Abstract
BackgroundA number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP), O6-methylguanine-DNA-methyltransferase (MGMT), and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting.ResultsAll the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector.ConclusionThe small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.
Highlights
A number of gene therapy applications would benefit from vectors capable of expressing multiple genes
The cDNAs encoding enhanced green fluorescent protein (eGFP), MGMT, and HOXB4 were used as model genes
MGMT incorporates an extra 23 amino-acid peptide fused to its C-terminus whilst eGFP has an additional 7 aminoacid peptide fused to its N-terminus
Summary
A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. Lentiviral vectors are efficient tools for gene transfer into various dividing and non-dividing target cells. Many potential gene transfer applications require vectors that express more than one protein These may include a therapeutic gene plus a selectable marker gene, multiple genes encoding different subunits of a complex protein or multiple independent genes that cooperate functionally. A number of strategies are employed in viral vectors to express multiple genes, including mRNA splicing, internal promoters, internal ribosomal entry sites, fusion proteins, and cleavage factors. The most commonly used strategy in the construction of two gene vectors is the insertion of an internal ribosome entry site (IRES) element between the two transgenes [8]. In many cases it has been reported that a gene transcribed upstream of an IRES is expressed strongly whereas a gene placed downstream is expressed at lower levels [9,10]
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