AAVs targeting human carbonic anhydrase IV enhance gene delivery to the brain

Gene therapies using natural adeno-associated virus (AAV) serotypes have restricted applications, particularly in the brain, due to their poor targeting and resulting safety concerns. Directed evolution has identified brain-enhanced engineered AAV capsids for various model organisms, but inter-species differences challenge their translation. Here, we engineer AAVs to target human carbonic anhydrase IV (CA-IV), a recently identified blood-brain barrier transcytosis receptor with several favorable properties. We perform in vitro on-column selection of an AAV library to exclude capsids that do not target human CA-IV. We subsequently screen the top 0.01% of variants in vivo in mice expressing human CA-IV in brain endothelial cells. Notably, two top-performing capsids in "humanized" mice are relatively lower ranked in vitro and outperform several top-ranked in vitro candidates. One of these, AAV-hCA4-IV77, achieves 100-fold-greater brain transduction than AAV9, with robust neuronal and astrocytic coverage across brain regions. These results advance our understanding of receptor-targeted capsid design and support the therapeutic potential of human CA-IV-engaging AAVs.