Fetal Hemoglobin Synthesis Research Articles

PGC-1α agonism induces fetal hemoglobin and exerts antisickling effects in sickle cell disease.

Sickle cell disease is a growing health burden afflicting millions around the world. Clinical observation and laboratory studies have shown that the severity of sickle cell disease is ameliorated in individuals who have elevated levels of fetal hemoglobin. Additional pharmacologic agents to induce sufficient fetal hemoglobin to diminish clinical severity is an unmet medical need. We recently found that up-regulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) can induce fetal hemoglobin synthesis in human primary erythroblasts. Here, we report that a small molecule, SR-18292, increases PGC-1α leading to enhanced fetal hemoglobin expression in human erythroid cells, β-globin yeast artificial chromosome mice, and sickle cell disease mice. In SR-18292-treated sickle mice, sickled red blood cells are significantly reduced, and disease complications are alleviated. SR-18292, or agents in its class, could be a promising additional therapeutic for sickle cell disease.

Potential Use of MicroRNA Technology in Thalassemia Therapy.

Thalassemia encompasses a group of inherited hemoglobin disorders characterized by reduced or absent production of the α- or β-globin chains, leading to anemia and other complications. Current management relies on lifelong blood transfusions and iron chelation, which is burdensome for patients. This review summarizes the emerging therapeutic potential of modulating microRNAs (miRNAs) to treat thalassemia. MiRNAs are small non-coding RNAs that regulate gene expression through sequence-specific binding to messenger RNAs (mRNAs). While they commonly repress gene expression by binding to the 3' untranslated regions (UTRs) of target mRNAs, miRNAs can also interact with 5'UTRs and gene promoters to activate gene expression. Many miRNAs are now recognized as critical regulators of erythropoiesis and are abnormally expressed in β-thalassemia. Therapeutically restoring levels of deficient miRNAs or inhibiting overexpression through miRNA mimics or inhibitors (antagomir), respectively, has shown preclinical efficacy in ameliorating thalassemic phenotypes. The miR-144/451 cluster is especially compelling for targeted upregulation to reactivate fetal hemoglobin synthesis. Advances in delivery systems are addressing previous challenges in stability and targeting of miRNA-based drugs. While still early, gene therapy studies suggest combinatorial approaches with miRNA modulation may provide synergistic benefits. Several key considerations remain including enhancing delivery, minimizing off-target effects, and demonstrating long-term safety and efficacy. While no miRNA therapies have yet progressed to clinical testing for thalassemia specifically, important lessons are being learned through clinical trials for other diseases and conditions, such as cancer, cardiovascular diseases, and viral. If limitations can be overcome through multi-disciplinary collaboration, miRNAs hold great promise to expand and transform treatment options for thalassemia in the future by precisely targeting pathogenic molecular networks. Ongoing innovations, such as advancements in miRNA delivery systems, improved targeting mechanisms, and enhanced understanding of miRNA biology, continue to drive progress in this emerging field towards realizing the clinical potential of miRNA-based medicines for thalassemia patients.

Exagamglogene Autotemcel for Transfusion-Dependent β-Thalassemia

BackgroundExagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis through ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing of the erythroid-specific enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs).MethodsWe conducted an open-label, single-group, phase 3 study of exa-cel in patients 12 to 35 years of age with transfusion-dependent β-thalassemia and a β0/β0, β0/β0-like, or non–β0/β0-like genotype. CD34+ HSPCs were edited by means of CRISPR-Cas9 with a guide mRNA. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was transfusion independence, defined as a weighted average hemoglobin level of 9 g per deciliter or higher without red-cell transfusion for at least 12 consecutive months. Total and fetal hemoglobin concentrations and safety were also assessed.ResultsA total of 52 patients with transfusion-dependent β-thalassemia received exa-cel and were included in this prespecified interim analysis; the median follow-up was 20.4 months (range, 2.1 to 48.1). Neutrophils and platelets engrafted in each patient. Among the 35 patients with sufficient follow-up data for evaluation, transfusion independence occurred in 32 (91%; 95% confidence interval, 77 to 98; P<0.001 against the null hypothesis of a 50% response). During transfusion independence, the mean total hemoglobin level was 13.1 g per deciliter and the mean fetal hemoglobin level was 11.9 g per deciliter, and fetal hemoglobin had a pancellular distribution (≥94% of red cells). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No deaths or cancers occurred.ConclusionsTreatment with exa-cel, preceded by myeloablation, resulted in transfusion independence in 91% of patients with transfusion-dependent β-thalassemia. (Supported by Vertex Pharmaceuticals and CRISPR Therapeutics; CLIMB THAL-111 ClinicalTrials.gov number, NCT03655678.)

