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BIVV003 is a gene-edited autologous cell therapy in clinical development for the potential treatment of sickle cell disease (SCD). Hematopoietic stem cells (HSC) are genetically modified with mRNA encoding zinc finger nucleases (ZFN) that target and disrupt a specific regulatory GATAA motif in the BCL11A erythroid enhancer to reactivate fetal hemoglobin (HbF). We characterized ZFN-edited HSC from healthy donors and donors with SCD. Results of preclinical studies show that ZFN-mediated editing is highly efficient, with enriched biallelic editing and high frequency of on-target indels, producing HSC capable of long-term multilineage engraftment in vivo, and express HbF in erythroid progeny. Interim results from the Phase 1/2 PRECIZN-1 study demonstrated that BIVV003 was well-tolerated in seven participants with SCD, of whom five of the six with more than 3 months of follow-up displayed increased total hemoglobin and HbF, and no severe vaso-occlusive crises. Our data suggest BIVV003 represents a compelling and novel cell therapy for the potential treatment of SCD., (© 2024. The Author(s).)

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34 + hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders., Competing Interests: Declaration of interests A.S. has received consultant fees from Spotlight Therapeutics, Medexus Inc., Vertex Pharmaceuticals, Sangamo Therapeutics, and Editas Medicine. He is a medical monitor for RCI BMT CSIDE clinical trials, for which he receives financial compensation. He has also received research funding from CRISPR Therapeutics and honoraria from Vindico Medical Education. A.S. is the St. Jude Children’s Research Hospital site principal investigator of clinical trials for genome editing of sickle cell disease sponsored by Vertex Pharmaceuticals/CRISPR Therapeutics (NCT03745287), Novartis Pharmaceuticals (NCT04443907), and Beam Therapeutics (NCT05456880). The industry sponsors provide funding for the clinical trial, which includes salary support paid to A.S.’s institution. A.S. has no direct financial interest in these therapies. J.S.Y. is an equity owner of Beam Therapeutics. M.J.W. is on advisory boards for Cellarity Inc., Novartis, and Forma Therapeutics. S.Q.T. is a co-inventor on licensed patents for CHANGE-seq and other genome engineering technologies. S.Q.T. is a member of the scientific advisory boards of Prime Medicine and Ensoma., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Sickle cell disease is the most common hereditary hemoglobinopathy followed by beta-thalassemia. Until recently, allogeneic stem cell transplantation was the only curative approach. Based on the Crispr-Cas9-technology enabling targeting specific genes of interest, fetal hemoglobin which is normally shut-off after birth can be switched on and sufficient levels can alleviate symptoms in sickle cell disease and avoid transfusions in beta-thalassemia. Two first-in-human clinical studies in sickle cell disease and beta-thalassemia aiming to increase the level of fetal hemoglobin by using Crispr-Cas9 to modify autologous hematopoietic stem cells in patients aged 12-35 years have proved safety and efficacy and have shown promising clinical outcomes., Areas Covered: The paper summarizes the outcome of the results of the two recently published clinical studies and compares them with the other available curative approaches., Expert Opinion: Based on the currently available safety and efficacy data of the two published clinical results on gene therapy with Crispr-Cas9 modified autologous stem cells (exagamglogene autotemcel), it can be anticipated that this approach will add significantly to the therapeutic options for patients with sickle cell disease and beta-thalassemia and can be considered for all patients above 12 years of age independent of a suitable allogeneic stem cell donor.

Hydroxyurea (HU) is the most common drug therapy for sickle cell disease (SCD). The clinical benefits of HU derive from its upregulation of fetal hemoglobin (HbF), which reduces aggregation of the mutated sickle hemoglobin protein (HbS) and reduces SCD symptoms and complications. However, some individuals do not respond to HU, or stop responding over time. Unfortunately, current understanding of the mechanism of action of HU is limited, hindering the ability of clinicians to identify those patients who will respond to HU and to optimize treatment for those receiving HU. Given that epigenetic modifications are essential to erythropoiesis and HbF expression, we hypothesize that some effects of HU may be mediated by epigenetic modifications, specifically DNA methylation. However, few studies have investigated this possibility and the effects of HU on DNA methylation remain relatively understudied. In this review, we discuss the evidence linking HU treatment to DNA methylation changes and associated gene expression changes, with an emphasis on studies that were performed in individuals with SCD. Overall, although HU can affect DNA methylation, research on these changes and their clinical effects remains limited. Further study is likely to contribute to our understanding of hematopoiesis and benefit patients suffering from SCD., (© 2024 The Author(s). European Journal of Haematology published by John Wiley & Sons Ltd.)

Background: Excessive expression of hemoglobin F (HbF) is a characteristic feature and important diagnostic marker of β 0 -thalassemia/HbE disease. However, some patients may exhibit low-HbF levels, leading to misdiagnosis and precluding genetic counseling. The genetic factors influencing these differences in HbF expression in this atypical disease are not completely understood., Aims: To investigate determinants contributing to the non-elevation of HbF expression in β 0 -thalassemia/HbE disease., Methods: We studied 231 patients with β 0 -thalassemia/HbE confirmed by DNA analysis; classified them into the low-HbF (n = 62) and high-HbF (n = 169) groups; analyzed hematological parameters and hemoglobin levels in both groups; and characterized mutations in β- and α-globin genes and genetic variants in γ-globin promoters., Results: Both groups showed similar rates of type β 0 -thalassemia mutations but significantly different proportions of α-globin mutations: approximately 88.7% (95% confidence interval [CI] = 66.8-115.5) and 39.1% (95% CI = 30.2-49.7) in the low- and high-HbF groups, respectively. The results revealed single-nucleotide polymorphisms (SNPs) at -158 (C>T) in the G γ-globin promoters and novel SNPs at the 5' untranslated region position 25 (G>A) in A γ-globin promoters. The distribution of CC genotypes of the G γ-globin promoter in the low-HbF group was significantly higher than that in the high-HbF group., Conclusions: Cases with HbE predominance with low-HbF levels and undetectable HbA may not be as conclusive as those with homozygous HbE until DNA analysis is performed. Concomitant inheritance of α-thalassemia is an important inherent factor modifying HbF expression in a typical β 0 -thalassemia/HbE, and SNPs with the CC genotype in the G γ-globin promoter may indicate unelevated HbF expression in patients with this disease., (Copyright © 2024 Instituto Mexicano del Seguro Social (IMSS). Published by Elsevier Inc. All rights reserved.)

