Trapani, Ivana (2014) Expanding AAV cargo capacity for gene therapy of Stargardt disease. [Tesi di dottorato]

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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.

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