Ginsenoside Rg1 promotes fetal hemoglobin production in vitro: A potential therapeutic avenue for ß-thalassemia.

ß-thalassemia, a globally prevalent genetic disorder, urgently requires innovative treatment options. Fetal hemoglobin (HbF) induction stands as a key therapeutic approach. This investigation focused on Ginsenoside Rg1 from the Panax genus for HbF induction. Employing K562 cells and human erythroid precursor cells (ErPCs) derived from neonatal cord blood, the study tested Rg1 at different concentrations. We measured its effects on γ-globin mRNA levels and HbF expression, alongside assessments of cell proliferation and differentiation. In K562 cells, Rg1 at 400 µM significantly increased γ-globin mRNA expression by 4.24 ± 1.08-fold compared to the control. In ErPCs, the 800 µM concentration was most effective, leading to an over 80% increase in F-cells and a marked upregulation in HbF expression. Notably, Rg1 did not adversely affect cell proliferation or differentiation, with the 200 µM concentration showing an increase in γ-globin mRNA by 2.33 ± 0.58-fold, and the 800 µM concentration enhancing HbF expression by 2.59 ± 0.03-fold in K562 cells. Our results underscore Rg1's potential as an effective and safer alternative for ß-thalassemia treatment. By significantly enhancing HbF levels without cytotoxicity, Rg1 offers a notable advantage over traditional treatments like Hydroxyurea. While promising, these in vitro findings warrant further in vivo exploration to confirm Rg1's therapeutic efficacy and to unravel its underlying mechanistic pathways.

Long Non-Coding RNA H19 Leads to Upregulation of γ-Globin Gene Expression during Erythroid Differentiation.

Long noncoding RNAs (lncRNAs) are important because they are involved in a variety of life activities and have many downstream targets. Moreover, there is also increasing evidence that some lncRNAs play important roles in the expression and regulation of γ-globin genes. In our previous study, we analyzed genetic material from nucleated red blood cells (NRBCs) extracted from premature and full-term umbilical cord blood samples. Through RNA sequencing (RNA-Seq) analysis, lncRNA H19 emerged as a differentially expressed transcript between the two blood types. While this discovery provided insight into H19, previous studies had not investigated its effect on the γ-globin gene. Therefore, the focus of our study was to explore the impact of H19 on the γ-globin gene. In this study, we discovered that overexpressing H19 led to a decrease in HBG mRNA levels during erythroid differentiation in K562 cells. Conversely, in CD34+ hematopoietic stem cells and human umbilical cord blood-derived erythroid progenitor (HUDEP-2) cells, HBG expression increased. Additionally, we observed that H19 was primarily located in the nucleus of K562 cells, while in HUDEP-2 cells, H19 was present predominantly in the cytoplasm. These findings suggest a significant upregulation of HBG due to H19 overexpression. Notably, cytoplasmic localization in HUDEP-2 cells hints at its potential role as a competing endogenous RNA (ceRNA), regulating γ-globin expression by targeting microRNA/mRNA interactions.

Identification and characterization of CHD4-associated eRNA as a novel modulator of fetal hemoglobin levels in ß-thalassemia.

Fetal-to-adult hemoglobin switching is controlled by programmed silencing of γ-globin while the re-activation of fetal hemoglobin (HbF) is an effective strategy for ameliorating the clinical severity of ß-thalassemia and sickle cell disease. The identification of enhancer RNAs (eRNAs) related to the fetal (α2γ2) to adult hemoglobin (α2ß2) switching remains incomplete. In this study, the transcriptomes of GYPA+ cells from six ß-thalassemia patients with extreme HbF levels were sequenced to identify differences in patterns of noncoding RNA expression. It is interesting that an enhancer upstream of CHD4, an HbF-related core subunit of the NuRD complex, was differentially transcribed. We found a significantly positive correlation of eRNA-CHD4 enhancer-gene interaction using the public database of FANTOM5. Specifically, the eRNA-CHD4 expression was found to be significantly higher in both CD34+ HSPCs and HUDEP-2 than those in K562 cells which commonly expressed high level of HbF, suggesting a correlation between eRNA and HbF expression. Furthermore, prediction of transcription binding sites of cis-eQTLs and the CHD4 genomic region revealed a putative interaction site between rs73264846 and ZNF410, a known transcription factor regulating HbF expression. Moreover, in-vitro validation showed that the inhibition of eRNA could reduce the expression of HBG expression in HUDEP-2 cells. Taken together, the findings of this study demonstrate that a distal enhancer contributes to stage-specific silencing of γ-globin genes through direct modulation of CHD4 expression and provide insights into the epigenetic mechanisms of NuRD-mediated hemoglobin switching.

