Recombinant (r)AAV2.5T was selected from the directed evolution of an AAV capsid library in human airway epithelium (HAE). The capsid gene of rAAV2.5T is a chimera of the N-terminal unique coding sequence of AAV2 VP1 unique (VP1u) and the VP2- and VP3-coding sequence of AAV5 with a single amino acid mutation of A581T. We conducted two rounds of genome wide CRISPR gRNA library screening for host factors limiting rAAV2.5T transduction in HeLa S3 cells. The screen identified several genes that are critical for rAAV2.5T transduction in HeLa S3 cells, including previously reported genes KIAA0319L , TM9SF2 , VPS51 , and VPS54 , as well as a novel gene WDR63 . We verified the role of KIAA0319L and WDR63 in rAAV2.5T transduction of polarized HAE by utilizing CRISPR gene knockouts. Although KIAA0319L, a proteinaceous receptor for multiple AAV serotypes, played an essential role in rAAV2.5T transduction of polarized HAE either from apical or basolateral side, our findings demonstrated that the internalization of rAAV2.5T was independent of KIAA0319L. Importantly, we confirmed WDR63 is an important player in rAAV2.5T transduction of HAE, while not being involved in vector internalization and nuclear entry. Furthermore, we identified that the basal stem cells of HAE can be significantly transduced by rAAV2.5T.
Significance: The essential steps of a successful gene delivery by rAAV include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63 , whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.