722. AAV Capsid Evolution for Enhanced Antibody Delivery to Human Skeletal Muscle for Use in Next-Generation HIV Vaccines and Muscle Gene Therapies
Authors: Nicole K. Paulk, Gregory Charville, Katie Maguire, Katja Pekrun, Yue Zhang, Matthew Tiffany, Jose Vilches-Moure, Gordon Lee, Joseph Shrager, Thomas Rando, Mark A. Kay
Journal: Molecular Therapy (2016)

Our goal is to develop an AAV vector that can transduce human skeletal muscle after intramuscular administration at levels sufficient to express therapeutic levels of antibodies with broad-spectrum protection against HIV. AAV has begun being used in passive vaccines for HIV and influenza. Yet for many indications, greater human skeletal muscle transduction is needed than has been achieved with existing AAV serotypes. To bioengineer novel capsids with unparalleled human skeletal muscle transduction, we utilized primary human skeletal muscle cells from surgical resections to screen libraries of replication-competent shuffled AAV capsids. Two screens were performed in pools of primary human skeletal muscle stem cells or myotubes from six patients. Six rounds of replicating selection were carried out and the five most highly selected variants from each screen were vectorized and validated against existing serotypes with muscle tropism (AAV1, 6 and 8). In primary human muscle stem cells, variants NP22, NP66 and NP94 had significantly increased transduction that ranged from a 65 to 284-fold improvement over AAV1, 27 to 118-fold over AAV6, and 10 to 45-fold over AAV8. In primary human myotubes, NP22, NP66 and NP94 again showed significantly increased transduction that ranged from a 13 to 464-fold improvement over AAV1, 25 to 871-fold over AAV8, and NP94 showed a 15-fold improvement over AAV6. To assess human skeletal muscle transduction capabilities in vivo, we injected xenografted humanized muscle NSG mice with shuffled or control capsids expressing luciferase by intramuscular injection (1E9/leg) and assessed transduction weekly by live imaging in time course studies over two months. AAV6 was the first to uncoat but NP66 produced the highest sustained transduction levels. To control for species-specific transduction, we performed the same time course experiment in strain- and gender-matched non-transplanted mice. Strikingly, AAV6 outperformed shuffled variants when no human muscle fibers were present, highlighting the importance of performing preclinical studies in human cells and xenograft systems whenever possible to prevent misleading results which are mouse-specific. To further demonstrate the superiority of our shuffled variants and more accurately predict eventual muscle transduction in humans, we transduced human skeletal muscle explants from surgical resections ex vivo. Four adult patients (two male and two female) had healthy skeletal muscle tissue removed for ex vivo transduction analyses with luciferase. In all 4 patients, NP22 and NP66 (and NP94 in 3/4 patients) had significantly increased transduction by live fiber luciferase imaging as well as luciferase assays on lysed fibers that ranged from a 4 to 116-fold improvement over all control serotypes. To support preclinical vaccine testing in non-human primates, we assessed transduction in rhesus macaque skeletal muscle explants ex vivo. Here again we showed that NP22 and NP66 showed a 30 to 57-fold improvement in transduction over control serotypes. Taken together, our results demonstrate that capsid variants NP22 and NP66 (and in some cases NP94), have highly significant increased human skeletal muscle fiber transduction when assessed in vitro, in vivo and ex vivo. These capsids represent powerful tools to express therapeutic quantities of human monoclonal antibodies for use in passive vaccines, or transgenes for various muscle disorders in gene therapy, specifically in humans.