Fetal Globin Research Articles

Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression

BCL11A-XL directly binds and represses the γ-globin (HBG1/2) gene promoters, using three Zinc-Finger domains (ZnF4, ZnF5, ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in de-repression of fetal globin and high HbF, but also affects Hematopoietic Stem and Progenitor Cells (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we employed both CRISPR/Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR/Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and the elevated HbF levels but impacted normal hematopoiesis. Far fewer side effects were observed with base editing, for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B-cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different impacts. Disrupting ZnF4 elevated HbF levels significantly, while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected as this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL required to repress γ-globin expression and provides a framework for exploring the introduction of natural mutations that may enable de-repression of single genes while leaving other functions unaffected.

RNA Activators of Stress Kinase PKR within Human Genes That Control Splicing or Translation Create Novel Targets for Hereditary Diseases.

Specific sequences within RNA encoded by human genes essential for survival possess the ability to activate the RNA-dependent stress kinase PKR, resulting in phosphorylation of its substrate, eukaryotic translation initiation factor-2α (eIF2α), either to curb their mRNA translation or to enhance mRNA splicing. Thus, interferon-γ (IFNG) mRNA activates PKR through a 5'-terminal 203-nucleotide pseudoknot structure, thereby strongly downregulating its own translation and preventing a harmful hyper-inflammatory response. Tumor necrosis factor-α (TNF) pre-mRNA encodes within the 3'-untranslated region (3'-UTR) a 104-nucleotide RNA pseudoknot that activates PKR to enhance its splicing by an order of magnitude while leaving mRNA translation intact, thereby promoting effective TNF protein expression. Adult and fetal globin genes encode pre-mRNA structures that strongly activate PKR, leading to eIF2α phosphorylation that greatly enhances spliceosome assembly and splicing, yet also structures that silence PKR activation upon splicing to allow for unabated globin mRNA translation essential for life. Regulatory circuits resulting in each case from PKR activation were reviewed previously. Here, we analyze mutations within these genes created to delineate the RNA structures that activate PKR and to deconvolute their folding. Given the critical role of intragenic RNA activators of PKR in gene regulation, such mutations reveal novel potential RNA targets for human disease.

Pharmacologic Therapies in Beta Hemoglobinopathies: Fetal Globin Gene Induction in the First Molecular Diseases

Beta hemoglobinopathies and thalassemias are caused by diverse molecular mutations of the βA globin chains and modulated by polymorphisms. These are important disorders in medical history, as they became prevalent globally in regions where P. falciparum malaria was endemic. Heterozygous or carrier states conferred a survival advantage; however, doubly heterozygous or homozygous states cause severe hemolytic anemia and multi-organ complications. These disorders are somewhat unique in that all humans have alternate genes for fetal globin chains, which are expressed in fetal life but are silenced on a developmental clock in infancy. An established approach to ameliorating conditions of abnormal adult globin genes is to reactivate, or increase expression, of the endogenous fetal globin genes to replace the missing protein chains in beta thalassemias or inhibit polymerization of hemoglobin S and reduce the red cell abnormalities. This review provides a high-level overview of cell and molecular mechanisms that mediate fetal to adult globin gene switching and pharmacologic therapies that can reactivate fetal globin through different actions. Intermittent or metronomic dosing regimens have overcome challenges of undesirable off-target effects by agents that cause erythroid cell growth arrest. Multiple pharmacologic candidates reactivate or increase the expression of fetal globin protein and proportions of red blood cells containing HbF (F-cells). Hydroxyurea maintains high levels of HbF if begun in infancy, with some decline in mid-childhood. It elicits lower responses in HbF in adult patients, but still has broad clinical benefit in reducing many complications. However, many adult patients do not tolerate optimal hydroxyurea dosing due to cytopenias. Parenterally administered therapies with differing molecular actions, such as demethylating agents and histone deacetylase inhibitors, have shown proof-of-principle in reactivating HbF. Orally bioavailable candidates with complementary molecular mechanisms are in trials. Combining fetal globin-inducing agents with other therapies with complementary mechanisms, such as recombinant erythropoietin that promotes the survival of red blood cells and therapeutics that promote erythroid cell metabolism, should have additive effects. These pharmaceutical candidates offer great clinical potential and global feasibility for ameliorating these serious diseases.

NR2F1 : A Novel Candidate for Gamma Globin Activation

The reactivation of human gamma globin holds tremendous promise as a potential treatment for hemoglobinopathies, including sickle cell disease and beta-thalassemia. Despite identifying various repressors of fetal globin genes in adult erythroid cells over the past decade, evidence concerning stage-specific factors that activate human fetal globin prior to the switch to adult beta-globin remains inadequately understood. In our study, we constructed an erythroid-specific gene regulatory network (GCN) by integrating multiple RNA-seq datasets from erythroid cells at different stages of differentiation and development. Our analysis unveiled both known HbF activators, like HIC2, and novel candidates. Among the novel contenders, NR2F1, a member of the steroid/thyroid hormone receptor superfamily, emerged as a prominent candidate. To validate our predictions, we overexpressed NR2F1 in Hudep2 cells and observed more than a four-fold increase (28% vs. 6%) in the proportion of F cells and a three-fold rise in HbF levels as measured by HPLC. In addition, the overexpression of NR2F1 did not appear to impact beta-globin expression or cell differentiation. Intriguingly, we observed a significant reduction in the protein level of BCL11A, a master regulator of HbF, despite no major change in the total RNA level of BCL11A. This observation suggests that NR2F1 plays a role in the post-transcriptional modification of BCL11A. Additionally, NR2F1 overexpression led to an upregulation of Lin28B expression in Hudep2 cells. Lin28B is known to directly bind BCL11A mRNA and prevent its effective translation without disturbing BCL11A transcription. Consequently, the observed upregulation of Lin28B in NR2F1-overexpressing cells implies a potential NR2F1-Lin28B-BCL11A axis involved in controlling human hemoglobin switching. In conclusion, our findings propose NR2F1 as a novel activator of gamma globin, representing an attractive target for gamma globin reactivation. Additional investigation is required to explore the molecular mechanisms through which NR2F1 regulates gamma globin expression and its role in the NR2F1-Lin28B-BCL11A axis.

