Adeno-associated Vector Research Articles

Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial

Neovascular, or wet, age-related macular degeneration causes central vision loss and represents a major health problem in elderly people, and is currently treated with frequent intraocular injections of anti-VEGF protein. Gene therapy might enable long-term anti-VEGF therapy from a single treatment. We tested the safety of rAAV.sFLT-1 in treatment of wet age-related macular degeneration with a single subretinal injection. In this single-centre, phase 1, randomised controlled trial, we enrolled patients with wet age-related macular degeneration at the Lions Eye Institute and the Sir Charles Gairdner Hospital (Nedlands, WA, Australia). Eligible patients had to be aged 65 years or older, have age-related macular degeneration secondary to active subfoveal choroidal neovascularisation, with best corrected visual acuity (BCVA) of 3/60-6/24 and 6/60 or better in the other eye. Patients were randomly assigned (3:1) to receive either 1 × 10(10) vector genomes (vg; low-dose rAAV.sFLT-1 group) or 1 × 10(11) vg (high-dose rAAV.sFLT-1 group), or no gene-therapy treatment (control group). Randomisation was done by sequential group assignment. All patients and investigators were unmasked. Staff doing the assessments were masked to the study group at study visits. All patients received ranibizumab at baseline and week 4, and rescue treatment during follow-up based on prespecified criteria including BCVA measured on the Early Treatment Diabetic Retinopathy Study (EDTRS) scale, optical coherence tomography, and fluorescein angiography. The primary endpoint was ocular and systemic safety. This trial is registered with ClinicalTrials.gov, number NCT01494805. From Dec 16, 2011, to April 5, 2012, we enrolled nine patients of whom eight were randomly assigned to receive either intervention (three patients in the low-dose rAAV.sFLT-1 group and three patients in the high-dose rAAV.sFLT-1 group) or no treatment (two patients in the control group). Subretinal injection of rAAV.sFLT-1 was highly reproducible. No drug-related adverse events were noted; procedure-related adverse events (subconjunctival or subretinal haemorrhage and mild cell debris in the anterior vitreous) were generally mild and self-resolving. There was no evidence of chorioretinal atrophy. Clinical laboratory assessments generally remained unchanged from baseline. Four (67%) of six patients in the treatment group required zero rescue injections, and the other two (33%) required only one rescue injection each. rAAV.sFLT-1 was safe and well tolerated. These results support ocular gene therapy as a potential long-term treatment option for wet age-related macular degeneration. National Health and Medical Research Council of Australia, Richard Pearce Bequest, Lions Save Sight Foundation, Brian King Fellowship, and Avalanche Biotechnologies, Inc.

Gene therapy for age-related macular degeneration

Age-related macular degeneration is the most common cause of untreatable blindness in the world, and its prevalence is increasing. 1 Bourne RR Stevens GA White RA et al. Causes of vision loss worldwide, 1990–2010: a systematic analysis. Lancet Glob Health. 2013; 1: e339-e349 Summary Full Text Full Text PDF PubMed Scopus (1034) Google Scholar Current therapies for neovascular age-related macular degeneration aim to prevent growth of the abnormal retinal blood vessels that could leak and cause rapid visual loss in the later stages of the disease. This growth inhibition is achieved by regular injections into the eye of molecules that block the activity of vascular-endothelial growth factor (VEGF). 2 Schmidt-Erfurth U Chong V Loewenstein A et al. Guidelines for the management of neovascular age-related macular degeneration by the European Society of Retina Specialists (EURETINA). Br J Ophthalmol. 2014; 98: 1144-1167 Crossref PubMed Scopus (358) Google Scholar These drugs are expensive, and the requirement for regular and indefinite intraocular injections puts a substantial strain on health-care resources. In The Lancet, Elizabeth Rakoczy and colleagues 3 Rakoczy EP Lai C-M Magno AL et al. Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial. Lancet. 2015; (published online Sept 30.)http://dx.doi.org/10.1016/S0140-6736(15)00345-1 PubMed Google Scholar present 1 year results from a phase 1 clinical trial to assess a single treatment, with gene therapy, as an alternative to regular injections for achieving long-term VEGF blockade. 3 Rakoczy EP Lai C-M Magno AL et al. Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial. Lancet. 2015; (published online Sept 30.)http://dx.doi.org/10.1016/S0140-6736(15)00345-1 PubMed Google Scholar Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trialrAAV.sFLT-1 was safe and well tolerated. These results support ocular gene therapy as a potential long-term treatment option for wet age-related macular degeneration. Full-Text PDF

