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Retroviral transduction of human CD34+ cells on fibronectin fragment CH-296 is inhibited by high concentrations of vector containing medium

Relander, Thomas LU ; Brun, Ann LU ; Hawley, Robert G.; Karlsson, Stefan LU and Richter, Johan LU (2001) In Journal of Gene Medicine 3(3). p.207-218
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
CD34+, retroviral, gene transfer, cord blood, NOD/SCID
in
Journal of Gene Medicine
volume
3
issue
3
pages
207 - 218
publisher
John Wiley & Sons
external identifiers
  • pmid:11437326
  • scopus:0035351273
  • wos:000169271800002
ISSN
1521-2254
DOI
10.1002/1521-2254(200105/06)3:3<207::AID-JGM183>3.0.CO;2-O
language
English
LU publication?
yes
id
9f53ca22-32e8-4b6f-b5b9-646e2be820da (old id 1120243)
date added to LUP
2008-07-10 11:08:38
date last changed
2018-01-09 12:54:43
@article{9f53ca22-32e8-4b6f-b5b9-646e2be820da,
  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.},
  author       = {Relander, Thomas and Brun, Ann and Hawley, Robert G. and Karlsson, Stefan and Richter, Johan},
  issn         = {1521-2254},
  keyword      = {CD34+,retroviral,gene transfer,cord blood,NOD/SCID},
  language     = {eng},
  number       = {3},
  pages        = {207--218},
  publisher    = {John Wiley & Sons},
  series       = {Journal of Gene Medicine},
  title        = {Retroviral transduction of human CD34+ cells on fibronectin fragment CH-296 is inhibited by high concentrations of vector containing medium},
  url          = {http://dx.doi.org/10.1002/1521-2254(200105/06)3:3<207::AID-JGM183>3.0.CO;2-O},
  volume       = {3},
  year         = {2001},
}