Characterization of a bioengineered AAV3B capsid variant with enhanced hepatocyte tropism and immune evasion.
Academic Article
Overview
abstract
Capsid engineering of adeno associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over 5 rounds of iterative selection in hepatocyte spheroid cultures. Here, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (1-3) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse passaged primary human hepatocytes as well as in humanized liver chimeric mice, as compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. 44% of serum samples with pre-existing Nabs to AAV3B had 5-20- fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only 9 amino acid substitutions, clustered in variable region IV (VR-IV) as compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.