In β‑thalassemia, excessive α‑globin chain impedes the normal development of red blood cells resulting in anemia. Numerous miRNAs, including miR‑6747‑3p, are aberrantly expressed in β‑thalassemia major (β‑TM), but there are no reports on the mechanism of miR‑6747‑3p in regulating red blood cell lineage development and fetal hemoglobin (HbF) expression. In the present study, RT‑qPCR was utilized to confirm miR‑6747‑3p expression in patients with β‑TM and the healthy controls. Electrotransfection was employed to introduce the miR‑6747‑3p mimic and inhibitor in both HUDEP‑2 and K562 cells, and red blood cell lineage development was evaluated by CCK‑8 assay, flow cytometry, Wright‑Giemsa staining and Benzidine blue staining. B‑cell lymphoma/leukemia 11A (BCL11A) was selected as a candidate target gene of miR‑6747‑3p for further validation through FISH assay, dual luciferase assay and Western blotting. The results indicated that miR‑6747‑3p expression was notably higher in patients with β‑TM compared with healthy controls and was positively related to HbF levels. Functionally, miR‑6747‑3p overexpression resulted in the hindrance of cell proliferation, promotion of cell apoptosis, facilitation of cellular erythroid differentiation and γ‑globin expression in HUDEP‑2 and K562 cells. Mechanistically, miR‑6747‑3p could specifically bind to the 546‑552 loci of BCL11A 3'‑UTR and induce γ‑globin expression. These data indicate that upregulation of miR‑6747‑3p affects red blood cell lineage development and induces HbF expression by targeting BCL11A in β‑thalassemia, highlighting miR‑6747‑3p as a potential molecular target for β‑thalassemia therapy.

Sickle cell disease (SCD) is an inherited blood disorder marked by homozygosity of hemoglobin S, which is a defective hemoglobin caused by a missense mutation in the β-globin gene. However, clinical phenotypes of SCD vary among patients. To investigate genetic variants associated with various clinical phenotypes of SCD, we genotyped DNA samples from 520 SCD subjects and used a genome-wide association study (GWAS) approach to identify genetic variants associated with phenotypic features of SCD. For HbF levels, the previously reported 2p16.1 locus (BCL11A) reached genome significance (rs1427407, P = 8.58 × 10 -10 ) in our GWAS as expected. In addition, we found a new genome-wide significance locus at 15q14 (rs8182015, P = 2.07 × 10 -8 ) near gene EMC7. GWAS of acute chest syndrome (ACS) detected a locus (rs79915189, P = 3.70 × 10 -8 ) near gene IDH2 at 15q26.1. The SNP, rs79915189, is also an expression quantitative trait locus (eQTL) of IDH2 in multiple tissues. For vasoocclusive episode (VOE), GWAS detected multiple significant signals at 2p25.1 (rs62118798, P = 4.27 × 10 -8 ), 15q26.1 (rs62020555, P = 2.04 × 10 -9 ) and 15q26.3 (rs117797325, P = 4.63 × 10 -8 ). Our findings provide novel insights into the genetic mechanisms of SCD suggesting that common genetic variants play an important role in the presentation of the clinical phenotypes of patients with SCD., (© 2024. The Author(s).)

Abstract: It has been known for over half a century that throughout ontogeny, humans produce different forms of hemoglobin, a tetramer of α- and β-like hemoglobin chains. The switch from fetal to adult hemoglobin occurs around the time of birth when erythropoiesis shifts from the fetal liver to the bone marrow. Naturally, diseases caused by defective adult β-globin genes, such as sickle cell disease and β-thalassemia, manifest themselves as the production of fetal hemoglobin fades. Reversal of this developmental switch has been a major goal to treat these diseases and has been a driving force to understand its underlying molecular biology. Several review articles have illustrated the long and at times arduous paths that led to the discovery of the first transcriptional regulators involved in this process. Here, we survey recent developments spurred by the discovery of CRISPR tools that enabled for the first time high-throughput genetic screens for new molecules that impact the fetal-to-adult hemoglobin switch. Numerous opportunities for therapeutic intervention have thus come to light, offering hope for effective pharmacologic intervention for patients for whom gene therapy is out of reach., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

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.

Activating transcription factor 4 (ATF4) is a fundamental basic region/leucine zipper transcription factor, responds to various stress signals, and plays crucial roles in normal metabolic and stress response processes. Although its functions in human health and disease are not completely understood, compelling evidence underscores ATF4 is indispensable for multiple stages and lineages of erythroid development, including the regulation of fetal liver hematopoietic stem cells, induction of terminal erythroid differentiation, and maintenance of erythroid homeostasis. β -Thalassemia is a blood disorder arising from mutations in the β -globin gene. Reactivating the expression of the γ -globin gene in adult patients has emerged as a promising therapeutic strategy for ameliorating clinical symptoms associated with β -thalassemia. Recent research has suggested that ATF4 contributes to decreased fetal hemoglobin (HbF) level through its binding to potent negative regulators of HbF, such as BCL11A and MYB. Notably, evidence also suggests a contrasting outcome where increased ATF4 protein levels are associated with enhanced HbF at the transcriptional level. Consequently, the identification of mechanisms that modulate ATF4-mediated γ -globin transcription and its effects on erythroid development may unveil novel targets for β -thalassemia treatment., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both β-thalassemia (β-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.