Initial Results in a Phase 1b Trial of PB-04 in Sickle Cell Disease Demonstrate Fetal Hemoglobin Induction, Additive Activity with Hydroxyurea, and Improved Red Blood Cell Sickling Parameters

Extensive evidence has shown fetal hemoglobin (HbF) is a major modulator of sickle cell disease (SCD) phenotype. Clinical benefit with any increment of HbF was shown in the NIH-sponsored Natural History Study and the Multicenter Study of Hydroxyurea (HU), with mean HbF increases of 3.6% above baseline (BL), to improve survival, reduce painful vaso-occlusive crises, and recently stroke prevention and improved cardiac and neurocognitive function with HU are reported. As some patients do not tolerate optimal HU doses due to cytopenias, additional inducers suitable for combined use are desirable. Proportions of F-cells and HbF-quantity/cell have been cited as important. PB-04, a repurposed drug with a well-known safety profile, is an oral fetal globin stimulant identified in high throughput screening, which suppresses 4 repressors of the HMG gene promoter, ( BCL-11A, LSD-1, HDAC-3, KLF-1). PB-04 induces fetal globin in nonhuman primates, transgenic mice, with additive effect when combined with HU in SCD erythroid progenitors, and was shown to reduce infarcts in multiple organs with preserved splenic function in transgenic sickle mice. An ongoing Phase 1b trial in beta thalassemia intermedia (BTI) ( NCT04432623) of 3 doses (1,3, or 5 mg/kg QD) given 3 (TIW) or 5 times/week (Wk) for 12 or 24 Wks in 18 courses in 10 subjects demonstrates a favorable safety profile, dose-proportional PK with 5 >3 mg/kg doses and 5 >3 doses/Wk, Median fold-increases from baseline (BL) in F-cells of 4.4x BL (range 2.3-7x), 2.8x F-reticulocytes (1.6-14.8x), and 6.5x (range 3-11x) increases in mean fluorescence intensity (MFI, representing amount of HbF/RBC). Mean increases in % HbF were 6.5%. Evaluation of PB-04 has now begun in 7 subjects with SCD, 19-51 yrs; 3 were receiving HU with HbF responses (12-22%). Adverse events, F-cells, F-reticulocytes, MFI (amount of HbF/RBC or HbF/reticulocyte) by FACS and ImageStream Analysis and % HbF are assessed Q 4 wks; flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM) and sickling kinetics are assessed q 12 wks. In 4 subjects on 3 mg/kg QD TIW for 24 wks, changes observed from BL values included mean increases of 6% F-cells and 27% F-reticulocytes (2-fold) . In 3 ongoing subjects receiving 5 mg/kg QD TIW for 12 wks mean changes above BL are currently 13% F-cells, 15% F-reticulocytes; HbF/cell or HbF/reticulocyte have increased up to 10-fold. Mean % HbF has increased above HU effects in 2/3 pts on the second dose by 5-12.6% (mean 8.8%), producing 81-95% F-cells, and 35-38% HbF. Decline in FA-WB-VCAM) was observed ± HU and with both doses in 3 subjects. Correlation of HbF quantity/cell with RBC survival and sickling dynamics is being explored. These early results with not-yet-optimized doses of PB-04 suggest additive activity with HU, producing significant HbF and F-cell levels, and support further PK-optimized studies in patients with SCD.

GATA-1 and BCL11A Collaboratively Regulate Erythroid Gene Expression through HDAC1 Dependent Manner