Activation of γ-globin expression by LncRNA-mediated ERF promoter hypermethylation in ß-thalassemia.

The mechanism that drives the switch from fetal to adult hemoglobin (Hb) provides a therapeutic target for ß-thalassemia. We have previously identified that hypermethylation of transcription factor ERF promoter reactivated γ-globin expression. To uncover the mechanism underlying the hypermethylation of ERF promoter, we performed RNA sequencing in ß0/ß0-thalassemia patients and identified an upregulated long noncoding RNA (RP11-196G18.23) associated with HbF production. RP11-196G18.23 bound to the ERF promoter and recruited DNA methyltransferase 3A to promote DNA hypermethylation-mediated ERF downregulation, thereby ameliorating ERF-induced γ-globin inactivation. The identification of RP11-196G18.23 provides an epigenetic mechanism for the reactivation of fetal γ-globin expression for ß-hemoglobinopathies.

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

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for ß-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (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 used 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 elevated the HbF levels but affected 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 effects. 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 because 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 that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

Bach1 inhibitor HPP-D mediates γ-globin gene activation in sickle erythroid progenitors.

Sickle cell disease (SCD) is the most common ß-hemoglobinopathy caused by various mutations in the adult ß-globin gene resulting in sickle hemoglobin production, chronic hemolytic anemia, pain, and progressive organ damage. The best therapeutic strategies to manage the clinical symptoms of SCD is the induction of fetal hemoglobin (HbF) using chemical agents. At present, among the Food and Drug Administration-approved drugs to treat SCD, hydroxyurea is the only one proven to induce HbF protein synthesis, however, it is not effective in all people. Therefore, we evaluated the ability of the novel Bach1 inhibitor, HPP-D to induce HbF in KU812 cells and primary sickle erythroid progenitors. HPP-D increased HbF and decreased Bach1 protein levels in both cell types. Furthermore, chromatin immunoprecipitation assay showed reduced Bach1 and increased NRF2 binding to the γ-globin promoter antioxidant response elements. We also observed increased levels of the active histone marks H3K4Me1 and H3K4Me3 supporting an open chromatin configuration. In primary sickle erythroid progenitors, HPP-D increased γ-globin transcription and HbF positive cells and reduced sickled erythroid progenitors under hypoxia conditions. Collectively, our data demonstrate that HPP-D induces γ-globin gene transcription through Bach1 inhibition and enhanced NRF2 binding in the γ-globin promoter antioxidant response elements.

An α-chain modification rivals the effect of fetal hemoglobin in retarding the rate of sickle cell fiber formation.

Adults with sickle cell disease bear a mutation in the ß-globin gene, leading to the expression of sickle hemoglobin (HbS; α2ßS2). Adults also possess the gene for γ-globin, which is a component of fetal hemoglobin (HbF, α2γ2); however, γ-chain expression normally ceases after birth. As HbF does not form the fibers that cause the disease, pharmacological and gene-modifying interventions have attempted to either reactivate expression of the γ chain or introduce a gene encoding a modified ß chain having γ-like character. Here, we show that a single-site modification on the α chain, αPro114Arg, retards fiber formation as effectively as HbF. Because this addition to the repertoire of anti-sickling approaches acts independently of other modifications, it could be coupled with other therapies to significantly enhance their effectiveness.

Effect of the LSD1 inhibitor RN-1 on γ-globin and global gene expression during erythroid differentiation in baboons (Papio anubis).

Elevated levels of Fetal Hemoglobin interfere with polymerization of sickle hemoglobin thereby reducing anemia, lessening the severity of symptoms, and increasing life span of patients with sickle cell disease. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the γ-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on γ-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.

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 ß-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 ß-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 ß-hemoglobinopathies.

Effects of Mithramycin on BCL11A Gene Expression and on the Interaction of the BCL11A Transcriptional Complex to γ-Globin Gene Promoter Sequences.

The anticancer drug mithramycin (MTH), has been proposed for drug repurposing after the finding that it is a potent inducer of fetal hemoglobin (HbF) production in erythroid precursor cells (ErPCs) from ß-thalassemia patients. In this respect, previously published studies indicate that MTH is very active in inducing increased expression of γ-globin genes in erythroid cells. This is clinically relevant, as it is firmly established that HbF induction is a valuable approach for the therapy of ß-thalassemia and for ameliorating the clinical parameters of sickle-cell disease (SCD). Therefore, the identification of MTH biochemical/molecular targets is of great interest. This study is inspired by recent robust evidence indicating that the expression of γ-globin genes is controlled in adult erythroid cells by different transcriptional repressors, including Oct4, MYB, BCL11A, Sp1, KLF3 and others. Among these, BCL11A is very important. In the present paper we report evidence indicating that alterations of BCL11A gene expression and biological functions occur during MTH-mediated erythroid differentiation. Our study demonstrates that one of the mechanisms of action of MTH is a down-regulation of the transcription of the BCL11A gene, while a second mechanism of action is the inhibition of the molecular interactions between the BCL11A complex and specific sequences of the γ-globin gene promoter.

