HBG Promoters Research Articles

Base editing of the HBG promoter induces potent fetal hemoglobin expression with no detectable off-target mutations in human HSCs

Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating β-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and β-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating β-hemoglobinopathies.

Direct and Indirect Functions of TR4 in the Control of Fetal Hemoglobin Silencing

Elevated levels of fetal hemoglobin (HbF) compensate for reduced normal β-globin chains and ameliorate clinical symptoms for patients with beta-globinopathies, such as sickle cell disease (SCD). The embryonic and fetal globin genes, but not the adult β-globin gene, have direct repeat (DR) elements in their promoters, which have been implicated in γ-globin gene repression (Tanabe et al., 2002). We previously purified a DR element-binding protein complex, which we named DRED, and was the first identified HBG gene repressor (Tanabe et al., 2002). DRED activity is conferred by a complex including LSD1, recruited by the nuclear receptors TR2/TR4 (Cui et al., 2011). BCL11A is a well-characterized transcription factor that independently represses the γ-globin genes through binding to their promoters (Liu et al., 2018). We have shown by co-immunoprecipitation that TR4 interacts with BCL11A in HUDEP2 cells. To understand how TR4 acts to repress the γ-globin gene and the relationship of TR4 to BCL11A in γ-globin repression, we characterized the chromatin occupancy of TR4 and BCL11A within the β-globin locus by CUT&RUN (CNR) in HUDEP2 cells. We established HUDEP2 cells bearing TR4 tagged with 3FLAG+3Myc epitopes (FM-TR4) and in the same cells, BCL11A tagged with 3HA+3Myc (HM-BCL11A) epitopes by genome editing. In addition, another HUDEP2 cell line bearing BCL11A tagged with the 3HA+3Myc (HM-BCL11A) epitope as well a cell line bearing TR4 tagged with the 3FLAG+3Myc epitopes (FM-TR4) were generated. The tagged cell lines not only improved the efficiency of antibody targeting, but also facilitated direct comparison of TR4 vs BCL11A chromatin occupancy by binding of the common Myc epitope in FM-TR4 and HM-BCL11A cell lines. Unbiased motif discovery of TR4 CNR binding of these epitopes in all 3 cell lines yielded the motif of a direct repeat consisting of 2 AGGTCA repeat elements with a 1 nt spacer. The exact match of BCL11A motif with one of the direct repeat elements of TR4 motif suggested that BCL11A and TR4 may be compete for binding to the γ-globin promoter. Overall, TR4 and BCL11A showed a similar pattern of occupancy within the globin locus, although of lower intensity. TR4 presented strong interaction with the locus control regions, among which HS2 and HS3 were the highest, consistent with the model of long-distance interaction through chromosomal looping. However, TR4 was found to be significantly less occupied at the γ-globin promoters than BCL11A. To test whether TR4 and BCL11A can bind in vitro to the motifs found in the γ-globin promoters, we performed TR4 and BCL11A electrophoretic mobility shift assays (EMSAs). We showed that TR4 binds to the DR1 element within the γ-globin promoters, which contains the distal TGACCA motif that was bound by BCL11A. Notably, mutation of the TGACCA motif disrupted both BCL11A and TR4 binding, while the other direct motif is indispensable for TR4 binding, supporting overlapping binding of BCL11A and TR4. Interestingly, DNA-TR4 protein complexes were competitively depleted by high concentrations of BCL11A and vice versa, suggesting competitive binding between the two factors. Studies of this mechanism in the erythroid cells are underway. In addition, unbiased motif discovery of TR4 CNR also enriched for KLF1, LRF and NF-Y motifs. Co-IP assays demonstrated interaction between TR4 and KLF1 and LRF, suggesting the presence of a large repressor complex. We also explore genome-wide occupancy of TR4 and discovered strong TR4 interactions at promoters of multiple HbF repressing transcription factors and corepressors, including NFIA and EHMT1, suggesting that the role of TR4 in HbF repression may be partially mediated by regulating expression of various HbF repressor genes. In summary, these data suggest that the function of TR4 in HbF silencing is mediated in part by direct repression of HBG promoters in a competitive manner with BCL11A, and in part by regulating the expression of multiple HbF repressor genes.

Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing.