Objective: The objective of this study was to investigate the therapeutic efficacy of thalidomide across various genotype presentations of β-thalassemia so as to facilitate the early screening of thalidomide-sensitive thalassemia cases and to understand the impact of iron overload on thalidomide., Methods: From our initial sample of 52 patients, we observed 48 patients with β-thalassemia for two years after administration of thalidomide. This cohort included 34 patients with transfusion-dependent thalassemia (TDT) and 14 patients with non-transfusion-dependent thalassemia (NTDT). We recorded the values of hemoglobin (Hb), fetal hemoglobin (HbF), and serum ferritin (SF) in the baseline period and at 1, 3, 6, 12, 18, and 24 months after enrollment, as well as the pre- and post-treatment blood transfusion volume in all 48 cases. According to the increase in Hb levels from baseline during the 6-month observation period, the response to thalidomide was divided into four levels: main response (MaR), minor response (MiR), slow response (SLR), and no response (NR). A decrease in serum ferritin levels compared to baseline was considered alleviation of iron overload. We calculated the overall response rate (ORR) as follows: ORR = MaR + MiR + SLR/number of observed cases., Results: The ORR was 91.7% (44/48 cases), and 72.9% showed MaR (35/48 cases). Among the 34 patients with TDT, 21 patients (61.8%) were free of blood transfusion, and the remaining 13 patients still required blood transfusion, but their total blood transfusion volume reduced by 31.3% when compared to the baseline. We found a total of 33 cases with 10 combinations of advantageous genes, which included 5 cases with βCD41-42/βCD17 and 6 cases with βCD41-42/β-28. Based on the treatment outcomes among the 48 cases in the observation group, there were 33 cases in the MaR group and 15 cases in the SLR/NR group. There was a difference in HbF between the two groups at baseline (P = 0.041). There were significant differences between the two groups in Hb and HbF at the time points of 6 and 12 months, respectively (P < 0.001). Compared to the baseline measurement, there was a significant decrease in the level of SF at months 12 and 24 (P < 0.001)., Conclusion: In this study, we identified 10 β-thalassemia gene combinations that were sensitive to thalidomide. These gene combinations can be used for initial screening and to predict the therapeutic effect of thalidomide in clinical practice. We examined the therapeutic response to thalidomide and found that the administration of thalidomide in combination with standardized iron removal was more beneficial in reducing iron overload., (© 2024. The Author(s).)

Genome-Wide Association Studies (GWASs) have identified a huge number of variants associated with different traits. However, their validation through in vitro and in vivo studies often lags well behind their identification. For variants associated with traits or diseases of biomedical interest, this gap delays the development of possible therapies. This issue also impacts beta-hemoglobinopathies, such as beta-thalassemia and sickle cell disease (SCD). The definitive cures for these diseases are currently bone marrow transplantation and gene therapy. However, limitations regarding their effective use restrict their worldwide application. Great efforts have been made to identify whether modulators of fetal hemoglobin (HbF) and, to a lesser extent, hemoglobin A2 (HbA 2 ) are possible therapeutic targets. Herein, we performed the post-GWAS in vivo validation of two genes, cyclin D3 ( CCND3 ) and nuclear factor I X ( NFIX ), previously associated with HbF and HbA 2 levels. The absence of Ccnd3 expression in vivo significantly increased g (HbF) and d (HbA 2 ) globin gene expression. Our data suggest that CCND3 is a possible therapeutic target in sickle cell disease. We also confirmed the association of Nfix with γ-globin gene expression and present data suggesting a possible role for Nfix in regulating Kruppel-like transcription factor 1 ( Klf1 ), a master regulator of hemoglobin switching. This study contributes to filling the gap between GWAS variant identification and target validation for beta-hemoglobinopathies.

Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in β-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.

A newly identified epigenetic modifier increases fetal hemoglobin in preclinical studies.

Background: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients' epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance., Results: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24-1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, r s  =  - 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, r s  =  - 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, r s  = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (p HBG2  = 0.011, r s  = 0.480/p ZBTB7A  = 0.026, r s  = 0.427) and showed prognostic value for PFS (p ZBTB7A  = 0.037, HR = 1.14, CI 0.34-3.8)., Conclusions: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action., (© 2024. The Author(s).)