Transcription factor GATA-1 plays important roles in hematopoiesis through interaction with other key transcription factors and epigenetic modulators. Previously, we established that GATA-1 can physically interact with HDAC1 at two arginine residues of the linker region between two zinc fingers. Arginine to alanine mutation at these sites blocks the HDAC1 interaction and results in constitutive GATA-1 acetylation, and reduced GATA-1 chromatin binding at the gene regulatory regions. In addition, the loss of HDAC1 interaction causes the blockage of erythroid and megakaryocytic differentiation in mice. Having established the importance of GATA-1 and HDAC1 interaction in GATA-1 function, next we aim to further investigate whether HDAC1-associated proteins are involved in regulating the function of GATA-1. We performed LC-MS/MS analysis using GATA-1 wild type and mutant expressing cells and identified that BCL11A was one of the major proteins that associates with GATA-1 through HDAC1. BCL11A is a master regulator of γ-globin gene silencing through association with the NuRD complex and blocking of NFYA recruitment to γ-globin gene promoters. The role of BCL11A in regulating gene expression during erythroid differentiation is not well studied. HDAC1 mediated GATA-1 and BCL11A association was validated by pull down and immunoprecipitation assay. Next, the BCL11A and HDAC1 interacting site was determined. The N-terminal region of BCL11A is found to be important for HDAC1 interaction by pull down assay. Through analysis of the secondary structure of BCL11A N-terminal, L12/K14 are predicted to be important for protein-protein interaction. Indeed, L12A/K14A mutation significantly reduced HDAC1 interaction as well as HDAC1 mediated GATA-1 and NuRD complex association. To assess the effects of the L12A/K14A mutation of BCL11A on gene expression, we performed RNA-seq analysis using BCL11A knock down (KD) or L12A/K14A mutant overexpressed in HUDEP-2 cells. The result shows that the L12A/K14A mutation de-repressed γ-globin expression and positively affects genes for erythrocyte development that is similar to the expression profile of BCL11A KD. Therefore, L12/K14 residues of BCL11A might have an important role in γ-globin silencing and erythropoiesis by mediating HDAC1 associated GATA-1 and NuRD complex. The Cut&Run assay was performed to study the recruitment of BCL11A, GATA-1, HDAC1, NuRD complex, and NFYA in WT, BCL11A KD, and Flag-BCL11A L12A/K14A mutant cells. There are over 7000 GATA-1 and BCL11A co-binding peaks detected in wild type cells. Within these peaks, about 2000 peaks are at BCL11A binding motif site. GATA-1, HDAC1 and NuRD binding were abolished at these sites in BCL11A KD or L12A/K14A mutant cells, while BCL11A binding was not affected. There are about 3000 peaks at GATA-1 binding motif sites. While GATA-1 binding is not affected at these sites in BCL11A KD or L12A/K14A mutant cells, BCL11A, HDAC1 and NuRD complex binding were significantly reduced. This study demonstrates that there are two modes of BCL11A and GATA-1 co-recruitment: BCL11A can recruit GATA-1 and GATA-1 can recruit BCL11A to their specific binding motifs, respectively. Interestingly, these two modes regulate differential gene targets. BCL11A recruits GATA-1 at genes mainly involved in chromatin organization and cell cycle, while GATA-1 recruits BCL11A to genes mainly involved in transcription, erythrocyte and megakaryocyte differentiation. At the β-globin locus, BCL11A KD or L12A/K14A mutation reduced GATA-1 and NuRD binding and enhanced NFYA binding at the γ-globin promoter, suggesting that BCL11A cooperates with GATA-1, HDAC1, and NuRD complex and prevents NFYA binding at the distal binding region to suppress γ-globin expression. At LCR hypersensitive site 2 (HS2) region, we also observed a reduction of GATA-1 and NuRD recruitment with BCL11A KD or L12A/K14A mutation while GATA-1 recruitment at other promoters or LCR regions were not changed, suggesting that the HS2 region may be involved in reactivating γ-globin in BCL11A KD or L12A/K14A mutation cells. Future study will continue to analyze how BCL11A KD or mutation affects BCL11A and GATA-1 co-binding and target gene expression, and NFYA or other factors recruitment. In summary, our study shows that BCL11A interacts with GATA-1 in a HDAC1 dependent manner and this interaction is important in regulating fetal globin expression and erythroid development.

Safety and Efficacy of RM-001 (Autologous HBG1/2 Promoter-modified CD34+ Hematopoietic Stem and Progenitor Cells) in Patients with Transfusion-Dependent β-Thalassemia

Background: Reactivating fetal globin (HbF) is a promising treatment for β-hemoglobinopathies. Natural mutations in the promoter region of γ-globin genes ( HBG1/2) that disrupt the binding of the transcriptional repressors BCL11A could lead to a lifelong persistence of fetal γ-globin expression. Using gene editing to mimic these mutations should reactivate γ-globin in patients with transfusion-dependent β-thalassemia (TDT) and ameliorate the symptoms of patients. RM-001 is a novel cell therapy that uses non-viral, ex vivo CRISPR-Cas9 gene editing in autologous hematopoietic stem and progenitor cells (HSPCs) at the promoter of the γ-globin genes ( HBG1/2) to disrupt the binding site of BCL11A. Aims: ChiCTR2100053406 and ChiCTR2100052858 are ongoing multi-center, first-in-human studies of RM-001 for TDT. Here, we present available safety and efficacy results from 7 patients that have been dosed with RM-001. Methods: Patients (6-35 y of age) with TDT receiving packed red blood cell (pRBC) transfusions of ≥100 mL/kg/y or ≥10 units/y in the previous 2ys were eligible. Peripheral CD34+ HSPCs were collected by apheresis after mobilization with G-CSF and plerixafor. CD34+ cells were edited with CRISPR-Cas9 using a guide RNA specific for the binding site of BCL11A on the HBG1/2 promoter. Prior to RM-001 product infusion (day 0), patients received myeloablative conditioning with Busulfan from day-7 to day-3. Patients were monitored for stem cell engraftment/hematopoietic recovery, adverse events (AEs), Hb production, HbF and F-cell expression, and pRBC transfusion requirements. Bone marrow cells were obtained at 3, 6, 12 and 24 months after RM-001 infusion to measure the on-target allelic editing frequency using next-generation sequencing. Results: Data presented here for 7 TDT patients have been treated with RM-001. As of July 31, 2023, patients were followed up from 1 to 20 months and 5 of them have been followed up more than 15 months. Six patients have β 0/β 0 genotype (CD17/CD41-42, n=1; CD41-42/CD41-42, n=5) and the other has β 0/β + genotype (CD41-42/IVS-II-654). In addition to β-thalassemia (CD41-42/CD41-42), two patients also carry a Southeast Asian deletion of α-globin genes (– SEA/αα). Patients had received a mean of 55.8 units/y pRBC transfusions (range: 39-79.6 units/y). All patients received a single dose of RM-001 cells, and achieved both neutrophil and platelet engraftments 2 to 3 weeks after RM-001 infusion (neutrophil: day 11-19, platelet: day 10-22). All patients ceased pRBC transfusions within 1 month after RM-001 infusion and remained transfusion-free through the reported period (Figure). For the 6 patients that have been followed up more than 6 months, HbF reached 9g/dL at 4 month post-RM-001 infusion and continuously maintained over this level through the reported period. From 6 month post-RM-001 infusion, hemoglobin in all patients consists of HbF (97.6%-99.8%) and HbA2 only, including the fifth patient who has a β0/β+ genotype (99.5% HbF). Five participants have remained transfusion independent more than 15 months and the mean HbF in the first 4 patients was 11g/dL(10.9-11.3 g/dL) at 18 month post-RM-001 infusion. The safety profile was generally consistent with busulfan myeloablation and autologous hematopoietic stem cell transplantation. No RM-001 related SAE report. Summary/Conclusion: This updated data reported here from 7 patients with TDT infused with RM-001 demonstrated clinically meaningful increases in total hemoglobin (Hb) and HbF levels. All patients stopped receiving pRBC transfusions within 1 month after RM-001 infusion and remained transfusion-free through the time of this analysis. The safety profile of RM-001 is generally consistent with myeloablative conditioning and autologous hematopoietic stem cell transplantation. These results strongly support continued investigation of RM-001 as a potential cure for patients with TDT. Data will be updated for the presentation. Submitted on behalf of the RM-001 Investigators.

