Therapeutic Silencing of MicroRNA-122

1、Therapeutic Silencing of MicroRNA-122in Primates with Chronic Hepatitis CVirus InfectionScience 327, 198 (2010)徐欢 2010-3-15Backgroud-whyProtocol -howDiscussion -whatInstruction-learn A type of non-coding small RNA (2125 nucleotides) produced by Dicer from a stem-loop structured RNA precursor (70-90

2、nts ong) (结构和来源结构和来源). miRNAs are widely expressed in animal and plant cells as RNAprotein complexes, termed miRISCs, and have been implicated in the control of development because they lead to the destruction or translational suppression of target mRNAs with homology to the miRNA (生物学功能和机制生物学功能和机制)

3、.microRNA-122: liver-specificIn 1989,studies on genetic alterations in Woodchuck(旱獭）旱獭） liver tumors 发现发现miRNA-122内源性转录本：内源性转录本：hcr基因基因 In 2002, systematic cloning and sequencing of small RNAs鉴定鉴定miRNA-122序列序列miRNA122 与与HCV 复制的关系复制的关系:Modulation of Hepatitis C Virus RNA Abundance by a Liver-Specific

4、 MicroRNAScience 309, 1577 (2005);表达靶基因筛选靶基因筛选结合位点的突变结合位点的突变 EMSAEMSACHIPCHIPWestern BlotWestern Blot功能鉴定功能鉴定作用机制研究作用机制研究生物信息学预测生物信息学预测反义核酸反义核酸抑制表达抑制表达Western Western blotblotNorthern Northern blot blot Nothern blotSEAP REPORTER一：一： 靶位点的预测靶位点的预测Two predicted binding sitesNorthern blotWesternblot122-

5、2OMemiR122eGFP-122Rand-2OMemiR-124eGFP-124Let7-2OMeBrainspecificeGFP sensor mRNAs contain sequences Complementary to miR-122 in their 3NCRIntroduce mutations into H77c cDNATransfection of H77c RNAs 3B, lane2lane9lane4lane11lane8Ectopic expreesion of miR-122 RNAs that Contain base complementary mutat

6、ions miR-122 antagonism SPC3649vSPC3649:locked nucleic acid (LNA)modified Phosphorothioate(磷硫酰） oligonucleotideTherapeutic Silencing of MicroRNA-122 in Primates with Chronic Hepatitis C Virus Infectionv实验动物：4只长期感染HCV1 型的大猩猩 Group 1：0513 0514Group 2:0358 0267High dose:5 mg/kg-1iv weeklyLow dose1 mg/k

7、g-1iv weeklySPC3649BaselineFREE-40121v2miR-122SPC3649 影响病毒复制影响病毒复制对自身代谢的影响对自身代谢的影响Real-time RT PCRHCV titersNorthern blotOf RNALiver mRNASIRGS expressionSerum cholesterol miR-122定量检测Sequencing of miR-122Binding site检测resistancemutationsChemokineIP-10Assess safetyvPharmacokinetic properities of spc3649ALT levels and Creatine levels首个首个miRNA药物进入临床试验药物进入临床试验v丹麦制药公司Santaris Pharma2008年6月宣布SPC3649 (LNA-antimiRTM-122)开始进入人体第一阶段的临床试验，这是世界上首个在人体中试验的microRNA药物。这项研究由哥本哈根PhaseOneTrials A/S组织管理，最多包含48名健康男性志愿者。公司同时宣布研究的第一批健康志愿者已经圆满的完成了治疗。SPC3649是由Santaris Pharma开发的丙型