An engineered AAV variant with high-efficiency outer retinal tropism for gene delivery

Abstract

Retinal degenerative diseases, such as Stargardt disease and age-related macular degeneration (AMD), are leading causes of irreversible blindness, and AAV-based gene therapy has become a key therapeutic option. For most natural AAV serotypes, however, efficient transduction of the outer retina, where photoreceptors reside, via intravitreal injection (IVI) remains a major challenge. Although several engineered capsids have been reported to achieve photoreceptor transduction after intravitreal delivery, precise outer-retinal gene delivery has predominantly relied on invasive subretinal injection, which risks retinal detachment and limits clinical adoption. We subjected the AAV5 capsid to rapid directed evolution by inserting random heptapeptides at residue Q574 within variable region VIII and performed iterative selection on the 661 W photoreceptor cell line, yielding the lead variant AAV5-AlyT. Transduction efficiency was assessed in vitro and in mice and rabbits following IVI. The therapeutic potential was evaluated by AAV5-AlyT-mediated delivery of the complement regulator Crry in ABCA4 −/− (Stargardt) mice and a light-induced retinal degeneration (dry AMD) model, using electroretinography, immunofluorescence, and histology. Pre-existing neutralizing antibody responses against AAV5 and AAV5-AlyT were measured in 300 human serum samples. AAV5-AlyT exhibited significantly higher transduction efficiency than parental AAV5, mediating robust, sustained transgene expression in the outer retina after a single IVI in mice (over six months) and efficient cross-layer transduction in rabbits. In Stargardt and light-damage models, AAV5-AlyT-Crry markedly rescued visual function, suppressed pathologic complement activation, and reduced RPE damage. AAV5-AlyT also showed a 34.9% lower pre-existing neutralizing antibody response than AAV5 in human sera. AAV5-AlyT is a potent, Minimally invasive vector for outer-retinal gene delivery via IVI, Offering a promising strategy for retinal degenerative diseases.