Gene editing for sickle cell disease and transfusion dependent thalassemias- A cure within reach.

Sickle cell disease (SCD) is associated with significant morbidity and shortened life expectancy. Similarly, patients with transfusion dependent beta thalassemia (TdT) require life-long transfusion therapy, chelation therapy and significant organ dysfunction. Allogeneic transplantation from a matched family donor provided the only curative option for patients with SCD and TdT. Unfortunately, less than 20% of patients have a fully matched related donor and results using unrelated donor transplant were associated with high rate of complications. Ex vivo gene therapy through globin gene addition has been investigated extensively and recent encouraging preliminary data resulted in regulatory approval in patients with TdT. Recent improvements in our understanding of the molecular pathways controlling erythropoiesis and globin switching from fetal hemoglobin to adult hemoglobin offer a new and exciting therapeutic options. Rapid and substantial advances in genome editing tools using CRISPR/Cas9, have raised the possibility of genetic editing and correction in patient derived hematopoietic stem and progenitor cells. We will review results of gene editing approach that can induce fetal hemoglobin production in patients with SCD and TdT.

The continuing global challenges of treating patients with beta-thalassemia.

In this issue, Hokland et al. offer an interesting view of the different approaches on how to treat a beta-thalassemia patient. The principal concerns, that this report reveals, are the very wide differences in the facilities and economic resources available for the care of patients. Management of thalassemia should become a word wide health care priority and may include at last those two steps: national and international registries; national programs for screening couples at risk and providing preventive measures to prevent births of patients with thalassemia. Commentary on: Hokland et al. Thalassaemia-A global view. Br J Haematol. 2023;201:208-223.

Psychometric evaluation of the NTDT-PRO questionnaire for assessing symptoms in patients with non-transfusion-dependent beta-thalassaemia.

OBJECTIVES: The non-transfusion-dependent beta-thalassaemia-patient-reported outcome (NTDT-PRO) questionnaire was developed for assessing anaemia-related tiredness/weakness (T/W) and shortness of breath (SoB) among patients with NTDT. Psychometric properties were evaluated using blinded data from the BEYOND trial (NCT03342404). DESIGN: Analysis of a phase 2, double-blind, randomised, placebo-controlled trial. SETTING: USA, Greece, Italy, Lebanon, Thailand and the UK. PARTICIPANTS: Adults (≥18 years) (N=145) with NTDT who had not received a red blood cell transfusion within 8 weeks prior to randomisation, with mean baseline haemoglobin level ≤100 g/L. MEASURES: NTDT-PRO daily scores from baseline until week 24, and scores at select time points for the 36-Item Short Form Health Survey version 2 (SF-36v2), Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) and Patient Global Impression of Severity (PGI-S). RESULTS: Cronbach's alpha at weeks 13-24 was 0.95 and 0.84 for the T/W and SoB domains, respectively, indicating acceptable internal consistency reliability. Among participants self-reporting no change in thalassaemia symptoms via the PGI-S between baseline and week 1, intraclass correlation coefficients were 0.94 and 0.92 for the T/W and SoB domains, respectively, indicating excellent test-retest reliability. In a known-groups validity analysis, least-squares mean T/W and SoB scores at weeks 13-24 were worse in participants with worse scores for the FACIT-F Fatigue Subscale (FS), SF-36v2 vitality or PGI-S. Indicating responsiveness, changes in T/W and SoB domain scores were moderately correlated with changes in haemoglobin levels, and strongly correlated with changes in SF-36v2 vitality, FACIT-F FS, select FACIT-F items and the PGI-S. Improvements in least-squares mean T/W and SoB scores were higher in participants with greater improvements in scores on other PROs measuring similar constructs. CONCLUSIONS: The NTDT-PRO demonstrated adequate psychometric properties to assess anaemia-related symptoms in adults with NTDT and can be used to evaluate treatment efficacy in clinical trials.

Pathogenic Mechanisms in Thalassemia I: Ineffective Erythropoiesis and Hypercoagulability.

Erythropoiesis is the physiological process that results in the production of red blood cells (RBCs). In conditions of pathologically altered erythropoiesis or ineffective erythropoiesis, as in the case of ß-thalassemia, the reduced ability of erythrocytes to differentiate, survive and deliver oxygen stimulates a state of stress that leads to the ineffective production of RBCs. We herein describe the main features of erythropoiesis and its regulation in addition to the mechanisms behind ineffective erythropoiesis development in ß-thalassemia. Finally, we review the pathophysiology of hypercoagulability and vascular disease development in ß-thalassemia and the currently available prevention and treatment modalities.

