Administration Of AAV Vectors Research Articles

Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2.

Significant progress has been made in enhancing recombinant adeno-associated virus (rAAV) for clinical investigation. Despite its versatility as a gene delivery platform, the inherent packaging constraint of 4.7 kb imposes restrictions on the range of diseases it can address. In this context, we present findings of an exceptionally compact and long-term promoter that facilitates the expression of larger genes compared to conventional promoters. This compact promoter originated from the genome of the alphaherpesvirus pseudorabies virus, latency-associated promoter 2 (LAP2, 404 bp). Promoter driving an mCherry reporter was packaged into single strand (ss) AAV8 and AAV9 vectors and injected into adult C57BL/6 mice at a dose of 5 × 1011 vg/mouse by single intravenous or intramuscular administration. An ssAAV8 and ssAAV9 vector with elongation factor-1α promoter (EF1α, 1264 bp) was injected side-by-side for comparison. After 400 days, we sacrificed the mice and examined mCherry expression in liver, kidney, heart, lung, spleen, pancreas, skeletal muscle, and brain. We found that LAP2 exhibited robust transgene expression across a wide range of cells and tissues comparable to the larger EF1α, which is currently recognized as a rather potent and ubiquitous promoter. The AAV8-LAP2 and AAV9-LAP2 constructs displayed strong transduction and transcription in liver, kidney, and skeletal muscle on both route of administration. However, no expression was detected in the heart, lung, spleen, pancreas, and brain. The outcomes of our investigation propose the viability of LAP2 for gene therapy applications demanding the expression of large or multiple therapeutic genes following a single viralvector administration.

Interneuronal In Vivo Transfer of Synaptic Proteins.

Neuron-to-neuron transfer of pathogenic α-synuclein species is a mechanism of likely relevance to Parkinson's disease development. Experimentally, interneuronal α-synuclein spreading from the low brainstem toward higher brain regions can be reproduced by the administration of AAV vectors encoding for α-synuclein into the mouse vagus nerve. The aim of this study was to determine whether α-synuclein's spreading ability is shared by other proteins. Given α-synuclein synaptic localization, experiments involved intravagal injections of AAVs encoding for other synaptic proteins, β-synuclein, VAMP2, or SNAP25. Administration of AAV-VAMP2 or AAV-SNAP25 caused robust transduction of either of the proteins in the dorsal medulla oblongata but was not followed by interneuronal VAMP2 or SNAP25 transfer and caudo-rostral spreading. In contrast, AAV-mediated β-synuclein overexpression triggered its spreading to more frontal brain regions. The aggregate formation was investigated as a potential mechanism involved in protein spreading, and consistent with this hypothesis, results showed that overexpression of β-synuclein, but not VAMP2 or SNAP25, in the dorsal medulla oblongata was associated with pronounced protein aggregation. Data indicate that interneuronal protein transfer is not a mere consequence of increased expression or synaptic localization. It is rather promoted by structural/functional characteristics of synuclein proteins that likely include their tendency to form aggregate species.

Abstract LB201: Optimizing adeno-associated viral vector-mediated delivery of trastuzumab to the central nervous system for the treatment of Her2 positive brain metastasis