Molecular understanding of unusual HbE-ß+-thalassemia with Hb phenotype similar to HbE heterozygote: simple and rapid differentiation using HbE levels.

BACKGROUND: Low HbF expression in HbE-ß+-thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the ß- and α-globin genes and the modifying factors related to HbF expression in patients with an Hb phenotype similar to that of HbE heterozygotes. Furthermore, screening tools for differentiating HbE-ß+-thalassemia from HbE heterozygotes have been investigated. PARTICIPANTS AND METHODS: A total of 2133 participants with HbE and HbA with varying HbF levels were recruited. Polymerase chain reaction-based DNA analysis and sequencing were performed to characterize ß- and α-globin genes. DNA polymorphism at position -158 nt 5' to Gγ-globin was performed by XmnI restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA2, HbE, and HbF levels for the differentiation of HbE-ß+-thalassemia from HbE heterozygotes were determined. RESULTS: Five ß+-thalassemia mutations trans to ßE-gene (ß-87(C>A), ß-31(A>G), ß-28(A>G), ß19(A>G), and ß126(T>G)) were identified in 79 patients. Among these, 54 presented with low HbF levels, and 25 presented with high HbF levels. ROC curve analysis revealed an excellent AUC of 1.000 (95% confidence interval:1.000-1.000) for HbE levels, and a cut-off point of ≥35.0% had 100.0% sensitivity, specificity, and Youden's index for differentiating HbE-ß+-thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-ß+-thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α2-globin genes were identified. The genotype and allele of the polymorphism at -158 nt 5' to Gγ-globin was found to be negatively associated with HbF expression. CONCLUSIONS: HbE-ß+-thalassemia cannot be disregarded until appropriate DNA analysis is performed, and the detection of α-thalassemia mutations should always be performed under these conditions. An HbE level ≥35.0% may indicate screening of samples for DNA analysis for HbE-ß+-thalassemia diagnosis.

HbE-ß⁺-thalassemia displays a wide range of HbF expression, which may lead to the misdiagnosis of HbE heterozygosity in patients whose Hb analysis shows HbE and HbA. α-Thalassemia may be a major factor associated with decreased secondary activation of HbF expression in the disease.HbE may be a potential indicator for effectively differentiating HbE-ß⁺-thalassemia from HbE heterozygotes.The high proportion and heterogeneity of α-thalassemia mutations found in patients with HbE-ß⁺-thalassemia evoke a complex thalassemia syndrome, requiring complete DNA analysis.

BCL11A-targeted γ-globin gene induction by triterpenoid glycosides of Fagonia indica: A preclinical scientific validation of indigenous herb for the treatment of ß-hemoglobinopathies.

Pharmacological induction of fetal hemoglobin has proven to be a promising therapeutic intervention in ß-hemoglobinopathies by reducing the globin chain imbalance and inhibiting sickle cell polymerization. Fagonia indica has shown therapeutic relevance to ß-thalassemia. Therefore, we study the ethnopharmacological potential of Fagonia indica and its biomarker compounds for their HbF induction ability for the treatment of ß-thalassemia. Here, we identify, compound 8 (triterpenoid glycosides) of F. indica. as a prominent HbF inducer in-vitro and in-vivo. Compound 8 showed potent erythroid differentiation, enhanced cellular proliferation, ample accumulation of total hemoglobin, and a strong notion of γ-globin gene expression in K562 cultures. Compound 8 treatment also revealed strong induction of erythroid differentiation and fetal hemoglobin mRNA and protein in adult erythroid precursor cells. This induction was associated with simultaneous downregulation of BCL11A and SOX6, and overexpression of the GATA-1 gene, suggesting a compound 8-mediated partial mechanism involved in the reactivation of fetal-like globin genes. The in vivo study with compound 8 (10 mg/kg) in ß-YAC mice resulted in significant HbF synthesis demonstrated by the enhanced level of F-cells (84.14 %) and an 8.85-fold increase in the γ-globin gene. Overall, the study identifies compound 8 as a new HbF-inducing entity and provides an early "proof-of-concept" to enable the initiation of preclinical and clinical studies in the development of this HbF-inducing agent for ß-thalassemia.

