Highly efficient therapeutic gene editing of human hematopoietic stem cells

Abstract

INTRODUCTORY Re-expression of the paralogous γ-globin genes ( HBG1/2 ) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α 2 γ 2 ) 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 dispensable in non-erythroid cells 2 – 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 nonhomologous as compared to microhomology mediated end-joining repair. Erythroid progeny of edited engrafting sickle cell disease (SCD) HSCs express therapeutic levels of fetal hemoglobin (HbF) and resist sickling, while those from β-thalassemia patients show restored globin chain balance. NHEJ-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.

Adenovirus vectors in hematopoietic stem cell genome editing

“…Hence, nucleases are commonly used to create DSBs, accompanied by the delivery of the donor cassette. Recently, with the remarkable advancement in genome editing techniques [17,164], the frequency of HDR has been improved dramatically. Recently, with the remarkable advancement in genome editing techniques [17,164], the frequency of HDR has been improved dramatically.…”

“…Knockout of the BCL11A gene is not a viable option due to its indispensable roles in development. Latest reports have shown more than 90% disruption of the BCL11A GATAA motif in HSCs by electroporation of Cas9 RNPs [164]. Strategies based on shRNA-and CRISPR-mediated down-modulation of BCL11A have entered clinical stage (NCT03282656 and NCT03655678).…”

“…However, the HDR pathway is still much less efficient that the NHEJ repair mechanism, particularly in quiescent stem cells [162,163]. Recently, with the remarkable advancement in genome editing techniques [17,164], the frequency of HDR has been improved dramatically. For ex vivo gene transfer into human CD34 + HSCs, zinc-finger nuclease mRNA and rAAV6-mediated donor template delivery resulted in~15% targeted integration into the AAVS1 locus [165].…”

Advances in the gene therapy of monogenic blood cell diseases

“…This trial aims at the treatment of HIV infection using ZFNs cleaving the CCR5 locus, that encodes a HIV receptor . In the field of hemoglobinopathies, the knock‐out of the BCL11A gene (Figure D)—a repressor of fetal globin—facilitated the reexpression of fetal globin in adults cells . This strategy will soon be used in gene editing trials of β‐thalassemia and SCD.…”

“…176 In the field of hemoglobinopathies, the knock-out of the BCL11A gene ( Figure 2D)-a repressor of fetal globin 177 -facilitated the reexpression of fetal globin in adults cells. 178 This strategy will soon be used in gene editing trials of β-thalasse- Based on advances achieved in the field of hematopoietic gene therapy there is no doubt that this new therapeutic modality will constitute part of the therapeutic arsenal for the treatment of complex and life-threatening diseases.…”