Development of CNS tropic AAV1-like variants with reduced liver-targeting following systemic administration in mice

Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery across the blood -brain barrier (BBB) to the CNS following intravenous ( IV) injection. In this study, we used a different approach by creating a shuf fl ed rAAV capsid library based on parental AAV serotypes 1 through 12. Following selection in mice, 3 novel variants closely related to AAV1, AAV-BBB6, AAVBBB28, and AAV-BBB31, emerged as top candidates. In direct comparisons with AAV9, our novel variants demonstrated an over 270 -fold improvement in CNS transduction and exhibited a clear bias toward neuronal cells. Intriguingly, our AAV-BBB variants relied on the LY6A cellular receptor for CNS entry, similar to AAV9 peptide variants AAVPHP.eB and AAV.CAP-B10, despite the different bioengineering methods used and parental backgrounds. The variants also showed reduced transduction of both mouse liver and human primary hepatocytes in vivo . To increase clinical translatability, we enhanced the immune escape properties of our new variants by introducing additional modi fi cations based on rational design. Overall, our study highlights the potential of AAV1-like vectors for ef fi cient CNS transduction with reduced liver tropism, offering promising prospects for CNS gene therapies.