重组2型腺相关病毒肿瘤坏死因子相关凋亡诱导配体基因治疗制剂的质量分析  

Analysis on quality of recombinant adeno-associated virus 2 encoding tumor necrosis factor-related apoptosis-induced ligand

作　　者：付志浩[1] 高凯[1] 李永红[1] 李响[1] 陶磊[1] 王兰[1] 丁有学[1] 郭莹[1] 饶春明[1] 

机构地区：[1]中国食品药品检定研究院,北京100050

出　　处：《中国生物制品学杂志》2015年第5期501-504,509,共5页Chinese Journal of Biologicals

基　　金：国家"863"计划课题(2012AA020805)

摘　　要：目的对重组2型腺相关病毒肿瘤坏死因子相关凋亡诱导配体(recombinant adeno-associated virus 2 encoding tumor necrosis factor-related apoptosis-induced ligand,r AAV2-TRAIL)基因治疗制剂进行质量分析,并针对其关键质量属性建立质控方法。方法采用PCR法对r AAV2-TRAIL基因治疗制剂中插入的启动子CAG和目的基因TRAIL进行鉴别;在腺病毒(Ad5)的辅助下,将r AAV2-TRAIL体外感染293T细胞后,采用ELISA法检测培养上清中目的蛋白TRAIL的表达量,检测r AAV2-TRAIL对神经胶质瘤U251细胞的体外杀伤活性;设计引物及探针,采用Q-PCR法检测r AAV2-TRAIL基因治疗制剂中的r AAV2-TRAIL滴度、可能存在的复制型rc AAV滴度以及残余辅助病毒1型单纯疱疹病毒(herpes simplex virus 1,HSV1)滴度。结果启动子CAG和目的基因TRAIL的PCR鉴定结果与理论及理化对照品相符;r AAV2-TRAIL感染293T细胞48 h后,上清中TRAIL表达量为(0.36±0.18)ng/ml,RSD为50.0%;r AAV2-TRAIL体外作用于神经胶质瘤细胞U251,细胞生长相对抑制率为(26.6±3.75)%,RSD为14.1%;制剂中r AAV2-TRAIL滴度为(4.72×1011±0.52×1011)copies/ml,RSD为11.0%,rc AAV滴度为(2.49×107±0.18×107)copies/ml,RSD为7.2%,HSV1滴度为(6.02×106±0.51×106)copies/ml,RSD为8.5%。r AAV2-TRAIL/rc AAV为1.90×104,r AAV2-TRAIL/HSV1为7.84×104。结论建立的质控方法可用于r AAV2-TRAIL的质量控制,为该产品质量标准的建立奠定了基础,同时对以AAV为载体的基因治疗制剂的质量研究提供参考。Objective To analyze the quality and develop the methods for essential quality control of recombinant adeno-associated virus 2 encoding tumor necrosis factor-related apoptosis-induced ligand（r AAV2-TRAIL）. Methods The inserted promoter CAG and target gene TRAIL in r AAV2-TRAIL were identified by PCR. The 293 T cells were infected by r AAV2-TRAIL with the help of adenovirus（Ad5）in vitro, and determined for the expression level of TRAIL in culture supernatant by ELISA. The killing activity in vitro of r AAV2-TRAIL to glioma U251 cells was determined. The titers of r AAV2-TRAIL, replication competent AAV（rc AAV）and residual herpes simplex virus 1（HSV1）in r AAV2-TRAIL were determined by Q-PCR. Results The identification results of CAG and TRAIL by PCR were consistent with those in theory and those of physiochemical reference. The expression level of TRAIL in supernatant of 293 T cells 48 h after infection with r AAV2-TRAIL was（0. 36 ± 0. 18） ng / ml, with a RSD of 50. 0%. The relative growth-inhibiting rate of U251 cells by r AAV2-TRAIL was（26. 6 ± 3. 75）%, with a RSD of 14. 1%. The titer of r AAV2-TRAIL was（4. 72 × 10^11 ±0. 52 × 10^11 copies / ml）, with a RSD of 11. 0%, while that of rc AAV was（2. 49 × 10^7 ± 0. 18 × 10^7）copies / ml, with a RSD of 7. 2%, and that of HSV1 was（6. 02 × 10^6 ± 0. 51 × 10^6）copies / ml, with a RSD of 8. 5%. The ratio of r AAV2-TRAIL to rc AAV was 1. 90 × 10^4, while that of r AAV2-TRAIL to HSV1 was 7. 84 × 10^4. Conclusion The developed method may be used for the quality control of r AAV2-TRAIL, which lays a foundation of development of quality standard for the drug and provides a reference for quality study of other AAV-vectored gene therapy products.

关 键 词：腺相关病毒 肿瘤坏死因子相关凋亡诱导配体 基因治疗 质量控制 

分 类 号：Q78[生物学—分子生物学] R730.53[医药卫生—肿瘤]