High-Riter Bicistronic Retroviral Vectors Employing Foot-and-Mouth Disease Virus Internal Ribosome Entry Site

Abstract

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

“…To address this issue, several groups have sought to identify alternative cellular and viral IRES elements that initiate protein expression more efficiently than EMCV IRES. 15,[19][20][21][22][23][24][25][26] Cellular IRES elements such as eukaryotic initiation factor 4G, 15 immunoglobulin heavy chain binding protein, 15 c-myc proto-oncogene, 15 vascular endothelial growth factor 15 and fibroblast growth factor-1 27 IRES have been reported to show higher efficiency than EMCV IRES. In this study, we searched to identify an IRES(s) with higher translation efficiency than the EMCV IRES both in vitro and in vivo.…”

Cotransduction of nondividing cells using lentiviral vectors

“…Strategies for achieving retrovirus-mediated, multi-gene transduction include use of internal ribosomal entry sequences (IREs). [1][2][3][4] The IRES vectors allow for the transduction of up to two genes simultaneously, in cis, on a single vector. Other retroviral vectors for transducing two genes into individual cells have contained internal promoters 1,5,6 to drive gene expression.…”

Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy

“…Because high titers of the retroviral vectors and/or increased infectivity of the particles are required for Gene Therapy acceptable transduction rates into long-term repopulating cells, 26 IRES elements might not be optimal for application in gene therapy of hematopoietic stem cells. Even though high vector-titer producing packaging cells can in principle be obtained using IRES-containing retroviral vectors, 27 it usually involves a time-consuming selection procedure and the screening of many single cell clones. Our data indicate that the 2A protein offers the possibility to express HoxB4 and a second cDNA as a selfseparating polyprotein from one retroviral vector without negatively affecting retroviral vector titers.…”