Α-globin Chains Research Articles

Disease burden, management strategies, and unmet needs in α-thalassemia due to hemoglobin H disease.

Alpha-thalassemia is an inherited blood disorder caused by impaired α-globin chain production, leading to anemia and other complications. Hemoglobin H (HbH) disease is caused by a combination of mutations generally affecting the expression of three of four α-globin alleles; disease severity is highly heterogeneous, largely driven by genotype. Notably, non-deletional mutations cause a greater degree of ineffective erythropoiesis and hemolysis, higher transfusion burden, and increased complication risks versus deletional mutations. There are limited treatment options for HbH disease, and effective therapies are needed. This review discusses the pathophysiology of HbH disease, current management strategies, unmet needs, and emerging treatment options.

Therapeutic Relevance of Inducing Autophagy in β-Thalassemia.

The β-thalassemias are inherited genetic disorders affecting the hematopoietic system. In β-thalassemias, more than 350 mutations of the adult β-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free α-globin. Possible experimental strategies for a reduction in excess free α-globin chains in β-thalassemia are CRISPR-Cas9-based genome editing of the β-globin gene, forcing "de novo" HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free α-globin chains in β-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the α-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for β-thalassemia.

Interplay between α-thalassemia and β-hemoglobinopathies: Translating genotype-phenotype relationships into therapies.

α-Thalassemia represents one of the most important genetic modulators of β-hemoglobinopathies. During this last decade, the ongoing interest in characterizing genotype-phenotype relationships has yielded incredible insights into α-globin gene regulationand its impact on β-hemoglobinopathies. In this review, we provide a holistic update on α-globin gene expression stemming from DNA to RNA to protein, as well as epigenetic mechanisms that can impact gene expression and potentially influence phenotypic outcomes. Here, we highlight defined α-globin targeted strategies and rationalize the use of distinct molecular targets based on the restoration of balanced α/β-like globin chain synthesis. Considering the therapies that either increase β-globin synthesis or reactivate γ-globin gene expression, the modulation of α-globin chains as a disease modifier for β-hemoglobinopathies still remains largely uncharted in clinical studies.

Increased Expression of α-Hemoglobin Stabilizing Protein (AHSP) mRNA in Erythroid Precursor Cells Isolated from β-Thalassemia Patients Treated with Sirolimus (Rapamycin).

Background/Objectives: in β-thalassemia, important clinical complications are caused by the presence of free α-globin chains in the erythroid cells of β-thalassemia patients. These free α-globin chains are present in excess as a result of the lack of β-globin chains to bind with; they tend to aggregate and precipitate, causing deleterious effects and overall cytotoxicity, maturation arrest of the erythroid cells and, ultimately, ineffective erythropoiesis. The chaperone protein α-hemoglobin-stabilizing protein (AHSP) reversibly binds with free α-globin; the resulting AHSP-αHb complex prevents aggregation and precipitation. Sirolimus (rapamycin) has been previously demonstrated to induce expression of fetal hemoglobin and decrease the excess of free α-globin chain in the erythroid cells of β-thalassemia patients. The objective of this study was to verify whether sirolimus is also able to upregulate AHSP expression in erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. Methods: the expression of AHSP genes was analyzed by measuring the AHSP mRNA content by real-time quantitative PCR (RT-qPCR) and the AHSP protein production by Western blotting. Results: AHSP gene expression was found to be higher in ErPCs of β-thalassemia patients in comparison to ErPCs isolated from healthy subjects. In addition, AHSP expression was further induced by treatment of β-thalassemia ErPCs with sirolimus. Finally, AHSP mRNA was expressed at an increased level in ErPCs of sirolimus-treated β-thalassemia patients participating in the NCT03877809 Sirthalaclin clinical trial. Conclusions: this exploratory study suggests that AHSP expression should be considered as an endpoint in clinical trials based on sirolimus.

Wide spectrum of novel and rare hemoglobin variants in the multi-ethnic Indian population: A review.

