PROJECT SUMMARY Adeno-associated virus (AAV) is the most commonly used gene therapy vector. Currently, FDA has approved two AAV-mediated therapies, and many more are in pipeline. However, there still are challenges to the effective use of AAV vectors. For example, high doses (~1014 vector particles) are necessary for most human applications. Such high vector doses cause tissue toxicity and elicit high levels of anti-transgene antibodies, limiting therapeutic efficacy. High doses also require large injection volumes and/or multiple injections to achieved desired levels of transgene expression. In addition, high manufacturing costs preclude widespread use of this approach. Each of these difficulties can be addressed by increasing the efficiency of AAV transduction and thereby reducing the number of AAV particles necessary to achieve a desired therapeutic outcome. The intramuscular route of vector delivery has key advantages. It helps circumvent the problem of pre- existing antibodies to the AAV capsid, which otherwise prevents efficient transduction. Expression of a transgene from muscle persists for years or decades due the longevity of this tissue. Finally, unlike other tissues targeted to express AAV transgenes such as the liver, the consequences of tissue damage through local inflammation are modest and manageable to address. However, the relative inefficiency with which AAV transduces human and non-human primate muscle tissues necessitates high vector doses. Therefore many challenges to the AAV-based therapies can be addressed by improving the efficiency of muscle-specific transduction. In the Preliminary Studies, we show that AAV9 modified in the variable region VIII with an insulin receptor-binding peptide is dramatically more efficient than wild-type AAV9 in transducing human differentiated muscle cells and mouse muscles in vivo. We propose in Aim 1 to further improve this vector through directed evolution and selection, and to assess the utility of this peptide in the context of other AAV serotypes. We also propose in Aim 2 to verify our hypotheses that by enhancing vector efficiency, we can reduce tissue toxicity, inflammation, and anti-transgene antibody production, thereby increasing the safety and therapeutic efficacy of these vectors. Together, these studies will identify an AAV vector that much more efficiently transduce muscle tissue, and in doing so, they will also address several challenges associated with AAV-mediated gene therapy.