BCL11A enhancer–edited hematopoietic stem cells persist in rhesus monkeys without toxicity
Selami Demirci, … , Daniel E. Bauer, John F. Tisdale
Selami Demirci, … , Daniel E. Bauer, John F. Tisdale
Published September 8, 2020
Citation Information: J Clin Invest. 2020;130(12):6677-6687. https://doi.org/10.1172/JCI140189.
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Research Article Hematology

BCL11A enhancer–edited hematopoietic stem cells persist in rhesus monkeys without toxicity

Abstract

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer–edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer– or control locus–targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.

Authors

Selami Demirci, Jing Zeng, Yuxuan Wu, Naoya Uchida, Anne H. Shen, Danilo Pellin, Jackson Gamer, Morgan Yapundich, Claire Drysdale, Jasmine Bonanno, Aylin C. Bonifacino, Allen E. Krouse, Nathaniel S. Linde, Theresa Engels, Robert E. Donahue, Juan J. Haro-Mora, Alexis Leonard, Tina Nassehi, Kevin Luk, Shaina N. Porter, Cicera R. Lazzarotto, Shengdar Q. Tsai, Mitchell J. Weiss, Shondra M. Pruett-Miller, Scot A. Wolfe, Daniel E. Bauer, John F. Tisdale

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Figure 4

Gene editing dynamics after BCL11A enhancer editing.

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Gene editing dynamics after BCL11A enhancer editing. Relative loss of en...
Relative loss of engrafted edits repaired by MMEJ in transplanted animals for (A) BCL11A enhancer and (B) AAVS1 alleles. Deletions of at least 8 bp were categorized as MMEJ, while all other indels were categorized as NHEJ. (C) Shannon diversity index (H) for edit distribution in input cell products and engrafted samples. Granulocytes and lymphoid lineage data are plotted symmetrically, with the former on the left (for animals ZL22 and ZL25, both BCL11A enhancer and AAVS1 data are reported; for ZM17 and ZM26, only BCL11A enhancer data are reported). (D) Correlation of Shannon diversity index (H) with infusion cell dose in all transplanted animals (12–13 weeks after transplantation). Granulocytes (circles), lymphocytes (squares), BCL11A enhancer (solid shapes), AAVS1 (open shapes). (E) Off-target analysis of granulocytes in edited animals transplanted with BCL11A enhancer–edited CD34+ HSPCs. Using CIRCLE-Seq, 26 potential genomic off-target sites for BCL11A enhancer guide #1617 were identified. Editing frequencies at the 26 sites were evaluated through amplicon deep sequencing and analysis. The samples include the infusion product (input) and granulocyte fraction of a later peripheral blood collection for each rhesus macaque. Each point represents a single replicate.