Abstract Breast cancer is the most common cause of brain metastases in women, affecting 15-30% of all patients. Despite the success of systemic Her2-targeted therapies, up to half of patients with Her2+ primary breast cancer develop breast cancer brain metastases (BCBM). Subtype switching from Her2- primary breast tumors account for an additional 15% of Her2+ BCBM, bringing total Her2 positivity in the BCBM setting to nearly 65% of cases. Even though direct delivery of Her2-targeted therapies to the central nervous system (CNS) has shown promising clinical results, long-term feasibility is limited, at least in part, by the need for repeated dosing to overcome the shorter drug half-life in the cerebrospinal fluid vs. systemic circulation (e.g., ~1 vs. 5 days, respectively). To achieve sustained CNS expression of trastuzumab without frequent dosing, we developed a gene therapy approach to treat BCBM comprising a single local administration of AAV vector encoding trastuzumab. Previous work from our group has shown that intracerebroventricular (ICV) injection of trastuzumab-expressing AAV doubled the life expectancy of Rag1 knockout (KO) mice bearing BT-474 breast cancer brain tumors. Rag1 KO mice who received AAV-trastuzumab prior to implantation of BT-474 tumor cells into the brain showed a dose-dependent delay in tumorigenesis. Following these proof-of-concept studies, we performed extensive gene therapy vector optimization and in vivo evaluation. The concentration of trastuzumab achieved in the brain parenchyma after a single ICV administration of the optimized vectors into Rag1 KO mice ranged from 10-165 ng/mg of brain protein by day 28. Previous studies utilizing CNS delivery of gene therapy vectors have shown vector leakage into the systemic circulation, which results in detectable transgene expression in the blood following peripheral transduction. To mitigate the potential risk of trastuzumab-induced cardiotoxicity, we investigated using intravenously administered, plasma-derived, pooled human immunoglobulin (IVIG) as a source of anti-AAV neutralizing antibodies to reduce peripheral transduction. Compared to controls that received naïve mouse serum, Rag1 KO mice receiving IVIG two hours prior to ICV administration of AAV-trastuzumab exhibited similar levels of trastuzumab within the brain while displaying a 10-fold reduction in serum trastuzumab concentrations. Vector biodistribution analysis suggested that reduced serum trastuzumab levels resulted from effective blockade of liver transduction following IVIG pre-treatment. These data suggest that AAV vector administration to the CNS to achieve sustained expression of trastuzumab may effectively treat Her2+ BCBM. Additionally, patients with pre-existing neutralizing antibodies to the AAV vector capsid need not be excluded from future clinical trials. Citation Format: Marcela Salazar Werner, Shweta Aras, Ashleigh Morgan, Nesteene J. Param, Jenny Greig, James M. Wilson. Optimizing adeno-associated viral vector-mediated delivery of trastuzumab to the central nervous system for the treatment of Her2 positive brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB201.

Review of: "Systemic and local immune response to intraocular AAV vector administration in non-human primates"

Review of: "Systemic and local immune response to intraocular AAV vector administration in non-human primates"

Macrophage Depletion via Clodronate Pretreatment Reduces Transgene Expression from AAV Vectors In Vivo

Adeno-associated virus is a popular gene delivery vehicle for gene therapy studies. A potential roadblock to widespread clinical adoption is the high vector doses required for efficient transduction in vivo, and the potential for subsequent immune responses that may limit prolonged transgene expression. We hypothesized that the depletion of macrophages via systemic delivery of liposome-encapsulated clodronate would improve transgene expression if given prior to systemic AAV vector administration, as has been shown to be the case with adenoviral vectors. Contrary to our expectations, clodronate liposome pretreatment resulted in significantly reduced transgene expression in the liver and heart, but permitted moderate transduction of the white pulp of the spleen. There was a remarkable localization of transgene expression from the red pulp to the center of the white pulp in clodronate-treated mice compared to untreated mice. Similarly, a greater proportion of transgene expression could be observed in the medulla located in the center of the lymph node in mice treated with clodronate-containing liposomes as compared to untreated mice where transgene expression was localized primarily to the cortex. These results underscore the highly significant role that the immune system plays in influencing the distribution and relative numbers of transduced cells in the context of AAV-mediated gene delivery.

