called JX-594 has engineered deletions, natural mutations and expresses a GM-CSF transgene, all of which contribute to its ability to infect and kill tumour tissues while leaving normal tissues unscathed. The results of our preclinical models and molecular studies have shown that JX-594 attacks cancers using a multi-pronged approach that includes tumour vascular targeting, immune stimulation and direct cancer cell lysis. JX-594 has now been tested in over 70 patients in a variety of different trial designs and consistently shows tumour targeting activity and a very strong safety profile. The results of our ongoing translational research efforts and clinical data from our phase I and II trials will be presented.
Session: Cancer Gene Therapy
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Prostate Cancer Stem Cells: A New Target for Therapy
Human prostate cancer is characterised by cellular heterogeneity; perhaps one reason for the resistance of prostate cancer to many medical therapies. Once a prostate cancer has failed on hormone-based therapies, the prognosis for the patients is poor. Our approach to tissue heterogeneity in the prostate has been to identify and purify individual cell populations from human prostate cancers and matched normal tissues. We have fractionated cancer epithelium into a majority luminal/secretory population (which comprises more than 99% of the cancer mass) and a minor population consisting of committed basal cells, trans-amplifying cells and a stem cell population. The latter cells are responsible for tumour initiation and in vitro colony formation. Affymetrix microarray analysis of these populations confirms a distinct phenotype for each cell type and functional expression of fate influencing genes. Further analysis has revealed populations of genes whose expression is tightly linked to the differentiation process, while others are associated with the cancerous properties of the cells. The tumour origin of the identified cells has been confirmed by xenografting into a highly immuno-compromised mouse strain at orthotopic, kidney capsule and subcutaneous sites with demonstrable metastases. Genes associated with prostate cancer are expressed in the cultures and their functional significance assessed by SiRNA knock-down in stem cells and in their daughter cells. Epigenetic control, in contrast to permanent genetic alterations or gross alterations involving a reprogramming of cells to express specific transcription factor sets, seems to be a predominant mechanism for an adaptable stem cell in prostate. A better knowledge of the phenotype of the prostate CSC's should permit therapeutic targeting. However, whilst stem cell directed therapies (to treat less than 0.1% of the tumour mass) are unlikely to have an immediate effect on tumour volume, one consequence of targeting the wrong cell types within a heterogeneous tumour is to perturb the natural history of the tumour and results in an amplification of CSC's, in agreement with cancer relapse patterns in man.
Session: Tumor Biology and (Cancer) Stem Cells
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Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
Dendritic Cells (DCs) are key players in the development of adaptive immunity. Their use to boost anti-tumor responses have reached phase III clinical trials but ex vivo production is costly and difficult to standardize. A novel concept for DC production consists of overnight lentiviral vector (LV) transduction of growth factors and full-length antigens into monocytes that results into induction of “SMART-DCs” (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors). This concept has been validated in the preclinical B16 melanoma mouse model for potency and safety with MART-1 and TRP2 as melanoma-associated antigens, overexpressed in 90% of the metastatic melanoma (Koya et al., 2007; Pincha et al., submitted). Third generation self-inactivating tricistronic lentiviral vectors containing interspacing 2A elements co-expressing human GM-CSF, IL-4 and MART-1 or TRP-2 were constructed. Cytokine preconditioning and 16h transduction of CD14 + monocytes with high titer LVs resulted in persistent (3 weeks) autocrine production of GM-CSF (average 10 ng/ml), IL-4 (average 12 ng/ml) and expression of MART-1 or TRP2 (detectable by intracellular staining). Transduced monocytes readily self-differentiated into SMART-DCs (CD209 + , MHCII + , CD80 + CD86+) and were stable in culture for 3 weeks. SMART-DCs/MART-1 were recognized by T cell clones specific for MART-1 in an HLA-restricted manner, assessed by IFN-γ-ELISPOT-Assay. PBMCs obtained from healthy donors that were primed/boosted in vitro with autologous SMART-DCs/MART-1 demonstrated the induction of MART-1-specific T cell responses assayed by IFN-γ-ELISPOT-Assay. SMART-DCs/MART-1 and SMART-DCs/TRP2 are currently being tested for stimulation of T cell responses in melanoma patients. A consortium for clinical development of SMART-DCs has been formed: GMP-grade batch of the tricistronic vectors is planned for additional preclinical testing (optimal vector dose, number of integrated copies, toxicity), monocytes will be transduced with LVs in a closed GMP-grade bag system, development of Standard Operating Procedures and establishment of identity and potency markers are underway.
Session: Cancer Immune Therapy and T-Cell Therapy
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Arming Immune Cells to Fight Cancer
Cancer cells frequently escape T cell recognition by repression of the antigen processing machinery making them invisible for a cytolytic T cell attack. Strategies in adoptive immunotherapy during the last decade aimed to overcome the situation by engineering T cells with a recombinant chimeric antigen receptor (CAR) which uses a single chain fragment of variable region (scFv) antibody for target binding and the intracellular CD3zeta domain for T cell activation. To provide full T cell activation, the CD3zeta chain was fused with the CD28 costimulatory domain in one polypeptide chain; other costimulatory moieties are likewise used. We here discuss the impact of CD28 costimulation on the threshold in T cell activation and on the redirected anti-tumor response in the presence of TGF-beta and regulatory T cells. We moreover introduce a novel generation of CARs with combined CD28-OX40 costimulation in order to harness central memory T cells for a redirected anti-tumor attack. Finally, we demonstrate the power of a redirected T cell attack in eradicating established tumor lesions by eliminating minor tumor cell subsets.
Session: Cancer Immune Therapy and T-Cell Therapy
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Department of Haematological Medicine, King's College London, Rayne Institute, London, UK
Immune mediated rejection of established tumours needs adequate stimulation of cell mediated immunity, as well as the suppression of inhibitory responses that are induced by chronic exposure to tumour associated antigens. Preclinical studies have demonstrated that tumour cells expressing immune costimulatory molecules and appropriate Th1 cytokines can induce immune mediated rejection of previously established tumours. This strategy is now being assessed in a Phase-I clinical trial for the vaccination of relapsed poor prognosis acute myeloid leukaemia (AML) with autologous AML cells that are genetically modified to express B7.1 (CD80) and IL-2. We have also shown that vaccination of advanced solid tumour patients, with dendritic cells that are pulsed with telomerase (hTERT) peptides, can result in the expansion of antigen specific T cells with some evidence of a modest and short-lived therapeutic benefit. In a further recently started Phase-I study we are now evaluating the safety, and potential efficacy of, hTERT peptide loaded dendritic cell vaccination in combination with cyclophosphamide mediated inhibition of immune suppression by regulatory T cells (Treg). A much less demanding alternative to the use of dendritic or tumour cell vaccines is the direct vaccination with tumour associated antigens, provided that the vaccine could indeed induce cell mediated immunity. With this goal in mind we have recently developed a new vaccination strategy based on the combined adjuvants for synergistic activation of cellular immunity (CASAC). CASAC contains different combinations of defined molecules that act synergistically to induce dendritic cell activation. Subcutaneous vaccination with two doses of a single peptide (OVA or Trp2) plus CASAC, induces IL-12 secretion, stimulation of Th1-biased CD4 T-cells, and high levels (routinely 50% by tetramer staining) of antigen specific cytolytic CD8 T cells. The magnitude of CASAC mediated immune stimulation is substantially greater than can be achieved by other adjuvants (e.g., about 100-fold greater than complete Freund's adjuvant). The antigen specific CTL activity induced by CASAC mediated peptide vaccination allows the in vivo lysis of greater than 90% of antigen positive tumour cells in mouse tumour models, resulting in long-lasting immunity with a robust recall response. This strategy will shortly entre clinical studies for WT1 peptide vaccination of myeloid leukaemia patients.
Session: Cancer Immune Therapy and T-Cell Therapy
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Vector Safety Data Leading to a Clinical Trial for the Gene Therapy of Wiskott Aldrich Syndrome: A Perspective on Future Developments
Hematopoietic gene therapy currently relies on the ex vivo gene correction of patient autologous hematopoietic stem cells. This approach has now been tested for over 10 years in pilot phase I/II trials in several inherited diseases. The domain is evolving and the use of lentiviral vectors derived from HIV-1 provide significant advantages in terms of genomic stability, biological properties but also in the area of pharmaceutical product characterization. The rare primary immunodeficiency Wiskott Aldrich syndrome (WAS) is a combined platelet and immune defect. In the perspective of developing a gene therapy for WAS, we have characterized an advanced generation rHIV lentiviral vector pseudotyped with VSVg and implemented a large-scale industrial process to produce and to control this vector under good manufacturing practices (GMP). Recently several European centers were approved to start gene therapy trials for WAS with this lentiviral vector. With a growing number of potential disease applications for lentiviral vector-mediated hematopoietic gene therapy, it seems that there is ground to integrate information on preclinical vector safety features particularly at the level of design, manufacture process or controls. We will discuss these various points through the different aspects leading to the development of WAS gene therapy.
Session: Pharmacology and Toxicology
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Lentivector Transduction of Novel Target Cells
Session: Pharmacology and Toxicology
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DG-GT 2010 Oral Presentations*
Department of Gene Therapy, University of Ulm, Ulm, Germany
Adenovirus (Ad) vectors are widely used for gene therapy approaches. Due to the high abundance of the natural adenovirus receptors CAR/integrins on a wide variety of cells numerous methods have been developed to redirect the virions to specific receptors on target cell surfaces. An increasing number of recent publications evidenced that the success of targeting strategies does not only depend on receptor binding and cellular uptake, but also on intracellular trafficking processes. Therefore, improved knowledge on the intracellular fate of targeted Ad vector particles is mandatory for a rational design of targeted Ad vectors. However, while in principle potent tools are available for fluorescent labeling of Ad vectors in order to analyze post-receptor trafficking, these tools suffer from significant limitations: (i) capsids can so far only be labeled upon amino groups present in all proteins all over the particle surface and this imposes the risk of interference with particle infectivity, (ii) capsomere-specific labeling requires extensive genetic modifications and has only been demonstrated at protein IX, (iii) two-color labeling approaches are not available. Here we present a robust and straight forward labeling procedure based on a genetic-chemical approach that overcomes these limitations. It allows for specific labeling of the capsomeres fiber, protein IX, or hexon and permits two-color labeling. Using this technology we analyzed by means of live cell imaging two different bioresponsive bonds that potentially can be used for the reversible attachment of shielding/targeting moieties to the capsids: disulfide and hydrazone bonds. In our experiments we could not observe significant reduction of disulfides within the observation time of 80 minutes post cell entry. Acid-responsive hydrazone bonds in contrast became quickly hydrolyzed after uptake of the virions (30 min) and may thus be favorable for the generation of bioresponsive vectors and potent tools to overcome the limitations in intracellular trafficking processes of targeted Ad vectors.
Session: Vector Development
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Clinic I of Internal Medicine and Center for Molecular Medicine Cologne (CMMC), University Hospital Cologne, University of Cologne, Cologne, Germany
Vectors based on the adeno-associated virus (AAV), a non-enveloped DNA virus, have emerged as one of the leading gene transfer systems in gene therapy. However, transduction efficiency of clinical relevant cell types such as keratinocytes is insufficient and seems to depend on donor-specific factors. We therefore conducted AAV peptide display selection on human primary foreskin keratinocytes in order to identify AAV2-based targeting mutants with improved efficacy and specificity of gene transfer into keratinocytes. Aiming to force a ligand-receptor interaction that is independent of heparan sulphate proteoglycan (HSPG) binding, a cell surface molecule that is expressed on a wide range of cell types, we depleted our AAV peptide library of mutants capable of HSPG binding. Following five selection rounds on human primary keratinocytes obtained from different donors, viral genomes were sequenced to identify peptide sequence(s) that mediated viral infection. Interestingly, all selected mutants displayed an integrin binding motif as peptide insertion at amino acid position 587 of the AAV2 capsid. Selected peptides were then introduced into the capsid of recombinant AAV vectors (rAAV) which were subsequently assayed for their transduction efficiency and cell type specificity. Compared to rAAV2 the targeting vectors showed an increased transduction efficiency of up to 15-fold in primary human keratinocytes and a dramatic decrease (up to 30-fold) in off-target transduction. Inhibitor studies confirmed HSPG-independent, ligand-mediated cell entry. Further characterization of the entry mode and its consequences of host-cell interaction is ongoing and results will be discussed.
Session: Vector Development
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Department of Virology, Max von Pettenkofer-Institute, LMU, Munich, Germany
Integration systems are promising and attractive tools for the development of new gene therapeutic strategies. Based on site-directed recombination the bacteriophage derived PhiC31 system provides outstanding opportunities for transgene integration into the host genome. However, due to unpredicted insertion events in the genome mediated by PhiC31, the optimization of activity and directing integration into specific target sites are important goals for the usage in therapeutic applications. Therefore, we constructed several fusion proteins of the integrase and different DNA binding domains (DBD) like the synthetic polydactyl zinc finger E2C and the AAV Rep protein. The motifs were either fused N- or C-terminal to the integrase separated by a short TS or a long GGS5 linker. Immunoblot and flow cytometry analysis revealed that the fusion proteins were expressed in comparable amounts after transfection into 293 cells. Excision and integration activity retained up to 80% of the wildtype integrase (WT) when the binding motifs were attached to the C-terminus of the integrase separated by the 45-bp long GGS5 interdomain. As a further step we generated fusion mutants lacking the native DBD of the integrase (ΔDBD). As expected, these were catalytically inactive when measuring recombination efficiencies between wild type PhiC31 integrase recognition sites. However, after performing initial colony forming assays to measure integration events mediated by the ΔDBD fusion constructs, we observed 31% activity of the WT. To further improve the PhiC31 integrase system we plan to build up on our previous study in which we identified hyperactive variants of the integrase showing up to 5.5-fold increased integration efficiency (Liesner et al., 2010). We also characterized critical amino acids involved either in attaching DNA or in the insertion process. Therefore, mutants which lost their native DNA binding properties and their restoration by fusing known binding motifs would be an interesting approach to develop novel tools for site-specific integration. In combination with hyperactive PhiC31 variants this approach could lead to a more specific and efficient PhiC31 integrase system.
Session: Vector Development
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Division of Clinical Biotechnology, CDBIM, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
Gene delivery using cationic polymers has attracted much attention due to their potential advantages, such as large DNA loading capacity, ease of large-scale production, and reduced immunogenicity. We are developing a polyplex system forming micelle from polyethyleneglycol (PEG) - poly[N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide] (PEG-PAsp(DET)) and plasmid DNA. By highly-regulated structure of a diameter of ≈ 100 nm with a PEG palisade, the nanomicelle shows increased tolerance under physiologic conditions with the remarkably low cytotoxicity. A key feature for safe and effective gene transfer is the biodegradability of PAsp(DET). PAsp(DET) was revealed to undergo rapid degradation by Gel permeation chromatography (GPC) and electrospray ionization mass spectrometry (ESI-MS) measurements. In contrast, a derivative polycation, N-substituted polyglutamide (PGlu(DET)), showed no degradability, indicating that the degradation of PAsp(DET) was induced by a specific self-catalytic reaction between the PAsp backbone and the sidechain amide nitrogen. Degradation products of PAsp(DET) caused no cytotoxicity, even at high concentrations in the culture medium. Repeated transfection by administering the polyplexes for every 24 h showed that biodegradable PAsp(DET) provided a continuous increase in transgene expression, while non-degradable PGlu(DET) showed a decrease in transgene expression after 48 h, coupled with fluctuations in expression profiles of endogenous genes. The biocompatibility of nanomicelles plays a significantly role in in vivo applications. By obtaining sustained transgene expressions with minimized toxicity and inflammatory responses such as cytokine induction, we achieved successful therapeutic outcomes by gene introduction using nanomicelles, including in vivo bone regeneration by introducing osteogenic factors to bone defect area and intratracheal gene transfer of adrenomedullin to treat pulmonary hypertension. Hydrodynamic gene introduction to skeletal muscle was also well achieved compared with naked pDNA. We are now trying various applications for disease treatment and tissue regeneration using model animals.
Session: Vector Development
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Physical Chemistry, Department of Chemistry, Ludwig-Maximilians-University, Munich, Germany
For the selective gene therapy of cancer cells in the human body, a tumor specific targeting is required. The EGF receptor provides a promising target as it is overexpressed in many human cancers. A number of anticancer therapeutics directed against the EGF receptor have been successfully developed so far. Polyplexes consisting of DNA complexed with a cationic polymer can be equipped with EGF as a ligand resulting in their fast, receptor-dependent uptake into cancer cells. However, full-length EGF has a high tendency to aggregate and is expensive in production. Furthermore, it activates the mitogenic signaling cascade of target cells, leading to undesirable cell proliferation. In this study, we therefore examined the alternative usage of GE11, a short artificial peptide with high affinity to the EGF receptor, for the specific targeting of polyplexes HUH7 cells. By live-cell imaging with highly sensitive fluorescence microscopy, we directly compared the internalization kinetics of GE11 polyplexes to the uptake of EGF coupled and untargeted polyplexes. To determine the extent of mitogenic signaling after polyplex binding to the receptor, phosphorylated signaling molecules were detected by western blotting. The correlation between receptor signaling and polyplex uptake was then examined in more detail by inhibitor studies with Erlotinib, a specific inhibitor of EGF receptor phosphorylation. Our results reveal that the uptake of GE11 polyplexes is slow compared to EGF polyplexes but much more efficient compared to untargeted polyplexes. After two hours of incubation, GE11- and EGF polyplexes reach similar internalization levels resulting in comparable transfection efficiencies on a long time scale. The activation of mitogenic signaling is significantly reduced for GE11- compared to EGF polyplexes. Further studies reveal that the different uptake kinetics of GE11 and EGF can be correlated to the activation of receptor signaling. Taken together, GE11 polyplexes are a promising candidate for clinical therapy as they show a high transfection efficiency, low aggregation properties and a reduced mitogenic activity.
Session: Vector Development
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Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-Universität München, Munich, Germany
Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce rAAV-mediated gene replacement therapy as a potential treatment for this disease in the CNGA3−/− mouse model. We show that such therapy can restore cone-specific visual processing in the CNS even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3−/− mice became able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular CNG channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cGMP levels in cones, reduced the inflammatory response of Müller glia cells that is typical of retinal degenerations and delayed cone cell loss. Furthermore, ganglion cells from treated, but not from untreated CNGA3−/− mice displayed cone-driven light-evoked spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated vision-guided behavior.
Session: On the Route to Clinical Application
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Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Hannover, Germany
The Wiskott Aldrich Syndrome (WAS) is a complex primary immunodeficiency disease affecting both the lymphoid and myeloid compartment and leading to the development of thrombocytopenia, eczema, autoimmunity and an increased risk of malignancies. In a German WAS clinical gene therapy trial, two patients were successfully treated by receiving autologous CD34 + cells transduced with a MLV-based vector encoding the WAS gene. By performing linear amplification-mediated PCR (LAM-PCR) combined with high-throughput sequencing, we were able to retrieve > 5700 unique integration sites (IS) for patient 1 (P1) and > 9500 unique IS for patient 2 (P2). In both patients, the gene-corrected hematopoiesis showed a polyclonal IS pattern up to 3 years after gene therapy. The analysis of the clonal composition revealed a high clustering of IS in specific genomic regions with more than 70 common integration sites (CIS) of ≥ 10th order. Strikingly, this high clustering could be found amongst others within or close to the proto-oncogenes MDS1-EVI1 (P1: 81 IS; P2: 94 IS), PRDM16 (P1: 10 IS; P2: 28 IS), LMO2 (P1: 13 IS; P2: 29 IS) and CCND2 (P1: 11 IS; P2: 18 IS). A quantitative analysis further revealed that the most active cell clones showed a vector integration upstream of CCND2 in P1 and within the MDS1 locus in P2 constantly contributing to the gene-corrected hematopoiesis. The analysis of sorted cell fractions showed a distinct IS distribution with the appearance of IS within or close to the MDS1-EVI1 and PRDM16 gene loci almost exclusively in the myeloid fraction, whereas IS within or close to the LMO2 and CCND2 gene loci were preferentially detected in the lymphoid compartment. Even though we detected several IS within or close to already known proto-oncogenes, our IS analysis shows a highly polyclonal reconstitution of the hematopoietic system until the latest time point analyzed (1108 and 1071 days after gene therapy, respectively) without any apparent signs of clonal outgrowth. The monitoring of the clonal inventory and a large scale tracking of individual clones is ongoing to further study the efficacy and safety of the vector used for this clinical WAS gene therapy trial.
Session: On the Route to Clinical Application
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Internal Medicine I, Klinikum Großhadern, LMU München, Munich, Germany
Thymosin β4 (Tβ4) is known to be involved in wound healing, coronary vessel formation and cardioprotection, alltogether implying an activated Protein Kinase B. We investigated the role of Thymosin β4 and AKT activation in angiogenesis and arteriogenesis using recombinant AAV2/9 (rAAV) gene transfer in a model of chronic hindlimb ischemia. We hypothesise a myovascular crosstalk in which the Tβ4 induced stimulation of myocytes results in an enhanced neovascularisation. Methods: Mice (n = 8) were subjected to 3 × 1012 r.AAV encoding for Tβ4, wt-AKT, AKT-DN, AKT-DN-Tet-off, GFP-Tet-off or Lac-Z 14 days before femoral artery ligation (d0). Recovery of blood flow was assessed by Laser Doppler flowmetry on day 3, 7 and 14 after ligation. To further study the reversibility of the AAV system, we generated Tet-off vectors encoding for GFP and AKTDN. Doxycycline (Dox) was applied from day 3 to day 14 after femoral ligation and thereby disabeling gene transcription. Capillary density was assessed via immunostaining for PECAM-1 (c/mf). Results: rAAV-based overexpression of Tβ4 (0.80 ± 0.13) or wt-AKT (0.79 ± 0.02) showed an increase in perfusion in flow measurements on day 7 in comparison to the control group (0.59 ± 0.1). The application of AKT-DN (0.43 ± 0.12) resulted in distinctly lower perfusion. Furthermore, capillary density was significantly increased in mice treated with Tβ4 (1.71 ± 0.05 c/mf) or wt-AKT (1.67 ± 0.06 c/mf) in comparison to the AKT-DN group (1.14 ± 0.08 c/mf) which stayed on control level (1.1 ± 0.01 c/mf). Using the inducible Tet-System GFP-positive myocytes were only detectable in the hindlimb muscles in the absence of Dox. Injection of rAAV.Tet-off-AKT reduced perfusion in the ischemic limb (0.3 ± 0.01) in the absence of Dox, whereas application of Doxycyline from day 3 on normalized recovery (0.73 ± 0.04). We conclude that rAAV-Tβ4 or rAAV.AKT-transduction of skeletal myoctes activates a myovascular crosstalk resulting in enhanced angiogenesis and arteriogenesis. The Tet-off system, switching off gene transcription in the presence of Doxycycline, elucidates the relevance and reversibility of early AKT-inhibition for functional neovascularization.
