Viruses have evolved genetically encoded responsive behaviors that allow for specific cell targeting, cell-entry, genome delivery, and replication. Harnessing and reprograming these behaviors for desired treatments is the goal of the burgeoning field of synthetic virology - allowing for the creation of novel virus-based nanoparticles to diagnose diseases or deliver targeted therapies. Adeno-associated virus (AAV) is a strong candidate for synthetic virology due to its relatively simple structure, broad tropism, and lack of pathogenicity. However, the broad tropism of AAV can be detrimental to the development of gene therapy treatments since off-target delivery can lead to cytotoxic effects. To address this problem, our lab has previously developed a protease-activatable vector (provector) platform based on AAV serotype 9 that responds to extracellular proteases present at disease sites. Building on this platform, I sought to develop a high-throughput pipeline for future provector development and engineer a provector targeting membrane-bound enzymes, such as MMP-14, that are upregulated in sites of pancreatic ductal carcinomas. Overall, this thesis details the exploration of the provector design using rational and library-screening techniques to develop a pathway for further high-throughput provector development.