Gene Therapy for Hemoglobinopathies.

β-Thalassemia and sickle cell disease are autosomal recessive disorders of red blood cells due to mutations in the adult β-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.

Inhibition of FGF23 is a therapeutic strategy to target hematopoietic stem cell niche defects in β-thalassemia.

Clinical evidence highlights a relationship between the blood and the bone, but the underlying mechanism linking these two tissues is not fully elucidated. Here, we used β-thalassemia as a model of congenital anemia with bone and bone marrow (BM) niche defects. We demonstrate that fibroblast growth factor 23 (FGF23) is increased in patients and mice with β-thalassemia because erythropoietin induces FGF23 overproduction in bone and BM erythroid cells via ERK1/2 and STAT5 pathways.

Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy.

The growing clinical success of hematopoietic stem/progenitor cell (HSPC) gene therapy (GT) relies on the development of viral vectors as portable "Trojan horses" for safe and efficient gene transfer. The recent advent of novel technologies enabling site-specific gene editing is broadening the scope and means of GT, paving the way to more precise genetic engineering and expanding the spectrum of diseases amenable to HSPC-GT. Here, we provide an overview of state-of-the-art and prospective developments of the HSPC-GT field, highlighting how advances in biological characterization and manipulation of HSPCs will enable the design of the next generation of these transforming therapeutics.

A single approach to targeting transferrin receptor 2 corrects iron and erythropoietic defects in murine models of anemia of inflammation and chronic kidney disease.

Anemia is a common complication of systemic inflammation. Proinflammatory cytokines both decrease erythroblast sensitivity to erythropoietin (EPO) and increase the levels of the hepatic hormone hepcidin, sequestering iron in stores and causing functional iron deficiency. Anemia of chronic kidney disease (CKD) is a peculiar form of anemia of inflammation, characterized by impaired EPO production paralleling progressive kidney damage.

Adenine base editor-mediated correction of the common and severe IVS1-110 (G>A) β-thalassemia mutation.

β-Thalassemia (BT) is one of the most common genetic diseases worldwide and is caused by mutations affecting β-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically-modified HSPCs is an attractive therapeutic option.

Transferrin receptor 2 (Tfr2) genetic deletion makes transfusion-independent a murine model of transfusion-dependent β-thalassemia.

β-thalassemia is a genetic disorder caused by mutations in the β-globin gene, and characterized by anemia, ineffective erythropoiesis and iron overload. Patients affected by the most severe transfusion-dependent form of the disease (TDT) require lifelong blood transfusions and iron chelation therapy, a symptomatic treatment associated with several complications. Other therapeutic opportunities are available, but none is fully effective and/or applicable to all patients, calling for the identification of novel strategies.

Community voices: sowing, germinating, flourishing as strategies to support inclusion in STEM.

Understanding gaps in academic representation while considering the intersectionality concept is paramount to promoting real progress towards a more inclusive STEM. Here we discuss ways in which STEM careers can be sown and germinated so that inclusivity can flourish.

Targeting the Hematopoietic Stem Cell Niche in β-Thalassemia and Sickle Cell Disease.

In the last decade, research on pathophysiology and therapeutic solutions for β-thalassemia (BThal) and sickle cell disease (SCD) has been mostly focused on the primary erythroid defect, thus neglecting the study of hematopoietic stem cells (HSCs) and bone marrow (BM) microenvironment. The quality and engraftment of HSCs depend on the BM microenvironment, influencing the outcome of HSC transplantation (HSCT) both in allogeneic and in autologous gene therapy settings. In BThal and SCD, the consequences of severe anemia alter erythropoiesis and cause chronic stress in different organs, including the BM.

Unique molecular and functional features of extramedullary hematopoietic stem and progenitor cell reservoirs in humans.

Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays.

Correction of β-thalassemia by CRISPR/Cas9 editing of the α-globin locus in human hematopoietic stem cells.

β-thalassemias (β-thal) are a group of blood disorders caused by mutations in the β-globin gene (HBB) cluster. β-globin associates with α-globin to form adult hemoglobin (HbA, α2β2), the main oxygen-carrier in erythrocytes. When β-globin chains are absent or limiting, free α-globins precipitate and damage cell membranes, causing hemolysis and ineffective erythropoiesis.

Gene therapy using haematopoietic stem and progenitor cells.

