Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

S. Yang, C. J. Cohen, P. D. Peng, Y. Zhao, L. Cassard, Z. Yu, Z. Zheng, S. Jones, N. P. Restifo, S. A. Rosenberg, R. A. Morgan

Research output: Contribution to journalArticlepeer-review

60 Scopus citations


In human gene therapy applications, lentiviral vectors may have advantages over γ-retroviral vectors in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and a potentially safer integration site profile. However, unlike γ-retroviral vectors it has been problematic to drive the expression of multiple genes efficiently and coordinately with approaches such as internal ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1 were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG) before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor gene expression. Furthermore, we determined that the furin cleavage site was recognized in lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with an efficiency of 20-30%, which could not be increased by addition of multiple furin cleavage sites. The novel bicistronic lentiviral vector developed herein afforded robust anti-melanoma activities to engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic activity. Such optimal vectors may have immediate applications in cancer gene therapy.

Original languageEnglish
Pages (from-to)1411-1423
Number of pages13
JournalGene Therapy
Issue number21
StatePublished - 2008
Externally publishedYes

Bibliographical note

Funding Information:
We thank FACS laboratory and TIL laboratory in Surgery Branch for providing technical support and maintenance of tumor cells from patients. This work is supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health.


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