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.

Peripheral blood circular RNA circ-0008102 may serve as a novel clinical biomarker in beta-thalassemia patients.

Circular RNA circ-0008102 has previously been found dysregulated in ß-thalassemia (ß-thal) in circRNAs microarray (GSE196682 and GSE241141). Our study is aimed at identifying whether circ-0008102 could be a novel biomarker in ß-thal. The peripheral blood of pediatric ß-thal patients with (n = 39) or without (n = 20) blood transfusion and healthy controls (n = 30) was selected. qRT-PCR, ROC curve analysis, Spearman correlation analysis, and FISH were used to analyze clinical value of circ-0008102. qRT-PCR confirmed that circ-0008102 expression in pediatric ß-thal patients without blood transfusion was significantly higher. ROC curves analysis showed that the AUC of circ-0008102 for differentiating patients without blood transfusion from patients with blood transfusion and healthy controls with an AUC of 0.733 and 0.711. Furthermore, circ-0008102 expression was positively correlated with the levels of RBC, HbF, ß-globin, and γ-globin mRNA, but was negatively corrected with the levels of HbA and Cr. circ-0008102 was mainly located in the cytoplasm. circ-0008102 could induce the activation of γ-globin and negatively regulate the expression of the five highest-ranking candidate miRNAs (miR-372-3p, miR-329-5p, miR-198, miR-152-5p, and miR-627-3p) in K562 cells. CONCLUSION: We demonstrate that peripheral blood upregulated circ-0008102 may serve as a novel clinical biomarker for pediatric ß-thal without blood transfusion. WHAT IS KNOWN: • CircRNAs are known to be involved in various human diseases, and several circRNAs are regarded as a class of promising blood-based biomarkers for detection of ß-thal. • CircRNAs exert biological functions by epigenetic modification and gene expression regulation, and dysregulated circRNAs in ß-thal might be involved in the induction of HbF in ß-thal. WHAT IS NEW: • Peripheral blood circ-0008102 maybe serve as a novel clinical biomarker for detection of pediatric ß-thal without blood transfusion. • Circ-0008102 participates in the pathogenesis of ß-thal through regulating γ-globin expression, and negatively regulates the expression of miR-372-3p, miR-329-5p, miR-198, miR-152-5p and miR-627-3p.

A critical review of therapeutic interventions in sickle cell disease: Progress and challenges.

Sickle cell disease (SCD) is an autosomal recessive genetic disorder that occurs due to the point mutation in the ß-globin gene, which results in the formation of sickle hemoglobin (HbS) in the red blood cells (RBCs). When HbS is exposed to an oxygen-depleted environment, it polymerizes, resulting in hemolysis, vaso-occlusion pain, and impaired blood flow. Still, there is no affordable cure for this inherited disease. Approved medications held promise but were met with challenges due to limited patient tolerance and undesired side effects, thereby inhibiting their ability to enhance the quality of life across various individuals with SCD. Progress has been made in understanding the pathophysiology of SCD during the past few decades, leading to the discovery of novel targets and therapies. However, there is a compelling need for research to discover medications with improved efficacy and reduced side effects. Also, more clinical investigations on various drug combinations with different mechanisms of action are needed. This review comprehensively presents therapeutic approaches for SCD, including those currently available or under investigation. It covers fundamental aspects of the disease, such as epidemiology and pathophysiology, and provides detailed discussions on various disease-modifying agents. Additionally, expert insights are offered on the future development of pharmacotherapy for SCD.

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.

Development of a double shmiR lentivirus effectively targeting both BCL11A and ZNF410 for enhanced induction of fetal hemoglobin to treat ß-hemoglobinopathies.

