Flying Under the Radar: Engineering Avian Adeno-Associated Virus for Immune-Evasive Gene Therapy

Abstract

Adeno-associated virus (AAV) is a small DNA virus that infects a wide range of vertebrate species and has emerged as a leading platform for in vivo gene therapy. Here, we uncover a novel mechanism for species-specific control of AAV transcription and leverage this finding to engineer diverse AAV capsids for immune-evasive gene therapy. Unlike primate AAV isolates, avian AAV is transcriptionally silenced in human cells, limiting cross-species infection. Through rational mutagenesis of the AAV capsid, we identify the VP1 N-terminal domain as a minimal structural determinant of viral transcription and host range. By swapping the N-terminal domain from primate isolates onto avian AAV, we rescue transcription and unlock robust transduction in human cells and mouse tissue. This effect is accompanied by increased chromatin accessibility and altered histone methylation. Proximity ligation analysis reveals that essential host factors are selectively recruited by the VP1 N-terminal domain of primate AAVs, but not avian AAV, facilitating species-specific transcriptional activation. N-terminal domain substitution proves to be a broadly effective strategy for unlocking the host range of diverse avian and reptile AAV isolates. Building upon these findings, we investigate whether non-mammalian AAVs can be used to overcome the immune barriers which limit safety and efficacy of primate-derived vectors. Currently, the high seroprevalence of endemic primate isolates restricts patient eligibility, and their broad antigenic cross-reactivity precludes therapeutic redosing. Non-mammalian AAVs, however, offer a promising solution due to their evolutionary distance and absence of natural infection in humans. Using a barcoded screen of divergent non-mammalian isolates, we identify AAV.div3A, a chimeric capsid with robust in vivo transduction, no 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 human serum or following initial vector dosing. Rational engineering via peptide surface display yields AAV.div3A-M1, a myotropic, liver-detargeted capsid with 14-fold higher cardiac transduction, 12-fold higher diaphragm transduction, and 93% lower liver tropism. In a Pompe disease model, redosing with AAV.div3A or AAV.div3A-M1 significantly increases therapeutic GAA expression, boosting transgene levels by 9-fold. Overall, we identify a novel mechanism for capsid-mediated control of AAV transcription, unlocking the potential of previously untapped AAV diversity. These mechanistic insights enable the development of non-mammalian AAV vectors that evade both pre-existing antibodies and vector-induced immunity, thereby expanding patient eligibility and enabling effective redosing.