AAV8 transduction capacity is reduced by prior exposure to endosome-like pH conditions

Abstract

Adeno-associated virus (AAV) is an essential instrument in the neuroscientist’s toolkit, which allows delivery of DNA to provide labeling with fluorescent proteins or genetic instructions to regulate gene expression. In the field of neural regeneration, the transduction of neurons enables the observation and regulation of axon growth and regeneration, and in the future will likely be a mechanism for delivering molecular therapies to promote sprouting and regeneration after central nervous system injury. Traditional formulations of AAV preparations permit efficient viral transduction under physiologic conditions, but an improved understanding of the mechanistic limitations of AAV transduction may facilitate production of more resilient AAV strains for investigative and therapeutic purposes. We studied AAV transduction in the context of prior exposure of AAV serotype 8 (AAV8) to environmental pH within the range encountered during endosomal endocytosis (pH 7.4 to pH 4.4), during which low pH-triggered structural and autoproteolytic changes to the viral capsid are believed to be necessary for endosome escape and virus uncoating. Due to the fundamental nature of these processes, we hypothesized that premature exposure of AAV8 particles to acidic pH would decrease viral transduction of HT1080 cells in vitro, as measured by fluorescent reporter gene expression using high-content imaging analysis. We found that increasingly acidic incubation conditions were associated with concomitant reductions in transduction efficiency, and that quantitative levels of reporter gene expression in transduced cells were similarly decreased. The biggest decrease in transduction occurred between pH 7.4 and pH 6.4, suggesting the possible co-occurrence of a pH-associated event and viral inactivation within that range. Taken together, these findings indicate that exposure of AAV8 to acidic pH for as little as 1 hour is deleterious to transduction ability. Future studies are necessary to understand the pH-associated causative mechanisms involved. This study was approved by the University of Miami Institutional Animal Care and Use Committee, USA (Protocol #18-108-LF) on July 12, 2018.

Keywords

• adeno-associated virus; autolytic; capsid; low ph; protease; proteolytic; temperature; trafficking; transduction; viral inactivation