Abstract
Department of Infection and Immunity, University of Sheffield , Sheffield, UK
T-Cell Engineering for Cancer Therapy Applications
Adoptive Immunotherapy of cancer has shown remarkable therapeutic efficacy. The difficulty in selecting and expanding T-cells specific for a broad range of cancer antigens has initially limited this approach to a small number of primarily virally driven cancers. Through the application of gene-therapy methods to facilitate T-cell receptor transfer, it is now possible to generate large numbers of T-cells specific for almost any antigen. For instance, transduction of T-cells with Chimeric Antigen Receptors (CARs) allows us to re-direct T-cells to recognize practically any surface antigen on cancer cells in an MHC un-restricted fashion. This approach has met with some early clinical success: T-cells expressing disialoganglioside recognizing CARs can result in clinical responses in patients with refractory neuroblastoma. We have further refined these receptors by identifying the optimal format for surface expression and robust antigen recognition, replacing murine antigen-recognizing antibody single chains with humanized version and introducing compound signalling domains which transmit not only activation but survival and proliferation signals. Genetic engineering of T-cells has opened up other avenues of modification of T-cell behaviour apart from re-directing antigen specificity: For example, we can introduce suicide genes which result in selective destruction of T-cells in the face of unacceptable toxicity upon administration of an activating drug. We have optimized co-expression of suicide genes with CARs. Further, we have engineered T-cells to become resistant to immunosuppressive agents, to release payloads and to home to sites of disease. Increasingly complex engineering of T-cells with interconnected components will allow us to develop complete new therapeutics.
uPAR Targeted Nanoparticles
Decoration of nanoparticle surfaces with receptor-specific ligands can improve functional drug delivery to cancer cells. Our ligand of interest is a short peptide, U11, sequenced to show high affinity for the urokinase Plasminogen Activator Receptor (uPAR), a receptor over-expressed on the surface of many tumors. In this work we investigate the presentation of the peptide ligand, along with the nature of the nanoparticle platform and we optimize nanoparticles for optimum targeting efficiency and ability to deliver the nucleic acids. U11 peptides were coupled onto PEGylated liposomal nanoparticle surfaces by covalent conjugation. In vitro pDNA transfection and cell uptake studies established the viability of the U11 peptide as a ligand for cancer cell targeting. Spectroscopy methods were used to examine conformations of the coupled peptides. siRNA encapsulation assays, and in vitro gene silencing experiments were used to optimise the targeted nanoparticle for in vivo delivery. In vivo siRNA experiments were performed on mice carrying uPAR-positive and uPAR-negative xenografts stably transfected to express luciferase. Biodistribution was examined by administrating microRNA-encapsulated nanoparticles. U11-targeted nanoparticles were able to enhance delivery of pDNA and siRNA to uPAR-positive cells. Nanoparticles of lower charge reduced non-specific electrostatic interactions with cell membranes, hence allowed enhanced exhibition of the ligand's targeting effect. In vivo, U11-nanoparticles showed enhanced siRNA silencing to animals bearing uPAR-positive tumours. 24-hours post-administration, accumulation of targeted nanoparticles into the liver and the spleen was higher compared to non-targeted nanoparticles.
Non-Viral Strategies for the Episomal Modification of Cells
Gene therapy vectors based on modified viruses are unquestionably the most effective gene delivery systems in use today. Their efficacy at gene transfer is however tempered by their potential toxicity. An ideal vector for human gene therapy must deliver sustainable therapeutic levels of gene expression without compromising the viability of the host (at either the cellular or somatic level) in any way. Permanently maintained extra-chromosomal gene expression vectors, which comprise entirely human elements, provide the most suitable method of achieving this. Non-viral vectors are attractive alternatives to viral gene delivery systems because of their low toxicity, relatively easy production and great versatility. However, their efficiency is still below the requirements for realistic in vivo gene therapy. Fundamental to this inadequacy is deficient delivery exacerbated by the merely transient gene expression of plasmid DNA in vivo. We have produced a DNA vector system, which addresses all of these issues providing persistent transgene expression without vector toxicity. The functional element on this plasmid is the scaffold/matrix attachment region (S-MAR) from the human β-interferon gene cluster. The utility that this element provides to plasmid molecules is threefold: (1) plasmids harbouring an S-MAR motif are rendered resistant to integration; (2) their expression cassettes are not subject to epigenetic silencing; (3) they exhibit extrachromosomal, mitotic stability. These properties are conferred without virally encoded proteins or selective pressure. We will present here the utility of this vector system for the episomal modification of cells ex vivo and discuss their therapeutic application in vivo.