Proteomics screening uncovers HMGA1 as a promising negative regulator for γ-globin expression in response to decreased ß-globin levels.

Reactivation of fetal hemoglobin (HbF) is a critical goal for the treatment of patients with hemoglobinopathies. ß-globin disorders can trigger stress erythropoiesis in red blood cells (RBCs). Cell-intrinsic erythroid stress signals promote erythroid precursors to express high levels of fetal hemoglobin, which is also known as γ-globin. However, the molecular mechanism underlying γ-globin production during cell-intrinsic erythroid stress remains to be elucidated. Here, we utilized CRISPR-Cas9 to model a stressed state caused by reduced levels of adult ß-globin in HUDEP2 human erythroid progenitor cells. We found that a decrease in ß-globin expression correlates with the upregulation of γ-globin expression. We also identified transcription factor high-mobility group A1 (HMGA1; formerly HMG-I/Y) as a potential γ-globin regulator that responds to reduced ß-globin levels. Upon erythroid stress, there is a downregulation of HMGA1, which normally binds -626 to -610 base pairs upstream from the STAT3 promoter, to downregulate STAT3 expression. STAT3 is a known γ-globin repressor, so the downregulation of HMGA1 ultimately upregulates γ-globin expression. SIGNIFICANCE: This study demonstrated HMGA1 as a potential regulator in the poorly understood phenomenon of stress-induced globin compensation, and after further validation these results might inform new strategies to treat patients with sickle cell disease and ß-thalassemia.

Potent and uniform fetal hemoglobin induction via base editing.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate ß-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

Precision Editing as a Therapeutic Approach for ß-Hemoglobinopathies.

Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the ß-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic ß- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with ß-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for ß-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies.

ZBTB7A regulates primed-to-naïve transition of pluripotent stem cells via recognition of the PNT-associated sequence by zinc fingers 1-2 and recognition of γ-globin -200 gene element by zinc fingers 1-4.

ZBTB7A, a transcription factor containing a tandem array of four Cys2-His2 zinc fingers (ZFs), is vital for multiple physiological events through directional binding to different genomic loci. Our previously determined crystal structure of ZBTB7A in complex with a GCCCCTTCCCC sequence revealed that all four ZFs (ZF1-4) are involved in binding to γ-globin -200 gene element to repress fetal haemoglobin expression. Recently, it has been reported that ZBTB7A drives primed-to-naïve transition (PNT) of pluripotent stem cells through binding to a 12-bp consensus sequence ([AAGGACCCAGAT], referred to as PNT-associated sequence). Here, we report a crystal structure of ZBTB7A ZF1-3 in complex with the PNT-associated sequence. The structure shows that ZF1 and ZF2 primarily contribute to recognizing the GACCC core sequence mimicking the half part (GCCCC) of γ-globin -200 gene element via specific hydrogen bonding and van der Waals contacts. The mutations of key residues in ZF1-2 remarkably reduce their binding affinities for the PNT-associated sequence in vitro and cannot restore epiblast stem cells to the naïve pluripotent state in vivo. Collectively, our studies demonstrate that ZBTB7A mainly employs its ZF1-2 to recognize the PNT-associated sequence but recognizes γ-globin -200 gene element via ZF1-4, providing insights into the molecular mechanism for the diversity of ZBTB7A's genomic localization.

Combinatorial targeting of epigenome-modifying enzymes with decitabine and RN-1 synergistically increases HbF.

Increased fetal hemoglobin (HbF) levels reduce the symptoms of sickle cell disease (SCD) and increase the lifespan of patients. Because curative strategies for bone marrow transplantation and gene therapy technologies remain unavailable to a large number of patients, the development of a safe and effective pharmacological therapy that increases HbF offers the greatest potential for disease intervention. Although hydroxyurea increases HbF, a substantial proportion of patients fail to demonstrate an adequate response. Pharmacological inhibitors of DNA methyltransferase (DNMT1) and lysine-specific demethylase 1A (LSD1), 2 epigenome-modifying enzymes associated with the multiprotein corepressor complex recruited to the repressed γ-globin gene, are powerful in vivo inducers of HbF. The hematological side effects of these inhibitors limit feasible clinical exposures. We evaluated whether administering these drugs in combination could reduce the dose and/or time of exposure to any single agent to minimize adverse effects, while achieving additive or synergistic increases in HbF. The DNMT1 inhibitor decitabine (0.5 mg/kg per day) and the LSD1 inhibitor RN-1 (0.25 mg/kg per day) administered in combination 2 days per week produced synergistic increases in F-cells, F-reticulocytes, and γ-globin messenger RNA in healthy baboons. Large increases in HbF and F-cells were observed in healthy, nonanemic, and anemic (phlebotomized) baboons. Combinatorial therapy targeting epigenome-modifying enzymes could thus be a useful strategy for producing larger increases in HbF to modify the clinical course of SCD.