The Need for Translational Epidemiology in Beta Thalassemia Syndromes: A Thalassemia International Federation Perspective.

Epidemiology is the practical tool to provide information on which policy makers should base planning of services. Epidemiological data for thalassemia is based on inaccurate and often conflicting measurements. This study attempts to demonstrate with examples the sources of inaccuracy and confusion. The Thalassemia International Foundation (TIF) suggests that congenital disorders, for which increasing complications and premature death are avoidable through appropriate treatment and follow-up, should be given priority based on accurate data and patient registries. Moreover, only accurate information about this issue, especially for developing countries, will move national health resources in the right direction.

Fertility and Pregnancy in Women with Transfusion-Dependent Thalassemia.

Because women with transfusion-dependent thalassemia are seeking pregnancy, ensuring the best outcomes for both mother and baby require concerted and collaborative efforts between the hematologist, obstetrician, cardiologist, hepatologist, and genetic counselor among others. Proactive counseling, early fertility evaluation, optimal management of iron overload and organ function, and application of advances in reproductive technology and prenatal screening are important in ensuring a healthy outcome. Many unanswered questions remain requiring further study, including fertility preservation, non-invasive prenatal diagnosis, chelation therapy during pregnancy, and indications and duration of anticoagulation.

Emerging Therapies in ß-Thalassemia.

Advances in understanding the underlying pathophysiology of ß-thalassemia have enabled efforts toward the development of novel therapeutic modalities. These can be classified into three major categories based on their ability to target different features of the underlying disease pathophysiology: correction of the α/ß globin chain imbalance, targeting ineffective erythropoiesis, and targeting iron dysregulation. This article provides an overview of these different emerging therapies that are currently in development for ß-thalassemia.

Clinical Challenges with Iron Chelation in Beta Thalassemia.

Conventional therapy for severe thalassemia includes regular red cell transfusions and iron chelation therapy to prevent and treat complications of iron overload. Iron chelation is very effective when appropriately used, but inadequate iron chelation therapy continues to contribute to preventable morbidity and mortality in transfusion-dependent thalassemia. Factors that contribute to suboptimal iron chelation include poor adherence, variable pharmacokinetics, chelator adverse effects, and difficulties with precise monitoring of response. The regular assessment of adherence, adverse effects, and iron burden with appropriate treatment adjustments is necessary to optimize patient outcomes.

Assessment of orthodontic treatment needs in patients with ß-thalassemia major and sickle cell disease.

Patients suffering from hemoglobinopathies may suffer from pathogic dental and orofacial features. This study aimed to assess the prevalence of malocclusion and the need for orthodontic treatment in patients with ß-thalassemia major (BTM) and sickle cell disease (SCD). The study was conducted on 311 blood transfusion-dependent patients with BTM or SCD and 400 healthy individuals aged 10 to 16. The types of malocclusion were evaluated based on Angle's classification and Dewey's modification, and their oral habits were recorded using a questionnaire. The need for orthodontic treatment was assessed through the Dental Health Component of the Index of Orthodontic Treatment Need (IOTN), and the data were compared with normal participants. The Index of Orthodontic Treatment Need-Dental Health Component (IOTN-DHC) assessment showed that patients had a higher prevalence of objective need for treatment (IOTN grades 4 and 5) compared to healthy children. The prevalence of class II malocclusion was significantly higher in patients. Patients showed significantly less Angle's Class I malocclusion compared to normal participants. Oral habits were presented in 61%, 64.15% and 62.4% of normal participants, BTM and SCD patients, respectively. The higher prevalence of Angle's class II malocclusion and higher percentage of IOTN grade 4 and 5 among BTM and SCD patients reveal the importance of early orthodontic assessment and intervention in children with BMT and SDC.

Gene Therapy for ß-Hemoglobinopathies: From Discovery to Clinical Trials.

