Long-term Correction of β-Thalassemia With Minimal Cellular Requirement and Transplantation Modalities

Determination of minimal criteria, pre-transplantation regimens, and infusion modalities for effective and reproducible bone marrow (BM) therapy in β-thalassemia is of fundamental importance for clinical application. In this study, using repopulation assays, we first established the minimal proportion of normal BM stem cells that would result in therapeutic benefit in this red blood cell (RBC) disorder. Eight groups of stable chimeric hemizygous β-thalassemic (hemi-βthal) mice (10–89%) were systematically subjected to cellular, molecular, and patho-physiologic analyses for ∼2 years. In the chimeric hemi-βthal groups containing 19–24% normal donor cells, all RBC parameters and consequent erythropoiesis were significantly improved. Mice in the 24% chimeric group and above had marked reduction in organ pathology including iron deposits, and survived to a normal lifespan. Altogether, these results established that a range of 19–24% normal BM cells is sufficient for long-term significant correction of the hemi-βthal phenotype. We also determined concomitantly the minimal myelosuppression radiation doses, the number of cells to be infused, and the number of infusions required in order to attain this therapeutic range in hemi-βthal mice. Importantly, with prior minimal myelosuppression with 1 or 2 Gy, and using cell doses of 40 or 60 millions, 100% of the recipients were successfully engrafted at therapeutic levels, provided the cells were administered in two doses. This study has therefore determined the therapeutic chimeric level as 19–24% of normal cells, and has also defined the minimal transplantation modalities necessary for the stable and successful correction of the hemi-βthal phenotype. Determination of minimal criteria, pre-transplantation regimens, and infusion modalities for effective and reproducible bone marrow (BM) therapy in β-thalassemia is of fundamental importance for clinical application. In this study, using repopulation assays, we first established the minimal proportion of normal BM stem cells that would result in therapeutic benefit in this red blood cell (RBC) disorder. Eight groups of stable chimeric hemizygous β-thalassemic (hemi-βthal) mice (10–89%) were systematically subjected to cellular, molecular, and patho-physiologic analyses for ∼2 years. In the chimeric hemi-βthal groups containing 19–24% normal donor cells, all RBC parameters and consequent erythropoiesis were significantly improved. Mice in the 24% chimeric group and above had marked reduction in organ pathology including iron deposits, and survived to a normal lifespan. Altogether, these results established that a range of 19–24% normal BM cells is sufficient for long-term significant correction of the hemi-βthal phenotype. We also determined concomitantly the minimal myelosuppression radiation doses, the number of cells to be infused, and the number of infusions required in order to attain this therapeutic range in hemi-βthal mice. Importantly, with prior minimal myelosuppression with 1 or 2 Gy, and using cell doses of 40 or 60 millions, 100% of the recipients were successfully engrafted at therapeutic levels, provided the cells were administered in two doses. This study has therefore determined the therapeutic chimeric level as 19–24% of normal cells, and has also defined the minimal transplantation modalities necessary for the stable and successful correction of the hemi-βthal phenotype. Introductionβ-thalassemias are among the most frequently occurring human monogenic diseases, leading to high morbidity and mortality. This disorder is characterized by an abnormal level of hemoglobin (Hb) (α2β2) production due to decreased levels or complete absence of β-globin chain synthesis.In humans, the degree of imbalance in the ratio of β-globin chain to α-globin chain in red blood cells (RBCs) appears to be directly linked to the severity of β-thalassemia. The presence of excess unpaired α-globin chains in RBCs gives rise to aggregates or Heinz bodies, and morphologic changes including hypochromia, microcytosis, poikilocytosis, and anisocytosis, resulting in osmotic fragility, shortened RBC half-life, and anemia. This imbalance also hinders erythroid precursor maturation and promotes precursor destruction, rendering erythropoiesis ineffective. As a consequence of this primary defect, patients with severe β-thalassemia display general systemic alterations: significant levels of iron accumulation in multiple organs and tissues (due to erythroid cell destruction), massive erythroid hyperplasia, and extramedullary hematopoiesis to compensate the RBC loss, in addition to various pathologies mainly affecting spleen, kidneys, and liver.Individuals with severe thalassemia are dependent on repeated transfusions. However, this therapy causes additional iron overload in several organs and becomes life-threatening in the absence of continuous iron chelation. On the other hand, hematopoietic stem cell transfer by bone marrow transplantation (BMT), either from allogeneic sources or autologous following genetic correction, is potentially curative for thalassemia. Allogeneic BMT treatment requires suitable histocompatible donors. Even with a compatible donor, the patient is vulnerable to graft-versus-host disease, with the risk of early mortality.1Giardini C Galimberti M Lucarelli G Bone marrow transplantation in thalassemia.Annu Rev Med. 1995; 46: 319-330Crossref PubMed Scopus (18) Google Scholar,2La Nasa G Giardini C Argiolu F Locatelli F Arras M De Stefano P et al.Unrelated donor bone marrow transplantation for thalassemia: the effect of extended haplotypes.Blood. 