Long-term metabolic correction of Wilson’s disease in a murine model by gene therapy

Wilson's disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in the ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs and zinc salts, which may cause side effects and do not restore normal copper metabolism. In this work we assessed the efficacy of gene therapy to treat this condition. We transduced the liver of the Atp7b(-/-) WD mouse model with an adeno-associated vector serotype 8 (AAV8) encoding the human ATP7B cDNA placed under the control of the liver-specific α1-antitrypsin promoter (AAV8-AAT-ATP7B). After vector administration we carried out periodic evaluation of parameters associated with copper metabolism and disease progression. The animals were sacrificed 6months after treatment to analyze copper storage and hepatic histology. We observed a dose-dependent therapeutic effect of AAV8-AAT-ATP7B manifested by the reduction of serum transaminases and urinary copper excretion, normalization of serum holoceruloplasmin, and restoration of physiological biliary copper excretion in response to copper overload. The liver of treated animals showed normalization of copper content and absence of histological alterations. Our data demonstrate that AAV8-AAT-ATP7B-mediated gene therapy provides long-term correction of copper metabolism in a clinically relevant animal model of WD providing support for future translational studies.

Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions

Recombinant adeno-associated virus (rAAV) serotype 2, 3 and 8 vectors are the most promising liver-tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies (Nabs) were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as well as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential influence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2 > AAV3 = AAVLK03 > AAV8. There was no difference between: 1) AAV3 and its variants; 2) male and female subjects; and 3) different age cohorts (≤ 35, 36–50, and ≥ 51 years old). People in the Qi-deficiency constitution had significantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.

Emerging Concepts in Gene Therapy for Osteoarthritis.

Osteoarthritis (OA) is a disease of the total joint, affecting cartilage, bone, meniscus, synovium, and ligaments. It is estimated that 27 million Americans were affected with OA in 2008,1 that 35 million are currently affected, and that 67 million will be affected by 2030.2 Despite the high incidence, there is no treatment that significantly alters the course of disease. Unlike many other diseases, OA is well suited for local, intra-articular treatment because the disease affects a limited number of joints and lacks obvious extra-articular manifestations. Among several efforts made to treat OA, gene therapy has merit. Gene therapy is the intentional modulation and transfer of a therapeutic gene into the cells of injured tissue to achieve sustained and well-regulated synthesis of therapeutic protein at the site of injury. Gene transfer can be achieved through ex vivo or in vivo approaches. The goal is to augment the expression of a therapeutic gene or inhibit the expression of a disease-associated gene in cells. Application of ex vivo gene therapy merges well with the tissue-engineering approach. Ex vivo gene therapy requires removal of cells from the body and genetic manipulation of the cells in vitro before reimplantation. In contrast, in vivo gene therapy provides delivery of the therapeutic genes directly into the cells of the synovial joint. In both approaches, the transfer of DNA into the cells can be made either by viral or nonviral vectors.3 In the nonviral approach, gene transfer is achieved through several nonionic, physical (electromagnetic) and biochemical modes. Although they have no obvious risks for immunogenicity/mutagenesis, nonviral vectors are less efficient than viral vectors. This barrier can be overcome with the use of responsive (self-limiting) promotors that are stimulated only when joint homeostasis is perturbed and express the therapeutic gene only when needed.4,5 In addition, vectors with two promoters (ie, dual expression vectors) are able to harbor two genes and provide an opportunity for augmented therapeutic potential and eliminate the limitation of single-gene selection.6,7 For example, an anticatabolic and a proanabolic gene can be selected to ameliorate inflammation, improve matrix synthesis, inhibit apoptosis, enhance joint lubrication, and improve healing of microfractures and chondral defects. Viral vectors, which have had the viral genes removed, provide a renewable source of gene expression. Although these vectors have been used in gene therapy trials,8 they have potential limitations such as immunogenicity, insertional mutagenesis, persistence, and sustainability of transgene expression and tissue/cell specificity. Recently, adeno-associated vectors have gained popularity as a gene therapy vector secondary to the commercialization of alipogene tiparvovec, an adeno-associated vector-based product for management of familial lipoprotein lipase deficiency. The lessons learned from gene therapy in the cancer field further dictate how adenoviruses can be used effectively for management of OA. Several strategies have been used to overcome the problems related to adenovirus gene therapy. Adenovirus serotype 5 has several unique attributes that make it suitable for gene therapy. To circumvent the problem of cell and tissue specificity, refractory bispecific adapter molecules have allowed modification of adenovirus tropism for targeted gene transfer.9 The use of unconventional immunoglobulins derived from the serum of camels and alpacas provides compatibility with the cytosolic biosynthesis of adenovirus capsid proteins, thus allowing for target cell specificity and ultimately making the use of these immunoglobulins possible for adenovirus-mediated gene therapy for a specific tissue in the joint.10 Similarly, to circumvent the broad negative effects of preexisting immunity to common human serotypes of adenoviruses, researchers have developed vectors based on chimpanzee-derived adenoviruses for gene therapy.11 As far as the selection of a therapeutic gene is concerned, there is no single gene that can treat OA or delay its progression; however, nuclear factor-κ B is an initial candidate for inhibition.12 We believe that technologies, such RNA sequencing and microarray, can provide a comprehensive list of genes that are altered in disease and may qualify for therapy or provide therapeutic targets. Great potential exists for the application of gene therapy to treat diseases such as OA. However, attention must be paid to safety, stability, cost-effectiveness, and appropriate timing of therapy. Such key considerations will guarantee approval of gene therapy products by the regulatory authorities. We believe that if gene therapy can delay joint arthroplasty (currently the only available surgical option for end-stage OA) for at least a decade, this would exert a significant impact on patients’ quality of life.

