ABSTRACT After a first myocardial infarction (MI), 36% of male and 47% of female patients die within 5 years. This illustrates the inadequacy of current therapeutic interventions. The long-term goal of our laboratory is to develop reparative pluripotent stem cell (PSC)-based therapies that are immune-tolerated and meaningfully improve patient health and quality of life. The overall objectives of this R01 application are to: 1) use CRISPR/Cas9 gene-editing approaches to target adhesion molecules (AMs) on human PSC-derived cardiovascular therapies (PSC-CVTs) to disrupt the adherence, infiltration, and destruction of vascularized grafts by allogeneic immune cells; and 2) define optimal cellular composition and immunogenicity profiles of next-generation hypoimmune PSC-CVT grafts to maximize their reparative capacity in the inflammatory setting of MI. Our central hypothesis is that targeted deletion of AM genes will facilitate immune tolerance of PSC- CVTs via two mechanisms: 1) diminished immune cell contact-mediated destruction; and 2) anti-inflammatory effects (e.g., secreted factor and gene expression changes) directly associated with genetically disrupting AM function. The rationale for this project is that hypoimmune PSCs will be key clinical platforms in the coming years and improved gene-editing approaches are needed to achieve effective immune tolerance of PSC grafts. Additionally, successful validation of AM gene-editing in this project will provide a new avenue for advancing future transplantation therapies for other diseases. To attain our objectives, we will pursue the following specific aims (SAs): SA1) Define the effects of AM ablation on immune cell contact-mediated PSC-CVT graft destruction; SA2) Define the inflammatory responses initiated by immune cell:PSC-CVT graft interactions; and SA3) Determine the in vivo reparative capacity and immune-tolerance potential of AM knockout PSC-CVT grafts in the inflammatory MI setting. This research is significant because it validates a new graft strategy and testing platform for hypoimmune PSC therapies, with great potential to save lives and improve quality of life for many patients with MI and other pathologies characterized by cellular dysfunction in immune-competent anatomical sites. It is innovative because: 1) of the new approach of targeting immune cell adhesion in a manner anticipated to impede both adaptive and innate immune cell-mediated graft destruction; 2) it uses a tri- cellular PSC-CVT graft optimized for superior reparative function and hypoimmunogenicity; and 3) we rigorously interrogate the human immune response using advanced assays and models developed in our lab. Ultimately, this work will develop a breakthrough cardiac therapy well-suited for clinical trials, with the potential to save lives and improve the quality-of-life for millions of patients.