Immunotherapy of acute myeloid leukemia (AML) has been challenging because the lack of tumor-specific antigens results in "on-target, off-tumor" toxicity.To unlock the full potential of AML therapies, we used CRISPR-Cas9 to genetically ablate the myeloid protein CD33 from healthy donor hematopoietic stem and progenitor cells (HSPCs), creating tremtelectogene empogeditemcel (trem-cel).Trem-cel is a HSPC transplant product designed to provide a reconstituted hematopoietic compartment that is resistant to anti-CD33 drug cytotoxicity.Here, we describe preclin. studies and process development of clin.-scale manufacturing of trem-cel.Preclin. data showed proof-of-concept with loss of CD33 surface protein and no impact on myeloid cell differentiation or function.At clin. scale, trem-cel could be manufactured reproducibly, routinely achieving >70% CD33 editing with no effect on cell viability, differentiation, and function.Trem-cel pharmacol. studies using mouse xenograft models showed long-term engraftment, multilineage differentiation, and persistence of gene editing.Toxicol. assessment revealed no adverse findings, and no significant or reproducible off-target editing events.Importantly, CD33-knockout myeloid cells were resistant to the CD33-targeted agent gemtuzumab ozogamicin in vitro and in vivo.These studies supported the initiation of the first-in-human, multicenter clin. trial evaluating the safety and efficacy of trem-cel in patients with AML (NCT04849910).