Abstract
Recombinant adeno-associated virus (rAAV) is a leading vector for retinal gene therapy due to its favorable safety profile demonstrated by the FDA-approved Luxturna for Leber congenital amaurosis. However, challenges with low transduction efficiency and immunogenicity, coupled with the invasiveness of subretinal injections, have driven efforts to engineer AAV capsids for minimally invasive intravitreal delivery. Intravitreal injections face the barrier of the inner limiting membrane (ILM), particularly with AAV2-based vectors. In this study, we displayed cell-penetrating peptides (CPPs) on AAV2 capsids to enhance retinal cell transduction via intravitreal injection. Through in vivo capsid screening, we identified AAV2.CPP1, which showed significantly improved pan-retinal expression and photoreceptor transduction in mice as well as a reduced immune response compared to the AAV2.7m8 vector. We also revealed that the CPP1 insertion reduced heparan sulfate binding, improving ILM penetration. These findings highlight AAV2.CPP1 as a promising candidate for retinal gene therapy via intravitreal injection, offering enhanced efficiency and a minimized immune response.