Amniotic Fluid Stem Cells For Therapy: Where Do We Stand?
Human amniotic fluid cells have been used as a diagnostic tool for the prenatal diagnosis of fetal genetic anomalies for more than 50 years. Evidence provided in the last few years, however, suggests that they can also harbour a therapeutic potential for human diseases, as different populations of fetal-derived stem cells have been isolated from amniotic fluid. Mesenchymal stem cells were the first to be described, which possess the higher proliferation and differentiation plasticity of adult mesenchymal stem cells and are able to differentiate towards mesodermal lineages. Amniotic fluid stem (AFS) cells have also more recently been isolated from humans and rodents. They are characterized by the expression of the surface antigen c-kit (CD117), the type III tyrosine kinase receptor of the stem cell factor. 1 AFS cells represent a novel class of broadly multipotent stem cells with intermediate characteristics between embryonic and adult stem cells, as they are able to differentiate into lineages representative of all three germ layers but do not form tumours when injected in vivo.1 Finally, c-Kit(+)Lin(−) cells derived from amniotic fluid displayed a multilineage hematopoietic potential. 2 These characteristics, together with the absence of ethical issues concerning their employment, suggest that stem cells present in the amniotic fluid might be promising candidates for gene and stem cell therapy of several human disorders.
Derivation and Utility of Cardiomyocytes from Human Pluripotent Stem Cells
We have demonstrated that functional cardiomyocytes can be derived from human embryonic stem cells, potentially offering a novel cell source for drug screening, disease modelling and cell replacement. However, before these goals can be realised, several issues must be tackled. We have sought to standardise feeder-free culture methods that function in 14 hESC lines derived in 5 different countries, impacting on the ability to improve downstream technologies. Thus, we have demonstrated industrial scale automation of hESC culture to meet demands of commerce. Standardised culture also provides a platform from which differentiation to the cardiac lineage can be improved and directed. Moreover, high efficiency genetic modification has been demonstrated in 11 hESC lines, potentially providing new routes to RNAi library screening for genome analysis. We have also generated transgenic hESC lines that express puromycin N-acetyltransferase from the cardiac specific MYH6 promoter, allowing enrichment of cardiomyocytes to close to 100% purity by incubation with the antibiotic puromycin. This set of technologies is now being applied to proof-of-principle studies in drug screening and engineering in vitro disease models produced either by genetic modification or by exploitation of induced pluripotency (iPS) technology.
Lentiviral Gene Therapy for X-linked Adrenoleukodystrophy
X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disease of the central nervous system (1:17.000). The X-ALD gene is named ABCD1 and codes for a peroxisomal half-ABC-transporter, the ALD protein or ALDP, which is involved in the transport of very-long-chain fatty acids across the peroxisomal membrane. The disease is characterized by the accumulation of very-long-chain fatty acids (VLCFAs) in all organs and plasma, due to the impaired oxidation of these fatty acids in peroxisomes. X-ALD includes two distinct phenotypes. Childhood cerebral ALD (CCALD) is characterized by progressive cerebral demyelination with, in a second stage, a strong inflammatory response in myelin that results clinically in rapid neurologic degradation, then death often before adolescence. Adrenomyeloneuropathy (AMN) affects adults and is characterized by a pure myelopathy and peripheral neuropathy. However about 35% of AMN patients develop cerebral demyelination, with the same poor prognosis as children with CCALD. Cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. More than 200 patients have received allogeneic HCT with positive results. However any complication of the procedure that delays hematopoietic reconstitution (like graft vs host disease) impairs further neurological outcome.