Session: Gene Therapy
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Department of Hepatology and Transplantation, King's College London, School of Medicine, London, UK
Adenoviral vectors delivering single or multiple pancreatic transcription factors can induce liver cells or pancreatic exocrine cells to transdifferentiate towards pancreatic beta-cells. Nonviral approaches for gene delivery would be much more clinically applicable, but the level and longevity of gene expression necessary are unknown. We therefore evaluated five DNA expression constructs with different functional elements for the level and longevity of expression of the rat transcription factor pdx1 delivered hydrodynamically to rat liver. We also evaluated the degree of transdifferentiation towards a pancreas phenotype. Groups of three or four rats were harvested at days 1, 3, 7, 14 and 28. Pdx1 from the plasmids and insulin 2 as a marker of transdifferentiation were assayed quantitatively using Taqman PCR. The pEPI plasmid, with a CMV promoter and a ≈2kb Scaffold/Matrix Attachment Region, gave strong pdx1 expression at day 1, but levels at day 3 were ≈150 fold lower. Substituting alpha1-antitrypsin promoters, with or without a ubiquitous chromatin opening element (UCOE), gave sustained pdx1 expression to day 3, with a more gradual decline thereafter. However, actual levels of expression were 20 to 300-fold lower than with the pEPI with CMV promoter. A CpG-depleted plasmid (InvivoGen) gave both high levels and sustained expression. Only the CpG-depleted plasmid induced a pancreatic phenotype in rat liver. Insulin 2 expression 30 to 70-fold higher than day 1 liver was found in two of three rats at day 14. To conclude, nonviral delivery of pdx1 alone to liver in a plasmid giving sustained pdx1 expression gave weak insulin expression in some rats. Multiple transcription factors are currently under investigation.
Session: Gene Therapy
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Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare genetic disorder that is caused by inactivating mutations in the Thrombopoietin receptor MPL. Children with CAMT develop severe thrombocytopenia and aplastic anemia leading to early death. With the aim to develop a gene therapy for CAMT we retrovirally overexpressed Mpl. First experiments expressing Mpl C57Bl/6 wt mice caused severe adverse reactions (SAE), e.g., uncontrolled expansion of stem and progenitor cells (CMPD), stem cell alterations (MDS like phenotype) and increased frequency of insertional leukemias. To avoid those SAE, we developed self-inactivating lentiviral vectors that expressed Mpl by the lineage-specific human and murine Mpl or human GPIba promoter. For comparison we used the ubiquitously active PGK promoter. In vivo expression was restricted to megakaryocytes (CD41+) and hematopoietic stem cells (LSK CD34−). We established a murine model for CAMT gene therapy based on the transplantation of Mpl−/− hematopoietic cells into Mpl−/− mice. Mpl expressed by the PGK promoter caused death 6 days post transplantation (6/7 mice) due to spleen rupture after erythroid hyperproliferation showing the extreme sensitivity to ectopic Mpl expression. Mice transplanted with bone marrow (BM) that express Mpl from the lineage specfic promoters survived long term while mice that were transplanted with eGFP transduced Mpl−/− BM cells showed increased lethality during establishment of long term hematopoiesis (>12 weeks). Both early post-transplant complications were prevented when using the lineage-specific promoters. Furthermore platelet production was restored in ≈ 50% of transplanted mice (n = 23) and multinucleated megakaryocytes were found in the BM 6-7 months after transplantation. In addition, the number LSK cells was significantly increased in Mpl-corrected animals. The engraftment of secondary Mpl−/− recipients with Mpl corrected BM of primary mice gave further evidence for the correction of long term HSC. Our results show the potential of newly developed transcriptionally regulated lentiviral vectors to circumvent genotoxic and transgene related, potentially lethal adverse reactions.
Session: Gene Therapy
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Applied Virology and Gene Therapy Unit, Georg-Speyer-Haus, Frankfurt am Main, Germany
Malignant glioma is the most frequent primary brain tumor and still has a very poor prognosis despite advances in neurosurgical resection and adjuvant radio- and chemotherapy. A promising new approach is the use of vesicular stomatitis virus (VSV)-based systems for glioma-targeted oncolytic virotherapy and/or gene therapy. To improve glioma specificity and to abrogate the VSV G-mediated VSV-inherent neurotropism, we pseudotyped VSV with the LCMV glycoprotein (LCMV GP). Retargeting to glioma cells while sparing healthy neurons was successfully shown in vitro, using a panel of different brain tumor cell lines, human neural as well as brain tumor stem cells and human primary neurons. The reduction of neurotropism was confirmed in vivo in a rat model after intracranial injection of replication-deficient VSV-WT and LCMV-GP pseudotype vectors, encoding the eGFP gene. Furthermore, we investigated different systems to address viral spread and microdistribution of LCMV GP-pseudotyped VSV within the tumor. Besides the generation of a replication-competent LCMV GP pseudotype rVSV(GP) as a pure virotherapy approach, we were able to show that tumor-infiltrating adult stem cells can be used as a delivery system and packaging cell line for non-cytopathic, replication-deficient VSV vectors encoding the HSV-TK as suicide gene. Efficacy of rVSVΔG–TK(GP) producing progenitor cells in mediating tumor killing after ganciclovir treatment was shown in vitro in a G62 spheroid model. If the reduced neurotoxicity can be confirmed in our current studies in vivo, rVSV(GP) would be an extremely promising candidate for oncolytic virotherapy of brain cancer.
Session: Cancer Gene Therapy
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Abstract Withdrawn
An α-Fetoprotein Promoter Driven, Conditionally Replicating Adenovirus that Expresses the Sodium Iodide Symporter (NIS) for Radiovirotherapy of HCC
We recently reported iodide accumulation and therapeutic efficacy of 131I in hepatocellular carcinoma (HCC) cells stably expressing the NIS gene under control of the tumor-specific α-fetoprotein promoter (AFP) for transcriptional targeting. As a next crucial step towards clinical application of this promising targeting technology we investigated in the current study the in vitro and in vivo efficacy of NIS gene transfer using a replication-selective oncolytic adenovirus. For this purpose NIS gene transfer was performed in vitro and in vivo in human HCC cell (HepG2) xenografts, using a replication-selective oncolytic adenoviral vector carrying the NIS gene linked to a mouse AFP-promoter construct (Ad5-E3-AFP-NIS). In vitro experiments with HepG2 cells and control cell lines demonstrated high transduction efficiency and tumor selectivity of Ad5-E3-AFP-NIS. Functional NIS protein expression was confirmed by measurement of in vitro uptake of 125I in HCC cells showing a 30-fold increase in perchlorate-sensitive iodide uptake activity as compared to control cell lines. A clonogenic assay with HCC cells infected with Ad5-E3-AFP-NIS (20 MOI) showed only 6.45 % survival after incubation with 0.8 mCi (29.6 MBq) 131I as compared to 100% survival of untreated HCC cells representing the high therapeutic potential of this gene therapy strategy. qPCR of NIS and fiber protein of the infected cells, a cytopathic effect assay and a spread assay showed corresponding results clearly demonstrating the replication selectivity due to the AFP promoter. In first in vivo experiments functional NIS protein expression after adenoviral NIS gene transfer was confirmed by 123I gamma-camera imaging as well as qPCR. HepG2 cell xenografts in nude mice injected intratumorally with 5 × 108 PFU Ad5-E3-AFP-NIS accumulated approx. 11% ID/g (percentage injected dose per gram tumor tissue) 48 h after virus injection with an average biological half-life of 5.5 h. These results clearly demonstrate the high potential of an α-fetoprotein promoter driven, conditionally replicating adenovirus for tumor-selective NIS gene delivery in liver cancer.
Session: Cancer Gene Therapy
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Department of Internal Medicine II, Klinikum Grosshadern, Ludwig-Maximilians-University Munich, Munich, Germany
The tumor-homing property of mesenchymal stem cells (MSC) has suggested their potential use as a delivery vehicle for cancer therapeutics. Tracking the delivery and engraftment of MSCs into human tumors is a crucial prerequisite for safety and effectiveness of MSCs as promising gene delivery vehicles. Due to its dual role as reporter and therapy gene, the sodium iodide symporter (NIS) allows non-invasive imaging of functional NIS expression by 123I-scintigraphy and 124I-PET imaging, and thereby provides an excellent means to elucidate the capacity of MSCs for tumor targeting of therapeutic genes. In the current study we stably transfected human bone marrow derived CD34− MSCs with a NIS-expressing plasmid (CMV-NIS-pcDNA3) (NIS-MSC) followed by analysis of NIS expression and cytotoxicity of 131I. In a human hepatocellular cancer (HCC) xenograft model (Huh7) we further investigated distribution and tumor recruitment of NIS-MSCs by I-123-scintigraphy and 124I-PET imaging. NIS-MSCs revealed a 12-fold increase in perchlorate-sensitive iodide uptake activity as compared to wild-type MSCs. Further, Western Blot analysis confirmed NIS protein expression in NIS-MSCs. In mixed populations of NIS-MSC and HCC cells (ratio 1:1) a clonogenic assay showed a 50% reduction of HCC cell survival after 131I application induced by its crossfire effect. After establishment of subcutaneous Huh7 xenografts in nude mice, NIS-MSCs were injected via the tail vein. Gamma camera and PET imaging revealed active MSC recruitment into the tumor stroma as shown by tumor-selective iodide accumulation (9.5% ID/g 123I, biol. half-life 4h). Immunhistochemistry and ex vivo 123I biodistribution analysis by gamma counter analysis confirmed active recruitment of NIS-MSCs into the tumor stroma while non-target organs showed no significant MSC recruitment. Taken together, our results convincingly show tumor-specific radioiodine accumulation after MSC-mediated NIS gene delivery demonstrating tumor stroma-selective MSC recruitment. These results open the exciting prospect of tumor-specific NIS-mediated radionuclide therapy of thyroidal or extrathyroidal tumors using MSCs as gene delivery vehicles.
Session: Tumor Biology and (Cancer) Stem Cells
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Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
Cellular identity is largely determined by transcription factor networks. The concerted action of 4 reprogramming factors (RFs: Oct4, Sox2, Klf4, c-Myc) leads to generation of induced pluripotent stem cells (iPSC), a promising resource for regenerative medicine and disease modelling. Here we constructed a modular versatile lentiviral vector system for mono- and polycistronic expression of these RFs (of either murine or human origin and optionally codon-optimized). To drive the reprogramming factors, we used the strong retroviral promoter SFFV, which enables high RF expression in most somatic cells and is frequently silenced in ES cells. To avoid the risks of residual permanent RF expression and insertional mutagenesis, we further improved our system by the inclusion of FRT, lox or rox sites to be able to remove the RF cassette through transient recombinase expression. Having the choice of 3 different recombinases offers the targeted shut off of RF at multiple desired time points. Using this system and taking advantage of an Oct4-GFP reporter model, we were able to generate murine and human iPS cells with high efficiency. To learn more about the kinetics of iPSC reprogramming and to understand better the underlying mechanisms, we developed color-coded RF vectors to follow expression kinetics qualitatively and quantitatively in relation to Oct4-GFP. Thereby, we could document the dynamic conversion of fibroblasts to pluripotent cells by fluorescence microscopy combined with long-term single cell tracking and alternatively high-definition structural analysis of single iPSC colonies. Filming the “birth” of iPSC generation, we obtained movies, which show the potential genetic mosaic of early iPSC colonies and also indicate the necessity of stochastic epigenetic changes during the process of iPSC development. With the growing insights in cell-specific transcription factor networks our versatile vector system will be a good candidate for direct reprogramming approaches into somatic cells (e.g., neurons). In summary, with the described vector system we have an efficient tool in hand for “excisable” and safe reprogramming strategies in regenerative medicine.
Session: Tumor Biology and (Cancer) Stem Cells
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Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
Expression of T cell receptors (TCR) as transgenic proteins in peripheral blood lymphocytes (PBL) enables T cells with defined tumor specificity to be generated in high numbers for patient-individualized therapy (TCR gene therapy). Hereby, the selection of tumor-associated antigens (TAA) that can be effectively targeted on tumor cells is of central importance. Survivin, a well-characterized inhibitor of apoptosis, has been suggested as a candidate TAA because of its potential role in oncogenicity and its broad expression in most tumors but absence from most normal adult tissues. To explore use of survivin as a target antigen for TCR gene therapy, we generated HLA-A2 allo-restricted survivin-specific T cells with high functional avidity. We isolated several high affinity TCR recognizing peptides derived from survivin presented by HLA-A2 molecules. Following transfer of these TCR into HLA-A2- recipient lymphocytes, we found that the effector cells displayed excellent specific killing of HLA-A2 + survivin + tumor cells, indicating their therapeutic potential. Surprisingly, when these TCR were expressed as transgenic proteins in HLA-A2 + recipient lymphocytes, as they would be applied in the clinical setting, we discovered that the recipient lymphocytes underwent extensive apoptosis over time. This demise was caused by HLA-A2-restricted fratricide that occurred due to survivin expression in recipient lymphocytes, which created ligands for transgenic TCR recognition. Furthermore, we could demonstrate that cytotoxic T cell clones of various specificities derived from HLA-A2 + donors were directly killed by survivin-specific TCR-modified PBL. Therefore, survivin-specific TCR gene therapy would be limited to application in HLA-A2-mismatched stem cell transplantation. However, these results raise a general question regarding cancer vaccines targeting proteins that are also expressed in activated lymphocytes, since induction of high avidity T cells that expand in lymph nodes following vaccination or later accumulate at tumor sites might limit themselves by self-MHC-restricted fratricide and also eliminate neighboring T cells of other specificities.
Session: Cancer Immune Therapy and T-Cell Therapy
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German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany
The genetic modification of immune cells and the generation of recombinant vectors encoding cancer antigens have highlighted the great potential of immunotherapy in treating cancer. Since the ability of a T cell to specifically recognize an antigen relies on the T cell receptor (TCR), a tumor reactive T cell repertoire can be generated by the genetic introduction of transgenes encoding TCR directed towards specific tumor antigens. We applied our experience in the comprehensive genome wide retrieval of gammaretroviral insertions sites (IS) to analyze the integration site distribution and the potential influences on the clonal fate of transduced cells after TCR gene transfer in order to assess their safety for clinical trials. We performed IS analysis combined with high-throughput sequencing on mouse CD4 and CD8 T cells transduced with a gamma retroviral vector encoding an ovalbumin-specific OT-I TCR. Mice receiving IL-2 administration after OT-I TCR transplantation developed Graft-versus-Host-Disease (GvHD). In total, we found 841 unique IS in OT-I transduced mice and 1063 unique IS in control GFP transduced mice with a higher clustering of IS in genes involved in the immune response (e.g., Cd28, Ctla4) in OT samples compared to the control samples. Results from the Ingenuity Pathways Analysis indicated the presence of specific gene classes, Hematological disease and Inflammatory response, preferentially targeted in mice transduced with the OT-I encoding vector compared to the mice transduced with the GFP vector (p = 7.84*10−4 and p = 2.13*10−2, respectively). In GvHD developing mice we observed a higher frequency of IS located in gene and in the surrounding 10 kb compared to the control counterpart (74% and 65%, respectively; p = 3*10−2) and an increased contribution of integration clones to gene-corrected hematopoiesis, indicating the occurrence of a potential in vivo skewing. Further sequencing and statistical analyses are ongoing. Our results provide important insights in IS distribution, clonal diversity and identification of genes preferentially targeted by TCR specific gamma retroviral vectors.
Session: Cancer Immune Therapy and T-Cell Therapy
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Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-University, Munich, Germany
Noninvasive, biooptical in vivo imaging is a powerful method to study the biodistribution and pharmacodynamics of labeled drugs in the living organism. For studies on nucleic acid delivery, the use of luciferase gene allows the highly sensitive detection of transgene expression by bioluminescence imaging (BLI). Here we describe a method, which allows both monitoring the growth of tumor metastases and the visualization of transgene expression after intravenous delivery of nonviral gene vectors in a syngeneic mouse tumor model. Murine neuroblastoma cells (Neuro2A) were stably transduced with a firefly luciferase and enhanced green fluorescent protein (EGFP) encoding lentiviral vector. EGFP sorted cells were injected either intravenously or intrasplenic into A/J mice and the tumor growth was monitored by BLI after intraperitoneal injection of luciferin, the substrate for firefly luciferase. After development of well vascularized tumor metastases, polyplexes containing a novel plasmid encoding for membrane-bound gaussia luciferase (Santos et al., 2009, Nature Medicine 15, 338–344) and a polycationic, biodegradable gene carrier were applied via the tail vein. Gaussia transgene expression was monitored after intravenous injection of its substrate coelenterazine. Due to the different substrates applied for firefly and gaussia luciferases, we could clearly distinguish between the firefly luciferase signal from the labeled tumor cells and the gaussia luciferase signal from cells expressing the gaussia transgene. After intravenous injection of polyplexes, gaussia transgene was only observed in areas were firefly luciferase labeled tumors were located, whereas in tumor-free animals no significant gaussia signal was observed. Due to a time lag between luciferase and coelenterazine injection, crosstalk between firefly and gaussia BLI signal was avoided. This study clearly shows, that polyplexes based on biodegradable polycations can selectively transfect well vascularized tumor metastases and dual BLI allows to distinguish between tumor localization and transgene expression.
Session: Pharmacology and Toxicology
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Experimental Hematology, Hannover Medical School, Hannover, Germany
The constitutive expression of therapeutic genes might interfere with cell fate in vivo. Therefore, regulated transgene expression is of major interest for gene therapy. Such a system needs to provide tightly controlled inducible promoters, as shown for the tetracycline regulatory system (tet-system). However, its application requires the introduction of two components into the target cell genome: the tet-responsive transactivator and the regulated expression cassette. In order to render the tet-system valuable for approaches in gene therapy, preselection of transactivator expressing cells has to be avoided, thus both components for regulated transgene expression have to be provided by a single vector. Up to date, published All-In-One vectors led to regulatory windows of around 500-fold, even after selection for single cell clones, thereby displaying a relatively high signal-to-noise ratio. In this study, we develop optimized All-In-One vectors which display tight transgene regulation without the need for clonal selection. Improvements in the dynamic range could be achieved through modifications of the vector architecture combined with the introduction of newly developed tet-responsive promoters, Ptet. These optimizations increased the dynamic range to levels up to 14,000-fold for retroviral and 25,000-fold for nonviral transposon vectors in non-clonal human cell lines, and up to 2,800-fold in murine hematopoietic cell lines. This improved regulation was based on a strong reduction of background expression in the off-state, even if cells were transduced at high multiplicity of infection, while induction remained at high levels. Our results indicate that an optimized signal-to-noise ratio of transgene expression in different target cells depends on vector architecture, the choice of the inducible promoter and the integration pattern.
Session: Pharmacology and Toxicology
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German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany
In several (pre)clinical gene therapy trials insertional mutagenesis by integrating gammaretroviral vectors was observed. In a clinical trial for the treatment of X-linked chronic granulomatous disease (X-CGD) insertional activation of MDS1/EVI1 has led to the clonal expansion of gene marked myeloid progenitor cells, triggering the development of monosomy 7 and a myelodysplastic syndrome in both patients enrolled. Therefore, a new self-inactivating (SIN) vector containing an internal cellular promoter (c-fes) and a codon-optimized transgene (SINfes.gp91s) was developed for the treatment of X-CGD improving the safety and efficacy of gene delivery (Manuel Grez, Georg-Speyer-Haus, Frankfurt). As part of the preclinical testing we performed high-throughput integration site (IS) analysis and deep sequencing (454/Roche) with associated bioinformatical tools on mice transplanted with gene modified lineage marker-negative hematopoietic stem cells. As a control a full LTR SFFV-promoter driven vector (SF91eGFP) – as it was used in the previous trial – was analyzed in parallel. In total, we found 1159 unique IS in SF91eGFP transduced mice (n = 4) and 2261 unique IS in SINfes.gp91s transduced mice (n = 5). While we did not observe any significant differences in the integration site distribution between the vectors, high-throughput sequencing revealed a less polyclonal IS pattern in SF91eGFP mice compared to the SINfes.gp91s transplanted mice (in average 281 vs. 442 unique IS/mouse). Furthermore, we found an increased frequency of (pre)dominant clones in mice transduced with the SF91eGFP vector than with SINfes.gp91s. Moreover, SF91eGFP integrants are more frequently found in/near EVI1/MDS1 than SINfes.gp91s integrants (9 of 1065 and 2 of 2115, respectively; p = 6.7*10−4). Finally, we could observe an increased in vivo skewing in secondary recipients of SF91eGFP transduced mice compared to SINfes.gp91s (p = 2.9*10−6). Overall, our results showed no signs of obvious vector-induced side-effects (no loss of polyclonality, no obvious skewing) for the SINfes.gp91s vector. *contributed equally
Session: Pharmacology and Toxicology
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Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK
Adenoviruses are commonly used vectors in gene therapy clinical trials, the majority of which are based on serotype 5. Following intravascular administration in vivo Ad5 exhibits high sequestration to the liver and spleen. Recent studies show that hepatocyte transduction by Ad5 is mediated by a high affinity interaction between coagulation factor X (FX) and the adenovirus 5 hexon proteins bridging the adenovirus capsid to the cell hepatocyte surface putatively through interaction with heparan sulfate proteoglycan receptors (HSPGs). Pharmacological blockade of the heparin-binding exosite in the serine protease (SP) domain of FX prevents cell surface binding and gene transfer mediated through FX1. However the exact HSPG-interacting residues in the SP domain of FX require further elucidation. Previous studies have indicated that the basic residues Arg-316, Lys-319, Arg-349, Arg-390, Lys-394, Lys-463 and Arg-467 of FX constitute the exosite in the SP domain2. We therefore sought to generate a FX mutant and assess the importance of this exosite. A human FX plasmid construct with mutations at Arg-316, Lys-319, Arg-349, Arg-390, Lys-394, Lys-463 and Arg-467 was generated and confirmed by sequence analysis. Recombinant FX (mutant and wildtype) was produced by plasmid transfection into 293T cells in the presence of vitamin K with collection of the supernatant. Enzyme-linked immunosorbent assay was used to quantify the rFX. In vitro assays demonstrated that the exosite mutations blocked the ability of FX to enhance transduction of Ad5 thus confirming the importance of this exosite in HSPG engagement. [1] Waddington 2008 Cell 132,397-409; [2] Rezaie 2000 JBC 132,3320-3327.