The hemoglobin (Hb) variants are qualitative abnormalities due to production of structurally abnormal globin proteins. They are categorized based on the type of mutation present in the α1, α2, β, Gγ, Aγ and δ globin genes. So far, more than 1550 Hb variants are reported in the database. They could lead to Hb polymerization, Hb instability, altered oxygen affinity and decreased oxygen-carrying capacity of Hb or have no clinical manifestations. In India, ethnic diversity, consanguinity, regional variations and migration result in the presence of different Hb variants. We have compiled all the variants of α, β and δ globin chains in heterozygous, homozygous and in compound heterozygous forms reported from India in the last 52 years. Of the 63 rare and novel hemoglobin variants reported from India, 22 were α-globin chain variants, 37 were β-globin chain variants and 4 were δ-globin chain variants. Twelve novel Hb variants (Hb J Rajappan, Hb Koya Dora, Hb Rampa, Hb Godavari, Hb Chandigarh, Hb D Agri, Hb Lucknow, Hb Vellore, Hb Midnapore, Hb Bijnor, Hb A2Tianhe and Hb A2Saurashtra) were identified among persons of Indian origin. Majority of them were picked up on HPLC. Some of the variants like Hb Titusville, Hb Shimonoseki, Hb Chandigarh, Hb D Agri, Hb Yaizu and Hb Vellore eluted in the HbS window whereas variants like HbD Iran, Hb St. Louis, Hb G Coushata, HbM Saskatoon, Hb Lucknow, Hb Grange-Blanche and Hb Tianshui showed falsely elevated HbA2. Hence, careful and systematic investigations are required to identify them.

Detection of α-thalassemia mutations by Multiplex ligation-dependent probe amplification

Vietnam is a country with a high rate of people carrying α-globin gene mutations, therefore, it is highly important to screen for α-thalassemia carriers, especially in couples with one diagnosed α-carrier, in order to prevent and avoid having α-thalassemia major offspring. α-thalassemia is an inherited autosomal recessive disease, of which the phenotype depends on the degree of α-globin chain deficiency. Research samples were collected and analysed gene mutations at the Gene - Protein Research Center, Hanoi Medical University. With the objective of screening for deletional and nondeletional α-globin gene by using Multiplex ligation-dependent probe amplification (MLPA) method on eighty five people, whose spouses were α-thalassemia carriers, the results of this study have successfully identified 38/85 carriers including thirty five deletional carriers (26 --SEA, 5 -α3.7, 1 -α4.2, 1 --THAI, 1 whole α-globin deletion, and 1 POLR3K-ITFG3 gene deletion) and 3 cases of non-deletional mutation (2 -αHbCs, 1 anti3.7). Deletional mutations account for 92.1% while non-deletional mutations account for only 7.9%.

Α-Globin Lentiviral Vectors for Hematopoietic Stem Cell Gene Therapy of α-Thalassemia

