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Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes

Relander, Thomas LU ; Karlsson, Stefan LU and Richter, Johan LU (2002) In Journal of Gene Medicine 4(2). p.122-132
Abstract
Background The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34(+)/CD38(low) and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes. Methods CB cells were transduced on Retronectin using an MSCV-based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13-MGIN (GALV) 293GPG-MGIN (VSV-G) or AM12-MGIN (amphotropic). Results Sorted CD34(+)/CD38(low) cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP-positive cells was 61.8+/-6.6% (PG13-MGIN), 26.9+/-3.5% (293GPG-MGIN), and 39.3+/-4.8% (AM12-MGIN). For... (More)
Background The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34(+)/CD38(low) and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes. Methods CB cells were transduced on Retronectin using an MSCV-based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13-MGIN (GALV) 293GPG-MGIN (VSV-G) or AM12-MGIN (amphotropic). Results Sorted CD34(+)/CD38(low) cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP-positive cells was 61.8+/-6.6% (PG13-MGIN), 26.9+/-3.5% (293GPG-MGIN), and 39.3+/-4.8% (AM12-MGIN). For transplantation experiments, CD34(+) cells were prestimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5 x 10(5) cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum-free medium and transduction of CD34(+) cells using VSV-G-pseudotyped vectors under serum-free conditions was very inefficient. In contrast, transduction with PG13-MGIN under serum-free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3+/-6.6%), and efficient gene transfer to SRCs (46.2+/-4.8%). Conclusions The best conditions for transduction and engraftment of CB SRCs were obtained with GALV-pseudo typed vectors using serum-free conditions. Copyright (C) 2002 John Wiley Sons, Ltd. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
keywords
VSV-G, GALV, gene transfer, CD34+, NOD/SCID, amphotropic
in
Journal of Gene Medicine
volume
4
issue
2
pages
122 - 132
publisher
John Wiley & Sons
external identifiers
  • wos:000175071000002
  • scopus:0036518338
ISSN
1521-2254
DOI
10.1002/jgm.246
language
English
LU publication?
yes
id
23fee20f-2f42-4257-bac3-0ae21e2d8ef3 (old id 339927)
date added to LUP
2007-11-12 10:35:38
date last changed
2017-01-01 05:00:31
@article{23fee20f-2f42-4257-bac3-0ae21e2d8ef3,
  abstract     = {Background The aim of this study was to investigate gene transfer to human umbilical cord blood (CB) CD34(+)/CD38(low) and NOD/SCID repopulating cells using oncoretroviral vectors and to compare the transduction efficiency using three different viral envelopes. Methods CB cells were transduced on Retronectin using an MSCV-based vector with the gene for GFP (MGIN), which was packaged into three different cell lines giving different envelopes: PG13-MGIN (GALV) 293GPG-MGIN (VSV-G) or AM12-MGIN (amphotropic). Results Sorted CD34(+)/CD38(low) cells were efficiently transduced after 3 days of cytokine stimulation and the percentage of GFP-positive cells was 61.8+/-6.6% (PG13-MGIN), 26.9+/-3.5% (293GPG-MGIN), and 39.3+/-4.8% (AM12-MGIN). For transplantation experiments, CD34(+) cells were prestimulated for 2 days before transduction on Retronectin preloaded with vector and with the addition of 1/10th volume of viral supernatant on day 3. On day 4, the expanded equivalent of 2.5 x 10(5) cells was injected into irradiated NOD/SCID mice. All three pseudotypes transduced NOD/SCID repopulating cells (SRCs) equally well in the presence of serum, but engraftment was reduced when compared with freshly thawed cells. Simultaneous transduction with all three vector pseudotypes increased the gene transfer efficiency to SRCs but engraftment was significantly impaired. There were difficulties in producing amphotropic vectors at high titers in serum-free medium and transduction of CD34(+) cells using VSV-G-pseudotyped vectors under serum-free conditions was very inefficient. In contrast, transduction with PG13-MGIN under serum-free conditions resulted in the maintenance of SRCs during transduction, high levels of engraftment (29.3+/-6.6%), and efficient gene transfer to SRCs (46.2+/-4.8%). Conclusions The best conditions for transduction and engraftment of CB SRCs were obtained with GALV-pseudo typed vectors using serum-free conditions. Copyright (C) 2002 John Wiley Sons, Ltd.},
  author       = {Relander, Thomas and Karlsson, Stefan and Richter, Johan},
  issn         = {1521-2254},
  keyword      = {VSV-G,GALV,gene transfer,CD34+,NOD/SCID,amphotropic},
  language     = {eng},
  number       = {2},
  pages        = {122--132},
  publisher    = {John Wiley & Sons},
  series       = {Journal of Gene Medicine},
  title        = {Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes},
  url          = {http://dx.doi.org/10.1002/jgm.246},
  volume       = {4},
  year         = {2002},
}