Rationally engineered novel AAV capsids for intra-articular gene delivery.

Intra-articular adeno-associated virus (AAV) gene therapy has been explored as a potential strategy for joint diseases. However, concerns of low transduction efficacy, off-target expression, and neutralizing antibodies (Nabs) still need to be addressed. In this study, we demonstrated that AAV6 was the best serotype to transduce joints after screening serotypes 1 to 9. To develop a more effective AAV vector, a set of novel AAV capsids were rationally engineered. The mutant AAV62 created by swapping variable region I (VRI) of AAV2 into AAV6 induced a higher transduction efficiency per AAV genome copy number. To further investigate the roles of specific amino acids in the transduction of AAV62 and AAV6, we found out that AAV6D with the deletion of threonine at residue 265 induced a 2-fold higher transduction than AAV6, while the transduction efficiency from AAV6M with the mutation of alanine to glutamine at residue 263 was 10-fold lower. AAV6D efficiently transduced both synoviocytes and chondrocytes with low AAV genome copy numbers in other tissues and less Nab formation. This study demonstrates that novel AAV mutants with rational engineering may enhance joint transduction after intra-articular administration in mice, with the potential to evade AAV Nabs and minimize off-target effects in the liver.