Genome editing (GE) is one of the most efficient and useful molecular approaches to correct the effects of gene mutations in hereditary monogenetic diseases, including β-thalassemia. CRISPR-Cas9 gene editing has been proposed for effective correction of the β-thalassemia mutation, obtaining high-level "de novo" production of adult hemoglobin (HbA). In addition to the correction of the primary gene mutations causing β-thalassemia, several reports demonstrate that gene editing can be employed to increase fetal hemoglobin (HbF), obtaining important clinical benefits in treated β-thalassemia patients. This important objective can be achieved through CRISPR-Cas9 disruption of genes encoding transcriptional repressors of γ-globin gene expression (such as BCL11A, SOX6, KLF-1) or their binding sites in the HBG promoter, mimicking non-deletional and deletional HPFH mutations. These two approaches (β-globin gene correction and genome editing of the genes encoding repressors of γ-globin gene transcription) can be, at least in theory, combined. However, since multiplex CRISPR-Cas9 gene editing is associated with documented evidence concerning possible genotoxicity, this review is focused on the possibility to combine pharmacologically-mediated HbF induction protocols with the "de novo" production of HbA using CRISPR-Cas9 gene editing.

Erythroid lineage chromatin accessibility maps facilitate identification and validation of NFIX as a fetal hemoglobin repressor

Human genetics has validated de-repression of fetal gamma globin (HBG) in adult erythroblasts as a powerful therapeutic paradigm in diseases involving defective adult beta globin (HBB)1. To identify factors involved in the switch from HBG to HBB expression, we performed Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq)2 on sorted erythroid lineage cells derived from bone marrow (BM) or cord blood (CB), representing adult and fetal states, respectively. BM to CB cell ATAC-seq profile comparisons revealed genome-wide enrichment of NFI DNA binding motifs and increased NFIX promoter chromatin accessibility, suggesting that NFIX may repress HBG. NFIX knockdown in BM cells increased HBG mRNA and fetal hemoglobin (HbF) protein levels, coincident with increased chromatin accessibility and decreased DNA methylation at the HBG promoter. Conversely, overexpression of NFIX in CB cells reduced HbF levels. Identification and validation of NFIX as a new target for HbF activation has implications in the development of therapeutics for hemoglobinopathies.

Therapeutic adenine base editing of human hematopoietic stem cells

In β-thalassemia, either γ-globin induction to form fetal hemoglobin (α2γ2) or β-globin repair to restore adult hemoglobin (α2β2) could be therapeutic. ABE8e, a recently evolved adenine base editor variant, can achieve efficient adenine conversion, yet its application in patient-derived hematopoietic stem cells needs further exploration. Here, we purified ABE8e for ribonucleoprotein electroporation of β-thalassemia patient CD34+ hematopoietic stem and progenitor cells to introduce nucleotide substitutions that upregulate γ-globin expression in the BCL11A enhancer or in the HBG promoter. We observed highly efficient on-target adenine base edits at these two regulatory regions, resulting in robust γ-globin induction. Moreover, we developed ABE8e-SpRY, a near-PAMless ABE variant, and successfully applied ABE8e-SpRY RNP to directly correct HbE and IVS II-654 mutations in patient-derived CD34+ HSPCs. Finally, durable therapeutic editing was produced in self-renewing repopulating human HSCs as assayed in primary and secondary recipients. Together, these results support the potential of ABE-mediated base editing in HSCs to treat inherited monogenic blood disorders.

Targeted Base Editing Strategies for Beta-Hemoglobinopathies

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult hemoglobin β-chain. In β-thalassemia, the reduced (β+) or absent (β0) production of β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent RBC sickling, vaso-occlusive crises, organ damage and reduced life expectancy. Transplantation of autologous, genetically modified hematopoietic stem/progenitor cells (HSPCs) is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors or CRISPR/Cas9 nuclease are not equally effective in all the patients and/or raise safety concerns. Base editing (BE) allows the introduction of point mutations (C>T by cytidine base editors, CBEs; A>G by adenine base editors, ABEs) without generating dangerous double strand breaks. In this study, we have developed BE strategies aiming either to correct the genetic defect or to target disease modifiers in patient cells, and provide a safe and effective treatment for β-hemoglobinopathies. First, we exploited the capacity of ABEs in combination with single guide RNAs (gRNAs) to correct prevalent β-thalassemic mutations (the CD39 and IVS2-1 β0 mutations and the IVS1-110 β+ mutation). RBCs derived from edited HSPCs exhibited high β-globin levels. The delayed erythroid differentiation typically observed in β-thalassemic cell cultures was corrected by our treatment. Measurement of Annexin+ cells by flow cytometry showed a substantially reduced apoptotic rate in edited β-thalassemic samples compared to untreated controls. Finally, off-target activity was minimal and by-stander edits (additional base conversions occurring close to the target base) did not impact the β-globin expression. In parallel, we used BE to target disease modifiers such as the fetal γ-globin genes. In fact, the co-inheritance of genetic mutations causing a sustained fetal β-like γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin (HbF), HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 eitherdisrupt the binding sites (BS) for transcriptional repressors (e.g., LRF) or generate BS for transcriptional activators (e.g., KLF1). BE allowed the introduction of HPFH mutations in the HBG promoters, which disrupt the LRF BS (via CBE) or generate the KLF1 BS (via ABE). This led to therapeutically relevant HbF levels in RBCs differentiated from edited β-thalassemia or SCD HSPCs, and rescued the pathological phenotypes (correcting ineffective erythropoiesis in β-thalassemia and the sickling phenotype in SCD RBCs). Finally, serial xenotransplantation experiments showed BE in long-term HSCs; however, the efficiency was reduced compared to input HSPCs. RNA-seq analysis showed no alteration for ABE-treated HSPCs, but activation of the p53 pathway and dysregulation of genes involved in HSC biology in CBE-treated samples. To increase the fitness of edited HSCs and editing efficiency, we optimized the RNA delivery of the BE machinery and used histone deacetylase, RNase and apoptosis inhibitors. Moreover, we developed a strategy to enrich edited cells. By way of example, two rounds of mRNA electroporation led to increased BE efficiency with minimal toxicity. Furthermore, highly processive base editors outperformed the original enzymes even upon only one round of electroporation. In conclusion, we developed efficient BE approaches to either correct highly prevalent mutations or induce HbF expression. Overall, our treatments led to correction of the pathological phenotypes and optimal BE efficiencies to finally enable the clinical development of base-edited HSPCs for β-hemoglobinopathies.

