309. Optimization of Dual AAV Vectors for Gene Therapy of Inherited Retinal Diseases

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV limited cargo capacity prevents its application to therapies of inherited retinal diseases (IRD) due to mutations in genes over 5 kb. Dual AAV vectors, each containing one of the two halves of a large gene expression cassette, are emerging as promising tools to overcome this limitation. Dual AAV trans-splicing and hybrid vectors transduce efficiently the mouse and pig retina and are effective in animal models of IRD. However, some of dual AAV limitations include lower levels of transgene expression compared to a single AAV vector and the production of proteins shorter than expected from either the 5'- or 3'-half AAV. Thus, further development of dual AAV vectors is required before their clinical translation. To increase dual AAV recombination we have exploited various regions of homology while to mediate the degradation of the proteins shorter than expected we have tested the ability of various degradation signals. We found that the levels of transgene expression achieved with the alternative regions of homology are similar to those achieved with dual AAV vectors carrying the AK region of homology we have previously shown to be effective. Notably, we have identified a degradation signal which mediates the degradation of proteins shorter than expected from dual AAV vectors. In conclusion, our study outlines optimized features of dual AAV vectors that improve their safety and efficacy. This represents a step towards the clinical translation of dual AAV for retinal gene therapy

Expanding AAV cargo capacity for gene therapy of Stargardt disease

Inherited retinal degenerations (IRDs), with an overall global prevalence of 1/2,000, represent a major cause of blindness worldwide. IRDs are mostly inherited as monogenic conditions that are caused by mutations in genes preferentially expressed in photoreceptors (PRs) and retinal pigment epithelium (RPE) cells. Gene therapy represents a promising therapeutic strategy for IRDs. Vectors derived from the adeno-associated virus (AAV) have been proven to be the most efficient and safe tools for gene delivery to the retina. Further, AAV vectors have been successfully used in pre-clinical models and clinical trials focused on the treatment of IRDs. The major limitation to the use of AAVs is their packaging capacity, which is considered to be restricted to the size of the parental genome (4.7 kb) and thus hinders the treatment of certain forms of IRDs caused by mutations in genes whose coding sequence exceeds 5 kb in length, including the gene mutated in Stargardt disease (STGD), ABCA4. Thus, different strategies to overcome AAV cargo limitation have been investigated. One strategy for large gene transfer is based on “forced” packaging of large genes into AAV capsids (oversize AAV); this strategy, however, results in the production of viruses with a heterogeneous genome and thus may not be easily translated to the clinical arena due to safety concerns. Alternatively, the inherent ability of AAV genomes to undergo intermolecular concatemerization can be exploited to transfer large genes by splitting the expression cassette into two halves (<5 kb in size), each independently packaged in one of two separate (dual) AAV vectors. Dual AAV vectors can reconstitute a large gene via either splicing (dual AAV trans-splicing), homologous recombination (dual AAV overlapping) or a combination of the two (dual AAV hybrid). Dual AAV strategies have been used to efficiently delivery large genes to a number of tissues, however, the efficacy of the dual AAV systems for gene delivery to the retina remains to be investigated. Therefore, the goals of my Ph.D. project were to: - develop dual AAV vector-based strategies for large gene delivery to the retina; - compare the efficiency of oversize to that of dual AAV vector strategies for the delivery of large genes both in vitro and in vivo, in the retina; - characterize the effect of AAV-mediated large gene expression in an animal model of STGD. To this aim, I generated oversize and dual AAV vectors encoding for either the reporter EGFP or the therapeutic ABCA4 protein to compare the efficacy of AAV vector systems in vitro using HEK293 cells and in vivo in mouse and pig eyes. I found that dual AAV vectors are more efficient than oversize vectors, both in vitro and in the retina. While dual AAV OV vectors are effective in transducing the RPE, dual AAV trans-splicing and hybrid approaches transduce efficiently PRs, in addition to RPE. Thus, as the PRs are the cell target for the treatment of STGD, I selected dual AAV trans-splicing and hybrid vectors as the candidate strategies to be tested in the mouse model of STGD (Abca4-/-). I injected subretinally one-month-old mice with dual AAV trans-splicing and hybrid vectors encoding for ABCA4 and found a significant improvement of the main STGD mice retinal abnormalities, including: lipofuscin accumulation, RPE thickening and recovery from light desensitization. In conclusion, my data show that dual AAV trans-splicing and hybrid vectors are an attractive strategy for gene therapy of STGD as well as other retinal diseases that require the delivery of large genes

Adeno-Associated Viral Vectors as a Tool for Large Gene Delivery to the Retina

Gene therapy using adeno-associated viral (AAV) vectors currently represents the most promising approach for the treatment of many inherited retinal diseases (IRDs), given AAV&#8217;s ability to efficiently deliver therapeutic genes to both photoreceptors and retinal pigment epithelium, and their excellent safety and efficacy profiles in humans. However, one of the main obstacles to widespread AAV application is their limited packaging capacity, which precludes their use from the treatment of IRDs which are caused by mutations in genes whose coding sequence exceeds 5 kb. Therefore, in recent years, considerable effort has been made to identify strategies to increase the transfer capacity of AAV vectors. This review will discuss these new developed strategies, highlighting the advancements as well as the limitations that the field has still to overcome to finally expand the applicability of AAV vectors to IRDs due to mutations in large genes

Seeing the Light after 25 Years of Retinal Gene Therapy

The retina has been at the forefront of translational gene therapy. Proof-of-concept that gene therapy could restore vision in a large animal led to the initiation of the first successful clinical trials and, in turn, to the recent approval of the first gene therapy product for an ocular disease. As dozens of clinical trials of retinal gene therapy have begun, new challenges are identified, which include delivery of large genes, counteracting gain-of-function mutations, and safe and effective gene transfer to diseased retinas. Advancements in vector design, improvements of delivery routes, and selection of optimal timing for intervention will contribute to extend the initial success of retinal gene therapy to an increasing number of inherited blinding conditions

Has retinal gene therapy come of age? From bench to bedside and back to bench

Retinal gene therapy has advanced considerably in the past three decades. Initial efforts have been devoted to comprehensively explore and optimize the transduction abilities of gene delivery vectors, define the appropriate intraocular administration routes, and obtain evidence of efficacy in animal models of inherited retinal diseases (IRDs). Successful translation in clinical trials of the initial promising proof-of-concept studies led to the important milestone of the first approved product for retinal gene therapy in both US and Europe. The unprecedented clinical development observed during the last decade in the field is however highlighting new challenges which will need to be overcome to bring gene therapy to fruition to a larger patients population within and beyond the realm of IRDs

Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina

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Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease

Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4-/- mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5'-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4-/- mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1