Gene therapy is the next evolution in the treatment of diseases, allowing for the correction of genetic mutations, induction of growth to facilitate recovery at injury sites, or the delivery of suicide genes to tumor cells. However, the translation of more gene therapies to the clinic has been hindered by the lack of delivery specificity and efficiency. Many viral vectors have been engineered in an attempt to increase transduction efficiency and targeting capabilities. Adeno-associated virus (AAV) has recently become popular as a gene delivery vector because it has the ability to transduce many cell types efficiently, it is non-pathogenic as well as replication deficient, and it is able to be genetically modified. AAV is a promising vector for gene therapy, but its broad tropism can be detrimental if the transgene being delivered is harmful when expressed in non-target tissues. Delivering the transgene of interest to target cells at levels high enough to be effective while maintaining safety by minimizing delivery to off target cells is a prevalent challenge in the field of gene therapy. To address this problem, our lab has developed a protease-activatable vector (provector) platform based on AAV9 that responds to extracellular proteases present at disease sites. This thesis details my work expanding the provector platform to target cysteine-aspartic proteases as well as matrix-metalloproteinases as stimuli for provector activation. These caspase-activatable provectors demonstrate up to 200-fold reduction in transduction ability in the OFF state compared to AAV9, reducing the virus’ ability to transduce healthy tissue. Following proteolysis by caspase-3, the provector shows a 90-fold increase in transduction compared to the OFF state. This provector has also been characterized in vivo, where compared to AAV9 the provector has significantly decreased delivery to off target organs with similar levels of delivery to the injured heart following a myocardial infarction. This work also details characterization of the of the MMP-activatable provector in disease models of acute heart failure, stroke, and chronic heart disease while also explores the therapeutic efficacy of delivering various therapeutic transgenes in murine MI models. To further increase the control of gene delivery with the provector platform, I also detail designs and in vitro testing of provectors requiring two inputs for activation. Preliminary results indicate the vectors perform as designed in vitro which could provide higher specificity of delivery in vivo. Overall, this thesis diversifies the provector platform to target new diseases and increases our understanding of the provector behavior in vitro and in vivo.