基因治疗药物AAV5-脂蛋白脂酶变异体临床前神经系统安全性评价

Nonclinical evaluation of safety of gene therapy drug AAV5 lipoprotein lipase variant(GC304)in the nervous system

目的通过评价AAV5-脂蛋白脂酶变异体(GC304)腺相关病毒注射液对C57BL/6N小鼠中枢神经系统的影响,以确定受试物可能关系到人的安全性问题。方法共使用40只小鼠,雌雄各半,分别单次尾静脉给予溶媒对照、1.0×10^(13)、5×10^(13)、1.5×10^(14)vg·kg^(-1)GC304腺相关病毒注射液,采用功能观测组合试验方法(FOB),分别在给药前和给药后1、4、24、48、96 h,7、14 d观察小鼠运动功能、感觉功能、自主活动等行为,评价受试物对小鼠中枢神经系统的影响,并在给药后第14天检测血清TG、白蛋白和总蛋白改变。结果本研究前期,采用荧光定量PCR方法检测小鼠脑中目的基因(LPL),其结果显示给予1.5×10^(14)vg·kg^(-1)GC3042周后,仍可在脑区内检测到目的基因(LPL)的拷贝数,基于上述数据开展了GC304行为学检测,其结果显示给予GC304后,可引起与药理作用相关的甘油三酯的明显降低,但在给药后2周内,GC304各剂量组动物笼内观察、手持观察、开放场观察、反射评价各指标均未见明显异常。结论GC304可进入脑区,且给药后14 d内,目的基因在脑区内仍有少量分布,但其不会诱导产生神经毒性,为临床用药提供了参考。

Objective To determine the safety of GC304 in humans by focusing on evaluation of the effect of GC304 on the central nervous system of C57BL/6N mice.Methods A total of 40 mice,half male and half female,were given vehicle control and 1.0×10^(13),5×10^(13),1.5×10^(14)vg·kg^(-1)GC304 through the caudal vein respectively using the function observational battery(FOB)test method.The motor function,sensory function,autonomous activity and other behaviors of mice were observed before administration and 1 h,4 h,24 h,48 h,96 h,7 d and 14 d after administration respectively.In addition,levels of TG,TP and ALB were detected 14 days after administration.Results The results of fluorescent quantitative PCR used in the early stage of this study showed that the copy number of the target genes could still be detected in the brain region two weeks after 1.5×10^(14)vg·kg^(-1)GC304 was given.Based on the above data,the behavioral study of GC304 was carried out.The results showed that after administration of GC304,triglycerides related to pharmacological effects could be significantly reduced.No obvious abnormalities were found during cage observation,hand-held observation,open field observation and ref lex evaluation of animals in any of the dose groups of GC304 two weeks after administration.Conclusion GC304 can be directed into the brain region and a small number of target genes are still distributed in the brain region 14 days after administration,but GC304 will not induce neurotoxicity.