TBC1D23-AAVR Interaction Drives Endosome-to-TGN Trafficking Required for rAAV Transduction

Area of Science:

• Molecular and Cell Biology
• Virology
• Gene Therapy

Background:

• Adeno-associated virus receptor (AAVR) mediates adeno-associated virus (AAV) entry into cells.
• The precise role of AAVR's cytosolic domain in AAV trafficking is not fully understood.
• Recombinant AAV (rAAV) vectors are vital tools for gene delivery, but their intracellular transport mechanisms require further elucidation.

Purpose of the Study:

• To identify host proteins interacting with the cytosolic tail of AAVR (AAVR-C).
• To elucidate the function of these interactions in AAV intracellular trafficking and transduction.
• To define the molecular mechanisms governing AAV retrograde transport.

Main Methods:

• Glutathione S-Transferase (GST)-pulldown assays using GST-AAVR-C to identify binding partners.
• Biolayer interferometry (BLI) to quantify binding affinity between AAVR-C and TBC1D23.
• CRISPR-mediated gene knockout of TBC1D23 in human cell lines and primary airway epithelium.
• Confocal microscopy to track AAV capsid localization and nuclear import.

Main Results:

• TBC1D23 was identified as a direct binding partner of AAVR-C.
• TBC1D23 mediates the retrograde transport of AAV from endosomes to the trans-Golgi network (TGN).
• Knockout of TBC1D23 significantly impaired rAAV transduction and nuclear import, particularly in polarized human airway epithelium.
• Specific acidic residues in AAVR-C are critical for TBC1D23 binding and subsequent AAV transport.

Conclusions:

• TBC1D23 acts as a critical host adaptor protein linking AAVR engagement to intracellular vesicle transport.
• The AAVR-C to TBC1D23 interaction represents a key checkpoint in AAV retrograde transport essential for productive gene delivery.
• Understanding this host-vector interface offers potential for optimizing rAAV vectors for gene therapy applications.