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Expression of PiT1 and PiT2 retroviral receptors and transduction efficiency of tumor cells*.

100%

Grabarczyk P., Wysocki P. J., Gryska K., Mackiewicz A.

vol. 49

issue 2

333-339

Recombinant retroviral vectors are still the most common gene delivery vehicles for gene therapy purposes, especially for construction of genetically modified tumor vaccines (GMTV). However, these vehicles are characterized by relatively low titre and in the case of many tumor cell lines, low transduction efficiency. We constructed bicistronic retroviral vector pseudotypes of amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GaLV), encoding enhanced green fluorescent protein (EGFP) as a rapid and easily detectable reporter gene. Transduction of five different human melanoma and four renal carcinoma cell lines by these two virus pseudotypes revealed differences in transduction efficiency, which wase markedly lower for the renal carcinoma cell lines. Stimulation of retroviral receptor expression (PiT1 and PiT2) by phosphate depletion induced a limited increase of receptor mRNA levels, but did not improve the gene transfer efficiency. In contrast, simultaneous transduction with both vector pseudotypes markedly increased the transduction efficiency, compared to GaLV or A-MuLV alone. The same effect could be achieved by several repeated exposures of target cells to fresh vector preparation. Overexpression of GaLV receptor (PiT1) in target cells significantly increased the transduction rate and enabled retrovirus mediated gene transfer into the cells which normally are not transducible by GaLV pseudotypes. We demonstrated that, using different transduction strategies, the relatively inefficient, widely used retroviral vector systems could be significantly improved.

Optimization of a retroviral vector for transduction of human CD34 positive cells

64%

Szyda A., Paprocka M., Krawczenko A., Lenart K., Heimrath J., Grabarczyk P., Mackiewicz A., Duś D.

Acta Biochimica Polonica

2006

vol. 53

issue 4

815-823

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°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.

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2 Acta Biochimica Polonica

1 2006

1 2002

2 Grabarczyk P.

2 Mackiewicz A.

1 Duś D.

1 Gryska K.

1 Heimrath J.

1 Krawczenko A.

1 Lenart K.

1 Paprocka M.

1 Szyda A.

1 Wysocki P. J.

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Expression of PiT1 and PiT2 retroviral receptors and transduction efficiency of tumor cells*.

Optimization of a retroviral vector for transduction of human CD34 positive cells