Background: Sickle cell disease (SCD) is a major heritable genetic disease in sub-Saharan Africa, including Mauritania. Fetal hemoglobin (HbF) can affect the pathophysiology, moderate the clinical course, and offer prospects for curative treatment of SCD. This study aimed to investigate the influence of single nucleotide polymorphisms (SNPs) in the BCL11A gene on the levels of HbF and hematological parameters in Mauritanian sickle cell ( HbSS ) patients., Methods: Complete blood count was assessed in 565 patients suspected to have SCD. Polymerase chain reaction (PCR)-restriction fragment length polymorphism was performed to identify the HbSS , and sequencing was used for genotyping three SNPs: rs4671393 ( A>G ) and rs11886868 ( C>T ) in the intron 2 and rs1052520 ( G>A ) in the 3'UTR regions of the BCL11A gene in 50 sickle cell patients., Results: The prevalence of HbSS among the study population was 8.8% (50/565), and the mean (± standard deviation) of HbF level was 15.0% (± 6.0%). Sequencing showed the presence of three genotypes: AA (13.6%), AG (46.6%), GG (39.6%) in rs4671393; CC (17.6%), CT (48.7%), and TT (33.6%) in rs11886868 . All samples from HbSS individuals displayed a wild-type genotype in the rs1052520 allele. The prevalence of minor alleles A ( rs4671393 ) and C ( rs11886868 ) were 37% and 39%, respectively. There was a statistically significant association ( p = 0.034) between rs4671393 SNP and elevated HbF (mean 12.72 ± 6.26%)., Conclusions: The study of three SNPs in the BCL11A locus in Mauritanian patients with SCD showed a significant association of rs4671393 allele with the HbF level. Further research is needed to explore additional SNPs in the BCL11A locus and investigate other genetic markers reported to modulate HbF levels, such as HBS1L-MYB and Xmn1-HBG2 , to improve the management of this potentially life-threatening condition in Mauritania., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Sickle cell disease (SCD) clinically manifests itself with a myriad of complications. Stroke, both ischemic and hemorrhagic, as well as silent white matter changes, occurs at a relatively high prevalence. Understanding why and in whom stroke is most likely to occur is critical to the effective prevention and treatment of individuals with SCD. Genetic studies, including genome- and exome-wide association studies (GWAS and EWAS), have found several key modifiers associated with increased stroke/stroke risk in SCD via mechanisms including Hemoglobin F (HbF) modulation, inflammation, cellular adhesion, endothelial disruption, and hemolysis. We present a review on the modifiers that have most clearly demonstrated an association to date. More studies are needed to validate other potential polymorphisms and identify new ones. Incorporating gene-focused screenings in clinical care could provide avenues for more targeted, more effective, and less toxic prevention of stroke in this population. The data from this review will be used to inform the initial GWAS performed by the International Hemoglobinopathy Research Network (INHERENT) consortium.

Background: Exagamglogene 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 ., Methods: We 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., Results: A 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., Conclusions: Treatment 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.)., (Copyright © 2024 Massachusetts Medical Society.)

Background: Exagamglogene 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)., Methods: We 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 β 0 /β 0 , β 0 /β 0 -like, or non-β 0 /β 0 -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., Results: A 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., Conclusions: Treatment 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.)., (Copyright © 2024 Massachusetts Medical Society.)

Abstract: The switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (β-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A-mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA-mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG)., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Recent advancements in gene editing illuminate new potential therapeutic approaches for Sickle Cell Disease (SCD), a debilitating monogenic disorder caused by a point mutation in the β-globin gene. Despite the availability of several FDA-approved medications for symptomatic relief, allogeneic hematopoietic stem cell transplantation (HSCT) remains the sole curative option, underscoring a persistent need for novel treatments. This review delves into the growing field of gene editing, particularly the extensive research focused on curing haemoglobinopathies like SCD. We examine the use of techniques such as CRISPR-Cas9 and homology-directed repair, base editing, and prime editing to either correct the pathogenic variant into a non-pathogenic or wild-type one or augment fetal haemoglobin (HbF) production. The article elucidates ways to optimize these tools for efficacious gene editing with minimal off-target effects and offers insights into their effective delivery into cells. Furthermore, we explore clinical trials involving alternative SCD treatment strategies, such as LentiGlobin therapy and autologous HSCT, distilling the current findings. This review consolidates vital information for the clinical translation of gene editing for SCD, providing strategic insights for investigators eager to further the development of gene editing for SCD., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In this short review we have presented and discussed studies on pharmacogenomics (also termed pharmacogenetics) of the drugs employed in the treatment of β-thalassemia or Sickle-cell disease (SCD). This field of investigation is relevant, since it is expected to help clinicians select the appropriate drug and the correct dosage for each patient. We first discussed the search for DNA polymorphisms associated with a high expression of γ-globin genes and identified this using GWAS studies and CRISPR-based gene editing approaches. We then presented validated DNA polymorphisms associated with a high HbF production (including, but not limited to the HBG2 XmnI polymorphism and those related to the BCL11A , MYB , KLF-1 , and LYAR genes). The expression of microRNAs involved in the regulation of γ-globin genes was also presented in the context of pharmacomiRNomics. Then, the pharmacogenomics of validated fetal hemoglobin inducers (hydroxyurea, butyrate and butyrate analogues, thalidomide, and sirolimus), of iron chelators, and of analgesics in the pain management of SCD patients were considered. Finally, we discuss current clinical trials, as well as international research networks focusing on clinical issues related to pharmacogenomics in hematological diseases.

β-Thalassemia is a single-gene disease caused by mutations in β-globin and has a distinct geographical characteristics. Current treatment for patients with moderate to severe thalassemia has mainly relied on long-term blood transfusion and/or hematopoietic stem cell transplantation. B cell lymphoma/leukemia 11A (BCL11A) as a transcriptional repressor plays a vital role in monitoring γ/β hemoglobin switching, maintaining the normal function of hematopoietic stem cells, and regulating erythrocyte differentiation and lymphocyte development. With the rapid progress in gene editing technology, the BCL11A as a therapeutic target for β-thalassemia has shown promising results. This article has systematically summarized the regulatory mechanism and therapeutic potential of the BCL11A, with an aim to provide new ideas for the treatment of β-thalassemia.

The aim of this study was to identify genetic markers in the HBB Cluster; HBS1L-MYB intergenic region; and BCL11A , KLF1 , FOX3 , and ZBTB7A genes associated with the heterogeneous phenotypes of Sickle Cell Anemia (SCA) using next-generation sequencing, as well as to assess their influence and prevalence in an Angolan population. Hematological, biochemical, and clinical data were considered to determine patients' severity phenotypes. Samples from 192 patients were sequenced, and 5,019,378 variants of high quality were registered. A catalog of candidate modifier genes that clustered in pathophysiological pathways important for SCA was generated, and candidate genes associated with increasing vaso-occlusive crises (VOC) and with lower fetal hemoglobin (HbF) were identified. These data support the polygenic view of the genetic architecture of SCA phenotypic variability. Two single nucleotide polymorphisms in the intronic region of 2q16.1, harboring the BCL11A gene, are genome-wide and significantly associated with decreasing HbF. A set of variants was identified to nominally be associated with increasing VOC and are potential genetic modifiers harboring phenotypic variation among patients. To the best of our knowledge, this is the first investigation of clinical variation in SCA in Angola using a well-customized and targeted sequencing approach.