701. Successful In Vivo Re-Administration of AAV with the Use of Two-Step AAV Injection

Introduction: The major challenge for a successful re-administration of AAV vectors is the presence of neutralizing antibodies (NABs) directed against AAV developed after the first administration of AAV vectors. Those serotype-specific neutralizing antibodies directed towards the viral capsid proteins prevent efficient gene transfer with AAV of the same serotype. The generation of a humoral immune response does not permit the use of “vector of choice” more than once which is a concern for life-long disorders for which re-administration has to be considered. Currently different methods are being explored in order to circumvent the presence of anti-AAV NABs at re-administration. One of those methods is based on the principle of antibody adsorption in AAV-based gene delivery. It was first observed in mice by Scallan et al. who reported that the administration of AAV vectors in the presence of empty AAV capsids significantly reduced the neutralization of AAV vectors by anti-AAV circulating NAB (Scallan et al. 2006). Additionally, Mingozzi et al. have also shown in mice and non-human primates that injection of therapeutic AAV vector together with empty AAV capsids allows liver transduction in the presence of high titers of anti-AAV NABs (Mingozzi et al. 2013). Goal: The goal of our study was to decrease the anti-AAV NABs to a level that would allow successful repeated gene delivery with the same AAV serotype. Study design and results: We have designed an approach to decrease the levels of circulating NABs in vivo by mean of anti-AAV NABs adsorption. In our study, for the re-administration of AAV in the presence of anti-AAV NABs, a two-step AAV vector injection was used. The first dose of AAV vector was used as a “decoy” AAV that captured circulating anti-AAV NABs. To allow a proper binding of circulating anti-AAV NABs by “decoy” AAVs, we delayed the second AAV injection. In our proof of concept experiments, mice were initially immunized with AAV5-GFP. Two weeks later the re-administration of AAV5 was performed. The re-administration procedure consisted of a first “decoy” AAV5-GFP injection followed by an injection with AAV5-hSEAP. In a first experiment, mice were injected with a “decoy” AAV5-GFP followed by AAV5-hSEAP at 30, 60 or 120 minutes after the first injection. A significant decrease of total anti-AAV5 antibody levels after injection of the “decoy” AAV5-GFP vector was observed. In the second study, we used a similar procedure with a 30 min time frame between ”decoy” AAV5-GFP and AAV5-hSEAP injection, using three different doses of the “decoy” AAV5-GFP. The decrease of total anti-AAV5 antibodies 30 min after “decoy” AAV5-GFP injection was inversely proportional to the dose of the “decoy” AAV5-GFP used and to the hSEAP activity levels that were achieved after AAV5-hSEAP injection. Conclusions: Based on hSEAP transgene activity levels, we were able to determine a cut-off level of anti-AAV5 antibodies that allow a successful re-administration of the AAV5. Overall, the two-step AAV injection approach as such is promising for AAV re-administration.

172. B-Type Natriuretic Peptide Gene Therapy as a Novel Early Treatment for Familial Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common familial cardiomyopathy, with the prevalence between 0.2 to 0.5%. HCM presents as an increase in left ventricular mass with thickening of the interventricular septum (IVS), myocellular disarray, interstitial fibrosis, heart rhythm abnormalities, and sudden death, necessitating therapeutic intervention. HCM is commonly caused by mutations in genes MYH7, MYBPC3, TNNT2, and TNNI3.The cardiac hormone, B-type natriuretic peptide (BNP) is a guanylyl cyclase A (GC-A) agonist. Physiological effects of BNP include natriuresis, vasodilation and blood pressure-lowering action. Additionally, BNP shows direct cardioprotective properties including anti-hypertrophic and anti-fibrotic effects, independently of its anti-hypertensive effects. We have recently found that genetic BNP ablation results in progressive cardiac hypertrophy with upregulation in HCM-associated genes in rats. In the current study, we tested the hypothesis that sustained BNP treatment with novel gene delivery of BNP may preserve cardiac function and structure in HCM in a mouse model of HCM.We used transgenic HCM mice expressing a mutated α-myosin heavy chain. Adeno-associated virus serotype 9 (AAV9) vectors were utilized to exploit their natural myocardial tropism. Three groups of mice were treated via tail-vein injections at four weeks of age (week one of study); Group 1, No HCM control (C57bl/6, no transgenic α-myosin heavy chain, n=6); Group 2, Untreated HCM mice with transgenic α-myosin heavy chain (PBS treated, n=10); Group 3, AAV9-BNP vector-treated HCM mice (n=6). To observe therapeutic benefit we assayed non-invasive blood pressure, myocardial architecture and remodeling (echocardiography), exercise capacity (treadmills), and any cardiorenal interplay through renal parameters. At 6, 9, 12, 15 and 25 weeks after AAV vector administration, left ventricular mass by echo was significantly higher in the untreated HCM group than the AAV-BNP-treated HCM and wildtype C57bl/6 mice. Significant increases in IVS and posterior wall thickness were also observed in control HCM group. At 11, 19, and 25 weeks after AAV vector administration, exercise capacity, monitored by treadmill tolerance, was significantly lower in the control HCM group than the AAV-BNP-treated HCM mice. No significant changes were seen in urine outputs. HCMAAV9-BNP treated mice showed a trend towards a lower blood pressure than both C57bl/6 and HCM mice.Our data demonstrate that AAV9-mediated BNP over-expression blocks progressive left ventricular enlargement and improves exercise capacity in a mouse model of HCM. The present study supports the BNP/GC-A/cGMP axis as a novel therapeutic target for the treatment of HCM.

