383. Ancestral AAV Demonstrates Unique Transduction Properties in Mouse and Primate

Gene therapy is a promising approach in the treatment of inherited and common complex disorders of the retina. Preclinical and clinical studies have validated the use of adeno-associated viral vectors (AAV) as a safe and efficient gene delivery vehicle for subretinal gene transfer. AAV can efficiently target RPE cells and rod and cone photoreceptors but other retinal cell types are more challenging targets. Despite the immune privileged status of the eye we still face some challenges with higher inflammation risks to vector or transgene antigens depending on route of delivery. Studies here aim to build on AAV as a validated platform while exploring new and optimized vector reagents to overcome current limitations for clinical use. We hypothesized that an ancestral AAV species would be less susceptible to pre-existing AAV immunity (PEI) in human populations and confer an improved clinical safety and efficacy profile.We used phylogenetic and bioinformatic methods to infer the ancestral state of AAV and most likely AAV capsid sequences libraries were synthesized and members were evaluated for viral assembly and DNA packaging. Lead candidates were selected and, following subretinal injection into C57Bl/6 mice, retinal tropism was characterized by (a) in vivo transduction by reporter gene expression from fundus imaging, (b) molecular analyses of expression levels, (c) onset of expression and (d) histological profile of targeted retinal cell types.Using synthetic biology methods, in silico reconstructed ancestral AAV capsid sequences were derived and ancestral AAV vectors produced using standard AAV production methods. Ancestral particles, named Anc80, were found to be biochemically similar to existing AAV, stable, and infectious in vitro. In vivo retinal transduction in mice demonstrated a high level of RPE and photoreceptor tropism that is quantitatively higher than AAV8. In addition, onset of expression of Anc80 was visible as early as day 1 post injection and accelerated compared to AAV8. Several variants of Anc80 were evaluated for inner retinal tropism with evidence for improved transduction of Muller glia, RGC, and other inner INL neurons. Preliminary studies in non-human primates also indicate an accelerated onset of expression compared to AAV9 and AAV5. Ancestral AAV have significantly increased resistance to PEI in humans and demonstrate enhanced retinal tropism.