8. Further Characterization of U2 snRNP Mediated Restriction of AAV Vector Transduction
Authors: Claire A. Schreiber, Yoshihiro Izumiya, Aravind Asokan, Yasuhiro Ikeda
Journal: Molecular Therapy (2016)

Improving adeno-associated virus (AAV) vector transduction efficiency is central to the development of its continued, widespread use in gene therapy. More effective AAV transduction would reduce vector doses required for efficient gene delivery, minimizing the risks associated with high dose AAV vectors. Several treatments have been reported to increase AAV vector transduction, including adenoviral co-infection. Through screening of a siRNA library, we recently identified U2 snRNP as a host restriction factor for AAV vector transduction. Disruption of U2 snRNP spliceosome and associated proteins, including PHF5A, SF3B1, SF3B2 and U2AF1, potently enhanced AAV vector transduction. Relevant to gene therapy applications of AAV vectors, meayamycin B, a powerful SF3B1 inhibitor, allowed for substantial increases in AAV vector transduction (up to 400-fold). This post-entry restriction appeared to occur after the second-strand synthesis but before transgene expression or accumulation of transgene transcripts and independently of the cellular splicing machinery. No notable changes were found in the cytoplasmic trafficking, nuclear entry, or genome release of AAV vector infection by U2 snRNP inhibition. Here, we further studied the mechanism(s) underlying the U2 snRNP-mediated block of AAV vectors. We first tested another commercially available U2 snRNP inhibitor, pladienolide B (PladB) and verified that PladB treatment also showed substantial dose dependent increases (up to 700-fold) in AAV vector transduction. We next studied the relationship between adenoviral co-infection and U2 snRNP inhibition. Adenoviral co-infection alone enhanced AAV vector transduction up to 30-fold, while adenoviral co-infection, in addition to genetic or pharmacological U2 snRNP inhibition, showed marginal additive effects, suggesting a common pathway targeted by adenoviral co-infection and U2 snRNP inhibition in enhanced AAV vector infection. Using a series of plasmids providing adenoviral helper functions, we have identified Ad5 E4, but not the E2A and VA RNA genes, as partially responsible for the enhanced AAV transduction by adenoviral co-infection through U2 snRNP inhibition. Since the effects of U2 snRNP inhibition are most likely on the regulation of transgene expression, we also assessed the epigenetic modifications of the AAV2 genome in the presence or absence of PladB. Through chromosome immunoprecipitation (ChIP) assays we mapped histone recruitment patterns at different regions of the AAV vector epigenome. PladB treatment reproducibly enhanced recruitment of histone H2b and H3 proteins to the AAV CMV promoter, luciferase transgene, and polyA regions up to 3-fold. Our preliminary study also suggested that U2 snRNP inhibition altered specific histone modification patterns on the AAV vector genome. We are in the process of verifying the influence of U2 snRNP inhibition, as well as AAV capsid variations, on epigenetic modifications of the AAV vector epigenome. Better understanding the underlying mechanism would provide novel insights into host-virus interactions and could lead to the rational design of next generation AAV vectors with improved transduction efficiencies and safety profiles.