We have thus decided to evaluate a gene therapy strategy based on the correction of autologous hematopoietic stem cells (CD34 + cells) using a lentiviral vector followed by reinfusion of corrected cells. Mobilized peripheral blood CD34 + cells were transduced ex vivo with a non-replicative HIV1-derived lentiviral vector expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter. Transduced cells were frozen to perform replication competent lentivirus (RCL) assays and other safety tests. After thawing, transduced CD34 + cells were infused to ALD patients after full myeloablation with cyclophosphamide and busulfan. Tests assessing vector-derived RCL and vector mobilization were negative up to the last follow-ups in the 3 patients. Integration of the vector was polyclonal as suggested by extensive integration profile analysis and high throughput sequencing of integration sites. Stable expression was documented in circulating leucocytes up to 36 months in P1 and 30 months in P2, 16 months in P3. HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that ex vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization and allows efficient reconstitution with transduced cells. There is no evidence of clonal expansion of the transduced ALD cells. These data support the use of this gene therapy strategy. Longer follow-up is needed to confirm tolerance and evaluate the clinical benefits in ALD patients.
Development of Novel Therapies in Murine Models for Gaucher Disease
Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder; caused by mutations in the glucosidase, beta, acid (GBA) gene that encodes the lysosomal enzyme glucosylceramidase (GCase). GCase deficiency leads to characteristic visceral pathology and in some patients, lethal neurological manifestations. Previous mouse models with GCase deficiency have either been lethal in the perinatal period or viable without displaying clinical features of GD. We have generated viable mice with characteristic clinical symptoms of type 1 GD (hepatosplenomegaly, anemia, etc.), by conditionally deleting GCase exons 9-11 upon induction postnatally. Both transplantation of wild type (wt) bone marrow (BM) and gene therapy through retroviral transduction of BM from GD mice prevented development of disease as well as corrected an already established GD phenotype. The gene therapy approach generated considerably higher GCase activity than transplantation of wt BM. Strikingly, both therapeutic modalities normalized glucosylceramide levels and practically no infiltration of Gaucher cells could be observed in BM, spleen and liver demonstrating correction at 5–6 months following treatment. The findings demonstrate for the first time the feasibility of gene therapy for type 1 GD in vivo.
Using similar approaches, we have generated mouse models for severe neuronopathic Gaucher disease. To circumvent the lethal skin phenotype observed in several of the previous GCase-deficient animals, we genetically engineered a mouse model with strong reduction in GCase activity in all tissues except the skin. These mice exhibit rapid motor dysfunction associated with severe neurodegeneration and apoptotic cell death within the brain, reminiscent of neuronopathic GD. In addition, we have created a distinct mouse model, in which GCase deficiency is restricted to neural- and glial cell progenitors and progeny. These mice develop similar pathology as the first mouse model, but with a delayed onset and slower disease progression, indicating that GCase deficiency within microglial cells which are of hematopoietic origin is not the primary determinant of the CNS pathology. These findings strongly suggest that normal microglial cells cannot rescue this serious neurodegenerative disease. These mouse models can be used to investigate pathological mechanisms and develop novel therapies for all types of Gaucher Disease. Recently, we have used non-myeloablative conditioning of type 1 Gaucher mice and transplanted them with normal bone marrow cells. The findings demonstrate that 10% or even less engraftment is enough to correct the pathology of the disease. Collectively, these findings indicate that gene therapy of type1 Gaucher disease is likely to be successful and lead to a cure of the disease.
Hematopoietic Stem Cell Gene Therapy for the Treatment of Lysosomal Disorders with Central Nervous System Involvement
Hematopoietic stem cell (HSC) transplantation from healthy donors has been employed in Lysosomal Storage Disorders (LSD) for delivering a functional hydrolase to the affected tissues. However, likely due to the slow kinetics of infiltration of the nervous system, the efficacy of the transplant is poor in LSD with severe neurologic manifestations. HSC gene therapy could ameliorate the outcome of allogeneic transplant in these LSD. Indeed, HSC can be genetically modified to express supra-normal levels of the therapeutic enzyme and become a more effective source of enzyme than normal donor's cells also within the nervous system. Moreover, the use of autologous HSC is associated with a significantly reduced transplant-related morbidity and mortality and these cells are immediately available, thus saving precious time in rapidly progressing forms. We are implementing a gene therapy approach based on the transplantation of gene corrected HSC for the treatment of LSD lacking alternative therapeutic opportunities. To this goal, we exploit the unique features of lentiviral vectors (LV), which transduce at high efficiency HSC with minimal manipulation and significantly alleviate the safety concerns associated with gamma-retroviral vectors integration into the genome. By using LV for HSC gene correction, we proved the therapeutic potential of HSC gene therapy in the murine model of metachromatic leukodystrophy (MLD), a severe dysmyelinating LSD and, according to these results, HSC gene therapy for MLD has now reached clinical testing. The approach has also been applied with success also to the murine models of type 1 Mucopolysaccharidosis and, employing a HSC-specific microRNA for transgene expression regulation, of globoid leukodystrophy.
