Project Summary CAR T cell therapies have been revolutionary for patients with relapsed/refractory hematologic malignancies; however, the relapse rates are unacceptably high, with ~60% of patients relapsing with leukemia and lymphoma, and for multiple myeloma (MM), where more follow up is needed, relapse is likely even higher. Notably, adult patients rarely develop long-term immunologic memory or functional long-term persistence of CAR T cells, with relapse predictably often occurring after the loss of functional adoptive anti-tumor immunity. One solution to short-lived adoptive immunity is to genetically modify hematopoietic stem cells (HSCs) with the transgene for the CAR. This would result in the CAR being expressed across hematopoiesis long-term. However, as cytokine release syndrome (CRS) is mediated by T and myeloid cells, this long-term pan-hematopoietic expression would put patients at great risk for cytokine-mediated or other toxicity. We propose an alternative concept, where HSCs are stably integrated with DNA encoding the CAR; however, transcription is only initiated once cells have differentiated into NK cells to generate safe and efficacious “HSC CAR-NK factories”. We selected NK cells as the risk of CRS is minimal, there is no need for thymic differentiation, and they have a proven track record of CAR-mediated killing; however, the defining limitation of CAR-NK cell therapies has been persistence, which this approach is designed to address via long-term continuous renewal. We hypothesize that including NK-specific regulatory elements controlling CAR transgene expression will generate NK-specific, highly efficacious long-term anti-cancer immunity. In preliminary data, we demonstrated that patient HSC-based CAR transduction and transplantation in the setting of murine models that generate minimal T cells can be highly active in preventing tumor growth; NK cells generated this way and isolated ex vivo maintain cytolytic anti-tumor function through the CAR. We further identified, via analysis of comparative ATAC-seq data, a library of NK-specific regulatory elements and found a lead assembly of these regulatory elements that reproducibly led to CAR expression highly enriched in NK cells; a finding that we validated in different in vivo HSC transplant/NK differentiation models using patient HSCs. Here, we will investigate if a naturally occurring NK-specific regulatory element assembly, (Aim 1) can be leveraged to develop a model HSC-based persistent adoptive cellular therapy platform for anti-tumor immunity, and (Aim 2) seek to understand the biology and maximize the efficacy of a “HSC CAR-NK cell factory” approach. Specifically, we will use three separate in vivo models that allow for NK development post-huHSC transplantation.