A Promoter-Specific TFO Prevents Liver Fibrosis

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DESCRIPTION (provided by applicant): In response to injury, an evolutionarily conserved wound healing process occurs. This response, if gone awry, can result in a pathologic process known as fibrosis, which is due to abnormal accumulation of extra cellular matrix (ECM) and is responsible for causing structural alterations and loss of function of the involved organ. Hepatic fibrosis, if not checked, can progress into cirrhosis and cause irreversible damage to the liver. The main component of the ECM is type I collagen, which, in the case of hepatic fibrosis, is synthesized by the activated stellate cells of the liver. Upon activation, stellate cells become more active in that they are proliferative, contractile due to a -smooth muscle actin and synthesize increased amounts of type I collagen. In our laboratory we have developed a triplex-forming oligodeoxyribonueleotide (TFO), which forms a triple helix structure with the C1 region (-170 to -141) of the a1(I) collagen gene promoter and inhibits transcription. Further we have shown that this TFO inhibits liver fibrosis, induced by administration of the chemical, dimethyl-nitorosamine (DMN), in rats. Now we would like to develop this antigene TFO as a potential antifibrotic agent. We hypothesize that this TFO selectively inhibits collagen synthesis in activated stellate cells. In this proposal, we describe experiments to address the mechanism by which the TFO inhibits collagen gene transcription. Whether the TFO blocks transcription by forming triplexes or by blocking events that lead to inflammation and activation of stellate cells will be studied. Experiments to develop a most efficacious TFO and to study its toxicity, stability, and biodistribution have been proposed. Further, the uptake by different tissues in rats and by different cell types, such as hepatocytes, stellate and Kupffer cells of the liver will also be studied. In these studies we will be using histochemical methods to monitor fibrosis, immunohistochemical methods to examine toxicity and inflammation and various biochemical and nucleic acid hybridization techniques to quantitate the levels of collagen mRNA in the liver tissues. In addition, we will assay for liver function to assess the extent of damage to the liver by the DMN and prevention by the TFO. The data generated from this project may lead to Phase I trials in humans for the treatment of liver fibrosis.