Gene Therapy (Glybera®) for Lipoprotein Lipase Deficiency
Lipoprotein lipase deficiency (LPLD) is a metabolic disorder that is caused by inactivating mutations in the gene encoding for lipoprotein lipase (LPL). Homozygous LPLD results in recurrent pancreatitis, glucose intolerance, and accumulation of triglycerides in skin, liver and spleen. Because triglyceride-lowering drug therapy is not effective and enzyme replacement is not feasible, current management of LPLD is limited to restriction of dietary lipid intake. However, patients that adhere to a very low-fat diet remain at risk for pancreatitis. AMT-010 and AMT-011 consist of a construct encoding a naturally occurring LPL variant that is packaged in an AAV-1 capsid. AMT-010 was produced in a mammalian cell (HEK293) system, whereas AMT-011 (Glybera®) is produced in a fully validated commercially scalable and GMP compliant baculovirus/SF+ (insect cell) system. AMT has conducted two long-term observational studies and three therapeutic intervention studies in patients with homozygous or compound heterozygous LPLD. Therapy was well tolerated and safe. At the therapeutic dose (1E12 gc/kg), administered as a one-time series of intramuscular injections) Glybera® resulted in a significant reduction of the fasting triglyceride concentration and a clinically important long-term reduction of the incidence of pancreatitis. In December of 2009 AMT has filed Glybera® at the EMA for market authorization.
A Hybrid Bacteriophage-Based Vector for Targeted Systemic Gene Delivery to Tumour Vasculature
The major hurdle of gene therapy has been the inability to deliver vectors at high enough efficiency via a systemic route to the target tissue. Unquestionably, eukaryotic viruses have been the vectors of choice for gene delivery to mammalian cells; but ligand-directed targeting of such vectors requires ablation of their native tropism for mammalian cells. In contrast, bacteriophages (phage) are prokaryotic viruses that infect bacteria but naturally have no tropism for mammalian cells. Importantly, if bacteriophages are engineered to display a targeting ligand on the capsid, they can deliver genes to mammalian cells. However, phage-based vectors have inherently been considered poor vectors for mammalian cells. We hypothesized that combining the favorable biological attributes of eukaryotic and prokaryotic viruses may yield chimeric particles with potential as targeted gene delivery vectors. We have reported a new generation of targeted systemic hybrid prokaryotic-eukaryotic vectors as chimeras between an adeno-associated virus (AAV) and targeted bacteriophage (termed AAV/phage; AAVP). Within this hybrid vector, a targeted bacteriophage capsid serves as a shuttle to deliver the AAV transgene DNA cassette, incorporated within the bacteriophage genome. We assessed the in vivo efficacy of vector in animal models of cancer by displaying on the phage capsid the cyclic Arg-Gly-Asp (RGD-4C) ligand that binds to av integrin receptors. These integrins are highly expressed on the abnormal angiogenic blood vessels of tumours but are absent or barely detectable in mature normal vasculature. After intravenous administration, the ligand-directed AAV-phage vector specifically and efficiently delivered imaging and therapeutic transgenes to the blood vessels of tumours in mice and rats, while sparing the normal organs. A recent study carried out under the direction of the National Cancer Institute of the USA elegantly demonstrates the potential of this vector. Targeted AAV-phage was used to deliver a cytokine, tumour necrosis factor-α (TNFα) to naturally occurring cancers diagnosed in dogs. In this study, single and repeated dosing using targeted AAV-phage-TNFα proved safe in treated animals. Remarkably, repeated therapy resulted in complete tumour eradication in a few dogs with aggressive cancers such as soft tissue sarcoma.
