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The development and characterization of a novel autogene based, dual promoter cytoplasmic expression system Finn, Jonathan Douglas


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 below the levels achieved by viral gene transfer systems. One strategy for increasing gene expression is to design a cytoplasmic expression system that does not require nuclear delivery for gene expression to occur. This can be achieved through the use of an autocatalytic cytoplasmic expression system using phage RNA polymerases. In this work, the design, construction, and characterization of a novel dual promoter, autogene based cytoplasmic expression system is described. The purpose of this work was to develop new plasmids capable of giving rise to increased gene expression levels over standard plasmids currently used in the field of either non-viral gene therapy, or general gene expression. In Chapter 2 the system is introduced and characterized. Chapter 3 is concerned with attempting to optimize expression levels, and determining what factors are limiting gene expression. In Chapter 4, the differences between a high expressing cell line (BHK) and a low expressing cell line (COS-7) are investigated. In Chapter 2, the ability of the autogene based expression system to give rise to exponential increases in gene expression is demonstrated, as well as the ability of the expression system to give rise to levels of gene expression in BHK cells that are 20 times higher per transfected cell than a standard CMV based nuclear expression plasmid. Further analysis determined that the mRNA transcripts made in the cytoplasm are not as stable as those made in the nucleus (most likely due to the lack of a 5' cap structure on the cytoplasmic transcripts), and that the cytoplasmic expression system gives rise to much higher mRNA levels than the nuclear system. In Chapter 3 an attempt is made to determine what factors are limiting the cytoplasmic expression system, in an effort to optimize the system to give rise to even higher levels of gene expression. After exploring various factors, such as the nuclear promoter, RNAP gene and IRES sequence, it was found that the critical factors responsible for limiting the cytoplasmic system is at the mRNA level, both the transcription and translation. It was found that the cytoplasmic transcripts are being translated 20 times less efficiently than their capped nuclear counterparts, and by measuring mRNA transcript levels, it was found that the autogene based cytoplasmic expression system appears to be saturating the cells ability to produce new mRNA, and that transgene transcript levels are reaching 30% of the total RNA in the cell, a level that is 10 times higher than the sum of every other mRNA transcript in the cell. In addition to this finding, insights into possible IRES function are gained. In Chapter 4, the differences between a cell line that gives rise to high levels of cytoplasmic gene expression (BHK) are compared with a cell line that gives poor cytoplasmic expression (COS-7). After investigating the individual components of the cytoplasmic expression system, such as the IRES activity and RNAP function, it was found that the reason for the difference in the two cell lines is most likely at the level of the intracellular processing of the DNA:cationic lipid complexes, as the COS-7 cells appear to have less intact plasmid per cell over time than the BHK cells. In summary, this work represents a substantial advance in the understanding of cytoplasmic expression systems, and demonstrates the potential of these systems to give rise to increased levels of gene expression. In addition, this study clarifies potential areas that need to be addressed in order to further optimize cytoplasmic expression systems.

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