In humans, specific aberrations in β-globin results in sickle cell disease and β-thalassemia, symptoms of which can be ameliorated by increased expression of fetal globin (HbF). Two recent CRISPR-Cas9 screens, centered on ∼1500 annotated sequence-specific DNA binding proteins and performed in a human erythroid cell line that expresses adult hemoglobin, uncovered four groups of candidate regulators of HbF gene expression. They are (1) members of the nucleosome remodeling and deacetylase (NuRD) complex proteins that are already known for HbF control; (2) seven C2H2 zinc finger (ZF) proteins, including some (ZBTB7A and BCL11A) already known for directly silencing the fetal γ-globin genes in adult human erythroid cells; (3) a few other transcription factors of different structural classes that might indirectly influence HbF gene expression; and (4) DNA methyltransferase 1 (DNMT1) that maintains the DNA methylation marks that attract the MBD2-associated NuRD complex to DNA as well as associated histone H3 lysine 9 methylation. Here we briefly discuss the effects of these regulators, particularly C2H2 ZFs, in inducing HbF expression for treating β-hemoglobin disorders, together with recent advances in developing safe and effective small-molecule therapeutics for the regulation of this well-conserved hemoglobin switch., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 β-thalassemia and sickle cell disease. The identification of enhancer RNAs (eRNAs) related to the fetal (α2γ2) to adult hemoglobin (α2β2) switching remains incomplete. In this study, the transcriptomes of GYPA + cells from six β-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Rationale: Compound heterozygotes for deletional β-thalassemia can be difficult to diagnose due to its diverse clinical presentations and no routine screenings. This can lead to disease progression and delay in treatment., Patient Concerns: We reported pedigree analysis and genetic research in a family with rare β-thalassemia., Diagnosis: Pedigree analysis and genetic research demonstrated that the patient was a compound heterozygote for β-thalassemia CD17/Southeast Asian hereditary persistence of fetal hemoglobin deletion, inherited from the parents. Magnetic resonance imaging T2* examination revealed severe iron deposition in the liver. Echocardiography revealed endocardial cushion defect., Interventions: The patient was treated with Deferasirox after receiving the final molecular genetic diagnosis. The initial once-daily dose of Deferasirox was 20 mg/kg/d., Outcomes: The patient discontinued the medication three months after the first visit. Two years later, the patient visited the Department of Hepatobiliary and Pancreatic Diseases. He was recommended to undergo splenectomy after surgical repair of the congenital heart disease. However, the patient refused surgical treatment because of the economic burden., Lessons: We report that fetal hemoglobin is a sensitive indicator for screening large deletions of the β-globin gene, which can be effectively confirmed by the multiplex ligation-dependent probe amplification assay. In non-transfusion-dependent thalassemia patients, iron status assessment should be regularly performed, and iron chelation treatment should be initiated early. This case will provide insights for the diagnosis of rare genotypes of β-thalassemia and has important implications for genetic counseling., Competing Interests: The authors have no other conflicts of interest to declare., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected., Competing Interests: Declaration of interests All of the authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exagamglogene autotemcel (Casgevy™) is a genetically modified autologous CD34 + cell enriched population. It contains human haematopoietic stem and progenitor cells edited ex vivo by CRISPR/Cas9 (a DNA double strand break-inducing nuclease system) to differentiate into erythroid cells that produce high levels of foetal hemoglobin. Developed by Vertex Pharmaceuticals and CRISPR Therapeutics, exagamglogene autotemcel received its first approval on 16 November 2023 in the UK for the treatment of transfusion-dependent β-thalassemia (TDT) in patients aged ≥ 12 years for whom haematopoietic stem cell (HSC) transplantation is appropriate and a human leukocyte antigen matched related HSC donor is not available. On the same day, it was also approved in the UK for the treatment of sickle cell disease (SCD) in patients aged ≥ 12 years with recurrent vasoocclusive crises (VOCs) who have the β S /β S , β S /β + or β S /β 0 genotype for whom HSC transplantation is appropriate and a human leukocyte antigen matched related HSC donor is not available. Subsequently, exagamglogene autotemcel was approved in the USA on 8 December 2023 for the treatment of SCD in patients aged ≥ 12 years with recurrent VOCs and received a positive opinion in the EU on 14 December 2023 for the treatment of TDT and SCD. A regulatory assessment of exagamglogene autotemcel is currently underway for the treatment of TDT in the USA. This article summarizes the milestones in the development of exagamglogene autotemcel leading to these first approvals., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications., (© 2024. The Author(s).)

The mechanism that drives the switch from fetal to adult hemoglobin (Hb) provides a therapeutic target for β-thalassemia. We have previously identified that hypermethylation of transcription factor ERF promoter reactivated γ-globin expression. To uncover the mechanism underlying the hypermethylation of ERF promoter, we performed RNA sequencing in β 0 /β 0 -thalassemia patients and identified an upregulated long noncoding RNA (RP11-196G18.23) associated with HbF production. RP11-196G18.23 bound to the ERF promoter and recruited DNA methyltransferase 3A to promote DNA hypermethylation-mediated ERF downregulation, thereby ameliorating ERF-induced γ-globin inactivation. The identification of RP11-196G18.23 provides an epigenetic mechanism for the reactivation of fetal γ-globin expression for β-hemoglobinopathies., (© 2024. The Author(s).)