Exagamglogene Autotemcel for Severe Sickle Cell Disease

BackgroundExagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis by means of ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A.MethodsWe conducted a phase 3, single-group, open-label study of exa-cel in patients 12 to 35 years of age with sickle cell disease who had had at least two severe vaso-occlusive crises in each of the 2 years before screening. CD34+ HSPCs were edited with the use of CRISPR-Cas9. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was freedom from severe vaso-occlusive crises for at least 12 consecutive months. A key secondary end point was freedom from inpatient hospitalization for severe vaso-occlusive crises for at least 12 consecutive months. The safety of exa-cel was also assessed.ResultsA total of 44 patients received exa-cel, and the median follow-up was 19.3 months (range, 0.8 to 48.1). Neutrophils and platelets engrafted in each patient. Of the 30 patients who had sufficient follow-up to be evaluated, 29 (97%; 95% confidence interval [CI], 83 to 100) were free from vaso-occlusive crises for at least 12 consecutive months, and all 30 (100%; 95% CI, 88 to 100) were free from hospitalizations for vaso-occlusive crises for at least 12 consecutive months (P<0.001 for both comparisons against the null hypothesis of a 50% response). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No cancers occurred.ConclusionsTreatment with exa-cel eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for a period of 12 months or more. (CLIMB SCD-121; ClinicalTrials.gov number, NCT03745287.)

Enzyme-free sensitive SERS biosensor for the detection of thalassemia-associated microRNA-210 using a cascade dual-signal amplification strategy

Backgroundβ-thalassemia is a blood disorder caused by autosomal mutations. Gene modulation therapy to activate the γ-globin gene to induce fetal hemoglobin (HbF) synthesis has become a new option for the treatment of β-thalassemia. MicroRNA-210 (miR-210) contributes to studying the mechanism regulating γ-globin gene expression and is a potential biomarker for rapid β-thalassemia screening. Traditional miRNA detection methods perform well but necessitate complex and time-consuming miRNA sample processing. Therefore, the development of a sensitive, accurate, and simple miRNA level monitoring method is essential. ResultsWe have developed a non-enzymatic surface-enhanced Raman scattering (SERS) biosensor utilizing a signal cascade amplification of catalytic hairpin assembly reaction (CHA) and proximity hybridization-induced hybridization chain reaction (HCR). Au@Ag NPs were used as the SERS substrate, and methylene blue (MB)- modified DNA hairpins were used as the SERS tags. The SERS assay involved two stages: implementing the CHA-HCR cascade signal amplification strategy and conducting SERS measurements on the resulting product. The HCR was started by the products of target-triggered CHA, which formed lengthy nicked double-stranded DNA (dsDNA) on the Au@Ag NPs surface to which numerous SERS tags were attached, leading to a significant increase in the SERS signal intensity. High specificity and sensitivity for miR-210 detection was achieved by monitoring MB SERS intensity changes. The suggested SERS biosensor has a low detection limit of 5.13 fM and is capable of detecting miR-210 at concentration between 10 fM and 1.0 nM. SignificanceThe biosensor can detect miR-210 levels in the erythrocytes of β-thalassemia patients, enabling rapid screening for β-thalassemia and suggesting a novel approach for investigating the regulation mechanism of miR-210 on γ-globin gene expression. In the meantime, this innovative technique has the potential to detect additional miRNAs and to become an important tool for the early diagnosis of diseases and for biomedical research.

