人β-半乳糖苷酶R299L突变体的获得与活性研究*

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。

Acquisition and Activity Exploration of Human β-Galactosidase R299L Mutant

Objective: GM1 gangliosidosis is a devastating lysosomal storage disease featured by progressive and fatal neurodegeneration, caused by a mutation in the galactosidase beta 1 (GLB1) gene that reduces the activity of human beta-galactosidase. Currently, no effective therapy exists for the affected individuals, and AAV gene therapy is viewed as the most promising method. The object of this research is to obtain a mutant of GLB1 with higher β-gal activity through genetic modification, so as to be used in subsequent AAV-mediated gene therapy. Methods: The sequence of galactosidase beta 1 gene from human and other six vertebrates was analyzed by multiple sequence alignment, and some amino acid sites were selected for modification. HEK-293 cells were transfected or infected with either the recombinant plasmid or rAAV9 carrying the mutant site, and the activity of the mutant was compared with that of the control. The rAAV9 virus carrying coGLB1-R299L was injected into GM1 mice to explore the expression of the mutant in vivo. Results: The coGLB1-R299L mutant was screened from 15 mutants and then transfected into HEK-293 cells. It displayed higher β-gal activity with a 30%~40% increase compared with that of the wild-type amino acid sequence. The β-gal activity of rAAV9-coGLB1-R299L group was 2.2 times higher than that of the cell control group in the AAV infection experiment in vitro. The results in vivo showed that rAAV9-coGLB1-R299L was widely expressed in the GM1 mice, and the β-gal enzyme activities in the heart, liver, spleen, lung and brain tissue were significantly increased. Conclusion: A mutant coGLB1-R299L with higher β-gal activity is obtained, and the in vitro expression and distribution of enzyme activity of rAAV9-coGLB1-R299L in model mice was preliminarily explored, laying a foundation for the application of this mutant in the treatment of AAV-mediated GM1 gangliosidosis.