One of the many advantages of a non-viral vector is that it is less toxic than the viral vectors. However, recent results from this and other labs have shown that plasmid DNA delivered systemically can induced an acute and systemic inflammatory response, which is dose limiting in some applications. High levels of pro-inflammatory cytokines, i.e. TNF-alpha, IL-6, IL-12 and gamma-INF, are detected in the serum of the injected mice few hours after the treatment. Besides the undesirable toxicity, high levels of cytokine production also decrease the extent and duration of gene expression in the target tissue. Most of the inflammatory activities come from the unmethylated CpG motifs, such as RRCGYY, in the plasmid DNA. The goal of this project is to investigate the relationship between DNA sequence and inflammation, with an ultimate hope to construct a plasmid vector that is substantially non-inflammatory and suitable to be used in non-viral gene therapy. In addition, the mechanism of action for these immunostimulating motifs will be studied. The project will be focused in the following four aims: (1) At least two oligonucleotides have been identified with a strong inhibitory activity. They contain sequences similar to the binding elements of two different transcriptional factors, NFKappaB and ETS-2. Since these elements are found in the upstream regulatory region of several cytokine genes, it is hypothesized that the oligonucleotides work as decoys to inhibit the transcriptional factors that are required for the expression of cytokines. We will look for additional decoy oligonucleotides along the same line of reasoning. (2) We will also investigate if the inhibitory motifs work as decoys for the transcriptional factors by performing gel shift and super shift assays. The transcriptional activity of several pro-inflammatory cytokine genes will be studied by Northern blot analysis and cDNA microarray. (3) The mechanism of action of the immunostimulating motifs will be studied. We will look for the putative binding protein(s) of RRCGYY by using yeast one hybrid cloning method. (4) We will construct a plasmid vector(s) with most, if not all, of the stimulating motifs mutated and with several inhibitory motifs added. The goal is to obtain a vector with much reduced inflammatory activity and suitable for use in non-viral gene therapy.
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