41. U2 snRNP Spliceosome Proteins Block Recombinant AAV Vector Transduction

Abstract

The recent approval, continued success and safety of recombinant adeno-associated viral (AAV) vectors in gene therapy clinical trials have paved the way for therapeutic modalities for a range of genetic disorders. However, efficient therapeutic gene expression by AAV vectors can often require high vector doses, presenting potential challenges related to production and dose-related toxicity. In this regard, a particularly well known barrier to AAV transduction is the host proteasomal machinery. To identify specific host restriction factors, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively increased transduction by multiple AAV serotypes. Notably, genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased AAV vector transduction, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further study indicated that U2 snRNP proteins suppress AAV vector transgene expression through recognition of incoming AAV capsid and enhanced histone recruitment to the vector genome, independently of the cellular splicing machinery. In summary, we postulate a novel cellular mechanism involving spliceosome U2 snRNP proteins in regulating AAV vector epigenetic modification and its manipulation to enhance transgene expression for human gene therapy.