Novel Vectors and Antigens for a Next Generation HIV-1 Vaccine
Rare serotype Ad vectors such as rAd26 and rAd35 are biologically substantially different than rAd5 vectors. We have evaluated rAd26 and rAd35 vectors expressing SIV antigens in immunogenicity and challenge studies in rhesus monkeys, and we have recently advanced a prototype rAd26 vector expressing HIV-1 Env into a phase 1 clinical trial. Importantly, this vector has proven safe and immunogenic in humans at doses of 109 vp, 1010 vp, and 1011 vp. We have also assessed the capacity of vector-specific CD4+ T lymphocytes to traffic to mucosal surfaces following rAd vaccination in rhesus monkeys, and we have observed that trafficking of vector-specific CD4+ T lymphocytes to colorectal mucosa does not occur more readily in monkeys with baseline vector immunity as compared with monkeys without baseline vector immunity. In addition, we have demonstrated that computationally optimized “mosaic” HIV-1 Gag/Pol/Env antigens substantially expand cellular immune breadth and depth as compared with consensus or natural sequence antigens in rhesus monkeys. Taken together, these data suggest that a rAd35/rAd26 prime-boost vector regimen expressing mosaic HIV-1 antigens should be evaluated in clinical studies.
Hopital Necker Enfants Malades, Université Paris Descartes , Inserm U781, Paris, France , 75015
Gene therapy treatments using retroviral and lentiviral vectors have revealed the genotoxicity associated with random insertion of vector DNA into the genome. Our goal is to design lentiviral vectors with which a therapeutic sequence is inserted at a universal safe locus in the human genome, using homologous recombination (HR). Lentiviral-mediated HR occurs at high frequency when a DNA recombination matrix is delivered to cells along with a site specific endonuclease that creates a locus-specific double strand break. Heterodimeric Zinc finger Nucleases as well as single chain meganucleases can be used for this purpose. However, for therapeutic applications, it is preferable to have an appropriate control of the amount of endonuclease in the target cells, and especially, to avoid sustained expression. One possibility is to vectorize the protein itself, instead of an expression cassette. We have shown that highly efficient HR is obtained when the nuclease is delivered as a protein incorporated in the lentiviral particles. I-SceI, a prototypic meganuclease from yeast, was incorporated into the virions as a fusion with Vpr, an HIV accessory protein. Non-integrating lentiviral vectors (NILV) containing a recombination matrix and the I-SceI fusion protein were produced at high titers. We have used a CHO-derived reporter cell line in which I-SceI mediated HR events result in the repair of a puromycin resistance gene. These cells were transduced by NILVs containing both I-SceI as a Vpr fusion and the recombination matrix, or by separate vectors encoding either the recombination matrix or I-SceI. Puromycin resistant cell clones were selected and analysed by PCR and Southern blot for targeting events. A majority of puromycin resistant clones contained the expected genomic structure and globally, our data indicate that the transfer of the endonuclease as a virion incorporated protein results in up to 5% of HR events, in a dose dependent manner. HR levels were lower when the fusion protein and the recombination matrix were provided in separate viral particles, suggesting that the physical association of the two in the same lentiviral pre-integration complex may be important for efficient targeting. Interestingly, the levels of HR obtained when I-SceI was encoded by the lentiviral vector were consistently lower.
Targeted Gene Addition by Adeno-Associated Virus
Adeno-associated viruses (AAV) are widely spread throughout the human population, yet no pathology has been associated with infection. However, our understanding of the intriguing features of this virus is far from exhausted and it is likely that the mechanisms underlying the viral life style will reveal possible novel strategies that can be employed in future gene transfer approaches. One such aspect is the unique mechanism AAV has evolved in order to establish latency. In the absence of a cellular milieu that will support productive viral replication, wild type AAV can integrate its genome site specifically into a locus on human chromosome 19 (termed AAVS1) where it resides without apparent effects on the host cell. Here we will introduce this unique viral strategy place relevant questions into the context of attempts to establish therapeutic gene addition approaches. Specifically, we will present a molecular model for the integration mechanism; we will discuss the concept of functional characterization of gene addition to the human genome and highlight potential applications that might benefit from this viral strategy.
