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- Relander, Thomas, et al.
(författare)
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Overexpression of Gibbon Ape Leukemia Virus (GALV) Receptor (GLVR1) on Human CD34(+) Cells Increases Gene Transfer Mediated by GALV Pseudotyped Vectors.
- 2002
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Ingår i: Molecular Therapy. - : Elsevier BV. - 1525-0024 .- 1525-0016. ; 6:3, s. 400-406
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Tidskriftsartikel (refereegranskat)abstract
- Retroviral transduction of CD34(+) cells on Retronectin using gibbon ape leukemia virus (GALV) pseudotyped vectors is inhibited by high concentrations of vector containing medium (VCM). Furthermore, this inhibitory activity is stable for at least 48 hours at 37 degrees C and partially blocks a second hit with a GALV pseudotyped vector. We hypothesized that this inhibition was due to interference at the receptor level between infectious and noninfectious vector particles and that it might be possible to overcome it by increasing receptor expression on target cells. Activation of protein kinase C in CD34(+) cells with the phorbol ester PMA (phorbol 12-myristate 13-acetate) increased the mRNA level of the GALV receptor (GLVR1) and the transduction efficiency (TE), and fully reversed the inhibition of transduction seen with high-titer GALV VCM. A murine stem cell virus (MSCV) vector with the GLVR1 receptor and green fluorescent protein cDNAs (MGLIG) was used to transduce fibroblasts, and clones expressing different levels of GLVR1 were isolated. The TE of these cells using a GALV vector correlated with the level of GLVR1 expression. When CD34(+) cells or K562 cells were first transduced with MGLIG and then with high-titer GALV VCM, no inhibition of transduction was seen. The low level of GLVR1 expression limits gene transfer to K562 and CD34(+) cells using GALV pseudotyped vectors, especially in the presence of high-titer VCMs.
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| 2. |
- Relander, Thomas, et al.
(författare)
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Retroviral transduction of human CD34+ cells on fibronectin fragment CH-296 is inhibited by high concentrations of vector containing medium
- 2001
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Ingår i: Journal of Gene Medicine. - 1521-2254. ; 3:3, s. 207-218
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The objective of the present study was to optimize conditions for retroviral transduction of human cord blood (CB) CD34+ cells and to reveal mechanisms which interfere with efficient gene transfer. METHODS: An MSCV based retroviral vector with the gene for enhanced green fluorescent protein (MGIN) produced by GP+envAM12 (amphotropic envelope), PG13 (gibbon ape leukemia virus envelope) or 293GPG (vesicular stomatitis virus envelope) cell lines was used to transduce cord blood CD34+ cells on Retronectin (fibronectin fragment CH-296) in three different ways: either in vector containing medium (VCM), in fresh medium on Retronectin pre-loaded with vector or in VCM on Retronectin pre-loaded with vector. RESULTS: Paradoxically, the transduction efficiency obtained with pre-load of vector onto Retronectin alone was higher than pre-load plus VCM for PG13-MGIN (67.9 +/- 6.0% vs 24.9 +/- 8.0%) and AM12-MGIN (47.5 +/- 5.8% vs 38.7 +/- 2.2%). Further experiments showed that transduction on Retronectin pre-loaded with PG13-MGIN or AM12-MGIN was inhibited by the presence of the same VCM at high concentrations, but not by the presence of a VCM with a different receptor specificity. If no pre-load of vector was performed, the highest transduction efficiencies were seen when VCMs were diluted 1:10 (MOIs of 3). The inhibitory effect of high titer PG13-MGIN VCM was confirmed in more primitive CD34+CD38low cells and in NOD/SCID repopulating cells, and was also seen in experiments with bone marrow CD34+ cells. CONCLUSIONS: Retroviral transduction of CB CD34+ cells on Retronectin is inhibited by high titer PG13 and GP+envAM12 vector containing medium. Efficient gene transfer to human hematopoietic cells can be obtained by preload alone of the vector onto Retronectin. These findings are of importance for the design of transduction protocols for repopulating hematopoietic cells.
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