Directed evolution of novel AAV variants using the MCMS library for enhanced CNS tropism and reduced liver targeting in mice.

Systemic delivery of adeno-associated virus serotype 9 (AAV9) to the central nervous system (CNS) is insufficient due to hindrance from the tight junctions of the blood-brain barrier (BBB). While peptide-display-based AAV engineering has advanced CNS-targeting capsid development, traditional strategies inserting or substituting a 7-mer peptide remain limited by low success rates and scarcity of efficient variants. To address these issues, we developed the Multiple Capsid Mutation Strategies (MCMS) library, which enhanced sequence diversity by incorporating random peptide insertions flanked by AAV9 or variant-derived residues and peptide substitutions within the VR-VIII of the AAV9 capsid protein. Following capsid selection in mice, the leading AAV variant BRC06 was identified and validated across different mouse strains. BRC06 exhibited approximately 1.9-fold higher brain transgene expression than AAV.PHP.eB in C57BL/6J mice. In BALB/c mice, BRC06 achieved a 1,482-fold brain enhancement with a 92-fold liver reduction relative to AAV9. Sequence analysis revealed that BRC06 was derived from the MCMS library's substitution strategies. Additionally, host factor screening revealed AAVR-dependent entry with accessory factors like Acp2 contributing to BRC06 transduction. Our results demonstrate that the MCMS library enables efficient selection of AAV capsids with improved BBB penetration, CNS tropism, and reduced liver targeting in mice.