Investigations to understand the function and control of the globin genes have led to some of the most exciting molecular discoveries and biomedical breakthroughs of the 20th and 21st centuries. Extensive characterization of the globin gene locus, accompanied by pioneering work on the utilization of viruses as human gene delivery tools in human hematopoietic stem and progenitor cells (HPSCs), has led to transformative and successful therapies via autologous hematopoietic stem-cell transplant with gene therapy (HSCT-GT). Due to the advanced understanding of the ß-globin gene cluster, the first diseases considered for autologous HSCT-GT were two prevalent ß-hemoglobinopathies: sickle cell disease and ß-thalassemia, both affecting functional ß-globin chains and leading to substantial morbidity. Both conditions are suitable for allogeneic HSCT; however, this therapy comes with serious risks and is most effective using an HLA-matched family donor (which is not available for most patients) to obtain optimal therapeutic and safe benefits. Transplants from unrelated or haplo-identical donors carry higher risks, although they are progressively improving. Conversely, HSCT-GT utilizes the patient's own HSPCs, broadening access to more patients. Several gene therapy clinical trials have been reported to have achieved significant disease improvement, and more are underway. Based on the safety and the therapeutic success of autologous HSCT-GT, the U.S. Food and Drug Administration (FDA) in 2022 approved an HSCT-GT for ß-thalassemia (Zynteglo™). This review illuminates the ß-globin gene research journey, adversities faced, and achievements reached; it highlights important molecular and genetic findings of the ß-globin locus, describes the predominant globin vectors, and concludes by describing promising results from clinical trials for both sickle cell disease and ß-thalassemia.

The Novel Role of the B-Cell Lymphoma/Leukemia 11A (BCL11A) Gene in ß-Thalassaemia Treatment.

ß-thalassaemia is a genetic disorder resulting in a reduction or absence of ß-globin gene expression. Due to the high prevalence of ß-thalassaemia and the lack of available treatment other than blood transfusion and haematopoietic stem cell (HSC) transplantation, the disease represents a considerable burden to clinical and economic systems. Foetal haemoglobin has an appreciated ameliorating effect in ß-haemoglobinopathy, as the γ-globin chain substitutes the ß-globin chain reduction by pairing with the excess α-globin chain in ß-thalassaemia and reduces sickling in sickle cell disease (SCD). BCL11A is a critical regulator and repressor of foetal haemoglobin. Downregulation of BCL11A in adult erythroblasts and cell lines expressing adult haemoglobin led to a significant increase in foetal haemoglobin levels. Disruption of BCL11A erythroid enhancer resulted in disruption of the BCL11A gene solely in the erythroid lineages and increased γ-globin expression in adult erythroid cells. Autologous haematopoietic stem cell gene therapy represents an attractive treatment option to overcome the immune complications and donor availability associated with allogeneic transplantation. Using genome editing technologies, the disruption of BCL11A to induce γ- globin expression in HSCs has emerged as an alternative approach to treat ß-thalassaemia. Targeting the +58 BCL11A erythroid enhancer or BCL11A binding motif at the γ-gene promoter with CRISPR-Cas9 or base editors has successfully disrupted the gene and the binding motif with a subsequent increment in HbF levels. This review outlines the critical role of BCL11A in γ-globin gene silencing and discusses the different genome editing approaches to downregulate BCL11A as a means for ameliorating ß-thalassaemia.

Longitudinal study of ICET-A on glucose tolerance, insulin sensitivity and ß-cell secretion in eleven ß-thalassemia major patients with mild iron overload.

BACKGROUND: Iron chelation therapy (ICT) is the gold standard for treating patients with iron overload, though its long-term effects are still under evaluation. According to current recommendations regarding  transfusion-dependent  (TD)  ß-thalassemia major (ß-TM) patients, their serum ferritin (SF) levels should be maintained below 1,000 ng/mL and ICT should be discontinued when the levels are <500 ng/mL in two successive tests. Alternatively, the dose of chelator could be considerably reduced to maintain a balance between iron input and output of  frequent transfusions. STUDY DESIGN: Due to the paucity of information on long-term effects of ICT  in ß-TM with low SF levels on glucose homeostasis, the International Network of Clinicians for Endocrinopathies in Thalassemia and Adolescence Medicine (ICET-A) promoted a retrospective and an ongoing prospective observational study with the primary aim to address the long-term effects of ICT on glucose tolerance and metabolism (ß-cell function and peripheral insulin sensitivity) in adult ß-TM patients with persistent SF level below 800 ng/mL. PATIENTS AND METHODS: 11 ß-TM patients (mean age: 35.5 ± 5.5 years; SF range: 345-777 ng/mL) with normal glucose tolerance test (OGTT) or abnormal glucose tolerance (AGT) for a median of 5.3(1.1-8.3) years. RESULTS: Abnormal glucose tolerance (AGT) was observed in 7 patients (63.6%) at first observation and ) persisted in 6 patients (54.5%) at last observation. None of them developed diabetes mellitus. AGT was reversed in two patients. One patient with NGT developed early glucose intolerance (1-h PG ≥155 and 2-h PG <140 mg/dL). Three out of  5 patients with isolated impaired glucose tolerance presented a variation of  ATG. Stabilization of low indices for ß-cell function and insulin sensitivity/resistance was observed. One patient developed hypogonadotrophic hypogonadism. Three out of 6 patients with SF below 500 ng/dL had hypercalciuria. CONCLUSION: Despite low SF level, the burden of endocrine complications remains a challenge in ß-TM patients. The ability to keep iron at near "normal" level with acceptable risks of toxicity remains to be established.