Recombinant AAV-mediated in vivo long-term expression and antitumour activity of an anti-ganglioside GM3(Neu5Gc) antibody.

The ganglioside GM3(Neu5Gc) has gained increasing attention as therapeutic target because of its selective expression in various human tumours, such as melanoma, breast and lung cancer. 14F7 is a mouse IgG1 with specific reactivity to GM3(Neu5Gc)-positive tumours. The therapeutic activity of 14F7 has also been demonstrated in vivo, through its repetitive passive administration in tumour-bearing animals. In this work we used an alternative strategy to deliver recombinant 14F7 in vivo and analysed the therapeutic efficacy of this approach. We engineered a recombinant adeno-associated vector to direct the expression of secretable recombinant 14F7 in BALB/c animals. A single administration of the rAAV induced efficient production and secretion of the antibody in the bloodstream, with an expression level reaching plateau at ∼3 weeks after injection and persisting for almost a year. Strikingly, upon challenge with GM3(Neu5Gc)-positive X63-AG8.653 myeloma cells, tumour development was significantly delayed in animals treated with rAAV-14F7 with respect to animals treated with a control rAAV codifying for an irrelevant antibody. Finally, no significant differences in survival proportion were detected in animals injected with rAAV-14F7 or treated by standard administration of repetitive doses of purified monoclonal antibody 14F7.

39. Modulation of Histone Dosage and Chromatin Dynamics Regulates AAV Transduction

Recombinant adeno-associated vectors (AAVs) have gained momentum due to their combined characteristics of safety and efficiency. Nonetheless, the identity of the host functions intercepting the AAV transduction pathway still needs to be thoroughly understood.To address this issue on a genome-wide scale, we previously performed an unbiased RNAi high throughput screening (HTS; 18,120 human target genes) and identified 710 negative and 414 positive regulators of AAV efficiency. Based on the results obtained and with the purpose to identify factors involved in single-stranded (ss) AAV genome processing, we compared, again in an HTS format, siRNAs affecting ssAAV, but dispensable for scAAV transduction. One of the genes identified was ERI-1. While the effects of this protein were negligible on scAAV transduction, its knock down or overexpression lead to a 5 fold decrease or 2-7 fold increase of ssAAV efficiency, respectively.ERI-1 is a 3’-exoribonuclease known to degrade endogenous miRNAs and histone mRNAs. Indeed, we observed that AAV transduction negatively correlated with histone mRNA levels. Chromatin immunoprecipitation (ChIP) studies aimed at assessing the extent of chromatinization of the AAV genome revealed that the overexpression of ERI-1 determined an over 10-fold, selective reduction on ssAAV genome association with H3 and H4, while changes were negligible for scAAV DNA and for control cellular genes. Consistent with chromatin exerting a repressive role on ssAAV transduction, we also noticed that the downregulation of the main replication-dependent histone chaperone CAF-1 induced an over 20-fold increase in transduction. Increase of ssAAV2 transduction by ERI-1 also decreased the association, with the viral DNA, of proteins of the cellular DNA damage response (DDR; e.g. Nbs1 and Mre11), which our previous work had indicated as inhibitory of AAV transduction.Interestingly, DNA damage per se induced downregulation of histone gene expression. In particular, hydroxyurea, a drug markedly increasing AAV transduction, also determined histone mRNA degradation, an effect that required integrity of ERI-1.These results underline the importance of chromatin and its dynamic regulation in determining the fate of productive AAV transduction. These findings can be exploited for the development of more effective AAV-mediated gene delivery strategies.

