38. Pharmacological Regulation of Vesicular Trafficking as a Strategy to Enhance Recombinant AAV Transduction

Abstract

Vector dose-related toxicity has been highlighted as a potential safety concern in recent preclinical studies and clinical trials with recombinant AAV vectors. Thus, investigation of strategies to enhance AAV transduction may allow for administration of lower vector doses, thereby mitigating toxicity. Several host cell factors have been shown to restrict viral infection during post-entry trafficking steps through a variety of mechanisms. In the case of AAV, the 26S proteasome is thought to reduce transduction efficiency by restricting nuclear entry via ubiquitin-dependent capsid degradation. The AAA+ ATPase p97/valosin-containing protein (VCP) is a segregase and unfoldase which is closely associated with the proteasomal degradation pathway and disassembles multimeric protein complexes for subsequent processing. VCP modulates numerous cellular processes ranging from endosomal sorting to autophagy and ER-associated degradation. In the current study, we sought to determine whether VCP plays a role as a restriction factor in the AAV infectious pathway. Chemical inhibition of VCP by Eeyarestatin I (EerI) increased AAV transduction by nearly an order of magnitude in a cell type and serotype independent manner. This effect was associated with increased recovery of viral genomes and capsid accumulation within the nucleus, but not cell surface binding or uptake. Interestingly, VCP appears to restrict AAV infection by regulating vesicular trafficking of viral particles. In particular, VCP inhibition redistributes AAV capsids to Rab7 and LAMP1 positive vesicles, while simultaneously disrupting capsid association with syntaxin-5 positive vesicles recently shown to be essential for Golgi transport. These results support the notion that AAV vesicular transport can be redirected to enhance transduction efficiency. In summary, our results identify VCP as a novel host restriction factor that regulates the post-entry trafficking of recombinant AAV vectors, and highlight VCP as a potential target for modulating AAV transduction in the clinic.