α-thalassemia is an inherited blood disorder caused by mutations in α-globin genes (HBA1 and HBA2) resulting in the reduction of α-globin chains, the subunit, along with β-globin chain, constituting adult hemoglobin (α 2β 2). While a reduction of α-globin expression of 40% presents asymptomatic phenotypes, expression levels decreasing to <20% or an α/β-globin mRNA ratio of <0.2, causes severe anima. Treatment for severe cases of α-thalassemia entails lifelong, biweekly blood transfusions with daily chelation therapy. While these therapies enable patients to live into mid- to late-adulthood, they continue to engender serious clinical manifestations, such as iron toxicity. To answer this clinical need, our laboratory has developed a stem cell gene therapy in which functional copies of α-globin gene integrate into the genome of patient's hematopoietic and progenitor stem cells (HSPCs) by lentiviral vectors (LVs) to normalize the globin chain imbalance and restore hemoglobin function in derived red blood cells (RBCs). The design of our α-globin LVs (AGLVs) is based on GLOBE β-globin LV utilized in a clinical trial for β-thalassemia (NCT02453477), which has achieved transfusion reduction and independence in patients with transfusion-dependence β-thalassemia major. We have constructed a series of short proviral length AGLVs for optimized titer production, HSPC infectivity, and lineage-specific α-globin gene expression. Twelve AGLVs varying in gene and regulatory element compositions were constructed and assessed for raw titer yields and characterized for gene transfer efficiency, mRNA expression and hemoglobin production in a α-globin knockout (KO) human erythroid cell line. Successfully, all AGLVs confer high raw titers ~ 1e7 TU/mL and enabled the production of adult hemoglobin (HbA). We identified two optimal AGLVs for further characterization in human primary HSPCs: 1) A2-UV for yielding highest raw titers and gene transfer efficiency, and 2) A2-Globe for producing the most α-globin mRNA. Both A2-UV and A2-Globe LVsharbor HBA2, are regulated by the β-globin promoter and enhanced by the core or large β-LCR enhancer region, respectively. To assess candidate AGLVs in human HSPCs, AGLVs were tagged at the transcription level to enable identification and quantification of vector-derived α-globin mRNA proceeding HSPCs transduction anderythroid differentiation. A2-UV demonstrated optimum CD34 + infectivity and gene transfer, and A2-Globe expressed a high ~30% of α-globin mRNA per total β-globin per vector copy number (VCN), achieving levels of one endogenous α-globin gene (~25% per total β-globin). Current studies examined A2-UV and A2-Globe in α-thalassemia major patient HSPCs - lacking all four α-globin genes - harboring a lethal homozygous 20kb deletion of HBA1 and HBA2, known as SEA –/– mutation. Patient HSPCs were transduced and differentiated into RBCs. A high CD34+ infectivity and gene transfer was obtained by both candidate vectors, without utilization of transduction enhancers. Furthermore, a correction of α/β-globin mRNA ratio of ~0.7 was obtained while retaining a low VCN (<3).RBCs were then analyzed at the protein level . Chromatograms of hemoglobin tetramers revealed that HbA represented 98% of all hemoglobin, compared to 0% in non-transduced patient RBCs. Single globin chain chromatograms successfully demonstrated α/β-globin chain ratio of ~0.4 or higher and successfully demonstrated that a low average of copies per cell (~3) restored 60% of adult hemoglobin when normalized to healthy donor RBCs. These data demonstrate α/β-globin correction and hemoglobin restoration in α-thalassemia patient cells with the most severe mutation. Three assays were conducted on patient cells, all with similar and highly encouraging results for disease amelioration in severe cases of α-thalassemia. Overall, this project is paving the way for the first development of a curative gene therapy approach for α-thalassemia.

Pharmacologic Inhibition of XPO1 By Selinexor Improves Late-Stage Erythropoiesis in Severely Affected β 0-Thalassemia/Hemoglobin E