443. A Comparison of Fetal Gene Therapy Outcomes in Early and Late Gestation

Fetal gene therapy (FGT) may eliminate clinical disease in monogeneic disorders by correcting aberrant protein production early in development. Early (eFGT) and late (lFGT) intervention suit different therapeutic aims. Critical assessment of long-term outcomes is required for clinical translation, which we have performed in a non-human primate (NHP) model using adeno-associated vector (AAV) expressing human Factor IX. Fetuses received 1-10E+12vg at 0.3-0.5G (eFGT) or 4E+12vg at 0.9G (lFGT). Long-term data is available up to 22 and 62 months for eFGT (n=6) and lFGT infants (n=6) respectively. AAV5 was used if the dam had pre-existing immunity to AAV8. Infants were monitored serially for protein levels and immune toxicity. To boost expression and test tolerance, subjects with protein levels <1% received 2E+11vg/kg of the same AAV serotype in a postnatal challenge.eFGT-AAV5 infants showed stable hFIX activity at 0-15%. AAV8 infants showed significantly higher activity at 10-37% in the first year, (median 15 vs 2%, p<10−3). Initial lFGT expression peaked in the first postnatal month and reached steady-state around day 60. Median expression was higher with AAV8, both during peak (246 vs 33%, p<10−4) and plateau phases (19 vs 8%, p<10−4). Steady-state expression was lower after eFGT with both AAV8 (16 vs 47%, p<10−4) and AAV5 (2 vs 12%, p<10−4). Males had higher expression regardless of serotype (24 vs 2%, p<10−3). eFGT infants showed a mild transient humoral response to AAV capsid while neutralizing antibody and T cell assays were negative. In contrast, lFGT resulted in more robust though transient AAV-IgG levels perinatally. In both groups AAV5 was the more immunogenic vector. Lowest hFIX levels corresponded to prolonged AAV-IgG response. Anti-hFIX IgG was negligible. Most infants maintained transgene expression despite the presence of binding antibodies.VCN in all organs was 1-3 log-folds lower in eFGT infants. Isolated gametes were transduced at a rate of 0.1vg/sperm cell (n=1/2 males) and 0.9vg/oocyte (n=1/3 females), only in lFGT infants. Brief transaminitis occurred without clinical toxicity. Postnatally-treated AAV5-eFGT infants (n=3) demonstrated brief hFIX increases to 3-10% over 60 days. Transient T cell activation occurred but neutralising antibodies were absent. AAV5-lFGT expression was raised to 1-2% without T cell activation. AAV8-lFGT showed no improvement after two postnatal doses; a robust cellular and humoral response was observed and maximum hFIX levels remained <1%. Both lFGT infants showed transient neutralising antibody expression. Outcomes of single-dose FGT thus depend on timing, dose, serotype, gender; immune reactions were not clinically damaging, and tolerance was observed. Germ-line transmission was observed with low frequency. FGT can be tailored to achieve the desired clinical outcomes in the target disease.

700. Stable Correction of Severe Metabolic Liver Disease Phenotypes in the Growing Murine Liver Using a Hybrid rAAV/piggyBac Transposon Gene Delivery System