A promising treatment for ß-hemoglobinopathies is the de-repression of γ-globin expression leading to increased fetal hemoglobin (HbF) by targeting BCL11A. Here, we aim to improve a lentivirus vector (LV) containing a single BCL11A shmiR (SS) to further increase γ-globin induction. We engineered a novel LV to express two shmiRs simultaneously targeting BCL11A and the γ-globin repressor ZNF410. Erythroid cells derived from human HSCs transduced with the double shmiR (DS) showed up to a 70% reduction of both BCL11A and ZNF410 proteins. There was a consistent and significant additional 10% increase in HbF compared to targeting BCL11A alone in erythroid cells. Erythrocytes differentiated from SCD HSCs transduced with the DS demonstrated significantly reduced in vitro sickling phenotype compared to the SS. Erythrocytes differentiated from transduced HSCs from ß-thalassemia major patients demonstrated improved globin chain balance by increased γ-globin with reduced microcytosis. Reconstitution of DS-transduced cells from Berkeley SCD mice was associated with a statistically larger reduction in peripheral blood hemolysis markers compared with the SS vector. Overall, these results indicate that the DS LV targeting BCL11A and ZNF410 can enhance HbF induction for treating ß-hemoglobinopathies and could be used as a model to simultaneously and efficiently target multiple gene products.

New Synthetic Isoxazole Derivatives Acting as Potent Inducers of Fetal Hemoglobin in Erythroid Precursor Cells Isolated from ß-Thalassemic Patients.

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying ß-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from ß-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for ß-thalassemia.

Molecular Determination of Vascular Endothelial Growth Factor, miRNA-423 Gene Abnormalities by Utilizing ARMS-PCR and Their Association with Fetal Hemoglobin Expression in the Patients with Sickle Cell Disease.

Recent studies have indicated that microRNA and VEGF are considered to be genetic modifiers and are associated with elevated levels of fetal haemoglobin HbF, and thus they reduce the clinical impact of sickle haemoglobin (HbS) patients. This cross-sectional study was performed on clinical confirmed subjects of SCD cases. miR-423-rs6505162 C>T and VEGF-2578 C>A genotyping was conducted by ARMS-PCR in SCD and healthy controls. A strong clinical significance was reported while comparing the association of miR-423 C>T genotypes between SCD patients and controls (p = 0.031). The microRNA-423 AA genotype was associated with an increased severity of SCD in codominant model with odd ratio (OR = 2.36, 95% CI, (1.15-4.84), p = 0.018) and similarly a significant association was observed in recessive inheritance model for microRNA-423 AA vs (CC+CA) genotypes (OR = 2.19, 95% CI, (1.32-3.62), p < 0.002). The A allele was associated with SCD severity (OR = 1.57, 95% CI, (1.13-2.19), p < 0.007). The distribution of VEGF-2578 C>A genotypes between SCD patients and healthy controls was significant (p < 0.013). Our results indicated that in the codominant model, the VEGF-2578-CA genotype was strongly associated with increased SCD severity with OR = 2.56, 95% CI, (1.36-4.82), p < 0.003. The higher expression of HbA1 (65.9%), HbA2 (4.40%), was reported in SCD patients carrying miR-423-AA genotype than miR-423 CA genotype in SCD patients carrying miR-423 CA genotype HbA1 (59.98%), HbA2 (3.74%) whereas SCD patients carrying miR-423 CA genotype has higher expression of HbF (0.98%) and HbS (38.1%) than in the patients carrying AA genotype HbF (0.60%), HbS (36.1%). ARMS-PCR has been proven to be rapid, inexpensive and is highly applicable to gene mutation screening in laboratories and clinical practices. This research highlights the significance of elucidating genetic determinants that play roles in the amelioration of the HbF levels that is used as an indicator of severity of clinical complications of the monogenic disease. Further well-designed studies with larger sample sizes are necessary to confirm our findings.

miRNA Expression Associated with HbF in Saudi Sickle Cell Anemia.

Background and Objectives: Sickle cell anemia (SCA) is a hereditary monogenic disease due to a single ß-globin gene mutation that codes for the production of sickle hemoglobin. Its phenotype is modulated by fetal hemoglobin (HbF), a product of γ-globin genes. Exploring the molecules that regulate γ-globin genes at both transcriptional and translational levels, including microRNA (miRNA), might help identify alternative therapeutic targets. Materials and Methods: Using next-generation sequencing we identified pre-miRNAs and mature miRNA expression signatures associated with different HbF levels in patients homozygous for the sickle hemoglobin gene. The involvement of identified miRNAs in potential SCD-related pathways was investigated with the DIANA TOOL and miRWalk 2.0 database. Results: miR-184 were most highly upregulated in reticulocytes. miR-3609 and miR-483-5p were most highly downregulated in sickle cell anemia with high HbF. miR-370-3p that regulates LIN28A, and miR-451a which is effective in modulating α- and ß- globin levels were also significantly upregulated. miRNA targeted gene pathway interaction identified BCL7A, BCL2L1, LIN28A, KLF6, GATA6, solute carrier family genes and ZNF genes associated with erythropoiesis, cell cycle regulation, glycosphingolipid biosynthesis, cAMP, cGMP-PKG, mTOR, MAPK and PI3K-AKT signaling pathways and cancer pathways. Conclusions: miRNA signatures and their target genes identified novel miRNAs that could regulate fetal hemoglobin production and might be exploited therapeutically.

