AAV载体介导的蓬佩病模型小鼠体内基因治疗研究

AAV Vector Mediated Gene Therapy in Pompe Model Mice

目的:蓬佩病是一种由酸性α-葡糖苷酶(GAA)缺乏引起的溶酶体糖原贮积症,病理特征是糖原在心脏、骨骼肌和中枢神经系统中累积。基因治疗有望成为治疗蓬佩病的突破性手段。采用AAV9载体在蓬佩病模型小鼠体内介导GAA基因转移,评估转基因干预后小鼠体内GAA酶活力变化、组织糖原累积及病理改变。方法:采用AAV9载体携带密码子优化的GAA基因(coGAA),通过杆状病毒生产工艺包装AAV病毒载体rAAV9-coGAA,分别以1.1×10^(13)vg/kg、3.0×10^(13)vg/kg、1.2×10^(13)vg/kg的剂量静脉单次注射给予成年蓬佩病模型小鼠,以3.0×10^(14)vg/kg的剂量静脉单次注射给予老龄蓬佩病模型小鼠。到达试验终点后安乐死小鼠,使用荧光分光光度法测定GAA酶活力、PAS染色观察糖原累积、HE染色检查病理变化。结果:成年模型小鼠给药5周后,各个组织能够广泛表达具有活性的GAA,其中心脏、肝脏表达水平较高,脑和脊髓表达水平较低。转基因干预后心肌、骨骼肌与脑中的糖原含量下降,心肌、骨骼肌的空泡样变性显著减少。老龄小鼠治疗后,组织酶活力与模型小鼠相比显著提升,心肌的空泡样变性和炎细胞浸润减少,但骨骼肌的病理改善有限。结论:静脉单次注射rAAV9-coGAA在蓬佩病模型小鼠中能够提升GAA酶活力,减少糖原累积并改善病理,治疗效果呈剂量依赖,对老龄小鼠也有一定治疗效果。为AAV9静脉递送GAA基因治疗蓬佩病的临床应用奠定了基础。

Objective:Pompe disease is a lysosomal glycogen storage disease caused by acidα-glucosidase(GAA)deficiency,which is characterized by glycogen accumulation in the heart,skeletal muscle,and central nervous system(CNS).AAV vector-mediated gene therapy is expected to be a breakthrough in the treatment of Pompe disease.In this study,AAV9 vector was used to mediate GAA gene transfer in Pompe disease model mice,and the changes of GAA protease activity,glycogen accumulation in tissues and pathological changes in mice after transgenic intervention were evaluated.Methods:Codon optimized GAA gene(coGAA)was carried by AAV9 vector,and the AAV vector was packaged by baculovirus production process.Adult Pompe model mice were given a single intravenous injection at the dose of 1.1×10^(13),3.0×10^(13),1.2×10^(13)vg/kg,and aged Pompe model mice were given a single intravenous injection at the dose of 3.0×10^(14)vg/kg.After reaching the end point of the experiment,the mice were euthanized,GAA protease activity was determined by fluorescence spectrophotometry,glycogen accumulation was observed by PAS staining,and pathological changes were detected by HE staining.Results:Five weeks after administration,GAA protein was widely expressed in all tissues of adult model mice,with higher expression levels in heart and liver,and lower expression levels in brain and spinal cord.After rAAV9-coGAA treatment,glycogen content in myocardium,skeletal muscle and brain decreased,and vacuolar degeneration in myocardium and skeletal muscle decreased significantly.After treatment,the tissue enzyme activity of the aged animals was significantly increased compared with that of the model mice.The vacuolar degeneration and inflammatory cell infiltration of the myocardium were decreased,but the pathological improvement of skeletal muscle was limited.Conclusion:A single intravenous injection of rAAV9-coGAA can enhance GAA enzyme activity,reduce glycogen accumulation and improve pathology in Pompe model mice.The therapeutic effect was dose-dependent,and the injection also had certain therapeutic effect on aged animals.This study laid a theoretical foundation for the clinical application of AAV9 mediated gene therapy via intravenous route in Pompe disease.