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.

HEXIM1 is an essential transcription regulator during human erythropoiesis

AbstractRegulation of RNA polymerase II (RNAPII) activity is an essential process that governs gene expression; however, its contribution to the fundamental process of erythropoiesis remains unclear. hexamethylene bis-acetamide inducible 1 (HEXIM1) regulates RNAPII activity by controlling the location and activity of positive transcription factor β. We identified a key role for HEXIM1 in controlling erythroid gene expression and function, with overexpression of HEXIM1 promoting erythroid proliferation and fetal globin expression. HEXIM1 regulated erythroid proliferation by enforcing RNAPII pausing at cell cycle check point genes and increasing RNAPII occupancy at genes that promote cycle progression. Genome-wide profiling of HEXIM1 revealed that it was increased at both repressed and activated genes. Surprisingly, there were also genome-wide changes in the distribution of GATA-binding factor 1 (GATA1) and RNAPII. The most dramatic changes occurred at the β-globin loci, where there was loss of RNAPII and GATA1 at β-globin and gain of these factors at γ-globin. This resulted in increased expression of fetal globin, and BGLT3, a long noncoding RNA in the β-globin locus that regulates fetal globin expression. GATA1 was a key determinant of the ability of HEXIM1 to repress or activate gene expression. Genes that gained both HEXIM1 and GATA1 had increased RNAPII and increased gene expression, whereas genes that gained HEXIM1 but lost GATA1 had an increase in RNAPII pausing and decreased expression. Together, our findings reveal a central role for universal transcription machinery in regulating key aspects of erythropoiesis, including cell cycle progression and fetal gene expression, which could be exploited for therapeutic benefit.

MiR-365-3p mediates BCL11A and SOX6 erythroid-specific coregulation: A new player in HbF activation

Hemoglobin switching is a complex biological process not yet fully elucidated. The mechanism regulating the suppression of fetal hemoglobin (HbF) expression is of particular interest because of the positive impact of HbF on the course of diseases such as β-thalassemia and sickle cell disease, hereditary hemoglobin disorders that affect the health of countless individuals worldwide. Several transcription factors have been implicated in the control of HbF, of which BCL11A has emerged as a major player in HbF silencing. SOX6 has also been implicated in silencing HbF and is critical to the silencing of the mouse embryonic hemoglobins. BCL11A and SOX6 are co-expressed and physically interact in the erythroid compartment during differentiation. In this study, we observe that BCL11A knockout leads to post-transcriptional downregulation of SOX6 through activation of microRNA (miR)-365-3p. Downregulating SOX6 by transient ectopic expression of miR-365-3p or gene editing activates embryonic and fetal β-like globin gene expression in erythroid cells. The synchronized expression of BCL11A and SOX6 is crucial for hemoglobin switching. In this study, weidentified a BCL11A/miR-365-3p/SOX6 evolutionarily conserved pathway, providing insights into the regulation of the embryonic and fetal globin genes suggesting new targets for treating β-hemoglobinopathies.

In vivo HSC prime editing rescues sickle cell disease in a mouse model

AbstractSickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the β-globin gene. Current gene therapy studies are mainly focused on lentiviral vector–mediated gene addition or CRISPR/Cas9–mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a nonintegrating, prime editor–expressing viral vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of ∼40% of βS alleles in HSCs. On average, 43% of sickle hemoglobin was replaced by adult hemoglobin, thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.

