Acquisition and Activity Exploration of Human β-Galactosidase R299L Mutant

doi:10.13523/j.cb.2203055

China Biotechnology

2022, Vol. 42

Issue (9): 39-49 DOI: 10.13523/j.cb.2203055

Acquisition and Activity Exploration of Human β-Galactosidase R299L Mutant

ZHANG Yan^1,²,MA Wen-hao²,ZHAO Tian-yi²,WU Xiao-bing^2,^**(

),SHENG Wang¹,YANG Yi-shu^1,^**(

)

1. Beijing University of Technology, Beijing 100124, China
2. Beijing GeneCradle Pharmaceutical Co., Ltd., Beijing 100023, China

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Abstract

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.

Key words： GM1 gangliosidosis Galactosidase beta 1 gene β-galactosidase Adeno-associated virus vector Site directed mutation

Received: 24 March 2022 Published: 10 October 2022

ZTFLH:

Q819

Corresponding Authors: Xiao-bing WU,Yi-shu YANG E-mail: wuxiaobing@bj;yishu-y@bjut.edu.cn

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	Yan ZHANG
	Wen-hao MA
	Tian-yi ZHAO
	Xiao-bing WU
	Wang SHENG
	Yi-shu YANG

Cite this article:

ZHANG Yan,MA Wen-hao,ZHAO Tian-yi,WU Xiao-bing,SHENG Wang,YANG Yi-shu. Acquisition and Activity Exploration of Human β-Galactosidase R299L Mutant. China Biotechnology, 2022, 42(9): 39-49.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2203055 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I9/39

Table 1 Primers used for constructing mutants

Fig.1 Design and construction of GLB1 mutants (a)β-Gal protein sequence alignment of human, zebrafish, mouse, cat, dog, pig, and rhesus monkey; arrows, selected 12 GLB1 gene mutation sites (b) Nucleic acid sequencing results (c) Schematic diagram of the plasmid carrying GLB1 gene

Fig.2 Activity of β-gal of GLB1 mutants (a) Activity of β-gal of various GLB1 mutants (b) Dose-effect relationship for R299L, control, untransfected HEK-293 cells

Fig.3 In situ staining of cells expressing β-gal with X-gal Control: Untransfected HEK-293 cells

Fig.4 Comparison of enzyme activities of four mutants at the 299^th amino acid (a) Expression of β-gal in transfected cells (b) Enzyme activity of HEK-293 cells transfected with mutants at the 299^th amino acid. Control: Untransfected HEK-293 cells

Fig.5 AAV9 mediates the in vitro expression of β-gal by coGLB1-R299L (a) Titer detection of rAAV9-coGLB1-R299L by dot blot. Standard: Plasmids with concentration of 2×10¹², 1×10¹², 5×10¹¹, 2.5×10¹¹, 1.25×10¹¹, 6.25×10¹⁰, 3.125×10¹⁰ vg/mL (b) HEK-293 cell β-gal activity infected by rAAV9-coGLB1-R299L. Control: Untransfected HEK-293 cells

Fig.6 AAV9 mediates the expression of β-gal by coGLB1 and R299L mutant in model mouse (a) X-gal staining of frozen section of liver tissue. Scale: 100 μm (b) The enzyme activities of mouse heart, liver, spleen, lung and kidney (c) The enzyme activities of mouse brain (d) The enzyme activities of mouse serum. WT+PBS: PBS injected wide type C57BL/6N; GM1 mice+PBS: PBS injected homozygous G455R model mouse; GM1 mice+AAV: rAAV9-coGLB1-R299L injected homozygous G455R model mouse


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