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 1

Durable autologous engraftment following BCL11A enhancer gene editing.

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Durable autologous engraftment following BCL11A enhancer gene editing. (...
(A) Schematic representation of electroporation of rhesus CD34+ HSPCs with ribonucleoprotein (RNP) complex composed of 2×NLS SpCas9 or 3×NLS SpCas9 protein, and either BCL11A enhancer targeting (#1617) or AAVS1 targeting sgRNA. The cells are either used for ex vivo analysis or autologous transplantation. (B) Editing efficiency measured by Sanger sequencing with TIDE analysis, and (C) β-like globin RNA expression by RT-qPCR normalized to α-globin expression in nonedited (Mock), AAVS1-, and BCL11A-edited rhesus CD34+ HSPCs in small scale (ZL24 and ZL25, 5 × 104 cells, 200 pmol for both SpCas9 and sgRNAs) and large scale (ZL57, 5 × 106 cells, 1000 pmol for both SpCas9 and sgRNAs) electroporation conditions (n = 3, 1-way ANOVA followed by the Tukey’s post hoc test, **P < 0.01). (D) β-like globin protein expression by RP-HPLC (n = 3, 1-way ANOVA followed by the Tukey’s post hoc test, ** P < 0.01). ZL25 and ZL22 were transplanted with AAVS1- and BCL11A enhancer–edited cells (1:1). The gray rectangle represents the phlebotomy course. Editing frequencies in granulocytes for (E) AAVS1 and (F) BCL11A enhancer in transplanted rhesus macaques. Slopes were calculated separately for the first 13 weeks of transplantation (early progenitor phase) and later time points (HSC phase) as indicated by the dashed line. (G) Distribution of monoallelic and biallelic edited colonies collected from methylcellulose plates for bone marrow mononuclear cells of ZL25 at 100 weeks and ZL22 at 13 weeks after transplantation. (H) γ-globin protein expression percentage in ZL25 and ZL22.