基因治疗药物AAV5-脂蛋白脂酶变异体在小鼠体内的毒性研究  

Toxicity of gene therapy drug AAV5-LPLS447X in mice

作　　者：侯田田 马思思 吴小兵 霍桂桃[1] 潘东升[1] 王超[1] 夏艳 刘艺 周晓冰[1] 刘国庆 耿兴超[1] HOU Tiantian;MA Sisi;WU Xiaobing;HUO Guitao;PAN Dongsheng;WANG Chao;XIA Yan;LIU Yi;ZHOU Xiaobing;LIU Guoqing;GENG Xingchao(National Center for Safety Evaluation of Drugs,National Institutes for Food and Drug Control,Beijing Key Laboratory for Safety Evaluation of Drugs,Beijing 100176,China;Genecradle Therapeutics Inc,Beijing 100176,China)

机构地区：[1]中国食品药品检定研究院国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京100176 [2]北京锦篮基因科技有限公司,北京100176

出　　处：《中国药物警戒》2023年第1期19-26,共8页Chinese Journal of Pharmacovigilance

基　　金：国家重点研发计划(2021YFA1101602)。

摘　　要：目的评估基因治疗药物AAV5-脂蛋白脂酶变异体(GC304)在小鼠体内的毒性。方法(1)急性毒性研究中,40只C57BL/6N小鼠随机分为2组,分别为溶媒组和受试物组,单次尾静脉注射后,进行连续15 d临床症状观察、体重和摄食量测定,于第15天剖检。(2)长期毒性研究中,440只小鼠随机分为4组,分别设置溶媒组,受试物低剂量组(1×10^(13)vg·kg^(-1))、中剂量组(5×10^(13)vg·kg^(-1))和高剂量组(1.5×10^(14)vg·kg^(-1)),单次尾静脉注射后,对动物体重、摄食量、血液学、血清生化、免疫毒性及免疫原性等毒性指标进行测定。给药后4周和6个月时解剖,动物进行组织病理学检查,并在2、4周,3、6个月采用q PCR的方法检测GC304在动物各个脏器的分布和表达情况。结果(1)急性毒性研究中,动物临床症状、体重、摄食量未见异常,未见与GC304相关的大体病理学改变。(2)长期毒性研究中,动物临床症状、体重、摄食量、细胞因子和T淋巴细胞亚群未见与GC304相关的明显异常。GC304能够降低血浆中甘油三酯以及与抗药抗体相关的白蛋白/球蛋白比值下降,能够诱导抗AAV5结合抗体和中和抗体的产生,并持续至给药后6个月。给予GC304后,未检测到抗脂蛋白脂肪酶(LPL)抗体。组织病理学结果显示,小鼠给药后4周,注射部位出现与药物相关的炎性细胞浸润,至给药后6个月可见恢复。与药物相关的改变为脾脏生发中心活跃伴易染体巨噬细胞增多、腹股沟淋巴结生发中心活跃伴易染体巨噬细胞增多。qPCR检测结果显示,GC304在小鼠外周血及脏器广泛分布和表达,但在肝脏中分布和表达显著高于其他脏器。结论C57BL/6N小鼠单次静脉给予GC304后,主要在肝脏中表达和分布,并且动物耐受性良好,未见明显毒性反应,为该药物进入临床试验提供参考。Objective To assess the toxicity of gene therapy drug AAV5-LPLS447X(GC304)in mice.Methods(1)Acute toxicity study:40 C57BL/6N mice were randomly divided into 2 groups:the vehicle group and the test substance group.After a single tail vein injection,clinical symptoms,body weight and food intake were measured for 15 consecutive days,and necropsy was performed on the 15thday.(2)Long-term toxicity study:440 mice were randomly divided into 4 groups:the vehicle group,low-dose group(1×10^(13)vg·kg^(-1)),middle-dose group(5×10^(13)vg·kg^(-1))and highdose group(1.5×10^(14)vg·kg^(-1)).After a single tail vein injection,such indicators of toxicity as body weight,food intake,hematology,serum biochemistry,immunotoxicity and immunogenicity were measured.The mice were dissected at 4 weeks and 6 months after administration before being examined histopathologically.The distribution and expression of GC304 in various organs of the animals were detected by qPCR at 2 weeks,4 weeks,3 months,and 6months.Results(1)In the acute toxicity study:the clinical symptoms,body weight and food intake of the animals were normal,and no gross pathological changes related to GC304 were found.(2)In the long-term toxicity study:no obvious abnormalities related to GC304 were found in clinical symptoms,body weight,food intake,cytokines or T lymphocyte subsets.GC304 reduced plasma triglycerides and the albumin/globulin ratio associated with anti-drug antibodies,and induced the production of anti-AAV5-binding antibodies and neutralizing antibodies,which persisted up to 6 months after administration.After administration of GC304,no anti-LPL antibodies were detected.Histopathological results showed that drug-related inf lammatory cell infiltration occurred at the injection site 4 weeks after administration in mice,and recovery was seen 6 months after administration.Drugrelated changes were an active germinal center in the spleen with increased predisposition macrophages,and an active germinal center in the inguinal lymph nodes with increased predispose

关 键 词：基因治疗 AAV5-脂蛋白脂酶变异体 毒性研究 小鼠 

分 类 号：R965.3[医药卫生—药理学]