Production of Adeno-Associated Virus Vector Serotype rh.10 and Optimization of Its Purification via Chloroform Extraction.

Abstract

Recombinant adeno-associated virus (AAV) vectors are widely used for manipulating gene expression. AAVrh.10 is a highly infectious AAV serotype for the central nervous system and various tissues. Owing to its potential use in research, we aimed to optimize the production strategy and develop a simple purification protocol for the AAVrh.10 vector. In this study, we explored a simple production and purification strategy for the AAVrh.10 vector via chloroform extraction and ultrafiltration. Initially, we evaluated the optimal conditions for AAVrh.10-CAG-GFP production using AAV-293 cells. AAVrh.10-CAG-GFP was successfully produced in a serum-free medium after plasmid transfection. Moreover, the culture medium contained a substantial amount of the virus. Therefore, both AAVrh.10-containing cell lysate and culture medium should be used to prepare the AAVrh.10 viral vector. To purify and concentrate AAVrh.10-CAG-GFP from the crude lysate and medium, we optimized the chloroform extraction and ultrafiltration strategies. Subsequently, purified AAVrh.10-CAG-GFP was used to infect HEK-293T cells. Overall, this study provides a simple and effective AAVrh.10 vector preparation strategy for basic and preclinical research.