Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes.
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 pre-stimulated 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 2.5x10(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-pseudotyped vectors using serum-free conditions.
Relander
Thomas
T
Department of Molecular Medicine and Gene Therapy, Lund University Hospital, Lund, Sweden.
Karlsson
Stefan
S
Richter
Johan
J
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000096:biosynthesis; D000943:Antigens, Differentiation / Q000096:biosynthesis; D001854:Bone Marrow Cells / Q000378:metabolism; D002469:Cell Separation; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005312:Fetal Blood / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D016513:Mice, SCID; D009244:NAD+ Nucleosidase / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics*; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000378:metabolism
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D016895:Culture Media, Serum-Free; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1