Liver-targeted gene therapy using adeno-associated virus-based vectors (rAAV) shows therapeutic promise in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver where rapid clearance of rAAV episomal genomes occurs in concert with hepatocellular proliferation. This phenomenon has been shown to underpin progressive loss of therapeutic efficacy, initially achieved in the neonatal period, in mouse models of early-onset urea cycle defects (UCDs). We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an EGFP expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks post-treatment compared to traditional rAAV gene delivery. We then modeled the therapeutic potential of the system in the context of severe early onset UCDs. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase (OTC) deficient Spfash mouse and neonatal lethal argininosuccinate synthetase (ASS) knock-out mouse. The system was next utilized in a murine model of chronic liver disease, progressive familial intrahepatic cholestasis type 3 (PFIC3), in which intervention prior to disease onset is necessary due to the development of chronic liver pathology which impedes vector delivery and hepatocyte transduction. Stable persistent expression extending into adulthood was achieved with increased mean biliary phosphatidylcholine concentration and dramatically reduced liver fibrosis. Finally, transposon integration patterns were analysed, revealing 127,286 unique integration sites which conformed to previously published piggyBac data. Conclusion: Using a hybrid rAAV/piggyBac transposon vector system we achieved stable therapeutic protection in two UCD mouse models and a murine model with chronic liver pathology. This strategy has the potential for treating a wide array of inherited liver diseases with early onset. A clinically conceivable early application of this technology in the management of severe UCDs could be as a bridging therapy while awaiting liver transplantation. Further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration.

108. Versican G1 Domain Stimulates Adenoviral Transgene Expression in a JAK/STAT Dependent Manner

Gene therapy using either adeno-associated (AAV) or adenoviral (AdV) vectors has been shown to be effective in the ocular environment. Understanding the mechanisms of this enhancement could benefit gene therapy protocols in general. Vitreous, the gelatinous material filling the posterior eye, enhances the expression of transgenes delivered by AdV and AAV. Vitreous has no effect on vector internalization but is associated with increased transgene mRNA levels and mediates enhancement in hyaluronan-dependent and independent-manners (Chaudhuri et al. Mol Therapy 2007). We hypothesize that versican, a hyaluronan-binding proteoglycan expressed in vitreous, has a role in both mechanisms. Furthermore, we define an intracellular pathway through which the increased gene expression occurs.Versican-containing supernatant (VCS) was produced by culturing versican-secreting ACHN cells or HepG2 cells transfected to transiently express the recombinant versican G1 domain either lacking (G1-) or containing (G1+) the hyaluronan-binding region. Y79 or Weri retinoblastoma cells, or SKNDZ neuroblastoma cells that lack a cellular hyaluronan binding receptor were transduced in the presence of bovine vitreous, VCS, or either G1 domain with AdV vectors delivering a luciferase reporter transgene.Incubation of target cells with vitreous enhanced AdV or AAV gene expression greater than 4-fold (p<0.001) when Y79 cells, Weri cells, or SKNDZ cells were used as targets. Boiling vitreous prevented this enhancement. Thus, heat-stable hyaluronan alone was not responsible. Incubation with dasatanib (a Src kinase inhibitor), VCS, G1+, or G1- mimicked the effects of vitreous. Small-molecule inhibition of JAK1/2 or STAT3/5 using ruxolitinib or C188-9 mitigated the enhancement of transgene expression mediated by vitreous, dasatanib, VCS or either G1 domain, while inhibition of the mTOR pathway using rapamycin or everolimus showed no effect on enhancement. Vitreous or VCS treatment enhanced STAT3 phosphorylation in comparison to untreated cells.These results support a model in which Src kinase activity inhibits AdV or AAV viral vector mediated transgene expression. This inhibition can be overcome by hyaluronan-independent and dependent mechanisms mediated by the G1 domain of versican through activation of the JAK/STAT signaling pathway. Therefore, the versican G1 domain could be used as an adjuvant to enhance gene therapy protocols.

127. Detection of Vector Integration Sites by Targeted Sequencing

For biosafety assessment and the understanding of viral integration mechanisms the determination of the exact position and distribution of viral integration sites in the host genome is crucial. Current methods for mapping of vector insertion sites are based on primer binding within the vector genome, its elongation into the host genome, ligation of a common adapter on the host part, and subsequent PCRs. Thus, these methods depend on the presence of the primer binding site in the viral genome and biases are introduced during restriction digest (if applied), ligation, and PCR. Here, we present results from a targeted sequencing approach (SureSelect, Agilent) in which we enriched for genomic regions including vector sequences. This approach has major advantages over primer based approaches like LAM PCR: •As less PCR cycles are needed, a more quantitative estimation of cell clonality (clones with the sample integration site) should be possible.•Genomic regions that are captured together with the vector allow for relative quantification of vector copies per genome.•Integrations of incomplete vectors (common for adeno-associated vectors) can be detected without the need of specific primers.•As the entire vector is sequenced, mutations within the vector (and transgene) can be detected.Using control samples we defined the sensitivity, background, and dynamic quantitative range of this method. As the entire vector is sequenced, mutations within the vector (and transgene) can be detected. In summary, targeted sequencing provides an important complementary tool to primer based approaches (like LAM-PCR) for mapping of vector/viral integration sites.

