PROJECT SUMMARY While immunotherapies have proved compelling efficacy against other leukemias, their application for acute myeloid leukemia (AML) is still hampered by the absence of tumor-restricted targets. The most suitable AML targets are shared with healthy hematopoietic stem/progenitor cells (HSPCs) or mature myeloid cells, leading to on-target/off-tumor toxicity and impairment of hematopoietic reconstitution. To address this issue, we hypothesized that epitope-engineering of donor HSPCs used for conventional bone marrow transplantation can endow hematopoietic lineages with selective resistance to CAR-T or monoclonal antibodies (mAb), without affecting protein function or regulation. This strategy allows targeting genes essential for leukemia survival regardless of shared expression on HSPCs, thus reducing the risk of tumor immune escape by antigen downregulation/loss. We have already identified single amino-acid (aa) changes that abrogate the binding of therapeutic mAb targeting FLT3, CD123, and KIT and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation capacity. We confirmed the in vivo resistance of epitope-edited hematopoiesis to CAR-T treatment and the concomitant eradication of patient-derived AML xenografts (Casirati et al., Nature 2023). Here, we will capitalize on these achievements and exploit state-of-the-art genetic engineering tools and in vivo modeling with the objectives to i) generate and functionally validate “stealth” FLT3, KIT, and IL3RA genes by multiplex base-editing; ii) identify the best- performing CAR configuration for multi-Ag targeting on AML samples and iii) validate resistance of edited HSPC to new immunotherapies directed against these targets. This project will provide fundamental advancement of new and more effective immunotherapy approaches for AML that should additionally have broad applicability to several other hematopoietic malignancies.