310. Analysis of AAV5 Biodistribution and Viral Shedding in the Presence or Absence of Neutralizing Antibodies

Neutralizing antibodies (NAbs) are the main obstacle that must be overcome to achieve a successful delivery of a viral vector to the target organ. NAbs acquisition can be induced naturally after infection with wild type AAV or after the first administration of AAV vector in the course of gene therapy treatment. Therefore, the objective of this project was to evaluate the efficacy and presence of the vector in the organism, as well as the response and vector elimination in the presence and absence of NAbs. We analyzed the presence of the reporter transgene (hFIX), humoral and cellular immune responses against the vector, viral shedding and biodistribution. The administration of AAV2/5 was successful in absence of NAbs. In contrast, vector administration in presence of specific NAbs was unsuccessful, probably due to the vector being completely depleted by anti-AAV2/5 NAbs. Our data demonstrate that while in the absence of antibodies the vector can be detected in serum and in other body fluids for 70 days after vector administration, the presence of antibodies immediately clears the vector from the organism. 24 hours after the AAV-hFIX administration 1X1008 gc/mL are detected in the serum in the absence of NAbs but in the presence of NAbs the concentration is reduced 1000-fold. In both groups the presence of NAbs increased upon infection without a cellular immune response against the vector. Finally, the biodistribution studies showed remarkable differences between the two groups: in the absence of NAbs vector genomes are detected mainly in liver, heart, spleen and inguinal LN, while in the presence of NAbs vector genomes are almost undetectable in all tissues analyzed.

164. Improvement of Gene Therapy for Wilson Disease

Wilson's disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs, which may cause side effects and do not restore normal copper metabolism. We have recently demonstrated that the administration of an AAV vector expressing ATP7B under the control of a liver specific promoter induces full restoration of copper homeostasis in a mouse model of Wilson's disease. However, the size of the vector genome surpasses the optimal size for AAV packaging, limiting the use of large promoters or additional regulatory sequences such as introns or bigger poly A sequences. In the present work we have designed two truncated versions of ATP7B protein (T1 and T2), and have analyzed their functionality and therapeutic efficacy. ATP7B contains 6 metal binding sites (MBS) in the N-terminal region. T1 presents a deletion of 1 MBS and a fragment of the adjacent one, whereas T2 lacks the first 4 MBS. Both proteins were active since they were able eliminate intracellular excess of copper in an in vitro system. The administration of recombinant AAV vectors carrying the truncated proteins under the control of a liver specific promoter was able to reduce liver damage and urinary copper excretion in the murine model, but T1 was less efficient than the WT ATP7B gene. In contrast, T2 showed full capacity to normalize these parameters. Furthermore, T2 was more efficient than WT in restoring ceruloplasmin oxidase activity in serum. In conclusion, we have identified a fully functional truncated version of ATP7B protein that will reduce the size of the therapeutic AAV vector genome and allow the introduction of additional regulatory elements.

82. Mapping the Humoral Immune Response to AAV by Molecular Docking and Cryo-Electron Microscopy for the Design of Next-Generation AAV Vectors

