Reliable generation of stable high titer producer cell lines for gene therapy.
OBJECTIVE
Retroviral vectors represent one of the most robust technologies for in vivo expression of heterologous gene sequences and are still the most commonly used vectors in clinical gene therapy trials. The production of high titer retroviral preparations, however, can be a problematic procedure for certain constructs.


METHODS
GALV- or RD114-pseudotyped retroviral particles carrying selectable fluorescence markers or drug resistance genes, such as the green fluorescent protein (GFP) or the O(6)-methylguanine-DNA-methyltransferase (MGMT) mutants, were used to stably transduce Phoenix-(FNX-)eco cells. Thereafter, a polyclonal population of producer cells was generated by enriching cells with high marker gene expression. In addition, single producer clones were selected by limiting dilution.


RESULTS
Retroviral titers were increased 1-2 logs by enriching for a polyclonal population of producer cells, and selection of single producer clones allowed another 1- to 2-log increase in titers. Using this method, reproducibly high titer viral preparations allowing efficient transduction of hematopoietic stem cells were generated.


CONCLUSIONS
A reliable and time-effective method to generate stable high titer producer cells based on the FNX-cell line for problematic retroviral vector constructs is described.
Rattmann
Ina
I
Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Duisburg-Essen Medical School, Essen, Germany. ina.rattmann@uni-essen.de
Kleff
Veronika
V
Feldmann
Anja
A
Ludwig
Carsten
C
Sorg
Ursula Regina
UR
Opalka
Bertram
B
Moritz
Thomas
T
Flasshove
Michael
M
D002460:Cell Line / Q000821:virology*; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006412:Hematopoietic Stem Cells; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000096:biosynthesis; D015203:Reproducibility of Results; D012190:Retroviridae / Q000254:growth & development*; D014161:Transduction, Genetic
D049452:Green Fluorescent Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase