Engineering novel AAV capsids by broadly attenuated and subsequent muscle-specific tropism in mice and NHPs

Recombinant adeno-associated virus (rAAV) vectors are a potent gene delivery tool, but their clinical application is restricted by poor transduction of target tissues and off-target toxicity. To address this, we employed a two-step capsid engineering strategy: broad attenuation of rAAV tropism followed by peptide-driven tissue-specific retargeting. We first generated AAV.Zero1, a capsid with markedly reduced transduction across tissues, by VR swapping from AAV9 into AAV2. Introducing an R585A substitution (AAV.Zero2) partially restored the transduction, while deletion of residues 585-587 (AAV.Zero3) abolished it. By inserting a myogenic peptide into the AAV.Zero3 backbone, we produced a novel capsid (AAV.eM), which drove robust muscle-specific transgene expression with minimal off-target transduction in the liver, lung, brain, and kidney. This favorable profile was consistent across two mouse strains and non-human primates. AAV.eM mediated expression levels comparable to the leading myotropic vector, MyoAAV 4A, but exhibited a superior safety profile. Importantly, AAV.eM was able to functionally rescue a mouse model of Duchenne muscular dystrophy following systemic delivery of a micro-dystrophin gene. These results establish AAV.eM as an improved myotropic vector with enhanced specificity and proof of concept for a platform to create capsids with specific properties that translate across species by addition of peptides onto low transduction backbones.