A specific G9a inhibitor unveils BGLT3 lncRNA as a universal mediator of chemically induced fetal globin gene expression

Sickle cell disease (SCD) is a heritable disorder caused by β-globin gene mutations. Induction of fetal γ-globin is an established therapeutic strategy. Recently, epigenetic modulators, including G9a inhibitors, have been proposed as therapeutic agents. However, the molecular mechanisms whereby these small molecules reactivate γ-globin remain unclear. Here we report the development of a highly selective and non-genotoxic G9a inhibitor, RK-701. RK-701 treatment induces fetal globin expression both in human erythroid cells and in mice. Using RK-701, we find that BGLT3 long non-coding RNA plays an essential role in γ-globin induction. RK-701 selectively upregulates BGLT3 by inhibiting the recruitment of two major γ-globin repressors in complex with G9a onto the BGLT3 gene locus through CHD4, a component of the NuRD complex. Remarkably, BGLT3 is indispensable for γ-globin induction by not only RK-701 but also hydroxyurea and other inducers. The universal role of BGLT3 in γ-globin induction suggests its importance in SCD treatment.

A Cell-Permeant Nanobody-Based Degrader That Induces Fetal Hemoglobin.

Proximity-based strategies to degrade proteins have enormous therapeutic potential in medicine, but the technologies are limited to proteins for which small molecule ligands exist. The identification of such ligands for therapeutically relevant but "undruggable" proteins remains challenging. Herein, we employed yeast surface display of synthetic nanobodies to identify a protein ligand selective for BCL11A, a critical repressor of fetal globin gene transcription. Fusion of the nanobody to a cell-permeant miniature protein and an E3 adaptor creates a degrader that depletes cellular BCL11A in differentiated primary erythroid precursor cells, thereby inducing the expression of fetal hemoglobin, a modifier of clinical severity of sickle cell disease and β-thalassemia. Our strategy provides a means of fetal hemoglobin induction through reversible, temporal modulation of BCL11A. Additionally, it establishes a new paradigm for the targeted degradation of previously intractable proteins.

Early Effects of a Repurposed Therapeutic PB-04 in Beta Thalassemia Intermedia and in Transgenic Sickle Mice

Background: Biochemical, epidemiologic, genetic, and clinical data over 7 decades have shown that renewed or persistent expression of fetal globin reduces clinical severity in beta-thalassemias (BTI) and hemoglobinopathies (sickle cell disease, SCD). Hydroxyurea (HU) and epigenetic therapies which act through HDAC and DNMT-1 inhibition have demonstrated beneficial clinical activity; butyrate, a Class I HDAC inhibitor which suppresses BCL11A, increased HbF by 3-fold and total hemoglobin (Hb) by 1 gm/dl in SCD and eliminated transfusions in beta thalassemia patients, and decitabine strongly induces HbF, increases total Hb, and reduces biomarkers of sickling. Active erythropoiesis, basal EPO levels, available iron, and select mutations or modifiers are associated with responses. Additional small molecule therapies, especially those with additive activity with HU, are considered desirable for many patients world-wide. Methods: Seeking oral noncytotoxic therapeutic candidates with low development risks, we utilized a high-throughput screen with a gamma globin promoter-reporter and cytotoxicity readout for unrecognized fetal globin-inducing activity in libraries of all FDA- and EMA-approved or clinical stage therapeutics (for any medical indication). PB-04, a candidate with a benign long-term safety profile (used to inhibit L-dopa metabolism in Parkinson's disease), does not inhibit proliferation of erythroid progenitors and suppresses or displaces 4 repressors of the fetal globin gene promoter, BCL11A, HDAC3, KLF1, LSD-1, indicating targeted molecular activity. PB-04 has additive effects with HU in inducing F-cells in sickle cell erythroid progenitors. PB-04 was evaluated in 3 predictive animal models and in 3 escalating dose cohorts in adult subjects with BTI. Results: In 3 animal models, PB-04 increased fetal globin mRNA by 12-to 20-fold and F-cells by 20% in anemic baboons, F-cells and mean Hb F/cell by 2 to 7-fold in beta-YAC mice, and fetal globin mRNA by 1.8-fold and % F-cells by 2.4-fold without toxicity in Townes SCD mice, similar to the changes induced by HU (both given 5 days/wk for 4 wks). In six beta thalassemia (BTI) subjects who completed ten 12-week courses at 1, 3, or 5 mg/kg doses given once/day, 3 times/wk, increases from baseline in F-reticulocytes (mean 5.2-fold, range 1.5-15.7), F-cells, (mean 4.4-fold, range 2.3 to 7.3-fold), and HbF/cell (MFI), (mean 6.6-fold, range 3 to 11.3-fold), were observed (p<0.03 for all parameters). Increases were observed even in subjects with low baseline HbF levels. HbF increases by HPLC were 2.7 to 14%. Treatment emergent adverse effects (TEAEs) have been Gr 1-2 and largely transient in these subjects receiving 12 weeks' therapy. Other dosing regimens are being investigated. Conclusions: Initial studies in beta globin disorders indicate PB-04 induces fetal globin protein expression assayed by F-reticulocytes, F-cells, and mean HbF/cell in BTI patients and in transgenic SCD mice without toxicity. These findings indicate PB-04, which has complementary activity to all the approved SCD therapeutics, merits clinical evaluation in patients with sickle cell disease.