Kruppel-like factor 1 (KLF1) is an essential erythroid-specific transcription factor. Several reports have shown that KLF1 gene mutations are associated with increased levels of Hb F and Hb A 2 . However, scarce population studies have analysed common KLF1 variations. This study examines the potential association with Hb F and Hb A 2 levels in β-thalassemia (β-thal) carriers of Portugueseancestry of the four common KLF1 gene variants: -251C>G (rs3817621) and -148G>A (rs79334031), in the promoter region; and c.115A>C (p.Met39Leu) (rs112631212) and c.304T>C (p.Ser102Pro) (rs2072597), in exon 2. Ninety-two Portuguese β-thal carriers (43 males and 49 females) aged 2 to 77 years old (mean 32.55 years) were engaged in the study. Hb F levels range from 0.2 to 12.5% and Hb A 2 was above the normal level, ranging from 3.6 to 6%. The Hb A 2 and Hb F levels were determined by high-performance liquid chromatography. Single-nucleotide polymorphisms were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Minor allele frequencies for SNPs rs3817621 (G), rs79334031 (A), rs112631212 (C) and rs2072597 (C) were 0.196, 0.016, 0.011 and 0.169, respectively. Basic simple linear regression in the total population showed no significant associations with the levels of Hb F ( P >0.05). For the low-frequency variant -148A, a statistically significant association was found with increased levels of Hb A 2 (β = 0.855; P = 0.017). In conclusion, an association signal with Hb A 2 levels was observed for the variant -148A>G (rs79334031). The complex pattern of SNP interactions related to their influence on the KLF1 transcriptional activity mayexplain the absence of association with Hb F levels.

In acute lymphoblastic leukaemia (ALL), elevated foetal haemoglobin (HbF) levels have been associated with the prognosis of patients. Genetic variants in HbF regulatory genes: BAF chromatin remodelling complex subunit ( BCL11A ), HBS1L-MYB transcriptional GTPase intergenic region ( HBS1L-MYB ), Krüppel-like factor 1 ( KLF1 ), haemoglobin gamma subunit 2 ( HBG2 ), haemoglobin gamma subunit 1 (HBG1), and haemoglobin subunit beta pseudogene 1 ( HBBP1 ) are often associatedwith elevatedHbF concentration. This study investigated the association of genetic variants in HbF regulatory genes with HbF concentration, unfavourable prognosis, and outcome in children with ALL.We quantified HbF concentration and genotyped 17 genetic variants in 48 patients with ALL and 64 children without ALL as a reference group. HbF concentrationwas higher in patients than in the reference group (4.4%vs 1.4%), and 75%( n = 36) of thepatientshadHbF>2.5%.Unfavourable prognosis ALL was established in 68.8% ( n = 33) of the patients. Variant HBG2 rs7482144 was associated with high HbF concentration ( P = 0.015); while HBS1L-MYB rs9399137 ( P = 0.001), HBG2 rs7482144 ( P = 0.001) and the β-globin genes HBG2 , HBG1 , and HBPP1 haplotypeTGC(P = 0.017) with unfavourable prognosisALL.Additionally, variantBCL11A rs4671393 showed a protective role (P = 0.0001). In conclusion, variants HBG2 rs7482144, HBS1L-MYB rs9399137 and BCL11A rs4671393 may play a significant role in ALL.

Sickle cell disease (SCD) is the most common β-hemoglobinopathy caused by various mutations in the adult β-globin gene resulting in sickle hemoglobin production, chronic hemolytic anemia, pain, and progressive organ damage. The best therapeutic strategies to manage the clinical symptoms of SCD is the induction of fetal hemoglobin (HbF) using chemical agents. At present, among the Food and Drug Administration-approved drugs to treat SCD, hydroxyurea is the only one proven to induce HbF protein synthesis, however, it is not effective in all people. Therefore, we evaluated the ability of the novel Bach1 inhibitor, HPP-D to induce HbF in KU812 cells and primary sickle erythroid progenitors. HPP-D increased HbF and decreased Bach1 protein levels in both cell types. Furthermore, chromatin immunoprecipitation assay showed reduced Bach1 and increased NRF2 binding to the γ-globin promoter antioxidant response elements. We also observed increased levels of the active histone marks H3K4Me1 and H3K4Me3 supporting an open chromatin configuration. In primary sickle erythroid progenitors, HPP-D increased γ-globin transcription and HbF positive cells and reduced sickled erythroid progenitors under hypoxia conditions. Collectively, our data demonstrate that HPP-D induces γ-globin gene transcription through Bach1 inhibition and enhanced NRF2 binding in the γ-globin promoter antioxidant response elements., Competing Interests: Declaration of competing interest The authors declared no conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

BCL11A, a zinc finger repressor, is a stage-specific transcription factor that controls the switch from fetal (HbF, α 2 γ 2 ) to adult (HbA, α 2 β 2 ) hemoglobin in erythroid cells. While BCL11A was known as a factor critical for B-lymphoid cell development, its relationship to erythroid cells and HbF arose through genome-wide association studies (GWAS). Subsequent work validated its role as a silencer of γ-globin gene expression in cultured cells and mice. Erythroid-specific loss of BCL11A rescues the phenotype of engineered sickle cell disease (SCD) mice, thereby suggesting that downregulation of BCL11A expression might be beneficial in patients with SCD and β-thalassemia. Common genetic variation in GWAS resides in an erythroid-specific enhancer within the BCL11A gene that is required for its own expression. CRISPR/Cas9 gene editing of the enhancer revealed a GATA-binding site that confers a large portion of its regulatory function. Disruption of the GATA site leads to robust HbF reactivation. Advancement of a guide RNA targeting the GATA-binding site in clinical trials has recently led to approval of first-in-man use of ex vivo CRISPR editing of hematopoietic stem/progenitor cells (HSPCs) as therapy of SCD and β-thalassemia. Future challenges include expanding access and infrastructure for delivery of genetic therapy to eligible patients, reducing potential toxicity and costs, exploring prospects for in vivo targeting of hematopoietic stem cells (HSCs), and developing small molecule drugs that impair function of BCL11A protein as an alternative option., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

