Retroviral transduction of human CD34+ cells on fibronectin fragment CH-296 is inhibited by high concentrations of vector containing medium.
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.
Relander
T
T
Department of Molecular Medicine and Gene Therapy, Lund University, Sweden.
Brun
A
A
Hawley
R G
RG
Karlsson
S
S
Richter
J
J
D018952:Antigens, CD34 / Q000096:biosynthesis; D002454:Cell Differentiation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay / Q000379:methods; D005312:Fetal Blood; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D017988:Gammaretrovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D014161:Transduction, Genetic
D018952:Antigens, CD34; D005353:Fibronectins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins