Discovering a Novel AAV2-Based Capsid for RPE Transduction Via Intravitreal Injection for Leber Congenital Amaurosis Type 2 Gene Therapy

Purpose: Several retinal degenerations initiate because of gene defects in the outermost retinal layer, the retinal pigment epithelium (RPE). Subretinal injection achieves optimal RPE transduction but has limitations, including iatrogenic damage. Here, we develop and evaluate the efficacy of a novel adeno-associated virus (AAV) capsid with intravitreal delivery, in an effort to overcome limitations of subretinal delivery for treatment of retinal degenerations that require precise targeting of the outer retina. Methods: We conducted a high-throughput screening of 1.28 × 109 AAV2 capsids, each with a different 7-mer peptide insertion to alter its binding properties, to be delivered with intravitreal (IVT) injection, a less invasive surgical procedure. A novel AAV variant, variant 3 (AAV2.Var3) was found to transduce RPE cells with CAG-GFP and Best1-GFP in mouse eyes. For comparison, a leading, clinical-stage vector that is also AAV2-based, 7m8, effectively transduces the outer retina but has limited success targeting RPE through IVT delivery. To test the therapeutic efficacy of variant 3, we packaged therapeutic gene, hRPE65, and injected it intravitreally in rd12 mice, a disease model for Leber congenital amaurosis type 2. Results: AAV2.Var3-hRPE65 treated mice cohorts showed an increase in RPE65 protein expression and function as well as visual function in response to light flashes. Translatability of AAV2.Var3 for intravitreal gene delivery was validated by AAV2.Var3-Best1-GFP vector induced expression of GFP in peripheral RPE cells in non-human primate macaque. Conclusions: Variant 3 is capable of RPE transduction with IVT injection, serving as a vehicle for less invasive IVT gene delivery to the RPE. Therapeutic gene delivery leads to functional results with efficiency limitations.