Base-editing-mediated dissection of a γ-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression

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.

Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

Base Editing-Mediated Dissection of the -200 Region of the γ-Globin Promoters to Induce Fetal Hemoglobin and Rescue Sickle Cell Disease and β-Thalassemia

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. DisclosuresEl Nemer: Hemanext: Consultancy.

Multiplex CRISPR/Cas9 genome editing in hematopoietic stem cells for fetal hemoglobin reinduction generates chromosomal translocations

Sickle cell disease and β-thalassemia are common monogenic disorders that cause significant morbidity and mortality globally. The only curative treatment currently is allogeneic hematopoietic stem cell transplantation, which is unavailable to many patients due to a lack of matched donors and carries risks including graft-versus-host disease. Genome editing therapies targeting either the BCL11A erythroid enhancer or the HBG promoter are already demonstrating success in reinducing fetal hemoglobin. However, where a single locus is targeted, reliably achieving levels high enough to deliver an effective cure remains a challenge. We investigated the application of a CRISPR/Cas9 multiplex genome editing approach, in which both the BCL11A erythroid enhancer and HBG promoter are disrupted within human hematopoietic stem cells. We demonstrate superior fetal hemoglobin reinduction with this dual-editing approach without compromising engraftment or lineage differentiation potential of edited cells post-xenotransplantation. However, multiplex editing consistently resulted in the generation of chromosomal rearrangement events that persisted in vivo following transplantation into immunodeficient mice. The risk of oncogenic events resulting from such translocations therefore currently prohibits its clinical translation, but it is anticipated that, in the future, alternative editing platforms will help alleviate this risk.

SIRT1 Increases the Expression of Fetal Hemoglobin Genes in Human Adult and Cord Blood Erythroblasts