SR-18292 Induces Fetal Hemoglobin Synthesis and Reduces Disease Pathology in Sickle Cell Mice

Sickle cell disease (SCD) is a commonly inherited blood disorder, which affects millions around the world. Reactivation of fetal hemoglobin (HbF) synthesis in adult erythroid cells can diminish the severity of many clinical features of the disease. Up-regulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has been shown to induce HbF levels in human primary erythroid progenitor CD34 + cells. Here we reported the in vitro and in vivo effects of a small molecular compound, SR-18292, on HbF induction via modulating PGC-1α. To determine whether SR-18292 can induce HbF in human CD34 + cells, we performed RT-qPCR, flow cytometry, and western blot to measure the mRNA and protein levels of PGC-1α and γ-globin. We found that mRNA levels of PGC-1α and γ-globin were significantly increased in cells treated with SR-18292 while β-globin mRNA levels were unaffected. Protein levels of γ-globin and the ratio of F-cells were increased in treated cells. Erythroid cellular differentiation markers (CD71/CD235a) and terminal differentiation markers (CD49d/CD233) were used to examine the differentiation status. There was no significant difference in the population of CD71 +CD235a + cells among all groups. However, SR-18292 treatment increased the percentage of CD49d -CD233 + cells at 5 or 7.5 μM. These results suggest that SR-18292 at higher concentrations can promote erythroid terminal differentiation. To evaluate whether the HbF induction by SR-18292 can reduce the SCD disease pathology in SCD mice or not, we intraperitoneally administered SR-18292 to SCD mice. HbF induction was determined by flow cytometric analysis using an anti-HbF antibody. And it was shown that the HbF-high cell population was induced in SCD mice after 4 weeks of treatment. And we used transferrin receptor CD71 and erythroid-specific marker Ter119 to determine whether SR-18292 could affect erythroid differentiation. There was no significant difference in the CD71 +Ter119 + population after 4 weeks of SR-18192 treatment compared with untreated animals. The mRNA levels of γ-globin and PGC-1α were increased in treated mice. And the increase of PGC-1α and γ-globin protein levels were confirmed by western blot. Reticulocyte is a sign of premature RBC destruction (hemolysis), and the SCD mice usually have high reticulocyte counts (&amp;gt;50%). We measured the reticulocyte in the peripheral blood of SR-18292 treated and untreated SCD mice by flow-cytometric analyses and found that reticulocyte was significantly reduced in SR-18292 treated animals (from 55% to 20%) after 4 weeks of treatment. Complete blood cell counts (CBC) showed that SR-18292 treated mice had slightly elevated numbers of red blood cells (RBCs) and more hemoglobin, but lower counts of white blood cells, lymphocytes, and neutrophils. In addition, the RBC distribution width (RDW) was significantly reduced after SR-18292 treatment, indicating that the size of circulating RBCs was more uniform in treated mice. In addition, erythroid cell morphology was evaluated by blood smears with Wright-Giemsa stain. The number of sickled RBCs were reduced in SCD mice treated with SR-18292 for 4 weeks. This is consistent with our observation that RDW was reduced in SCD mice treated with SR-18292. Furthermore, SR-18292 treated mice had a statistically significant reduction in spleen size and weight in contrast to the spleens of untreated SCD mice. The indices of RBCs were improved in accompany with reduced hemolysis and a higher fraction of circulating mature erythrocytes in treated SCD mice. There were no observable adverse effects on other hematopoietic cell lineages. We also performed single-cell RNA-seqs in human CD34 + cells treated with SR-18292 and found that some known HbF repressors (such as BCL11A and ZBTB7A) were decreased in treated groups. Besides that, there were many differentially expressed genes (DEGs), which might be novel HbF regulators. We are verifying those DEGs genes by qPCR and using CRISPR/cas9 to examine their functions in globin regulation. In summary, our findings suggest that SR-18292 might be a promising therapeutic agent to treat SCD and modulating PGC-1α activity or the signaling pathways that it regulates might benefit therapeutically patients with SCD.

MicroRNA29B induces fetal hemoglobin via inhibition of the HBG repressor protein MYB in vitro and in humanized sickle cell mice.