The Novel Role of the B-Cell Lymphoma/Leukemia 11A (BCL11A) Gene in ß-Thalassaemia Treatment.

ß-thalassaemia is a genetic disorder resulting in a reduction or absence of ß-globin gene expression. Due to the high prevalence of ß-thalassaemia and the lack of available treatment other than blood transfusion and haematopoietic stem cell (HSC) transplantation, the disease represents a considerable burden to clinical and economic systems. Foetal haemoglobin has an appreciated ameliorating effect in ß-haemoglobinopathy, as the γ-globin chain substitutes the ß-globin chain reduction by pairing with the excess α-globin chain in ß-thalassaemia and reduces sickling in sickle cell disease (SCD). BCL11A is a critical regulator and repressor of foetal haemoglobin. Downregulation of BCL11A in adult erythroblasts and cell lines expressing adult haemoglobin led to a significant increase in foetal haemoglobin levels. Disruption of BCL11A erythroid enhancer resulted in disruption of the BCL11A gene solely in the erythroid lineages and increased γ-globin expression in adult erythroid cells. Autologous haematopoietic stem cell gene therapy represents an attractive treatment option to overcome the immune complications and donor availability associated with allogeneic transplantation. Using genome editing technologies, the disruption of BCL11A to induce γ- globin expression in HSCs has emerged as an alternative approach to treat ß-thalassaemia. Targeting the +58 BCL11A erythroid enhancer or BCL11A binding motif at the γ-gene promoter with CRISPR-Cas9 or base editors has successfully disrupted the gene and the binding motif with a subsequent increment in HbF levels. This review outlines the critical role of BCL11A in γ-globin gene silencing and discusses the different genome editing approaches to downregulate BCL11A as a means for ameliorating ß-thalassaemia.

Role of microRNA in hydroxyurea mediated HbF induction in sickle cell anaemia patients.

Hydroxyurea (HU) is found to be beneficial in sickle cell anaemia (SCA) patients, due to its ability to increase foetal haemoglobin (HbF), however, patients show a variable response. Differences in HbF levels are attributed to many factors; but, the role of miRNA in HbF regulation is sparsely investigated. In this study, we evaluated the effect of miRNA expression on HbF induction in relation to hydroxyurea therapy in 30 normal controls, 30 SCA patients at baseline, 20 patients after 3 and 6 months of hydroxyurea (HU) therapy. HbF levels were measured by HPLC. Total RNA and miRNA were extracted from CD71+ erythroid cells and the expression was determined using Taqman probes. The mean HbF level increased 7.54 ± 2.44 fold, after 3 months of HU therapy. After the HU therapy 8 miRNAs were significantly up-regulated while 2 were down-regulated. The increase in miR-210, miR16-1, and miR-29a expression and decrease in miR-96 expression were strongly associated with the HU mediated HbF induction. Post HU therapy, decreased miR-96 expression negatively correlate with HbF and γ-globin gene while increased expression of miR-210, miR-16-1 and miR-29a post HU therapy positively corelate with HbF and γ-globin gene. Thus, suggest that miR-210, miR-16-1 and miR-29a are positive regulator of γ-globin gene and miR-96 is negative regulator of γ-globin gene. The study suggests the role of miR-210, miR16-1, miR-29a, and miR-96 in γ-globin gene regulation leading to HbF induction. Identification of the relevant protein targets might be useful for understanding the HU mediated HbF induction.

The rs368698783 (G>A) Polymorphism Affecting LYAR Binding to the Aγ-Globin Gene Is Associated with High Fetal Hemoglobin (HbF) in ß-Thalassemia Erythroid Precursor Cells Treated with HbF Inducers

The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5'-GGTTAT-3') is located within the 5'-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in ß-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify ß-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in ß-thalassemia patients. The most striking association was found using rapamycin as an HbF inducer. The results presented here could be considered important not only for basic biomedicine but also in applied translational research for precision medicine in personalized therapy of ß-thalassemia. Accordingly, our data suggest that the rs368698783 polymorphism might be considered among the parameters useful to recruit patients with the highest probability of responding to in vivo hydroxyurea (HU) treatment.