Thalassaemia-A global view.

The thalassaemias are a group of genetic disorders of haemoglobin which are endemic in the tropics but are now found worldwide due to migration. Basic standard of care therapy includes regular transfusions to maintain a haemoglobin level of around 10 g/dL, together with iron chelation therapy to prevent iron overload. Novel therapies, bone marrow transplantation, and gene therapy are treatment options that are unavailable in many countries with stressed economies. This Wider Perspectives article presents the strategies for management of an adolescent refugee patient with beta thalassaemia, as it would be performed by expert haematologists in six countries: Italy, Lebanon, Oman, the Sudan, Thailand and the United States. The experienced clinicians in each country have adapted their practice according to the resources available, which vary greatly. Even in the current modern era, providing adequate transfusions and chelation is problematic in many countries. On the other hand, ensuring adherence to therapy, particularly during adolescence, is a similar challenge seen in all countries. The concluding section highlights the disparities in available therapies and puts the role of novel therapies into a societal context.

T helper 17 and regulatory T-cell profile and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in pediatric patients with beta-thalassemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment option for hereditary hemoglobin disorders, such as beta-thalassemia; However, this procedure is not without constraints, mainly engendering complications such as acute graft-versus-host disease (aGvHD), chronic GvHD (cGvHD), and susceptibility to infections. The clinical outcomes of allo-HSCT are highly dependant on the quality and quantity of T-cell subsets reconstitution. Following the allo-HSCT of six pediatric patients afflicted with beta-thalassemia, their mononuclear cells were isolated, and then cultured with a combination of phorbol myristate acetate (PMA)/ionomycin and Brefeldin A. The content of CD4 T-cell subsets, including T helper 17 (Th17) cells and regulatory T cells (Tregs), were determined by specific conjugated-monoclonal antibodies three and six months post-HSCT. An increased frequency of total CD4 T-cells, Tregs and Th17 cells was observed at day 90 and 180 after allo-HSCT, albeit the numbers were still lower than that of our healthy controls. In patients who developed cGvHD, a lower Th17/Treg ratio was observed, owing it to a decreased proportion of Th17 cells. In conclusion, creating balance between Th17 and Treg subsets may prevent acute and chronic GvHD in patients after allo-HSCT.

Successful outcome in a compound heterozygote haemoglobin E/beta-thalassaemia in pregnancy.

Haemoglobin E (HbE) affects at least 1 million people around the world. The carrier frequency of HbE/beta-thalassaemia (HbE/ß-thalassaemia) is highest in Southeast Asia. In India, the highest frequency is observed in the northeast region. Distinguishing between homozygous HbE disease and HbE/ß-thalassaemia is a challenge to the haematopathologist as well as to the treating obstetrician because both are clinically and haematologically similar, posing a difficulty in managing anaemia and assessing the fetal risk for the same disease. This article reports a case of compound heterozygote HbE/ß-thalassaemia in pregnancy and its successful outcome.

Foetal haemoglobin inducers for reducing blood transfusion in non-transfusion-dependent beta-thalassaemias.

