Adeno-associated virus (AAV) has emerged as a promising gene delivery vector because of its non-pathogenicity, simple structure and genome, and low immunogenicity compared to other viral vectors. However, its adoption as a safe and effective gene therapy treatment for disease relies on targeting the vector to deliver transgenes to desired cell populations. To this end, our lab has developed a protease-activatable virus (PAV) model based on AAV that responds to elevated protease activity commonly found in diseased tissue microenvironments. This thesis explores the expansion of this platform from the original AAV2-based prototype into AAV serotype 9 to allow for greater clinical translation, specifically for cardiac disease applications. These AAV9-based PAVs have been characterized in vitro, and the activatability of these PAVs ranges from 2.5x to 5.4x differences between the “locked” vs. “unlocked” states. The PAVs have also been characterized in vivo in a murine MI model. Compared to wild-type AAV9, PAV-L001 is able to deliver a reporter transgene site-specifically to the injury region of the heart, with decreased delivery to off-target organs. We are currently investigating the therapeutic efficacy of the PAV packaging the YAP5SA transgene in the murine MI model. This work also explores a new PAV lock format that incorporates different protease cleavage motifs besides MMPs in an attempt to make the PAV response to MMPs more modular and predictable. While this lock format does not display the desired behavior, it reveals the need for further characterization of the PAV lock behavior. To answer some of the remaining questions about the PAV lock behavior, I also demonstrate a method for the quantification of the kinetics of PAV cleavage by MMPs through the development of a simplified virus-like particle model, and preliminary results suggest that the C-terminal motif of the lock may be cleaved slightly faster than the N-terminal motif by MMP-9. Preliminary results also suggest that both sides of the lock must be cleaved off AAV9-based PAVs for the virus to resume binding. Overall, this thesis expands the protease-activatable virus toolkit for new disease applications and advances our understanding of PAV behavior.