Overcoming Barriers to AAV Gene Transfer

Recombinant adeno-associated virus (AAV) has emerged as a leading platform for therapeutic gene transfer. Despite significant progress and a string of FDA approvals, significant challenges that prevent the broader application of AAV gene therapy exist. High systemic dosing of AAV vectors in the clinic poses potential risk of severe and adverse side-effects due to anti-capsid immunity. While different immune modulatory regimens (IMR) are being explored, there is an urgent need for continued development of effective strategies to improve the safety of AAV gene therapies. In chapter 2 of this dissertation, I describe the discovery of a novel enzyme (IceM) that cleaves human IgM, a key trigger in the anti-AAV immune cascade. We then engineer a fusion enzyme (IceMG) with dual proteolytic activity against human IgM and IgG. IceMG cleaves B cell surface antigen receptors (BCR) and inactivates phospholipase gamma signaling in vitro. Importantly, IceMG is more effective at inhibiting complement activation compared to an IgG cleaving enzyme alone. Administration of IceMG in rhesus macaques enables robust and reversible clearance of both circulating IgM and IgG. Antisera from macaques treated with IceMG shows significantly decreased AAV neutralization as well as complement activation. Finally, we demonstrate that pre-treatment with IceMG restores AAV vector transduction in mice passively immunized with human antisera containing anti-AAV neutralizing antibodies. Thus, IgM cleaving enzymes show promise in simultaneously addressing multiple aspects of anti-AAV immunity mediated by B cells, circulating antibodies and complement. These studies have implications for improving safety of AAV gene therapies and more broadly, for use in organ transplantation and autoimmune diseases. In parallel, unraveling the biology of AAV entry and trafficking is central to developing improved AAV vectors. In chapter 3 of this dissertation, I delve into the biology of host factors that limit AAV transduction. We identify furin as a host factor that significantly restricts the transduction of AAV4-like serotypes. Through the interrogation of different steps and attachment factors in the AAV infectious pathway, we demonstrate that AAV cellular binding and uptake are significantly increased in a sialic acid-dependent manner. We postulate that furin likely plays a key role in regulating expression of cellular sialoglycans, which in turn, can influence permissivity to AAVs and possibly other viruses.