Site-directed mutagenesis improves the transduction efficiency of capsid library-derived recombinant adeno-associated virus vectors

Abstract Recombinant adeno-associated virus (rAAV) vectors selected from capsid libraries present enormous advantages in high selectivity of tissue-tropism and their potential use in human gene therapy applications. For example, rAAV-LK03, was used in a gene therapy trial for hemophilia A (NCT03003533). However, high doses in patients resulted in severe adverse events and subsequent loss of FVIII expression. Thus, additional strategies are needed to enhance the transduction efficiency of capsid library-derived rAAV vectors such that improved clinical efficacy can be achieved at low vector doses. Here, we characterized two commonly used library-derived rAAV vectors, rAAV-DJ and rAAV-LK03. It was concluded that rAAV-DJ shared similar transport pathways (cell surface binding, endocytosis-dependent internalization, and cytoplasmic trafficking, etc.) with rAAV serotype 2, whilst rAAV-LK03 and rAAV serotype 3 shared similar transport pathways. We then performed site-directed mutagenesis of surface-exposed tyrosine (Y), serine (S), aspartic acid (D), and tryptophan (W) residues on rAAV-DJ and rAAV-LK03 capsids. Our results demonstrated that rAAV-DJ-S269T and rAAV-LK03-Y705+731F variants had significantly enhanced transduction efficiency compared to wild-type counterparts. Our studies suggest that the strategy of site-directed mutagenesis should be applicable to other non-natural AAV variants for their optimal use in human gene therapy.