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测量GNE基因强度和骨肌肉中的基因替换的测试方法的开发

Deborah A Zygmunt¹, Patricia Lam¹, Anna Ashbrook¹

¹Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, Children's Drive, Columbus, OH, USA.

Journal of neuromuscular diseases

|July 17, 2023

概括

使用腺相关病毒 (AAV) 载体的基因疗法通过恢复肌肉细胞中酸 (Sia) 生产来治疗GNE肌肉病的前景. 有构成性促进体的AAV载体有效地传递GNE基因用于Sia生物合成,为这种严重的肌肉疾病提供了潜在的治疗方法.

科学领域:

分子生物学分子生物学
基因治疗基因治疗
生物化学生物化学

背景情况:

基因内核肌病 (GNEM) 是一种严重的遗传性肌肉疾病,由UDP-GlcNAc-2-epimerase/ManNAc-6-kinase (GNE) 基因的突变引起.
GNE基因编码了一种关键的酶,用于酸 (Sia) 生物合成,这对肌肉功能至关重要.
缺陷的Sia生产导致了在GNE肌肉病变中观察到的病理.

研究的目的:

开发和验证测试,以评估腺相关病毒 (AAV) 基因治疗载体在恢复Sia生物合成中的疗效.
确定AAV载体的最佳剂量,以在骨肌肉中用人类GNE替代内源性小鼠GNE基因表达.
通过AAV载体比较不同促进剂在驱动GNE基因表达和Sia产生方面的有效性.

主要方法:

建立并利用GNE缺陷细胞系 (Lec3MyoDI和人类肌肉细胞系) 来评估AAV载体功效.
使用Sia特异性乳素结合试验 (MAA和SNA) 来量化Sia的产量.
在野生型小鼠的静脉输送后,使用qPCR和qRT-PCR进行量化AAV生物分布和GNE基因表达.

主要成果:

AAV载体成功地增加了GNE缺乏细胞中的Sia产量,由剂量依赖的MAA增强的乳素结合证明.
与肌肉特异性促进剂 (MCK,MHCK7) 相比,构成性CMV促进剂显示出较高的Sia产生的诱导.
一种特定的AAV载体 (rAAVrh74.CMV.GNE) 以1x10^13vg/kg的剂量在肌肉中实现了人类GNE表达的治疗水平,超过了肌肉特异性促进剂.

结论:

缺乏GNE的细胞系是评估Sia生产中AAV载体的功效的有效工具.
系统输送的rAAVrh74.CMV.GNE在骨肌肉中提供有效的GNE基因替代,而不会在非肌肉组织中过度表达.
使用构成性促进体的AAV载体代表了基因疗法治疗GNE肌病的有希望的策略.

关键词:

在 AAV AAV AAV 中.在GNE神经病变上.基因治疗的基因疗法肌肉发育不良肌肉发育不良肌肉发育不良酸是酸的一种.

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