Introduction: High fetal hemoglobin (HbF, α2γ2) levels ameliorate the clinical manifestations of sickle cell disease and β thalassemia. The mechanisms that repress HbF expression and silence γ-globin genes (HBG) in adults are incompletely characterized and only a single HbF inducer, hydroxyurea, is approved for treatment, and only in patients with sickle cell disease. Many efforts have been made to develop new HbF inducers. HDAC1 and HDAC2, the class I histone deacetylases, suppress HbF gene transcription, and histone deacetylase inhibitors induce HbF expression. We therefore wanted to study whether SIRT1, a NAD-dependent class III histone deacetylase, also played a role in suppressing HBG expression. Unexpectedly, we found that SIRT1 was an inducer of HBG expression.Methods: We evaluated the role of SIRT1 in fetal globin gene regulation by ectopic expression or knockdown of SIRT1 and treatment with small molecule SIRT1 activators SRT2104 and SRT1720, in primary erythroid progenitor cells isolated from both cord blood and adult peripheral blood, CD34+ HUDEP-1 and HUDEP-2 cell lines that express fetal and adult hemoglobins, and K562 cells, using qRT-PCR, flow cytometry, immunoblotting, chromatin immunoprecipitation, and chromatin conformation capture assays.Results: We identified SIRT1 as a new inducer of γ-globin. Ectopic expression of SIRT1 in cord blood CD34+ cells increased HBG mRNA 4-fold and the g-globin protein level 2.5-fold compared with vector control (p<0.05). SIRT1 knockdown in cord blood CD34+ cells decreased HBG mRNA 8-fold and γ-globin protein level 5-fold compared with a scrambled shRNA control (p<0.05). Stable SIRT1 knockdown in K562 cells decreased HBG mRNA levels by 11-fold (p<0.01).To evaluate the effect of small molecule SIRT1 agonists on HbF gene expression, we treated erythroid progenitor cells from cord blood or normal adult blood and HUDEP-1 and HUDEP-2 cells and analyzed HBG mRNA, HbF-cells and g-globin protein levels. In cord erythroid progenitor cells, SRT12104 at 100nM, 500nM, and 2uM concentrations induced a mean increase of 2.4-fold, 3.4-fold and 8.1-fold in HBG mRNA, respectively, compared with vehicle treated controls (p<0.01). SRT1720 at 500nM and 5uM concentrations induced a mean increase of 2.1-fold and 4.4-fold in HBG mRNA levels above the controls, respectively (p<0.05). In adult progenitor cells, SRT2104 at 500nM and 2uM induced a mean increase of 2.6-fold and 4.1-fold HBG mRNA respectively (p<0.05), and induced an increase of 2.7% and 6.5% HbF-cells respectively (p<0.01). g-Globin was increased 5-fold and 3-fold by exposure to SRT2104 at 2uM and SRT1720 at 2.5uM respectively. Incubation of HUDEP-1 cells with 500nM and 2uM SRT2104 induced HBG expression 3.3-fold (p<0.01) and 6.2-fold (p<0.05) respectively; SRT1720 at 2.5uM induced HBG expression 8.4-fold (p<0.01), compared with controls. F-cells were elevated by 15.0 % and 17.4 % with SRT2104 treatment at 500nM and 2uM respectively (p<0.05), compared with controls. HUDEP-2 cells treated with SRT2104 at 500 nM and 2 uM had 1.8-fold and 2.5-fold induction of HBG expression (p<0.05). Treatment with SRT1720 at 2.5uM induced HBG expression 4.2-fold (p<0.05) compared with controls. These findings show that SIRT1 is an HBG inducer and that SIRT1 activators can reactivate silenced HBG expression in adult progenitor cells.SIRT1 binds in the β-globin gene cluster locus control region (LCR) and HBG promoters, and promotes the looping of the LCR to HBG promoter, and increases the binding of RNA polymerase II and H4K16Ac in the HBG promoter. SIRT1 suppressed the expression of the HBG suppressors BCL11A, KLF1, HDAC1 and HDAC2 . SIRT1 did not change the proliferation of human erythroid progenitor cells or the expression of differentiation marker CD235a.Conclusions: SIRT1 is an activator of HBG transcription. Our data suggest that SIRT1 activates HBG expression through facilitating LCR looping to the HBG promoter, inhibiting the expression of transcriptional suppressors of HBG, and indirectly increasing histone acetylation in the HBG promoter. SIRT1 is a potential therapeutic target for γ-globin induction, and small molecule SIRT1 activators might serve as a lead compound for the development of new HbF inducers. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic inactivation of ERF reactivates γ-globin expression in β-thalassemia

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of β-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from β-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of β-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in β-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in β-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for β-hemoglobinopathies.

A Small Key for a Heavy Door: Genetic Therapies for the Treatment of Hemoglobinopathies.

Throughout the past decades, the search for a treatment for severe hemoglobinopathies has gained increased interest within the scientific community. The discovery that ɤ-globin expression from intact HBG alleles complements defective HBB alleles underlying β-thalassemia and sickle cell disease, has provided a promising opening for research directed at relieving ɤ-globin repression mechanisms and, thereby, improve clinical outcomes for patients. Various gene editing strategies aim to reverse the fetal-to-adult hemoglobin switch to up-regulate ɤ-globin expression through disabling either HBG repressor genes or repressor binding sites in the HBG promoter regions. In addition to these HBB mutation-independent strategies involving fetal hemoglobin (HbF) synthesis de-repression, the expanding genome editing toolkit is providing increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin (HbA) restoration for a personalized treatment of hemoglobinopathies. Moreover, besides genome editing, more conventional gene addition strategies continue under investigation to restore HbA expression. Together, this research makes hemoglobinopathies a fertile ground for testing various innovative genetic therapies with high translational potential. Indeed, the progressive understanding of the molecular clockwork underlying the hemoglobin switch together with the ongoing optimization of genome editing tools heightens the prospect for the development of effective and safe treatments for hemoglobinopathies. In this context, clinical genetics plays an equally crucial role by shedding light on the complexity of the disease and the role of ameliorating genetic modifiers. Here, we cover the most recent insights on the molecular mechanisms underlying hemoglobin biology and hemoglobinopathies while providing an overview of state-of-the-art gene editing platforms. Additionally, current genetic therapies under development, are equally discussed.

