Leukemia https://doi.org/10.1038/s41375-018-0277-8 BRIEF COMMUNICATION Acute myeloid leukemia Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33 exon 2 Olivier Humbert 1 ● George S. Laszlo 1 ● Sophie Sichel 1 ● Christina Ironside 1 ● Kevin G. Haworth 1 ● Olivia M. Bates 1 ● Mary E. Beddoe 1 ● Ray R. Carrillo 1 ● Hans-Peter Kiem 1,2 ● Roland B. Walter 1,3 Received: 7 May 2018 / Revised: 17 August 2018 / Accepted: 10 September 2018 © Springer Nature Limited 2018 CD33 has long been pursued as immunotherapeutic target in acute myeloid leukemia (AML) [1, 2]. Improved survival with gemtuzumab ozogamicin (GO) validates this approach [3]. Partly stimulated by GO’s success, several investiga- tional CD33-directed therapeutics are currently in clinical testing [4]. However, CD33 expression on normal hema- topoietic cells leads to “on-target, off-leukemia” toxicity with significant morbidity/mortality from profound cyto- penias, limiting the use of CD33-directed immunotherapies [4]. This toxicity should be minimal if normal blood cells did not express the epitope targeted by these antibodies. Supporting the feasibility of CD33-engineering the hema- topoietic system are the findings that CD33-deficient mice have a very mild phenotype and show no difference in cellular response to pro-inflammatory stimuli compared to wild-type animals, indicating functional degeneracy between CD33 and other proteins [5]. Moreover, recent studies have shown that CRISPR/Cas9-mediated disruption of the CD33 coding region in CD34+ hematopoietic stem and progenitor cells (HSPCs) may not affect engraftment [6], suggesting that the generation of CD33-manipulated hematopoiesis is a clinically viable strategy to protect from “off-leukemia” cell toxicity of CD33-directed immu- notherapy. Here we have investigated an alternative, precise CD33 genome-editing approach that would only eliminate exon 2 and therefore the V-set immunoglobulin-like domain, which is the target of all current clinical CD33- directed approaches. Our editing strategy is expected to result in expression of a naturally occurring shorter isoform of CD33 (CD33 ΔE2 ) but not full-length CD33 (CD33 FL ), which may minimize potential adverse effects associated with disruption of the entire CD33 locus. We used CRISPR/ Cas9 [7–10] to accomplish this goal and functionally assessed genome-edited human hematopoietic cells in vitro and in immunodeficient mice. Human myeloid ML-1 cells and human fetal liver CD34 + HSPCs were used for our studies. ML-1 cells were maintained as described [11]. Human fetal liver CD34+ cells were enriched by immunomagnetic separation from tissue obtained from Advance Bioscience Resources Inc. (ABR, Alameda, CA). Cells were cultured in StemSpan SFEMII media (StemCell Technologies, Cambridge, WA) supplemented with penicillin/streptomycin (Life Technolo- gies, Carlsbad, CA), Stem cell factor , Thrombopoietin (both PeproTech, Rocky Hill, NJ), and FLT3-L (Miltenyi Biotec, Auburn, CA). CRISPR/Cas9-editing was carried out by electroporation of purified Cas9 protein (TrueCut Cas9 V2; ThermoFisher Scientific, Waltham, MA) complexed with synthetic guide RNAs (sgRNAs; Supplementary Table 1), which were modified at the 5′ and 3′ ends with 2′- O-methyl-3′-phosphorothiate (Synthego, Redwood City, CA) using the ECM 380 Square Wave Electroporation system (Harvard Apparatus, Cambridge, MA) [12]. For evaluation of colony-forming units (CFUs), 1500 CD34+ cells were seeded in 3.5 mL ColonyGEL 1402 (ReachBio, Seattle, WA) and scored after 12–14 days. CFU DNA was extracted in QuickExtract (Epicentre, Madison, WI). We quantified drug-induced cytotoxicity as described previously [11, 13]. Briefly, parental and CRISPR- engineered ML-1 cells were incubated in 96-well round These authors contributed equally: Olivier Humbert, George S. Laszlo and Hans-Peter Kiem, Roland B. Walter * Olivier Humbert ohumbert@fredhutch.org 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 2 Division of Medical Oncology, Department of Medicine, University of Washington, Seattle, WA, USA 3 Division of Hematology, Department of Medicine, University of Washington, Seattle, WA, USA Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41375-018-0277-8) contains supplementary material, which is available to authorized users. 1234567890();,: 1234567890();,: