97. Stability and Inactivation of AAV Serotype 1 Vectors

Abstract

Adeno-associated viral (AAV) vectors have become a commonplace tool for gene delivery ranging from cell lines to human gene therapy. One feature that makes AAV desirable for gene delivery is its relative safety owing to no known disease associated with wild-type AAV. For this reason, it is often used with BSL1 practices negating the need for dedicated BSL2 facilities. Another useful property of AAV is its stability, both the duration of transgene expression and retention of infectivity as a viral particle. In the current study, we examine the stability of AAV serotype 1 vectors which are capable of transducing human cells and are routinely produced and used in our facility. First, we examined stability at 4° C for up to 7 weeks and found <10% decrease in transduction efficiency compared to a freshly-thawed aliquot. After 10 freeze-thaw cycles, 25% loss in transduction efficiency was observed. These data indicated that AAV serotype 1 is a highly stable viral particle with regards to retaining infectivity. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, we demonstrate that infectious AAV vector expressing Cre-recombinase can be reconstituted and used up to 7 days at room temp, although there was a clear decrease in transgene expression over time. The stability of AAV is a desired feature as a tool; however, it is important that the vector can be inactivated to avoid transgene delivery to end users or cross-contamination of non-disposable instruments. AAV decontamination procedures can vary. We tested multiple disinfectants commonly used in the laboratory for their ability to inactivate an AAV serotype 1 vector. The vector was added directly into solution for 5 or 30 minutes at a 1:10 ratio. It was then diluted, dialyzed, concentrated, and tested for transduction on primary neurons. Of the decontamination procedures tested, only autoclaving, 3.9% peracetic acid, iodine or 10% Clorox bleach completely prevented the subsequent detection of transgene expression. Although the nature of the transgene (e.g. proto oncogene or toxin) can elevate an AAV vector to BSL2, our data show that when working with AAV serotype 1 vectors, it's important that safety procedures for handling and decontaminating AAV are effective to avoid inadvertent transgene expression in human cells or cross contamination of AAV vectors used in non-disposable instruments. Other serotypes have not yet been tested.