Sharma et al. screened a panel of guide RNAs (gRNAs) that tile the promoters of HBG1 and HBG2 for those that mediate derepression of γ-globin expression; gRNAs were introduced with Cas9 into donor-derived CD34+ HSPCs for CRISPR–Cas9 gene editing. gRNA-68 produced the greatest increase in the proportion of fetal hemoglobin-immunostaining erythroblasts (F cells) among the erythroid progeny of CD34+ HSPCs differentiated in vitro or transplanted into immunodeficient mice. The predominant outcome of gRNA-68-mediated editing (which occurred at 49–87% of alleles) was an approximately 5-kb deletion that fused the HBG2 promoter to the HBG1 coding sequence, disrupting a repressor element and inducing γ-globin production.
Moving into a phase 1–2 clinical study, three individuals with severe sickle cell disease received a single transfusion of autologous gRNA-68-edited HSPCs after myeloablative treatment with busulfan. At 6–18 months follow-up, these patients showed a sustained increase in levels of total and fetal hemoglobin and F cells as well as partial amelioration of sickle cell disease symptoms, such as fewer vaso-occlusive crises. However, mild hemolysis persisted in all participants, indicating that the polymerization of sickle hemoglobin was not completely abrogated. Although further optimization of the gene-editing frequency and improved delivery of autologous stem cells could yield greater clinical improvement, the positive results of this preliminary clinical trial suggest that autologous gRNA-68-edited HSPCs are a safe option for attenuating severe sickle cell disease.
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