The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes Journal Article


Authors: Gallardo, H. F.; Tan, C.; Sadelain, M.
Article Title: The internal ribosomal entry site of the encephalomyocarditis virus enables reliable coexpression of two transgenes in human primary T lymphocytes
Abstract: 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 active 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 μM ganciclovir, respectively, establishing that the EMCV IRES ensures efficient and sufficient expression of the two genes in human primary T cells.
Keywords: controlled study; human cell; nonhuman; t lymphocyte; t-lymphocytes; animal cell; mouse; animals; mice; cell division; gene expression; gene transfer; genetic transduction; animalia; cell surface marker; cell isolation; transgene; simplexvirus; cell culture techniques; fibroblasts; adoptive cell therapy; thymidine kinase; herpes simplex virus; herpes; ganciclovir; transgenes; marker gene; antiviral agents; expression vector; gene transfer techniques; gene expression regulation, viral; suicide gene; nerve growth factor receptor; ribosome; lymphocyte culture; cistron; unidentified retrovirus; humans; human; priority journal; article; encephalomyocarditis virus; internal ribosomal entry site; retrovirus-mediated gene transfer; gene coexpression; primary t lymphocyte
Journal Title: Gene Therapy
Volume: 4
Issue: 10
ISSN: 0969-7128
Publisher: Nature Publishing Group  
Date Published: 1997-10-01
Start Page: 1115
End Page: 1119
Language: English
PUBMED: 9415319
PROVIDER: scopus
DOI: 10.1038/sj.gt.3300506
DOI/URL:
Notes: Article -- Export Date: 17 March 2017 -- Source: Scopus
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MSK Authors
  1. Michel W J Sadelain
    538 Sadelain
  2. Cuiwen   Tan
    11 Tan