Ineffective erythropoiesis due to accumulation of excess α-globin chains profounds unwarranted consequences in β-thalassemia. In severe β-thalassemia, the excess α-globin chains trap HSP70-the guardian of GATA1-within the cytoplasm. Thus, in the nucleus, GATA1 is degraded and leads to late-stage erythropoiesis arrest. XPO1 is a nuclear exporter that exports HSP70 from the nucleus to the cytoplasm. Previous study showed that chemical downregulation of XPO1 in β-thalassemia major using an old generation of XPO1 inhibitor (KPT-251) could improve late-stage in vitro erythropoiesis. Moreover, XPO1 inhibitor treatments in clinical trials revealed improvement of transfusion burden and hemoglobin levels in some patients with myelofibrosis. Together suggested that inhibition of XPO1 might be considered as a possible option for β-thalassemia treatment. Here, we investigated the effects of pharmacological XPO1 inhibition using old and new generation of XPO1 inhibitors, and natural products in human CD34 + hematopoietic stem and progenitor cells (HSPCs) derived from β 0-thalassemia/HbE with severe phenotypes. Coinheritance of common α-thalassemia were ruled out in all patients and severity score was computed using Sripichai scoring system (Sripichai et al. Am J Hematol 2008). HSPCs were purified from peripheral blood and subjected to 3-phase erythroid culture system. Nine compounds with known XPO1 inhibitory effect including five synthetic (KPT-185, KPT-276, KPT-330 or selinexor, KPT-335 or verdinexor and KPT-8602 or eltanexor) and four natural active compounds (curcumin, oridonin, piperlongumine and plumbagin) were evaluated in patient-derived HSPCs (n=1) for their potency and efficacy. Of these, selinexor exhibited the maximal efficacy in which the accumulation of CD71 Low/GPA High cell population was predominate. To affirm this finding, HSPCs derived from six independent β 0-thalassemia/HbE patients were treated with selinexor (5 and 10 nM) at early-stage of the in vitro erythropoiesis and maintained throughout the culture system. After treatments (n=3), XPO1 was downregulated in both cytoplasmic (5 nM; 1.5±0.1-fold, p < 0.005 and 10 nM; 1.7±0.5-fold, p < 0.05) and nuclear compartments (5 nM; 1.1±0.3-fold and 10 nM; 1.3±0.2-fold, p < 0.05). No significant cytotoxic effects were observed. In addition, effects of selinexor on erythroid differentiation and maturation were assessed by flow cytometry using CD71/GPA immunostaining and Wright-Giemsa staining. The results revealed that selinexor treatments could augment late-stage in vitro erythropoiesis (orthochromatic+enucleated/basophilic+polychromatic erythroid cell) in dose-dependent manner (5 nM; 1.4±0.4-fold and 10 nM; 3.5±1.8-fold, p < 0.05). This finding was correlated with erythroid cell morphology under a light microscope. We further explored whether XPO1 inhibition by selinexor (n=3) affects HSP70 and GATA1 expression. Homogenous erythroid cell population (CD71 High/GPA High) were collected and subjected for western blot analysis. We found that HSP70 was upregulated in the cytoplasm (5 nM; 1.8±1.0-fold and 10 nM; 2.1±1.1-fold). However, HSP70 and GATA1 in the nucleus were insignificant changed. Our finding demonstrated that manipulations of XPO1-HSP70 axis in the cytoplasm by selinexor were associated with accelerated late-stage erythropoiesis in severely affected β 0-thalassemia/HbE HSPCs. We then hypothesized whether selinexor functioned through an accumulation of HbF. Therefore, Hb profiles were investigated using HPLC. However, non-significant changes of Hb profiles and HbF levels were observed. With this finding, we purposed that selinexor functioned independently of HbF regulatory pathways and precise mechanisms underlying the regulation of late-erythropoiesis by selinexor are required. According to our preliminary data, an FDA approval compounds for multiple myeloma-selinexor-is a potent compound for improving in vitro late-stage erythropoiesis in HSPCs derived from severe β 0-thalassemia/HbE. Therefore, repurposing of selinexor to improve ineffective erythropoiesis in β-thalassemia is compelling. Moreover, combination treatments of selinexor with HbF inducers and/or SMAD2/3 blockers could be further validated to possibly achieve greater therapeutic benefits for β-thalassemia treatments.

The Spectrum of α-Thalassemia Mutations in Syrian Patients

α-Thalassemia (α-thal) is a globally prevalent genetic disorder of hemoglobin (Hb) structure where the rate of α-globin chain synthesis is reduced or absent due to the presence of α-globin mutation(s). The aim of this study is to define the spectrum of α-globin gene mutations and evaluate their allele frequency in a group of α-thal carriers. A total of 55 individuals with possible α-thal patients were referred from the thalassemia centers in Syria. They have unexplained hypochromia and microcytosis. All patients were genetically tested for 21 common α-globin gene mutations using reverse hybridization kit. Seven different α-globin gene mutations and 13 different genotypes were detected in 55 patients. The two most frequently encountered mutations were -α3.7 deletion (47.1%) and --MED mutation (21.4%). The most commonly observed genotype was -α3.7/αα (40%), followed by --MED/αα genotype (21.8%). We determined the most common α thalassemia mutations in the Syrian patients. α-Thalassemia mutations with deletions were mostly observed in our study.

Characterization of New Alpha Zero (α0) Thalassaemia Deletion (--GB) among Malays in Malaysian Population.

Malaysia is a multicultural and multiethnic country comprising numerous ethnic groups. From the total population of 32.7 million, Malays form the bulk of the Bumiputera in Malaysia comprise about 69.9%, followed by Chinese 22.8%, Indian 6.6%, and others 0.7%. The heterogeneous population and increasing numbers of non-citizens in this country affects the heterogeneity of genetic diseases, diversity, and heterogeneity of thalassaemia mutations. Alpha (α)-thalassaemia is an inherited haemoglobin disorder characterized by hypochromic microcytic anaemia caused by a quantitative reduction in the α-globin chain. A majority of the α-thalassaemia are caused by deletions in the α-globin gene cluster. Among Malays, the most common deletional alpha thalassaemia is -α3.7 deletion followed by --SEA deletion. We described the molecular characterization of a new --GB deletion in our population, involving both alpha genes in cis. Interestingly, we found that this mutation is unique among Malay ethnicities. It is important to diagnose this deletion because of the 25% risk of Hb Bart's with hydrops fetalis in the offspring when in combination with another α0- thalassaemia allele. MLPA is a suitable method to detect unknown and uncommon deletions and to characterize those cases which remain unresolved after a standard diagnostic approach.

Decrease in α-Globin and Increase in the Autophagy-Activating Kinase ULK1 mRNA in Erythroid Precursors from β-Thalassemia Patients Treated with Sirolimus.

The β-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the β-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including β-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from β-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5-2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for β-thalassemias.

Hemoglobin H hastalığında genetik danışmanlık önemli midir?

Alpha thalassemia is a genetic disease characterized by insufficient expression or definite absence of the α-globin chain. Three large deletions (thal-1; 26.5 kb or MedII, 20.5 kb and 17.4 kb or MedI) and two small deletions (thal-2; 4.2 kb and 3.7 kb) have been characterized in our country. In addition, two different PolyA mutations (PA1: AATAAA>AATAAG and PA2: AATAAA>AATGA) on the α2-globin gene (αα/αPAα), 5nt deletion (αα/α5ntα), and unstable Hb variant (CD 59; GGC→GAC) synthesized by the α1-globin gene (αα/ααCD59) have been reported. More than ten different combinations of α-thal-1 and α-thal-2 (--/-α) or HbH genotypes with point mutations (--/αPAα or --/ααCD59) were determined. In this study, which was carried out in Çukurova region, it is aimed to emphasize the importance of giving genetic counseling to families with alpha thalassemia carriers and to determine genotype combinations. DNA was isolated from blood samples taken from 5 children and their families who were admitted to Çukurova University Balcalı Hospital and diagnosed with severe anemia (Hb

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

Background: Increased production of fetal hemoglobin (HbF) can be beneficial for β-thalassemia patients and several clinical trials in β-thalassemia and/or sickle-cell disease (SCD) are ongoing using HbF inducers, such as NCT01245179 (based on the HDAC inhibitor Panobinostat), NCT00790127 (based on 2,2-dimethylbutyrate, HQK-1001) and NCT03877809 (based on the mTOR inhibitor sirolimus). Methods: In this context, new molecules able to induce HbF are needed. In this communication, we have studied in deep a new class of promising HbF inducers characterized by an isoxazole chemical skeleton. These derivatives are 3,4-isoxazolediamide compounds recently synthesized in our laboratories. The original structures of two natural molecules, geldanamycin and radicicol, known to be natural Hsp (Heat Shot Protein) inhibitors, were modified to lead to a novel class of synthetic compounds, containing the isoxazole nucleus, and displaying potent and selective inhibition of Hsp90. Here we used erythroid precursors cells (ErPCs) isolated from β-thalassemic patients and treated with appropriate concentrations of 8 isoxazole derivatives previously demonstrated to be able to induce erythroid differentiation of K562 cells. Results: We demonstrated the high efficacy of the analyzed isoxazoles in inducing HbF. Some derivatives demonstrated an activity even higher than other HbF inducers used as positive controls, such as Hydroxyurea (HU) or Sirolimus. Most of the isoxazole derivatives displayed high effects in decreasing the excess of free α-globin chains in treated ErPCs. All the compounds were assayed for genotoxicity through the Ames test, employing the histidine-requiring Salmonella typhimurium mutant TA 97A, TA98, TA100 and TA1535 strains that allow to check frameshift mutation and base-pair substitution, with and without metabolic activation induced by S9 Mix. These assays excluded any drawbacks related to genotoxicity. Summary - Conclusion: We may therefore conclude that these novel isoxazoles could be proposed for clinical studies for possible therapeutic application for the treatment of β-thalassemia. These compounds have been recently patented as potential therapeutic agents for β-thalassemia (US Patent 11077116). Acknowledgements This study was sustained by the Wellcome Trust (innovator award 208872/Z/17/Z), by AIFA (AIFA-2016-02364887) and by the UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Βeta-Thalassemia; no. 306201-FP7-HEALTH-2012-INNOVATION-1). We thank A.L.T. (Associazione per la lotta alla Talassemia) “Rino Vullo” - Ferrara, and A.V.L.T. (Associazione Veneta per la Lotta alla Talassemia) “Elio Zago” - APS – Rovigo.

