Abstract
Current gene therapeutic protocols directed towards the treatment of inherited disorders (eg ADA-SCID) and viral infections (eg AIDS), as well as adoptive immunotherapy approaches are based on the use of genetically modified lymphocytes. Since only insufficient transduction of T cells is obtained using existing techniques, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 × 106/ml) by retroviral infection. Using retroviral vectors encoding a truncated human low-affinity nerve growth factor receptor (ΔLNGFR) as a gene transfer marker, we obtained transduction frequencies of more than 70% of CD3+ cells after two cycles of infection. Our protocol is based on the use of FBS-free media for both the production of retrovirus-containing supernatant and the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large-scale conditions, it may be easily adapted to clinical needs and ‘good manufacturing practice’ (GMP) standards.
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Acknowledgements
We wish to thank Claudia Kielburger for excellent technical assistance. We are indebted to the blood banks of the University Hospital Eppendorf and DRK Bad Kreuznach as well as Richard Morgan and his group at the NIH for kind co-operation. This work was supported by a grant of the Bundesministerium für Bildung und Forschung (01KV9531). BF’s stay at the NIH was supported by a travel grant from the German American Academic Council.
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Ayuk, F., Li, Z., Kühlcke, K. et al. Establishment of an optimised gene transfer protocol for human primary T lymphocytes according to clinical requirements. Gene Ther 6, 1788–1792 (1999). https://doi.org/10.1038/sj.gt.3300999
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DOI: https://doi.org/10.1038/sj.gt.3300999
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