Sickle cell disease and β-thalassemia affect the production of the adult β-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the −200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy – even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating β-hemoglobinopathies.
Beta-like globin gene expression is developmentally regulated during life by transcription factors, chromatin looping and epigenome modifications of the β-globin locus. Epigenome modifications, such as histone methylation/demethylation and acetylation/deacetylation and DNA methylation, are associated with up- or down-regulation of gene expression. The understanding of these mechanisms and their outcome in gene expression has paved the way to the development of new therapeutic strategies for treating various diseases, such as β-hemoglobinopathies. Histone deacetylase and DNA methyl-transferase inhibitors are currently being tested in clinical trials for hemoglobinopathies patients. However, these approaches are often uncertain, non-specific and their global effect poses serious safety concerns. Epigenome editing is a recently developed and promising tool that consists of a DNA recognition domain (zinc finger, transcription activator-like effector or dead clustered regularly interspaced short palindromic repeats Cas9) fused to the catalytic domain of a chromatin-modifying enzyme. It offers a more specific targeting of disease-related genes (e.g., the ability to reactivate the fetal γ-globin genes and improve the hemoglobinopathy phenotype) and it facilitates the development of scarless gene therapy approaches. Here, we summarize the mechanisms of epigenome regulation of the β-globin locus, and we discuss the application of epigenome editing for the treatment of hemoglobinopathies.
β-hemoglobinopathies are caused by mutations affecting the adult hemoglobin production. In sickle cell disease (SCD), the β6 Glu→Val substitution leads to sickle hemoglobin (HbS) polymerization and red blood cell (RBC) sickling. In β-thalassemia, reduced β-globin production leads to precipitation of uncoupled α-chains causing ineffective erythropoiesis and the production of poorly hemoglobinized RBCs. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. The clinical severity of β-hemoglobinopathies is alleviated by the co-inheritance of mutations causing hereditary persistence of fetal Hb (HPFH). HPFH mutations clustering 200 nucleotides upstream of the TSS of the fetal γ-globin (HBG) genes either disrupt the binding site (BS) of the fetal Hb (HbF) repressor LRF or generate a de novo BS for the KLF1 activator. To reactivate γ-globin expression, nuclease-based approaches have been explored. However, nucleases generate double-strand breaks (DSBs), raising safety concerns for clinical applications. Base editing (BE) allows the introduction of point mutations without generating DSBs. In this study, we designed BE systems to introduce a variety of HPFH or HPFH-like mutations in the -200 region of the HBG promoters. First, we screened in erythroid cell lines known and novel BEs, and we selected combinations of BEs and guide RNAs that edit alternative bases of the -200 region. We then developed a clinically-relevant protocol based on RNA-transfection to deliver the BE system to HSPCs. The expression profile of genes activated by RNA stimuli revealed no immune response in HSPCs. A progenitor assay indicated no alteration in the growth and multilineage differentiation of edited HSPCs. We applied this protocol to SCD and β-thalassemia HSPCs, achieving editing efficiencies up to ~70% of the HBG promoters. In RBCs differentiated from edited SCD HSPCs, RT-qPCR, HPLC and flow cytometry showed a potent γ-globin reactivation with a high frequency of HbF + cells and a concomitant decrease in the HbS content/cell. Importantly, the pathological RBC sickling phenotype was corrected in the samples derived from edited HSPCs. Similarly, in β-thalassemia samples, RT-qPCR and HPLC analyses showed strong γ-globin induction and decrease of the α-globin precipitates. HbF expression rescued the delay in erythroid differentiation and ineffective erythropoiesis characterizing β-thalassemia, as demonstrated by the increased RBC enucleation rate and the reduced apoptosis and oxidative stress. We then compared BE strategies that either disrupt the LRF BS or create a de novo KLF1 BS in single colonies derived from erythroid progenitors. Generation of the KLF1 BS was associated with higher levels of HbF compared to the LRF BS disruption. These results suggest that eviction of the LRF repressor is sufficient to reactivate HBG genes, but recruitment of an activator is more effective to achieve high levels of gene expression. HbF expression induced by both LRF BS disruption and KLF1 BS generation was sufficient to rescue the SCD cell phenotype, but higher HbF levels - achieved only through KLF1 BS generation - were necessary to fully correct the β-thalassemia phenotype. In the majority of cases, we detected no DSB-induced insertions, deletions, or large genomic rearrangements in base-edited samples. Accordingly, DSB-induced DNA damage response (DDR) was absent in base-edited HSPCs, as measured by evaluating the expression of p21, a readout of p53-induced DDR. DNA off-target activity was assessed by GUIDE-seq and targeted sequencing of the potential off-target sites in edited HSPCs, while RNA off-target activity was evaluated by RNA-seq in HSPCs. Finally, BE-treated HSPCs were transplanted in immunodeficient mice to evaluate the engraftment and differentiation capability of edited HSCs. We detected good frequencies of human cells with up to ~60% of edited promoters in the peripheral blood of transplanted mice. In conclusion, we developed a clinically-relevant strategy to perform efficient BE in the HBG promoters that led to therapeutically-relevant HbF levels and rescued both the SCD and β-thalassemia phenotypes, thus providing sufficient proof of efficacy and safety to enable the clinical development of base-edited HSPCs for the therapy of β-hemoglobinopathies. Disclosures El Nemer: Hemanext: Consultancy.
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