Good engraftment after reduced intensity targeted busulfan-based conditioning and matched related donor hematopoietic cell transplantation in hemoglobinopathies.

BACKGROUND: Hematopoietic cell transplantation (HCT) is an established curative therapy for transfusion-dependent thalassemia (TDT) and sickle cell disease (SCD). The latest American Society of Hematology guidelines recommend myeloablative preparative regimen in patients under 18 years of age. PROCEDURE: The objective was to demonstrate safety and efficacy of a reduced intensity conditioning (RIC) regimen including high-dose fludarabine, anti-thymocyte globulin, and targeted busulfan as a single alkylator to sub-myeloablative exposures. RESULTS: Between 2012 and 2021, 11 patients with SCD and five patients with TDT and matched related donor (MRD) HCT were included. The median age at transplantation was 8.3 years (range: 3.7-18.8 years). The median administered busulfan AUC was 67.4 mg/L×h (range: 60.7-80 mg/L×h). Overall survival was 93.8% and event-free survival 87.5% with one engrafted SCD patient with pre-existing moyamoya disease succumbing after drainage of a subdural hematoma. One SCD patient developed a secondary graft failure and was treated with a second HCT. Myeloid chimerism was full in all other patients with a median follow-up time of 4.1 years (range: 2.0-11.1 years), whereas T-cell donor chimerism was frequently mixed. CONCLUSION: This RIC conditioning followed by MRD HCT is sufficiently myeloablative to cure pediatric patients with hemoglobinopathies without the need for additional total body irradiation or thiotepa.

Autologous gene therapy for hemoglobinopathies: From bench to patient's bedside.

In recent years, a growing number of clinical trials have been initiated to evaluate gene therapy approaches for the treatment of patients with transfusion-dependent ß-thalassemia and sickle cell disease (SCD). Therapeutic modalities being assessed in these trials utilize different molecular techniques, including lentiviral vectors to add functional copies of the gene encoding the hemoglobin ß subunit in defective cells and CRISPR-Cas9, transcription activator-like effector protein nuclease, and zinc finger nuclease gene editing strategies to either directly address the underlying genetic cause of disease or induce fetal hemoglobin production by gene disruption. Here, we review the mechanisms of action of these various gene addition and gene editing approaches and describe the status of clinical trials designed to evaluate the potentially for these approaches to provide one-time functional cures to patients with transfusion-dependent ß-thalassemia and SCD.

Transfusion in hemoglobinopathies and red blood cell alloimmunization: data from Sicily, Sardinia and Malta.

BACKGROUND: Hemoglobinopathies are a group of diseases that include those due to globin gene mutations, such as thalassemia major (TM) and thalassemia intermedia (TI) or due to alteration of hemoglobin structure such as sickle cell disease (SCD), as well as a combination of these conditions such as thalasso-drepanocytosis (TD). They constitute the most frequent hereditary anemias requiring blood transfusion. MATERIALS AND METHODS: In April 2022, a questionnaire was sent to the Transfusion Services (TS) of Sicily, Sardinia and the Maltese National Blood Transfusion (MNBT) service. The questionnaire was divided into a generic part including the number of patients followed and the type of hemoglobinopathy, and a section relating to transfusion therapy, including the number of units transfused, whether red blood cells (RBC) were washed and, finally, a section relating to the presence or absence of alloantibodies and their identification. RESULTS: Data was retrieved for 2,574 patients: 68.6% TM, 15.4% TI, 10.3% TD, 4.1% SCD, and 1.6% other hemoglobinopathies (OHA). The number of RBC units transfused was 76,974, equivalent to 24.5% of all the RBCU transfused from the total number of patients followed. The number of washed RBCU was 21.1% of all the units used; 337 patients (37%) were diagnosed with alloantibodies, the majority of which were patients with SCD (20.6%). Of the 485 alloantibodies found, 90.3% were identified. The antibodies found most frequently were related to the Kell system (41.7%) followed by antibodies to the Rhesus system (37.9%); 29.7% of patients had more than one antibody. DISCUSSION: From our study, certain indications can be formulated: complete the National Registry for patients with hemoglobinopathies; create a Registry of alloimmunized patients to ensure transfusion therapy is as safe as possible, considering antibody evanescence; and 3) increase the recruitment of blood donors of diverse ethnicities.

