AAV Vector Mediated Gene Therapy in Pompe Model Mice

doi:10.13523/j.cb.2202040

China Biotechnology

2022, Vol. 42

Issue (7): 24-34 DOI: 10.13523/j.cb.2202040

AAV Vector Mediated Gene Therapy in Pompe Model Mice

Zhi-jie WU^1,²,Wen-hao MA²,Zhe-yue DONG²,Xiao-bing WU^2,^**(

),Yi-shu YANG^1,^**(

),Wang SHENG¹

1. Beijing University of Technology, Beijing 100124,China
2. Beijing GeneCradle Pharmaceutical Co Ltd, Beijing 100176,China

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Abstract

Objective: Pompe disease is a lysosomal glycogen storage disease caused by acid α-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 1.1×10¹³, 3.0×10¹³, 1.2×10¹⁴ vg/kg, and aged Pompe model mice were given a single intravenous injection at the dose of 3.0×10¹³ 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.

Key words： Pompe disease Lysosomal acid alpha-glucosidase Gene therapy AAV9 Model mice

Received: 24 February 2022 Published: 03 August 2022

ZTFLH:

Q819

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

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	Zhi-jie WU
	Wen-hao MA
	Zhe-yue DONG
	Xiao-bing WU
	Yi-shu YANG
	Wang SHENG

Cite this article:

Zhi-jie WU,Wen-hao MA,Zhe-yue DONG,Xiao-bing WU,Yi-shu YANG,Wang SHENG. AAV Vector Mediated Gene Therapy in Pompe Model Mice. China Biotechnology, 2022, 42(7): 24-34.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2202040 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I7/24

Fig.1 In vitro expression of GAA by rAAV9-coGAA (a)Structure diagram of recombinant AAV virus vector (b)Western blot of GAA in BHK-21 cells infected with rAAV9-coGAA (c)GAA activity in BHK-21 cells infected with rAAV9-coGAA

Fig.2 GAA activity in Pompe model mice after administration of rAAV9-coGAA (a) GAA activity in adult Pompe model mice (b) GAA activity in aged Pompe model mice GAA^-/-:Pompe model mice;WT:Wild-type B6.129 mouse;BQL:Below detection limit

Fig.3 Immunofluorescence staining in adult Pompe model mice at 5 weeks after administration of rAAV9-coGAA GAA^-/-:Pompe model mice;PND:Postnatal day;PI:Post-injection; Blue fluorescence: DAPI positive nucleus; Red fluorescence:GAA immunofluorescence

Fig.4 PAS staining in adult Pompe model mice at 5 weeks after administration of rAAV9-coGAA (a)PAS staining in quadriceps femoris (b)PAS staining in heart (c)PAS staining in brain tissue GAA^-/-:Pompe model mice;PND:Postnatal day;PI:Post-injection

Fig.5 HE staining in Pompe model mice after administration of rAAV9-coGAA (a)HE staining in heart 5 weeks after administration of rAAV9-coGAA (b)HE staining in quadriceps femoris 5 weeks after administration of rAAV9-coGAA (c) HE staining in aged Pompe model mice 8 weeks after administration GAA^-/-:Pompe model mice;PND:Postnatal day;PI:Post-injection


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