Genotarget LLC, Skolkovo Innovation Center, 121205 Moscow, Russia.
PJSC Human Stem Cells Institute, 129110 Moscow, Russia.
Int J Mol Sci. 2023 Aug 31;24(17):13551. doi: 10.3390/ijms241713551.
Dysferlinopathy treatment is an active area of investigation. Gene therapy is one potential approach. We studied muscle regeneration and inflammatory response after injection of an AAV-9 with a codon-optimized DYSF gene. A dual-vector system AAV.DYSF.OVERLAP with overlapping DYSF cDNA sequences was generated. Two AAV vectors were separately assembled by a standard triple-transfection protocol from plasmids carrying parts of the DYSF gene. Artificial myoblasts from dysferlin-deficient fibroblasts were obtained by MyoD overexpression. RT-PCR and Western blot were used for RNA and protein detection in vitro. A dysferlinopathy murine model (Bla/J) was used for in vivo studies. Histological assay, morphometry, and IHC were used for the muscle tissue analysis. Dysferlin was detected in vitro and in vivo at subphysiological levels. RT-PCR and Western Blot detected dysferlin mRNA and protein in AAV.DYSF.OVERLAP-transduced cells, and mRNA reached a 7-fold elevated level compared to the reference gene (GAPDH). In vivo, the experimental group showed intermediate median values for the proportion of necrotic muscle fibers, muscle fibers with internalized nuclei, and cross-sectional area of muscle fibers compared to the same parameters in the control groups of WT and Bla/J mice, although the differences were not statistically significant. The inverse relationship between the dosage and the severity of inflammatory changes in the muscles may be attributed to the decrease in the number of necrotic fibers. The share of transduced myofibers reached almost 35% in the group with the highest dose. The use of two-vector systems based on AAV is justified in terms of therapeutic efficacy. The expression of dysferlin at a subphysiological level, within a short observation period, is capable of inducing the restoration of muscle tissue structure, reducing inflammatory activity, and mitigating necrotic processes. Further research is needed to provide a more detailed assessment of the impact of the transgene and viral vector on the inflammatory component, including longer observation periods.
肌营养不良症的治疗是一个活跃的研究领域。基因治疗是一种潜在的方法。我们研究了注射经过密码子优化的 DYSF 基因的 AAV-9 后肌肉再生和炎症反应。生成了具有重叠 DYSF cDNA 序列的双载体系统 AAV.DYSF.OVERLAP。通过标准的三转染方案从携带 DYSF 基因部分的质粒上分别组装两个 AAV 载体。通过 MyoD 过表达从肌营养不良蛋白缺陷型成纤维细胞中获得人工成肌细胞。体外使用 RT-PCR 和 Western blot 检测 RNA 和蛋白质。使用肌营养不良症小鼠模型（Bla/J）进行体内研究。组织学检测、形态计量学和免疫组化用于肌肉组织分析。在亚生理水平检测到体外和体内的肌营养不良蛋白。RT-PCR 和 Western blot 在转导 AAV.DYSF.OVERLAP 的细胞中检测到 dysferlin mRNA 和蛋白质，与参照基因（GAPDH）相比，mRNA 水平升高了 7 倍。在体内，实验组与 WT 和 Bla/J 小鼠的对照组相比，坏死肌纤维、内化核肌纤维和肌纤维横截面积的比例表现出中等中位数，尽管差异无统计学意义。肌肉中炎症变化的剂量与严重程度之间的反比关系可能归因于坏死纤维数量的减少。在最高剂量组中转导的肌纤维比例达到近 35%。使用基于 AAV 的双载体系统在治疗效果方面是合理的。在短观察期内以亚生理水平表达 dysferlin 能够诱导肌肉组织结构的恢复，降低炎症活性，并减轻坏死过程。需要进一步的研究来更详细地评估转基因和病毒载体对炎症成分的影响，包括更长的观察期。
