ß-Globin sleeping beauty transposon reduces red blood cell sickling in a patient-derived CD34(+)-based in vitro model.

Sjeklocha LM, Wong PY, Belcher JD, Vercellotti GM, Steer CJ
Source: PLoS ONE
Publication Date: (2013)
Issue: 8(11): e80403
Research Area:
Immunotherapy / Hematology
Basic Research
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
4D-Nucleofector™ X-Unit
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-ß(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-ß(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.