利用CRISPR/Cas9基因编辑靶向下调FGFR2缓解Apert综合征小鼠颅缝早闭  

CRISPR/Cas9 mediated FGFR2 down-regulation alleviates premature closure of cranial suture in an Apert syndrome mouse model

作　　者：罗凤涛[1,2] 谢杨丽 李灿[1,2] 谭乔燕 杜晓兰[1,2] 陈林 Luo Fengtao;Xie Yangli;Li Can;Tan Qiaoyan;Du Xiaolan;Chen Lin(Department of Wound Repair and Rehabilitation,Daping Hospital,Third Military Medical University,Chongqing 400042,China;State Key Laboratory of Trauma,Burn and Combined Injury,Third Military Medical University,Chongqing 400038,China)

机构地区：[1]第三军医大学大坪医院,战伤组织修复与康复医学研究室,重庆400042 [2]第三军医大学创伤、烧伤与复合伤国家重点实验室,重庆400038

出　　处：《生物医学转化》2021年第2期63-71,共9页Biomedical transformation

基　　金：国家自然科学基金面上项目(81672125);重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0112,cstc2018jcyjAX0704)。

摘　　要：Apert综合征(Apert Syndrome,AS)是一类常染色体显性遗传疾病,主要表现为颅缝早闭、中面部发育不良、并指(趾)、智力发育障碍等,多由FGFR2分子功能增强型点突变引起。目前,此疾病主要靠手术矫形来恢复功能,其中大脑等病变部位手术难度大,难以实施,且通常需要多次手术。CRISPR/Cas9基因编辑技术在人类遗传病治疗中具有巨大潜力。本研究筛选到靶向FGFR2基因的gRNA,与Cas9经慢病毒包装后作用于Apert小鼠(FGFR2^(+/P253R))的原代颅骨细胞、体外培养的颅骨以及活体小鼠颅骨中,可敲低FGFR2的表达,改善Apert原代颅骨细胞的分化程度。CRISPR/Cas9处理体外培养的颅骨及注射至Apert小鼠头颅,颅骨冠状缝早闭状态有效缓解,异常降低的颅骨骨量、骨密度等也显著改善,表明CRISPR/Cas9基因编辑可缓解Apert小鼠头颅异常。本研究为Apert综合征的基因治疗提供了实验依据,有望为其它骨骼遗传病治疗提供新的策略与借鉴。Apert syndrome(AS) is characterized by synostosis of coronal sutures, midfacial hypoplasia,abnormity of brain, syndactyly of hands and feet. Majority of AS is caused by gain-of-function mutation of fibroblast growth factor receptor 2(FGFR2). To date, the treatment of AS is surgical correction for the deformed skull.The reconstruction of sutures is often executed several times, because of constant premature of sutures. Some diseased tissues, such as brain, are difficult to treat with surgery. CRISPR/Cas9 gene editing technology exhibits powerful prospect in the treatment of human genetic diseases. In this study, small guiding RNA(gRNA) specifically targeting the Fgfr2 gene was screened and lentivirus mediated Cas9/gRNA was employed to treat mice(Fgfr2+/P253R) that mimicking human Apert syndrome in primary calvarial cells, calvarial explants or calvarial bone. In cultured primary calvarial cells, Cas9/gRNA reduced the phosphorylation of ERK1/2 and P38, and resultantly inhibited osteoblastic differentiation and matrix mineralization. Besides, Cas9/gRNA attenuated the premature closure of coronal suture and the decreased calvarial bone volume calvarial explants and AS mice. This study provides experimental basis of gene therapy for Apert syndrome, and is prospective to provide new strategies for the treatment of skeletal genetic diseases.

关 键 词：颅缝早闭 APERT综合征 2型成纤维细胞生长因子受体 CRISPR/Cas9基因编辑 

分 类 号：R459.9[医药卫生—治疗学] Q789[医药卫生—临床医学]