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Abstract
Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.
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1 Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA
2 Glenn Center for Biology of Aging and Department of Neurology & Neurological Sciences, Stanford University, Stanford, CA 94305, USA; Department of Pathology, Stanford University, Stanford, CA 94305, USA
3 Glenn Center for Biology of Aging and Department of Neurology & Neurological Sciences, Stanford University, Stanford, CA 94305, USA
4 Department of Comparative Medicine, Stanford University, Stanford, CA 94305, USA
5 Department of Surgery, Division of Plastic & Reconstructive Surgery, Stanford University, Stanford, CA 94305, USA
6 Department of Cardiothoracic Surgery, Division of Thoracic Surgery, Stanford University and VA Palo Alto Health Care System, Stanford, CA 94305, USA
7 Glenn Center for Biology of Aging and Department of Neurology & Neurological Sciences, Stanford University, Stanford, CA 94305, USA; Neurology Service and Rehabilitation Research and Development Center of Excellence, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA