应用CRISPR/Cas13b编辑技术治疗TNNT2R141W转基因小鼠的扩张型心肌病  

Treatment of the Dilated Cardiomyopathy in the TNNT2R141W Transgenic Mice by Using the CRISPR/Cas13b Editing System

作　　者：匡歆 王玉瑶[1] 聂宇[2] 李昊[2] 陈显达 廉虹[2] 刘立会 李佳成 郭睿[1] KUANG Xin;WANG Yu-Yao;NIE Yu;LI Hao;CHEN Xian-Da;LIAN Hong;LIU Li-Hui;LI Jia-Cheng;GUO Rui(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030000,China;State Key Laboratory of Cardiovascular Disease,Fuwai Hospital,National Center for Cardiovascular Disease,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100037,China)

机构地区：[1]山西医科大学生物化学与分子生物学教研室,太原030000 [2]中国医学科学院北京协和医学院国家心血管病中心,阜外医院心血管疾病国家重点实验室,北京100037

出　　处：《中国生物化学与分子生物学报》2020年第1期49-60,共12页Chinese Journal of Biochemistry and Molecular Biology

基　　金：中国医学科学院医学与健康科技创新工程项目(2016-I2M-1-015);国家自然科学基金项目(No.81500364和No.31801068);山西省研究生教育创新项目(No.2019SY265);北京市自然科学基金(No.7182140)资助~~

摘　　要：应用CRISPR/Cas13b系统对TNNT2R141W转基因扩张型心肌病(dilated cardiomyopathy,DCM)小鼠进行探索性治疗,尝试发现治疗扩张型心肌病的一种新方式,为CRISPR/Cas13b系统在体内应用提供实验基础。随机设计11种Cas13b-TNNT2 gRNA并成功构建表达质粒,把它和人源TNNT2过表达质粒共同转染到293T细胞中,通过实时定量PCR(Q-PCR)检测人源TNNT2 mRNA的表达水平。结果显示,gRNA2引导Cas13b敲低目标基因的效率最高,达到80%(P<0.0001)。把gRNA2表达质粒包装到慢病毒载体中,转导出生后1 d的DCM小鼠原代心肌细胞。Q-PCR检测结果表明,CRISPR/Cas13b系统对人源TNNT2 mRNA敲低效率达到55%(P<0.01)。把PspCas13b和gRNA2的表达载体分别包装到AAV9病毒载体中,再将200μL约1×1012AAV9病毒颗粒通过尾静脉注射到4月龄DCM小鼠体内,待注射小鼠发育至5月龄时。Q-PCR检测结果显示,AAV9+DCM组TNNT2R141W表达水平较未注射组对照明显下降至40%(P<0.01)。对5月龄野生型(WT)、DCM(未注射病毒组)和AAV9+DCM(基因组编辑工具注射组)三组小鼠的心脏形态、心功能、心肌纤维化和心力衰竭等表型的观察结合显示:DCM小鼠的心血管形态异常,而AAV9+DCM小鼠心血管形态趋于正常。对3组小鼠的心脏进行超声心动图,并对心功能指标进行统计发现,DCM组较WT组小鼠的左心室射血分数(left ventricular percent ejection fraction,LV EF%)、左心室短轴缩短率(left ventricular percent fractional shortening,LV FS%)分别下降50.4%(P<0.0001),55.1%(P<0.0001),而AAV9+DCM组较DCM组小鼠的LV EF%、LV FS%分别上升66.5%(P<0.01),77.0%(P<0.01)。通过Q-PCR和天狼星红染色检测3组小鼠的心肌纤维化程度。结果显示,DCM组较WT组小鼠的Col3a1和Postn两种纤维化基因,分别高表达5.2倍(P<0.001)、4.5倍(P<0.01),而AAV9+DCM组较DCM组小鼠两种基因表达分别下降2.0倍(P<0.05)、1.4倍(NS)。天狼星红染色结果显示,纤维化区域明显下降;通过Q-PCR和蛋白质免疫In this study the CRISPR/Cas13 b system was used to treat TNNT2 R141 Wtransgenic dilated cardiomyopathy(DCM)mice,aiming to find a new way to treat DCM mice,and providing experimental basis for the application of the CRISPR/Cas13 b system in vivo.Eleven Cas13 b-TNNT2 gRNAs were randomly designed and successfully constructed.They were co-transfected into 293 T cells with human TNNT2 over-expression plasmids.The expression level of human TNNT2 mRNA was detected by quantitative real-time PCR(qPCR).The results showed that the knockdown efficiency of gRNA 2 was the highest,reaching 80%(P<0.0001).The original generation cardiomyocytes of DCM mice were extracted,the gRNA 2 plasmid was packed as lentivirus,and the primary generation cardiomyocytes were transduced by lentivirus.The knockdown efficiency of the CRISPR/Cas13 b system on human TNNT2 mRNA was 55%(P<0.01)by qPCR.The PspCas13 b and gRNA 2 were packed as AAV9,respectively,and injection of 200μL 1×1012 AAV9 through tail veins to four-month-old DCM mice.The expression of TNNT2 R141 Win the hearts of the DCM group and AAV9+DCM group were detected by qPCR and immunofluorescence staining(human TNNT2 andα-actinin).The results showed that the expression level of TNNT2 R141 WmRNA in the AAV9+DCM group was 40%(P<0.01).The phenotypes of heart morphology,cardiac function,myocardial fibrosis and heart failure of 5 M wild type(WT),DCM and AAV9+DCM mice were observed.The heart morphology of the three groups of mice was photographed.We observed that the heart morphology of DCM mice was aberrent,while that of AAV9+DCM mice tended to be normal.The heart of the three groups of mice was echocardiogram and the cardiac function indexes were statistically analyzed.Compared with WT,the DCM group was better.In the AAV9+DCM group,the left ventricular percent ejection fraction(LV EF%)and left ventricular percent shortening(LV FS%)decreased by 50.4%(P<0.0001)and 55.1%(P<0.0001),respectively,while in the AAV9+DCM group,the LV EF%and LV FS%increased by 66.5%(P<0.01)and 77.0%(P<0.01),respec

关 键 词：扩张型心肌病 CRISPR/Cas13b RNA编辑技术 TNNT2R141W基因突变 

分 类 号：R542.2[医药卫生—心血管疾病]