Treating Alcoholic Liver Fibrosis by Reversal of Type I Collagen

描述(申请人提供)：在西方发达国家，酗酒是导致肝纤维化/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)在肝脏过度积聚。I型胶原是细胞外基质的主要成分，由I型胶原11(I)基因编码。肝星状细胞(HSCs)是I型胶原过度产生的主要来源，肝纤维化肝脏I型胶原的产生是正常肝脏的70倍左右。I型胶原在肝纤维化中的增加主要是由于其mRNA的半衰期从静止的HSC的1.5小时增加到激活的HSC的24小时以上。研究发现，在激活的HSC中，11(I)型胶原基因的3‘端由PCBP2基因编码的RNA结合蛋白1CP稳定，而在静止状态的HSCs中则不稳定。因此，阻断PCBP2的表达是一种潜在的治疗途径，可以降低11(I)型胶原mRNA的稳定性，最终逆转酒精性肝纤维化肝脏中积聚的I型胶原。具有高度序列特异性的靶基因的有效敲除使siRNA成为一种非常有希望的沉默PCBP2基因的治疗策略。我们设计并鉴定了一种能够显著沉默PCBP2基因，从而降低11(I)mRNA水平的siRNA。将对该siRNA进行骨架修饰和胆固醇结合，以增加其细胞摄取，并实现向纤维化肝脏的目标输送。我们的总体假设是，I型胶原蓄积的逆转是治疗酒精性肝纤维化的关键步骤。具体的假设是：i)下调1型胶原的表达将导致酒精性肝纤维化的恢复；ii)用siRNA阻断PCBP2的表达将导致稳定的11(I)型胶原mRNA的降解，从而诱导积聚的I型胶原的逆转；iii)siRNA的骨架修饰将在不中断其活性的情况下增加其稳定性；iv)结合胆固醇与siRNA将增加其肝脏摄取，从而达到抗肝纤维化的作用。本研究的目的是开发一种有效的针对PCBP2的siRNA治疗药物，以降低11(I)mRNA的稳定性，从而逆转酒精性肝纤维化中积聚的I型胶原。 公共卫生相关性：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。该项目的成功完成将为治疗酒精性肝纤维化提供一种有效的治疗方法，通过诱导堆积的I型胶原降解来治疗酒精性肝纤维化。

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.