Pregnancy and assisted reproductive technology use in Australian female transfusion-dependent haemoglobinopathy patients: a 20-year retrospective analysis.

BACKGROUND: In the last few decades, the life expectancy of patients with transfusion-dependent thalassaemia (TDT) and sickle cell disease (SCD) has improved significantly, in part because of improved iron chelation. Fertility challenges and pregnancy complications have historically limited reproductive options in this group; however, improved multi-disciplinary care has made infertility a chronic disease complication requiring attention. Despite this, there are very few reports and no Australian data describing fertility and pregnancy outcomes in this population. AIMS: To identify the rate of assisted reproductive technologies (ART) utilisation in our female transfusion-dependent haemoglobinopathy patients and to establish the nature of maternal and neonatal complications in this cohort. METHODS: A 20-year retrospective analysis (1997-2017) at an Australian centre captured data on conception rates, use of assisted reproductive techniques (ART), and pregnancy and neonatal outcomes in female transfusion-dependent haemoglobinopathy patients. RESULTS: Conception was attempted in 14 women (11 TDT and three SCD) during the study period. A total of 28 pregnancies resulting in 25 live births were recorded. ART supported 13 conceptions. A positive association was not identified between elevated mean serum ferritin and ART use; however, all patients with an established diagnosis of hypogonadotropic hypogonadism (HH) required ART. Maternal complications included gestational diabetes mellitus and post-partum haemorrhage. There were no cardiac complications. Two-thirds of women underwent lower segment caesarean section, with prematurity complicating 20% of births. There were no neonatal or maternal deaths. CONCLUSION: Pregnancy is an achievable goal for women with transfusion-dependent haemoglobinopathies, although the support of ART may be required in a subset of patients.

Evaluation of Mono- and Bi-Functional GLOBE-Based Vectors for Therapy of ß-Thalassemia by HBBAS3 Gene Addition and Mutation-Specific RNA Interference.

Therapy via the gene addition of the anti-sickling ßAS3-globin transgene is potentially curative for all ß-hemoglobinopathies and therefore of particular clinical and commercial interest. This study investigates GLOBE-based lentiviral vectors (LVs) for ßAS3-globin addition and evaluates strategies for an increased ß-like globin expression without vector dose escalation. First, we report the development of a GLOBE-derived LV, GLV2-ßAS3, which, compared to its parental vector, adds anti-sickling action and a transcription-enhancing 848-bp transcription terminator element, retains high vector titers and allows for superior ß-like globin expression in primary patient-derived hematopoietic stem and progenitor cells (HSPCs). Second, prompted by our previous correction of HBBIVSI-110(G>A) thalassemia based on RNApol(III)-driven shRNAs in mono- and combination therapy, we analyzed a series of novel LVs for the RNApol(II)-driven constitutive or late-erythroid expression of HBBIVSI-110(G>A)-specific miRNA30-embedded shRNAs (shRNAmiR). This included bifunctional LVs, allowing for concurrent ßAS3-globin expression. LVs were initially compared for their ability to achieve high ß-like globin expression in HBBIVSI-110(G>A)-transgenic cells, before the evaluation of shortlisted candidate LVs in HBBIVSI-110(G>A)-homozygous HSPCs. The latter revealed that ß-globin promoter-driven designs for monotherapy with HBBIVSI-110(G>A)-specific shRNAmiRs only marginally increased ß-globin levels compared to untransduced cells, whereas bifunctional LVs combining miR30-shRNA with ßAS3-globin expression showed disease correction similar to that achieved by the parental GLV2-ßAS3 vector. Our results establish the feasibility of high titers for LVs containing the full HBB transcription terminator, emphasize the importance of the HBB terminator for the high-level expression of HBB-like transgenes, qualify the therapeutic utility of late-erythroid HBBIVSI-110(G>A)-specific miR30-shRNA expression and highlight the exceptional potential of GLV2-ßAS3 for the treatment of severe ß-hemoglobinopathies.

Gene Therapy for Hemoglobinopathies.

ß-Thalassemia and sickle cell disease are autosomal recessive disorders of red blood cells due to mutations in the adult ß-globin gene, with a worldwide diffusion. The severe forms of hemoglobinopathies are fatal if untreated, and allogeneic bone marrow transplantation can be offered to a limited proportion of patients. The unmet clinical need and the disease incidence have promoted the development of new genetic therapies based on the engineering of autologous hematopoietic stem cells. Here, the steps of ex vivo gene therapy development are reviewed along with results from clinical trials and recent new approaches employing cutting edge gene editing tools.

