TBC1D23–AAVR interaction drives endosome-to-TGN trafficking required for rAAV transduction

Abstract The adeno-associated virus receptor (AAVR; also known as KIAA0319L) is the proteinaceous receptor required for the transduction of multi-serotype adeno-associated viruses (AAVs). While the extracellular polycystic kidney disease (PKD) domains of AAVR directly bind AAV capsids, the function of its C-terminal cytosolic domain (AAVR-C) in governing AAV internalization and intracellular trafficking remains undefined. Using targeted pulldown of AAVR-interacting host proteins with a glutathione S-Transferase (GST)-fused AAVR-C recombinant protein (GST-AAVR-C) as a bait, we identified that TBC1 domain family member 23 (TBC1D23), a specialized intracellular trafficking adaptor and bridging factor, binds AAVR via the AAVR-C and mediates AAV’s endosome-to- trans -Golgi network (TGN) transport. Biolayer interferometry (BLI) demonstrates nanomolar-affinity binding between AAVR-C and the C-terminal scaffold domain of TBC1D23 (TBC1D23-C). CRISPR-mediated knockout of TBC1D23 severely impairs rAAV transduction across multiple human cell types, including polarized human airway epithelium (HAE). Loss of TBC1D23 disrupts convergence of internalized AAV capsids at the TGN, leading to diminished nuclear import of AAV vectors. Mutational analyses of AAVR-C reveal that its acidic residue cluster is required for TBC1D23 binding and AAV retrograde transport from the endosome to the TGN. Together, these findings define TBC1D23 as a receptor-proximal trafficking module that couples AAV-AAVR engagement to vesicle transport, revealing a missing core regulatory step in AAV retrograde transport that is essential for productive rAAV transduction. Significance Recombinant adeno-associated viruses (rAAVs) are widely used gene delivery vectors, yet the intracellular trafficking steps that enable productive transduction remain incompletely defined. We identify TBC1D23 as a critical host adaptor that directly binds the cytosolic tail of the AAV receptor (AAVR) and mediates retrograde transport of AAV vectors from endosomes to the trans -Golgi network (TGN). Disruption of the AAVR acidic residue cluster abolishes TBC1D23 binding, blocks TGN trafficking, impairs nuclear import, and severely reduces transduction across multiple serotypes and in polarized human airway epithelium. These findings reveal a receptor-proximal trafficking checkpoint essential for rAAV gene delivery and define a host–vector interface with potential for rational vector engineering and therapeutic modulation.