Session: Pharmacology and Toxicology
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DG-GT 2010 Poster Presentations*
Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University Munich, Munich, Germany
Polyplexes based on linear polyethylenimine (LPEI) are efficient non-viral gene delivery systems but the requirement for freshly prepared formulations prior to administration due to the instability in aqueous suspension is a major drawback. Therefore, the aim was to establish an up-scaled and reproducible method for the preparation of standardized batches of well-defined LPEI polyplexes and to develop isotonic lyophilized polyplex formulations with long-term stability. Polyplexes consisting of pCMVLuc plasmid and 22 kDa LPEI were prepared by classical hand pipetting or using an up-scaled micro-mixer method at different mixing speeds. Freeze-thawing and lyophilization were performed on a laboratory scale freeze-drier. Several excipients at varying concentrations were tested for their cryo- and lyoprotectant potential. To evaluate long-term stability, lyophilized samples were stored at 2-8°C, 20°C and 40°C for 6 weeks. The z-average diameter and polydispersity index (PdI) of the polyplexes was measured by dynamic light scattering. Transfection efficiency was evaluated in murine neuroblastoma (Neuro 2A) cells and metabolic activity as an indicator for cell toxicity was analyzed. By using the up-scaled micro-mixer method with varied mixing speeds, size (59–197 nm) and PdI (0.05-0.19) of the polyplexes can be directly controlled. When formulated with 14% lactosucrose, 10% HP-beta-CD/6.5% sucrose or 10% povidone/6.3% sucrose at isotonic concentrations, the increase in polyplex size was less than 50% after freeze-thawing, freeze-drying and during storage. Transfection efficiency and metabolic activity was high when polyplexes were formulated with lactosucrose or HP-beta-CD/sucrose but decreased when PVP/sucrose was used as stabilizer. The possibility to reproducibly manufacture large standardized batches of well-defined, transfection efficient polyplexes with long-term stability by using the established up-scaled preparation method followed by lyophilization is an important step closer from promising technology to clinical application.
Session: Vector Development
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Recombinant Influenza C Virus: A New Prototype of a Melanoma-Specific Oncolytic Virus
The ability to genetically engineer viruses contributes greatly to our understanding of their life cycle and allows creation of virus-derived vectors for vaccine development, gene therapy or oncolysis. In the past decade, several reverse-genetics systems have been developed to manipulate the genomes of influenza A and B viruses. Here, we introduce a system for the generation of recombinant influenza C viruses from seven plasmids.The seven full-length viral cDNAs were cloned into an RNA pol I/pol II-based bidirectional vector. Transfection of Vero cells and subsequent amplification on MDCK cells yielded viral HA titres of 128. With this efficient method, mutations or foreign genes may be easily incorporated into the influenza C virus genome. Next, the influenza C virus protein NS1 was identified as a factor antagonising the cellular interferon (IFN) signalling, and the regions of NS1 necessary for IFN inhibition were mapped. Several melanoma cell lines have a defective IFN signalling and express the cellular receptor for influenza C virus, which is 9-O-acetyl sialic acid. Thus NS1-deficient recombinant influenza C viruses could be engineered to serve as gene delivery vectors and/or tumour-specific oncolytic viruses in the future.
Session: Vector Development
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Physical Chemistry, Department of Chemistry, Ludwig-Maximilians-University, Munich, Germany
We investigate the uptake of targeted and untargeted polymeric gene vectors (polyplexes) by highly sensitive fluorescence microscopic methods by live cell imaging on a single cell level. The epidermal growth factor receptor (EGFR) is overexpressed on a high percentage of human carcinomas and is therefore an attractive therapeutic target for tissue-specific targeting by non-viral vectors in cancer gene therapy. By comparing uptake kinetics and internalization dynamics, single particle tracking in combination with quenching experiments revealed typical three-phase dynamics of the uptake process independent of targeting. Phase I was characterized by slow, actin-cytoskeleton-mediated movement of the particles with drift and included the internalization process. During phase II, particles displayed increased velocities with confined and anomalous diffusion in the cytoplasm. Phase III was characterized by fast active transport along microtubules. Targeting of polyplexes for receptor-mediated endocytosis by the EGF receptor resulted in shortening of phase I and strongly accelerated internalization. Targeted as well as untargeted particles were transported in early endosomes marked by Rab5-GFP and accumulated in late endosomes marked by Rab9-GFP. The endosomal release dynamics of polyplexes consisting of DNA condensed with the cationic polymers linear polyethyleneimine (LPEI), poly-(
Session: Vector Development
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Generation of the Novel, Synthetic Hybrid SCE-Promoter for Gene-Therapeutical Applications
A major factor for successful gene therapy is strong and enduring expression of the transgene. Commonly used promoters for expression of transgenes are the CMV-immediate early (CMV-IE), chicken β-actin, ubiquitin C or the EF1α promoter, each with their own advantages and disadvantages. Here we present the generation of novel synthetic hybrid promoter, based on the CMV-IE and the EF1α promoter. It was obtained by generating shuffled consensus sequences of both promoters, which were analyzed by promoter prediction software. One sequence, the shuffle CMV/EF1α promoter (SCE), was synthesized and tested in vitro and in vivo. The SCEP sequence features an identity of 83% with the CMV and 85% with the EF1α promoter. The sequence was CpG-free designed to circumvent inflammatory reactions towards CpG isles and promoter methylation leading to silencing. In several cell lines a 2-7 fold stronger expression of the SCE promoter compared to the EF1α was observed; when compared to CMV-IE, SCE promoter activity was almost similar. All promoters were within a CpG-free plasmid featuring a human CMV-IE enhancer and luciferase as transgene. In immunocompetent Balb/c mice we observed a ≈3 fold stronger transgene expression of SCEP compared to the EF1α promoter after hydrodynamic gene delivery to the liver for a time period of 30 days. Hence the SCE promoter is qualified for further applications in biotechnology or gene therapy, when strong and enduring transgene expression and evasion of promoter silencing is needed.
Session: Vector Development
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pH-Labile Degradable Pseudodendritic Polycations for In Vitro and In Vivo DNA Delivery into Cancer Cells
Polycationic dendritic structures show good properties for DNA delivery into cancer cells. For example branched polyethylenimines (bPEI) are known to have a good transfection ability but also high toxicity. Former work of our group (Russ et al., Gene Therapy 2008) indicated that pseudodendrimers containing degradable ester linkages have similar transfection efficiency but lower toxicity. Within this work we designed novel pseudodendrimers based on different polyamines such as polypropylenimine and oligoethylenimine as core structure. They were modified with the acid degradable bisacrylate acetal linker BAA and oligoethylenimine (OEI 800Da) as polycationic surface structure. We could show that these dendritic molecules have the ability to form stable polyplexes with DNA and are degradable under incubation at endosomal pH. Cell testing confirmed that this degradability leads to a lower intracellular toxicity, while keeping the transfection ability both in vitro as well as in vivo in a neuroblastoma mouse tumor model.
Session: Vector Development
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Biodegradable Pseudodendritic Oligoamines for siRNA Delivery
Searching for efficient synthetic vectors for siRNA delivery, a series of pseudodendritic oligoamines with different hydrophilic/lipophilic properties was synthesized. The polymers consist of an 800 Da oligoethylenimine (OEI) core and various surface molecules (spermine, spermidine, OEI, etc.), attached to the core via ester bond containing linkers. Out of this series, the HD-S polymer most effectively facilitates siRNA delivery, silencing luciferase expression in stably transfected Neuro2A-luc cells. All presented polymers are biodegradable because of the incorporated ester bonds, which can be cleaved by esterases ubiquitously present in living organisms. Ineffective polymers like the HD-O can be transformed into successful delivery agents by crosslinking the surface amines of the polyplexes with DSP (Dithiobis-succinimidyl-propionate). This lateral stabilization of polyplexes boosts the delivery efficiency of HD-O, which is demonstrated on Neuro2A-luc cells and HUH-7-luc hepatoma cells. Enhanced cellular uptake of the crosslinked particles is proven by FACS analysis and gene down regulation is analyzed on mRNA level by qPCR. The disulfide bond in DSP maintains the biodegradability of the polymers and ensures sufficient release of the siRNA out of the stabilized polyplexes due to cleavage of disulfide bonds in the reductive cytosolic environment.
Session: Vector Development
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Succinylated PEI as Carrier for siRNA Delivery in Tumor Xenograft Models: Evaluation by RT-qPCR
Developing polymers for tumor targeted delivery of siRNA, succinylated 25 kDa PEI has emerged as highly effective carrier system. Mechanistic in vitro study demonstrated that lower toxicity of the polymer compared to standard 25 kDa PEI is mainly responsible for the enhanced siRNA delivery efficacy. Higher amounts of polymer can be applied, which result in improved endosomal escape characteristics. For in vivo knockdown studies in xenograft tumor models the establishment of accurate and reliable mRNA detection methods is important. For this purpose we optimized Reverse Transcription (RT) quantitative Polymerase Chain Reaction (qPCR). Variations in expression levels of reporter genes (luciferase), therapeutic genes (RAN, KSP) and housekeeping genes (ACTB, GAPDH, AHA) were evaluated in tumor samples in vitro and in vivo. Human and mouse specific primer probe sets were designed. Those species specific primer probe sets offer the possibility to accurately detect levels of the human tumor target RNA in xenograft tumor models in the presence of mouse tissue. In our studies ACTB has emerged to be a more reliable housekeeping gene than GAPDH due to less variation in mRNA levels. Knockdown of therapeutic genes (such as KSP), which are cell cycle dependant, showed higher variations in analysis of cell culture and in vivo tumor samples. In addition silencing target genes which affect tumor survival complicate the evaluation due to cell death of transfected cells. Thus, targeting of non-essential gene targets seems to be more feasible for measuring silencing efficiency.
Session: Vector Development
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Klinikum rechts der Isar, TU München, Institute für Experimentelle Onkologie, Munich, Germany
In recent years microbubbles technology has gained lot of interest in the field of gene and drug delivery. Based on the “Magnetofection” concept [1], besides the development of magnetic acoustically active lipospheres [2, 3], we have been able to prepare lipid monolayer shelled microbubbles loaded with highly positively charged naked magnetic nanoparticles (composed of iron oxide) through electrostatic and matrix affinity interactions. These magnetic microbubbles show strong ultrasound contrast. Treatment of cancer cells with these microbubbles using ultrasound exhibited strong dose-dependent cytotoxic effects, although ultrasound alone, lipid microbubbles alone, magnetic nanoparticles or magnetic microbubbles alone at the corresponding concentrations did not affect the cell viability. On the other hand, when these magnetic microbubbles were mixed with plasmid DNA encoding a reporter gene, we achieved gene delivery to cultured adherent cells only when ultrasound was applied. Gene transfer efficiency was strongly dependent on the application of a gradient magnetic field to sediment the microbubbles on the target cell membranes. From the preliminary experiments we conclude that magnetic microbubbles could be used as magnetically targeted diagnostic agents for real-time ultrasound as well as magnetic resonance imaging. At the same time, such magnetic microbubbles may be useful for therapeutic purposes such as in cancer therapy, vascular thrombolysis and gene therapy. However, further improvements are required to control their cytotoxicity. References [1] C. Plank, U. Schillinger, F. Scherer, C. Bergemann, J-S. Remy, F. Krötz, M. Anton, J. Lausier, J. Rosenecker. The magnetofection method: using magnetic force to enhance gene delivery. Biol. Chem. 2003; 384 (5): 737–747. [2] Plank C, Vlaskou D, Schillinger U, Mykhaylyk O, Brill T, Rudolph C, Huth S, Krötz F, Hirschberger J, Bergemann C. Localized nucleic acid delivery using magnetic nanoparticles. European Cells and Materials. 2005; 10(Suppl. 5):8. [3] Vlaskou D, Mykhaylyk O, Giunta R, Neshkova I, Hellwig N, Krötz F, Bergemann C, Plank C. Magnetic Microbubbles: New Carriers for Localized Gene and Drug Delivery. Mol Ther. 2006; 13(Suppl. 1):S290.
Session: Vector Development
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Sleeping Beauty-Mediated Transposition in Human Cells Is Enhanced in RNA Interference Knockdown Cells
Integrating nonviral vectors based on transposable elements and bacteriophage derived integrases are widely used for genetically engineering mammalian cells, in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs. This may lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and finally to silencing of the transgene. Therefore, in the present study we generated and characterized RNAi knockdown mammalian cell lines and analyzed transposition in these cells. RNAi knockdown HEK293 cell lines were generated by stably introducing the RNAi suppressor protein P19 from the tomato bushy stunt virus which binds and inhibits 21 nucleotide long double stranded siRNAs. In order to characterize our RNAi knockdown cell lines we used the marker gene HoxB8 which, in differentiated cells, is suppressed by the miRNA miR196a. Using a quantitative Real-Time PCR based approach we detected up to 10-fold higher HoxB8 mRNA levels in the RNAi knockdown cell lines compared to the parental cell line clearly demonstrating that P19 is a sufficient inhibitor of the RNAi pathway. After performing colony forming assays measuring SB transposition events we could show that transposition was increased up to 4.5-fold in our RNAi knockdown cell lines compared to parental HEK293 cells, providing clear evidence for the first time that SB transposition is regulated by the RNAi machinery in mammalian cells. In contrast another transposable element, the Frog Prince transposon that displays only modest transcriptional activity, a 1.2-fold increase in transposition events was observed. To investigate integration of non-viral vectors in a more global manner, we also quantified bacteriophage-derived integrase PhiC31 mediated integration events, and found that integration was enhanced up to 3-fold. In conclusion, our results indicate that transposition in mammalian cells and potentially also the PhiC31 integrase system are regulated by the endogenous RNAi machinery.
Session: Vector Development
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Micro RNA Knockdown Significantly Enhances Adenovirus Replication and Vector Production
The use of microRNAs (miRNAs) fundamentally improved the generation of viral vectors for gene therapy. For instance the introduction of miRNA target sequences into lentiviral vectors excluded expression of the respective protein from an unwanted tissue (Brown et al., 2006). However, the effect of the RNA interference (RNAi) pathway itself on gene therapy vectors remains an obstacle. In this study we used our recently established RNAi knockdown cell line B6 (Rauschhuber and Ehrhardt, submitted) to analyze the influence of the RNAi pathway on adenoviral vectors. We first investigated replication of wildtype adenovirus 5 (wtAd5) and first generation adenovirus (FgAd) on a genome level. An up to 10-fold increase in viral genome copy numbers 24 hours and 48 hours post infection could demonstrate that there is significantly enhanced viral DNA replication under RNAi knockdown conditions. To analyze whether we can also achieve higher viral titers in the B6 cell line, we infected with a commonly used FgAd expressing firefly luciferase (FgAdluc). After re-infection of HEK293 cells we measured up to 5-fold increased luciferase activity indicating a higher titer of FgAdluc derived from B6 cells. To reach even stronger effects on virus replication we hypothesized that the amount of the RNAi suppressor protein P19 may be the limiting factor. Thus, we applied our novel adenovirus cloning technology based on bacterial artificial chromosomes and generated a recombinant adenovirus expressing p19 under the control of the fiber promoter for which the amount of P19 directly correlates with adenovirus genome replication. We detected up to 10-fold increased genome copy numbers compared to wtAd5 and the corresponding control virus. Following our new method of adenovirus type switch we also generated a helper-virus expressing p19 for helper-dependent adenovirus (HC-AdV) production. In contrast to a conventionally used helper-virus we detected up to 12-fold increased HC-AdV titers. In conclusion, we provide new insights into regulation of adenoviral vectors by RNAi and our RNAi knockdown cell line B6 could be used as a novel producer cell line for adenoviral vectors.
Session: Vector Development
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A Transient Cell Cycle Arrest Enhances AAV and ZFN-Mediated Gene Targeting
Vectors based on adeno-associated virus (AAV) are efficient tools to modify complex genomes by gene targeting. Gene targeting is based on the homologous recombination (HR) pathway, which is mainly available during the G2 phase of the cell cycle. We have shown that insertion of a DNA double strand break (DSB) in the target locus stimulates gene targeting up to 1000-fold, most likely by activating the HR repair pathway. To assess the effect of the cell cycle on AAV and DSB-mediated gene targeting, we established three human cell lines (HeLa, HT-1080, U-2 OS) that carry a copy of a mutated eGFP gene flanked by recognition sites for the meganuclease I-SceI and zinc-finger nucleases (ZFNs). After treatment with different cytostatic drugs (including hydroxyurea, vinblastine, indirubin), cells were transduced with AAV vectors that encode the nuclease and an HR donor with the purpose of rescuing eGFP expression by gene targeting. The cell cycle profile, the extent of cytotoxicity and the frequency of gene targeting were assessed by flow cytometry. We show that a transient cell cycle arrest before the creation of a DSB in the target locus increased AAV-mediated gene targeting up to 6-fold. Although this effect could be partially attributed to an increase in AAV transduction, a significant increase in gene targeting was also observed by plasmid-based eGFP rescue, implying that a drug-induced transient cell cycle arrest is a general means to augment DSB-mediated gene targeting. Depending on the cell line and the AAV vector dose, we achieved targeted gene editing in up to 12% of transduced cells. Interestingly, the ratio between AAV vectors used for gene targeting vs. illegitimate integration was strongly cell type dependent and ranged between 30:1 and 1:9. Also, drug-induced transient cell cycle arrest had only minor effects on this ratio. In conclusion, cytostatic drugs can be used to enhance DSB-mediated gene targeting in different cell lines. The combined beneficial effects of site-specific DSBs and transient cell cycle arrest allowed us to lower the AAV vector dose – and hence illegitimate integration of the vectors – without compromising on the gene targeting efficacy.
Session: Vector Development
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Novel Heterospecific FRTs Enable Gene Targeting of Several Loci by Flp-Multiplex-RMCE
Since 48 bp Flp-recombinase sites (FRTs) represent unique targets in all eukaryotic genomes recombinase-mediated cassette exchange (Flp-RMCE) is not hampered by the occurence of genomic pseudosites. We explored the feasibility of exchanging two distinct genomically anchored cassettes, each flanked by an unique pair of heterospecific FRT sites. For successful RMCE novel constructed mutant FRTs had to meet two major prerequisites (a) given self-recognition potential comparable to a pair of FRT wildtype sites (b) negligible crossinteraction among heterospecific sites. We applied a two-step strategy to characterize various newly created FRT spacer mutants for these properties. As a result of our screening steps we identified combinations F3/F and F13/F14 of sites, which were successfully applied to simultaneous Flp-mediated genomic targeting (“multiplexing”) reactions. The emergence of novel heterospecific FRTs can play a key role for specified gene targeting approaches, for instance the sequential elaboration of larger gene domain subunits by RMCE. Reference: Turan S, Kuehle J, Schambach A, Baum, C and Bode, J (2010) RMCE-multiplexing: versatile extensions of the Flp-Recombinase-Mediated Cassette-Exchange technology. J. Mol. Biol. In press.
Session: Vector Development
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Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-University, Munich, Germany
Recombinant adenovirus type 5 (Ad5) is a highly efficient vector for cancer gene therapy approaches, but due to its natural tropism for the coxsackie- and adenovirus receptor (CAR) it lacks specificity for tumor cells and can induce severe inflammatory responses. Although present on a broad range of tissues, CAR is often down-regulated in several tumor types. Combining the high transfection efficiency of Ad with synthetic polymers can potentially allow de-targeting from CAR positive tissues and re-targeting to tumor cells. We have coated Ad5 with cationic polymers like PAMAM dendrimer or linear polyethylenimine (LPEI) by electrostatic interaction with the negatively charged Ad capsid. Size and surface charge of polymer coated Ad5 were measured by laser light scattering, particle shape by transmission electron microscopy (TEM). Uptake and intracellular transport was studied by fluorescence microscopy and flow cytometry using fluorescently labeled Ad (Alexa488) and polymer (Cy5). Transgene expression studies in vitro were carried out using either luciferase gene or the sodium iodide symporter gene (NIS), were the latter leads to cellular uptake of radioactive iodine. With TEM it was possible to show that the virus is encapsulated by the polymer forming a shell around the virus with a rough surface structure. The virus surface charge changed from a net negative value of naked virus (−17mV) to + 4 mV when coated with PAMAM dendrimer, and the average particle size increased with increasing amounts of polycation added. Recharging the virus led to pronounced cellular binding and uptake of virus. On high CAR expressing cells transfection efficiency was reduced after polymer coating, whereas on cell lines with low CAR expression increased transgene expression was measured. In addition, we covalently attached a peptide ligand to the polymer binding to the epidermal growth factor receptor (EGFR). This further promoted uptake in EGFR positive tumor cells. The obtained data suggests that a combination of viral and nonviral gene vector systems could compensate the limitations each approach has on its own and lead to superior success in cancer gene therapy.
