Identification of SLC35A1 as an essential host factor for the transduction of multi-serotype recombinant adeno-associated virus (AAV) vectors.

We conducted a genome-wide CRISPR/Cas9 screen in suspension 293-F cells transduced with rAAV5. The highly selected genes revealed after two rounds of screens included the previously reported , , and , along with a cluster of genes involved in glycan biogenesis, Golgi apparatus localization and endoplasmic reticulum penetration. In this report, we focused on solute carrier family 35 member A1 (), a Golgi apparatus-localized cytidine 5'-monophosphate-sialic acid (CMP-SIA) transporter. We confirmed that knockout (KO) significantly decreased rAAV5 transduction to a level lower than that observed in or KO cells. Although KO drastically reduced the expression of α2,6-linked SIA on the cell surface, the expression of α2,3-linked SIA, as well as the cell binding and internalization of rAAV5, were only moderately affected. Moreover, KO significantly diminished the transduction of AAV multi-serotypes, including rAAV2 and rAAV3 which do not utilize SIAs for primary attachment. Notably, the SLC35A1 KO markedly increased transduction of rAAV9 and rAAV11, which primarily attach to cells via binding to galactose. Further analyses revealed that KO significantly decreased vector nuclear import. More importantly, although the C-terminal cytoplasmic tail deletion (ΔC Tail) mutant of SLC35A1 did not drastically decrease SIA expression, it significantly decreased rAAV transduction, as well as vector nuclear import, suggesting the C-tail is critical in these processes. Furthermore, the T128A mutant significantly decreased SIA expression, but still supported rAAV transduction and nuclear import. These findings highlight the involvement of the CMP-SIA transporter in the intracellular trafficking of rAAV vectors post-internalization.