Induction of therapeutic levels of HbF in genome-edited primary β0 39-thalassaemia haematopoietic stem and progenitor cells.

Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of β-thalassaemia. The homozygous mutation c.-196 C>T in the Aγ-globin (HBG1) promoter, which causes Sardinian δβ0 -thalassaemia, is able to completely rescue the β-major thalassaemia phenotype caused by the β0 39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from β0 -thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the -196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous β0 39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the -196 position has the potential to induce therapeutic levels of HbF in patients with most types of β-thalassaemia irrespective of the β-globin gene (HBB) mutations.

Multiplex CRISPR/Cas9 Genome Editing Targeting the BCL11A/HBG Axis Maximizes Fetal Hemoglobin Reinduction but Generates Chromosomal Translocations Which Persist In Vivo

Sickle cell disease and β-thalassemia are among the most common monogenic disorders globally, causing significant morbidity and early mortality. The only curative option available is allogeneic hematopoietic stem cell transplantation, which is limited by a lack of matched donors and risks, including graft versus host disease and secondary malignancy. Retroviral gene transfer is being explored in clinical trials, but an alternative approach is more targeted CRISPR/Cas9 genome editing, to recapitulate naturally occurring hereditary persistence of fetal hemoglobin (HPFH) which ameliorates disease. HbF reinduction is achieved by disrupting either transcription factor binding sites within the HBG promoter, or an erythroid enhancer sequence within the HbF repressor, BCL11A. Results from preclinical studies have suggested that HbF levels may remain suboptimal when each locus is targeted individually. We thus investigated the feasibility of a dual editing approach, targeting both loci simultaneously or sequentially, to maximize HbF production. G-CSF-mobilized human peripheral blood hematopoietic stem and progenitor cells (HSPCs) underwent CRISPR/Cas9 genome editing at either HBG promoter, BCL11A erythroid enhancer, or both loci targeted either simultaneously or sequentially. Immunodeficient mice were transplanted with edited cells. HSPCs cultured in differentiation media demonstrated comparable levels of editing at each locus in the single edited arms (by TIDE).The dual editing approach did not impair editing efficiency at each site when conducted sequentially and only slightly reduced efficiency in the simultaneously-edited reactions. HbF reinduction was also greatest in sequentially double-edited reactions, with HbF/HbA ratio as high as 3.9 times that seen in mock reactions by flow cytometry and 4.1 times by HPLC. In single-edited arms, HbF reinduction was slightly greater with HBG promotor than BCL11A erythroid enhancer editing. Two separate chromosomal translocation events encompassing both loci were detected in each double-edited arm and quantified using digital droplet PCR. Both were more frequent in simultaneous (mean of 1.0% and 0.6%) than sequential reactions (means &amp;lt;0.2%, n=3, p=0.0420 and p=0.0296). Analysis of single BFU-E colonies grown on methocult media revealed markedly different indel patterns at the 2 target loci. Concurrent hemoglobin fraction analysis of these clonal populations by HPLC demonstrated greatest HbF proportion in double-edited colonies. Where ≥60% editing was reported at both loci mean HbF was 71.3%, compared to 47.1% with ≥60% editing at HBG only, and 29.6% with ≥60% editing at BCL11A only (p=0.0373). Following transplant into mice, engraftment and lineage differentiation were comparable among all experimental arms. At necropsy, bone marrow populations of human CD45+ cells, CD34+CD38low HSPCs and, within these, HSC-enriched CD90+CD45RA- subpopulation, were present at comparable levels indicating that the dual editing approach did not impair engraftment. Translocation events were detected, albeit at frequencies of less than 0.25%, in the bone marrows of all 4 mice transplanted with simultaneously dual-edited cells and in 5 of 6 mice transplanted with sequentially dual-edited cells. Bone marrow cells cultured ex vivo demonstrated greatest HbF from mice transplanted with sequentially double-edited cells. In summary, we present evidence of maximized HbF reinduction with sequentially applied multiplex genome editing at BCL11A erythroid enhancer and HBG promoter, with no impairment of engraftment or differentiation. However, chromosomal translocation events were consistently seen in double-edited reactions, even when edits at each locus were applied sequentially, and persisted in vivo after engraftment, thus rendering this approach inappropriate for clinical applications. However, multiplex editing and targeting both the BCL11A erythroid enhancer and HBG promoter to maximize fetal hemoglobin induction may be a promising strategy for alternative platforms such as base editors which are expected to greatly reduce or completely eliminate the occurrence of translocations. Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Enochian: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees.

