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Treatment of Erythroid Precursor Cells from β-Thalassemia Patients with Cinchona Alkaloids: Induction of Fetal Hemoglobin Production.

ABSTRACT: β-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding β-globin, causing an absence or low-level production of adult hemoglobin (HbA). Induction of fetal hemoglobin (HbF) is considered to be of key importance for the development of therapeutic protocols for β-thalassemia and novel HbF inducers need to be proposed for pre-clinical development. The main purpose on this study was to analyze Cinchona alkaloids (cinchonidine, quinidine and cinchonine) as natural HbF-inducing agents in human erythroid cells. The analytical methods employed were Reverse Transcription quantitative real-time PCR (RT-qPCR) (for quantification of γ-globin mRNA) and High Performance Liquid Chromatography (HPLC) (for analysis of the hemoglobin pattern). After an initial analysis using the K562 cell line as an experimental model system, showing induction of hemoglobin and γ-globin mRNA, we verified whether the two more active compounds, cinchonidine and quinidine, were able to induce HbF in erythroid progenitor cells isolated from β-thalassemia patients. The data obtained demonstrate that cinchonidine and quinidine are potent inducers of γ-globin mRNA and HbF in erythroid progenitor cells isolated from nine β-thalassemia patients. In addition, both compounds were found to synergize with the HbF inducer sirolimus for maximal production of HbF. The data obtained strongly indicate that these compounds deserve consideration in the development of pre-clinical approaches for therapeutic protocols of β-thalassemia.

SUBMITTER: Zuccato C

PROVIDER: S-EPMC8706579 | biostudies-literature |

REPOSITORIES: biostudies-literature

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5613423 TREATMENT OF ERYTHROID PRECURSOR CELLS FROM β-THALASSEMIC PATIENTS WITH ISOXAZOLE DERIVATIVES: POTENT INDUCTION OF FETAL HEMOGLOBIN

Project description: Not available

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Induction of fetal hemoglobin: Lentiviral shRNA knockdown of HBS1L in β0-thalassemia/HbE erythroid cells.

Project description:Imbalanced globin chain output contributes to thalassemia pathophysiology. Hence, induction of fetal hemoglobin in β-thalassemia and other β-hemoglobinopathies are of continuing interest for therapeutic approaches. Genome-wide association studies have identified three common genetic loci: namely β-globin (HBB), an intergenic region between MYB and HBS1L, and BCL11A underlying quantitative fetal hemoglobin production. Here, we report that knockdown of HBS1L (all known variants) using shRNA in early erythroblast obtained from β0-thalassemia/HbE patients triggers an upregulation of γ-globin mRNA 1.69 folds. There is modest perturbation of red cell differentiation assessed by flow cytometry and morphology studies. The levels of α- and β-globin mRNAs are relatively unaltered. Knockdown of HBS1L also increases the percentage of fetal hemoglobin around 16.7 folds when compared to non-targeting shRNA. Targeting HBS1L is attractive because of the potent induction of fetal hemoglobin and the modest effect on cell differentiation.

| S-EPMC9994754 | biostudies-literature

Pharmacological Induction of Fetal Hemoglobin in β-Thalassemia and Sickle Cell Disease: An Updated Perspective.

Project description:A significant amount of attention has recently been devoted to the mechanisms involved in hemoglobin (Hb) switching, as it has previously been established that the induction of fetal hemoglobin (HbF) production in significant amounts can reduce the severity of the clinical course in diseases such as β-thalassemia and sickle cell disease (SCD). While the induction of HbF using lentiviral and genome-editing strategies has been made possible, they present limitations. Meanwhile, progress in the use of pharmacologic agents for HbF induction and the identification of novel HbF-inducing strategies has been made possible as a result of a better understanding of γ-globin regulation. In this review, we will provide an update on all current pharmacological inducer agents of HbF in β-thalassemia and SCD in addition to the ongoing research into other novel, and potentially therapeutic, HbF-inducing agents.

| S-EPMC9227505 | biostudies-literature

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Project description:In the present report, we carried out clinical-scale editing in adult mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs) using zinc-finger nuclease-mediated disruption of BCL11a to upregulate the expression of ?-globin (fetal hemoglobin). In these cells, disruption of the erythroid-specific enhancer of the BCL11A gene increased endogenous ?-globin expression to levels that reached or exceeded those observed following knockout of the BCL11A coding region without negatively affecting survival or in vivo long-term proliferation of edited HSPCs and other lineages. In addition, BCL11A enhancer modification in mobilized CD34+ cells from patients with ?-thalassemia major resulted in a readily detectable ?-globin increase with a preferential increase in G-gamma, leading to an improved phenotype and, likely, a survival advantage for maturing erythroid cells after editing. Furthermore, we documented that both normal and ?-thalassemia HSPCs not only can be efficiently expanded ex vivo after editing but can also be successfully edited post-expansion, resulting in enhanced early in vivo engraftment compared with unexpanded cells. Overall, this work highlights a novel and effective treatment strategy for correcting the ?-thalassemia phenotype by genome editing.

| S-EPMC6120587 | biostudies-literature

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

Project description: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 β-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify β-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 β-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 β-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.

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Metformin induces FOXO3-dependent fetal hemoglobin production in human primary erythroid cells.

