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

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.

Therapeutic base editing of human hematopoietic stem cells.

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and ß-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.