Gene Mutation Spectrum among Alpha-Thalassaemia Patients in Northeast Peninsular Malaysia.

(1) Background: Alpha (α)-thalassaemia is a genetic disorder that affects 5% of the world population. Deletional or nondeletional mutations of one or both HBA1 and HBA2 on chromosome 16 will result in reduced production of α-globin chains, a component of haemoglobin (Hb) that is required for the formation of red blood cells (RBCs). This study aimed to determine the prevalence, haematological and molecular characterisations of α-thalassaemia. (2) Method: The parameters were based on full blood count, high-performance liquid chromatography and capillary electrophoresis. The molecular analysis involved gap-polymerase chain reaction (PCR), multiplex amplification refractory mutation system-PCR, multiplex ligation-dependent probe amplification and Sanger sequencing. (3) Results: With a total cohort of 131 patients, the prevalence of α-thalassaemia was 48.9%, leaving the remaining 51.1% with potentially undetected α gene mutations. The following genotypes were detected: -α3.7/αα (15.4%), -α4.2/αα (3.7%), --SEA/αα (7.4%), αCSα/αα (10.3%), αAdanaα/αα (0.7%), αQuong Szeα/αα (1.5%), -α3.7/-α3.7 (0.7%), αCSα/αCSα (0.7%), -α4.2/αCSα (0.7%), -SEA/αCSα (1.5%), -SEA/αQuong Szeα (0.7%), -α3.7/αAdanaα (0.7%), --SEA/-α3.7 (2.2%) and αCSα/αAdanaα (0.7%). Indicators such as Hb (p = 0.022), mean corpuscular volume (p = 0.009), mean corpuscular haemoglobin (p = 0.017), RBC (p = 0.038) and haematocrit (p = 0.058) showed significant changes among patients with deletional mutations, but not between patients with nondeletional mutations. (4) Conclusions: A wide range of haematological parameters was observed among patients, including those with the same genotype. Thus, a combination of molecular technologies and haematological parameters is necessary for the accurate detection of α-globin chain mutations.

Production and Characterization of K562 Cellular Clones Hyper-Expressing the Gene Encoding α-Globin: Preliminary Analysis of Biomarkers Associated with Autophagy.

One of the most relevant pathophysiological hallmarks of β-thalassemia is the accumulation of toxic α-globin chains inside erythroid cells, which is responsible for their premature death (hemolysis). In this context, the availability of an experimental model system mimicking the excess in α-globin chain production is still lacking. The objective of the present study was to produce and characterize K562 cellular clones forced to produce high amounts of α-globin, in order to develop an experimental model system suitable for studies aimed at the reduction of the accumulation of toxic α-globin aggregates. In the present study, we produced and characterized K562 cellular clones that, unlike the original K562 cell line, stably produced high levels of α-globin protein. As expected, the obtained clones had a tendency to undergo apoptosis that was proportional to the accumulation of α-globin, confirming the pivotal role of α-globin accumulation in damaging erythroid cells. Interestingly, the obtained clones seemed to trigger autophagy spontaneously, probably to overcome the accumulation/toxicity of the α-globin. We propose this new model system for the screening of pharmacological agents able to activate the full program of autophagy to reduce α-globin accumulation, but the model may be also suitable for new therapeutical approaches targeted at the reduction of the expression of the α-globin gene.

