303. AAV Capsid Evolution for Enhanced Antibody Delivery To Human Muscle for Use in Next-Generation HIV Vaccines

Abstract

Our goal is to develop a recombinant adeno-associated virus (AAV) vector that is both resilient to neutralization by pre-existing human antibodies, and can efficiently transduce human muscle after systemic or intramuscular administration at levels sufficient to express therapeutic quantities of human monoclonal antibodies with broad-spectrum protection against HIV for use as a prophylactic vaccine and as a treatment for HIV-infected individuals. We hypothesized that capsid library screens in human skeletal muscle cells both in vitro and in vivo would be an ideal route to accomplish these goals. We utilized wild-type replicating AAV libraries of 10e5 variants via DNA shuffling of eleven different parental AAV capsids. Our libraries selectively replicate in human cells when co-administered with wild-type adenovirus, making chimeric humanized muscle mice and human muscle cell cultures excellent tools to allow for selection of capsids with tropism for human muscle cells. Two in vivo screens are ongoing in chimeric humanized muscle mice. These screens involve transplanting immunodeficient mice with pooled primary human muscle stem cells FACS-purified from skeletal muscle biopsies from five human donors. Human muscle progenitors fuse with those from the mouse and create chimeric myofibers expressing human muscle proteins for AAV selection. AAV capsid libraries are used to serially infect xenotransplanted mice and are then replicated with either Adenovirus-19b or 36. Two in vitro screens were performed in either primary human muscle stem cells isolated and purified from patient biopsies, or differentiated human myotubes. Completed screens were carried out for six rounds of replicating selection with Adenovirus-5 and the five most highly selected variants from each screen were sequenced and vectorized into EF1a-driven GFP-luciferase dual expression vectors. We are validating these 10 highly selected variants with comparisons to parental serotypes with some known muscle tropism (AAVs 1,6,8,9), as well as establishing neutralizing antibody levels against pooled human immunoglobulins. Vectors are being tested in humanized muscle mice and various human muscle cell lines (both healthy and diseased) by four methods of administration (intra-muscular, intra-arterial, intra-venous and intra-peritoneal) at a variety of doses for robust transgene expression. Best candidates will be used to package broadly neutralizing antibodies against HIV for further pre-clinical validation as a vaccine delivery agent. More generally, we hope to provide the muscle gene therapy community with a panel of optimized capsids with enhanced tropism for numerous muscle cell types with broad potential applications.