Successful Treatment of a Child with Hemoglobin Hammersmith with Hematopoietic Stem Cell Transplantation.

Hemoglobin (Hb) Hammersmith, formed by serine substitution for phenylalanine at residue 42 in the beta-globin chain, is a very rare variant of unstable hemoglobin with low oxygen affinity. For patients with hemoglobinopathies, it is well-established that hematopoietic stem cell transplantation provides a complete cure, but the literature on its role for those with Hb Hammersmith is limited. A seven-month-old girl who was examined for anemia and splenomegaly was followed up for congenital hemolytic anemia. The patient with visible cyanosis of the lips and whose p50 was low in blood gas was diagnosed with Hb Hammersmith through the DNA sequence analysis. During the follow-up, frequent blood transfusions had to be given due to anemia aggravated by infections. Following a successful hematopoietic stem cell transplant from an HLA-matched sibling, the patient completely recovered from Hb Hammersmith. The case is presented because of its rarity.

Case report: Curing a rare, unstable hemoglobin variant Hb Bristol-Alesha using haploidentical hematopoietic stem cell transplantation.

Unstable hemoglobinopathies are a rare, heterogeneous group of diseases that disrupt the stability of hemoglobin (Hb), leading to chronic hemolysis and anemia. Patients with severe phenotypes often require regular blood transfusions and iron chelation therapy. Although rare, studies have reported that hematopoietic stem cell transplantation (HSCT) seems to be an available curative approach in transfusion-dependent patients with unstable hemoglobinopathies. Here, we describe successful haploidentical HSCT for the treatment of an unstable Hb variant, Hb Bristol-Alesha, in a 6-year-old boy with severe anemia since early childhood. Two years after transplantation, he had a nearly normal hemoglobin level without evidence of hemolysis. DNA analysis showed complete chimerism of the donor cell origin, confirming full engraftment with normal erythropoiesis.

Precision Editing as a Therapeutic Approach for ß-Hemoglobinopathies.

Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the ß-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic ß- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with ß-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for ß-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies.

A systematic review of clinical trials for gene therapies for ß-hemoglobinopathy around the world.

BACKGROUND AIMS: Amidst the success of cell therapy for the treatment of onco-hematological diseases, the first recently Food and Drug Administration-approved gene therapy product for patients with transfusion-dependent ß-thalassemia (TDT) indicates the feasibility of gene therapy as curative for genetic hematologic disorders. This work analyzed the current-world scenario of clinical trials involving gene therapy for ß-hemoglobinopathies. METHODS: Eighteen trials for patients with sickle cell disease (SCD) and 24 for patients with TDT were analyzed. RESULTS: Most are phase 1 and 2 trials, funded by the industry and are currently recruiting volunteers. Treatment strategies for both diseases are fetal hemoglobin induction (52.4%); addition of wild-type or therapeutic ß-globin gene (38.1%) and correction of mutations (9,5%). Gene editing (52.4%) and gene addition (40.5%) are the two most used techniques. The United States and France are the countries with the greatest number of clinical trials centers for SCD, with 83.1% and 4.2%, respectively. The United States (41.1%), China (26%) and Italy (6.8%) lead TDT trials centers. CONCLUSIONS: Geographic trial concentration indicates the high costs of this technology, logistical issues and social challenges that need to be overcome for gene therapy to reach low- and middle-income countries where SCD and TDT are prevalent and where they most impact the patient's health.

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.

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.

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.

Therapeutic perspective for children and young adults living with thalassemia and sickle cell disease.