BACKGROUND: Non-transfusion-dependent ß-thalassaemia (NTDßT) is a subset of inherited haemoglobin disorders characterised by reduced production of the ß-globin chain of haemoglobin leading to anaemia of varying severity. Although blood transfusion is not a necessity for survival, it may be required to prevent complications of chronic anaemia, such as impaired growth and hypercoagulability. People with NTDßT also experience iron overload due to increased iron absorption from food sources which becomes more pronounced in those requiring blood transfusion. People with a higher foetal haemoglobin (HbF) level have been found to require fewer blood transfusions, thus leading to the emergence of treatments that could increase its level. HbF inducers stimulate HbF production without altering any gene structures. Evidence for the possible benefits and harms of these inducers is important for making an informed decision on their use. OBJECTIVES: To compare the effectiveness and safety of the following for reducing blood transfusion for people with NTDßT: 1. HbF inducers versus usual care or placebo; 2. single HbF inducer with another HbF inducer, and single dose with another dose; and 3. combination of HbF inducers versus usual care or placebo, or single HbF inducer. SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search date was 21 August 2022. SELECTION CRITERIA: We included randomised controlled trials (RCTs) or quasi-RCTs comparing single HbF inducer with placebo or usual care, with another single HbF inducer or with a combination of HbF inducers; or comparing different doses of the same HbF inducer. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methods. Our primary outcomes were blood transfusion and haemoglobin levels. Our secondary outcomes were HbF levels, the long-term sequelae of NTDßT, quality of life and adverse events. MAIN RESULTS: We included seven RCTs involving 291 people with NTDßT, aged two to 49 years, from five countries. We reported 10 comparisons using eight different HbF inducers (four pharmacological and four natural): three RCTs compared a single HbF inducer to placebo and seven to another HbF inducer. The duration of the intervention lasted from 56 days to six months. Most studies did not adequately report the randomisation procedures or whether and how blinding was achieved. HbF inducer against placebo or usual care Three HbF inducers, HQK-1001, Radix Astragali or a 3-in-1 combined natural preparation (CNP), were compared with a placebo. None of the comparisons reported the frequency of blood transfusion. We are uncertain whether Radix Astragali and CNP increase haemoglobin at three months (mean difference (MD) 1.33 g/dL, 95% confidence interval (CI) 0.54 to 2.11; 1 study, 2 interventions, 35 participants; very low-certainty evidence). We are uncertain whether Radix Astragali and CNP have any effect on HbF (MD 12%, 95% CI -0.74% to 24.75%; 1 study, 2 interventions, 35 participants; very low-certainty evidence). Only medians on haemoglobin and HbF levels were reported for HQK-1001. Adverse effects reported for HQK-1001 were nausea, vomiting, dizziness and suprapubic pain. There were no prespecified adverse effects for Radix Astragali and CNP. HbF inducer versus another HbF inducer Four studies compared a single inducer with another over three to six months. Comparisons included hydroxyurea versus resveratrol, hydroxyurea versus thalidomide, hydroxyurea versus decitabine and Radix Astragali versus CNP. No study reported our prespecified outcomes on blood transfusion. Haemoglobin and HbF were reported for the comparison Radix Astragali versus CNP, but we are uncertain whether there were any differences (1 study, 24 participants; low-certainty evidence). Different doses of the same HbF inducer Two studies compared two different types of HbF inducers at different doses over two to six months. Comparisons included hydroxyurea 20 mg/kg/day versus 10 mg/kg/day and HQK-1001 10 mg/kg/day, 20 mg/kg/day, 30 mg/kg/day and 40 mg/kg/day. Blood transfusion, as prespecified, was not reported. In one study (61 participants) we are uncertain whether the lower levels of both haemoglobin and HbF at 24 weeks were due to the higher dose of hydroxyurea (haemoglobin: MD -2.39 g/dL, 95% CI -2.80 to -1.98; very low-certainty evidence; HbF: MD -10.20%, 95% CI -16.28% to -4.12%; very low-certainty evidence). The study of the four different doses of HQK-1001 did not report results for either haemoglobin or HbF. We are not certain if major adverse effects may be more common with higher hydroxyurea doses (neutropenia: risk ratio (RR) 9.93, 95% CI 1.34 to 73.97; thrombocytopenia: RR 3.68, 95% CI 1.12 to 12.07; very low-certainty evidence). Taking HQK-1001 20 mg/kg/day may result in the fewest adverse effects. A combination of HbF inducers versus a single HbF inducer Two studies compared three combinations of two inducers with a single inducer over six months: hydroxyurea plus resveratrol versus resveratrol or hydroxyurea alone, and hydroxyurea plus l-carnitine versus hydroxyurea alone. Blood transfusion was not reported. Hydroxyurea plus resveratrol may reduce haemoglobin compared with either resveratrol or hydroxyurea alone (MD -0.74 g/dL, 95% CI -1.45 to -0.03; 1 study, 54 participants; low-certainty evidence). We are not certain whether the gastrointestinal disturbances, headache and malaise more commonly reported with hydroxyurea plus resveratrol than resveratrol alone were due to the interventions. We are uncertain whether hydroxyurea plus l-carnitine compared with hydroxyurea alone may increase mean haemoglobin, and reduce pulmonary hypertension (1 study, 60 participants; very low-certainty evidence). Adverse events were reported but not in the intervention group. None of the comparisons reported the outcome of HbF. AUTHORS' CONCLUSIONS: We are uncertain whether any of the eight HbF inducers in this review have a beneficial effect on people with NTDßT. For each of these HbF inducers, we found only one or at the most two small studies. There is no information on whether any of these HbF inducers have an effect on our primary outcome, blood transfusion. For the second primary outcome, haemoglobin, there may be small differences between intervention groups, but these may not be clinically meaningful and are of low- to very low-certainty evidence. Data on adverse effects and optimal doses are limited. Five studies are awaiting classification, but none are ongoing.