Session: Vector Development
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II. Medizinische Klinik und Poliklinik, Klinikum rechts der Isar, TU München, Munich, Germany
Vesicular stomatits virus (VSV) is an oncolytic virus, which shows promise as a therapeutic agent for cancer, however, toxic collateral effects on the brain and the liver occur when it is administered at high doses. We hypothesized that by employing a novel strategy, termed microRNA-engineering, we could eliminate off-target replication and improve the therapeutic index. This approach involves the incorporation of target sequences of a specific miRNA (miRTs) into the viral genome, resulting in degradation of viral gene transcripts in cells in which this miRNA is highly expressed, without interfering with virus replication in cells not expressing the relevant miRNA. We screened a panel of candidate miRNAs to select for those with high expression levels in the brain and/or liver and low levels in primary human HCC and CRC and cell lines. We then confirmed the miRNA-mediated regulation of the candidates in a reporter gene assay. Four tandem repeats of perfect complementary miRTs were cloned into the 3′-UTR of a luciferase reporter gene, and these plasmids were transfected into a panel of normal and tumor cells. We then selected only those miRTs which resulted in a reduction of luciferase expression in primary hepatocytes and neurons but not in tumor cells. Based on these preliminary experiments, we chose three miRTs which best fit our selection criteria, and created recombinant VSV vectors containing four tandem repeats of the miRTs in the 3′-UTRs of crucial endogenous viral genes. After testing of these vectors in tumor and non-tumor cells in vitro, thorough toxicity and efficacy studies will be performed in mouse models of HCC and CRC. We ultimately aim to engineer next-generation viruses with one or more viral gene regulated by multiple miRTs. Because saturation of endogenous miRNAs in the target tissues could result in “leaky” virus replication, we hypothesize that the incorporation of multiple miRTs could overcome this limitation and result in an even safer vector. Thus, this strategy has the potential to provide a basis for clinical application of VSV vectors for the treatment of primary and metastatic liver disease, as well as other cancers, in the future.
Session: Vector Development
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Institut für Experimentelle Onkologie und Therapieforschung, Klinikum rechts der Isar, TUM, Munich, Germany
A multifunctional magnetic liposome formulation, which is designed to combine features of biological (folate receptor mediated) and physical (magnetic field assisted) drug targeting for use in magnetic hyperthermia triggered drug release for cancer therapy has been developed. The magnetic liposomes (MagFolDox) composed of DPPC/Cholesterol/DSPE-PEG2000/DSPE-PEG2000-Folate at 80:20:4.5:0.5 molar ratio released 52% doxorubicin at 43°C after 60 minutes incubation in 50% fetal bovine serum. The MagFolDox, when physically targeted to tumor cells in culture by a permanent magnetic field yielded a several fold increase in cellular uptake of doxorubicin as compared to Caelyx® (a stealth liposomal formulation of doxorubicin), nonmagnetic folate-targeted liposomes and free doxorubicin in folate receptor expressing tumor cell lines. Consequently, the MagFolDox liposomes enabled improved tumor cell killing. Moreover, MagFolDox and magnetic hyperthermia (at 42.5-43.5°C) showed synergistic cytotoxic effect in KB cells. Also, the distribution of magnetic liposomes in vivo can be monitored by MRI due to their contrast enhancement properties. In vivo real time MRI monitoring in mice confirmed significantly higher blood circulation time for MagFolDox compared with free magnetic particles. This also confirmed that the biodistribution of the MagFolDox can be monitored in vivo by MRI. Thus, the multifunctionality of the magnetic liposomes has been successfully demonstrated.
Session: Vector Development
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Melanoma-Killing Adenovirus Derived by Direct Evolution
Viral oncolysis is a strategy for targeted treatment of cancer by tumor-restricted virus infection. Human Adenoviruses (Ad) are promising oncolytic agents, but showed insufficient therapeutic efficiency in clinical trials. Therefore, the key challenge for virotherapy research is to develop Ad with enhanced viral lysis. In this regard, I focus on developing effective oncolytic Ad for malignant melanoma by directed evolution in order to derive adenoviruses with increased lytic activity. First, we investigated the efficiency of a panel of Ad serotypes from species B, C, and D in melanoma cells in comparison to the most potent virus for this type of cancer, Ad5/3 (Ad5 with the cell-binding domain of Ad3). Our results indicate that none of those serotypes are more potent than Ad5/3 in melanoma cells. However, the oncolysis of the melanoma cells is still strongly attenuated in comparison with bronchial epithelial cells, which are the natural host cells. Therefore, although Ad5/3 is the most potent Ad in melanoma an improvement is still needed. To enhance the lytic activity of Ad5/3, the viruses were treated with nitrous acid sodium (NaNO2) which induces random mutations in the virus genome. Subsequently, the originated mutations were fixed and the individual mutants were amplified in melanoma cells. Finally, with the objective of harvesting the most rapid spread viruses, the bioselection rounds of the mutagenized Ad5/3 were performed by passaging the supernatant (at early stage of infection) repeatedly on the melanoma cells. After the 20th round of bioselection, our results show that mutagenized Ad5/3 were 10-fold more potent in melanoma cells in comparison with the parenteral virus. This result suggests that the most potent virus generated after NaNO2 treatment replicate, lyse the host and are released into the supernatant before the majority of virus population. Currently, I am characterizing the mutants to restrict their replication in melanoma cells by a tumor-specific promoter. In summary, our results indicated that directed evolution is a strategy which allows the enrichment of viruses' mutants with enhanced replication and spread for malignant melanoma cells.
Session: Vector Development
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Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
Zinc-finger nucleases (ZFNs) are an innovative tool for targeted genome engineering. They consist of a catalytic nuclease domain and a designed DNA binding domain and can be employed for either targeted deletion or gene disruption by harnessing the non-homologous end joining DNA repair pathway or, in the presence of a properly designed donor DNA, gene targeting by activating homology directed repair (HDR). Here, we demonstrate the versatility of ZFN-mediated genome editing when combined with vectors derived from adeno-associated virus type 2 (AAV2). For proof-of-concept studies, we generated four different human reporter cell lines. For assessing gene disruption and targeted deletion of an integrated provirus, a lentiviral EGFP vector containing ZFN target sites in the LTRs was used to generate an EGFP-positive cell line. To evaluate therapeutic gene correction, cells harboring a mutated EGFP or Luciferase reporter were produced. The frequencies of targeted genome editing and the extent of cytotoxicity were determined by flow cytometry and/or luminometry at different time points after transduction. Disruption of the EGFP locus was achieved in 18% of cells infected with vectors encoding EGFP-specific ZFNs. Upon transduction with AAV vectors coding for LTR-specific ZFNs, the lentiviral provirus was excised in 10% of cells. Furthermore, after co-infection of cells with vectors that encode locus-specific ZFNs or serve as donors for gene targeting, AAV-mediated correction of the mutated marker genes restored reporter activity in up to 6% of cells. The frequency of genome editing was depending on the vector dose and the specificity of the ZFNs. Significant signs of cell death were not observed, suggesting minor toxicity associated with these approaches. In conclusion, the combined ZFN–AAV vector technology is a flexible tool to efficiently modify the human genome ad libitum. Because AAV vectors can transduce multiple human tissues, including induced pluripotent stem cells and mesenchymal stem cells, the ex vivo delivery of ZFNs and HDR donors via AAV vectors will be of great interest for the treatment of several disorders in regenerative medicine.
Session: Vector Development
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Progress in Minicircle Manufacturing and Performance Testing
Plasmid DNA is commonly used in vaccination, gene- or cell therapy but also as a basic substance in viral vector production. The dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences present in such plasmids may have profound detrimental effects. Additionally, unmethylated CpG motifs have been shown to contribute to silencing of episomal transgene expression. Therefore, an important goal in vector development is to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle DNA. PlasmidFactory's minicircle production technology facilitates the large-scale production of highly pure minicircle DNA for applications in gene therapy and vaccination as well as, e.g., virus production. The production technology is based on two processes: (1) An inducible, sequence specific, and very efficient in vivo recombination process; (2) A chromatographic purification technology for the isolation of the minicircle DNA. The resulting minicircle DNA only consists of the gene of interest and a tiny residual sequence stretch including one of the two recombination sites. The chromatographic purification results in an exceptional purity, proven by various QC tests, which is extremely important since even small amounts of contaminants can produce dramatic experimental artefacts. For first efficacy studies, reporter genes for different types of analyses within various tissues, cells, animals and for testing the mode of administration (e.g., electro gene transfer, sonoporation, lipofection, magnetofection, etc.) have been used. Also biodistribution studies using these constructs have been performed. Additionally, minicircle constructs for vaccines, virus production, etc., are currently investigated. The results demonstrate a significant increase of gene expression of minicircle DNA in comparison to a standard plasmid. As a result of the work presented here, PlasmidFactory has launched several commercially available minicircle DNA products.
Session: Vector Development
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Minicircle – An Overview
For future gene therapy and even genetic vaccination approaches it is crucial to develop safe and highly efficient vector systems to be transferred into the target cells. Currently, viral and non-viral vectors are used, both having their advantages and limitations. The dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences present in plasmid DNA may have profound detrimental effects. Additionally, unmethylated CpG motifs have been shown to contribute to silencing of episomal transgene expression. Hence, it seems obvious that the removal of bacterial backbone DNA can greatly improve the safety and efficiency of plasmid DNA used in gene therapy and vaccination. Here, we give an overview on earlier and recent approaches for the production, purification, and application of such minimal constructs, devoid of bacterial backbone sequences. Different approaches have been described so far, from plasmids where the antibiotics resistance gene has been replaced by another marker to minicircle DNA consisting only of the gene of interest and a tiny residual sequence stretch from the recombination. Minicircles can be produced by in vivo site-specific recombination of a so-called parental plasmid resulting in a miniplasmid and the minicircle. This recombination can be achieved using different enzymes which need specific target sequences as recombination sites. The most important difference between these approaches is the efficiency of the recombination step as well as the purification procedure used in order to remove the miniplasmid (with the bacterial sequence motifs) and residual amounts of the parental plasmid if necessary. In addition to their improved safety profile, minicircles have been shown to greatly increase the efficiency of transgene expression in various in vitro and in vivo studies. Scale-up of such technology was recently possible in fermentation and results in an extremely pure DNA product.
Session: Vector Development
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Genetic Manipulation of High-Capacity Adenoviral Vectors Utilizing Bacterial Artificial Chromosomes and Rapid Small-Scale Vector Preparations
First-generation adenoviral vectors (FG-AdVs) are easy to produce and therefore representing the best option to analyse capsid-modifications or new expression systems. On the other hand high-capacity adenoviral vectors (HC-AdVs) lacking all viral coding sequences are the most attractive option for therapeutic approaches. Therefore, there is a great interest in developing a method for adoption of evaluated vector-modifications or expression systems in FG-AdVs towards HC-AdVs including fast generation of small scales of purified HC-AdV for initial experiments and subsequent large-scale amplification. Utilizing a novel platform based on homologous recombination of bacterial artificial chromosomes (BACs) we generated a BAC from a plasmid containing an HC-AdV genome by replacing the plasmid-backbone with the BAC-backbone in a single cloning step. After this backbone-exchange we incorporated an eGFP-expression cassette, generated the respective HC-AdV and showed eGFP-expression. To adopt FG-AdVs to the BAC-platform we demonstrated that BACs with FG-AdV genomes can be constructed either by homologous recombination with adenoviral DNA isolated from purified virions or by backbone-exchange. Furthermore, we showed that our BAC technology can be used to switch from FG-AdVs to BACs with a helper virus (HV) genome for HC-AdV amplification, wild-type adenovirus or oncolytic adenovirus in a single cloning step. To highlight the impact of our BAC platform on adenovirus vectorology we exemplified adenovirus vector type switches by generating a HV with the established fiber mutant fib5/35 as well as a HV with an eGFP/Fluc expression cassette in the E3 region and a new hexon mutant with precisely exchanged hypervariable regions from human adenovirus serotype 12. All viruses were reconstituted and amplified. Notably, we observed that HC-AdVs can be first amplified in small amounts (4-6 × 20 ml) and purified by column (Vivapure AdenoPACK 20, Sartorius). Yielded HC-AdV was used for initial proof-of-principle experiments and to optimize large-scale amplification making this technology amenable also for researchers without experiences in HC-AdV production.
Session: Vector Development
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A Novel Adenoviral Hybrid-Vector System Carrying a Plasmid Replicon for the Safe and Efficient Gene Therapy of Human Diseases
In dividing cells the two primary aims a gene therapy approach should accomplish are defined as the nuclear distribution and retention of therapeutic DNA. Because conventional monosystems fail to fulfil both tasks with equal efficiency, hybrid-vector systems are most promising in facing the challenges of modern molecular medicine. Our hybrid-vector system HDAdV-pEPito synergizes the helper-dependent adenoviral vector technology (HDAdV) with the plasmid pEPito containing the therapeutic gene and special DNA sequence SMAR(Scaffold/Matrix Attachment Region) for episomal retention and replication. Thus, our technique provides a powerful tool for stable maintenance of the transgene reducing the risk of insertional mutagenesis. Adenoviral hybrid-vectors HDAdV-pEPito and the respective SMAR deleted control (HDAdV-pEPito-ΔSMAR) were generated with a novel BAC-technology established in our laboratory (Hausl et al., in preparation). Both contain an eGFP-IRES-BSD-cassette under control of promoter hCMV/EF1. In FACS analysis 83% of A549-cells infected with a MOI of 50 were positive for eGFP-expression. Since adenoviral vectors carry linear DNA, pEPito has to be released from their genomes with Flpe-recombinase to function as a plasmid. A specific PCR proved this recombination event for both constructs. The episomal status of recombined plasmids could be verified with bacterial rescue experiments at day 4 after coinfecting A549-cells with our constructs and Flpe-expressing virus HDAdV-mSB-Flpe. More bacterial colonies grew after transformation of genomic DNA containing HDAdV-pEPito with Flpe (n = 281) than of DNA containing HDAdV-pEPito-ΔSMAR with Flpe (n = 25), indicating replication of excised plasmids. Ongoing experiments are the comparison of HDAdV-pEPito and HDAdV-pEPito-ΔSMAR concerning their long-term persistence in mice and their in vitro establishment efficiency in a colony forming assay (CFA). Previous CFAs performed with the original plasmids inserted into our two viruses seemed to show a tendency towards SMAR-positive pEPito (48 surviving colonies) compared to SMAR deleted pEPito-ΔSMAR (27 surviving colonies) after 4 weeks of blasticidin selection.
Session: Vector Development
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Transient Cold Shock Enhances Zinc-Finger Nuclease-Mediated Gene Disruption – Rule or Exception?
Zinc-finger nucleases (ZFNs) are a promising tool for precise editing of the human genome and hence of great interest in the field of gene therapy. ZFNs are designer nucleases consisting of the catalytic domain of the FokI endonuclease and an engineered DNA binding domain. Three major platforms are available to generate zinc-finger based DNA-binding domains: modular assembly, context-specific selection (OPEN), and the proprietary Sangamo platform. Recently, Doyon et al. (Nat Methods 7, 459-60; 2010) reported that transient hypothermia generally and robustly increased the frequency of ZFN-induced gene disruption. Here, we aimed to work out the mechanism behind enhanced ZFN activity at 30°C and to elucidate whether enhanced ZFN activity upon cold shock is a generally applicable phenomenon. To this end, we employed six different ZFN pairs – Sangamo-based ZFNs directed against the human AAVS1, CCR5 and IL2RG loci and OPEN-based ZFN that recognize the CFTR, EGFP and HOXB13 genes – and compared their activities at 30°C and 37°C in vitro and in human cell lines. In vitro cleavage assays revealed no difference in ZFN activity when comparing ZFNs of the different platforms at 30°C vs. 37°C, implying that the temperature does neither affect DNA binding nor the catalytic activity. On the other hand, incubation of the cells at 30°C induced an M phase arrest and assessment of the ZFN levels by immunoblotting revealed increased protein levels, suggesting that the cold shock either enhances ZFN expression or stabilizes the enzyme. However, the higher ZFN levels in transfected cells did not correlate with a significant increase in gene disruption at 30°C at the analyzed genomic loci, neither in HeLa nor in 293T cells. Moreover, analysis of ZFN-mediated EGFP disruption by flow cytometry in a U2OS.EGFP cell line did not reveal augmented knockout efficiency for cells that underwent transient cold shock treatment. In summary, our data suggest that although mild hypothermic conditions let to a robust increase in ZFN protein levels, enhanced gene disruption activity of ZFNs at 30°C may not be a general phenomenon but rather dependent on the cell type and the genomic target locus.
Session: Vector Development
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Gene Therapy Research Group, Section of Molecular and Medicine, National Heart and Lung Institute, Imperial College London, London, UK
We previously developed a Scaffold Matrix Attachment Region (S/MAR) plasmid system for in vivo application (1). We demonstrated about 40% hepatocyte transfection and sustained in vivo luciferase transgene expression in the mouse liver for at least six months following a single hydrodynamic administration. Subsequently, we observed sustained transgene expression for the lifetime of the animal, but this dropped appreciably over time. It has been shown that bacterial sequences present in plasmid DNA are responsible for vector toxicity and silencing of expression in vivo. We therefore hypothesised that by eliminating these extraneous bacterial components from our vectors we might improve their performance in vivo. We describe here the development of a minimally sized S/MAR vector using Cre/Lox recombinase (2), to generate a minicircle devoid of bacterial sequences and comprising only an expression cassette and an S/MAR moiety. Upon administration to mice by hydrodynamic delivery we found it to be maintained episomally (Southern blot analysis) and to provide sustained levels of luciferase expression for up to three months (end of experiment), as measured by a bioluminescent imager (XENOGEN). In addition, luciferase expression did not decline over time, as seen with our original S/MAR vector (1) containing the bacterial backbone. At the final time point, luciferase expression from the minicircle vector was approximately two orders of magnitude higher than both the control minicircle vector (no S/MAR) and the original S/MAR vector containing the bacterial backbone. In a further experiment, we performed partial hepatectomy on S/MAR minicircle treated mice, and observed a rapid drop of expression, indicative of absence of plasmid replication. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression in the liver. (1) Argyros et al., Gene Ther. (2008), 15:1593-1605. (2) Bigger et al., J. Biol. Chem. (2001), Jun 22;276(25):23018-23027.
Session: Vector Development
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Gene Therapy Research Group, Section of Molecular and Medicine, National Heart and Lung Institute, Imperial College London, London, UK
Nonviral vectors have not been extensively investigated in neonatal mice due to the poor efficiency of the delivery methods available. Understanding the effects of nonviral vectors during early development is vital to develop safe gene therapy treatments where irreversible pathological processes may be avoided by early gene reconstitution. Here we describe a simple and effective method for the systemic administration of nonviral vectors via the superior temporal vein of mouse pups at 1.5 days of age. We show that injection of polyethylenimine (PEI) complexed plasmid DNA (pDNA) intravenously results in effective transfection in the liver, lung, heart and spleen. We also investigate the specific targeting of transgene expression to the proliferating neonate liver using a liver-specific S/MAR plasmid, which has previously been shown to confer long-term expression in adult mouse liver. Using bioluminescent imaging, a gradual increase in transgene expression was observed peaking at days 11–12, before the reduction of expression to background levels by day 25, suggestive of vector copy number loss. We conclude that nonviral vectors can successfully be used for systemic delivery to neonatal mice but that further optimization of the S/MAR nonviral systems is required for longer-term maintenance of expression during neonatal growth and development.
Session: Vector Development
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Klinikum rechts der Isar, TU München, Munich, Germany
Session: On the Route to Clinical Application
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Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany
Hepatitis C virus (HCV) is the main cause of chronic hepatitis and an effective therapeutic approach should effectively result in long-term immunologic eradication of the virus. Recombinant viruses and DNA vaccines are in the development for adjunct therapy along with pegylated interferon alpha (IFN-α) plus ribavirin. We propose to develop potent antigen-presenting cells (APCs), such as dendritic cells (DCs) capable to directly mimic the IFN-α therapy concurrently with optimized antigen presentation. We developed a novel technology for production of DC consisting of one overnight lentiviral vector (LV) transduction of GM-CSF and IL4 plus full-length antigens into DC precursors that results into induction of “SMART-DCs” capable to self-differentiate directly into long-lived DCs in vivo, and thus bypass in vitro culturing. This concept has been tested and validated in preclinical human and mouse tumor models for potency (Koya et al., Molecular Therapy, 2007). Recently, we have demonstrated that monocyte-derived DCs transduced with lentiviral vectors expressing the HCV nonstructural cluster result into multiantigenic anti-HCV responses in vitro (Jirmo et al., Vaccine, 2010). Therefore, we are currently validating multicistronic LVs that coexpress GM-CSF, IFN-α and a conserved and potent HCV antigen (NS3) in inducing a novel modality of engineered APCs, namely self-differentiated myeloid derived and lentivirus-induced DCs (SMYLE-DCs). Cotransduction of GM-CSF and IFN-α genes into CD14 + monocytes with a bicistronic LV resulted high levels of GM-CSF (1-2 ng/ml) and IFN-α (4 ng/ml) production, which were persistently detectable for 21 days. The SMYLE-DCs showed similar morphological and immunophenotypic features of DCs (MHCII + , CD80 + CD86 + and CCR5+) and autonomous viability for 21 days of culture. The SMYLE-DCs coexpressing NS3 will be subsequently tested in in vitro assays and in humanized mouse models for the stimulation of CD4 + T cell responses against NS3 class II epitopes, which have been correlated with virus clearance. Ultimately, we propose clinical development of programmed DC vaccines for HCV immunotherapy with high capabilities of standardization.
Session: On the Route to Clinical Application
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Internal medicine I, Klinikum Großhadern, LMU München, Munich, Germany
In patients suffering from ischemic cardiomyopathy, lacking interventional or surgical treatment options, induction of neovascularisation via vascular growth factor application and cell therapy is a novel therapeutic approach. In a preclinical pig model model of chronic ischemia, we investigated the efficacy of regional application of murine eEPCs or an AAV-mediated longterm Thymosin β4 (Tβ4) overexpression. Methods: In vitro, human microvascular endothelial cells (HMECs) were embedded in matrigel and tube formation was obtained. Cells were stimulated with conditioned media (CM) from eEPCs ± Tβ4shRNA. Tubes like structures per low power field were evaluated. In pigs (n = 6) a reduction stent graft was implanted into the circumflex artery, leading to complete occlusion at day 28. Retroinfusion of saline, 5 × 106 murine eEPCs or recombinant AAV2/9 rAAV.Tβ4 (5 × 1012 viral particles) was performed at d28. At days 28 and 56, global myocardial function (LVEDP) was assessed. Regional myocardial function (SES) and post mortem angiography were obtained at day 56. Tissue samples from the ischemic and non-ischemic tissue were harvested for capillary staining. Results: In vitro, HMECs incubated with conditioned media of the eEPCs revealed an increase of tube formation which was comparable to the stimulation with VEGF (16 ± 1 control vs. 34 ± 2 VEGF stimulation and 31 ± 2 with eEPC CM tube-like structures/low power field). The reduction of Tβ4 expression in the eEPCs via shRNA abolished this effect (11 ± 1). In the pig model, retroinfusion of eEPCs induced an increase of capillaries (200 ± 12 vs. 112 ± 6 C/F in controls) and collaterals (4.4 ± 0.4 vs. 1.2 ± 0.2 in controls), followed by improvement of the global and regional myocardial function (LVEDP: 14 ± 1 vs. 19 ± 1.2 mmHg in control group; SES 44 ± 4 vs. 10 ± 6% in control group). rAAV.Tβ4 displayed a similar gain of neovascularization and myocardial function. (223 ± 14 C/F; 6.4 ± 0.5 collaterals; 14 ± 0.5mmHg LVEDP; 56 ± 7% SES). Murine embryonic EPCs are capable of inducing angiogenesis and arteriogenesis in a Tβ4 dependent manner. The combination of AAV vector and Tβ4 may offer therapeutic potential in patients with ischemic cardiomyopathy.
