UNCOVERING STRUCTURAL COMPONENTS OF THE ADENO-ASSOCIATED VIRAL CAPSID THAT CAN BE MODIFIED TO IMPROVE CLINICAL GENE THERAPY OUTCOMES

Warischalk, Jayme

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March 19, 2019

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Warischalk, Jayme
- Affiliation: School of Medicine, Department of Pharmacology

Abstract

Recombinant adeno-associated virus (rAAV) has become increasingly employed as a gene delivery vector in clinical trials. Despite several notable instances of success using rAAV-based gene therapy, a number of obstacles must be overcome before such therapy will be widely utilized for the treatment of disease. These include the limited transduction efficiency of rAAV, the promiscuity of viral biodistribution following systemic administration, and the prevalence of neutralizing antibodies against the rAAV capsid that exists within the population at large, hindering the use of gene therapy in many patients. Each of these issues ultimately necessitates the administration of high doses of vector to achieve therapeutic effect - a costly prospect when feasible. Engineering the rAAV capsid protein to overcome these hurdles is an emerging strategy to better optimize the virion for translational studies. To develop new logical approaches to capsid engineering, it is necessary to uncover the structure-function relationships of various features of the rAAV capsid protein. In this dissertation, computational analysis of capsid crystal structure data was used to guide the molecular dissection of a single loop on the capsid surface, termed variable region 1 (VR1). We discovered that the destabilization of intra-loop hydrogen bond networks in rAAV serotype 1 (rAAV1) via select amino acid deletions in VR1 increased transduction efficiency by orders of magnitude in skeletal muscle following intramuscular injection. We further applied this strategy to numerous additional serotypes to create capsids harboring mutations designed to disrupt VR1 stability. These novel capsids specifically and efficiently target cardiac and skeletal muscle tissue following intravenous administration. This work is the first demonstration of which we are aware of a rational engineering strategy that can create muscle-targeted vectors in a conserved manner across serotypes. Our method expands the pool of reagents available to treat cardiac and musculoskeletal disease and offers an attractive pathway towards patient-personalized gene therapy. The ability to optimize numerous capsids using a single, easily accessible mutational strategy facilitates the pairing of a given serotype to a given patient based on preexisting variables such as neutralizing antibody titers, and removes the reliance on a single serotype for a given application.

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