title: β-Globin sleeping beauty transposon reduces red blood cell sickling in a patient-derived CD34(+)-based in vitro model.
abstract: The ultimate goal of gene therapy for sickle cell anemia (SCA) is an improved phenotype for the patient. In this study, we utilized bone marrow from a sickle cell patient as a model of disease in an in vitro setting for the hyperactive Sleeping Beauty transposon gene therapy system. We demonstrated that mature sickle red blood cells containing hemoglobin-S and sickling in response to metabisulfite can be generated in vitro from SCA bone marrow. These cells showed the characteristic morphology and kinetics of hemoglobin-S polymerization, which we quantified using video microscopy and imaging cytometry. Using video assessment, we showed that delivery of an IHK-β(T87Q) antisickling globin gene by Sleeping Beauty via nucleofection improves metrics of sickling, decreasing percent sickled from 53.2 ± 2.2% to 43.9 ± 2.0%, increasing the median time to sickling from 8.5 to 9.6 min and decreasing the maximum rate of sickling from 2.3 x 10(-3) sickling cells/total cells/sec in controls to 1.26 x 10(-3) sickling cells/total cells/sec in the IHK-β(T87Q)-globin group (p < 0.001). Using imaging cytometry, the percentage of elongated sickled cells decreased from 34.8 ± 4.5% to 29.5 ± 3.0% in control versus treated (p < 0.05). These results support the potential use of Sleeping Beauty as a clinical gene therapy vector and provide a useful tool for studying sickle red blood cells in vitro.
authors.affiliation: Department of Medicine, Division of Gastroenterology, Hepatology and Nutrition, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.
authors.forename: Lucas M
authors.initials: LM
authors.lastname: Sjeklocha
authors.forename: Phillip Y-P
authors.initials: PY
authors.lastname: Wong
authors.forename: John D
authors.initials: JD
authors.lastname: Belcher
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Vercellotti
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Steer
mesh_terms: D000755:Anemia, Sickle Cell / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D018952:Antigens, CD34 / Q000378:metabolism*; D001853:Bone Marrow / Q000378:metabolism / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D004912:Erythrocytes / Q000378:metabolism* / Q000502:physiology*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006451:Hemoglobin, Sickle / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019076:Transgenes / Q000235:genetics; D055544:beta-Globins / Q000235:genetics*
chemical_list: D018952:Antigens, CD34; D004251:DNA Transposable Elements; D006451:Hemoglobin, Sickle; D055544:beta-Globins