Editing the LRF Repressor Binding Site in the γ-Globin Promoters Induces Therapeutically Relevant Fetal Hemoglobin Levels for the Treatment of β-Hemoglobinopathies

β-hemoglobinopathies are genetic anemias caused by a reduced or abnormal synthesis of the adult β-globin chain. In β-thalassemia, the reduced (β+) or absent (β0) production of adult β-chains causes α-globin precipitation and death of red blood cell (RBC) precursors. In sickle cell disease (SCD), a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) βS-chain causes Hb polymerization with consequent red blood cell (RBC) sickling, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) reduces the clinical severity of β-hemoglobinopathies. HPFH mutations in the promoter of the two γ-globin genes, HBG1 and HBG2 disrupt the binding sites (BS) for transcriptional repressors (e.g., BCL11A and LRF). Recently, we demonstrated that CRISPR/Cas9-mediated disruption of the LRF BS in the HBG promoters via non-homologous end joining and microhomology-mediated end joining (MMEJ) repair mechanisms mimics the effect of HPFH mutations by impairing the LRF binding and re-activating the γ-globin expression (Weber, Frati et al., Science Advances, 2020). Efficient editing of the LRF BS (≥ 3 γ-globin promoters in &amp;gt;70% of SCD hematopoietic stem/progenitor cells (HSPCs)) resulted in a robust HbF reactivation and a concomitant reduction in βS-globin levels recapitulating the phenotype of asymptomatic SCD-HPFH patients. RBCs derived from edited HSPCs displayed HbF levels sufficient to correct the SCD cell phenotype. Similarly, LRF BS targeting in β0-thalassemic cells results in HbF reactivation potentially correcting the α/β-like globin imbalance. Xenotransplantation of human HSPCs edited using several gRNAs targeting the LRF BS showed a robust engraftment of edited cells that were capable to differentiate into multiple lineages. HBG editing in engrafted cells ranged from 26% to 76% with a decrease (-33%) of editing events compared to the input HSPCs, partially due to a reduced occurrence of MMEJ-mediated events in hematopoietic stem cells (HSCs). Erythroid progenitors (BFU-E) obtained ex vivo from engrafted human cells, showed a relevant γ-globin expression (~40% of the total β-like chain) despite of the reduction in the number of edited promoters per BFU-E after transplantation. Moreover, mature RBCs ex vivo differentiated from edited human cells ensure therapeutically relevant HbF levels. Sequencing of top-scoring off-targets identified by GUIDE-seq showed a relatively high off-target activity within an intergenic site devoid of known regulatory elements both in vitro and in vivo in primary human cells treated with one of the gRNAs targeting the LRF BS. Although the occurrence of this off-target event in repopulating cells suggests that it has no detrimental effect on HSC engraftment and multilineage differentiation, we tested high fidelity Cas9 variants to reduce off-target activity in primary HSPCs. Finally, we used CAST-Seq assay to evaluate the potential chromosomal rearrangements in edited primary human cells in vitro and in vivo. To minimize the potential genotoxicity associated to Cas9 nuclease-mediated double-strand breaks (DSBs) in the genome, we explored the base editing system to introduce C&amp;gt;T point mutations in the LRF BS without generating DSBs. The absence of the canonical SpyCas9 NGG PAM close to the LRF BS, prompted us to use known and novel base editors containing non-NGG Cas9 variants that allowed the editing of up to 6 out of 8 cytosines of the LRF BS in erythroid cell lines and in primary HSPCs from SCD patients. These C&amp;gt;T conversions include not only known HPFH mutations but also mutations that can further impair LRF binding. In the majority of cases, we detected no insertions or deletions in base-edited samples, as compared to nuclease-edited samples. The 4.9-kb deletion that can be generated upon cleavage of the two identical HBG promoters by the Cas9 nuclease was barely detected in base-edited samples. Importantly, disruption of the LRF BS by non-NGG enzymes led to HbF de-repression, without affecting erythroid differentiation. This work identifies the LRF BS as an effective and safe therapeutic target for the treatment of β-hemoglobinopathies. Disclosures Casini: Alia Therapeutics: Current Employment, Current equity holder in publicly-traded company. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.

Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), resulting in ABE8e. ABE8e contains eight additional mutations that increase activity (kapp) 590-fold compared with ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disrupting regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by introducing additional mutations in the TadA-8e domain. Finally, we show that ABE8e can efficiently edit natural mutations in a GATA1 binding site in the BCL11A enhancer or the HBG promoter in human cells, targets which were poorly edited with ABE7.10. ABE8e broadens the effectiveness and applicability of adenine base editing.

