β-globin sleeping beauty transposon reduces red blood cell sickling in a patient-derived CD34+-based in vitro model

Lucas M. Sjeklocha, Phillip Y.P. Wong, John D Belcher, Gregory M Vercellotti, Clifford J Steer

Research output: Contribution to journalArticle

11 Citations (Scopus)

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-βT87Q 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-βT87Q- 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.

Original languageEnglish (US)
Article numbere80403
JournalPloS one
Volume8
Issue number11
DOIs
StatePublished - Nov 18 2013

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Beauty
Gene therapy
Globins
transposons
Sickle Hemoglobin
Blood
erythrocytes
Erythrocytes
Cells
Bone
gene therapy
Imaging techniques
Genetic Therapy
sickle cell anemia
cells
Sickle Cell Anemia
Microscopic examination
bone marrow
Genes
Polymerization

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β-globin sleeping beauty transposon reduces red blood cell sickling in a patient-derived CD34+-based in vitro model. / Sjeklocha, Lucas M.; Wong, Phillip Y.P.; Belcher, John D; Vercellotti, Gregory M; Steer, Clifford J.

In: PloS one, Vol. 8, No. 11, e80403, 18.11.2013.

Research output: Contribution to journalArticle

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