Despite constant improvements in gene therapy with AAV vectors, including use of new serotypes, routes of administration, and transgene cassette engineering, immune responses continue to be a major obstacle to therapeutic translation. Neutralizing antibodies (NAb) against the capsid generated by prior viral infection or AAV vector administration significantly reduce not only the effective patient population but also the overall efficacy of an AAV-based gene therapy. Here, by cloning out and evaluating anti-AAV antibodies from singly-sorted memory B cells from seropositive individuals, we have designed an approach that allows us to look at the humoral immune response globally, to hone in on the immunogenic regions of the capsid itself, and to compare responses between individuals in an unbiased and therapeutically-relevant setting. In this study, we screened a panel of 30 normal human donors, selected one with high pre-existing NAb titers (1:320 for AAV2, 1:40 for AAV3B), then sorted out switched memory B cells by negative selection, seeding on irradiated ms3T3-CD40L feeder cells and culturing for 2 weeks in the presence of IL-2 and IL-21. Following supernatant screening for anti-AAV antibody production, over 100 AAV-reactive clones were identified. After isolation and cloning using nested PCR, antibodies were evaluated for AAV capsid binding as well as neutralizing capacity. To date, all antibodies demonstrated binding to AAV2 and AAV3B as well as a panel of additional AAV serotypes (8, 9, rh10, rh32.33), suggesting that AAV-binding, yet non-neutralizing antibodies may possess broad serotype specificity.To identify the epitopes for these anti-AAV antibodies, we first took a high-throughout, predictive approach. Antibody variable region sequences were placed into a generic antibody scaffold and their three-dimensional structure modeled using Kotai Antibody Builder and RosettaAntibody followed by validation using COOT. The resulting structures were then iteratively docked onto the published structure of AAV3B using PIPER to identify the most energetically-favorable binding conformation. Thus far, the vast majority of footprint residues lie in variable regions of the capsid comprising and surrounding the 3-fold spikes. For a number of antibodies, initial prediction-directed capsid alanine scanning experiments have shown decreased antibody binding at the predicted residues, supporting the use of this approach. More comprehensive mutagenesis experiments are underway to further validate the approach and more completely map immunogenic epitopes of the AAV capsid proteins. In addition, cryo-EM analysis is currently underway for a number of these Fab-AAV complexes for additional, direct observation of the repertoire of binding footprints. These studies will provide information critical to understanding the antibody response to AAV and guide future attempts to rationally design next-generation capsids that are able to evade the anti-AAV antibody response.

AAV viral vector delivery to the brain by shape-conforming MR-guided infusions

Gene transfer technology offers great promise as a potential therapeutic approach to the brain but has to be viewed as a very complex technology. Success of ongoing clinical gene therapy trials depends on many factors such as selection of the correct genetic and anatomical target in the brain. In addition, selection of the viral vector capable of transfer of therapeutic gene into target cells, along with long-term expression that avoids immunotoxicity has to be established. As with any drug development strategy, delivery of gene therapy has to be consistent and predictable in each study subject. Failed drug and vector delivery will lead to failed clinical trials. In this article, we describe our experience with AAV viral vector delivery system, that allows us to optimize and monitor in real time viral vector administration into affected regions of the brain. In addition to discussing MRI-guided technology for administration of AAV vectors we have developed and now employ in current clinical trials, we also describe ways in which infusion cannula design and stereotactic trajectory may be used to maximize the anatomical coverage by using fluid backflow. This innovative approach enables more precise coverage by fitting the shape of the infusion to the shape of the anatomical target.

Gene therapy for haemophilia: a very modern success story

Abstract AAV-mediated gene therapy has the potential to be paradigm shifting as a treatment for haemophilia. A single administration of AAV vector can result in safe and consistent long-term expression of transgene (>5 years), reduction in spontaneous bleeding episodes, reduction in clotting factor usage and an improvement in quality of life. There is huge commercial interest in this approach, with the expectation that an AAV gene therapy product for haemophilia B will be licensed by 2020.

