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

Abstract

In human gene therapy applications, lentiviral vectors may have advantages over g-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 g-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 antimelanoma 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.

Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling

“…We first generated a single vector dualexpression system, using an internal ribosome entry site (IRES) sequence to drive the expression of the second, C-TEV, fragment; however, this suffered from low expression levels of the second fragment and poor activity upon dimerization. Next, we used a 2A "self-cleaving" peptide sequence to drive cistronic translation of N-TEV and C-TEV in two orientations (31). This 2A configuration yielded robust protein expression levels and inducible activity, with the C-Tev_T2A_N-Tev being the better of the two.…”

Adenovirus co-expressing CD40 ligand and interleukin (IL)-2 contributes to maturation of dendritic cells and production of IL-12

“…IRES is a commonly used method for producing double gene expression vectors (28,29); however, the expression levels of the two genes connected via the IRES is not completely consistent, and often the gene expression level close to the promoter is high, while the gene expression level downstream of the IRES is low (30). In conclusion, the present study indicates that CD40L and IL-2 connected by IRES may be highly expressed, and IRES and IL-2 may be connected by NcoI locus (ccatgg), with the Kozak sequence (accatgg) being formed in favor of the expression of IL-2.…”

Engineered Drug Resistant Cell-Mediated Immunotherapy

“…Several CTLs harboring transgenic TCRs have now been developed that are directed specifically to tumor antigens, such as MART-1, gp100, NY-ESO-1 and CEA, resulting in tumor elimination in animal models (Abad, et al, 2008;Morgan, et al, 2003;Kessels et al, 2001;Stanislawski et al, 2001;Wargo et al, 2009;Xue et al, 2005). Bicistronic viral vectors encoding cDNA sequences for both α and β chains have been successfully incorporated into retroviral based strategies to transfer αβ transgenic TCR into T lymphocytes (Yang et al, 2008). The functional efficacies of the engineered CTLs have also been improved by codon optimizing α and β sequences that result in increased surface expression of the transgenic TCRs (Jorritsma et al, 2007;Scholten et al, 2006).…”