HEXIM1 Overexpression Alters GATA1 Activity and Promotes Fetal Globin Expression

Erythropoiesis is a highly organized process whereby multipotent HSCs become committed to the erythroid linage and mature into enucleate erythrocytes. This process is associated with significant changes in gene expression as non-erythroid genes are switched off while expression of erythroid genes is maintained. HEXIM1 is a key regulator of RNA Polymerase II (RNPII) activity that is essential for erythropoiesis. Following transcription initiation, RNPII transcribes 30-50 base pairs before pausing. Phosphorylation of the c-terminal domain of RNPII by the dimer pTEFb (positive transcription elongation factor beta) is necessary for RNPII to resume active transcription elongation. HEXIM1 associates with pTEFb in the 7SK complex and renders it inactive. Phosphorylation of HEXIM1 releases pTEFb from the 7SK complex, allowing it to act on RNPII. As 7SK can be targeted to specific sequences, HEXIM1/7SK mediated pTEFb delivery is a crucial mechanism to facilitate on-site pTEFb activation and RNPII pause release. Recent work from our lab demonstrated that HEXIM1 is essential in the regulation of erythroid proliferation, viability and gene expression, and also promotes the expression of GATA1 target genes (Murphy Blood 2021). New analyses of HEXIM1 overexpression (OE) in HUDEP2 cells revealed a significant increase of gamma globin production, with a higher proportion of cells expressing fetal hemoglobin. These changes were accompanied by downregulation of beta globin and multiple key repressors of fetal hemoglobin, including BCL11A, MYB, and SOX6. These phenotypes were dependent on pTEFb activity, as the pTEFb (CDK9) inhibitor NVP-2 rescued the proliferative advantage granted by HEXIM1 OE and greatly decreased both gamma globin levels and the portion of fetal hemoglobin expressing cells. Further suggesting the effects of HEXIM1 OE are pTEFb-dependent, overexpression of HEXIM1 with a tyrosine to alanine mutation (Y271A) that prevents phosphorylation of HEXIM1 and subsequent release of pTEFb cannot enhance proliferation or gamma globin production. To gain insights into the mechanisms underlying the increased fetal globin expression, we performed CUT&RUN assays for Ser5 phosphorylated and total RNPII in HEXIM1 OE and control cells. Surprisingly, OE of HEXIM1 had only modest impacts on RNPII pausing, but instead lead to changes in the genes occupied by RNPII. Notably, the gene that lost the most RNPII was HBB and the gene that gained the most RNPII was HBG. Of note, GATA1 motifs were highly enriched in regions that gained RNPII occupancy. CUT&RUN identified peaks of GATA1 in the HEXIM1 OE cells at regions of increased RNPII occupancy that were not present in the EV or Y271A controls. These novel GATA1 sites occurred in regions of previously closed chromatin, suggesting that HEXIM1 OE enhanced the pioneer ability of GATA1. Similar to RNPII, GATA1 occupancy shifted from the beta globin gene in cells transduced with EV or HEXIM Y271A to the gamma globin genes in cells transduced with WT HEXIM1. Novel RNPII and GATA1 peaks were also identified in other genes associated with a fetal erythroid phenotype, most notably ARID3A and LIN28B. Together, these results support a model where elevated HEXIM1 levels promote the pioneer activity of GATA1, and facilitate the expression of a fetal transcriptional program in adult cells in a pTEFb-dependent manner.

Gene Editing for Inherited Red Blood Cell Diseases.

Today gene therapy is a real therapeutic option to address inherited hematological diseases that could be beneficial for thousands of patients worldwide. Currently, gene therapy is used to treat different monogenic hematological pathologies, including several red blood cell diseases such as β-thalassemia, sickle cell disease and pyruvate kinase deficiency. This approach is based on addition gene therapy, which consists of the correction of hematopoietic stem cells (HSCs) using lentiviral vectors, which integrate a corrected version of the altered gene. Lentivirally-corrected HSCs generate healthy cells that compensate for the deficiency caused by genetic mutations. Despite its successful results, this approach lacks both control of the integration of the transgene into the genome and endogenous regulation of the therapeutic gene, both of which are important aspects that might be a cause for concern. To overcome these limitations, gene editing is able to correct the altered gene through more precise and safer approaches. Cheap and easy-to-design gene editing tools, such as the CRISPR/Cas9 system, allow the specific correction of the altered gene without affecting the rest of the genome. Inherited erythroid diseases, such as thalassemia, sickle cell disease and Pyruvate Kinase Deficiency, have been the test bed for these gene editing strategies, and promising results are currently being seen. CRISPR/Cas9 system has been successfully used to manipulate globin regulation to re-activate fetal globin chains in adult red blood cells and to compensate for hemoglobin defects. Knock-in at the mutated locus to express the therapeutic gene under the endogenous gene regulatory region has also been accomplished successfully. Thanks to the lessons learned from previous lentiviral gene therapy research and trials, gene editing for red blood cell diseases is rapidly moving from its proof-of-concept to its first exciting results in the clinic. Indeed, patients suffering from β-thalassemia and sickle cell disease have already been successfully treated with gene editing, which will hopefully inspire the use of gene editing to cure erythroid disorders and many other inherited diseases in the near future.