Human Embryonic Stem Cell-Derived RPE for the Treatment of Age-Related Macular Degeneration
The London Project to Cure Blindness was launched at the UCL Institute of Ophthalmology in June 2007, and aims to make the most of human embryonic stem cells to prevent blindness and restore sight in patients with Age-related Macular Degeneration (AMD) by 2012. Our goal is to replace cells essential for “seeing” lost by disease at the back of the eye. We aim to repair and regenerate the aged, diseased eye using human embryonic stem cells which have been transformed into the cells affected in AMD: the support cells for the photoreceptors (retinal pigment epithelium) and the photoreceptors. The cells will be surgically implanted into a clinical population of AMD patients.
Human Immune Responses in AAV-Mediated Gene Transfer: Implications for Safety and Efficacy
A clinical trial of AAV-2 expressing human Factor IX, infused into the hepatic artery in men with severe hemophilia B, resulted in short-term expression of Factor IX at therapeutic levels. A gradual decline in F.IX levels was accompanied by an asymptomatic transaminase elevation and expansion of a population of capsid-specific CD8 + T cells. These findings have not been seen in animal models. We sought to analyze the processing of AAV capsid in transduced hepatocytes. To track peptide-MHC complexes on the surface of transduced cells, we cloned an AAV capsid-specific T cell receptor. This reagent retained the specificity of a bona fide TCR. Using a labeled multimerized soluble TCR, and confocal microscopy with quantitative image analysis, we detected peptide-MHC complexes on the surface of transduced human hepatocytes. In subsequent experiments we demonstrated that the number of peptide-MHC complexes was sufficient to trigger T cell activation. Moreover, we showed that AAV transduction was sufficient to flag hepatocytes for destruction by HLA-matched capsid-specific CD8 + T cells. In subsequent studies, we sought to determine whether the proteasome inhibitor bortezomib could reduce presentation of capsid-derived peptide-MHC complexes on the surface of transduced cells. Using a T cell line transduced with a capsid-specific T cell receptor as the reporter, we showed that addition of gradually increasing levels of bortezomib reduced capsid-derived peptide-MHC complexes on the cell surface of AAV-transduced human hepatocytes to undetectable levels. This suggests that there may be a pharmacologic approach to reduction of capsid antigen presentation after AAV transduction.
Magnetic Targeting and Imaging of Stem Cells Using Nanoparticles
One of the current challenges in the biomedical sciences is the localization of stem cells to the sites of interest for the repair of tissue damage or the delivery of therapies. Cellular therapies are increasingly applied in clinical trials, and in recent years the utilization of hematopoietic progenitors as has been the focus of considerable attention. In the context of ischemic heart disease, these efforts have led to modest success. However, delivery to specific targets within the body generally remains a difficult task, in part due to low uptake at the site of injury. As such, cell therapies would greatly benefit from methodologies aimed at targeting and monitoring cell trafficking. Superparamagnetic iron oxide nanoparticles (SPIO) offer attractive possibilities in biomedicine as they can be incorporated into cells affording a controllable means of ‘tagging’. These particles lead to a marked decrease in the magnetic resonance imaging (MRI) parameter T2* and the possibility of visualizing their localization non-invasively on T2*-weighted MR images. Furthermore, the magnetic properties of SPIOs allow them to be manipulated mechanically by a magnetic field gradient. This ‘action at a distance’, combined with the intrinsic penetrability of magnetic fields into human tissue, opens up potential applications involving the transport of magnetically tagged biological entities.
Therapeutic Approaches for Dominantly Inherited Retinal Degenerations
Mutational heterogeneity represents a significant barrier to therapeutic development for many dominantly inherited diseases. For example, over 100 mutations in the rhodopsin (RHO) gene have been identified in patients with Retinitis Pigmentosa (RP). To overcome mutational heterogeneity while still correcting the primary genetic defect, therapeutics comprising two elements, gene suppression in conjunction with gene replacement, are being explored. Using this approach, suppression is targeted to a site independent of the mutation and hence both mutant and wild type alleles are suppressed. In parallel with suppression a codon-modified replacement gene refractory to suppression is provided. RNA interference (RNAi) has been utilised to achieve potent in vivo suppression (greater than 90%) of RHO in photoreceptors using adenoassociated virus (AAV) for delivery. Codon-modifed RHO replacement genes have been shown to express functional wild type protein in the presence of targeting RNAi molecules. Notably therapeutic benefit associated with AAV-delivered suppression and replacement therapies has been obtained in transgenic mouse models of rhodopsin-linked RP. Results indicate that suppression and replacement can provide a therapeutic solution for dominantly inherited disorders such as RHO-linked RP and can be employed to circumvent mutational heterogeneity.
