Development of Novel Multigene Lentiviral Vectors.

Lentiviral vectors are efficient in transducing dividing and non-dividing cells. Many gene transfer applications require vectors that express more than one protein which include but not limited to therapeutic gene plus a selectable marker gene, multiple genes encoding different subunits of a complex protein or multiple independent genes that cooperate functionally. Previous strategies in constructing multigene lentiviral vectors by other groups included splicing, multiple internal promoters, internal ribosome entry site (IRES), and fusion proteins. In this study we explored the feasibility of expressing homeobox B4 (HOXB4), O⁶-methylguanine-DNA-methyltransferase (MGMT), and enhanced green fluorescent protein (EGFP) under the control of a single promoter with the use of IRES sequence from encephalomyocarditis (EMCV) virus and/or foot and mouth disease virus (FMDV) 2A cleavage factor in a HIV-1 based self-inactivating lentiviral vector. In bicistronic vectors the small FMDV 2A sequence can be used efficiently as an alternative to IRES. In case of tricistronic vectors the 2A sequence can be used in combination with IRES to obtain simultaneous expression of three cDNAs under the control of a single internal promoter. Monocistronic, bicistronic and tricistronic vectors were constructed and a three-plasmid expression system was used to generate vesicular stomatitis virus glycoprotein pseudotyped lentiviral vector particles. Viral titers were measured by quantification of HIV p24 gag by ELISA. The relative efficiency of transgene expression in transduced cells by various vectors was compared by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. All the multigene vectors produced high titer viral particles and were able to simultaneously express two or three transgenes in transduced cells. However, the expression of EGFP from monocistronic vector as measured by mean fluorescence intensity was 3–8 times and 10–20 times higher than that of bicistronic and tricistronic vectors respectively. Notably expression of second gene encoded by the bicistronic vector containing FMDV 2A was 2–3 fold higher than that of IRES-based vector. Expression of MGMT in monocistronic and bicistronic constructs was also 4 to 20 times higher than tricistronic vectors. The FMDV 2A cleavage factor efficiently mediated the generation of the expected cleavage products from the artificial fusion protein. These vectors can be used in a wide range of applications including the expression of: multiple subunits of a functional protein, tumor antigen and co-stimulatory molecule(s), multiple tumor antigens for immunotherapy applications, and co-expression of selectable marker gene/s with therapeutic gene/s.