14. Next Generation AAV Vectors for Limiting Systemic Leakage and Improving Safety Following CNS Administration

Intracranial or intrathecal administration of certain AAV vectors for CNS gene transfer is accompanied with systemic leakage into off-target organs such as the liver and spleen. Both preclinical and clinical studies have highlighted potential concerns related to high vector dose-related immunotoxicity and more recently, hepatic genotoxicity in mouse models. In order to address these potential safety issues and reduce the effective dose required to achieve efficient transgene expression in the CNS, we have rationally engineered next generation AAV vectors that show robust CNS spread and efficient transduction, while demonstrating minimal leakage into the systemic circulation. Direct CNS administration or intrathecal infusion of AAV9 results in highly efficient gene expression in neuronal and glial cellular populations in neonatal and adult mice in vivo. However, AAV9 vectors are also disseminated into the blood circulation accompanied by broad vector biodistribution and reporter gene expression in the heart, liver, spleen and kidney. CNS-to-liver and CNS-to-spleen ratios of vector genome copy numbers ranging from 0.3 to 1 were observed. A prototype, engineered AAV strain demonstrated similar potential for spread and high transduction efficiency in neonatal and adult mice. However, transgene expression was primarily restricted to neurons and virtually no leakage into systemic organs was observed regardless of CNS injection route. Preliminary studies in rhesus macaques also confirm the ability of the engineered AAV strain to spread and globally transduce the primate brain. Additional biodistribution data from rodent and primate models is forthcoming. These studies provide a roadmap for addressing clinical gene therapy challenges through continued vector development and confirm that natural AAV isolates are excellent platforms for building next generation vectors with robust transduction efficiency and improved safety profiles.

221. The Utilization of Capsid-Optimized Adeno-Associated Virus (AAV) Vectors for Cancer Immunotherapy

Cancer, a leading cause of death in the human population today, is generally being treated with chemotherapeutic methods, which have adverse toxic side effects. Immunotherapy represents an attractive alternative to common treatment since it is based on activation of host immune system.In current studies we assessed the ability the of AAV vectors expressing antigen to activate a specific T-cell response in vivo. The ovarian albumin (OVA), commonly used in mouse models as an antigen for immunization studies was used to evaluate immune response. AAV6-WT-OVA and capsid-optimized AAV6-S662V+T492V-OVA vectors were injected intramuscularly in C57BL/6 mice with a dose of 5×10e11 vgs/mouse. Enchased green fluorescent protein (AAV6-EGFP) was used as negative control. In two weeks after injection, blood was collected and a number of OVA-specific T-cells were analyzed by stain with iTAg MHC Class I Murine Tetramer. The data shown suggest that the administration of AAV vectors expressing OVA led to a robust activation (approximately 9%) of specific T-cells compared to the mock and AAV-WT treated animals (less than 1%).Next, we evaluated the killing ability of these OVA specific T-cell. Splenocytes from C57BL/6 mice i.m. injected with AAV6-WT and AAV6-mutant vectors were isolated in 14 days after injection. OVA-CD8 cells were expanded in vitro in the presence of a predominant for C57BL/6 mice, SIINFEKL peptide. Stimulated T-cells were used for killing assay against mouse prostate cell line RM1 stably expressing OVA. Two-color fluorescence assay of cell-mediated cytotoxicity was used as described above to estimate percentage of dead/alive target cells and generate a killing curve with different effectors to target cell ratio. Results of these experiments suggest that OVA-CD8 cells isolated from mice injected with AAV-S662V+T492V-OVA vectors have higher killing activity compared with OVA-CD8 cells from AAV-WT-OVA injected mice. Control T-cell do not show significant cytotoxicity which eliminates the possibility of autoreactive response.Finally, we evaluated the ability of capsid-optimized AAV6 vectors to initiate a protective anti-cancer immune response. Prostatic acid phosphatase (PAP), a gene up regulated in both human and mouse prostate cancer, was used as a specific target. C57BL/6 mouse subcutaneously injected prostate cancer cell line, RM1, was used as an animal model. We genetically modified this cell line for a stable expression of firefly luciferase (FLuc) to monitor progression of tumor growth in live animals. First, animals were immunized with AAV6-S663V+T492-PAP or AAV6-WT-PAP vectors. Two weeks later mice were challenged with RM1-FLuc cells by subcutaneous injection. The effect of treatment on tumor growth was evaluated by whole body life imaging. Results indicate suppression of tumor growth by AAV6-S663V+T492-PAP for approximately four weeks in comparison to one week and two weeks for negative control AAV6-GFP and AAV6-WT-PAP treated mice, respectively.In conclusion, successful inhibition of tumor growth in this artificial animal model would set the stage for potential clinical application.

Vitreal delivery of AAV vectored Cnga3 restores cone function in CNGA3-/-/Nrl-/- mice, an all-cone model of CNGA3 achromatopsia.

