Optimization of a retroviral vector for transduction of human CD34 positive cells.
Human stem and progenitor cells have recently become objects of intensive studies as an important target for gene therapy and regenerative medicine. Retroviral vectors are among the most effective tools for genetic modification of these cells. However, their transduction efficiency strongly depends on the choice of the ex vivo transduction system. The aim of this study was to elaborate a system for retroviral vector transduction of human CD34 positive cells isolated from cord blood. The retroviral vector pMINV EGFP was chosen for transduction of two human erythroblastoid cell lines: KG-1a (CD34 positive) and K562 (CD34 negative). For vector construction, three promoters and two retroviral vector packaging cell lines were used. To optimize the physicochemical conditions of the transduction process, different temperatures of supernatant harvesting, the influence of centrifugation and the presence of transduction enhancing agents were tested. The conditions elaborated with KG-1a cells were further applied for transduction of CD34 positive cells isolated from cord blood. The optimal efficiency of transduction of CD34 positive cells with pMINV EGFP retroviral vector (26% of EGFP positive cells), was obtained using infective vector with LTR retroviral promoter, produced by TE FLY GA MINV EGFP packaging cell line. The transduction was performed in the presence of serum, at 37 degrees C, with co-centrifugation of cells with viral supernatants and the use of transduction enhancing agents. This study confirmed that for gene transfer into CD34 positive cells, the detailed optimization of each element of the transduction process is of great importance.
Szyda
Anna
A
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland.
Paprocka
Maria
M
Krawczenko
Agnieszka
A
Lenart
Katarzyna
K
Heimrath
Jerzy
J
Grabarczyk
Piotr
P
Mackiewicz
Andrzej
A
Duś
Danuta
D
D018952:Antigens, CD34; D005312:Fetal Blood / Q000166:cytology; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D008722:Methods; D011401:Promoter Regions, Genetic; D012190:Retroviridae; D014161:Transduction, Genetic / Q000379:methods*
D018952:Antigens, CD34