Haematopoietic Stem Cell Transplantation in Thalassaemia Major: A Narrative Review

Thalassaemia constitutes an especially prevalent human monogenic illness caused by a lack of synthesis of the α- or β-globin chains. The clinical impact of β-thalassaemia is worse since it consists of the same pair gene configuration, thalassaemia major, causing significant health discouragement and loss of life due to life threateningly insufficient haemoglobin (Hb) levels. Only a few nations have successfully reduced the prevalence of β-thalassaemia major, even though comprehensive screening, group counselling, pre-natal detection and public education can all be used. Since over ½ century ago, the fundamental elements of treatment for thalassaemia major have been iron chelation and hypertransfusion. The globin chain that makes up the adult Hb molecule is missing or synthesised at a reduced rate in β-thalassemia. The aberrant buildup of the α-globin chain and faulty formation of red blood cells (RBCs) leading to RBC haemolysis are the outcomes of this genetic abnormality. Since allogeneic haematopoietic stem cell transplantation (Allo-HSCT) has been a well-established gene replacement therapy for individuals with thalassaemia major for several years, it has had very successful outcomes for patients with access to it. Over the past 20 years, the consequences for more susceptible patients have also steadily improved, leading to 80%–90% longer-term life expectancy amongst this group of patients. However, providing Allo-HSCT as a treatment for these patients globally presents numerous difficulties. Replacing genes in autologous HSCs employing viral vectors has been possible in recent years.

Applying Multiplex ligation-dependent probe amplification technique to identify α-thalassemia carriers

α-thalassemia disease is mostly caused by mutations in the HBA1 and HBA2 genes that lead to the deficiency in the α-globin chain, which builds up the haemoglobin molecule. Depending on the number of missing α chains, the clinical manifestations of the disease are at different levels. This disease is inherited in an autosomal recessive manner, hence identifying healthy individuals carrying mutations in the α-thalassemia gene is essential for prenatal and premarital genetic counselling to reduce the incidence in the community. Multiplex ligation-dependent probe amplification (MLPA) technique was used to determine 15 people who were suspected of carrying the α-thalassemia gene based on their complete blood count. This study identified that therewere 12/15 people carrying --SEA mutation, 1/15 carrier having -α3.7 mutation, 1/15 carrier having -α4.2 mutation and 1/15 person carrying point mutation Hb constant spring -αHbCs.

Alpha-Thalassemia: Diversity of Clinical Phenotypes and Update on the Treatment

One of the more common single-gene disorders worldwide is α-thalassemia, carriers of which are found at variable frequencies (>1%) across all tropical and subtropical countries. Two linked α-globin genes on each allele of chromosome 16 regulate α-globin chain production. Deletion of one or more α-globin genes is the most frequent molecular defect found in α-thalassemia, whereas non-deletional mutations also occur, leading to unstable α-globin chains. HbH is the most common clinically important α-thalassemia disease and occurs when three α-globin genes are deleted/mutated, leaving only one copy of the gene intact. HbH can be divided into deletional (--/-α) and non-deletional genotypes (--/αTα). Whereas clinical phenotypes of the former are usually homogenously mild to moderate, those of the latter can be diverse. As HbH disease is particularly prevalent in Southeast Asia and some parts of the Mediterranean region, where β-thalassemia is also prevalent, affected patients are sometimes left undertreated. Therefore, hematologists and general physicians need to be educated to provide optimal disease monitoring and early identification of those with more severe phenotypes. Some issues regarding transfusion and iron chelation management differ from those of β-thalassemia, and these need to be recognized. Hb Bart’s hydrops fetalis syndrome (BHFS) is the most severe form of α-thalassemia; affected patients lack production of α-globin chains. Recent advances in fetal medicine and neonatal intensive care have made it possible for BHFS to no longer constitute a universally fatal disorder. Transfusion and chelation strategies for rare survivors are distinct and require updating.