Haematopoietic stem and progenitor cell (HSPC) gene therapy has emerged as an effective treatment modality for monogenic disorders of the blood system such as primary immunodeficiencies and β-thalassaemia. Medicinal products based on autologous HSPCs corrected using lentiviral and gammaretroviral vectors have now been approved for clinical use, and the site-specific genome modification of HSPCs using gene editing techniques such as CRISPR-Cas9 has shown great clinical promise. Preclinical studies have shown engineered HSPCs could also be used to cross-correct non-haematopoietic cells in neurodegenerative metabolic diseases.

Conditioning Regimens in Long-Term Pre-Clinical Studies to Support Development of Gene Therapy: Review of Nonproliferative and Proliferative Changes.

Hematopoietic stem cell gene therapy has become a successful therapeutic strategy for some inherited genetic disorders. Pre-clinical toxicity studies performed to support the human clinical trials using viral-mediated gene transfer and autologous hematopoietic stem and progenitor cell (HSPC) transplantation are complex and the use of mouse models of human diseases makes interpretation of the results challenging. In addition, they rely on the use of conditioning agents that must induce enough myeloablation to allow engraftment of transduced and transplanted HSPC.

Correcting β-thalassemia by combined therapies that restrict iron and modulate erythropoietin activity.

β-Thalassemia intermedia is a disorder characterized by ineffective erythropoiesis (IE), anemia, splenomegaly, and systemic iron overload. Novel approaches are being explored based on the modulation of pathways that reduce iron absorption (ie, using hepcidin activators like Tmprss6-antisense oligonucleotides [ASOs]) or increase erythropoiesis (by erythropoietin [EPO] administration or modulating the ability of transferrin receptor 2 [Tfr2] to control red blood cell [RBC] synthesis). Targeting Tmprss6 messenger RNA by Tmprss6-ASO was proven to be effective in improving IE and splenomegaly by inducing iron restriction.

Hematopoietic stem cell function in β-thalassemia is impaired and is rescued by targeting the bone marrow niche.

Hematopoietic stem cells (HSCs) are regulated by signals from the bone marrow (BM) niche that tune hematopoiesis at steady state and in hematologic disorders. To understand HSC-niche interactions in altered nonmalignant homeostasis, we selected β-thalassemia, a hemoglobin disorder, as a paradigm. In this severe congenital anemia, alterations secondary to the primary hemoglobin defect have a potential impact on HSC-niche cross talk.

NCOA4-mediated ferritinophagy in macrophages is crucial to sustain erythropoiesis in mice.

Nuclear receptor coactivator 4 (NCOA4) promotes ferritin degradation and Ncoa4-ko mice in a C57BL/6 background show microcytosis and mild anemia, aggravated by iron deficiency. To understand tissue-specific contributions of NCOA4-mediated ferritinophagy we explored the effect of Ncoa4 genetic ablation in the iron-rich Sv129/J strain. Increased body iron content protects these mice from anemia and, in basal conditions, Sv129/J Ncoa4-ko mice show only microcytosis; nevertheless, when fed a low-iron diet they develop a more severe anemia compared to that of wild-type animals.

Bone marrow stromal cells from β-thalassemia patients have impaired hematopoietic supportive capacity.

Background: The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. β-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs.

Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia.

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (β) or absent (β) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%.

Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-Thalassemia.

Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for β-thalassemia, we performed and studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes.

Transferrin receptor 2 is a potential novel therapeutic target for β-thalassemia: evidence from a murine model.

β-thalassemias are genetic disorders characterized by anemia, ineffective erythropoiesis, and iron overload. Current treatment of severe cases is based on blood transfusion and iron chelation or allogeneic bone marrow (BM) transplantation. Novel approaches are explored for nontransfusion-dependent patients (thalassemia intermedia) who develop anemia and iron overload.

Gene therapy and gene editing strategies for hemoglobinopathies.

Gene therapy for hemoglobinopathies is currently based on transplantation of autologous hematopoietic stem cells genetically modified with an integrating lentiviral vector expressing a globin gene under the control of globin transcriptional regulatory elements. Studies and safety works demonstrated the potential therapeutic efficacy and safety of this approach, providing the rationale for clinical translation. The outcomes of early clinical trials, although showing promising results, have highlighted the current limitations to a more general application.