Therapeutic strategies aimed at reactivating HBG gene transcription and fetal hemoglobin (HbF) synthesis remain the most effective strategy to ameliorate the clinical symptoms of sickle cell disease (SCD). We previously identified microRNA29B (MIR29B) as a novel HbF inducer via targeting enzymes involved in DNA methylation. We provided further evidence that the introduction of MIR29B into KU812 leukemia cells significantly reduced MYB protein expression. Therefore, the aim of this study was to determine the extent to which MIR29B mediates HbF induction via targeting MYB in KU812 leukemia cells and human primary erythroid progenitors and to investigate the role of MIR29B in HbF induction in vivo in the humanized Townes SCD mouse model. Human KU812 were cultured and normal CD34 cells (n = 3) were differentiated using a two-phase erythropoiesis culturing system and transfected with MIR29B (50 and 100 nM) mimic or Scrambled (Scr) control in vitro. A luciferase reporter plasmid overexpressing MYB was transfected into KU812 cells. Luciferase activity was quantified after 48 h. Gene expression was determined by quantitative real-time PCR. In vivo studies were conducted using Townes SCD mice (6 per group) treated with MIR29B (2, 3, and 4 mg/kg/day) or Scr control by 28-day continuous infusion using subcutaneous mini osmotic pumps. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes at weeks 0, 2, and 4. Flow cytometry was used to measure the percentage of HbF-positive cells. In silico analysis predicted complementary base-pairing between MIR29B and the 3'-untranslated region (UTR) of MYB. Overexpression of MIR29B significantly reduced MYB mRNA and protein expression in KU812 cells and erythroid progenitors. Using a luciferase reporter vector that contained the full-length MYB 3'-UTR, we observed a significant reduction in luciferase activity among KU812 cells that co-expressed MIR29B and the full-length MYB 3'-UTR as compared to cells that only expressed MYB 3'-UTR. We confirmed the inhibitory effect of a plasmid engineered to overexpress MYB on HBG activation and HbF induction in both KU812 cells and human primary erythroid progenitors. Co-expression of MIR29B and MYB in both cell types further demonstrated the inhibitory effect of MIR29B on MYB expression, resulting in HBG reactivation by real-time PCR, Western blot, and flow cytometry analysis. Finally, we confirmed the ability of MIR29B to reduce sickling and induce HbF by decreasing expression of MYB and DNMT3 gene expression in the humanized Townes sickle cell mouse model. Our findings support the ability of MIR29B to induce HbF in vivo in Townes sickle cell mice. This is the first study to provide evidence of the ability of MIR29B to modulate HBG transcription by MYB gene silencing in vivo. Our research highlights a novel MIR-based epigenetic approach to induce HbF supporting the discovery of new drugs to expand treatment options for SCD.

Molecular characterization of a novel homozygous deletion in β-globin cluster causing (δβ)0-Thalassemia among Tunisian family.

Deletions in the β-globin cluster are uncommon and cause thalassemia (thal) with hereditary persistence of fetal hemoglobin. They constitute a heterogenous group of disorders characterized by absent or reduced synthesis of adult hemoglobin (Hb A) and increased synthesis of fetal hemoglobin (Hb F). Although the clinical severity of these disorders are asymptomatic owing to the increased Hb F levels, the molecular basis is very heterogenous due to the large deletions in the β-globin cluster spanning both HBD and HBB genes. Here, we describe a Tunisian family carrying a novel deletion mutation causing (δβ)°-thalassemia. The amounts of hemoglobin fractions were measured by capillary electrophoresis of hemoglobin. Amplification and sequencing of different regions on the β-gene cluster were performed by Sanger method. Family study and genetic analysis revealed a large deletion mutation in the β-globin cluster of 14.5 kb (NG_000,007.3:g. 58,253 to g.72837del14584) at the homozygous state in the patient and at heterozygous state at the other members of the family. This deletion removes the HBD and HBB genes. In our knowledge, this new large deletion is described for the first time in the Tunisian population and in the world, designed Tunisian(δβ)0 in Ithanet database (IthaID: 3971). Therefore, it is important to identify the deletion leading to δβ-thalassemia carriers at the molecular level, to highlight the importance of recognizing the clinical features and implementing appropriate testing to clarify the diagnosis and manage the condition.

Трансфузионнонезависимая талассемия: традиционное и новое лечение

This literature review attempts to describe modern approaches to the diagnosis and therapy of non transfusion dependent thalassaemia (NTDT). NTDT has a wide clinical spectrum. The clinical polymorphism of the disease is due to genetic heterogeneity. There are three major factors, which are responsible for the clinical manifestations of NTDT: ineffective erythropoiesis, chronic anemia, and iron overload. Untreated NTDT is the cause of various complications: splenomegaly, gallstones, extramedullary erythropoiesis, kidney stones, lower limbs trophic ulcers, thrombophilia, pulmonary hypertension, endocrine complications, iron overload, bone abnormalities, osteoporosis. Traditional therapy for NTDT include splenectomy, transfusion therapy, stimulation of fetal hemoglobin (HbF) synthesis, and bone marrow transplantation. However, due to the limitations and challenges associated with available conventional therapies, novel methods are currently being developed. These include: JAK2 inhibition, hepcidin modulation, TMPRSS6 inhibition, apo-transferrin, HIF2 inhibition, Activin receptor-II trap ligands, ferroportin inhibitors.