Session: On the Route to Clinical Application
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Internal medicine I, Klinikum Groβhadern, LMU München, Munich, Germany
In patients suffering from ischemic cardiomyopathy or peripheral artery disease, lacking interventional or surgical treatment options, induction of neovascularization via vascular growth factor application is a novel therapeutic concept. Here we tested an approach by combining hVEGF-A with hPDGF-B and prolonging the expression period by using a AAV as viral vector.
Session: On the Route to Clinical Application
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Inhibition of G-Protein Coupled Receptor Kinase 2 Prevents Myocardial Hypertrophy in a Mouse Model for Dystrophin-Deficient Cardiomyopathy
Severe cardiomyopathy is a common feature in Duchenne muscular dystrophy (DMD) and has critical impact on mortality of these patients. Although, to date efficient treatment strategies to prevent cardiac complications in DMD remain elusive, cardiac-specific adeno-associated virus (AAV)-based gene therapy strategies represent a promising tool. In DMD, absence of dystrophin causes instability of the dystrophin-glycoprotein-complex and increased vulnerability of cardiomyocytes towards contraction-induced damage. It is unclear to what extend impairment of the cardiac beta-adrenergic receptor (BAR) system contributes to progressive cardiac damage in DMD. It has been shown in transgenic mice that the inhibition of G-protein coupled receptor kinase 2 (GRK2) through the carboxyl-terminus of GRK2 (βARKct) has beneficial effects with an attenuation of heart failure symptoms. Therefore, we have investigated the efficiency of AAV9- mediated cardiac overexpression of βARKct to prevent the development of cardiomyopathy in dystrophin-deficient (mdx) mice. AAV-9 vectors containing βARKct cDNA under transcriptional control of the CMV-MLC promoter (AAV9/βARKct) were created. 1012 AAV9/βARKct vector particles were intravenously injected into the tail vein of 8 week-old mdx mice before the onset of any signs of cardiomyopathy. In some mice AAV9 with enhanced green fluorescent protein (AAV9/EGFP) was injected as control vector. At the age of 12 months echocardiography to assess contractility (fractional shortening (FS)) and diastolic left ventricular posterior wall thickness (PWTd) was performed. Untreated and AAV9/EGFP-treated mdx mice showed extensive myocardial hypertrophy and reduced contractility (PWTd 16.5 + 0.04 and 16.3 + 0.14 mm; FS 45 + 9% and 45 + 8%, respectively (values + standard error)). Treatment with AAV9/βARKct attenuated myocardial hypertrophy and improved contractility (PWTd 12.6 + 0.08 mm and FS 71 + 3%) in mdx mice. We suggest that cardiac inhibition of GRK2 through AAV-mediated overexpression of βARKct is a valuable strategy limiting cardiac damage in muscular dystrophy-associated cardiomyopathy.
Session: On the Route to Clinical Application
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Non-Integrating Lentiviral Gene Transfer of E2F2 to Induce Corneal Endothelial Cell Replication
The osmotic activity of corneal endothelial cells (CEC) maintains the clarity of the eye's cornea. CECs being non-replicative in humans, a decrease of CEC density due to aging, injury or inherited disease causes corneal swelling and loss of transparency. E2F2 is a transcription factor regulating progression from G1 to S phase of the cell cycle. We investigate whether gene transfer of E2F2 is able to induce cell proliferation in CEC. We constructed a non-integrating HIV-based lentiviral vector delivering E2F2 under a CMV promoter (LNT-E2F2). A lentiviral vector delivering GFP (LNT-GFP) served as control. In 293T cells, exposure to LNT-E2F2 resulted in a > 200-fold increase of E2F2 mRNA compared to uninfected or LNT-GFP infected controls, as detected by quantitative PCR. E2F2 overexpression was detected by Western blot and immunohistochemistry. Samples of human corneal tissue were exposed to either LNT-E2F2 or LNT-GFP or left uninfected. Exogenous E2F2 protein was detectable only in LNT-E2F2 infected samples by Western blot and immunohistochemistry. 5-bromodeoxyuridine (BrdU) incorporation and Ki-67 immunostaining was used to detect G1 to S phase progression or cells in any active cell cycle phase, respectively. CECs infected with LNT-E2F2 showed both BrdU and Ki67 nuclear staining. Lentivirus mediated E2F2 overexpression could be used to increase CEC density in donor corneas ex vivo before transplantation. Safety measures are now being investigated to enable clinical use.
Session: On the Route to Clinical Application
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Dept. of Hygiene, Microbiology and Social Medicine, Section Virology, Medical University Innsbruck, Innsbruck, Austria
C peptides derived from the HIV envelope glycoprotein gp41 (e.g., T-20, C46) are highly efficient inhibitors of virus entry. Here, we analysed a membrane-anchored (maC46) as well as a secreted (sC46) variant of the peptide C46 for gene therapy of HIV infection. Membrane-anchored or secreted C46 peptides were expressed from gammaretroviral or lentiviral vectors in B and T cell lines as well as in primary human T cells. Cultures were analysed for expression of the peptides and antiviral effect. Furthermore, a humanized mouse model was estabished to test these antiviral peptides in vivo. The sC46 peptides were expressed and secreted in different lymphoid cells and exerted high antiviral activity against a variety of HIV envelope glycoproteins. In mixed cell cultures peptides secreted from transduced cells produced a bystander effect and prevented infection of non-modified cells. In the mouse model, we observed a substantial increase of maC46 + CD4 + T cells in blood as well as in spleen, apparently due to the selective pressure of ongoing HIV infection. This increase of CD4 + T cells was neither seen in uninfected control mice nor with a control vector. The clear accumulation of maC46 + cells in HIV-infected humanized mice indicates that these cells are protected from HIV infection in vivo. Further studies in this model with maC46 and sC46 will allow us to analyze the conditions that determine efficacy of T cell based immuno/gene therapy for HIV-infection.
Session: Gene Therapy
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Department of Virology, Medical University Innsbruck, Innsbruck, Austria
Antiviral entry-inhibitory C-peptides, a novel class of antivirals in HIV standard therapy (HAART), are derived from the C-heptad motif in the extracellular domain of the gp41 transmembrane protein. C-peptides (such as T-20 or C46) bind to a fusion intermediate state of gp41 and thereby block viral entry with an IC50 in the low nM range. T-20 is the only marketed C-peptide and is administered twice daily via subcutaneous injection. Major drawbacks of this drug are (i) the inconvenient route of administration and (ii) rapid viral resistance emergence. To circumvent these drawbacks, our group focuses on the development of an entry-inhibitory gene therapy based on an elongated variant of T-20, the ‘in vivo secreted antiviral entry inhibitory C-peptide’ (iSAVE). In comparison to T-20, iSAVE also targets a very conserved membrane proximal region, which cannot be mutated without massive viral replication deficiencies. Additionally, a gene therapy ideally requires only one administration of antiviral and thus reduces the need for constant patient adherence. In our gene therapeutical model, iSAVE expression can be targeted to several sites of production. Besides the lymphatic tissue as sites of viral replication, the liver is a very promising organ for iSAVE expression, since (i) hepatocytes are prone to secrete high amounts of protein and (ii) the liver is known to be able to induce a tolerogenic response towards transgenes. Here, we present first results of iSAVE delivered by a self-complementary rAAV8 vector to the liver. iSAVE is expressed to moderate nM plasma concentrations, while its IC50 is in the low nM range promising a sustained antiviral effect upon in vivo challenge. Furthermore, no humoral response against the iSAVE peptide could be detected in immunocompetent mice, pointing to an potential tolerogenic transgene response. Currently, we are investigating the antiviral effect in a humanized mouse model for HIV-infection. In conclusion, scAAV8-iSAVE is a promising candidate for HIV gene therapy and will be further analyzed for its antiviral activity and transgene-induced immunologic responses.
Session: Gene Therapy
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Institute of Experimental Oncology and Therapy Research, Technische Universität München, Munich, Germany
TricOs is a resorbable bone substitute which is already used in clinical applications. This material is a biphasic calcium phosphate ceramic composed of a mixture of hydroxyapatite and β-tricalcium phosphate which combines structural stability with osteogenic capacity. For faster and better healing of large bone defects the optimization of the biological properties of this substitute would be an advantage. This could be achieved by localized gene delivery using vectors coding, for e.g., a growth factor such as BMP-2. Based on our knowledge of localized gene delivery systems using copolymer protected gene vectors (COPROGs) and organic matrices such as collagen sponges or fibrin glue, we examined whether a combination of COPROGs and the inorganic calcium ceramic for matrix mediated gene delivery is possible. For the in vitro studies we used a novel reporter gene, Metridia luciferase, a protein which is secreted in the tissue culture supernatant. We could demonstrate that our gene vector formulation immobilized on TricOs revealed Metridia luciferase expression over a period of 48 days with a maximum at day 5. Detection of GFP expression exhibited a bright fluorescene of cells which colonized the TricOs granules. For in vivo studies we implanted TricOs granules loaded with COPROGs coding either for BMP-2 or firefly luciferase as control in the musculus latisimus dorsi in rats. At different time points the bone substitutes were explanted and the formation of newly formed bone was examined by micro-CT, conventional CT and PETscan. In contrast to the luciferase implants BMP-2 gene vector loaded granules exhibited a better turnover of the bone substitute into newly formed bone and a higher bone density. The finding that ectopic bone formation in the muscle of rats suggests that matrix mediated gene delivery is useful in bone regeneration applications.
Session: Gene Therapy
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Institute für Experimentelle Onkologie, Klinikum rechts der Isar, TU München, Munich, Germany; 2. Klinik für Plastische Chirurgie und Handchirurgie, Klinikum rechts der Isar, TU München, Munich, Germany
Session: Gene Therapy
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Max von Pettenkofer-Institute, Department of Virology, Ludwig-Maximilians-University of Munich, Munich, Germany
Recombinant adeno-associated viral (rAAV) vectors predominantly persist as extrachromosomal genomes. However, in dividing cells, rAAV vector genome copy numbers and transgene expression levels decline rapidly. Herein, we inserted the hyperactive Sleeping Beauty (SB) transposase variants HSB5 and the novel mutant SB100 into our AAV-based two-viral-vector system displaying 10- and 100-fold increased integration efficiencies compared to wild-type SB, respectively. Our previous work showed that transposition only works sufficiently from circular substrates. However, once inside the cell rAAV genomes form various episomal DNA forms including circular and linear DNA molecules. Thus, in initial experiments we addressed whether Flp recombinase-mediated circularization of the transposon from rAAV genome is a precondition of our system. Our data suggested that Flp-mediated circularization may not be required for AAV-delivered transposase activity significantly reducing the complexity of our system. In order to avoid co-transduction of two AAV-vectors into the same cell we generated a stably SB100 expressing cell line. To analyze integration efficiencies we performed colony forming assays and found that after infection with an rAAV with a neomycin encoding transposon (MOI 10.000), the integration efficiencies in SB100 cells was 5-fold increased compared to the control cell line with inactive SB (mSB). This demonstrated that inserting the SB transposase system into rAAV increases integration efficiency significantly. We are in the process to determine sites of insertion after SB100-mediated integration from the rAAV vector into the host genome by using a plasmid rescue strategy. To address the question whether the new version of the rAAV hybrid-vector system also results into stable transgene expression levels in vivo, we coinjected C57Bl/6 mice with the rAAV transposon donor vector expressing the coagulation factor IX and HSB5 delivered by an adenoviral vector. After induction of rapid cell cycling in mouse liver, transgene expression levels were more stable in mice which received HSB5 compared to the control mSB group indicating that transposition works from rAAV.
Session: Gene Therapy
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Internal medicine I, Klinikum Großhadern, LMU München, Munich, Germany
Thymosin β4, an endogenously occurring peptide of 5 kDa, is cardioprotective after ischemia and reperfusion in a Protein Kinase B (AKT) dependent manner. Furthermore it is essential for coronary vessel development and provides angiogenesis during wound healing of the adult organism. Here we tested if long-term overexpression of Thymosin β4 with an adeno-associated virus is beneficial for therapeutic neovascularisation in a chronic in vivo model of hindlimb ischemia.
Session: Gene Therapy
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National Center for Tumour Diseases (NCT) & German Cancer Research Center (DKFZ), Heidelberg, Germany
Lentiviral vectors (LV) possess a high stability of expression in vivo and are often used to investigate genes and their functions. Lentiviral gene transfer is considered to be a promising less time consuming technology to facilitate the generation of transgenic mice with a higher yield of transgenic offspring as compared to the commonly used DNA microinjection. We applied LV to generate a mouse model transgenic for SETBP1 and eGFP. Lentiviral particles were injected into the perivitelline space of early stage embryos. LV integration was detected in newborn animals (F0) using PCRs specific for either the SETBP1 transgene or for the WPRE element of the construct. Lentiviral integration sites were detected in 65% of 31 analyzed F0 mice by highly sensitive LAM-PCR. 5 out of 9 F0 mice showed germline transmission, revealing a total of 33% vector positive offspring. However, no ectopic transcription and overexpression of neither SETBP1 nor eGFP could be detected in transgenic mice. We therefore analyzed the methylation status of the internal SFFV promoter (SFFVp) by bisulfite sequencing. 18 of 18 analyzed CpGs within the promoter region were extensively methylated in F0 animals and in all progeny determined (n = 12). To exclude transgene effects on epigenetic silencing of SFFVp sequences in self-inactivating LVs, we transduced mES cells with LV.SFFV.Setbp1.IRES.eGFP or the corresponding eGFP-expressing control vector. eGFP expression decreased 1.8 fold and 3.5 fold after differentiation of ES cells infected with the transgene vector and SFFV driven control vector, respectively. Further, we compared the methylation status of SFFVp sequences of gammaretroviral (RV) and LV in peripheral blood of bone marrow transplanted mice (n = 7) 3 months after transplantation. Strikingly, LV transplanted mice showed a higher degree of methylation. Here, we demonstrate that the commonly used SFFV promoter is highly methylated with remarkable strength and frequency during development in vivo and differentiation in vitro. We conclude that LV using an internal SFFV promoter are not suitable for the generation of transgenic mice or constitutive expression studies in hematopoietic cells.
Session: Gene Therapy
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BHF Glasgow Cardiovascular Research Centre, Division of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK
Proliferation and migration of vascular smooth muscle cells (VSMCs) is a hallmark of neointima formation (NI) associated with acute vascular injury. Nogo-B is a member of the reticulon 4 family of proteins. Nogo-B is highly expressed in vascular endothelial cells and VSMCs and regulates vascular remodeling. Previous studies have demonstrated that adenoviral-mediated overexpression of Nogo-B reduced NI after acute vascular injury in animal models. This effect was mediated through inhibition of VSMC migration and proliferation. Lentiviral vectors (LVs) are efficient at targeting VSMCs and the recent development of integration-deficient lentiviral vectors (IDLVs) offers additional potential for LVs in vascular gene delivery. Here, we have assessed Nogo-B overexpression in VSMCs mediated by IDLVs in vitro, as a potential therapeutic strategy for the prevention of NI in vivo. Immunofluorescence and western blot analysis indicated that IDLVs expressing Nogo-B efficiently increased Nogo-B expression in VSMCs, compared to IDLVs expressing GFP and no virus controls. Assessment of proliferation demonstrated that IDLVs overexpressing Nogo-B, led to a significant decrease in VSMC proliferation compared to IDLVs expressing GFP (multiplicity of infection (MOI) 25: 0.14 ± 0.0084 absorbance (abs) at 570 nm vs. 0.28 ± 0.01 abs, p < 0.05). A wound-mediated cell migration assay demonstrated that IDLV delivery of Nogo-B significantly reduced the migration of VSMCs, compared to the IDLVs expressing GFP (MOI 25: 55 ± 4.9 μm vs. 113 ± 7.6 μm, p < 0.05). Taken together, our study demonstrates that IDLVs are efficient in mediating overexpression of Nogo-B in VSMCs, leading to phenotypic effects on migration and proliferation. This has important implications for the use of IDLVs as a potential therapeutic vector for the prevention of NI during acute vascular injury.
Session: Gene Therapy
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Helmholtz University Group Oncolytic Adenoviruses, DKFZ and Department of Dermatology, Heidelberg University, Heidelberg, Germany
Oncolytic viruses which selectively kill tumor cells are emerging tools for cancer treatment. We seek to combine adenoviral oncolysis and therapeutic antibody expression in order to further increase anti-cancer efficacy through antibody-mediated toxicity. Therefore, we created a model system by arming our adenoviruses with a recombinant single-chain antibody directed against the well established carcinoembryonic cancer antigen (CEA) fused to the constant domain of IgG2a (scFvCEA-Fc). To ensure cancer cell specific replication, replication-competent viruses were engineered to bear a 24 bp deletion in the E1A region and a chimeric 5/3 fiber to further increase infectivity for various cancer entities. Furthermore, we used different genomic positions and genetic tools for our scFvCEA-Fc transgene insertion. Among these we investigated splice acceptor sites derived from the beta-actin gene, the adenoviral pIII gene, and the Ad40 long fiber gene and compared resulting transgene levels to expression via established tools such as an internal ribosomal entry site or a nonreplicating adenovirus containing a cytomegalovirus early promoter. Infection of various CEA + and CEA − cell lines with any of our recombinant adenoviruses led to detectable amounts of scFvCEA-Fc in cell culture supernatants and cell pellets. However, transgene levels were strongly varying depending on the genomic loci and genetic tool used, giving the highest amount with the Ad40 splice acceptor. The expressed scFvCEA-Fc was then further characterized in flow cytometry, SDS-page, ELISA, and cytotoxicity assays. Our results show that scFvCEA-Fc binds only to cells expressing CEA or to purified antigen in a dose-dependent way and that transgene expression/antibody mediated cytotoxicity does not interfere with adenoviral replication and hence oncolysis. Currently, we are investigating anti-tumoral efficacy of immune cells in presence of scFvCEA-Fc in vitro and eventually in vivo. In the end, we want to create a highly efficient oncolytic adenovirus platform that combines virus-induced tumor cell killing and expression of therapeutic antibodies with systemic activity directed against various cancer markers.
Session: Cancer Gene Therapy
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Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-University, Munich, Germany
Tumor necrosis factor alpha (TNF) is a highly potent cytokine involved in endothelial cell activation, inflammation and tumor cell killing. As systemic application of TNF protein can be highly toxic, tumor localized expression of the cytokine after gene delivery is a potentially suited approach. We could recently show that systemic TNF gene delivery promoted tumor accumulation of liposomally encapsulated doxorubicine (Caelyx). Here we present the therapeutic effect of this treatment on subcutaneous Neuro2A murine neuroblastoma and enhanced Caelyx accumulation in HUH7 human hepatocellular carcinoma. A/J mice bearing subcutaneous, syngeneic Neuro2A tumors were treated with polyplexes based on a biodegradable polymer (G3-HD-OEI), HUH7 hepatoma xenografts in SCID mice received TNF polyplexes based on linear polyethylenimine carrying a peptide (GE11) selectively binding to the epidermal growth factor receptor, which is highly overexpressed in HUH7. When pretreating mice with TNF polyplexes, improved tumor accumulation of Caelyx was observed in both tumor models as compared to control polyplexes plus Caelyx. With the help of live bioluminescense imaging, enhanced tumor accumulation of Caelyx fluorescently labeled with the lipid DiR emitting in the near infrared was observed in living animals. This suggests a TNF mediated increase of tumor blood vessel permeability augmenting the accumulation of liposomal chemotherapeutics. In the Neuro2A model, three rounds of systemic TNF polyplex application and subsequent Caelyx treatment led to tumor growth inhibition lasting until eight days after the last treatment. Regrowth of treated tumors could be inhibited by a second treatment cycle indicating that no resistance towards combined TNF/Caelyx treatment occurred. Histological staining of cryosection showed significant decrease of CD31 expression in the re-treated group, which indicates a decreased vessel density caused by TNF/Caelyx treatment. In conclusion, combining liposomal chemotherapeutic with TNF gene therapy leads to a synergistic effect on tumor reduction in vivo.
Session: Cancer Gene Therapy
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Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-University, Munich, Germany
A major challenge in cancer therapy is to achieve high levels of specificity and efficacy preventing cancer cells to escape apoptosis, which otherwise leads to uncontrolled proliferation. For targeted cancer gene therapy a tumor cell specific promoter would be essential, which is inactive in nontransformed tissue and at the same allows for high levels of transgene expression in tumor cells. Moreover, the tumor-specific activity of such a promoter can be utilized for tumor detection as well as measuring anti-tumoral effects. In gastrointestinal tumors, the constitutive activation of the wnt-pathway leads to malignancy due to high levels of the transcription factor beta-catenin in the nucleus, which in turn constitutively activates target genes causing enhanced proliferation, invasion and metastasis. Here we describe the use of the synthetic beta-catenin dependent promoter CTP4 for specific expression of toxic transgenes, like the VP3 subunit of the chicken anemia virus (Apoptin). In addition, CTP4 driven luciferase expression allowed us to evaluate the effect of RNAi mediated knockdown of wnt components on beta-catenin mediated transcription. Early passages of human colorectal cancer cell lines and control cell lines (HeLa and U87MG glioblastoma) were either transiently transfected with CTP4-luciferase plasmid or stably transduced with a lentiviral vector encoding for CTP4 driven luciferase. Only cell lines with a deregulated wnt/beta-catenin signaling showed transgene expression, whereas in control cell lines transgene expression was below the level of the control vector without promoter. Knockdown of beta-catenin with siRNA clearly reduced CTP4 driven luciferase expression, and in a similar way other factors were targeted by siRNA transfection. For plasmid based therapies we were able to show that in wnt/beta-catenin deregulated colon carcinoma cells CTP4 driven apoptin expression led to more prominent cell killing when compared to control cells. Taken together, these results provide rationales for further preclinical in vivo testing of transcriptionally targeted plasmid vectors and siRNA based therapies for colorectal cancer.