Interrogating the Architecture of Cancer Genomes
Cancer is driven by mutation. Using massively parallel sequencing technology, we can now sequence the entire genome of cancer samples, allowing the generation of comprehensive catalogues of somatic mutations of all classes. Bespoke algorithms have been developed to identify somatically acquired point mutations, copy number changes and genomic rearrangements, which require extensive validation by confirmatory testing. The findings from our first handful of genomes illustrate the potential for next-generation sequencing to provide unprecedented insights into mutational processes, cellular repair pathways and gene networks associated with cancer development. I will also review possible applications of these technologies in a diagnostic and clinical setting, and the potential routes for translation.
(presenting)
PlasmidFactory GmbH & Co. KG , Bielefeld, Germany
For gene therapy and genetic vaccination it is crucial to develop safe and efficient vector systems to be transferred into the target cells. 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 contribute to silencing of episomal transgene expression. Hence, it seems obvious that the removal of bacterial backbone DNA can greatly improve the vector safety and efficiency 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. Different approaches have been described so far, from plasmids where the antibiotics resistance gene has been replaced by another marker to minicircle DNA consisting almost only of the gene of interest. 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. 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.
(presenting)
CIEMAT , Avenida Complutense 22, Madrid, Spain
Fanconi anemia (FA) is a heterogeneous recessive disease generated by mutations in any of the thirteen FANC genes. In contrast to the efficacy of gene therapy in patients with X1-SCID or ADA-SCID, no clinical benefits have been so far reported in FA. Indeed, the hematopoietic stem cell (HSC) defects observed in FA patients should account for these discrepancies. In order to develop improved gene therapy approaches for FA, self-inactivating lentiviral vectors have been developed. Additionally, new ex vivo manipulation conditions have been proposed for the genetic correction of FA HSCs, based on the short transduction of not purified bone marrow (BM) cells. Since the low number of HSCs in the BM FA patients may limit the efficacy of conventional protocols of gene therapy, we investigated whether combined procedures of lentiviral-mediated gene therapy and cell reprogramming allowed the generation of corrected hematopoietic progenitors from the skin of FA patients. ES-like colonies, fulfilling all the criteria required for iPS cells were generated from reprogrammed FA fibroblasts and keratinocytes. After in vitro differentiation in OP9 cells, iPS-derived CD34 + cells were generated, which produced erythroid and myeloid colonies in methylcellulose cultures. Significantly, these colonies were resistant to MMC and showed nuclear foci of FANCD2, indicating that these cells were disease-free. Our results open new perspectives for the therapy of FA and for other BM failure syndromes of genetic etiology.
(presenting)
Cellectis , 102 avenue Gaston Roussel, 93235 Romainville Cedex, France
Targeted approaches have emerged today as an alternative to current random insertion strategies for gene therapy. Such approaches include the targeted insertion of therapeutic transgenes into chosen loci (“safe harbour” strategy), the precise editing of deleterious mutation (gene correction), as well as various gene deletion and knock-out strategies. A variety of different tools has been envisioned, including integrases, recombinases, transposases and endonucleases. Meganucleases, the most specific natural endonucleases represent ideal tools for precise genome editing, or “genome surgery”. Natural as well as engineered can be used to induce up to 20% of gene insertion into chosen human genes. However, meganucleases can also be used in “knock out” or deletion approaches. Many chronic viral infections are due to double-stranded DNA viruses or viruses that involve a double-stranded DNA intermediate during their replicative cycle. Such DNA forms can be targeted and destroyed by meganucleases. For example, we have shown that natural and engineered meganucleases can efficiently prevent the infection of COS cells by a recombinant HSV1 virus. Eventually, the use of these proteins for therapeutic applications will depend on their intrinsic properties (activity/specificity) as well as on the use or identification of appropriate vectorization methods, and we will present an update of these two aspects.