Chromatin Accessibility Mapping of Primary Erythroid Cell Populations Leads to Identification and Validation of Nuclear Factor I X (NFIX) As a Novel Fetal Hemoglobin (HbF) Repressor

Human beta-like globin gene expression is developmentally regulated. Erythroblasts (EBs) derived from fetal tissues, such as umbilical cord blood (CB), primarily express gamma globin mRNA (HBG) and HbF, while EBs derived from adult tissues, such as bone marrow (BM), predominantly express beta globin mRNA (HBB) and adult hemoglobin. Human genetics has validated de-repression of HBG in adult EBs as a powerful therapeutic paradigm in diseases involving defective HBB, such as sickle cell anemia. To identify novel factors involved in the switch from HBG to HBB expression, and to better understand the global regulatory networks driving the fetal and adult cell states, we performed transcriptome profiling (RNA-seq) and chromatin accessibility profiling (ATAC-seq) on sorted EB cell populations from CB or BM. This approach improves upon previous studies that used unsorted cells (Huang J, Dev Cell 2016) or that did not measure chromatin accessibility (Yan H, Am J Hematol 2018). CD34+ cells from CB and BM were differentiated using a 3-phase in vitro culture system (Giarratana M, Blood 2011). Fluorescence-activated cell sorting and the cell surface markers CD36 and GYPA were used to isolate 7 discrete populations, with each sorting gate representing increasingly mature, stage-matched EBs from CB or BM (Fig 1A, B). RNA-seq analysis revealed expected expression patterns of the beta-like globins, with total levels increasing during erythroid maturation and primarily composed of HBB or HBG transcripts in BM or CB, respectively (Fig 1C). Erythroid maturation led to progressive increases in chromatin accessibility at the HBB promoter in BM populations. In CB-derived cells, erythroid maturation led to progressive increases in chromatin accessibility at the HBG promoters through the CD36+GYPA+ stage (Pops 1-5). Chromatin accessibility shifted from the HBG promoters to the HBB promoter during the final stages of differentiation (Pops 6-7), suggesting that HBG gene activation is transient in CB EBs (Fig 1D). Hierarchical clustering and principal component analysis of ATAC-seq data revealed that cell populations cluster based on differentiation stage rather than by BM or CB lineage, suggesting most molecular changes are stage-specific, not lineage-specific (Fig 2A, B). To identify transcription factors driving cell state, and potentially beta-like globin expression preference, we searched for DNA binding motifs within regions of differential chromatin accessibility and found NFI factor motifs enriched under peaks that were larger in BM relative to CB (Fig 2C). Transcription factor footprinting analysis showed that both flanking accessibility and footprint depth at NFI motifs were also increased in BM relative to CB (Fig 2D). Increased chromatin accessibility was observed at the NFIX promoter in BM relative to CB populations, and in HUDEP-2 relative to HUDEP-1 cell lines (Fig 2E). Furthermore, accessibility at the NFIX promoter correlated with elevated NFIX mRNA in BM and HUDEP-2 relative to CB and HUDEP-1, respectively. Together these data implicated NFIX in HbF repression, a finding consistent with previous genome-wide association and DNA methylation studies that suggested a possible role for NFIX in regulating beta-like globin gene expression (Fabrice D, Nat Genet 2016; Lessard S, Genome Med 2015). To directly test the hypothesis that NFIX represses HbF, short hairpin RNAs were used to knockdown (KD) NFIX in primary erythroblasts derived from human CD34+ BM cells (Fig 3A). NFIX KD led to a time-dependent induction of HBG mRNA, HbF, and F-cells comparable to KD of the known HbF repressor BCL11A (Fig 3B-D). A similar effect on HbF was observed in HUDEP-2 cells following NFIX KD (Fig 3E). Consistent with HbF induction, NFIX KD also increased chromatin accessibility and decreased DNA methylation at the HBG promoters in primary EBs (Fig 3F, G). NFIX KD led to a delay in erythroid differentiation as measured by CD36 and GYPA expression (Fig 3H). Despite this delay, by day 14 a high proportion of fully enucleated erythroblasts was observed, suggesting NFIX KD cells are capable of terminal differentiation (Fig 3H). Collectively, these data have enabled identification and validation of NFIX as a novel repressor of HbF, a finding that enhances the understanding of beta-like globin gene regulation and has potential implications in the development of therapeutics for sickle cell disease. Disclosures Chaand: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Johnston:Syros Pharmaceuticals: Employment, Equity Ownership. Moon:Syros Pharmaceuticals: Employment, Equity Ownership. Carulli:Syros Pharmaceuticals: Employment, Equity Ownership. Shearstone:Syros Pharmaceuticals: Employment, Equity Ownership.

