ABSTRACT This is a competitive renewal of R01EY024280, “Engineering AAV for safe and efficient gene delivery to the human retina”. FDA approval of an Adeno associated virus (AAV)- based gene therapy for RPE65-Leber congenital amaurosis (LCA2) solidified gene therapy’s place in current medical practice. However, in the 6 years since, there have been no additional approvals, and multiple hurdles associated with retinal gene therapy have come to light. The development of therapies that safely and efficiently target the human retina remains a significant, unmet need. In previous funding cycles, we asked questions grounded in both basic and translational science. We have i) elaborated upon biological determinants of retinal transduction by AAV via different routes of administration, ii) identified novel variants with increased potency and neutralizing antibody evasion relative to benchmark vectors and iii) identified a novel variant with the ability to spread laterally beyond the margins of the subretinal injection bleb. Notably, we have also translated our findings- our laterally spreading capsid is being used in an ongoing Phase I/II clinical trial to treat X-linked retinoschisis (NCT05878860). The scope of our proposed studies is broadened based on the advances we’ve made, and our evolving understanding of challenges in the field. A significant portion of the proposed work will be performed in macaques as these challenges are most effectively modeled in intact eyes of animals with ocular characteristics and immune systems most similar to humans. In Aim 1, we explore the biological determinants of retinal transduction by AAV. We will use a novel in-situ visualization approach to analyze retinal glycans and their interactions with the AAV capsid across species, evaluate the role of Müller glia in retinal transduction following intravitreal injection, and determine the retinal distribution of ‘universal AAV’ host cell factors, AAVR and GPR108 and their roles in retinal transduction via multiple routes of administration. In Aim 2, we will enhance transduction and safety of AAV capsids delivered by intravitreal injection. Capitalizing on strong preliminary data, this Aim expands capsid library screening to non-AAV2- based capsids. In Aim 3, we will enhance the biodistribution and safety of AAV capsids delivered by subretinal injection. We propose to block capsid interactions with host cellular factors to limit transduction of peripheral tissues and/or endogenous antigen presenting cells, while simultaneously enhancing PR and/RPE transduction. Vectors and methods investigated in this proposal will have an immediate impact on planned clinical trials to address inherited retinal diseases as well as non-orphan indications such as AMD. Development of these tools by academia (rather than industry) will ensure the availability of shared resources with the broader scientific community.