1、Therapeutic Silencing of MicroRNA-122in Primates with Chronic Hepatitis CVirus InfectionScience 327, 198 (2010)徐歡 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) (結(jié)構(gòu)和來(lái)源結(jié)構(gòu)和來(lái)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 (生物學(xué)功能和機(jī)制生物學(xué)功能和機(jī)制)

3、.microRNA-122: liver-specificIn 1989,studies on genetic alterations in Woodchuck(旱獺）旱獺） liver tumors 發(fā)現(xiàn)發(fā)現(xiàn)miRNA-122內(nèi)源性轉(zhuǎn)錄本：內(nèi)源性轉(zhuǎn)錄本：hcr基因基因 In 2002, systematic cloning and sequencing of small RNAs鑒定鑒定miRNA-122序列序列miRNA122 與與HCV 復(fù)制的關(guān)系復(fù)制的關(guān)系:Modulation of Hepatitis C Virus RNA Abundance by a Liver-Specific

4、 MicroRNAScience 309, 1577 (2005);表達(dá)靶基因篩選靶基因篩選結(jié)合位點(diǎn)的突變結(jié)合位點(diǎn)的突變 EMSAEMSACHIPCHIPWestern BlotWestern Blot功能鑒定功能鑒定作用機(jī)制研究作用機(jī)制研究生物信息學(xué)預(yù)測(cè)生物信息學(xué)預(yù)測(cè)反義核酸反義核酸抑制表達(dá)抑制表達(dá)Western Western blotblotNorthern Northern blot blot Nothern blotSEAP REPORTER一：一： 靶位點(diǎn)的預(yù)測(cè)靶位點(diǎn)的預(yù)測(cè)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實(shí)驗(yàn)動(dòng)物：4只長(zhǎng)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 影響病毒復(fù)制影響病毒復(fù)制對(duì)自身代謝的影響對(duì)自身代謝的影響Real-time RT PCRHCV titersNorthern blotOf RNALiver mRNASIRGS expressionSerum cholesterol miR-122定量檢測(cè)Sequencing of miR-122Binding site檢測(cè)resistancemutationsChemokineIP-10Assess safetyvPharmacokinetic properities of spc3649ALT levels and Creatine levels首個(gè)首個(gè)miRNA藥物進(jìn)入臨床試驗(yàn)藥物進(jìn)入臨床試驗(yàn)v丹麥制藥公司Santaris Pharma2008年6月宣布SPC3649 (LNA-antimiRTM-122)開(kāi)始進(jìn)入人體第一階段的臨床試驗(yàn)，這是世界上首個(gè)在人體中試驗(yàn)的microRNA藥物。這項(xiàng)研究由哥本哈根PhaseOneTrials A/S組織管理，最多包含48名健康男性志愿者。公司同時(shí)宣布研究的第一批健康志愿者已經(jīng)圓滿(mǎn)的完成了治療。SPC3649是由Santaris Pharma開(kāi)發(fā)的丙型