Hemoglobinopathies, including thalassemias and sickle cell disease, are the most common monogenic diseases worldwide, with estimated annual births of more than 330,000 affected infants. Hemoglobin disorders account for about 3.4% of deaths in children under 5 years of age. The distribution of these diseases is historically linked to current or previously malaria-endemic regions; however, immigration has led to a worldwide distribution of these diseases, making them a global health problem. During the last decade, new treatment approaches and novel therapies have been proposed, some of which have the potential to change the natural history of these disorders. Indeed, the first erythroid maturation agent, luspatercept, and gene therapy have been approved for beta-thalassemia adult patients. For sickle cell disease, molecules targeting vaso-occlusion and hemoglobin S polymerization include crizanlizumab, which has been approved for patients ≥ 16 years, voxelotor approved for patients ≥ 12 years, and L-glutamine for patients older than 5 years.    Conclusion: We herein present the most recent advances and future perspectives in thalassemia and sickle cell disease treatment, including new drugs, gene therapy, and gene editing, and the current clinical trial status in the pediatric populations. What is Known: • Red blood cell transfusions, iron chelation therapy and hematopoietic stem cell transplantation have been the mainstay of treatment of thalassemia patients for decades. • For sickle cell disease, until 2005, treatment strategies were mostly the same as those for thalassemia, with the option of simple transfusion or exchange transfusion. In 2007, hydroxyurea was approved for patients ≥ 2 years old. What is New: • In 2019, gene therapy with betibeglogene autotemcel (LentiGlobin BB305) was approved for TDT patients ≥ 12 years old non ß0/ß0 without matched sibling donor. • Starting from 2017 several new drugs, such as L-glutamine (approved only by FDA), crizanlizumab (approved by FDA and EMA for patients ≥ 16 years), and lastly voxelotor (approved by FDA and EMA for patients ≥ 12 years old).

Epigenetic Regulation of ß-Globin Genes and the Potential to Treat Hemoglobinopathies through Epigenome Editing.

Beta-like globin gene expression is developmentally regulated during life by transcription factors, chromatin looping and epigenome modifications of the ß-globin locus. Epigenome modifications, such as histone methylation/demethylation and acetylation/deacetylation and DNA methylation, are associated with up- or down-regulation of gene expression. The understanding of these mechanisms and their outcome in gene expression has paved the way to the development of new therapeutic strategies for treating various diseases, such as ß-hemoglobinopathies. Histone deacetylase and DNA methyl-transferase inhibitors are currently being tested in clinical trials for hemoglobinopathies patients. However, these approaches are often uncertain, non-specific and their global effect poses serious safety concerns. Epigenome editing is a recently developed and promising tool that consists of a DNA recognition domain (zinc finger, transcription activator-like effector or dead clustered regularly interspaced short palindromic repeats Cas9) fused to the catalytic domain of a chromatin-modifying enzyme. It offers a more specific targeting of disease-related genes (e.g., the ability to reactivate the fetal γ-globin genes and improve the hemoglobinopathy phenotype) and it facilitates the development of scarless gene therapy approaches. Here, we summarize the mechanisms of epigenome regulation of the ß-globin locus, and we discuss the application of epigenome editing for the treatment of hemoglobinopathies.

A Hematopoietic Stem Cell Transplantation Startup in Iraqi Kurdistan: Results in Thalassemia Patients and Analysis of the Methodology.

In hemoglobinopathy-prone regions, like the Middle East, thalassemia is the most prevalent noncommunicable life-threatening disorder of children and is highly curable by hematopoietic stem cell transplantation (HSCT). Moreover, transplantation is very cost-effective, and HSCT programs can be established directly in middle-income countries (MICs) at a reduced cost while maintaining quality standards and outcomes consistent with international ones. The aim of the present study was to review and verify the efficacy of the applied methodology through the analysis of 47 consecutive matched-related HSCTs in children with thalassemia. In 2016, the first HSCT unit for adults and children with both malignant and nonmalignant diseases was developed in Iraqi Kurdistan, thanks to a capacity building project funded by the Italian Agency for Development Cooperation. Data on clinical activity were obtained from a cohort of patients treated in the newly established HSCT unit. Primary endpoints were overall survival (OS) and thalassemia-free survival (TFS). Startup of the HSCT unit was completed over a 3-year period. Assessing and meeting minimum requirements were crucial for the startup; moreover, a team of international health care professionals (HCPs), all experts in the field of HSCT, conducted the education and training phase, involving all the clinical and nonclinical professionals in the program. At a median follow-up of 2.6 years, the 3-year TFS and OS were 82.8% (SE, 5.5%) and 87.1% (SE, 4.9%), respectively. TFS and graft-versus-host-disease-free composite survival was 80.6% (SE, 5.8%). At present, the HSCT service is completely autonomous, and more than 250 transplants have been done in both adults and children. The minimal essential requirements for an HSCT startup may be affordable in many MICs. Our results for thalassemia are comparable with international data. A twinning program with an international group of experts and a capacity-building approach is crucial for the success of the program, a strategy that allows for rapid development of HSCT units.