Genetic modifiers of fetal hemoglobin affect the course of sickle cell disease in patients treated with hydroxyurea.

The course of sickle cell disease (SCD) is modified by polymorphisms boosting fetal hemoglobin (HbF) synthesis. However, it has remained an open question how these polymorphisms affect patients who are treated with the HbF-inducing drug hydroxyurea/ hydroxycarbamide. The German SCD registry offers the opportunity to answer this question, because >90% of patients are treated according to national guidelines recommending the use of hydroxyurea in all patients above 2 years of age. We analyzed the modifying effect of HbF-related genetic polymorphisms in 417 patients with homozygous SCD >2 years old who received hydroxyurea. HbF levels were correlated with higher total hemoglobin levels, lower rates of hemolysis, a lower frequency of painful crises and of red blood cell transfusions. The minor alleles of the polymorphisms in the γ-globin promoter (rs7482144), BCL11A (rs1427407) and HMIP (rs66650371) were strongly associated with increased HbF levels. However, these associations did not translate into lower frequencies of vaso-occlusive events which did not differ between patients either carrying or not carrying the HMIP and BCL11A polymorphisms. Patients on hydroxyurea carrying the γ-globin promoter polymorphism demonstrated substantially higher hemoglobin levels (P<10-4) but also higher frequencies of painful crises and hospitalizations (P<0.01) when compared to patients without this polymorphism. Taken together, these data indicate that the γ-globin, HMIP and BCL11A polymorphisms correlate with increased HbF in SCD patients on hydroxyurea. While HbF is negatively correlated with the frequency of painful crises and hospitalizations, this was not observed for the presence of known HbF-boosting alleles.

Novel Conjugates of Butyrate and δ-Aminolevulinate Increase γ-Globin Gene Expression and Fetal Hemoglobin Synthesis in Erythroid Progenitors

Pharmacologic induction of fetal hemoglobin (HbF) expression reduces the clinical severity of sickle cell disease (SCD) and improves long-term survival. Research efforts to develop effective and safe therapies have focused on a wide variety of agents however, Hydroxyurea is the only FDA-approved drug proven to mediate HbF induction in about 50% of adult SCD patients. Our group previously reported potent HbF-induction by sodium butyrate through p38 MAPK activation and CREB1 binding to the Gγ-globin cyclic AMP response element (Sangerman et al., Blood 2006). However, oral administration of butyrate derivatives to SCD patients was ineffective due to rapid metabolic inactivation by the liver. Therefore to address this critical barrier of limited treatment options for SCD, we investigated the prodrugs 1-(butyryloxy)ethyl 5-amino-4-oxopentanoate (AN233) and 1-(butyryloxy)butyl 5-amino-4-oxopentanoate (AN908), esters of δ-aminolevulinate (ALA) and butyric acid as HbF inducers in erythroid cells. Oral administration of AN233 and AN908 to BALB/c mice increased total hemoglobin levels without causing tissue toxicity (Rephaeli et al., Eur J Pharm Sci 2016).To test the ability of prodrugs to induce HbF, both K562 and KU812 cells and the immortalized human umbilical cord blood-derived erythroid progenitor 1 (HUDEP1) model were used. Treatment of K562 cells with 0.125-0.50 mM concentrations of AN233 produced cell toxicity at the 0.50 mM concentration, however >90% viability was observed by Trypan blue exclusion for 0.25 mM AN233 without evidence of caspace-3 cleavage. Using this concentration of AN233, a 1.5-fold and 6-fold (p<0.05) increase in γ-globin mRNA and HbF levels respectively, were observed supporting robust intracellular activation of AN233. Flow cytometry analysis confirmed a 1.5-fold increase in the percent of HbF positive cells (F-cells). Preliminary studies in HUDEP1 cells showed 0.25 mM AN233 increased γ-globin expression by 1.5 fold and HbF protein synthesis by 2.2 fold. Mechanistically, AN233 mediated a 53% decrease in phosphorylation levels of the kinase, eIF2α (eukaryotic initiation factor 2α) which favors conditions for protein synthesis. As further evidence that both prodrugs induce HbF synthesis in different cell types, KU812 cells expressing the γ-globin and β-globin genes were treated with AN908 (0.125-0.50 mM). In contrast to AN233, cell toxicity was not observed at the 0.50 mM AN908 concentration where 93% cell viability was maintained. Treating KU812 cells with 0.25 mM AN908 for 48 h produced a 1.4-fold increase in γ-globin transcription and HbF synthesis by western blot. In addition, F-cell levels increased from 45% to 70% and HbF levels (by mean fluorescence intensity) 2.2-fold (p<0.05). Interestingly, when 0.25 mM AN908 was combined with hydroxyurea (100 μM) we observed an additive increase in HbF and F-cells levels.Collectively, these findings support the oral active prodrugs AN233 and AN908 as HbF inducers in erythroid cells. Furthermore, the combination of AN908 and hydroxyurea produced an additive effect without increasing cell toxicity. Studies are underway to determine the EC50 of both prodrugs in normal and sickle primary erythroid progenitors and mechanisms of γ-globin regulation through epigenetic modifications. DisclosuresNo relevant conflicts of interest to declare.