δβ-thalassemia is a rare type of thalassemia characterized by increased Hb F levels, including mainly Chinese G γ( A γδβ) 0 -thalassemia, Yunnanese G γ( A γδβ) 0 -thalassemia, Cantonese G γ( A γδβ) 0 -thalassemia in China. Due to the low rate of δβ-thalassemia carriers, there are few reports of δβ-thalassemia combined with β-thalassemia causing β-thalassemia major. Herein, we described the combination of Chinese G γ( A γδβ) 0 -thalassemia and β-thalassemia leading to β-thalassemia major in a Chinese patient. Hemoglobin analysis was performed by capillary electrophoresis (CE). Routine genetic analysis was carried out by gap-polymerase chain reaction (Gap-PCR) and PCR and reverse dot blot (PCR-RDB). Multiple ligation-dependent probe amplification (MLPA) was used to detect the large deletion, and Gap-PCR confirmed the deletion. A CE result showed an elevated Hb F level of 98.7% and 11.7% in the proband and her mother, but the proband was diagnosed with β CD17M /β CD17M using routine genetic analysis. However, her father was heterozygous for CD17 in β-globin, and her mother was detected as SEA heterozygous. The further analysis presented that the proband had actually missed the diagnosis of Chinese G γ( A γδβ) 0 -thalassemia by MLPA and PCR-RDB. Finally, the genotype of the proband was corrected from β CD17M /β CD17M to β CD17M /β Gγ(Aγδβ)0 . This is the first report of Chinese G γ( A γδβ) 0 -thalassemia combined with β-thalassemia resulting in β-thalassemia major in China. Screening for δβ-thalassemia by Hb analysis could be an effective method.

Elevated levels of Fetal Hemoglobin interfere with polymerization of sickle hemoglobin thereby reducing anemia, lessening the severity of symptoms, and increasing life span of patients with sickle cell disease. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the γ-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on γ-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN., Competing Interests: The authors have declared no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying β-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 β-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 β-thalassemia.

Reactivating silenced γ-globin expression through the disruption of repressive regulatory domains offers a therapeutic strategy for treating β-hemoglobinopathies. Here, we used transformer base editor (tBE), a recently developed cytosine base editor with no detectable off-target mutations, to disrupt transcription-factor-binding motifs in hematopoietic stem cells. By performing functional screening of six motifs with tBE, we found that directly disrupting the BCL11A-binding motif in HBG1/2 promoters triggered the highest γ-globin expression. Via a side-by-side comparison with other clinical and preclinical strategies using Cas9 nuclease or conventional BEs (ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif at the HBG1/2 promoters triggered the highest fetal hemoglobin in healthy and β-thalassemia patient hematopoietic stem/progenitor cells while exhibiting no detectable DNA or RNA off-target mutations. Durable therapeutic editing by tBE persisted in repopulating hematopoietic stem cells, demonstrating that tBE-mediated editing in HBG1/2 promoters is a safe and effective strategy for treating β-hemoglobinopathies., Competing Interests: Declaration of interests J.C., L. Yang, B.Y., and H.Y. are the scientific cofounders of CorrectSequence Therapeutics, a company that uses gene-editing technologies. X.M., L.W., Y.W., and H.M. are the employees of CorrectSequence Therapeutics. J.C., B.Y., L. Yang, W.H., S.S., and Y.Z. filed a provisional patent application related to this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Sickle Cell Anemia (SCA) is the most common genetic disease worldwide caused by a single mutation in the gene HBB . The disease severity is very variable and depends on many factors. We evaluated the clinical and biological profile of sickle cell anemia children in rural Central Africa., Methods: This cross-sectional study was conducted in the Hôpital Saint Luc de Kisantu, located 120 km away from Kinshasa-DR Congo in an area of 35 km around Kisantu with a population of roughly 80 000 individuals. We included SCA patients aged 6 months to 18 years. We collected clinical and hematological data. The SCA scoring system proposed by Adegoke et al. in 2013 was applied to determine the disease severity. We searched for factors associated to the disease severity., Results: This study included 136 patients, 66 males and 70 females (sex-ratio M/F 0.94). The mean severity score was 8.21 ± 5.30 (ranges 0-23). Fifty-nine (43.4%) children had mild disease, 62 (45.6%) moderate and 15 (11%) severe disease. Girls had higher levels of HbF than boys ( p  = 0.003). An inverse correlation was observed between fetal hemoglobin and the disease severity ( p  = 0.005, r -0.239, IC 95% -6.139; -1.469). Some factors such age influence the occurrence of certain chronic complications such as avascular bone necrosis., Conclusion: In conclusion, the disease severity of SCA depends on multiple factors. In this study, fetal hemoglobin was the main modulator of the disease severity. These data may also serve as a baseline to initiate HU treatment in this setting.