Session: Cancer Gene Therapy
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Division of Medical Biotechnology, Paul-Ehrlich-Insitut, Langen, Germany
Primary and secondary cancers of the liver are a significant health problem with limited treatment options. We sought to develop an oncolytic measles virus (MV) preferentially activated in liver tumor tissue thus reducing infection and destruction of healthy tissue. Making cell entry of measles virus (MV) dependent on tumour-associated matrix metalloproteases (MMPs) restricts virus propagation to tumour cells and improves the virus safety in xenograft tumour models. We documented with specimen of 44 patients that in primary tumor tissue urokinase type plasminogen activator (uPA) and especially matrix metallproteinase-2 (MMP-2) are significantly more active than in adjacent non-tumorous tissue. When allocated to specific tumor entities, the overall tendency observed in the whole collective was confirmed. We then generated variants of the MV fusion protein by inserting different MMP-substrate motifs at the protease cleavage site, and identified the motif PQGLYA as the most efficient cleavage site as determined by syncytia formation on protease positive tumor cells. The corresponding MMP-activatable oncolytic MV-MMPA1 virus was rescued and shown to be strongly restricted on primary human hepatocytes and healthy human liver tissue, while remaining as effective as the parental MV in tumor tissue sections on tissue slices of 10 patients. Thereby, a strong trend (p = 0.09) for improved tumor targeting of MV-MMPA1 was revealed, which correlated to the up-regulation of MMP-2 activity in tumorous tissue (r = 0.7388). These data underline the clinical potency of the MMP-activation concept to generate safer oncolytic viruses for the treatment of primary and secondary cancers of the liver.
Session: Cancer Gene Therapy
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Department of Vectorology and Experimental Gene Therapy, Biomedical Research Center, University of Rostock, Rostock, Germany
Differential mRNA splicing and alternative promoter usage of the TP73 tumor suppressor gene results in the expression of multiple NH2-truncated isoforms that act as oncogenes. Abundant levels of these p73 variants in a variety of human cancers correlate with adverse clinical prognosis and response failure to conventional therapies, underscoring their relevance as marker for disease severity and target for cancer intervention. With respect to an equally important role for amino-truncated p73 splice forms (DeltaTAp73) and DeltaNp73 (summarized as DNp73) in the tumorigenic process, we designed locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers against individual species that were complementary to DeltaEx2 and DeltaEx2/3 splice junctions and a region in exon 3B unique for DeltaN′ and DeltaN. Treatment of cancer cells with these ASOs resulted in a strong and specific reduction of tumorigenic p73 transcripts and proteins, importantly, without abolishing the wild-type p73 tumor suppressor form as observed with p73-shRNA. The specific antisense oligonucleotides rescued cells from apoptosis inhibition due to overexpression of their corresponding amino-truncated p73 isoform and decreased tumor cell proliferation. Furthermore, ASO-116 against DeltaEx2/3 coupled to magnetic nanobead polyethyleneimine (MNB/PEI) carriers significantly inhibited malignant melanoma growth, which correlated with a shift in the balance between endogenous TAp73 and DeltaEx2/3 towards apoptotic full-length p73. Our study demonstrates the successful development of LNA-ASOs that selectively differentiate between the closely related p73 oncoproteins, and provide new tools to further delineate their biological properties in different human malignancies and for therapeutic cancer targeting.
Session: Cancer Gene Therapy
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Oncolytic Enhancement of VSV Through Inhibition of Host Proinflammatory Innate Immune Responses by Vector-Mediated Expression of NFκB Super-Repressor
Vesicular stomatitis virus (VSV) vectors harboring the MD51 mutation in the endogenous matrix (M) protein are potent inducers of interferon (IFN) in non-neoplastic cells, making them safer oncolytic agents than the wild-type virus. However, we have observed substantial attenuation of intratumoral replication of these vectors in our immune-competent, orthotopic rat model of HCC, significantly limiting the therapeutic efficacy. Following a peak in rVSV(MD51) titers in infected tumors after only 24 hours, a rapid and logarithmic reduction occurs, which is coincidental with the massive infiltration of inflammatory cells to the infected tumor sites. We hypothesized that intratumoral virus replication, and hence the oncolytic potency of rVSV(MD51) could be significantly enhanced by blocking anti-viral inflammatory responses triggered by induction of the NF-κB pathway following viral infection without substantially interfering with the vector's intrinsic interferon (IFN) induction capacity to protect non-tumor cells. To test this hypothesis, we incorporated srIkBalpha, the so-called NF-κB super-repressor into the full-length VSV(MD51) vector. In vitro characterization revealed that expression of srIκBalpha as an additional transcription unit in the rVSV(MD51) backbone successfully blocked virus-mediated activation of NF-κB in primary human hepatocytes. Although reporter assays demonstrated that srIκBalpha expression partially interferes with the robust IFN induction and response to the rVSV(MD51) vector, the rVSV(MD51)-srIkB vector is nevertheless able to induce IFN significantly better than wild-type VSV. The advantage of such an intermediate vector is that it would maintain an improved safety profile over wild-type, while maintaining high levels of replication within the tumor. We predict that this strategy will prolong the kinetics of rVSV(M51R) replication, resulting in an improved oncolytic agent. We further hypothesize that a general reduction of inflammation could potentially allow us to safely administer higher doses of virus. These questions will be answered through comprehensive toxicity and efficacy studies, which are underway in HCC tumor-bearing immune-competent rats.
Session: Cancer Gene Therapy
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II. Medizinische Klinik und Poliklinik, Klinikum rechts der Isar, TU München, Munich, Germany
Oncolytic viruses are currently under intense development as alternative cancer therapies. Preclinical and clinical studies will benefit significantly from the development of noninvasive imaging modalities to repetitively monitor key parameters of tumor response. As oncolytic viruses can kill tumor cells by distinct mechanisms from conventional therapies, they require new approaches to accurately evaluate responses. We hypothesized that the combination of positron emission tomography (PET) and magnetic resonance (MR) imaging could informatively monitor the effects of vesicular stomatitis virus (VSV) treatment of HCC tumors. To this end, VSV or control buffer were infused through the hepatic arteries of multifocal HCC-bearing rats, and the animals were imaged by PET and MRI on days − 1, 1, and 3 post-treatment. In addition, a subset of animals was euthanized at each time-point in order to validate the imaging data with histology. For PET imaging, we utilized the 18F-fluorodeoxyglucose ([18F]-FDG) tracer, which is an analog of glucose that is taken up by metabolically active cells, such as tumor cells. Analysis of the isocontour 50 (50% of maximum) of tumor intensities revealed a significant decrease in [18F]-FDG uptake following VSV treatment, indicating a reduction of metabolic activity, as compared to buffer controls. To determine whether this reduction correlated with increased tumor necrosis, histological sections of corresponding tumors were subjected to morphometric analysis of necrotic areas. Interestingly, a strong correlation of the two parameters existed only for the buffer control group. Further investigation by analysis of MRI and histological data revealed complex processes underway at early time-points post-viral therapy, such as necrosis, inflammation, and repair of necrotic tissue, all of which result in changes in [18F]-FDG uptake. Together, these experiments indicate that [18F]-FDG-PET data should be carefully considered at early time-points after VSV therapy, as changes in tracer uptake can be easily misinterpreted. This data strongly supports the rationale for multimodal imaging to assess tumor responses to oncolytic viral therapy.
Session: Cancer Gene Therapy
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Klinikum rechts der Isar der TUM, Institut für Experimentelle Onkologie und Therapieforschung, Munich, Germany
Oncolytic adenoviruses (AdV) are recently gaining interest in gene therapy, as they have the ability to lyse 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 combined action of oncolysis and HSV1-sr39tk-Ganciclovir (GCV) mediated cytotoxicity in human U87MG glioma cells, and if this AdV is suitable for in vivo imaging studies. As a nuclear reporter gene HSV1-sr39tk is favoured over the wild-type form because of its improved in vivo imaging properties. YB-1-dependent oncolytic AdV have so far not been combined with suicide genes in attempt to boost their therapeutic effect. U87MG cells were infected with armed oncolytic AdV expressing HSV1-sr39tk in the E3 region and control AdV under varying conditions. DNA, RNA and protein levels of HSV1-sr39tk were determined by Southern blot, qRT-PCR and Western blot analyses, respectively. The oncolytic and cytotoxic effects of the virus were analysed by Sulforhodamine B staining or XTT assay and compared to the activity of other oncolytic and replication-deficient AdV. The enzymatic function of HSV1-sr39tk was measured by uptake of the [18F]-FHBG, a radiolabelled prodrug-analogue, 2 days after infection. Increased AdV DNA and HSV1-sr39TK DNA and protein levels over time were observed. The virus displayed an enhanced overlapping oncolytic and HSV1-sr39tk-GCV-mediated cytotoxic effect (>95%) in glioma cells, even at a low viral dosage and at 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 when infected cells were mixed with uninfected cells. Furthermore, HSV1-sr39TK displayed enhanced accumulation of radioactive tracer as compared to control AdV expressing HSV1-sr39tk in the E3 region. 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.
Session: Cancer Gene Therapy
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Department of Internal Medicine II, Klinikum Großhadern, Ludwig-Maximilians-University Munich, Munich, Germany
We have recently demonstrated induction of significant tumor-selective uptake and therapeutic efficacy of radioiodine in neuroblastoma tumors after systemic nonviral polyplex-mediated NIS gene delivery. The aim of the current study was to evaluate the efficacy of novel nanoparticle vectors based on linear polyethylenimine (LPEI), shielded by polyethylene glycol (PEG), and coupled with the synthetic peptide GE11 as an epidermal growth factor (EGF) receptor-specific ligand for targeting the NIS gene to EGFR-expressing human HCC (Huh7) cells. We used LPEI-PEG-GE11 to form targeted polyplexes with a NIS-expressing plasmid (pCpG-EF1-NIS) to transfect Huh7 cells followed by analysis of functional NIS expression in vitro and in vivo. In vitro incubation of Huh7 cells with NIS-conjugated LPEI-PEG-GE11 resulted in a 22-fold increase in iodide uptake activity. After establishment of subcutaneous Huh7 tumors in nude mice, NIS-conjugated nanoparticle vectors or control vectors were injected via the tail vein followed by analysis of radioiodine biodistribution after i.p. injection of 123 I using γ-camera imaging and ex vivo γ-counting. While injection of control vectors did not result in tumoral iodide accumulation, Huh7 tumors showed a perchlorate-sensitive iodide uptake of 6–9 % ID/g 123 I with an eff. half-life of approx. 6 h. In contrast, non-target organs like liver, lungs and kidneys showed no significant iodide uptake activity. After application of the EGFR-specific antibody cetuximab 24 h prior to administration of NIS-conjugated LPEI-PEG-GE11 tumoral iodide uptake activity and NIS mRNA expression were markedly reduced confirming the specificity of EGFR-targeted nanoparticle vectors. After 4 cycles of polymer application followed by therapeutic application of 131 I, tumor growth was significantly reduced as compared to control groups. In conclusion, these results clearly demonstrate that systemic in vivo NIS gene transfer using novel synthetic nanoparticle vectors coupled with an EGFR-targeting ligand is capable of inducing tumor-specific iodide uptake, which represents a promising innovative strategy for systemic NIS gene therapy in metastatic cancers.
Session: Cancer Gene Therapy
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National Centre for Biomedical Engineering Science, Molecular Therapeutics Group, National University of Ireland, Galway, Ireland
Disseminating tumour cells appear to be one of the biggest problems in oncological medicine. Here, we combined the tumour-specific apoptosis-inducing activity of TRAIL with the ability of mesenchymal stem cells (MSCs) to infiltrate both tumour and lymphatic tissues. Using this approach it was our aim to target primary tumours as well as disseminated cancer cells in a human pancreatic cancer mouse model. Targeting XIAP by applying RNAi inside the cancer cells further optimized this approach, because it is known that knocking down XIAP has an apoptosis sensitizing and anti-metastatic effect on targeted cancer cells. Furthermore, it has recently been published that the chemokine receptor CXCR4 and its ligand, the stromal-cell-derived factor (SDF-1), enhanced the migratory abilities of MSCs, following the tumour-derived SDF-1 chemotactic axis. We generated MSCs expressing a trimeric soluble wild-type TRAIL (MSC.sTRAIL). MSC.sTRAIL triggered limited apoptosis in pancreatic cancer cells that were resistant to soluble recombinant TRAIL, which is most likely due to the enhanced effect of the direct, cell-mediated delivery of trimeric TRAIL. MSC.sTRAIL-mediated cell death was markedly increased by concomitant knockdown of XIAP by RNAi in the cancer cells. These findings were confirmed in xenograft models. Using MSC.sTRAIL on XIAP silenced xenografts it was possible to force the primary tumour to go into remission and to block the growth of lung metastasis in MSC.sTRAIL treated animals. Furthermore, to enhance the directed migration of sTRAIL-loaded MSCs towards tumour cells, these cells were adenovirally transducted to overexpress the chemokine receptor CXCR4. The natural secretion of SDF-1 by tumour cells builds up a chemotactic axis and it could be demonstrated that CXCR4 overexpressing MSC.sTRAIL utilize this gradient for enhanced migration towards the tumour cells. In summary, this is the first demonstration that a combined approach using systemic MSC-mediated delivery of sTRAIL and overexpression of CXCR4 together with XIAP inhibition suppresses metastatic growth of pancreatic carcinoma.
Session: Tumor Biology and (Cancer) Stem Cells
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Hertie Institute for Clinical Brain Research and Center Neurology, Laboratory for Molecular Neuro-Oncology, Tübingen, Germany
Recent studies have demonstrated the existence of a small fraction of glioma cells endowed with features of primitive progenitor cells and tumor-initiating function. Such cells have been defined as glioma-like cancer stem cells (GCSC). GCCS contribute to glioma progression due o their properties to overcome cell death induced by chemo- and radiation-therapy, to self-renew and to promote angiogenesis. For this reason, GCSC might never be completely eradicated using standard therapy and will be responsible for the recurrence of glioma. It has been shown that the Y-box protein YB-1 is an oncogenic transcription/translation factor associated with the development of cancer. By upregulation of the detoxification membrane channel protein MRP and MDR, YB-1 induces drug resistance. Again, YB-1 itself is regulated via the stem cell-specific transcription factors SLUG, SNAIL and TWIST. Third, YB-1 is overexpressed in GCSC, but is barely detectable in normal human stem cells. Fourth, YB-1 plays an important role in the adenovirus half-life by targeting the adenoviral E2-late promoter. This knowledge promoted us to develop an YB-1 based glioma virotherapy. We investigated the therapeutic potential of the YB-1 dependent oncolytic adenovirus Ad-Delo3-RGD in GCSC. We demonstrated that in vitro Ad-Delo3-RGD replicates in GCSC, kills highly temozolomide (TMZ)-resistant GCSC and moderately rendered these cells susceptible towards TMZ-treatment. In vivo, using an orthotopic mouse xenograft glioma model, infection of GCSC-derived tumors with Ad-Delo3-RGD significantly prolonged survival. Taken together all data, an YB-1 based adeno-virotherapy might be a promising treatment strategy against glioma.
Session: Tumor Biology and (Cancer) Stem Cells
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Tet-Regulated, Lentivirally Mediated BMP-2 Expression in Primary Cells
Therapy of cartilage defects is challenging due to poor self-healing capacity. A combined gene and cell therapeutic approach using transduced primary chondrocytes and pluripotent mesenchymal stem cells (MSC) was chosen. MSC are able to differentiate into a variety of cell types among them osteocytes and chondrocytes and are therefore considered for the treatment of osteochondral defects. Regulated gene expression of growth factor bone morphogenetic protein 2 (BMP-2) was achieved using the Tet-on System, delivered by VSV-G pseudotyped lentiviral SIN vectors (LV). Primary rabbit chondrocytes were infected with 1-vector Tet-on constructs, expressing the reverse Transactivator (rtTA) and eGFP or BMP 2 under control of the Tet-responsive element (TRE). Transgene expression was induced by doxycycline (dox). The efficacy of the Tet-on induction system was optimized using eGFP. Using BMP-2-expressing vectors resulted in secretion of 15–16 ng/ml BMP-2 into the medium. After withdrawal of dox BMP-2 expression decreased to background levels and was re-inducible for various cycles without loss of efficacy. Additionally, prolonged tet-regulated BMP-2 expression was feasible. Produced BMP-2 was functional as evidenced by proteoglycan synthesis. Transduction efficiency of rabbit bone marrow derived MSC by eGFP expressing LV was 65%. After lentiviral infection cells retained full differentiation potential. Transduction of MSC with the BMP-2 Tet-on vectors resulted in inducible BMP-2 expression with a 36-fold increase of BMP-2 expression on RNA level as well as a 113-fold increase on protein level compared to uninduced cells. The lentivirally delivered Tet-on System allows for regulated expression of BMP-2 in primary rabbit chondrocytes as well as MSCs. The amount of BMP-2 produced by chondrocytes after induction in vitro is sufficient for proteoglycan synthesis, a marker for chondrogenesis and levels are comparable to results achieved using a constitutively expressing retroviral vector.
Session: Tumor Biology and (Cancer) Stem Cells
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Neutrality and Non-Neutrality in Clonal Development
Tissue regeneration and maintenance are generally driven by a population of stem cells. These cells are considered to have closely similar functional capabilities and contribute to the production of mature somatic cells by differentiation and continuous proliferation. Clonal marking studies in the hematopoietic system revealed that the peripheral blood is generally composed of the ancestry of multiple hematopoietic stem cells at each point in time. However, in certain pathological situations (i.e., in leukemias) this balance is upset and the system converts to a monoclonal, often malign situation. Similar results are confirmed in vivo and in vitro using a wide range of viral markers. However, the general questions remain how this clonal dominance is generated and how it can be potentially detected at early stages. Using a range of mathematical modeling approaches we characterize the temporal development of multiple clones under different assumptions about the relative fitness. In particular we use these models to study the case of neutral competition for certain in vitro and in vivo settings and extend our studies to the characterization of clonal dominance. Based on our model simulations we make predictions about the variability of clone sizes as a function of time for the situation of neutral and non-neutral clonal competition. Our results suggest that clonal conversion is inevitable even in the neutral situation and is only accelerated if dominating clones exist. We furthermore suggest experimental strategies for a quantitative characterization of the conversion process.
Session: Tumor Biology and (Cancer) Stem Cells
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Institute of Virology (CBF), Charité Medical School, Berlin, Germany
Murine embryonic stem cells (mESCs) have been an excellent model system for introducing specific modifications into the genome to study gene function in vitro or to generate knockout mouse models. However, gene targeting in mESCs using standard techniques is quite inefficient and typically reaches frequencies of 10-7 to 10-6. In consequence, the usage of complex positive/negative selection strategies to isolate gene targeted clones has been crucial. A promising strategy to introduce specific changes into complex genomes is based on zinc-finger nucleases (ZFNs). A ZFN subunit consists of a nonspecific endonuclease domain fused to a specific DNA-binding domain composed of engineered zinc-finger motifs that tether the nuclease domain to a preselected chromosomal site. Upon dimerization of two ZFN subunits at the target site, the ZFN pair specifically cleaves the DNA to trigger the ensuing DNA damage response, which can be harnessed for targeted genome engineering. Here, we used an EGFP-positive mESC line in order to explore the potential of ZFNs to generate a genetic knockout in the absence of antibiotic selection. EGFP-specific ZFNs carrying different previously described endonuclease variants were initially tested in an EGFP-positive human cell line and in EGFP + mESC to identify ZFN variants that combine high activity with minimal toxicity. Under optimized conditions, the single copy EGFP locus was disrupted in 9% of EGFP + mESC after lipofection of the ZFN expression vectors and 3% upon electroporation. Importantly, the percentage of knockout cells remained stable over time, suggesting minor ZFN-associated toxicity. For two independent clones the EGFP knockout status was verified on the genome level. Currently, these clones are being tested for their pluripotent status by a teratoma formation assay. In the advent of applying patient-derived induced pluripotent stem cells in regenerative medicine, this approach serves as a paradigm for knocking out a dominant mutant allele with minimal genomic intervention and without the need of introducing an antibiotic selection marker.
Session: Tumor Biology and (Cancer) Stem Cells
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Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar, TU Munich, Munich, Germany
The goal of this work was providing magnetic nanoparticles (MNPs) and their formulations for efficient viral genetic modification of cell lines and primary cells in vitro and ex vivo. MNPs of the core-shell type with magnetite cores of about 10 nm stabilized and decorated with surfactants and charged polymers were selected from our library of in-house synthesized MNPs for self-assembly with viral particles. To achieve maximal vector binding magnetic vectors were optimized with account for the vector association and magnetic sedimentation with MNPs. Measuring the time course of the turbidity of suspensions of the virus-MNP complexes in defined magnetic fields was used for the evaluation of the magnetophoretic mobility. We have learned that for magnetic viral vectors, it is reasonable to express the composition in terms of iron weight per PHYSICAL virus particle, and NOT per infectious virus particle, taking into account that both infectious and non-infectious virus particles are associated with appropriate MNPs. We suggest a “rule” to formulate virus magnetic complexes with suitable MNPs, based on the association and magnetic sedimentation of the virus with MNPs as well as with account for the functionality of gene delivery. The optimal complex composition of 2.5-20 fg iron per physical virus particle, depending on the MNPs used, is applicable to both adenoviral and lentiviral vectors. Optimized magnetic virus complexes were stable in 50% FCS. Electron and atomic force microscopy data showed structurally intact viruses surrounded by multiple MNPs. Genetic modification of cells on a cell separation column modified with formulated magnetic viral vectors (magselectofection) is highly efficient in hematopoietic stem cells and mesenchymal stem cells from human umbilical cord, hUC-HSCs and hUC-MSCs, respectively. Under optimized transduction conditions, viral magselectofection of hUC-MSCs with SO-Mag2 lentivirus complexes at MOI as low as 0.5 pfu/cell resulted in 60-100% transduced cells. We have also found a two-fold increase in the percentage of the reporter gene expressing cells post-magselectofection of the hUC-MSCs prelabelled with 20 pg Fe/cell of MNPs.
