Enhancing adeno-associated virus cellular entry through receptor engineering

Adeno-associated viruses (AAV) are approved for gene therapy of several genetic disorders; however, key aspects of AAV cellular entry remain poorly understood. We previously identified carboxypeptidase D (AAVR2) as an AAV receptor distinct from the multi-serotype AAV receptor KIAA0319L (AAVR). In this study, we investigate the molecular mechanisms and biological roles of AAVR and AAVR2 in mediating AAV gene transfer. Using proximity-dependent biotin identification (BioID), we defined the interactome of AAVR in the presence or absence of AAV8 and identified various proteins involved in viral entry including AAVR2. We confirmed a direct physical interaction between AAVR and AAVR2, mediated by non-AAV interacting regions in the C-termini. Further, we identified functional motifs within the carboxy-terminal tails of both receptors to facilitate the engineering of chimeric receptors with enhanced activity. Functional assays demonstrated that the overexpression of AAVR or AAVR2 enhances the cellular attachment and entry of AAV in a serotype-specific manner. Finally, we generated a stable cell line expressing a minimal functional AAVR2 with increased sensitivity for in vitro potency testing for AAVR2-engaging serotypes like AAV8. Collectively, these findings reveal significant functional similarities in AAV receptor biology and establish a framework for engineering receptor-guided modalities.