Project description:Induction of red blood cell (RBC) fetal hemoglobin (HbF; α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by reducing the concentration of sickle hemoglobin (HbS; α2βS2) to inhibit its polymerization. Hydroxyurea (HU), the only US Food and Drug Administration (FDA)-approved drug for SCD, acts in part by inducing HbF; however, it is not fully effective, reflecting the need for new therapies. Whole-exome sequence analysis of rare genetic variants in SCD patients identified FOXO3 as a candidate regulator of RBC HbF. We validated these genomic findings through loss- and gain-of-function studies in normal human CD34+ hematopoietic stem and progenitor cells induced to undergo erythroid differentiation. FOXO3 gene silencing reduced γ-globin RNA levels and HbF levels in erythroblasts, whereas overexpression of FOXO3 produced the opposite effect. Moreover, treatment of primary CD34+ cell-derived erythroid cultures with metformin, an FDA-approved drug known to enhance FOXO3 activity in nonerythroid cells, caused dose-related FOXO3-dependent increases in the percentage of HbF protein and the fraction of HbF-immunostaining cells (F cells). Combined HU and metformin treatment induced HbF additively and reversed the arrest in erythroid maturation caused by HU treatment alone. HbF induction by metformin in erythroid precursors was dependent on FOXO3 expression and did not alter expression of BCL11A, MYB, or KLF1. Collectively, our data implicate FOXO3 as a positive regulator of γ-globin expression and identify metformin as a potential therapeutic agent for SCD.

| S-EPMC6053951 | biostudies-literature

Genome editing strategies for fetal hemoglobin induction in beta-hemoglobinopathies.

Project description:Genome editing to correct a defective β-globin gene or induce fetal globin (HbF) for patients with beta-hemoglobinopathies has the potential to be a curative strategy available to all. HbF reactivation has long been an area of intense interest given the HbF inhibition of sickle hemoglobin (HbS) polymerization. Patients with HbS who also have high HbF tend to have less severe or even minimal clinical manifestations. Approaches to genetically engineer high HbF include de novo generation of naturally occurring hereditary persistence of fetal hemoglobin (HPFH) mutations, editing of transcriptional HbF repressors or their binding sites and/or regulating epigenetic intermediates controlling HbF expression. Recent preclinical and early clinical trial data show encouraging results; however, long-term follow-up is lacking, and the safety and efficacy concerns of genome editing remain.

| S-EPMC7673473 | biostudies-literature

Fetal hemoglobin induction in sickle erythroid progenitors using a synthetic zinc finger DNA-binding domain.

Project description: Not available

| S-EPMC6119143 | biostudies-literature

Fetal Hemoglobin Induction by Epigenetic Drugs.

Project description:Fetal hemoglobin (HbF) inhibits the root cause of sickle pathophysiology, sickle hemoglobin polymerization. Individuals who naturally express high levels of HbF beyond infancy thus receive some protection from sickle complications. To mimic this natural genetic experiment using drugs, one guiding observation was that HbF is increased during recovery of bone marrow from extreme stress. This led to evaluation and approval of the cytotoxic (cell killing) drug hydroxyurea to treat sickle cell disease. Cytotoxic approaches are limited in potency and sustainability, however, since they require hematopoietic reserves sufficient to repeatedly mount recoveries from stress that destroys their counterparts, and such reserves are finite. HbF induction even by stress ultimately involves chromatin remodeling of the gene for HbF (HBG), therefore, a logical alternative approach is to directly inhibit epigenetic enzymes that repress HBG-implicated enzymes include DNA methyltransferase 1, histone deacetylases, lysine demethylase 1, protein arginine methyltransferase 5, euchromatic histone lysine methyltransferase 2 and chromodomain helicase DNA-binding protein 4. Clinical proof-of-principle that this alternative, noncytotoxic approach can generate substantial HbF and total hemoglobin increases has already been generated. Thus, with continued careful attention to fundamental biological and pharmacologic considerations (reviewed herein), there is potential that rational, molecular-targeted, safe and highly potent disease-modifying therapy can be realized for patients with sickle cell disease, with the accessibility and cost-effective properties needed for world-wide effect.

| S-EPMC6029722 | biostudies-literature

Role of XmnIgG Polymorphism in Hydroxyurea Treatment and Fetal Hemoglobin Level at Isfahanian Intermediate β-Thalassemia Patients.

Project description:BACKGROUND:β-thalassemia is the most common monogenic disorder in human. The (C-->T) polymorphism at -158 upstream region of the γG-globin gene and pharmacological factors such as hydroxyurea have been reported to influence γ-globin gene expression and the severity of clinical symptoms of β-thalassemia. METHODS:In the present study, 51 β-thalassemia intermediate patients were studied. Xmn1γG polymorphism genotype was determined using Tetra-Primer ARMS-PCR technique. Hemoglobin (Hb) and fetal hemoglobin (HbF) levels were determined by gel electrophoresis. RESULTS:Of 51 patients, 35 (68.6%) patients were heterozygous (CT) and 16 (31.4%) patients were homozygous (CC). Of 30 patients under treatment by hydroxyurea, 20 (66.7%) patients were heterozygous (CT) and 10 (33.3%) patients were homozygous (CC). Our results demonstrated that in the heterozygous (CT) genotype, the Hb (9.58 ± 1.25 gm/dl) and HbF (89.30 ± 21.87) levels were significantly higher in comparison with homozygous (CC) genotype (7.94 ± 1.34 gm/dl and 70.32 ± 40.56, respectively). Furthermore, we observed that after drug usage, the Hb and HbF levels in patients with heterozygous (CT) genotype (0.7 ± 1.26 gm/dl and 5.95 ± 14.8, respectively) raised more in comparison with homozygous (CC) genotype (0.26 ± 1.43 gm/dl and 0.8 ± 1.31, respectively). CONCLUSION:Hb and HbF levels in the patients carrying T allele are increased significantly, and they also response to hydroxyurea treatment.

| S-EPMC4571014 | biostudies-literature

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