Gene transfer and cellular therapies for the cure of ß-thalassemia and sickle cell anemia
Preclinical and clinical studies demonstrate that it is possible to treat ß-thalassemia and sickle cell anemia by lentiviral-mediated transfer of the human ß-globin gene. However, all these studies did not address whether lentiviral vectors will correct the synthesis of hemoglobin in erythroid cells irrespective of the different mutations and amounts of endogenous hemoglobin produced. Therefore, clinical trials would greatly benefit from a simple analysis using progenitor erythroid cells to determine the relationship between the number of copies of the lentiviral vector integrated and the total amount of hemoglobins produced. This approach would provide an efficient way to predict the outcome of the gene transfer before the patients undergo a partial or total myeloablation and bone marrow transplant. We have constructed novel lentiviral gene delivery systems that carry the human ß-globin gene and activated fetal hemoglobin expression. We demonstrated that these vectors can achieve long-term correction of mice affected by hemoglobinopathies. We also characterized these new lentiviral vectors in ß-thalassemic and sickle cell anemia patient cells in vitro as a preclinical test for potential gene therapy trial, showing correction of the hemoglobin synthesis defect and phenotype. Our current goals are to devise new strategies to control and increase the number of hematopoietic stem cells transduced with our vectors in vitro, before the cells are infused into the bone marrow of the patients.
Recent advances in stem cell biology have demonstrated that it is possible to derive induced pluripotent stem cells (iPSCs) from thalassemic or sickle cell disease (SCD) human somatic cells. Furthermore, homologous recombination experiments demonstrated that it is possible to correct the mutated ß-globin gene in iPSCs derived from patients. In other words, it is now possible to reprogram the patient’s own blood cells into stem cells and correct their ß-globin gene. The ultimate goal would be to engraft patients with these corrected iPSC-derived bone marrow stem cells and generate healthy red cells. However, generating corrected iPSCs using the current technologies entails several potential drawbacks, including potential disruption of oncogenes and tumor suppressors, and further manipulation of the iPSC clones to correct mutations in the ß-globin gene. Unfortunately that means that we could theoretically cure the SCD and ß-thalassemia but might cause the patients to develop cancer or other diseases. We propose a novel, single-step approach to generate corrected iPSCs that overcomes all of these issues. Furthermore, in order to simplify this procedure, we established a protocol to expand blood progenitor cells from a small amount of blood (~30 mL) to generate curative iPSCs. Additional studies are undergoing to generate hematopoietic stem cells from iPSCs with the ability to engraft and repopulate the hematopoietic compartment.
Ramos P, Casu C, Gardenghi S, Breda L, Crielaard BJ, Guy E, Marongiu MF, Gupta R, Ghaffari S, Levine RL, Abdel-Wahab O, Ebert BL, Van Rooijen N, Ghaffari S, Grady RW, Giardina PJ and Rivella S. "Macrophages support pathological erythropoiesis in Polycythemia Vera and Beta-Thalassemia", Nature Medicine. March 17, 2013.
Guo S, Casu C, Gardenghi S, Booten S, Watt A, Freier S, Monia BP and Rivella S. "Reducing TMPRSS6 ameliorates hemochromatosis and beta-thalassemia in mice". Journal of Clinical Investigation. 2013 Mar 25.
Casanovas G, Vujiç Spasiç M, Casu C, Rivella S, Strelau J, Unsicker K, Muckenthaler MU. "The murine growth differentiation factor 15 is not essential for systemic iron homeostasis in phlebotomized mice". Haematologica. 2012 Sep 14.
Breda L, Casu C, Gardenghi S, Bianchi N, Cartegni L, Narla M, Yazdanbakhsh K, Musso M, Manwani D, Little J, Gardner LB, Kleinert DA, Prus E, Fibach E, Grady RW, Giardina PJ, Gambari R, Rivella S. "Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients". PLoS One. 2012;7(3):e32345.
Parrow NL, Gardenghi S, Ramos P, Casu C, Grady RW, Anderson ER, Shah YM, Li H, Ginzburg YZ, Fleming RE, Rivella S. "Decreased hepcidin expression in murine β-thalassemia is associated with suppression of Bmp/Smad signaling". Blood. 2012 Mar 29;119(13):3187-9.
Kobayashi H, Gilbert V, Liu Q, Kapitsinou PP, Unger TL, Rha J, Rivella S, Schlöndorff D, Haase VH. "Myeloid Cell-Derived Hypoxia-Inducible Factor Attenuates Inflammation in Unilateral Ureteral Obstruction-Induced Kidney Injury". Journal of Immunology. 2012 Apr 6.
Zuccato C, Breda L, Salvatori F, Breveglieri G, Gardenghi S, Bianchi N, Brognara E, Lampronti I, Borgatti M, Rivella S, Gambari R. "A combined approach for β-thalassemia based on gene therapy-mediated adult hemoglobin (HbA) production and fetal hemoglobin (HbF) induction". Ann Hematol. 2012 Mar 31.
Wang L, Rosenberg J, De B, Ferris B, Wang R, Rivella S, Kaminsky S, Crystal R. "In Vivo Gene Transfer Strategies to Achieve Partial Correction of Von Willebrand Disease". Hum Gene Ther. 2012 Apr 6.