PURIFICATION METHOD FOR ADENO-ASSOCIATED VIRAL VECTOR SEROTYPE 9 USING CERAMIC HYDROXYAPATITE CHROMATOGRAPHY AND ITS QUALIFICATION

Abstract

ABSTRACTAdeno-associated virus (AAV) vectors can efficiently transduce exogenous genes into various tissuesin vivo. Owing to their convenience, high efficiency, long-term stable gene expression, and minimal side effects, AAV vectors have become one of the gold standards for investigating gene functionsin vivo, especially in non-clinical studies. However, challenges persist in efficiently preparing a substantial quantity of high-quality AAV vectors that comprise only genome-packaged full particles. Commercial AAV vectors are typically associated with high costs. Further, in-laboratory production is hindered by the lack of specific laboratory equipment, such as ultracentrifuges. Therefore, a simple, quick, and scalable preparation method for AAV vectors is needed for proof-of-concept experiments. Herein, we present an optimized method for producing and purifying high-quality AAV serotype 9 (AAV9) vectors using standard laboratory equipment and chromatography. Using ceramic hydroxyapatite as a mixed-mode chromatography medium can markedly increase the quality of purified AAV vectors. Basic protocols and optional methods for evaluating purified AAV vectors are also described.