Emerging Genetic Therapy for Sickle Cell Disease.

The genetic basis of sickle cell disease (SCD) was elucidated >60 years ago, yet current therapy does not rely on this knowledge. Recent advances raise prospects for improved, and perhaps curative, treatment. First, transcription factors, BCL11A and LRF/ZBTB7A, that mediate silencing of the ß-like fetal (γ-) globin gene after birth have been identified and demonstrated to act at the γ-globin promoters, precisely at recognition sequences disrupted in rare individuals with hereditary persistence of fetal hemoglobin. Second, transformative advances in gene editing and progress in lentiviral gene therapy provide diverse opportunities for genetic strategies to cure SCD. Approaches include hematopoietic gene therapy by globin gene addition, gene editing to correct the SCD mutation, and genetic manipulations to enhance fetal hemoglobin production, a potent modifier of the clinical phenotype. Clinical trials may soon identify efficacious and safe genetic approaches to the ultimate goal of cure for SCD.

GATA zinc finger domain-containing protein 2A (GATAD2A) deficiency reactivates fetal haemoglobin in patients with ß-thalassaemia through impaired formation of methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex.

Reactivation of fetal haemoglobin (HbF) expression is an effective way to treat ß-thalassaemia and sickle cell anaemia. In the present study, we identified a novel GATA zinc finger domain-containing protein 2A (GATAD2A) mutation, which contributed to the elevation of HbF and ameliorated clinical severity in a patient with ß-thalassaemia, by targeted next-generation sequencing. Knockout of GATAD2A led to a significant induction of HbF in both human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) and human cluster of differentiation (CD)34+ cells with a detectable impact on erythroid differentiation. Furthermore, heterozygous knockout of GATAD2A impaired recruitment of chromodomain helicase DNA-binding protein 4 (CHD4) to the methyl-binding domain protein 2 (MBD2)-containing nucleosome remodelling and deacetylation (NuRD) complex. Our present data suggest that mutations causing the haploinsufficiency of GATAD2A might contribute to amelioration of clinical severity in patients with ß-thalassaemia.

MicroRNA-2355-5p regulates γ-globin expression in human erythroid cells by inhibiting KLF6.

Krüppel-like factors (KLFs) are a highly conserved family of transcription factors. We analysed expression profile data of KLFs and identified KLF6 as a new potential regulator of erythropoiesis. Knocking down the expression of KLF6 significantly raised γ-globin mRNA and protein levels in the erythroid cell line HUDEP-2 and haematopoietic progenitor (CD34+ ) cells. We found that overexpression of microRNA (miR)-2355-5p in HUDEP-2 and CD34+ cells correlated with increased γ-globin synthesis by suppressing expression of KLF6. Our discovery that the interaction between miR-2355-5p and KLF6 affects the expression of γ-globin may provide more information for the clinical management of ß-thalassaemia patients.

Long noncoding RNA HBBP1 enhances γ-globin expression through the ETS transcription factor ELK1.

ß-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, α2ß2), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat ß-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, α2γ2) in ß-thalassemia patients. However, the mechanism underlying HBBP1-mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in ß-thalassemia.

Comparison of efficacy and safety of thalidomide vs hydroxyurea in patients with Hb E-ß thalassemia - a pilot study from a tertiary care Centre of India.

