AAV Production Everywhere: A Simple, Fast, and Reliable Protocol for In-house AAV Vector Production Based on Chloroform Extraction

Abstract

Recombinant adeno-associated virus (rAAV) is a mammalian virus that has been altered to be used as a gene delivery vehicle. Several changes to the viral genome have made them replication deficient so that this aspect of the viral infection cycle is under full control of the experimenter, while maintaining gene expression machinery. Over the last decades, rAAVs have become the gold standard for studying in vivo gene function and are especially favorable for gene transfer in the central nervous system. AAVs have been proven safe and provide stable gene expression over a long period of time. They are extensively used in preclinical experiments and show great potential for clinical applications. However, the use of AAVs in preclinical settings are often held back due to availability. Waiting lines are long at commercial production facilities, and in-lab production is hindered due to lack of specific laboratory equipment needed. Here we present a novel production method that can be carried out in any molecular biology laboratory using standard laboratory equipment. We provide a simple, fast, and streamlined protocol for production that can result in titers comparable with the more time-consuming iodixanol gradient ultracentrifugation method. The yield using this protocol is high enough for any type of study where AAV is the vector of choice. © 2020 The Authors.

Keywords: AAV vector; PEI transfection; adeno associated virus; chloroform extraction; gene expression; gene therapy; vector production.