人体肝脏靶向性新型AAV血清型载体的研发  

Development of a Novel AAV Serotype Vector for Human Targeted Liver

作　　者：宁秦洁 陈颖 闫梦迪 胡泽岚 吴侠 杜增民 郑静[1] 肖啸 NING Qinjie;CHNE Ying;YAN Mengdi;HU Zelan;WU Xia;DU Zengmin;ZHENG Jing;XIAO Xiao(Laboratory of Molecular Neuropharmacology,School of Pharmacy,East China University of Science and Technology,Shanghai 200237,China)

机构地区：[1]华东理工大学药学院,神经分子药理学实验室,上海200237

出　　处：《华东理工大学学报（自然科学版）》2020年第3期404-410,共7页Journal of East China University of Science and Technology

基　　金：中央高校基本科研业务费专项资金资助。

摘　　要：通过DNA shuffling技术选择8种AAV(AAV1~AAV4,AAV6~AAV9)cap基因序列进行cap基因的DNA改组,获得AAV突变体基因库。将突变体基因库插入到含有AAV2Rep基因和ITR序列的质粒中,最终获得了大于107的独立克隆(具有全套AAV基因组序列)。通过转化实验获得随机感染质粒文库(pIRC)。将所得质粒经过共转染HEK293细胞,72 h后收集纯化病毒,获得感染性AAV文库。利用野生型5型腺病毒(Wild Type Ad5,wt Ad5)辅助感染人肝癌Hep G2细胞,进行筛选后,最终在肝细胞中富集获得1种新型AAV血清载体AAVXL12。通过将携带GFP基因的不同AAV血清型载体感染Hep G2细胞,观察到不同程度的绿色荧光的表达,最终显示筛选到的AAV血清型载体AAVXL12介导的GFP基因表达效果最好,荧光强度最强。In recent years,the number of clinical trials of adeno-associated virus(AAV)vectors for gene transfer in vivo has steadily increased.Excellent safety and efficient transduction of a wide range of target tissues make AAV vectors to be the platform of choice for gene therapy in vivo.However,due to the poor selectivity of AAV vector serotypes and target cells,the presence of AAV neutralizing antibodies and the continuous occurrence of immune responses,the effect of gene therapy is seriously affected.DNA shuffling was employed to produce an AAV cap gene library based on eight different AAV(AAV1-AAV4,AAV6-AAV9)capsid genes.DNA sequences in the mutant gene library were inserted into the plasmid containing the AAV2 Rep gene and the ITR sequence,and the resulting plasmid(pIRC)has a full set of AAV genomic sequences.The purified virus was collected after cotransfection with HEK 293 cells for 72 h hours to obtain an AAV mutant library.Human liver cancer Hep G2 cells were assisted by wild type 5 adenovirus(wt Ad5).After screening,we finally obtained a capsid protein mutant AAVXL12 in hepatocytes.By infecting Hep G2 cells with different serotypes of recombinant AAV virus carrying the GFP gene,we observed different degrees of green fluorescence expression,which finally showed that the serotype vector AAVXL12 mediated GFP gene expression was the best.Therefore,the new AAV vector,which is highly infectious to human liver cells,has enhanced the efficiency of gene transduction and is expected to provide a novel therapeutic vector for AAV for gene therapy.

关 键 词：腺相关病毒(AAV) 基因治疗 新型AAV血清型 DNA shuffling 靶向 肝脏细胞 

分 类 号：R450[医药卫生—治疗学]