Complete neutralizing antibody evasion by serodivergent non-mammalian AAVs enables gene therapy redosing

The use of adeno-associated virus (AAV) as a gene therapy vector is significantly limited by pre-existing immunity. The high seroprevalence and broad antigenic cross-reactivity of primate-derived AAVs restrict patient eligibility and preclude therapeutic redosing. Here, we harness the phylogenetic diversity of non-mammalian dependoparvoviruses to engineer serologically distinct AAV capsids for immune evasion. A barcoded screen of divergent Dependoparvovirus isolates identifies AAV.div3A, a chimeric capsid with robust transduction, zero antigenic cross-reactivity, and undetectable seroprevalence. Derived from a phylogenetically distant Muscovy duck isolate, AAV.div3A fully evades neutralization in mice, even after passive immunization with NAb+ human serum or following initial vector dosing. Further engineering yields AAV.div3A-M1, a myotropic, liver-detargeted capsid with enhanced cardiac and diaphragm transduction. In a Pompe disease model, redosing with AAV.div3A or div3A-M1 significantly increases therapeutic GAA levels. Overall, our work leverages untapped dependoparvoviral diversity to overcome pre-existing and vector-induced immunity, enabling expansion of patient eligibility and effective redosing.