74. Factors Affecting Long Terminal Repeat (LTR)-Driven Gene Expression Using VL30 Retro-Vectors and DNA Plasmid Vectors

Abstract

Retro-vectors can be used to drive expression of foreign genes by a variety of mechanisms, including: the long terminal repeat (LTR) promoter; internal promoter; spliced message; and use of internal ribosome entry sites (IRES). However, the confines of the baggage space between LTRs (~8 kb) can create difficulties due to promoter cross-talk, processing anomalies, and adventitious start codons, among other things. Starting with the mouse VL30 retrotransposon NVL-3 vector, pVLTRAP, we created a series of modifications, designed to test effects on green fluorescent protein (GFP) expression, in the context of a retro-vector. Expression was enhanced by three of the modifications, including: 1) use of a short, synthetic IRES sequence to drive selectable marker expression (in place of the SV40 early promoter); 2) use of an SV40 early region enhancer sequence to boost LTR expression; and 3) deletion of non-essential stuffer sequences of the 5'-untranslated region (which contained ATG codons). These modifications improved GFP protein expression approximately 1 fold, 2 fold, and 1.4 fold, respectively, compared to the basic vector. Experiments are in progress to determine whether these design elements can now be combined to achieve an additive effect. We also examined several modifications that could be used in a DNA plasmid (vaccine) vector bearing the same (NVL-3) promoter, but which could not easily be incorporated into a retro-vector backbone. By combining favorable 5'- and 3'-untranslated sequences, intron, and polyadenylation signal, cumulative expression was increased by 5-6 fold compared to the basic VLTRAP vector. This improved backbone expressed GFP, in PA317 cells, comparably to the optimized vector bearing the SV40-enhanced CMV promoter in place of the NVL-3 promoter, and is currently being developed as an alternative DNA vaccine platform.