HEK293-based production platform for γ-retroviral (self-inactivating) vectors: application for safe and efficient transfer of COL7A1 cDNA.
The clinical application of self-inactivating (SIN) retroviral vectors requires an efficient vector production technology. To enable production of γ-retroviral SIN vectors from stable producer cells, new targetable HEK293-based producer clones were selected, providing amphotropic, GALV, or RD114 pseudotyping. Viral vector expression constructs can reliably be inserted at a predefined genomic locus via Flp-recombinase-mediated cassette exchange. Introduction of a clean-up step, mediated by Cre-recombinase, allows the removal of residual sequences that were required for targeting and selection, but were dispensable for the final producer clones and eliminated homology-driven recombination between the tagging and the therapeutic vector. The system was used to establish GALV and RD114 pseudotyping producer cells (HG- and HR820) for a clinically relevant long terminal repeat-driven therapeutic vector, designed for the transfer of a recombinant TCR that delivered titers in the range of 2×10(7) infectious particles (IP)/ml. Production capacity of the amphotropic producer cell (HA820) was challenged by a therapeutic SIN vector transferring the large COL7A1 cDNA. The final producer clone delivered a titer of 4×10(6) IP/ml and the vector containing supernatant was used directly to functionally restore primary fibroblasts and keratinocytes isolated from recessive dystrophic epidermolysis bullosa patients. Thus, the combinatorial approach (fc-technology) to generate producer cells for therapeutic γ-retroviral (SIN) vectors is feasible, is highly efficient, and allows their safe production and application in clinical trials.
1 EUFETS GmbH , 55743 Idar-Oberstein, Germany .
Katharina
K
Hennig
Lars
L
Raasch
Carolin
C
Kolbe
Sibylle
S
Weidner
Matthias
M
Leisegang
Wolfgang
W
Uckert
Matthias
M
Titeux
Alain
A
Hovnanian
Klaus
K
Kuehlcke
Rainer
R
Loew
D024143:Collagen Type VII / Q000235:genetics* / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics* / Q000302:isolation & purification; D017988:Gammaretrovirus / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans
C569053:COL7A1 protein, human; D024143:Collagen Type VII; D004274:DNA, Recombinant