A Promoter-Specific TFO Prevents Liver Fibrosis

描述(由申请人提供)：作为对损伤的反应，发生了一个进化保守的伤口愈合过程。如果这种反应出错，可能会导致一种称为纤维化的病理过程，这是由于细胞外基质(ECM)的异常堆积，并导致相关器官的结构变化和功能丧失。肝纤维化，如果不加以控制，可能会发展为肝硬变，对肝脏造成不可逆转的损害。细胞外基质的主要成分是I型胶原，在肝纤维化的情况下，它是由激活的肝脏星状细胞合成的。一旦激活，星状细胞变得更加活跃，因为它们由于α-平滑肌肌动蛋白而增殖、收缩，并合成更多的I型胶原。我们实验室开发了一种三链形成的寡核苷酸(TFO)，它与A1(I)胶原基因启动子的-170到-141区域形成三螺旋结构，并抑制转录。此外，我们还发现该TFO可抑制由二甲基亚硝胺(DMN)所致的大鼠肝纤维化。现在，我们想开发这种抗基因的TFO作为一种潜在的抗纤维化药物。我们推测，这种转铁蛋白选择性地抑制激活的星状细胞中胶原的合成。在这个提案中，我们描述了TFO抑制胶原基因转录的机制的实验。TFO是通过形成三联体还是通过阻止导致星状细胞炎症和激活的事件来阻止转录将被研究。已经提出了开发最有效的TFO并研究其毒性、稳定性和生物分布的实验。此外，还将研究大鼠不同组织和不同类型细胞的摄取，如肝细胞、肝星状细胞和枯否细胞。在这些研究中，我们将使用组织化学方法来监测纤维化，免疫组织化学方法来检测毒性和炎症，以及各种生化和核酸杂交技术来定量肝组织中胶原mRNA的水平。此外，我们将进行肝功能检测，以评估DMN对肝脏的损害程度和TFO的预防作用。该项目产生的数据可能导致在人类身上进行治疗肝纤维化的第一阶段试验。

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.