HEK293-based production platform for γ-retroviral (SIN-) 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 {gamma}-retroviral SIN-vectors from stable producer cells, new targetable HEK293-based producer clones were selected, providing either 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 LTR-driven therapeutic vector, designed for the transfer of a recombinant TCR which delivered titers in the range of 2x10e7 infectious particles (IP)/ml. Production of the amphotropic producer cell (HA820) was challenged by a therapeutic SIN-vector transferring the large COL7A1 cDNA. The titer of the finally selected producer clone of around 4x10e6 IP/ml was used to functionally restore primary fibroblasts and keratinocytes isolated from the skin of Recessive Dystrophic Epidermolysis Bullosa (RDEB) patients. Thus, the combinatorial approach (fc-technology) to generate producer cells for therapeutic {gamma}-retroviral (SIN-) vectors is feasible, highly efficient and allows their safe production and application in clinical trials.