The CNGA3(-/-)/Nrl(-/-) mouse is a cone-dominant model with Cnga3 channel deficiency, which partially mimics the all cone foveal structure of human achromatopsia 2 with CNGA3 mutations. Although subretinal (SR) AAV vector administration can transfect retinal cells efficiently, the injection-induced retinal detachment can cause retinal damage, particularly when SR vector bleb includes the fovea. We therefore explored whether cone function-structure could be rescued in CNGA3(-/-)/Nrl(-/-) mice by intravitreal (IVit) delivery of tyrosine to phenylalanine (Y-F) capsid mutant AAV8. We find that AAV-mediated CNGA3 expression can restore cone function and rescue structure following IVit delivery of AAV8 (Y447, 733F) vector. Rescue was assessed by restoration of the cone-mediated electroretinogram (ERG), optomotor responses, and cone opsin immunohistochemistry. Demonstration of gene therapy in a cone-dominant mouse model by IVit delivery provides a potential alternative vector delivery mode for safely transducing foveal cones in achromatopsia patients and in other human retinal diseases affecting foveal function.

O.6 Restricting MTM1 transgene expression to skeletal muscle in AAV-mediated gene therapy for myotubular myopathy

Myotubular myopathy (XLMTM) is a severe congenital disease that affects skeletal musculature, which is characterized by the presence of small myofibers with frequent occurrence of central nuclei. The disease is due to mutations in the MTM1 gene, encoding a phosphoinositide phosphatase named myotubularin, and specific treatment is currently unavailable. We have previously demonstrated the efficacy of local administration of AAV vectors carrying the Mtm1 cDNA to treat the disease, and have more recently extended these studies to the whole body. In order to express MTM1 preferentially in skeletal muscles after systemic gene delivery, we have constructed several AAV vectors that contain the Mtm1 cDNA under the potent desmin promoter and miRNA target sequences for cardiac detargeting. We show that intravenous delivery of these vectors leads to myotubularin expression in skeletal muscles scattered throughout the body, including the diaphragm, and reduced expression in heart. We have selected one of these vectors for gene therapy in XLMTM mice and show the results in survival, pathology and contractile force. We demonstrate that this strategy is very efficient to drive selected expression of transgenes in skeletal muscles, avoiding potential side effects in heart, and is useful for the treatment of disorders that mainly affect the skeletal musculature, such as myotubular myopathy.

Preclinical Potency and Safety Studies of an AAV2-Mediated Gene Therapy Vector for the Treatment of MERTK Associated Retinitis Pigmentosa

Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

AAV-Mediated Gene Replacement, Either Alone or in Combination with Physical and Pharmacological Agents, Results in Partial and Transient Protection from Photoreceptor Degeneration Associated with βPDE Deficiency

Mutations in the PDE6B gene cause recessive, severe retinitis pigmentosa (RP). PDE6B encodes the β subunit of the rod-specific phosphodiesterase (βPDE), which, when absent, results in toxic levels of intracellular Ca(2+) and photoreceptor cell death. Ca(2+) blockers, such as nilvadipine, as well as light restriction, slow photoreceptor degeneration in animal models of βPDE deficiencies. The goal of the study was to evaluate the efficacy of AAV2/5- or AAV2/8-mediated gene replacement in combination with nilvadipine and/or with light restriction in the rd10 mouse bearing homozygous pde6b mutations. AAV vectors encoding either βPDE or EGFP were subretinally administered at postnatal day (P)2. Nilvadipine was administered from P7 to P28. For light restriction, pregnant rd10 mice were kept in a dark environment until their pups were 28 days old. All functional and histologic analyses were performed at P35. Significant morphologic photoreceptor protection was observed after subretinal administration of AAV vectors encoding EGFP. This protection further increased after administration of AAV2/8 or -2/5 encoding for βPDE and was not associated with significant functional improvement. Photoreceptor protection was higher after AAV2/8- than after AAV2/5-mediated delivery and was not significantly augmented by additional drug therapy and/or light restriction. The protective effect was lost after P35. In conclusion, more efficient gene transfer tools than those used in this study, as well as a better understanding of the disease pathogenesis, should be explored to increase the effect of gene replacement and to design gene-based strategies that block the apoptotic pathways activated by βPDE deficiency.

