Abstract
Adeno-associated virus (AAV) has emerged as the predominant viral vector in clinical gene therapy. However, its widespread application confronts critical challenges, including pre-existing neutralising antibodies in 40%-80% of the population, species-dependent therapeutic discrepancies, and suboptimal tropism specificity. While current AAV capsid modification strategies (e.g., directed evolution and rational design) have advanced the field, their implementation has been hampered by incomplete mechanistic understanding and persistent translational roadblocks, necessitating the need for the discovery of novel AAV capsids. In this study, we systematically captured 1925 natural AAV variants from non-human primate (NHP) tissues by integrating multiple Polymerase Chain Reaction (PCR) primers and deep long-read sequencing technology, significantly expanding the natural capsid library by more than 20-fold and identifying 1274 representative AAV11 family variants. Based on the co-evolution analysis of these natural AAV11 variants, we designed the engineered variant AAV11.P5V6, which showed significantly enhanced transduction efficiency in human and NHP primary hepatocytes in vitro and achieved efficient targeting in a mouse central nervous system model. In addition, AAV11 and its variants maintain a strong antibody escape ability in human serum and immune animal models, exhibiting unique serological characteristics with almost no cross-neutralisation reaction with AAV8 and AAV9, confirming its low serum prevalence and immune evasion advantages. This study established a systematic framework of 'natural discovery-evolutionary analysis-functional optimization', providing a new paradigm for the development of next-generation AAV vectors with clinical-grade tissue specificity, low immunogenicity, and cross-species compatibility.