314. mRNA Production Is the Major Factor Limiting Autogene-Based Cytoplasmic Expression

Abstract

The relatively low levels of transfection that can be achieved by current gene delivery systems have limited the therapeutic utility of gene transfer. This is especially true for non-viral gene delivery systems, where the levels of gene expression achieved are usually well below the levels achieved by viral gene transfer systems. Previous work from our laboratory describes an enhanced dual promoter autogene-based cytoplasmic expression system that gives rise to levels of gene expression 20 fold higher than that of a CMV nuclear expression plasmid control. Here we describe various strategies to increase the levels of autogene based gene expression by changing variables such as the type of nuclear promoter, phage RNAP gene, and IRES sequence. It was found that none of these changes demonstrated a significant increase in gene expression. However determination of the mRNA levels achieved using quantitative RNase protection assays and immunofluorescence experiments, revealed transgene mRNA levels up to 10 times higher than all other mRNA in the transfected cell combined. It follows that mRNA production is a major factor limiting autogene based cytoplasmic expression.