Barcodes in the forward PCR primers (bc2).

AAV capsid serotypes isolated from nature have been widely used in gene delivery and gene therapy. Recently, more than 1,000 distinct AAV capsids were identified from human clinical samples by high-throughput, long-read DNA sequencing. In this study, we tap into this broad natural biodiversity of AAV capsids to develop liver-tropic AAV capsids. We initially screened a subset of variants derived from AAV8 (n = 159) for packaging efficiency. The high-yielding variants were subjected to a barcoded vector library screen in mice and ferrets for their ability to mediate liver gene transfer. Although no variant surpassed AAV8 for liver targeting, several exhibited a liver detargeting phenotype. Among these, we focused on the N271D variant (AAV8 VP1 numbering), located in the variable region I (VR-1), which has been previously implicated in influencing liver tropism. The liver detargeting phenotype of AAV8.N271D was confirmed by single vector administration in mice. Additionally, we grafted the N271D variant onto AAV9 and MyoAAV capsids (N270D by AAV9 VP1 numbering). The AAV9.N270D and MyoAAV.N270D vectors showed a similar liver-detargeting phenotype, although muscle targeting was moderately reduced. Although we did not identify any capsid variants that outperform AAV8 in liver transduction, this study reinforces the important role of VR-1 in modulating liver tropism and highlights the potential of engineering VR-1 residues to reduce liver gene transfer and associated toxicity observed in several gene therapy studies.