Session: Tumor Biology and (Cancer) Stem Cells
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Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
Adoptive therapy with genetically engineered T cells carrying redirected antigen specificity (TCR gene therapy) is a new option to treat cancer, especially melanoma. However, this treatment is not yet available for metastatic renal cell carcinoma (RCC), due to the scarcity of therapeutically useful reagents. We analyzed tumor-infiltrating lymphocytes (TIL) from RCC to identify T cells with shared tumor-specific recognition for the generation of T cell receptor (TCR)-engineered T lymphocytes for TCR gene therapy of RCC. We established a T cell clone from TIL that recognized an HLA-A2-restricted tumor antigen. The TCRalpha- and beta-chain genes (TCR53) were isolated, modified by codon optimization and murinization, and retrovirally transduced into peripheral blood lymphocytes (PBL). TCR53-engineered PBL recapitulated the specificity of the TIL and demonstrated tumor-specific, HLA-A2-restricted effector activites (IFN-γ, TNF-α, IL-2, MIP-1β, cytotoxicity). TCR53-engineered PBL of healthy donors and RCC-patients exhibited similar TCR expression levels, expansion, and polyfunctional profile. Based on murine B3Z cells, a TCR53-expressing indicator line (B3Z-TCR53) was established for the screening of the antigen prevalence in RCC, in other malignancies, and in normal cell counterparts. Using B3Z-TCR53 cells, 130 tumor and normal cells were analyzed and shared TCR53 peptide:MHC expression was found in more than 60% of RCC and 25% of tumor cell lines of other histology, while normal tissue cells were not recognized. To date, TCR53 is the only TCR with shared HLA-A2-restricted recognition of RCC. It fulfills important criteria for utilization in TCR gene therapy and could advance T cell-based immunotherapy to patients with RCC and other malignancies expressing the TCR-ligand.
Session: Cancer Immune Therapy and T-Cell Therapy
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Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar, TU Munich, Munich, Germany
Feline fibrosarcoma is an everyday challenge in veterinary practice. Despite aggressive pre or post-operative treatment it has a high relapse rate of aprox. 75 % within 6 months after surgical resection. To obtain a better therapeutic outcome, novel strategies are necessary. Hence, we established immunostimulatory therapy by magnetofection. Here we report preliminary results from a comparative clinical study where the genes for feline GM-CSF, IFN-γ and IL-2, feline GM-CSF alone or human GM-CSF were administered. The study design is prospective, randomized, placebo-controlled ( = standard therapy) and includes four arms: (1) standard therapy, i.e., surgery alone; (2) nonviral magnetofection of the triple-combination of feline GM-CSF, IFN-γ and IL-2 genes into the tumor before surgery or nonviral administration by magnetofection of feline (3) or human GM-CSF (4) gene alone. Preliminary clinical endpoints of the studies are relapse-free survival. Nonviral magnetofection, a procedure developed in our laboratory, is gene therapy by plasmids associated with magnetic nanoparticles under the influence of a magnetic field. The magnetic field was applied to achieve improved retention of the injected vector dose in the tumor. All gene-therapeutic treatments were well tolerated and led to significantly prolonged relapse-free survival. Recent results from FACS-analysis of the primary tumor cells from the treated groups showed increased MHC-II expression compared to those of control cats. This is encouraging concerning future use in veterinary practice as this treatment can be easily administered. The results are promising with respect to their potential in human medicine, as they have been obtained in real patients instead of experimental tumor models.
Session: Cancer Immune Therapy and T-Cell Therapy
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Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
HER2 is over-expressed in 25–40% of all breast cancers and in a variety of other tumors such as ovarian cancer and hematological malignancies. Due to the selective over-expression in malignant tissue, HER2 is considered one of the most attractive targets for therapeutic interventions, such as monoclonal antibodies, kinase inhibitors, and cancer vaccines. However, tumor regression following immunization with vaccines is rare, likely a consequence of HER2 being a self antigen and therefore inducing tolerance. To overcome tolerance, adoptive T cell transfer (ATT) strategies have been developed. However, clinical application of ATT is limited, because isolation and characterization of HER2-reactive T cells is laborious. Yet, a promising approach is the transfer of TCRαβ genes, which have been isolated from non-tolerant settings, into T cells. In this study, we isolated the TCR genes of a HER2-reactive, allo-HLA-A2-restricted CTL clone and introduced the genes into a retroviral vector. Efficient cell surface expression of the TCR and improved functional avidity of the gene-modified T cells were achieved after murinization (replacement of the human TCR constant regions by mouse counterparts), codon-optimization and application of the P2A gene linker (HER2-TCR-opt). Thus, the TCR expression in transduced T cells increased from 1.5% to 41% measured by A2/HER2-multimer staining. The ability to secrete IFN-γ of HER2-TCR-opt transduced T cells was comparable to that of the CTL clone, which showed a half maximum IFN-γ secretion at 10-7 M towards HER2 peptide-loaded T2 cells. Furthermore, HER2-TCR-opt-transduced T cells lysed HER2-expressing tumor cell lines as efficient as the parental clone (lysis of 58% at a E:T of 30:1). The TCR showed a cross-reactivity to HER3 and HER4 that was similar to the parental CTL clone. Extensive testing with HER2/3/4 negative cell lines did not reveal any further epitopes that were recognized by the TCR. Our results contribute to the development of a TCR-based approach for the treatment of HER2-positive breast cancer, as well as of other malignancies expressing HER2, HER3 and/or HER4.
Session: Cancer Immune Therapy and T-Cell Therapy
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Department of Pathology, University of Frankfurt, Frankfurt am Main, Germany
The oncogenic potential of human hematopoietic stem cells (HSCs) in the context of retroviral gene therapy, as reported in patients treated for X-linked severe combined immunodeficiency (X-SCID) or X-linked chronic granulomatous disease (X-CGD), is now well described. Syngeneic mouse models for the development of leukemia/lymphoma are established as well as the reconstitution of immunodeficient mice with human stem cells. The combination of both systems, a mouse model for the development of human leukemia/lymphoma is still missing. To address this issue, we aim to test different humanized mouse models. Currently, the most promising mouse strains available for the engraftment of human cells are NOD-SCID γc −/− (NOG) and Rag2−/− γc −/− mice. These strains will be repopulated with human HSCs or mature T cells expressing potent (proto-)oncogenes after retroviral/lentiviral gene transfer. To further enhance the repopulation dynamics of mature T cells, human HSCs will be transduced with mouse strain-specific murine T-cell receptors (TCRs). After successful repopulation the strain-adapted human, mature T cells will be isolated, in vitro transduced with potent (proto-)oncogenes and transplanted into secondary recipients. A second strategy to overcome the mismatch between the human TCR and the murine major histocompatibility complex (MHC) is to utilize huMHC-transgene mice. Due to the lack of functional lymph nodes in the immunodeficient mouse strains in a third strategy we want to use the so-called trimera mouse model: lethally irradiated and cyclophosphamid treated immunocompetent mice will be reconstituted with bone marrow of highly immunodeficient mice to generate an optimal environment for the repopulation with retrovirally/lentivirally modified human, mature lymphocytes. Moreover, all described mouse models will be used to investigate established human leukemia/lymphoma cell lines in vivo. The future goals of the project are on the one hand to compare expression profiles of healthy and neoplastic lymphocytes and on the other hand the identification of leukemic stem cells (LSCs) of leukemia/lymphoma cell lines.
Session: Cancer Immune Therapy and T-Cell Therapy
<
Department of Pathology, University of Frankfurt, Frankfurt am Main, Germany
Retroviral insertional mutagenesis in hematopoietic progenitor cells can activate neighboring proto-oncogenes and thus contribute to leukemia development. This genotoxic activity of integrating viruses is highly relevant for the risk assessment of stem cell gene therapy and also has been the basis for the identification of several proto-oncogenes. In previous studies, we analyzed the susceptibility of mature T cells to transformation (Newrzela et al., Blood, 2008). Surprisingly, T-cell transplanted animals showed no sign of leukemia/lymphoma development during a follow-up of more than 500 days. These results show that polyclonal mature T cells are less susceptible to transformation in vivo by known T-cell oncogenes than progenitor cells. In contrast, TCR-monoclonal T lymphocytes expressing oncogenes developed T-cell leukemia/lymphoma in recipient mice. We thus postulate that clonal competition may control leukemogenesis in mature T-cell populations. To analyze the mechanisms that control the outgrowth of malignant clones in TCR polyclonal, but not in TCR monoclonal mice, clonal fluctuation within the T-cell population will be analyzed during the manifestation of mature T-cell leukemia/lymphoma. DNA samples from peripheral blood T cells will be frozen in regular intervals from mice transplanted with polyclonal or monoclonal T cells expressing specific (proto-)oncogenes or a control gene. As the gammaretroviral vector used for gene transfer integrates randomly into the host genome, individual T-cell clones are marked by their specific integration sites. Integration site analysis by LM-PCR will reveal dominant clones within the T-cell population. In order to visualize the clonal fluctuation and progressing dominance of certain clones with a growth advantage due to insertional mutagenesis, these T-cell clones will be investigated retrospectively by integration-site specific PCR. In addition, the TCR diversity in the polyclonal setting will be analyzed on a genomic level during leukemia/lymphoma development/suppression to correlate the homeostatic clonal competition and the outgrowth of malignant T-cell clones.
Session: Cancer Immune Therapy and T-cell Therapy
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Department of Gene Therapy, University of Ulm, Ulm, Germany
Pancreatic cancer is the fifth leading cause of cancer-related death in Germany. Pancreatic adenocarcinomas (PDACs) are characterised by rapid progression, early metastasis, diagnosis at an advanced stage, and high resistance to standard therapy. Thus, patients suffering from PDACs have a very poor prognosis. PDACs are composed of infiltrating tumor cells surrounded by extracellular components and other non-neoplastic cell types. Very frequently, tumor cells can be acounted for significantly less than half of the cellular mass within the tumor. The complex mutual interaction between tumor cells and non-neoplastic human pancreatic stellate cells (hPSCs – the major stromal cell type present in pancreatic cancer) is highly regulated through secretion of different growth factors (e.g., TGF-β), strongly influencing growth and malignancy of PDACs. In mouse experiments coinjection of tumor cells and hPSCs resulted in an advanced tumor progression. For treatment of pancreatic cancer with adenoviral (Ad) vectors, it would be therefore of advantage to target these vectors to both cell types. To date the systemic admininstration of Ad vectors, however, is characterised by two major drawbacks: the low transduction of tumor cells and the severe damage of healthy tissues (e.g., liver). To address these obstacles a hexon-modified Ad vector has been developed displaying the TGF-β receptor binding peptide CSK17 on the hypervariable region 5 of hexon resulting in AdhCKS17. Subsequent in vitro studies have shown a significantly enhanced transduction of early tumor cells and primary hPSCs after infection with AdhCKS17. As a consequence the hexon-modification resulted in an increased burst size and cytotoxicity in both cell types. A preliminary biodistribution study indicates a decreased liver uptake of this hexon-modified vector compared to the control vector indicating detargeting from liver, thereby potentially reducing liver damage. Subsequent in vivo investigations are directed at analysing this vector within the human tumor in an ex vivo culture system and in murine xenotransplantation models bearing composite tumors consisting of human pancreatic cancer cells and stroma cells.
Session: Pharmacology and Toxicology
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Universität Witten/Herdecke, Institut für Zellbiologie, Witten/Herdecke, Germany
The vector pEPI is the first non-viral autonomous replicon that was constructed for mammalian cells. It represents a minimal model system to study the epigenetic regulation of replication and transcription. Its function relies on a transcription unit linked to an S/MAR sequence. In gene therapy, situations exist in which transgenes have to be expressed for a limited time and thus it would be of considerable interest not only to silence the expression of the transgene but to construct a vector which can be removed from the cell at any time. Therefore we followed two approaches: (I) We constructed the inducible episomal vector pEPI-Tet in which transcription running into the S/MAR is regulable with doxycycline. We found that for vector replication and long-term maintenance an ongoing transcription running into the S/MAR element is required. Once established, the vector is lost from the cell upon switching off transcription from the gene linked to the S/MAR. (II) Secondly, we used the siRNA approach: Cells in which pEPI-eGFP has been stably established were transfected with a vector expressing siRNA directed against eGFP. As a consequence, due to the abrogated transcription of the transgene running in to the S/MAR, reduced plasmid copies per cell were detected using quantitative PCR. Furthermore, when comparing cells transfected with an eGFP-siRNA with those transfected with a nonsense-siRNA, we found that active chromatin markers, e.g., H3K36me or H3K4me3, got lost.
Session: Pharmacology and Toxicology
<
Department of Virology, Max von Pettenkofer-Institute, LMU, Munich, Germany
High-capacity adenoviral vectors (HC-AdVs) devoid of all viral coding sequences offer high transduction efficiencies, large packaging capacities (up to 35 kb) and reduced toxicity. To further improve the persistence of transgene expression and the maintenance of the vector genome in cycling cells, we recently developed an adeno/transposase hybrid-vector-system utilizing a hyperactive Sleeping Beauty transposase HSB5 for somatic integration (Hausl, Zhang et al., Molecular Therapy, accepted). However, with respect to preclinical evaluation this hybrid-vector system needs to be characterized in more detail. Herein, we optimized the virus dose required for long-term effects without toxicity and we performed a vector fate analysis. Based on our established hybrid-vector system, female and male C57Bl/6 mice were co-injected at three dosages [1.6 × 108 8 × 108 and 4 × 109 transducing units (TU) total per mouse] with a canine factor IX (cFIX) transposon donor vector and a second vector encoding the hyperactive SB transposase HSB5 and Flp recombinase (n = 5 per group). In groups which received the highest dose, toxicity was observed in mice of both genders. At the lowest dose, only low levels of serum cFIX were detected ( ≈ 100 ng/ml). This was in sharp contrast to the middle-dose group in which we measured ≈ 1000 ng/ml serum cFIX levels without toxicity. We concluded that an efficient non-toxic dose for our hybrid-vector lies between 8 × 108 to 4 × 109 TUs per mouse. Notably, at the middle dose we observed a gender influence on transgene expression levels. In order to address issues related to genotoxicity after HSB5-mediated somatic integration, transposition events were analyzed by a PCR-based high-throughput sequencing method. For each individual we generated 4 genomic DNA libraries using different restriction enzymes. Of the 73 transposition events identified in liver of HSB treated mice, 74% (54/73) were found in non-gene areas and 26% (19/73) in genes indicating a random integration pattern. Of the transposition events detected in genes, 17 insertion sites were in introns and only 2 in exons. From all animals, no integration site was found in or near putative oncogenes.
Session: Pharmacology and Toxicology
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Institute for Medical Informatics and Biometry, Faculty of Medicine “Carl Gustav Carus”, Dresden University of Technology, Dresden, Germany
Leukemia caused by insertional mutagenesis is a major risk of gene therapy with HSCs. In contrast, polyclonal mature T cells have turned out to be surprisingly resistant to leukemogenic insertional mutagenesis. Newrzela et al. showed that leukemia/lymphoma did not occur upon transplantation of wild-type mature T cells with polyclonal T-cell receptors (TCR) being transduced with high copy numbers of gammaretroviral vectors encoding potent T- cell oncogenes into RAG1-1-deficient recipients [1]. However, further studies demonstrated that the transplantation of T cells from TCR-monoclonal OT1 mice that were transduced with the same protocol resulted in leukemia/lymphoma. The underlying mechanisms that prevent oncogenesis in the polyclonal situation and permit the outbreak of leukemia in the monoclonal situation are currently unclear. Using a mathematical modelling approach, we challenge the arising hypothesis that polyclonality induces competition within the T-cell repertoire, which in turn suppresses the emergence of a leukemic clone. As a first step, we developed a mathematical model of T-cell homeostasis that is derived from a similar niche-based based model of hematopoiesis [2]. The key assumption of the novel model is that T-cell survival is critically dependant on the interaction of the clone-specific TCR with self-peptide-MHC-complexes and therefore subject to competition between different T-cell clones. Our model consistently reproduces the polyclonal pattern observed in T-cell homeostasis, and responds in an adequate manner to perturbations of the system such as infection. Furthermore, based on our modelling results, we speculate how the deregulation of T-cell receptor affinities supports the formation of dominant clones. The modelling results underscore the possibility that clonal competition can prevent the outbreak of overt mature T-cell leukemia/lymphoma. References: [1] Newrzela S et al. Resistance of mature T cells to oncogene transformation. Blood. 2008;112(6):2278–2286. [2] Roeder I., Loeffler M. A novel dynamic model of hematopoietic stem cell organization based on the concept of within-tissue plasticity. Exp. Hematol. 2002;30(8):853-861.
Session: Pharmacology and Toxicology
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Institute for Transfusion Medicine, University of Ulm, Ulm, Germany
Targeted gene repair with single-stranded oligodeoxynucleotides as repair matrix often yields low rates of corrected cells, but these rates can be enhanced by the concomitant induction of a DNA double-strand break (DSB) close to the mutation. As a tool to achieve defined DSBs, customisable zinc-finger nucleases (ZFNs) are studied. They consist of a specific DNA-binding domain (designed zinc-finger motifs) linked to a nonspecific FokI-derived dimer-forming nuclease domain. Here, off-target activities of two ZFNs (GZF1 and GZF3) were investigated using three complementing approaches. To reduce toxicity, both ZFNs already harbour a variant dimer interface to favour heterodimerisation. For in vitro cleavage assays, the ZFN proteins were incubated with target locus DNA. Off-target activity was detected at two positions due to unwanted GZF1 homodimerisation, where one ZFN bound its perfect 9-mer site while the partner functionally attached to a mismatched sequence. To examine whether this might happen in a 293 cell line with a transgenic single-copy target locus, one expression vector (for GZF1 or GZF3) was transfected to then analyse target locus DNAs for signatures of DSB repair (e.g., nonhomologous end joining (NHEJ)). At the regular GZF1/GZF3 site, one NHEJ-derived modification per ≈ 15,000 alleles was observed when only GZF1 was present. Since similar binding site variants were found also for GZF3 homodimers, related off-target activity—not yet detected—might also occur. In silico analyses (Ensembl BLASTN on haploid genome) predicted 28,100 and 12,695 additional perfect sites for GZF1 and GZF3, respectively. Based on sequence analyses from the in vitro and in cellula assays, ≈ 6% of these sites were classified as potential off-targets with canonical 5–7 base-pair spacers and second binding sites (9-mers) with 6–8 matches. At half of these sites, the favoured heterodimers might form. In conclusion, our multifaceted platform consisting of genome-wide in silico predictions combined with in vitro and in cellula assays represents a potent tool to evaluate ZFN specificities and can be used to estimate biological activities of any ZFN with known DNA binding parameters.
Session: Pharmacology and Toxicology
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Pathology of the Goethe-University Frankfurt, Frankfurt am Main, Germany
In safety studies in a mouse model, we found that in hematopoietic stem cells high copy numbers of gammaretroviral vectors expressing T-cell oncogenes readily induced T-cell leukemia/lymphoma in recipient mice. However, mature polyclonal T cells could not be transformed (Newrzela et al., Blood 2008). Yet, and quite surprisingly, T-cell receptor (TCR) monoclonal T lymphocytes derived from OT-I mice were transformed with the same kinetics and efficiency as stem cells. This propensity of monoclonal T-cell populations to develop malignancies is highly relevant for evaluating the safety of therapeutic gene transfer into mature T cells, especially the adoptive transfer of therapeutic TCRs. On the other hand, these observations imply that polyclonal T-cell populations can control the outgrowth of malignant T-cell clones. The population-dynamics of T cells will be analyzed with respect to the diversity of the T-cell repertoire. The generated data on T-cell dynamics will furthermore help to validate mathematical models on the normal and malignant T-cell homeostasis, which will be developed in the group of Ingo Roeder, Dresden. In addition, the molecular mechanisms that control T-cell malignancy in the polyclonal, but enhance leukemogenesis in the monoclonal setting will be identified. The mechanisms involved in the pathogenesis of mature T-cell malignancies are addressed in this study.
Session: Pharmacology and Toxicology
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National Center for Tumor Diseases Heidelberg, Department of Translational Oncology, German Cancer Research Center, Heidelberg, Germany
Long-term gene expression can be achieved following recombinant adeno-associated virus (rAAV) vector delivery in vivo. Previously, we have shown that rAAV8 delivery to newborn rat liver results in low frequent integrated rAAV genomes in a minority of cells. However, even the rare integration frequency of the rAAV vector raises concerns about its clinical safety. To evaluate the safety of different AAV-serotypes and vector doses in preclinical settings we estimated rAAV integration characteristics in rat liver after single-stranded (ss)AAV or self-complementary (sc)AAV injection of different vector doses. The persistence of ssAAV and scAAV was analysed in transduced rat liver 14 months and 13 months post-rAAV injection. In rats transduced with scAAV partial hepatectomy (HP) was performed 13 months post-injection and the integration frequency was evaluated 3 months post HP. LAM-PCR was performed on 250-500 ng of total DNA derived from 12 ssAAV1, 6 scAAV1 and 6 scAAV8 transduced rats and combined with deep sequencing allowing simultaneous and efficient identification of complex rAAV concatemeric forms and proviruses. Sequencing of > 68.000 LAM-amplicons revealed 67 unique insertion sites (IS), in ssAAV transduced rats up to seven per sample and in scAAV transduced ones up to eight per sample. The averaged number of IS was higher in rat liver transduced with 1.2 × 1012 TU/kg ssAAV1 compared to a vector dose of 8 × 1012 TU/kg (4.6 IS vs. 0.5 IS). Semi-quantitative measurements of rAAV concatemers showed that in rat liver transduced with 8 × 1012 TU/kg ssAAV1 up to 100% of the analysed sequences were due to concatemers while in rat liver transduced with 1.2 × 1012 TU/kg up to 3/4 of all analysed sequences could be referred to IS. In addition, sequencing of rAAV LAM-amplicons derived from ssAAV and scAAV transduced rat liver was used to show the feasibility of complete ITR sequencing in vivo. In partial ITR no preferential ITR breakpoints were detected in ssAAV transduced rat liver while in scAAV transduced ones preferential ITR breakpoints were detected. These insights are of clinical relevance for potential vector genotoxicity of different rAAV serotypes and doses.
