Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, 121205, Russia; Atlas Biomed Group Limited, Tintagel House, 92 Albert Embankment, Lambeth, SE1 7TY, London, United Kingdom.
Laboratory of Modeling and Gene Therapy of Hereditary Diseases, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia; Marlin Biotech LLC, Moscow, 143026, Russia.
Exp Cell Res. 2020 Jul 15;392(2):112033. doi: 10.1016/j.yexcr.2020.112033. Epub 2020 Apr 29.
AAV-delivered microdystrophin genes hold great promise for Duchenne muscular dystrophy (DMD) treatment. It is anticipated that the optimization of engineered dystrophin genes will be required to increase the efficacy and reduce the immunogenicity of transgenic proteins. An in vitro system is required for the efficacy testing of genetically engineered dystrophin genes. We report here on the proof of concept for an in vitro assay based on the assessment of sarcolemma damage after repetitively applied electrical stimuli. The primary cell culture of myoblasts was established from wild-type C57BL/10ScSnJ and dystrophin-deficient mdx mice. The preparation parameters and the differentiation of contractile myotubes were optimized. DAPI and TO-PRO-3 dyes were used to assess myotubular membrane permeability in response to electrical pulse stimulation (EPS). Myotubes derived from mdx mice exhibited a greater increase in membrane damage, as assessed by TO-PRO-3-measured permeability after EPS, than was exhibited by the healthy control myotubes. AAV-DJ particles carrying the microdystrophin gene were used to transduce mdx-derived differentiated myotubes. Microdystrophin delivery ameliorated the disease phenotype and reduced the EPS-induced membrane damage to a level comparable to that of the healthy controls. Thus, the in vitro system was shown to be capable of supporting studies on DMD gene therapy.
腺相关病毒（AAV）递送的微小肌营养不良蛋白基因在杜氏肌营养不良症（DMD）的治疗中具有巨大的应用前景。预计需要对工程化的肌营养不良蛋白基因进行优化，以提高转基因蛋白的疗效并降低其免疫原性。需要体外系统来测试基因工程化肌营养不良蛋白基因的疗效。我们在此报告了一种基于反复电刺激后评估肌膜损伤的体外检测方法的概念验证。从小鼠的野生型 C57BL/10ScSnJ 和肌营养不良蛋白缺陷型 mdx 中建立了成肌细胞的原代细胞培养。优化了制备参数和收缩性肌管的分化。使用 DAPI 和 TO-PRO-3 染料评估电脉冲刺激（EPS）后肌管膜通透性的变化。与健康对照组相比，来自 mdx 小鼠的肌管在 EPS 后通过 TO-PRO-3 测量的通透性显示出更大的膜损伤增加。携带微小肌营养不良蛋白基因的 AAV-DJ 颗粒被用于转导 mdx 来源的分化肌管。微小肌营养不良蛋白的传递改善了疾病表型，并将 EPS 诱导的膜损伤降低到与健康对照组相当的水平。因此，体外系统被证明能够支持 DMD 基因治疗的研究。
