domingo, 8 de abril de 2012

PLoS ONE: Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

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PLoS ONE: Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Therapeutic Hemoglobin Levels after Gene Transfer in β-Thalassemia Mice and in Hematopoietic Cells of β-Thalassemia and Sickle Cells Disease Patients

Laura Breda1, Carla Casu1, Sara Gardenghi1, Nicoletta Bianchi2, Luca Cartegni3, Mohandas Narla4, Karina Yazdanbakhsh4, Marco Musso5, Deepa Manwani6, Jane Little7, Lawrence B. Gardner8, Dorothy A. Kleinert1, Eugenia Prus9, Eitan Fibach9, Robert W. Grady1, Patricia J. Giardina1, Roberto Gambari2, Stefano Rivella1,10*
1 Department of Pediatrics, Division of Hematology-Oncology, Children's Blood and Cancer Foundation Laboratories, Weill Cornell Medical College, New York, New York, United States of America, 2 Dipartimento di Biochimica e Biologia Molecolare, Universita' di Ferrara, Ferrara, Italy, 3 Department of Molecular Pharmacology and Chemistry, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America, 4 Red Cell Physiology Laboratory, New York Blood Center, New York, New York, United States of America, 5 Centro della Microcitemia e Anemie Congenite, Ospedali Galliera, Genova, Italy, 6 Department of Pediatrics, Albert Einstein College of Medicine, Children's Hospital at Montefiore, Bronx, New York, United States of America, 7 Department of Medicine, Montefiore Medical Center, Bronx, New York, United States of America, 8 Department of Pharmacology, Langone Medical Center, New York University, New York, New York, United States of America, 9 Department of Hematology, Hadassah–Hebrew University Medical Center, Jerusalem, Israel, 10 Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, New York, United States of America

Abstract 

Preclinical and clinical studies demonstrate the feasibility of treating β-thalassemia and Sickle Cell Disease (SCD) by lentiviral-mediated transfer of the human β-globin gene. However, previous studies have not addressed whether the ability of lentiviral vectors to increase hemoglobin synthesis might vary in different patients.
We generated lentiviral vectors carrying the human β-globin gene with and without an ankyrin insulator and compared their ability to induce hemoglobin synthesis in vitro and in thalassemic mice. We found that insertion of an ankyrin insulator leads to higher, potentially therapeutic levels of human β-globin through a novel mechanism that links the rate of transcription of the transgenic β-globin mRNA during erythroid differentiation with polysomal binding and efficient translation, as reported here for the first time. We also established a preclinical assay to test the ability of this novel vector to synthesize adult hemoglobin in erythroid precursors and in CD34+ cells isolated from patients affected by β-thalassemia and SCD. Among the thalassemic patients, we identified a subset of specimens in which hemoglobin production can be achieved using fewer copies of the vector integrated than in others. In SCD specimens the treatment with AnkT9W ameliorates erythropoiesis by increasing adult hemoglobin (Hb A) and concurrently reducing the sickling tetramer (Hb S).
Our results suggest two major findings. First, we discovered that for the purpose of expressing the β-globin gene the ankyrin element is particularly suitable. Second, our analysis of a large group of specimens from β-thalassemic and SCD patients indicates that clinical trials could benefit from a simple test to predict the relationship between the number of vector copies integrated and the total amount of hemoglobin produced in the erythroid cells of prospective patients. This approach would provide vital information to select the best candidates for these clinical trials, before patients undergo myeloablation and bone marrow transplant.

 

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