Reactivation of γ-globin expression through Cas9 or base editor to treat β-hemoglobinopathies

Abstract

z Dear Editor, Mutations in the β-globin gene, the essential component of adult hemoglobin (HbA; α2β2), results in either a production of aberrant sickle hemoglobin (HbS) leading to sickle cell disease (SCD) or an insufficient β-globin synthesis leading to β-thalassemia. These two major forms of β-hemoglobinopathies cause impaired erythropoiesis and life-threatening anemia. Clinical evidence has suggested that reactivation of fetal γ-globin (HBG) gene expression which is normally silenced after birth by certain genetic mutations can ameliorate the clinical course of β-hemoglobinopathies. 1,2 In β-thalassemia, elevated levels of fetal γ-globin interact with α-globin to form fetal hemoglobin (HbF; α2γ2) restoring the α/β-like globin ratio and in SCD the γ-globin reduces HbS polymerization. There are two major strategies for re-activation of HBG expression: reducing the expression of critical trans-acting repression factors (such as BCL11A) 3,4 or deletion of inhibitory cis-regulatory elements in the HBG1/2 promoter region. To develop a practical strategy for clinical implementation, we leveraged electroporation of a Cas9:sgRNA RNP to edit the HBG1/2 promoter in hematopoietic stem/progenitor cells (HSPCs). We successfully achieved an average editing efficiency of 85% in β-thalassemia patient-derived HSPCs, leading to increased γ-globin mRNA expression (up to 126% relative to α-globin) and an improved terminal erythroid differentiation rate. Importantly, we discovered that the BCL11A binding site (TGACCA: −114 to −119), which is critical for the repression of γ-globin, 5 is an ideal target for base editor hA3A-BE3 induced mutation and elevation of HBG expression.To mimic the effect of the naturally occurring Δ13 bp allele in the HBG1 promoter (−102 to −114) which was identified in patients with hereditary persistence of fetal hemoglobin (HPFH), we synthesized two sgRNAs (with or without 2′-O-methyl 3′ phosphorothioate modifications at three terminal nucleotides at both the 5′ and 3′ ends) (sgRNA1, sgRNA2) targeting the HBG promoter region (Fig. 1a). After electroporation of either RNP complex into immortalized erythroid precursor HUDEP-2 cells, 4 we found that chemical modification 6 of sgRNA enhanced editing efficiency and that sgRNA1 was more efficient than sgRNA2 (Supplementary information, Fig. S1a). The enhancement effect of chemical modification on sgRNAs was also confirmed in T cell receptor (TCR) and beta-2-microglobulin (B2M) loci in human primary T cells (Supplementary information, Fig. S1b). Hence, unless specified, we used the chemically modified sgRNA1 in the following experiments. After titration of the optimal RNP concentration in the HUDEP-2 cell line and in human CD34 + HSPCs, we successfully achieved editing efficiency over 80% in both cells (Supplementary information, Fig. S1c, d). After erythroid differentiation, the γ-globin mRNA level in the edited HUDEP-2 cells reached a mean of 22.7% relative to the total β-like globin mRNA, which showed a 4-fold increase compared with controls (mean 5.6%) (Su...

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An overview of development in gene therapeutics in China

“…The author concluded that 10% or more expression of the exogenous normal β-globin gene was capable of reducing the degree of anemia in their β-thalassemia mouse model [29]. Recently, Wang et al reactivated the expression of γ-globin using RNPs of Cas9 and base editor to modify the hemoglobin subunit gamma (HBG) promoter region mimicking the effect of naturally occurring Δ13 bp allele in patients with hereditary persistence of fetal hemoglobin [34].…”