Innovative Treatments for Rare Anemias.

Rare anemias (RA) are mostly hereditary disorders with low prevalence and a broad spectrum of clinical severity, affecting different stages of erythropoiesis or red blood cell components. RA often remains underdiagnosed or misdiagnosed, and treatment options have been limited to supportive care for many years. During the last decades, the elucidation of the molecular mechanisms underlying several RA paved the way for developing new treatments. Innovative treatments other than supportive care and allogeneic bone marrow transplantation are currently in clinical trials for β-thalassemias, sickle cell disease (SCD), and congenital hemolytic anemias. Recently, luspatercept, an activin receptor ligand trap targeting ineffective erythropoiesis, has been approved as the first pharmacological treatment for transfusion-dependent β-thalassemia. L-glutamine, voxelotor, and crizanlizumab are new drugs approved SCD, targeting different steps of the complex pathophysiological mechanism. Gene therapy represents an innovative and encouraging strategy currently under evaluation in several RA and recently approved for β-thalassemia. Moreover, the advent of gene-editing technologies represents an additional option, mainly focused on correcting the defective gene or editing the expression of genes that regulate fetal hemoglobin synthesis. In this review, we aim to update the status of innovative treatments and the ongoing trials and discuss RA treatments’ future directions. Interestingly, several molecules that showed promising results for treating one of these disorders are now under evaluation in the others. In the near future, the management of RA will probably consist of polypharmacotherapy tailored to patients’ characteristics.

Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at G\u03b3-globin gene promoter

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for β-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from β-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG − 53, − 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in β-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

Retraction

Background Small interfering RNAs (siRNAs) specifically target mRNAs and induce silencing of protein-coding genes. MicroRNAs are single-stranded RNA molecules that can target several genes. This study evaluated microRNA expression following the inhibition of γ to β-globin gene switching and examined the increased expression of γ genes through the effect of siRNAs on the B-cell lymphoma/leukemia 11A (BCL11A) gene. Methods Stem cells were isolated from peripheral blood mononuclear cells using the Ficoll-Paque method. The cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) for differentiation into erythroid cells over 28 days and stored under optimal conditions. On the 21st day, siRNAs were inserted into BCL11A. The expression levels of miR-15a, miR-16, miR-23a, miR-27a, and miR-486 were evaluated before and after siRNA treatment. The comparative CT method (2-ΔΔCT) was used to compare miRNA expression. For data analysis, Student's t-tests were performed, with P<0.05 considered statistically significant. Results The five miRNAs in this study showed significant differences (P<0.01). The relative expression of BCL11A after siRNA transmission decreased, while the expression of γ gene showed an increasing trend (1.2, 2.1, and 2.3, respectively). Conclusions The expression levels of miR-15a, miR-16, miR-23a, miR-27a, and miR-486 changed before and after γ-to-β gene switching. Therefore, this may be an effective intervention for controlling the switching and synthesis of fetal hemoglobin.