INTRODUCTION: Hemoglobin (Hb)-F inducers are known to improve Hb level and transfusion dependence in thalassemia. This pilot study was conducted to assess the efficacy and safety of Hb-F inducer thalidomide compared to hydroxyurea (HU) in Hb E-ß thalassemia patients. METHODS: This was a prospective interventional single-centre study with 45 Hb E-beta thalassemia patients equally divided into group-I (thalidomide+folic acid), group-II (HU + folic acid) and group-III (folic acid). Response was assessed at various time intervals with 12-months follow up period. Primary end points were increment in Hb, Hb-F level and improvement in transfusion requirement; secondary end point were tolerability and safety. RESULTS: There was 100% responder (R: Hb-increment ≥1 g/dl) in group-I with 66.67% major responder (MaR: Hb-increment ≥2 g/dl), while there were 40% and 0% responder in group-II and III respectively. Hb-increment was significantly (p-value <0.0001) better in thalidomide arm compared to HU. The Hb-increment was attributable to both increase in Hb-F levels and reduction in ineffective erythropoiesis in thalidomide arm. Transfusion reduction was significantly better in group-I compared to group-II (100% vs 34%). No severe adverse effects was reported by patients of any group. CONCLUSION: Thalidomide showed a persistent significant Hb-increment and transfusion independence in Hb E-ß thalassemia patients compared to HU.

Sickle cell disease and COVID-19: Susceptibility and severity.

We surveyed published papers and an international sickle cell disease (SCD) registry to detect susceptibility and clinical course of coronavirus disease 2019 (COVID-19) in SCD patients. COVID-19 presentation was mild in children and moderate in many SCD adults. Regarding increased comorbidities with age, it seems severe COVID-19 to be more common in older SCD patients. Although the overall outcome of COVID-19 was favorable in SCD children, a high rate of pediatric intensive care unit admission should be considered in managing these patients. To explain COVID-19 outcome in SCD patients, the possible benefits of hydroxyurea therapy could be considered. The obtained results should be interpreted, considering low cases from sub-Saharan people, younger age of SCD patients compared to general population, a bias toward registry of the more severe form of disease, the effect of pre-existing comorbidities with multisystem organ damage, and the role of health socio-economic determinants.

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

ß-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding ß-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 ß-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 ß-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 ß-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 ß-thalassemia.

The Role of miRNAs as Therapeutic Tools in Sickle Cell Disease.

Background and Objectives: Sickle cell disorder (SCD) is a paradigmatic example of a complex monogenic disorder. SCD is characterized by the production of abnormal hemoglobin, primarily in the deoxygenated state, which makes erythrocytes susceptible to intracellular hemoglobin polymerization. Functional studies have affirmed that the dysregulation of miRNAs enhances clinical severity or has an ameliorating effect in SCD. miRNAs can be effectively regulated to reduce the pace of cell cycle progression, to reduce iron levels, to influence hemolysis and oxidative stress, and most importantly, to increase γ-globin gene expression and enhance the effectiveness of hydroxyurea. Results: This review highlights the roles played by some key miRNAs in hemoglobinopathies, especially in hematopoiesis, erythroid differentiation, and severity of anemia, which make miRNAs attractive molecular tools for innovative therapeutic approaches. Conclusions: In this era of targeted medicine, miRNAs mimics and antagomirs may be promising inducers of HbF synthesis which could ameliorate the clinical severity of SCD.

[Anti-interference hemoglobin analysis system by high performance liquid chromatography].

High performance liquid chromatography (HPLC) is currently the mainstream technology for detecting hemoglobin. Glycated hemoglobin (HbA1c) is a gold indicator for diagnosing diabetes, however, the accuracy of HbA1c test is affected by thalassemia factor hemoglobin F (HbF)/hemoglobin A2 (HbA2) and variant hemoglobin during HPLC analysis. In this study, a new anti-interference hemoglobin analysis system of HPLC is proposed. In this system, the high-pressure three-gradient elution method was improved, and the particle size and sieve plate aperture in the high-pressure chromatography column and the structure of the double-plunger reciprocating series high-pressure pump were optimized. The system could diagnose both HbA1c and thalassemia factor HbF/HbA2 and variant hemoglobin, and the performance of the system was anti-interference and stable. It is expected to achieve industrialization. In this study, the HbA1c and thalassemia factor HbF/HbA2 detection performance was compared between this system and the world's first-line brand products such as Tosoh G8, Bio-Rad Ⅶ and D10 glycosylated hemoglobin analysis system. The results showed that the linear correlation between this system and the world-class system was good. The system is the first domestic hemoglobin analysis system by HPLC for screening of HbA1c and thalassemia factor HbF/HbA2 rapidly and accurately.

