摘要
遗传视网膜退行性疾病是全球范围内最主要的致盲疾病.该疾病主要的特征是视网膜感光细胞退化导致了不可逆的视觉能力丧失.视网膜色素变性(Retinitis pigmentosa,RP)便是其中的一大亚类,目前临床上尚无有效的治疗手段.本研究首次利用腺相关病毒(Adeno Associated Virus,AAV)介导的CRISPR/SaCa9技术,成功实现原位敲除目的基因RHO,建立了非人灵长类RP模型.研究结果发现,RHO敲除的猕猴视网膜表现出典型的RP感光细胞变性,视杆和视锥细胞渐行性死亡.在分子水平上,RHO基因成功地被敲除.RHO蛋白表达量也大幅度下降.免疫组化和电镜结果揭示了突变视杆细胞外段缺失或者变短,再现了RP最典型的特征.生理功能检测发现,RHO敲除视网膜的视网膜电流图(Electroretinogram,ERG)反应大幅度下降,证明了其视觉能力的受损.该动物模型为研究人类RP的发病机理以及制定相应治疗策略提供了重要的类人模型.
Retinitis pigmentosa(RP)is a form of inherited retinal degenerative diseases that ultimately involves the macula,which is present in primates but not in the rodents.Therefore,creating nonhuman primate(NHP)models of RP is of critical importance to study its mechanism of pathogenesis and to evaluate potential therapeutic options in the future.Here we applied adeno-associated virus(AAV)-delivered CRISPR/SaCas9 technology to knockout the RHO gene in the retinae of the adult rhesus macaque(Macaca mulatta)to investigate the hypothesis whether non-germline mutation of the RHO gene is sufficient to recapitulate RP.Through a series of studies,we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression.More importantly,the mutant macaque retinae displayed clinical RP phenotypes,including photoreceptor degeneration,retinal thinning,abnormal rod subcellular structures,and reduced photoresponse.Therefore,we suggest somatic editing of the RHO gene is able to phenocopy RP,and the reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.
作者
李守振
胡英周
李云琴
胡敏
王文超
马玉乾
才源
卫敏
姚艺川
王云
董凯
顾永昊
赵欢
鲍进
仇子龙
章梅
胡新天
薛天
Shouzhen Li;Yingzhou Hu;Yunqin Li;Min Hu;Wenchao Wang;Yuqian Ma;Yuan Cai;Min Wei;Yichuan Yao;Yun Wang;Kai Dong;Yonghao Gu;Huan Zhao;Jin Bao;Zilong Qiu;Mei Zhanga;Xintian Hu;Tian Xue(Hefei National Laboratory for Physical Sciences at the Microscale,Eye Center at The First Affiliated Hospital of USTC,School of Life Sciences,Division of Life Sciences and Medicine,University of Science and Technology of China,Hefei 230026,China;Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences&Yunnan Province.Kunming Institute of Zoology.Chinese Academy of Sciences,Kunming 650223,China;Second People's Hospital of Yunnan Province,Yunnan Eye Institute,Key Laboratory of Yunnan Province for the Prevention and Treatment of Ophthalmology,Kunming 650223,China;Kunming Primate Research Center,Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming 650223,China;Center for Excellence in Brain Science and Intelligence Technology,Chinese Academy of Sciences,Shanghai 200031,China;Department of Biological and Environmental Engineering,Hefei University,Hefei 230601,China;Institute for Stem Cell and Regeneration,Chinese Academy of Sciences,Beijing 100101,China;Institute of Neuroscience,State Key Laboratory of Neuroscience,Chinese Academy of Sciences,Shanghai 200031,China;National Research Facility for Phenotypic&Genetic Analysis of Model Animals(Primate Facility),Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming 650107,China;Neurodegenerative Disorder Research Center,CAS Key Laboratory of Brain Function and Disease,University of Science and Technology of China,Hefei 230026,China)
基金
the National Key Research and Development Program of China(2020YFA0112200,2016YFA0400900,and 2018YFA0801403)
the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA16020603,XDB39000000,and XDB32060200)
the National Natural Science Foundation of China(81925009,81790644,61890953,31322024,81371066,91432104,81900855,31900712,and 31800901)
Guangdong Provincial Key Research and Development Program(2019B030335001 and 2018B030338001)
Anhui Provincial Natural Science Foundation(1808085MH289 and 1908085MC66)
the Fundamental Research Funds for the Central Universities(WK2070000174 and WK2090050048)。