Disrupting the adult globin promoter alleviates promoter competition and reactivates fetal globin gene expression

AbstractThe benign condition hereditary persistence of fetal hemoglobin (HPFH) is known to ameliorate symptoms of co-inherited β-hemoglobinopathies, such as sickle cell disease and β-thalassemia. The condition is sometimes associated with point mutations in the fetal globin promoters that disrupt the binding of the repressors BCL11A or ZBTB7A/LRF, which have been extensively studied. HPFH is also associated with a range of deletions within the β-globin locus that all reside downstream of the fetal HBG2 gene. These deletional forms of HPFH are poorly understood and are the focus of this study. Numerous different mechanisms have been proposed to explain how downstream deletions can boost the expression of the fetal globin genes, including the deletion of silencer elements, of genes encoding noncoding RNA, and bringing downstream enhancer elements into proximity with the fetal globin gene promoters. Here we systematically analyze the deletions associated with both HPFH and a related condition known as δβ-thalassemia and propose a unifying mechanism. In all cases where fetal globin is upregulated, the proximal adult β-globin (HBB) promoter is deleted. We use clustered regularly interspaced short palindromic repeats-mediated gene editing to delete or disrupt elements within the promoter and find that virtually all mutations that reduce ΗΒΒ promoter activity result in elevated fetal globin expression. These results fit with previous models where the fetal and adult globin genes compete for the distal locus control region and suggest that targeting the ΗΒΒ promoter might be explored to elevate fetal globin and reduce sickle globin expression as a treatment of β-hemoglobinopathies.

Therapeutic approaches in patients with β-thalassemia

Beta-thalassemia (β-thal) is a congenital hemoglobinopathy explained by a decreased level (β+) or absence (βο) of β-globin gene expression. Microcytic hypochromic anemia and various clinical symptoms comprising severe anemia to clinically nonsymptomatic features. Treatment with an ordered blood transfusion and iron chelator agents can decrease transfusion iron overload that causes normal maturation. These patients also are at high risk for secondary iron overload because of erythropheron (GF15–TWSG1) release from erythroblasts resulting in erythroid hyperplasia. Based on the previous studies, chemicals such as hydroxyurea and 5-azacytidine are useful in treating β-hemoglobinopathy, including β-thal and sickle cell disease (SCD). Regarding both side effects and lifelong treatment of these chemical components, researchers have recently regarded gene-based treatments. These techniques, such as micro RNA gene silencing, viral-mediated gene editing, and clustered regulatory interspaced short palindromic repeats (CRISPR)-CAS9 systems, are the most commonly used gene therapy methods. Nowadays, ɣ-globin (fetal globin) gene reactivation is one of the most popular treatments for β-thal. Researches showed that these gene modification methods for γ-globin gene reactivation are also useful in increasing hemoglobin F (HbF) and helping patients with β-thal. In this review study, new therapeutic approaches to manage this disorder are regarded.

The Benefits Trial of PB-04 to Evaluate Safety, PK, and Preliminary Efficacy in Inducing Fetal Globin Expression in Beta Thalassemia Intermedia and Sickle Cell Disease

Increased expression of fetal globin (HBG) has been shown to reduce clinical severity of beta-globin disorders and increase survival in sickle cell disease (SCD), through improved globin chain balance in beta thalassemia and reduced HbS polymerization. Pharmacologic approaches are considered most feasible for a majority of patients. Both proportions of F-cells and quantity of hemoglobin F (HbF)/cell are important for therapeutic effects. PB-04 was identified in a high-throughput screen of US and EU-approved drugs to activate HBG gene transcription, without cytotoxicity, to induce fetal globin expression (gamma globin mRNA, F-cells, HbF/cell, HbF), and to suppress or displace 4 repressors of the fetal globin gene promoter (BCL11A, LSD-1, KLF-1, and HDAC-3) in hemoglobinopathy patients' erythroid progenitors. PB-04 enhanced HbF in progenitors from hydroxyurea (HU)-treated patients with sickle cell disease. In in vivo studies, PB-04 induced fetal globin expression >20-fold with oral dosing in anemic baboons, and by 3.5-fold in mice transgenic for 2 copies of the human β-globin gene locus. This agent has been approved and in broad use for Parkinson's Disease treatment for 5 decades in Europe and Canada, to enhance the PK of an active Parkinson's agent, solely in a combination formulation. Because of its safety with chronic dosing up to 1300 mg/day, PB-04 is therefore of interest for repurposing in the treatment of thalassemia and SCD.This Ph1b trial (NCT004432623) will evaluate safety, tolerability, PK, and preliminary efficacy (fetal globin expression) with at least 3 escalating dose cohorts in up to 24 beta thalassemia intermedia (BTI) patients and 12 patients with sickle cell disease, > 18 years, both genders, with 12 weeks of treatment and 4 weeks of follow-up. Doses to be explored were selected from safe and active human equivalent doses in preclinical toxicology and efficacy studies in 2 species. Primary inclusion criteria include clinical diagnosis of BTI, including HbE beta thalassemia, with at least one beta globin gene mutation, or diagnosis of HbSS or HbSb thalassemia (after dose selection); total Hb levels 6-10 gm/dL. Exclusion criteria include red blood cell (RBC) transfusion within 2 months prior to administration of study medication, receiving regular transfusions, and hepatic or renal function > 3 x institutional ULN.Primary endpoints include occurrence, severity, and duration of adverse events by CTCAE v5.0 criteria and PK parameters. Secondary endpoints include change from baseline in assays of HbF expression, total Hb, markers of hemolysis . Exploratory endpoints include select polymorphisms associated with basal fetal globin expression and in vitro responses to the study drug. Statistical analysis will assess the rate of adverse events, PK parameters by dose, and changes in F-cells, F-reticulocytes, HbF/cell, HbF (% and total), compared to 2 averaged baseline values using the Wilcoxon signed rank test. Changes will be analyzed by mixed effect models during the 12-week dosing and 4-week follow-up period. Currently 3 dose cohorts with BTI are enrolled. This study, if successful, will provide a rational basis for definitive trials of an established safe oral therapeutic, with no overlapping toxicities with other experimental or approved agents, for combined use with HU and other therapeutics which enhance red cell viability in the globin disorders.The expertise and contributions of Jim Cradock of NCATS TRND to this work before his passing are gratefully acknowledged. DisclosuresKuo: Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Apellis: Consultancy; Bluebird Bio: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria. Sheth: CRISPR: Consultancy; Bluebird bio: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; Chiesi: Consultancy; Imara: Research Funding; Agios: Consultancy; Dispersol: Research Funding. Al-Samkari: Moderna: Consultancy; Amgen: Research Funding; Rigel: Consultancy; Argenx: Consultancy; Novartis: Consultancy; Dova/Sobi: Consultancy, Research Funding; Agios: Consultancy, Research Funding. Pace: Imara Inc.: Consultancy. Kuypers: Forma Therapeutics, Inc.: Research Funding. Nouraie: Phoenicia BioScience Inc.: Consultancy. Perrine: Agios Pharmaceuticals: Consultancy; Emmaus Medical: Consultancy.