702. Long Term Metabolic Correction of Wilson Disease

Wilson disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in the ATP7B gene resulting in impaired biliary copper excretion and hepatic copper accumulation. The increased hepatic copper concentration causes hepatocellular injury of variable intensity from acute liver failure to chronic hepatitis evolving to cirrhosis. Most often the first manifestation of the disorder is the hepatic disease which may be followed by copper deposition in the brain with resulting neurological damage. Current treatments are based on copper chelators that promote urinary excretion of the metal. These therapies should be maintained lifelong, may cause side effects and do not restore normal copper metabolism. In this work we assessed the efficacy of gene therapy to treat this condition. We transduced the liver of Atp7b-/- mice (a model of WD) with an adeno-associated vector serotype 8 (AAV8) encoding the human ATP7B cDNA placed under the control of the liver-specific a1-antitripsin promoter. After vector administration we performed serial determinations of serum transaminases, serum holoceruloplasmin concentration and ferroxidase activity and urinary copper excretion. In addition we assessed liver cooper concentration, oxidative status of hepatic proteins and histological liver damage in samples obtained at 6 months after therapy. We observed a dose-dependent therapeutic effect manifested by the reduction of transaminasemia and urinary copper excretion and normalization of serum holoceruloplasmin. Furthermore, we documented complete reversal of all hepatic alterations, including copper content, histopathological changes and protein oxidation. Conclusion: our data demonstrate that gene therapy provides long term correction of WD in a clinically relevant animal model of this disorder.

576. A Novel Gene Editing-Based Strategy for Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is an autosomal dominant muscular disease caused by an expansion of the CTG trinucleotide repeat located in the 3’ untranslated region (3’ -UTR) of the DMPK gene. The size of the expansion correlates with disease severity and to date there is no effective treatment for patients. Mutated DMPK transcripts aberrantly interact with proteins in the nucleus and form stable complexes where splicing factors are sequestered. As a consequence, the alternative splicing of numerous transcripts is pathogenically misregulated.We aim at developing a strategy to delete the CTG expansion from the human DMPK locus by using the CRISPR/Cas9 system. We have generated several constructs which express a small size Cas9 nuclease and guide-RNAs (gRNAs), and show their ability to excise the targeted DNA region in various cell lines. We are currently producing adeno-associated vectors for in vivo delivery of these constructs in a DM1 mouse model.

Liver-directed gene therapy of chronic hepadnavirus infection using interferon alpha tethered to apolipoprotein A-I

Current hepatitis B virus (HBV) management is challenging as treatment with nucleos(t)ide analogues needs to be maintained indefinitely and because interferon (IFN)-α therapy is associated with considerable toxicity. Previously, we showed that linking IFNα to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFNα. Here, we analyse the antiviral potential of an adeno-associated vector encoding IFNα fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFNα (AAV-IFN) in two animal models of chronic hepadnavirus infection. In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic hepatitis virus (WHV) infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA), experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN, while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchucks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult to treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogues represents a promising approach for the treatment of HBV infection in highly viremic patients.

Chronic exposure to IFNα drives medullar lymphopoiesis towards T-cell differentiation in mice.

Interferon-α is a potent antiviral agent and a vigorous adjuvant in the induction of T-cell responses but its use is limited by hematologic toxicity. Interferon-α alters hematopoietic stem cell dormancy and impairs myelocytic and erythrocytic/megakaryocytic differentiation from hematopoietic progenitors. However, the effect of chronic interferon-α exposure on hematopoietic precursors has still not been well characterized. Here, we transduced the liver of mice with an adenoassociated vector encoding interferon-α to achieve sustained high serum levels of the cytokine. The bone marrow of these animals showed diminished long-term and short-term hematopoietic stem cells, reduction of multipotent progenitor cells, and marked decrease of B cells, but significant increase in the proportion of CD8(+) and CD4(+)CD8(+) T cells. Upon adoptive transfer to RAG(-/-) mice, bone marrow cells from interferon-α-treated animals generated CD4(+) and CD8(+) T cells while CD19(+), CD11b(+) and NK1.1(+) lineages failed to develop. These effects are associated with the transcriptional downregulation of transcription factors involved in B-cell differentiation and modulation of key factors for T-cell development. Thus, sustained interferon-α exposure causes hematopoietic stem cells exhaustion and drives common lymphoid progenitors towards T-cell generation.