Rabbit Anti-Thymocyte Globulin (rATG) Administrated Concomitantly with Liver Delivery of AAV2-hFIX Can Promote Inhibitor Formation In Rhesus Macaques.

AbstractAbstract 3765Adeno-associated viral (AAV) vectors are one of the most extensively studied vector platforms for gene therapy applications. Our group is currently developing AAV vectors for the therapeutic treatment of hemophilia B (HB) in humans. The first clinical trial using an AAV2 vector to express human Factor IX (hFIX) (AAV2-hFIX16) from the liver of HB patients revealed a cytotoxic T lymphocyte (CTL) response directed against AAV capsid that occurred 4–6 weeks following treatment that was associated with a decline in transgene expression. Thus, immunosuppressive (IS) therapies may be required during AAV2 vector administration at high doses to prevent or to halt the immune mediated destruction of transduced hepatocytes. Previous work in murine and non-human primate (NHP) models has shown that sustained AAV-mediated expression of transgenes can induce tolerance, and that this is in part, dependent on CD4+ CD25+ FoxP3+ regulatory T cells (Tregs). Here we investigate the safety of a Treg sparing anti-T cell IS regimen in the context of liver mediated AAV2 gene transfer. Rabbit anti-thymocyte globulin (rATG) is an immune suppressive drug that is used in solid organ transplant and autoimmune disease. rATG has been shown to dramatically deplete the majority of T-cells, however some studies have shown that rATG spares Tregs and can induce tolerance in human T cells. rATG was administered to rhesus macaques (along with an 8-week course of Mycophenolate Mofetil (MMF) and sirolimus) either at the time of AAV vector administration (AAV2-hFIX16), or 5 weeks post-vector administration (rescue therapy). The administration of ATG at week 5 had no detrimental effect on hFIX expression and was not associated with inhibitor formation (n=3) indicating that rATG might be safe to use as an IS ‘rescue’ agent, after the detection of an ongoing immune response against transduced cells. Interestingly we observed that early administration of rATG prevented tolerance induction and resulted in inhibitor formation in 2 of 3 animals upon withdrawal of IS. The inhibitor formation was associated with transient elevations in circulating levels of IL-2, IL-4, IL-10 and IFN-g. These results are comparable to previous findings in NHP using an anti-CD25 IS regimen (Daclizumab) at the time of vector administration (Blood 2007, 110(7):2334-41). We conclude that the timing of IS regimens is critical, and that IS regimens that alter the numbers, frequency, and/or function of T-cells at the time of vector administration can result in neutralizing antibodies (inhibitors) to the transgene product (hFIX). These data suggest that there might be multiple mechanisms responsible for maintaining tolerance in this model, and that Tregs alone might not be sufficient. This study highlights the critical need for safety studies in large animal models of potential immune suppressive regimens in the context of gene transfer before translating to the clinic. Disclosures:High:Genzyme, Inc: Consultancy, Patents & Royalties; Third Rock Ventures: Consultancy; Novo-Nordisk: Consultancy; Shire, Inc.: Consultancy.

Systemic Correction of Storage Disease in MPS I NOD/SCID Mice Using the Sleeping Beauty Transposon System

The Sleeping Beauty (SB) transposon system is a nonviral vector that directs transgene integration into vertebrate genomes. We hydrodynamically delivered SB transposon plasmids encoding human alpha-L-iduronidase (hIDUA) at two DNA doses, with and without an SB transposase gene, to NOD.129(B6)-Prkdc(scid) IDUA(tm1Clk)/J mice. In transposon-treated, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with mucopolysaccharidosis type I (MPS I), plasma IDUA persisted for 18 weeks at levels up to several hundred-fold wild-type (WT) activity, depending on DNA dose and gender. IDUA activity was present in all examined somatic organs, as well as in the brain, and correlated with both glycosaminoglycan (GAG) reduction in these organs and level of expression in the liver, the target of transposon delivery. IDUA activity was higher in the treated males than in females. In females, omission of transposase source resulted in significantly lower IDUA levels and incomplete GAG reduction in some organs, confirming the positive effect of transposition on long-term IDUA expression and correction of the disease. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I in mice, including thickening of the zygomatic arch, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium, implying potential effectiveness of this approach in treatment of human MPS I.