Editing aberrant splice sites efficiently restores ß-globin expression in ß-thalassemia.

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from ß-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of ß-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent ß-thalassemia genotypes with currently available gene-editing technology.

Dissecting ELANE neutropenia pathogenicity by human HSC gene editing.

Severe congenital neutropenia (SCN) is a life-threatening disorder most often caused by dominant mutations of ELANE that interfere with neutrophil maturation. We conducted a pooled CRISPR screen in human hematopoietic stem and progenitor cells (HSPCs) that correlated ELANE mutations with neutrophil maturation potential. Highly efficient gene editing of early exons elicited nonsense-mediated decay (NMD), overcame neutrophil maturation arrest in HSPCs from ELANE-mutant SCN patients, and produced normal hematopoietic engraftment function. Conversely, terminal exon frameshift alleles that mimic SCN-associated mutations escaped NMD, recapitulated neutrophil maturation arrest, and established an animal model of ELANE-mutant SCN. Surprisingly, only -1 frame insertions or deletions (indels) impeded neutrophil maturation, whereas -2 frame late exon indels repressed translation and supported neutrophil maturation. Gene editing of primary HSPCs allowed faithful identification of variant pathogenicity to clarify molecular mechanisms of disease and encourage a universal therapeutic approach to ELANE-mutant neutropenia, returning normal neutrophil production and preserving HSPC function.

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.

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.

Highly efficient therapeutic gene editing of human hematopoietic stem cells.

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe ß-globin disorders sickle cell disease (SCD) and ß-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with ß-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.