Comprehensive biophysical characterization of AAV-AAVR interaction uncovers serotype- and pH-dependent interaction

After more than two decades of research and development, adeno-associated virus (AAV) has become one of the dominant delivery vectors in gene therapy. Despite the focused research, the cell entry pathway for AAV is still not fully understood. Universal AAV receptor (AAVR) has been identified to be involved in cellular entry of different AAV serotypes. With the unveiling of the high-resolution AAV-AAVR complex structure by cryogenic electron microscopy, the atomic level interaction between AAV and AAVR has become the focus of study in recent years. However, the serotype dependence of this binding interaction and the effect of pH have not been studied. Here, orthogonal approaches including bio-layer interferometry (BLI), size-exclusion chromatography coupled to multi-angle laser scattering (SEC-MALS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) were utilized to study the interaction between selected AAV serotypes and AAVR under different pH conditions. A robust BLI method was developed and the equilibrium dissociation binding constants (K_D) between different AAV serotypes (AAV1, AAV5 and AAV8) and AAVR was measured. The binding constants measured by BLI together with orthogonal methods (SEC-MALS and SV-AUC) all confirmed that AAV5 has the strongest binding affinity followed by AAV1 while AAV8 binds the weakest. It was also observed that lower pH promotes the binding between AAV and AAVR and neutral or slightly basic conditions lead to very weak binding. These data indicate that for certain serotypes, AAVR may play a prominent role in trafficking AAV to the Golgi rather than acting as a host cell receptor. Information obtained from these combinatorial biophysical methods can be used to engineer future generations of AAVs to have better transduction efficiency.