Evidences of the PI5P Increasing the Expression of HBG and Gamma Globin Concentrations

Efforts have been made to identify modulators of increasing fetal hemoglobin (HbF - α2ɣ2) that may act as promising targets for hemoglobinopathies, such as in sickle cell disease and beta thalassemia. The inositide PI5P is a second messenger from the inositol pathway that comprehends in the main phosphorylation target of the enzyme phosphatidylinositol-4-phosphate-5-kinase-II-alpha (PIPKIIα), found in high concentrations in the erythrocyte. Some evidences have postulated a relation between this enzyme and the expression of the hemoglobin (Hb) genes (1,2). More recently, PI5P was found to stabilize UHRF1 protein (3), which has been reported as a possible repressor of HBG (from ɣ globin gene), in the switch from ɣ to β in the transition HbF- HbA (4). To evaluate the effects of the inositide PI5P (substrate of PIPKIIα) in globins, a preliminary time course experiment was performed. KU812 cells (ATCC CRL-2099), which express all three globin genes (HBA, HBB and HBG), were treated with 1μM of PI5P (Thermo Fischer Scientific, USA) in triplicate (4, 8 and 12 hours of treatment) - time 0h used as control. Gene expression and protein concentrations were determined by qPCR - SYBR Green Master Mix amplification and detection kit (Thermo Fischer Scientific, USA, in equipment StepOnePlus Real Time PCR System, Applied Biosystems - USA) using 2DDCT method with the ACTB and GAPDH genes as internal controls - and immunoblotting. Primers were design according to Zacariotto et al (2). No additional phosphate was imputed into the cell culture and the ATP consumption was monitored by ATP Determination Kit (Thermo Fisher Scientific, EUA). PI5P treatment resulted in significant increasement of HBG RNA transcripts (p= 0.001; 8 hours / treatment) and decrease of PIP4K2A (from PIPKIIα enzyme) (p=0.02; 12 hours / treatment), with no other significant changes into globin genes (HBA and HBB). At the protein level, there was a prominent increase in ɣ globin concentrations according to time course. No other consistent change in the concentrations of PIPKIIα or globin chains were observed. Due to protein similarities between the subfamilies of phosphatidylinositol-phosphate kinases (PIPKins) (5), the gene expression of these enzymes was also monitored by qPCR. There was a significant reduction of PIP5K1A (from PIPKIα enzyme) (p=0.01 - 4h; p=0.01 - 8h, and p=0.009 - 12h) as well as PIP4K2B (from PIPKIIβ enzyme) (p=0.03 - 4 hours/ treatment). No significant changes were observed in the other PIPKins (PIPKIβ, PIPKIɣ, PIPKIIɣ and PIPKIII). Considering that a significant consumption of intracellular ATP was observed in time course (p=0.006) it is possible to infer that the high concentrations of PI5P have shifted the inositol pathway resulting in the downregulation of its own PIPKin genes and culminating into the upregulation of HBG, reflected into the protein level. The mechanisms involving the inositide as second messenger, the role of PIPKins or other genes into the modulation of hemoglobin genes, particularly in HBG, should be further investigated. However, despite preliminary, these results reinforce the involvement of the PIPKins and its molecular targets (mainly PIPKIα and PI5P) in globin gene regulation and could represent a promising target for future therapeutic target for hemoglobinopathies. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp.1.Wenning MR, Mello MP, Andrade TG, et al. PIP4KIIA and beta-globin: transcripts differentially expressed in reticulocytes and associated with high levels of Hb H in two siblings with Hb H disease. Eur J Haematol. 2009; 83: 490-3.2.Zaccariotto TR, Lanaro C, Albuquerque DM, Santos MN, Bezerra MA, Cunha FG, et al. Expression profiles of phosphatidylinositol phosphate kinase genes during normal human in vitro erythropoiesis. Genet Mol Res. 2012; 11: 3861-8.3.Gelato KA, Tauber M, Ong MS, Winter S, et al. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate. Mol Cell. 2014;54(6):905-19.4.Ruopeng Feng, Phillip A Doerfler, Yu Yao XT, et al. The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of H BG Promoter DNA Methylation. Blood. 2018;132:410.5.Heck JN, Mellman DL, Ling K, et al. A conspicuous connection: structure defines function for the phosphatidylinositol-phosphate kinase family. Crit Rev Biochem Mol Biol. 2007; 42: 15-39. DisclosuresCosta: Novartis: Consultancy.