Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels.
Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. (c)2002 Elsevier Science (USA).
Kühlcke
Klaus
K
EUFETS AG, Idar Oberstein, 55743, Germany.
Fehse
Boris
B
Schilz
Andrea
A
Loges
Sonja
S
Lindemann
Carsten
C
Ayuk
Francis
F
Lehmann
Friederike
F
Stute
Norbert
N
Fauser
Axel A
AA
Zander
Axel R
AR
Eckert
Hans-Georg
HG
D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002460:Cell Line; D002498:Centrifugation; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D012190:Retroviridae / Q000235:genetics*; D013234:Stem Cells / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*