Adeno-associated viruses (AAVs) are non-pathogenic, replication defective viruses that can facilitate long-term gene transfer to a variety of tissues in vivo. However, the limited DNA packaging capacity of AAV (~5 kb) poses challenges for the transfer of large genes. Additionally, while AAV is a minimally immunogenic viral vector, the risk of potential immune response against exogenous, AAV-delivered transgene products isn’t fully understood. Here I describe two in vivo AAV-mediated gene transfer projects that utilize engineered, miniaturized versions of larger genes to overcome the AAV packaging capacity for the disparate purposes of gene therapy for Duchenne muscular dystrophy and vaccination against SARS-CoV-2. Duchenne muscular dystrophy (DMD) is a degenerative X-linked muscle disease caused by mutations that result in the absence of the giant, rod-shaped structural protein dystrophin. While we have previously demonstrated that systemic delivery of an AAV-deliverable miniaturized form of the dystrophin paralog utrophin (AAV-Utrophin) can complement for absence of dystrophin without the immunogenicity observed against similarly designed AAV-Dystrophin, we believe further improvements can be made to the design of the miniaturized utrophin proteins to improve their functionality and durability. Here we reconstructed the evolution of striated muscle as well as the piecemeal evolution of the dystrophin gene. Based on this evolutionary reconstruction of dystrophin’s origin, we propose a structural model for how the spectrin repeats within the rod domain of dystrophin and utrophin interact to form a continuous, interlocking structure. Using this structural model of the dystrophin/utrophin rod domain, we engineered a new AAV-deliverable utrophin protein (nano4-Utrophin) with a chimeric spectrin repeat capable of maintaining tensile strength along the entire truncated rod domain. We then demonstrate that following systemic administration using AAV9, nano4-Utrophin localizes to the sarcolemma, restores sarcolemmal localization of -Sarcoglycan, and prevents ongoing muscle regeneration in mdx mouse model of DMD. While this project utilized a paralogous protein approach to avoid transgene product immunogenicity, my second project capitalizes on AAV-delivered exogenous transgene immunogenicity. I demonstrate that a single intramuscular injection of AAV6 or AAV9 encoding a modified, N-terminal domain deleted spike protein induces robust cellular immunity and provides long-term protection in k18-hACE2 transgenic mice from lethal SARS-CoV-2 challenge, associated weight loss and pneumonia independent of vaccine-induced neutralizing humoral immunity. In both mice and macaques, vaccine-induced cellular immunity results in the clearance of transduced muscle fibers coincident with macrophage and CD8+ cytotoxic T cell infiltration at the site of immunization. Additionally, mice demonstrate a strong Type-1 polarized cellular immunophenotype and equivalent ex vivo T cell reactivity to peptides of wt and alpha (B.1.1.7) variant spike. These studies demonstrate not only that AAV6 and AAV9 can function as effective vaccine platforms, but also that vaccines can provide long-term efficacy primarily through the induction of cellular immunity.