Pharmacological induction of fetal hemoglobin has proven to be a promising therapeutic intervention in β-hemoglobinopathies by reducing the globin chain imbalance and inhibiting sickle cell polymerization. Fagonia indica has shown therapeutic relevance to β-thalassemia. Therefore, we study the ethnopharmacological potential of Fagonia indica and its biomarker compounds for their HbF induction ability for the treatment of β-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 β-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 β-thalassemia., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background and Aims: High hemoglobin F determinants can be classified into hereditary persistence of fetal hemoglobin (HPFH) and δβ-thalassemia with different phenotype. We report the molecular basis and hematological features in a large cohort of deletional high Hb F determinants in Thailand., Materials and Methods: Subjects (n = 28,177) encountered during 2015-2022 were reviewed, and those with phenotypically suspected of having high Hb F determinants were selected. Combined PCR, multiplex ligation-dependent probe amplification, next-generation sequencing, and DNA sequencing were used to identify the mutations., Results: Among 28,177 subjects investigated, 300 (1.06 %) were found to carry deletional high Hb F determinants in a total of 302 alleles, including heterozygote, compound heterozygote with β-hemoglobinopathies, and homozygote. DNA analysis identified eight different DNA deletions, including δβ 0 -thalassemia (12.6 kb deletion) (73.8 %), HPFH-6 (14.9 %), Indian deletion-inversion A γδβ 0 -thalassemia (3.6 %), Thai deletion-inversion-insertion A γδβ 0 -thalassemia (3.0 %), SEA-HPFH (3.0 %), Chinese A γδβ 0 -thalassemia (1.0 %), Thai δβ 0 -thalassemia (11.3 kb deletion) (0.3 %), and a novel δβ 0 -thalassemia (137.1 kb deletion) (0.3 %). In addition, three novel genetic interactions, including Chinese A γδβ 0 -thalassemia/Hb E, δβ 0 -thalassemia/Indian deletion-inversion A γδβ 0 -thalassemia, and homozygous δβ 0 -thalassemia were found. Hematological features and Hb analysis results of 20 different genotypes were recorded. Multiplex gap-PCR assays for detection of these genetic determinants were described., Conclusions: Deletional high Hb F determinants are common and heterogeneous in Thailand. Data on the prevalence, molecular spectrum, phenotypic expression, and complex interactions of these genetic determinants should prove useful in the study and a prevention and control program of hemoglobinopathies in the region., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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

Importance: Sickle cell disease (SCD) is a monogenic disorder, yet clinical outcomes are influenced by additional genetic factors. Despite decades of research, the genetics of SCD remain poorly understood., Objective: To assess all reported genetic modifiers of SCD, evaluate the design of associated studies, and provide guidelines for future analyses according to modern genetic study recommendations., Data Sources: PubMed, Web of Science, and Scopus were searched through May 16, 2023, identifying 5290 publications., Study Selection: At least 2 reviewers identified 571 original, peer-reviewed English-language publications reporting genetic modifiers of human SCD phenotypes, wherein the outcome was not treatment response, and the comparison was not between SCD subtypes or including healthy controls., Data Extraction and Synthesis: Data relevant to all genetic modifiers of SCD were extracted, evaluated, and presented following STREGA and PRISMA guidelines. Weighted z score meta-analyses and pathway analyses were conducted., Main Outcomes and Measures: Outcomes were aggregated into 25 categories, grouped as acute complications, chronic conditions, hematologic parameters or biomarkers, and general or mixed measures of SCD severity., Results: The 571 included studies reported on 29 670 unique individuals (50% ≤ 18 years of age) from 43 countries. Of the 17 757 extracted results (4890 significant) in 1552 genes, 3675 results met the study criteria for meta-analysis: reported phenotype and genotype, association size and direction, variability measure, sample size, and statistical test. Only 173 results for 62 associations could be cross-study combined. The remaining associations could not be aggregated because they were only reported once or methods (eg, study design, reporting practice) and genotype or phenotype definitions were insufficiently harmonized. Gene variants regulating fetal hemoglobin and α-thalassemia (important markers for SCD severity) were frequently identified: 19 single-nucleotide variants in BCL11A, HBS1L-MYB, and HBG2 were significantly associated with fetal hemoglobin (absolute value of Z = 4.00 to 20.66; P = 8.63 × 10-95 to 6.19 × 10-5), and α-thalassemia deletions were significantly associated with increased hemoglobin level and reduced risk of albuminuria, abnormal transcranial Doppler velocity, and stroke (absolute value of Z = 3.43 to 5.16; P = 2.42 × 10-7 to 6.00 × 10-4). However, other associations remain unconfirmed. Pathway analyses of significant genes highlighted the importance of cellular adhesion, inflammation, oxidative and toxic stress, and blood vessel regulation in SCD (23 of the top 25 Gene Ontology pathways involve these processes) and suggested future research areas., Conclusions and Relevance: The findings of this comprehensive systematic review and meta-analysis of all published genetic modifiers of SCD indicated that implementation of standardized phenotypes, statistical methods, and reporting practices should accelerate discovery and validation of genetic modifiers and development of clinically actionable genetic profiles.

We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications. Here we report results of exploratory single-cell analysis of patients in which BCL11A is targeted molecularly and compare results with cells of patients treated with hydroxyurea (HU), the current standard of care. We use single-cell assays to assess HbF, HbS, oxygen saturation, and hemoglobin polymer content in RBCs for nine gene therapy trial subjects (BCL shmiR , median HbF% = 27.9) and compare them to 10 HU-treated subjects demonstrating high and comparable levels of HbF (HU High Responders, median HbF% = 27.0). All BCL11A patients achieved the primary endpoint for NCT03282656, which was defined by an absolute neutrophil count greater than or equal to 0.5 × 10 9 cells/L for three consecutive days, achieved within 7 weeks following infusion. Flow cytometric assessment of single-RBC HbF and HbS shows fewer RBCs with high HbS% that would be most susceptible to sickling in BCL shmiR vs. HU High Responders: median 42% of RBCs with HbS%>70% in BCL shmiR vs. 61% in HU High Responders (p = 0.004). BCL shmiR subjects also demonstrate more RBCs resistant to HbS polymerization at lower physiologic oxygen tension: median 32% vs. 25% in HU High Responders (p = 0.006). Gene therapy-induced BCL11A down-regulation reverses the fetal-to-adult hemoglobin switch and induces RBCs with higher HbF%, lower HbS%, and greater resistance to deoxygenation-induced polymerization in clinical trial subjects compared with a cohort of highly responsive hydroxyurea-treated subjects., (© 2023. Springer Nature Limited.)