Comparative analysis of lentiviral gene transfer approaches designed to promote fetal hemoglobin production for the treatment of ß-hemoglobinopathies.

ß-Hemoglobinopathies are among the most common single-gene disorders and are caused by different mutations in the ß-globin gene. Recent curative therapeutic approaches for these disorders utilize lentiviral vectors (LVs) to introduce a functional copy of the ß-globin gene into the patient's hematopoietic stem cells. Alternatively, fetal hemoglobin (HbF) can reduce or even prevent the symptoms of disease when expressed in adults. Thus, induction of HbF by means of LVs and other molecular approaches has become an alternative treatment of ß-hemoglobinopathies. Here, we performed a head-to-head comparative analysis of HbF-inducing LVs encoding for: 1) IGF2BP1, 2) miRNA-embedded shRNA (shmiR) sequences specific for the γ-globin repressor protein BCL11A, and 3) γ-globin gene. Furthermore, two novel baboon envelope proteins (BaEV)-LVs were compared to the commonly used vesicular-stomatitis-virus glycoprotein (VSV-G)-LVs. Therapeutic levels of HbF were achieved for all VSV-G-LV approaches, from a therapeutic level of 20% using γ-globin LVs to 50% for both IGF2BP1 and BCL11A-shmiR LVs. Contrarily, BaEV-LVs conferred lower HbF expression with a peak level of 13%, however, this could still ameliorate symptoms of disease. From this thorough comparative analysis of independent HbF-inducing LV strategies, we conclude that HbF-inducing VSV-G-LVs represent a promising alternative to ß-globin gene addition for patients with ß-hemoglobinopathies.

Pathophysiology and recent therapeutic insights of sickle cell disease.

Sickle cell disease (SCD) is an autosomal recessive blood disorder which occurs due to point mutation in the ß-globin chain of hemoglobin. Since the past decades, various therapies have been put forth, which are based on obstructing pathophysiological mechanisms of SCD including inhibition of Gardos channel and cation fluxes which in turn prevents sickle erythrocyte destruction and dehydration. The pharmacological approaches are based on the mechanism of reactivating γ-globin expression by utilizing fetal hemoglobin (HbF)-inducing drugs such as hydroxyurea. In SCD, gene therapy could be considered as a promising tool which involves modifying mutation at the gene-specific target by either promoting insertion or deletion of globins. Although there are various therapies emerged so far in the treatment of SCD, many of them have faced a major setback in most of developing countries in terms of cost, unavailability of expertise, and suitable donor. Therefore, in addition to pathophysiological aspects, this review will discuss new advancements and approaches made in the therapeutic domain of SCD including a viewpoint of modulating hemoglobin in SCD by the intervention of probiotics.

miR-30a regulates γ-globin expression in erythoid precursors of intermedia thalassemia through targeting BCL11A.

Patients with ß-thalassemia suffer from a lack or absence of the beta-globin chain of normal hemoglobin (Hb). Therefore, an increase in fetal Hb (HbF) levels could improve the clinical status of these patients. Downregulation of BCL11A, a key regulatory transcription factor, could ameliorate the clinical status of thalassemic patients by increasing HbF levels. miR-30a expression and its relationship with the BCL11A gene in erythroid precursors was explored in patients with ß-thalassemia. The relevance of miR-30a to clinical parameters was also investigated. We evaluated the expressions of miR-30a, BCL11A, and γ-globin genes by quantitative real-time PCR (qRT-PCR) on isolated erythroid precursors from peripheral blood samples of ß-thalassemia intermedia (TI) patients and in bone marrow samples from healthy individuals as controls. The correlation between miR-30a expression and clinical indices that included HbF levels, ferritin, and the frequency of blood transfusions were assessed. We observed increased expression of miR-30a in conjunction with decreased BCL11A expression and elevated γ-globin and HbF levels. Patients with elevated miR-30a expression had a higher percentage of HbF and a lower level of ferritin. In addition, we observed that overexpression of miR-30a in erythroid precursor cells led to reduced BCL11A expression and was associated with elevated γ-globin expression. Our findings showed the importance of miR-30a in BCL11A and HbF regulation, and in the clinical status of patients with ß-thalassemia.