Adenovirus-mediated delivery into myocytes of muscle glycogen phosphorylase, the enzyme deficient in patients with glycogen-storage disease type V.
Adenovirus-mediated delivery into myocytes of muscle glycogen phosphorylase, the enzyme deficient in patients with glycogen-storage disease type V.
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腺病毒介导将肌糖原磷酸化酶输送到肌细胞中，肌糖原磷酸化酶是 V 型糖原储存病患者体内缺乏的酶。
作者：
S. Baqué;C. Newgard;R. Gerard;J. Guinovart;A. M. Gómez
已经检查了使用腺病毒作为用于将糖原磷酸化酶活性引入肌细胞的载体的可行性。我们使用C2 C12成肌细胞系来测定磷酸化酶基因转移对肌细胞糖原代谢的影响，并在体外重现提出的用于治疗肌肉遗传性疾病的两种策略，成肌细胞移植和直接DNA递送。在这项研究中，一个重组腺病毒含有肌糖原磷酸化酶cDNA转录的巨细胞病毒启动子（AdCMV-MGP）被用来培养分化成肌细胞和非分裂成熟肌管细胞。肌糖原磷酸化酶mRNA水平和总磷酸化酶活性增加，在两种细胞类型的病毒治疗后，虽然更有效地在分化的肌管。磷酸化酶活性的增加在成肌细胞中是短暂的（15天），而在肌管中达到更高水平的磷酸化酶基因表达和活性，其在研究期间（20天）保持高于对照水平。肌管中肌磷酸化酶的引入增强了它们的糖原分解能力。AdCMV MGP转导的肌管在基础条件下具有较低的糖原水平。此外，这些工程细胞显示出更广泛的糖原分解反应肾上腺素，刺激糖原磷酸化酶磷酸化，和羰基氰间氯苯腙，代谢解偶联剂。总之，肌糖原磷酸化酶cDNA转移到肌管赋予增强和可调节的糖原分解能力。因此，该系统可能是有用的肌肉糖原磷酸化酶和肌细胞糖原分解的肌肉磷酸化酶缺乏症（麦卡德尔病）患者的恢复交付。
The feasibility of using adenovirus as a vector for the introduction of glycogen phosphorylase activity into myocytes has been examined. We used the C2C12 myoblast cell line to assay the impact of phosphorylase gene transfer on myocyte glycogen metabolism and to reproduce in vitro the two strategies proposed for the treatment of muscle genetic diseases, myoblast transplantation and direct DNA delivery. In this study, a recombinant adenovirus containing the muscle glycogen phosphorylase cDNA transcribed from the cytomegalovirus promoter (AdCMV-MGP) was used to transduce both differentiating myoblasts and nondividing mature myotube cells. Muscle glycogen phosphorylase mRNA levels and total phosphorylase activity were increased in both cell types after viral treatment although more efficiently in the differentiated myotubes. The increase in phosphorylase activity was transient (15 days) in myoblasts whereas in myotubes higher levels of phosphorylase gene expression and activity were reached, which remained above control levels for the duration of the study (20 days). The introduction of muscle phosphorylase into myotubes enhanced their glycogenolytic capacity. AdCMV MGP-transduced myotubes had lower glycogen levels under basal conditions. In addition, these engineered cells showed more extensive glycogenolysis in response to both adrenaline, which stimulates glycogen phosphorylase phosphorylation, and carbonyl cyanide m-chlorophenylhydrazone, a metabolic uncoupler. In conclusion, transfer of the muscle glycogen phosphorylase cDNA into myotubes confers an enhanced and regulatable glycogenolytic capacity. Thus this system might be useful for delivery of muscle glycogen phosphorylase and restoration of glycogenolysis in muscle cells from patients with muscle phosphorylase deficiency (McArdle's disease).