Base Editing for Therapeutic Induction of Fetal Hemoglobin in β-Thalassemia

Abstract

β-thalassemia, one of the most common inherited genetic disorders worldwide, is caused by more than 300 different mutations in the β-globin locus (HBB), all of which result in quantitative deficiency of β-globin protein with severe anemia and reduced or absent HbA (α2β2). Additionally, free α-globin protein forms toxic intracellular inclusions resulting in hemolysis of mature red blood cells, maturation arrest and apoptosis of erythroid precursors. The only approved cure for severe β-thalassemia is allogeneic hematopoietic stem cell transplantation. However, many patients lack ideal histocompatible donors and immune complications such as graft rejection and graft-versus-host disease can occur. These problems can be avoided by "gene therapies” in which the patient's own hematopoietic stem cells (HSCs) are isolated, genetically modified ex vivo and reinfused after administration of bone marrow conditioning to facilitate engraftment of the modified cells. Most β-thalassemia mutations can be treated by genetic manipulations that de-repress fetal-expressed γ-globin genes (HBG1 and HBG2) to induce fetal hemoglobin production (HbF, α2γ2) postnatally. Thus, Cas9 disruption of an erythroid enhancer in the γ-globin repressor gene BCL11A shows promising results in an ongoing clinical trial. However, multiple other strategies to induce HbF exist and the best one is not known. Compared to conventional Cas9 nuclease, base editors offer theoretical advantages, including the ability to create precise base pair substitutions without requiring genotoxic double strand DNA breaks. We used the adenine base editor ABE8e to generate the naturally occurring γ-globin -175 A>G variant, which causes hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition that alleviates co-inherited β-thalassemia. We edited human CD34+ hematopoietic stem and progenitor cells (HSPCs) from two individuals with severe transfusion dependent β-thalassemia by electroporating ribonucleoprotein (RNP) complexes of ABE8e-NG protein + single guide RNA (sgRNA) designed to install the γ-globin -175 A>G variant, followed by in vitro erythroid differentiation. On-target editing rates were 48-53% for donor 1 (n=3 replicates) and 22.9% for donor 2 (n=1). Analysis of burst forming unit erythroid (BFU-E) colonies revealed at least one on-target edit in 78% of donor 1 HSPCs and 63% of those from donor 2. Reverse phase HPLC of erythroid progeny showed increased β-like/α globin protein ratios: 0.92 ± 0.04 vs 0.53 ± 0.03 in unedited controls (p<0.0001) for donor 1, and 0.8 vs 0.5 in unedited controls for donor 2. Quantitative RT-PCR showed 6-8-fold increased γ-globin mRNA in the ABE-treated β-thalassemia cells. Ion exchange HPLC analysis of erythroid cell lysates showed reductions in free α-globin levels after ABE treatment: from 21.5 ± 2% of total globin protein in unedited cells to undetectable levels after editing in donor 1 and from 18% to 2.5% for donor 2. Maturation to the reticulocyte stage was also increased in base edited cells: 14.6 ± 2% vs 3.3 ± 1% in unedited controls for donor 1 (P<0.0001); and 26% vs 6.9% in unedited controls for donor 2. Reduction in apoptosis (37.4 ± 2.2% vs 23.6 ± 1.5% in unedited control cells, p=0.0003) and reactive oxygen species measured by DCFDA (MFI: 690 ± 97 vs 266 ± 9 in unedited control cells, p=0.009) was also seen in the ABE edited groups. To study editing outcomes in vivo, CD34+ HSPCs from donor 2 were treated with RNP and transplanted into immunodeficient NBSGW mice. After 16-18 weeks, human donor-derived cells were isolated from mouse recipient bone marrow and analyzed (n=4 mice per group). On-target editing frequencies in CD235a+ erythroblasts and CD34+ hematopoietic stem/progenitors were 17.5 ± 5% and 19.5 ±3%, respectively, compared to 22.9% in the input CD34+ cells. Similar to what we observed after in vitro differentiation, base edited erythroid cells generated in vivo exhibited improved globin chain balance with β-like/α globin protein ratios of 0.86 ± 0.12 vs 0.69 ± 0.04 in unedited controls, (p=0.04), reduced apoptosis (16.8 ± 4.2 % vs 26.6 ± 2.9 % in unedited control cells, p=0.004) and enhanced maturation into enucleated reticulocytes (14.4 ± 4.7 % vs 2.87 ± 1.4 % in unedited control cells, p=0.005). Our study provides proof of concept that ABE8e generation of the γ-globin -175 A>G HPFH variant in β-thalassemic HSCs represents a new therapeutic approach for β-thalassemia.