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. 2023 Aug 30;621(7978):404–414. doi: 10.1038/s41586-023-06496-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 9 — a. Peripheral blood lineage composition of NBSGW mice xeno-transplanted with AAVS1^BE and FLT3^N399 HSPCs at 8 weeks. Mean ± SD. AAVS1^BE N = 13, FLT3^N399 N = 18. Unpaired t-tests. b. Percent of AML cells (by mNeonGreen fluorescence) on total BM-derived CFUs plated with samples from the experiment reported in Fig. 4. Mean ± SD. One-way ANOVA. c. Percent of immature granulocytes (CD33/66b+14-10-11c-SSChigh) within CD33/66b+ BM cells. Same experiment reported in Fig. 4. Mean ± SD. One-way ANOVA. d. Lineage-negative CD34+ progenitors are depleted by 4G8 CAR-T in vivo and protected by FLT3^N399 editing (experiment reported in Fig. 4). (Left) Representative flow cytometry plots of lineage-neg cells (mNeonGreen-CD3-19-14-11c-56-) with gating of CD34+ progenitors (% reported within the gate). (Right) % of lin-CD34+ cells within hCD45+3-mNeonGreen- BM cells. Mean ± SD. One-way ANOVA. e. Relative composition of BM lin-CD34+ of mice from the experiment reported in Fig. 4. Mean ± SD. two-way ANOVA with multiple comparison (only significant AAVS1 vs FLT3^N399 comparisons are reported). HSC, hematopoietic stem cells; MPP, multipotent progenitors; LMPP, lymphoid-primed multipotent progenitors; CMP, common myeloid progenitors; GMP, granulo-mono progenitors; MEP, mega-erythroid progenitors. f. FLT3 expression (MFI) on myeloid (left) and lymphoid (right) BM subsets at experimental endpoint. LMPP, MPP and HSC from 4G8 CAR treated AAVS1^BE conditions are not evaluable (NE) due to low cell number. Mean ± SD. Multiple unpaired t-tests. g. CAR-T cell phenotype by flow cytometry in the BM of mice from Fig. 4. CD45RA+62L+, Naïve; CD45RA-62L+, central memory, CM; CD45RA-62L-, effector memory, EM; CD45RA+62L, terminally differentiated EM cells re-expressing CD45RA, EMRA. h. Bar plots reporting (from left to right) % of EGFRt+ within BM CD3+ cells, PD1 (CD279) MFI and CD69 MFI on BM CD8+ and CD4+ CAR T cells. Mean ± SD. Comparisons between AAVS1^BE and FLT3^N399 conditions by one-way ANOVA are reported. i. Peripheral blood lineage composition of NBSGW mice xeno-transplanted with AAVS1^BE and CD123^S59 HSPCs at 10 weeks (experiment reported in Extended Data Fig. 10). Mean ± SD (N = 11). Unpaired t-tests.