Identification of non-toxic AAV capsid variants with enhanced deep retinal transduction and extended distribution following subretinal delivery

Subretinal injection remains the standard method for delivering genes to the retina. However, diffusion into deeper retinal layers is often limited by sequestration of wild-type AAV2 vectors at the retinal pigment epithelium (RPE). In this study, we used in vitro phage display strategy to identify novel AAV capsids with enhanced retinal transduction efficiency. We addressed the discrepancy between phage enrichment rankings and actual transduction potency by systematically screening top-tier candidates under low multiplicity of infection conditions. This approach precisely identified variants with superior transduction potencies. Following subretinal administration, the lead engineered capsid demonstrated a non-toxic profile, overcoming physical barriers to efficiently transduce the inner nuclear layer (INL), a region typically inaccessible to conventional AAV2 vectors. Together, these results suggest the existence of a next-generation AAV platform capable of deep retinal transduction, offering a promising solution for gene therapies requiring broad retinal coverage. • Phage enrichment rankings do not correlate with AAV transduction efficiency. • Peptide insertions can induce severe retinal toxicity despite high infectivity. • NMKQLTS overcomes RPE restriction to achieve deep retinal transduction.