2002; 99: 4350-4356Crossref PubMed Scopus (120) Google Scholar In principle, autologous BMT could prevent these complications. However, success in autologous BMT depends on the ability to achieve persistent therapeutic gene expression stable with a In BMT that complete bone marrow (BM) is necessary in order to thalassemia. 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Introductionβ-thalassemias are among the most frequently occurring human monogenic diseases, leading to high morbidity and mortality. This disorder is characterized by an abnormal level of hemoglobin (Hb) (α2β2) production due to decreased levels or complete absence of β-globin chain synthesis.In humans, the degree of imbalance in the ratio of β-globin chain to α-globin chain in red blood cells (RBCs) appears to be directly linked to the severity of β-thalassemia. The presence of excess unpaired α-globin chains in RBCs gives rise to aggregates or Heinz bodies, and morphologic changes including hypochromia, microcytosis, poikilocytosis, and anisocytosis, resulting in osmotic fragility, shortened RBC half-life, and anemia. This imbalance also hinders erythroid precursor maturation and promotes precursor destruction, rendering erythropoiesis ineffective. As a consequence of this primary defect, patients with severe β-thalassemia display general systemic alterations: significant levels of iron accumulation in multiple organs and tissues (due to erythroid cell destruction), massive erythroid hyperplasia, and extramedullary hematopoiesis to compensate the RBC loss, in addition to various pathologies mainly affecting spleen, kidneys, and liver.Individuals with severe thalassemia are dependent on repeated transfusions. However, this therapy causes additional iron overload in several organs and becomes life-threatening in the absence of continuous iron chelation. On the other hand, hematopoietic stem cell transfer by bone marrow transplantation (BMT), either from allogeneic sources or autologous following genetic correction, is potentially curative for thalassemia. Allogeneic BMT treatment requires suitable histocompatible donors. Even with a compatible donor, the patient is vulnerable to graft-versus-host disease, with the risk of early mortality.1Giardini C Galimberti M Lucarelli G Bone marrow transplantation in thalassemia.Annu Rev Med. 1995; 46: 319-330Crossref PubMed Scopus (18) Google Scholar,2La Nasa G Giardini C Argiolu F Locatelli F Arras M De Stefano P et al.Unrelated donor bone marrow transplantation for thalassemia: the effect of extended haplotypes.Blood. 2002; 99: 4350-4356Crossref PubMed Scopus (120) Google Scholar In principle, autologous BMT could prevent these complications. However, success in autologous BMT depends on the ability to achieve persistent therapeutic gene expression stable with a In BMT that complete bone marrow (BM) is necessary in order to thalassemia. However, of the of patients with stable BM G M et of β-globin stable in patients with thalassemia bone marrow 1995; Google M Lucarelli G P et of patients with persistent bone marrow PubMed Scopus Google Scholar the for on this hematopoietic on the of to achieve In order to the of autologous or allogeneic BMT and a of the of cells to be of the regimens, and the of transfer of the cells to the patients to be a or of β-thalassemia P thalassemia of the β-globin PubMed Scopus Google for PubMed Scopus Google for 1995; PubMed Scopus Google of human of the and in stem 1995; PubMed Scopus Google Scholar and most of these for of gene therapy et and correction of β-thalassemia by multiple in hematopoietic stem 2002; 99: PubMed Scopus Google C G et in β-thalassemic mice human PubMed Scopus Google C M treatment of β-thalassemia by transfer of the human β-globin 2002; 99: PubMed Scopus Google The degree of correction of β-thalassemia following transfer of a human gene is by and PubMed Scopus Google C M of and by human β-globin gene PubMed Scopus Google Scholar these on the importance of to the cells on the minimal level of cells necessary for long-term correction of the from the β-globin as hemizygous β-thalassemic (hemi-βthal) is the most for with that severe thalassemia of human of the and in stem 1995; PubMed Scopus Google Scholar of the of hematopoiesis and erythropoiesis in hemi-βthal mice of hematopoietic cells and early erythroid consequent to the shortened RBC and M and in in and β-globin hemizygous PubMed Scopus Google Scholar are to a for normal or RBCs the hemi-βthal we a and long-term study of the number of cells to be of the regimens, and modalities of The results from a repopulation established the minimal level of normal donor cells at 19–24% normal RBC to and leading to significant long-term in order to attain the required of as as stable and reproducible for correction of the we determined the minimal of and the effect of the of the of gene therapy BMT in human β-thalassemia.

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