Development of a rapid, robust, and universal picogreen-based method to titer adeno-associated vectors.

Recombinant adeno-associated viruses (rAAVs) are promising vectors in preclinical and clinical assays for the treatment of diseases with gene therapy strategies. Recent technological advances in amplification and purification have allowed the production of highly purified rAAV vector preparations. Although quantitative polymerase chain reaction (qPCR) is the current method of choice for titrating rAAV genomes, it shows high variability. In this work, we report a rapid and robust rAAV titration method based on the quantitation of encapsidated DNA with the fluorescent dye PicoGreen®. This method allows detection from 3×10(10) viral genome/ml up to 2.4×10(13) viral genome/ml in a linear range. Contrasted with dot blot or qPCR, the PicoGreen-based assay has less intra- and interassay variability. Moreover, quantitation is rapid, does not require specific primers or probes, and is independent of the rAAV pseudotype analyzed. In summary, development of this universal rAAV-titering method may have substantive implications in rAAV technology.

PTEN Depletion Decreases Disease Severity and Modestly Prolongs Survival in a Mouse Model of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is the second most common genetic cause of death in childhood. However, no effective treatment is available to halt disease progression. SMA is caused by mutations in the survival motor neuron 1 (SMN1) gene. We previously reported that PTEN depletion leads to an increase in survival of SMN-deficient motor neurons. Here, we aimed to establish the impact of PTEN modulation in an SMA mouse model in vivo. Initial experiments using intramuscular delivery of adeno-associated vector serotype 6 (AAV6) expressing shRNA against PTEN in an established mouse model of severe SMA (SMNΔ7) demonstrated the ability to ameliorate the severity of neuromuscular junction pathology. Subsequently, we developed self-complementary AAV9 expressing siPTEN (scAAV9-siPTEN) to allow evaluation of the effect of systemic suppression of PTEN on the disease course of SMA in vivo. Treatment with a single injection of scAAV9-siPTEN at postnatal day 1 resulted in a modest threefold extension of the lifespan of SMNΔ7 mice, increasing mean survival to 30 days, compared to 10 days in untreated mice. Our data revealed that systemic PTEN depletion is an important disease modifier in SMNΔ7 mice, and therapies aimed at lowering PTEN expression may therefore offer a potential therapeutic strategy for SMA.

Efficient central nervous system AAVrh10-mediated intrathecal gene transfer in adult and neonate rats.

Intracerebral administration of recombinant adeno-associated vector (AAV) has been performed in several clinical trials. However, delivery into the brain requires multiple injections and is not efficient to target the spinal cord, thus limiting its applications. To assess widespread and less invasive strategies, we tested intravenous (IV) or intrathecal (that is, in the cerebrospinal fluid (CSF)) delivery of a rAAVrh10-egfp vector in adult and neonate rats and studied the effect of the age at injection on neurotropism. IV delivery is more efficient in neonates and targets predominantly Purkinje cells of the cerebellum and sensory neurons of the spinal cord and dorsal root ganglia. A single intra-CSF administration of AAVrh10, single strand or oversized self-complementary, is efficient for the targeting of neurons in the cerebral hemispheres, cerebellum, brainstem and spinal cord. Green fluorescent protein (GFP) expression is more widespread in neonates when compared with adults. More than 50% of motor neurons express GFP in the three segments of the spinal cord in neonates and in the cervical and thoracic regions in adults. Neurons are almost exclusively transduced in neonates, whereas neurons, astrocytes and rare oligodendrocytes are targeted in adults. These results expand the possible routes of delivery of AAVrh10, a serotype that has shown efficacy and safety in clinical trials concerning neurodegenerative diseases.

Effectiveness of gene delivery systems for pluripotent and differentiated cells

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases, both to study them and to explore therapies. However, a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study, we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV), adenoviruses and lentiviral vectors in hESC, hiPSC, and the derived cardiomyocytes. In undifferentiated cells, adenoviral and lentiviral vectors were superior, whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes, 1, 2, 6, and 9, of which 2 and 6 were superior in their transduction efficiency. Interestingly, we observed that AAVs severely diminished the viability of undifferentiated cells, an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore, we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally, adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion, adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines, whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells.

Gene therapy to treat inherited and complex retinal degenerative diseases

Gene therapy to treat inherited and complex retinal degenerative diseases