Development of a β-Globin Gene Replacement Strategy As a β-Thalassemia Therapy

Background: β-thalassemia is a genetic disorder with high unmet need worldwide. Over 300 genetic mutations that result in the reduction, or loss, of β-globin (HBB) expression have been characterized to date. Patients with thalassemic mutations in both HBB alleles often experience severe anemia resulting from ineffective erythropoiesis due to the loss of adult hemoglobin (HbA) tetramers (α2β2) and imbalanced excess of α-globin chains. One challenge is creating a genome-editing strategy that replaces the diversity of disease-causing mutations in the HBB gene while restoring physiological HbA expression. Therefore, a gene-editing platform using homology-directed repair (HDR) that could replace the entire HBB gene in situ and under the control of normal regulatory elements would be an ideal strategy. Methods: CD34+ hematopoietic stem and progenitor cells (HSPCs) were edited using high-fidelity Cas9, HBB guide RNA, and different AAV6 DNA donor templates and then differentiated into red blood cells in vitro. We evaluated HBB expression in 39 AAV6 donors engineered with heterologous introns and different polyadenylation signals first using a flow cytometry screening assay and later HPLC. Candidates that met or exceeded wild-type HBB expression were moved forward for further comparison. Next, we edited HSPCs with the 8 highest expressing AAV6 donors, performed in vitro differentiation, and evaluated HDR frequencies by droplet digital polymerase chain reaction (ddPCR) and HbA formation by HPLC. The candidate that demonstrated the highest conversion of donor knock-in to HbA was further engineered to boost expression and reduce construct size. Two final candidates were moved forward to further characterize their ability to rescue loss of HbA in CD34+ HSPCs isolated from patients with sickle cell disease (SCD) at the protein and RNA level. Additionally, we performed DNA analysis on the gene-edited cells using ddPCR and a targeted PCR-free long-range sequencing approach. Results: Adding heterologous introns to the HBB coding sequence significantly increased HBB expression relative to the no-intron construct containing only coding DNA. The results from our flow cytometry screen and HPLC analysis were positively correlated, and the top DNA donors with heterologous introns resulted in physiologic HbA expression. Using this gene-replacement strategy, we achieved HDR frequencies of up to 40% in CD34+ HSPCs. Further optimization resulted in a smaller DNA template, maintenance of HbA conversion rates, and a lower rate of unintended recombinations. Finally, using HSPCs from patients with SCD as a therapeutically relevant model, we found, by HPLC and RNA sequencing, the optimized DNA donor can successfully replace the entire nonfunctional HBB gene, thereby restoring physiologic HbA expression. Future experiments are investigating in vitro gene correction in cells isolated from patients with β-thalassemia and additional studies evaluating in vivo stem cell repopulation capacity of gene-edited HSPCs. Conclusion: We developed an HBB AAV6 donor engineered with heterologous introns that demonstrates efficient HBB gene replacement and HbA rescue in the erythroid progeny of CD34+ HSPCs isolated from patients with SCD, offering a potential differentiated approach for treating β-thalassemia.

The Roles of Mitophagy and Autophagy in Ineffective Erythropoiesis in β-Thalassemia.

β-Thalassemia is one of the most common genetically inherited disorders worldwide, and it is characterized by defective β-globin chain synthesis leading to reduced or absent β-globin chains. The excess α-globin chains are the key factor leading to the death of differentiating erythroblasts in a process termed ineffective erythropoiesis, leading to anemia and associated complications in patients. The mechanism of ineffective erythropoiesis in β-thalassemia is complex and not fully understood. Autophagy is primarily known as a cell recycling mechanism in which old or dysfunctional proteins and organelles are digested to allow recycling of constituent elements. In late stage, erythropoiesis autophagy is involved in the removal of mitochondria as part of terminal differentiation. Several studies have shown that autophagy is increased in earlier erythropoiesis in β-thalassemia erythroblasts, as compared to normal erythroblasts. This review summarizes what is known about the role of autophagy in β-thalassemia erythropoiesis and shows that modulation of autophagy and its interplay with apoptosis may provide a new therapeutic route in the treatment of β-thalassemia. Literature was searched and relevant articles were collected from databases, including PubMed, Scopus, Prospero, Clinicaltrials.gov, Google Scholar, and the Google search engine. Search terms included: β-thalassemia, ineffective erythropoiesis, autophagy, novel treatment, and drugs during the initial search. Relevant titles and abstracts were screened to choose relevant articles. Further, selected full-text articles were retrieved, and then, relevant cross-references were scanned to collect further information for the present review.