The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes.
It is essential for the improvement of adoptive cell therapies to generate efficiently large populations of human primary T cells that reliably express a suicide gene conferring drug sensitivity, such as herpes simplex virus thymidine kinase (HSVtk). We show here that an optimized dicistronic vector containing the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) is functional in human primary. T lymphocytes that bear on average one integrated vector copy per cell. We demonstrate reliable coexpression of the marker NTP, an inactive mutant of the human low-affinity nerve growth factor receptor and HSVtk. In the dicistronic vector NIT, NTP is expressed as a cap-dependent marker and HSVtk as a nonselectable IRES-dependent gene. Cell-surface expression of NTP is sufficient to allow for the efficient and rapid enrichment of the transduced cells to high purity. Of these purified lymphocytes, 97 +/- 4% and 92 +/- 6% are selectively eliminated when cultured in the presence of 1.0 or 0.1 microM ganciclovic respectively, establishing that the EMCV IRES ensures efficient and sufficient expression of two genes in human primary T cells.
Gallardo
H F
HF
Department of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Tan
C
C
Sadelain
M
M
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D018929:Cell Culture Techniques; D002455:Cell Division / Q000187:drug effects; D004686:Encephalomyocarditis virus / Q000235:genetics*; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology; D015774:Ganciclovir / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D018014:Gene Transfer Techniques; D006801:Humans; D051379:Mice; D013601:T-Lymphocytes / Q000187:drug effects / Q000502:physiology*; D019076:Transgenes
D000998:Antiviral Agents; D015774:Ganciclovir