Session: Pharmacology and Toxicology
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Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
The use of fluorescent proteins (FPs) as markers expressed by gene transfer vectors facilitates fast and unambiguous identification of gene-modified cells. However, to select cells based on their expression of FPs, fluorescence-activated cell sorting would have to be used. This complex and expensive technique is not available in all laboratories, bears a certain contamination risk and might be too stressful for some delicate cell types. To overcome these limitations we have recently developed ten fusion proteins as bifunctional markers composed of a variety of fluorescent proteins (Cerulean, eGFP, Venus, dTomato, mCherry) fused to drug resistance proteins (NeomycinR, ZeocinR, PuromycinR, HygromycinR, BlasticidinR). Now we present the second generation of bifunctional markers which are greatly improved in brightness by inserting a 2A peptide in between the fluorescent protein and the resistance protein. In fact, the brightness of the so far dimmest marker (eGFP fused to PuromycinR) was increased by a factor of seven, now reaching 90% of the brightness of unfused eGFP. All bifunctional markers have been tested in lentiviral vectors (
Session: Pharmacology and Toxicology
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From Insertion Site Profiles to Clonal Contributions
Initially, our goals were to test if viral insertion is nonrandom and, specifically, to clarify whether host and disease-specific integration patterns exist. We developed the bioinformatic applications IntegrationSeq, IntegrationMap and QuickMap, and others (manuscript in revision) which allow high throughput analyses (next generation sequencing) of insertion sites of viral vectors (QuickMap available at
Session: Pharmacology and Toxicology
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Next Generation Sequencing Data Management for Integration Site Analyses
Integration site analyses have become a valuable and powerful tool to detect clonal repopulation kinetics in clinical gene therapy, to address the biosafety of a specific gene transfer approach or to uncover vector-induced side effects. With a combinatorial approach of standard or nonrestrictive (nr) linear amplification-mediated (LAM) PCR followed by next generation sequencing, time- and cost-efficiency of integration site sequence retrieval can be improved by several logs compared to standard Sanger sequencing technologies. Using our implemented analyses tools, such as Quickmap (
Session: Pharmacology and Toxicology
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BHF GCRC, University of Glasgow, Glasgow, UK
The spleen is an important site for the induction of immune responses to adenoviral (Ads) vectors, especially following intravascular delivery. Ads have been shown to interact with macrophage populations and dendritic cells in the spleen, and high level splenic uptake has been associated with vector-induced toxicity in vivo. However, the precise immune effectors which determine the release of characteristic inflammatory cytokines in response to Ad remain to be fully identified. Using a panel of viruses, including Ad5, Ad48 and hexon-modified Ad5, we assessed their interactions with cell markers in the spleen (CD11b, CD11c, SIGNR1, MARCO, CD169 and F4/80) at low and high doses of Ad, and at early and late time-points post-injection. In order to fully analyse these interactions and their contribution to inflammatory responses, we administered a cohort of mice with clodronate-encapsulated liposomes and assessed the effects on macrophage and other immune cell populations in the spleen. We quantified serum cytokines 6 hr post-injection using a 20-plex luminex assay, for clodronate treated and untreated animals. Fluorescently labeled Ads (Ad5, Ad48 and hexon-modified Ad5) were detected by immunohistochemistry in co-localisation with MARCO + and Moma-1 + cells, within the marginal zone surrounding the white pulp of the spleen, and with F4/80 + red pulp macrophages (1 h). These macrophage populations were almost completely absent in animals treated with clodronate liposomes and subsequently the spatial distribution of virus within the spleen was altered. The defining splenic cell markers for virus transgene-expressing cell types at later time-points (48 h) were not the same as those detected at early time-points. These currently remain to be identified. Further studies will be required to fully characterise the time-course of adenoviral uptake in the spleen and the contribution of the various cellular effectors to the induction of inflammation.
Session: Pharmacology and Toxicology
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DG-GT 2010 Abstract Author Index*
Acosta-Sanchez, Abel, Inv 12
Al-Ghaili, Nabil, P 64
Alba, Raoul, P 38, P 69
Albarenque, Stella, P 48
Ali, Robin R, Inv 14, P 30
Almarza, David Gomez, Or 2
Aloysius, Mark, Inv 23
Altomonte, Jennifer, P 14, P 44, P 45
Anton, Martina, Inv 2, Or 18, P 41, P 46, P 50
Antoniou, Michael, Or 9
Apfel, Sibylle, P 14
Appelt, Uwe, P 65, P 67, P 68
Arens, Anne, Or 7, P 65, P 67, P 68
Argyros, Orestis, P 23, P 24
Armeanu, Sorin, P 42
Arzumanov, Andrey A, Inv 11
Bahadur, Dhirendra, P 15
Bai, Lin, Or 6
Baier, Ruth, P 18, P 19
Baiker, Armin, P 21
Bainbridge, JWB, Inv 14
Baker, Andrew H, Inv 10, Or 21, P 38, P 69
Bakshi, Rakesh, P 26
Ball, Claudia R, Or 7, P 37
Ballmaier, Matthias, Or 10
Banerjee, Rinti, P 15
Banfi, Andrea, P 28
Bartholomae, Cynthia C, P 67, P 68
Bauer, Ralf, P 29
Baum, Christopher, Or 10, Or 15, Or 19
Becirovic, Elvir, Or 6
Beck, Susanne C, Or 6
Bednarski, Christien, P 17, P 22
Belay, Eyayu, Inv 12
Bell, John C, Inv 18, Inv 19
Benda, Veronika, P 55
Bendle, Gavin M, Or 17
Benihoud, Karim, P 59
Benz, Alexander, P 57
Bergemann, Christian, P 8
Bernhard, Helga, P 56
Bhalla, Joti, Inv 23
Bhattacharya, SS, Inv 14
Biel, Martin, Or 6
Bies, Laura, Or 17
Bitzer, Michael, P 42
Blaesen, Markus, P 18, P 19
Blankenstein, Thomas, P 54
Blasczyk, Rainer, Or 7
Bock-Marquette, Ildiko, Or 8, P 27, P 36
Bode, Juergen, P 12
Boekstegers, Peter, P 27, P 28
Böhm, Marie, Or 7
Böning, Guido, Or 14
Bosma, Piter, P 65
Boztug, Kaan, Or 7
Bradke, Frank, P 14
Bradshaw, Angela C, Or 21, P 69
Braren, Rickmer, P 45
Bräuchle, Christoph, Or 5, P 3
Breitbach, Caroline, Inv 18
Brewer, James M., P 69
Brill, Thomas, P 55
Brugman, Martijn, Or 10, Or 15
Buch, Prateek, P 30
Buchholz, Christian J, P 42
Buck, Andreas K, P 45
Buckley, Suzanne MK, Inv 13
Büning, Hildegard, Or 2, Or 6, P 32
Busch, Dirk H, P 56
Büsche, Guntram, Or 10
Cantz, Tobias, Or 15, P 52, P 52
Carpentier, A, Inv 15
Cathomen, Toni, Or 3, P 11, P 17, P 22, P 52, P 52, P 63
Cattaneo, Roberto, Inv 17, P 42
Cengizeroglu, Arzu, Or 18, P 41
CGD Consortium, Inv 16
Chan, Jerry, Inv 13
Chan, Lucas, Inv 23
Charrier, Sabine, Inv 24
Chick, Helen, P 38
Chuah, Marinee KL, Inv 12
Cichutek, Klaus, Inv 25, P 42
Cim, Abdullah, Or 9
Collins, Louise, Or 9
Conrad, Heinke, P 56
Cooper, Jonathan D, Inv 13
Corjon, Stephanie, Or 1
Cornils, Kerstin, P 64
Cornu, Tatjana I, P 17
Coughlan, Lynda, P 69
Coutelle, Charles, P 23, P 24
Cowled, Christopher, Inv 23
Dannemann, Nadine, P 22
de Bruin, Karla, P 3
Deedigan, Laura, P 48
Deichmann, Annette, P 68
Deindl, Elisabeth, Or 8
Deutsch, Daniela, P 41
Diaz, Margarita, Or 20
Diepolder, Helmut, P 26
DiMaio, Michael, P 27
Döblinger, Markus, P 53
Dohmen, Christian, P 5
Dorer, Dominik E, P 16, P 39
Draganovici, Dan, Or 14
Dreyer, Anne-Kathrin, P 22
Duffy, Margaret R, Or 21
Dumler, Katja, P 46, P 50
Edinger, Daniel, P 7
Egana, Tomas J, P 34
Egerer, Lisa M, P 31, P 32
Ehrhardt, Anja, Or 3, P 9, P 10, P 20, P 21, P 35, P 61
El-Aouni, Chiraz, P 28
Ellwart, Joachim, Or 18
Emmrich, Stephan, P 43
Engels, Boris, P 54
Engler, Tatjana, Or 1
Eremin, Oleg, Inv 23
Espenlaub, Sigrid, Or 1, P 13
Fahl, Edda, Or 6
Farkasova, Katarina, Or 18, P 7, P 41
Farzaneh, Farzin, Inv 21, Inv 23
Fedonidis, Constantinos, P 23
Fehse, Boris, P 64, P 66
Fella, Carolin, Or 1
Ferizi, Mehrije, P 55
Fernández Ulibarri, Inés, P 16
Ferry, Nicolas, P 65
Filipczyk, Adam, Or 15
Fischer, Cornelia, P 55
Fischer, M Dominik, Or 6
Fitzke, FW, Inv 14
Flageul, Maude, P 65
Foralosso, Ruggero, P 5
Frankenberger, Bernhard, Or 16
Frenz, Jessica, P 25
Frieβ, Wolfgang, P 1
Fröhlich, Thomas, P 5, P 6
Fulda, Simone, P 48
Galla, Melanie, Or 15
Galy, Anne, Inv 24
Gänsbacher, Bernd, P 33, P 46, P 50, P 55
Garritsen, Henk, Inv 21
Garside, Paul, P 69
Gaudet, D, Inv 15
Gellhaus, Katharina, P 11, P 17
Geraerts, Martine, Inv 12
Gerdes, Sebastian, P 51, P 62, P 64
Gildehaus, Franz Josef, Or 14, P 47
Giordano, Frank A, P 67, P 68
Giri, Jyotsnendu, P 15
Giroglou, Tsanan, Or 11
Glauche, Ingmar, P 51, P 62
Glimm, Hanno, Or 7, Or 17, Or 20, P 37, P 68
Globisch, Franziska, P 28, P 36
Göke, Burkhard, Or 13, Or 14, P 47
Gollisch, Tim, Or 6
Gottlieb, Elena, P 36
Greentree, S, Inv 15
Grez, Manuel, Inv 16, Or 20
Grünwald, Geoffrey K, Or 13, P 47
Guhl, Eva, P 52, P 52
Gustafson, Derek, P 68
Gutzmer, Ralf, Inv 21
Habib, Nagy, Inv 23
Hagedorn, Claudia, P 60
Hallek, Michael, Or 2
Händel, Eva M, P 17
Hanschmann, Kay-Martin O, P 42
Hansmann, Martin-Leo, P 57, P 58, P 64
Harbottle, Richard, P 23, P 24
Hardwick, Nicola, Inv 23
Harms, Nina, Or 3, P 35
Hartmann, Marianne, P 31
Hartmann, Sylvia, P 57, P 58, P 64
Hartog, Christoph, P 25
Hatzopoulos, Antonis K, P 27
Hausl, Martin, P 10, P 20, P 21, P 35, P 61
Heckl, Dirk, Or 10
Heilbronn, Regine, P 11, P 17, P 52, P 52
Heinrich, Tim, P 57, P 58, P 62, P 64
Heinz, Niels, Or 19
Hensel, Karin, P 8
Herbst, Friederike, P 37
Hermann, Felix G, P 31
Hinkel, Rabea, Inv 3, Or 8, P 27, P 28, P 36
Hirschberger, Johannes, P 55
Hoeffer, Klemens, Inv 13
Holder, GE, Inv 14
Holm, Per Sonne, Or 13, P 13, P 46, P 49, P 53
Hopfner, Ursula, P 34
Horstkotte, Jan C, P 27
Howe, Steven, P 23, P 24
Huber, Gesine, Or 6
Ingram, Wendy, Inv 23
Itaka, Keiji, Or 4
Ivics, Zoltán, Inv 12, P 35
Izsvák, Zsuzsanna, Inv 12, P 35
Janicki, Hanna, P 32
John, Katja, P 43
Jones, Peter, Or 9
Jun, Tye Gee, Inv 23
Kahle, Joerg, P 31
Kampik, Daniel, P 30
Kane, NM, P 38
Kasper, Julia C, P 1
Kataoka, Kazunori, Or 4
Kathöfer, Astrid, P 25
Katus, Hugo A, P 29
Kay, Mark A, Inv 8
Khan, Kafaitullah, P 17, P 52, P 52
Kimpel, Janine, P 31, P 32
Kirn, David H, Inv 18
Klein, Christoph, Or 7
Klibanov, Alexander L, P 8
Kloz, Ulrich, P 37
Klump, Hannes, Or 15
Klutz, Kathrin, Or 13, Or 14, P 47
Kneiske, Inna, Or 11
Knoop, Kerstin, Or 14
Koch, Christian, P 15, P 33, P 34
Koch, Susanne, Or 6
Kochanek, Stefan, Or 1, P 59
Koenig, Frauke M, Or 5
Kolk, Andreas, P 33
Kolokythas, Marie, Or 14
Kondratenko, Irina, Or 7
Kontermann, Roland, P 39
Köstlin, Roberto, P 55
Kostova, Youlia, P 46
Kreppel, Florian, Or 1, P 13
Krishnamoorthy, Vidhyasankar, Or 6
Kritz, AB, P 38
Kuehle, Johannes, P 12
Kühlcke, Klaus, Or 7
Kühle, Johannes, Or 15
Kuhs, Sandra, Inv 21, P 26
Kupatt, Christian, Inv 3, Or 8, P 27, P 28, P 36
Larkin, Daniel F.P., P 30
Lauer, Ullrich, P 42
Laufs, Stephanie, P 67, P 68
Lebherz, Corinna, Or 8, P 27, P 28, P 36
Leisegang, Matthias, Or 16, P 54, P 56
Li, Wenzhong, P 43
Linnemann, Carsten, Or 17
Lipps, Hans J., Or 9, P 60
Loew, Rainer, Or 19
Lucas, Tanja, P 59
Luhmann, Ulrich FO, P 30
Lulay, Christina, Or 7
Lütge, Fabienne, P 52, P 52
Lyko, Frank, P 37
Machens, Hans G, P 25, P 34
Magnusson, Terese, P 4
Mailänder, Peter, P 25
Maitland, Norman J, Inv 20
Mantwill, Klaus, P 46, P 49
Maródi, Laszló, Or 7
Marozin, Sabrina, P 44
Mátés, Lajos, Inv 12, P 35
Mátrai, Janka, Inv 12
Mattar, Citra, Inv 13
Mätzig, Tobias, Or 15
Mayr, Juliane, P 2
McDonald, Robert A, P 38, P 69
McVey, JH, Or 21
Merten, Otto-Wilhelm, Inv 24
Meyer, Johann, Or 10
Meyerhuber, Peter, P 56
Michalakis, Stylianos, Or 6
Milosevic, Slavoljub, Or 16
Miranda, Paula, P 65
Modlich, Ute, Or 10
Mohr, Andrea, P 48
Momma, Stefan, Or 11
Montazami-Astaneh, Kaweh, P 11
Moore, AT, Inv 14
Muehlebach, Michael D, P 42
Mufti, Ghulam, Inv 23
Mühlfriedel, Regine, Or 6
Muik, Alexander, Or 11
Müller, Oliver J, P 29
Müller-Lerch, Felix, P 17
Müther, Nadine, P 35
Mykhaylyk, Olga M, P 8, P 15, P 53
Naundorf, Sonja, Or 7
Nelson, Peter J, Or 14
Nettelbeck, Dirk M, P 16, P 39
Newrzela, Sebastian, P 31, P 57, P 58, P 62, P 64
Nicklin, Stuart A, Or 21, P 69
Noble, Alistair, Inv 23
Noske, Nadja, Or 3
Nowrouzi, Ali, Or 7, P 65
Nößner, Elfriede, P 54
Oppenheim, David, Inv 23
Osiak, Anna, P 52
Ott, Michael, P 26
Paquet-Durand, François, Or 6
Parker, A.L., Or 21
Paruzynski, Anna, Or 7
Perabo, Luca, Or 2
Petkova, Mina, P 64
Petry, Harald, Inv 15
Pfosser, Achim, Or 8, P 28, P 36
Philipp, Alexander, P 6, P 7
Plank, Christian, P 8, P 15, P 33, P 34, P 53, P 55
Pradhan, Pallab, P 8, P 15
Preuß, Ellen, P 66
Pützer, Brigitte M, P 43
Radecke, Sarah, P 52, P 63
Rahim, Ahad A, Inv 13
Rahman, Shamim H, P 11
Rauschhuber, Christina, Inv 1, P 9, P 10, P 20
Reckhenrich, Ann K, P 34
Reidy, Mairead, P 48
Rengstl, Benjamin, P 57, P 58
Robins, Adrian, Inv 23
Rödl, Wolfgang, P 13, P 47
Roeder, Ingo, P 51, P 62, P 64
Rosenecker, Josef, Inv 7
Rubin, GS, Inv 14
Rudolph, Carsten, Inv 4
Ruggiero, Eliana, Or 17
Rupprecht, Sina, P 48, P 60
Ruthardt, Nadia, Or 5, P 3
Rutz, Miriam, P 55
Ruzsics, Zsolt, P 20
Ruβ, Verena, P 6
Salguero, Gustavo, P 26
Sallach, Jessica, Or 2
Samara-Kuko, Ermira, Inv 12
Sampaolesi, Maurillio, Inv 12
Sanchez-Antequera, Yolanda, P 53
Sancho-Bru, Pau, Inv 12
Sawyer, Greta, Or 9
Schaffert, David, P 1, P 47
Schambach, Axel, Or 10, Or 15, Or 20, P 12
Schaser, Thomas, P 42
Schendel, Dolores J, Or 16, P 54
Scheu, Christina, P 13
Schiedlmeier, Bernhard, Or 19
Schillinger, Ulrike, P 55
Schinkel, Stefanie, P 29
Schleef, Martin, P 18, P 19
Schmeer, Marco, P 18, P 19
Schmidt, Manfred, Or 7, Or 17, Or 20, P 37, P 65, P 67, P 68
Schmitz, Georg, P 8
Schöler, Hans, Or 15
Scholz, Simone J, Or 20
Schroeder, Timm, Or 15
Schumacher, Ton NM, Or 17
Schwarz, Klaus, P 52, P 52, P 63
Seeliger, Mathias W, Or 6
Senekowitsch-Schmidtke, Reingard, Or 13, P 47
Sessa, WC, P 38
Seymour, Len W, Inv 6, Inv 9
Seznec, Janina, P 49
Slobodianski, Alex, P 25
Smith, Alexander J, Inv 14, P 30
Spitzweg, Christine, Inv 5, Or 13, Or 14, P 13, P 47
Spranger, Stefani, Or 16
Stachel, Georg, Or 8, P 36
Stärck, Lilian, P 56
Stein, Stefan, Or 20
Steingoetter, Andreas, P 15
Stiess, Michael, P 14
Stockman, A, Inv 14
Stripecke, Renata, Inv 21, P 26
Stroes, E, Inv 15
Su, Baowei, P 40
Su, Haibin, Or 9
Sultana, Shahana, Or 8, P 36
Sundarasetty, Bala Sai, P 26
Thalhammer, Stefan, P 53
Thielecke, Lars, P 51
Thormann, Michael, P 28
Thrasher, AJ, P 38
Tolmachov, Oleg, P 23
Trenkwalder, Teresa, Or 8, P 36
Tuorto, Francesca, P 37
Turan, Sören, P 12
Turqueti-Neves, Adriana, P 54
Twisk, J, Inv 15
van der Hoeven, Franciscus, P 37
van Rooijen, Nico, P 69
VandenDriessche, Thierry, Inv 12
Verfaillie, Catherine, Inv 12
Vermeesch, Joris, Inv 12
Vetter, Alexandra, P 13
Vigh, Balasz, P 33
Virdi, Kulpreet Singh, P 13
Vlasak, Reinhard, P 2
Vlaskou, Dialechti, P 8
Vogt, Stephan, P 50
Voigtlander, Richard, P 20, P 21
Volk, Andreas, P 31, P 32
Volz, Christian, P 29
von Kalle, Christof, Or 7, Or 17, Or 20, P 37, P 65, P 67, P 68
von Laer, Dorothee, Or 11, P 31, P 32, P 57, P 58, P 62, P 64
Wagner, Ernst, Or 1, Or 5, Or 14, Or 18, P 1, P 3, P 4, P 5, P 6, P 7, P 13, P 40, P 41, P 47
Walsch, Florian, P 55
Wang, Wei, P 37
Wang, Weiwei, P 43
Warlich, Eva, Or 15
Weber, Christian, P 65
Weber, Kristoffer, P 66
Wedemeyer, Heiner, P 26
Weingarten, Lisa, P 66
Wells, James, Inv 23
Wester, Hans-Jürgen, P 46
Wexel, Gabriele, P 50
Wicke, Daniel, Or 10
Wilde, Susanne, Or 16
Willhauck, Michael J, Or 13, Or 14, P 47
Wolf, Anja, P 46
Wolf, Nicola M, P 35
Wong, Andrew MS, Inv 13
Wong, Suet-Ping, P 23, P 24
Wood, Matthew, Inv 11
Wortmann, Andreas, P 59
Wübbenhorst, Daniela, P 50
Wuchrer, Alexander, P 27, P 28
Wunderlich, Nathalie, Or 13, Or 14, P 47
Yin, Haifang, Inv 11
Yu, Rui, P 48
Yzer, S, Inv 14
Zhang, Wenli, P 35, P 61
Zhang, Xiahong, Or 9
Zhang, Ziyang, P 25
Zimmer, Gert, Or 11
Zimmermann, Martina, P 42
Zong, Xiangang, Or 6
Zwacka, Ralf, P 48
Footnotes
*
Presentors are underlined for all abstracts.
*
Invited abstracts are found on pages 1170–1178, Oral abstracts on Pages 1179–1187, and Poster abstracts on Pages 1189–1217.