Engineering a highly durable adeno-associated virus receptor for analytical applications.

Adeno-associated virus (AAV) is a major viral vector used in gene therapy. There are multiple AAV serotypes, and many engineered AAV serotypes are developed to alter their tissue tropisms with capsid modification. The universal AAV receptor (AAVR) is an essential receptor for multiple AAV serotypes. Since most AAV serotypes used in gene therapy infect cells via interaction with AAVR, the quantification of the vector-binding ability of AAV to AAVR could be an important quality check for therapeutic AAV vectors. To enable a steady evaluation of the AAV-AAVR interaction, we created an engineered AAVR through mutagenesis. Engineered AAVR showed high durability against acid while retaining its AAV-binding activity. An affinity chromatography column with the engineered AAVR was also developed. This column enabled repeated binding and acid dissociation measurements of AAVR with various AAV serotypes. Our data showed that the binding affinities of AAV2 to AAVR were diverse among serotypes, providing insight into the relationship with the infection efficiency of AAV vectors. Thus, this affinity column can be used in process development for quality checks, quantitating capsid titers, and affinity purification of AAV vectors. Furthermore, this column may serve as a useful tool in novel AAV vector capsid engineering.