Treating Alcoholic Liver Fibrosis by Reversal of Type I Collagen

描述（由申请人提供）：酒精滥用是西方发达国家肝纤维化/肝硬化的最常见原因之一。酒精性肝纤维化的特征是细胞外基质（ECM）在肝脏中过度积聚。I型胶原蛋白是ECM的主要成分，它由胶原蛋白11（I）基因编码。肝星状细胞（HSC）是负责过度生产I型胶原的主要生产者，与正常肝脏相比，纤维化肝脏中I型胶原的产量增加了约70倍。纤维化肝脏中I型胶原蛋白的增加主要是因为其mRNA的半衰期从静止HSC中的1.5小时增加到活化HSC中的大于24小时。研究发现，在活化的HSC中，胶原11（I）mRNA被RNA结合蛋白1CP（由PCBP 2基因编码）稳定，所述RNA结合蛋白1CP结合于胶原11（I）mRNA的3'末端，而在静止的HSC中不稳定。因此，阻断PCBP 2表达是降低胶原11（I）mRNA稳定性并最终逆转酒精性纤维化肝脏中积累的I型胶原的潜在治疗方法。具有高序列特异性的靶基因的有效敲低使得siRNA成为沉默PCBP 2基因的非常有前途的治疗策略。我们设计并鉴定了一种siRNA，它可以显著沉默PCBP 2基因，从而降低11（I）mRNA的水平。将对该siRNA进行骨架修饰和胆固醇缀合，以增加其细胞摄取并实现靶向递送至纤维化肝脏。我们的总体假设是，逆转积累的I型胶原是治疗酒精性肝纤维化的关键步骤。具体假设如下：i）1型胶原蛋白表达的下调将导致酒精性肝纤维化的恢复; ii）用siRNA阻断PCBP 2表达将导致稳定的胶原蛋白11（I）mRNA的降解，并因此诱导积累的I型胶原蛋白的逆转; iii）siRNA的主链修饰将增加其稳定性而不中断其活性; iv）胆固醇与siRNA的缀合将增加其肝摄取并因此实现抗纤维化作用。目的是开发一种有效的靶向PCBP 2的siRNA治疗剂，以降低11（I）mRNA的稳定性，从而诱导酒精性肝纤维化中I型胶原积聚的逆转。 酒精性肝纤维化是一个全球性的健康问题，尤其是在发达国家。本课题的成功完成将为通过诱导Ⅰ型胶原降解来治疗酒精性肝纤维化提供一种有效的治疗方法。

DESCRIPTION (provided by applicant): Alcohol abuse is one of the most common causes of liver fibrosis/cirrhosis in western developed countries. Alcoholic liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) in the liver. Type I collagen is the major component of ECM and it is encoded by the collagen 11(I) gene. Hepatic stellate cells (HSCs) are the main producers responsible for the excessive production of type I collagen in a ~70-fold increase in fibrotic liver compared to the normal liver. The increase of type I collagen in the fibrotic liver is primarily because of the increased half-life of its mRNA from 1.5 hours in quiescent HSCs to greater than 24 hours is activated HSCs. It has been found that the collagen 11(I) mRNA is stabilized by the RNA-binding protein 1CP (encoded by PCBP2 gene) which binds to the 3' end of collagen 11(I) mRNA in activated HSCs, but not in the quiescent HSCs. Therefore, blocking PCBP2 expression is a potential therapeutic approach to decrease the stability of collagen 11(I) mRNA and finally reverse the accumulated type I collagen in the alcoholic fibrotic liver. Potent knockdown of the target gene with high sequence specificity makes siRNA a very promising therapeutic strategy to silence the PCBP2 gene. We have designed and identified one siRNA which can significant silence the PCBP2 gene and consequently decrease the level of 11(I) mRNA. Backbone modification and cholesterol conjugation will be conducted for this siRNA to increase its cellular uptake and achieve the target delivery to the fibrotic liver. Our overall hypothesis is that the reversal of accumulated type I collagen is the critical step in the treatment of alcoholic liver fibrosis. The specific hypotheses are: i) down-regulation of type 1 collagen expression will lead to the recovery of alcoholic liver fibrosis; ii) blocking PCBP2 expression with siRNA will lead to the degradation of stabilized collagen 11(I) mRNA and accordingly induce the reversal of accumulated type I collagen; iii) backbone modification of siRNA will increase its stability without interrupting its activity; iv) conjugation of cholesterol with siRNA will increase its hepatic uptake and consequently achieve the antifibrotic effect. The objective is to develop an effective siRNA therapeutics targeting PCBP2 to decrease the stability of 11(I) mRNA and then induce the reversal of accumulated type I collagen in the alcoholic liver fibrosis. Public health relevance: Alcoholic liver fibrosis is a global health problem, especially in developed countries. Successful accomplishment of this project will provide an effective therapeutics to treat alcoholic liver fibrosis by inducing degradation of the accumulated type I collagen.