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

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

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

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

Monoterpenes as therapeutic candidates to induce fetal hemoglobin synthesis and up-regulation of gamma-globin gene: An in vitro and in vivo investigation

Pharmacologically induced production of fetal hemoglobin (HbF) is a pragmatic therapeutic strategy for the reduction of globin chain imbalance and improving the clinical severities of patients with β-hemoglobinopathies. To identify highly desirable new therapeutic HbF-inducing agents, we screened functionally diverse ten monoterpenes, as molecular entities for their potent induction and erythroid differentiation ability in human erythroleukemia cell line (K562) and transgenic mice. Benzidine hemoglobin staining demonstrated six compounds to have significantly induced erythroid differentiation of K562 cells in a dose and time-dependent manner. This induction paralleled well with the optimal accumulated quantity of total hemoglobin in treated cultures. The cytotoxic studies revealed that three (carvacrol, 3-carene, and 1,4-cineole) of the six compounds with their maximal erythroid expansion ability did not affect cell proliferation and were found non-toxic. Four compounds were found to have high potency, with 4-8-fold induction of HbF at both transcriptional and protein levels in vitro. Subsequently, an in vivo study with the three active non-cytotoxic compounds showed significant overexpression of the γ-globin gene and HbF production. Carvacrol emerged as a lead HbF regulator suggested by the increase in expression of γ-globin mRNA content (5.762 ± 0.54-fold in K562 cells and 5.59 ± 0.20-fold increase in transgenic mice), accompanied by an increase in fetal hemoglobin (F-cells) levels (83.47% in K562 cells and 79.6% in mice model). This study implicates monoterpenes as new HbF inducing candidates but warrants mechanistic elucidation to develop them into potential therapeutic drugs in β-thalassemia and sickle cell anemia.

Efficacy and safety of oral hydroxyurea in transfusion-dependent β-thalassaemia: a protocol for randomised double-blind controlled clinical trial

IntroductionDespite being one of the first diseases to be genetically characterised, β-thalassaemia remains a disorder without a cure in a majority of patients. Most patients with β-thalassaemia receive only supportive...

Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype.

Sickle cell disease (SCD) is caused by a single amino acid change in the adult hemoglobin (Hb) β chain that causes Hb polymerization and red blood cell (RBC) sickling. The co-inheritance of mutations causing fetal γ-globin production in adult life hereditary persistence of fetal Hb (HPFH) reduces the clinical severity of SCD. HPFH mutations in the HBG γ-globin promoters disrupt binding sites for the repressors BCL11A and LRF. We used CRISPR-Cas9 to mimic HPFH mutations in the HBG promoters by generating insertions and deletions, leading to disruption of known and putative repressor binding sites. Editing of the LRF-binding site in patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in γ-globin derepression and correction of the sickling phenotype. Xenotransplantation of HSPCs treated with gRNAs targeting the LRF-binding site showed a high editing efficiency in repopulating HSPCs. This study identifies the LRF-binding site as a potent target for genome-editing treatment of SCD.

Embryonic and Fetal Human Hemoglobins: Structures, Oxygen Binding, and Physiological Roles.

During the past two decades, significant advances have been made in our understanding of the human fetal and embryonic hemoglobins made possible by the availability of pure, highly characterized materials and novel methods, e.g., nano gel filtration, to study their properties and to correct some misconceptions. For example, whereas the structures of the human adult, fetal, and embryonic hemoglobins are very similar, it has generally been assumed that functional differences between them are due to primary sequence effects. However, more recent studies indicate that the strengths of the interactions between their subunits are very different leading to changes in their oxygen binding properties compared to adult hemoglobin. Fetal hemoglobin in the oxy conformation is a much stronger tetramer than adult hemoglobin and dissociates to dimers 70-times less than adult hemoglobin. This property may form the basis for its protective effect against malaria. A major source of the increased strength of fetal hemoglobin resides within the A-helix of its gamma subunit as demonstrated in studies with the hybrid hemoglobin Felix and related hybrids. Re-activating fetal hemoglobin synthesis in vivo is currently a major focus of clinical efforts designed to treat sickle cell anemia since it inhibits the aggregation of sickle hemoglobin. The mechanisms for both the increased oxygen affinity of fetal hemoglobin and its decreased response to DPG have been clarified. Acetylated fetal hemoglobin, which makes up 10-20% of total fetal hemoglobin, has a significantly weakened tetramer structure suggesting a similar role for other kinds of protein acetylation. Embryonic hemoglobins have the weakest tetramer and dimer structures. In general, the progressively increasing strength of the subunit interfaces of the hemoglobin family during development from the embryonic to the fetal and ultimately to the adult types correlates with their temporal appearance and disappearance in vivo, i.e., ontogeny.