Editing a γ-Globin Repressor Binding Site Restores Fetal Hemoglobin Synthesis and Corrects the Phenotype of Sickle Cell Disease Erythrocytes

Sickle cell disease (SCD) is a severe, life-threatening disorder caused by a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) β-chain that causes Hb polymerization with consequent red blood cell (RBC) rigidity, anemia, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) decreases sickling and significantly reduces the clinical severity of SCD. HPFH mutations cluster at several loci in the promoters of the two γ-globin genes, HBG1 and HBG2, and, disrupt binding sites for transcriptional repressors (e.g., BCL11A and LRF), leading to elevated levels of fetal hemoglobin (HbF) representing up to 40% of total hemoglobin tetramers. In addition, SNPs at position -158 of both γ-globin promoters are associated with enhanced γ-globin expression and may identify a putative transcriptional repressor binding site. Here, we used the CRISPR/Cas9 system to mimic HPFH mutations in the HBG promoters by generating insertion and deletions (InDels) leading to disruption of known and putative binding sites for transcriptional repressors via non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) DNA repair mechanisms. Efficient editing of the LRF binding site (≥ 3 γ-globin promoters/cell in &gt;70% of cells) in SCD patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in a robust, virtually pancellular HbF reactivation and a concomitant reduction in βS-globin levels, recapitulating the phenotype of asymptomatic SCD-HPFH patients. Of note, in RBCs derived from edited HSPCs, HbF levels exceeded 40% of total Hb tetramers, suggesting that CRISPR/Cas9 mediated disruption of the LRF binding site is even more potent than naturally occurring HPFH point mutations in reactivating HbF expression. RBCs derived from edited HSPCs displayed HbF levels sufficient to substantially correct the SCD cell phenotype (~80% of non-sickling RBCs). Although larger deletions (in part generated likely by MMEJ) disrupt more efficiently the LRF binding site, shorter InDels mainly produced by NHEJ, the most active repair mechanism in bona fide hematopoietic stem cells (HSCs), were still able to induce a potent γ-globin reactivation and correct the SCD cell phenotype. Interestingly, upon targeting of the -158 site, we observed HBG de-repression, which, however, was mild, indicating that this region contains a sequence that moderately inhibits γ-globin expression in adult cells, consistently with the mild increase in HbF associated with the -158 SNP. To evaluate the efficiency and the safety of this approach in long-term repopulating HSCs, untreated and edited HSPCs were injected into NSG immunodeficient mice. 16 weeks after transplantation, the engraftment of control and HBG-edited cells was comparable, as analyzed in bone marrow, spleen, thymus and blood. Edited HSCs were capable to differentiate into multiple hematopoietic cell types, with no skewing towards any particular lineage. Importantly, xenotransplantation of HSPCs edited using the gRNAs targeting the LRF binding site demonstrated a high editing efficiency in long-term repopulating HSCs (up to 75%) and a modest reduction in the frequency of deletions likely generated by MMEJ. Overall, this study identifies the binding site for the LRF repressor as a novel, potent and safe target for a genome editing-based treatment of SCD. Disclosures El Nemer: Hemanext: Other: Other. Cavazzana:SmartImmune: Other: Founder.

MIR29B mediates epigenetic mechanisms of HBG gene activation.

SummarySickle cell disease (SCD) affects over 2 million people worldwide with high morbidity and mortality in underdeveloped countries. Therapeutic interventions aimed at reactivating fetal haemoglobin (HbF) is an effective approach for improving survival and ameliorating the clinical severity of SCD. A class of agents that inhibit DNA methyltransferase (DNMT) activity show promise as HbF inducers because off‐target effects are not observed at low concentrations. However, these compounds are rapidly degraded by cytidine deaminase when taken by oral administration, creating a critical barrier to clinical development for SCD. We previously demonstrated that microRNA29B (MIR29B) inhibits de novo DNMT synthesis, therefore, the goal of our study was to determine if MIR29 mediates HbF induction. Overexpression of MIR29B in human KU812 cells and primary erythroid progenitors significantly increased the percentage of HbF positive cells, while decreasing the expression of DNMT3A and the HBG repressor MYB. Furthermore, HBG promoter methylation levels decreased significantly following MIR29B overexpression in human erythroid progenitors. We subsequently, observed higher MIR29B expression in SCD patients with higher HbF levels compared to those with low HbF. Our findings provide evidence for the ability of MIR29B to induce HbF and supports further investigation to expand treatment options for SCD.