Novel adeno-associated virus (AAV) vectors for improved retinal gene therapy

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Recombinant adeno-associated virus (AAV) vectors have become the “gold standard” gene delivery tool for targeting retinal cells. AAV-based vectors were tested in a number of human clinical trials and have proven safe for the use in the retina. The most advanced gene therapy is AAV-RPE65, a AAV vector for gene supplementation treatment of RPE65-linked Leber congenital amaurosis (LCA2). In clinical studies subretinal delivery of AAV-RPE65 has proven to be safe and showed some efficacy in improving vision. However, the AAV-RPE65 treatment was not able to halt or slow photoreceptor degeneration in LCA2. The reasons for this are not known, but could include disease-specific factors or issues related to the AAV vector like inadequate levels of transduction and expression. For efficient transduction, conventional AAV vectors require direct application to the target cell surface and interaction with receptors for internalization. Most inherited blinding retinal diseases are due to mutations in genes that are specifically expressed in photoreceptors or in retinal pigment epithelium (RPE). Targeting these cells requires surgical detachment of the neuroretina from the RPE and subretinal injection of the AAV vectors into a temporally formed cavity. This procedure can be deleterious to an already compromised retina. In addition, subretinal injections result only in a very localized transduction of retinal cells within the subretinal bleb area. Therefore, there is a critical unmet need to develop novel AAV vectors with improved transduction properties that enable transretinal gene expression through less invasive routes of delivery such as intravitreal (IVT) injection. The major goal of the present grant proposal is to further develop and characterize innovative AAV vectors that show great promise for targeting retinal photoreceptors after IVT administration. The work program includes in vitro characterization of the novel AAVs in 661w cone photoreceptor-like cells and human iPS-derived retinal organoids. In vivo experiments in wild type and retinal degeneration mice with vector versions expressing eGFP driven by cell type-specific promoters will assess their transduction efficiency of mouse rod or cone photoreceptors. These experiments will be followed by analogous experiments in dogs and non-human primates (NHP) to confirm the ability of the vectors to transduce rods and cones from the vitreous in large animal models and to define the optimal dose range in large eyes. The NHP and/or dog studies will also assess any immune response to the novel capsids, effects of re-administration of the vectors to the same eye and their basic biodistribution profile. Finally, the optimal vectors will be tested regarding their efficiency to restore vision and delay degeneration by IVT-delivered gene supplementation therapy in the Cnga3 knockout (KO) mouse model of achromatopsia and in the Cngb1 KO mouse model of retinitis pigmentosa.