(presenting)
Hannover Medical School , Germany
Zinc-finger nucleases (ZFNs) hold great promise to pave the way for targeted genome engineering strategies in human gene therapy. ZFNs consist of a zinc-finger based DNA-binding domain and a non-specific endonuclease domain. Upon binding to the target site, ZFNs dimerize and introduce a DNA double strand break (DSB). The activated DNA repair pathways can be harnessed subsequently either to disrupt a gene or to correct an inborn mutation. We have developed a ZFN-mediated gene targeting system based on vectors derived from adeno-associated virus (AAV) and show that transient cell cycle arrests increased AAV-mediated gene targeting up to 6-fold. Dependent on the cell line and the AAV vector dose, we reached gene-targeting frequencies of up to 34%. To expand the application range of ZFNs, we developed autonomously working ZFN pairs that allow us to direct two ZFN pairs simultaneously to two different target sites without cross-reactivity. To this end, we used a panel of in silico, in vitro, and in vivo assays to characterize the consequence of structure-based changes in the ZFN dimer interface. By assaying for activity and ZFN-associated toxicity, we identified variants that combine high cleavage activity with low toxicity, and found two variant ZFN pairs that did not cross-react with each other. Expression of two variant ZFN pairs designed to recognize two adjacent sites on an endogenous human locus demonstrated that it was possible to induce targeted chromosomal deletions in about 10% of transfected cells, validating the utility of autonomous ZFN variants in rational genome engineering.
(presenting)
The Royal Veterinary College , London NW1 0TU
The majority of mutations that lead to Duchenne muscular dystrophy, a lethal X-linked muscle wasting disorder, disrupt the open reading frame of the DMD gene. In contrast, the allelic condition Becker muscular dystrophy, that is generally a much milder disorder, involves mutations that retain the open reading frame allowing the production of an internally deleted but at least partially functional dystrophin protein. Antisense oligonucleotides can be used to sterically mask splicing protein binding sites and hence modify the splicing of the primary transcript. In most DMD cases removal of one or more exons will restore the open reading frame. Exon skipping has been demonstrated with animal and human cells in culture and in vivo in the mdx mouse and CXMD dog models of DMD. Recent work in our laboratory using the mdx mouse has demonstrated that resistance to force drop associated with lengthening (eccentric) contractions is proportional to the number of dystrophin positive fibres and this is a better predictor of functional improvement that changes in muscle force or quantity of dystrophin by western blot. Systemic delivery by intravenous or subcutaneous injection is relatively inefficient and at least for the phosphorodiamidate morpholino oligomers (PMO) this inefficiency appears to be due to poor passage across the capillary endothelium. A variety of strategies to improve efficiency have been developed. Two antisense chemistries for exon skipping, PMO and 2′-O-methyl phosphorothioate have been taken to clinical trial in DMD patients with both local and systemic delivery and the current status of the systemic trials will be discussed.
(presenting)
Department of Molecular Cell Biology, Leiden University Medical Center , PO Box 9600, 2300 RC Leiden, the Netherlands
Many new oncolytic viruses are created by reverse-genetics strategies. Although powerful, the progress with this approach comes relatively slow. Recent studies reported on directed evolution-type strategies for the improvement of viruses as oncolytic agents. With adenovirus (Ad) the evolutionary approaches entailed chemical mutagenesis of Ad genomes followed by selection for improved oncolytic potency. Here, we sought to explore the possibility of whether the mutagenesis step in such bioselection schemes could be executed by an error-prone polymerase. For this purpose, we set out to generate a panel of candidate mutator Ad polymerases, each member of which contained a single amino acid substitution at a position implicated in governing polymerization fidelity. Next, by way of employing a polymerase complementation system, a subset of these mutant polymerases was identified as being able to fully support replication of Ad. Those polymerases found capable of such were subsequently analyzed for their proneness to cause mutations during viral replication. With employment of high-throughput sequencing technology the mutation loads in multiple-clone virus pools were determined. Finally, a selection of promising mutant polymerases was used in a bioselection scheme involving multi-passage growth on tumour cells. Importantly, this procedure proved to yield Ad clones with increased tumour cell killing capacity. These findings thus validate the use of mutant Ad polymerases for bioselection purposes. In a first series of experiments new Ad mutants were bioselected with enhanced oncolytic activity in SKOV3 cells, formally demonstrating the feasibility of this approach.