Therapeutic genetics and disease modeling in LAMA2-CMD

摘要 肌营养不良影响了美国估计25万名患者，导致每年的医疗费用增加。 支出超过460亿元。经济损失在先天性肌肉萎缩症中更大 营养不良（CMD），在生命的早期表现，由于患者无法进入劳动力市场和/或他们的 过早死亡LAMA 2缺陷型先天性肌营养不良症（LAMA 2-CMD），是最常见的 CMD由编码LAMA 2蛋白的LAMA 2基因突变引起。缺乏LAMA 2蛋白 导致骨骼肌退化和受损的雪旺细胞髓鞘形成，导致级联反应， 继发性事件包括凋亡、炎症和纤维化，这些事件最终促使疾病发生。 为患者提供疾病症状管理，如辅助通气、门诊服务， 和物理疗法来减少挛缩，但没有治愈的选择。 由于这种疾病的遗传性质，纠正突变将是一种有希望的治疗方法， LAMA2-CMD。我们先前已经描述了使用CRISPR/Cas9来校正在人乳腺癌细胞中的剪接位点突变。 在LAMA 2-CMD小鼠模型中，Lama 2基因的表达和疾病表型的改善。但 在患者人群中发现了600多种致病突变，这严重阻碍了未来的 任何LAMA 2突变校正策略的翻译。 与此相反，通过靶向调节疾病修饰因子的表达来减弱疾病致病性， 基因将有益于受LAMA 2-CMD影响的所有个体。我们是第一个开发CRISPR的人 一种基于激活的方法来在出生后上调疾病修饰基因Lama 1，该基因在结构上 在小鼠模型中与Lama 2功能相似。这些发现导致了一种假设，即靶向LAMA 1 基因激活可以作为LAMA 2-CMD的突变非依赖性治疗方法。 我们将通过对以下药物的疗效和安全性进行专门的临床前评价来检验这一假设： 严重LAMA 2-CMD小鼠模型中的Lama 1上调。此外，我们还将翻译 从小鼠到患者相关模型的策略，并评估人LAMA 1上调对 细胞损伤，包括线粒体呼吸、迁移和髓鞘形成。成功完成 这些实验将为开发不依赖突变的治疗干预铺平道路 LAMA 2-CMD和潜在的其他肌营养不良症。

ABSTRACT Muscular dystrophies impact an estimated 250,000 patients in the United States, resulting in an annual medical expenditure of over $46 billion. The economic loss is more substantial in the congenital-onset muscular dystrophies (CMDs), which manifest very early in life, due to patients' inability to enter the workforce and/or their premature death. LAMA2-deficient congenital muscular dystrophy (LAMA2-CMD), the most common form of CMD, is caused by mutations in the LAMA2 gene encoding the LAMA2 protein. The lack of LAMA2 protein causes degeneration of skeletal muscle and impaired Schwann cell myelination, resulting in a cascade of secondary events that include apoptosis, inflammation, and fibrosis, which ultimately precipitate the disease. Patients are provided with disease symptom management, such as assisted ventilation, ambulatory services, and physiotherapy to reduce contractures, but there is no curative option. Owing to the genetic nature of the disease, the correction of mutations would be a promising treatment for LAMA2-CMD. We have previously described the use of CRISPR/Cas9 to correct a splice site mutation in the Lama2 gene and showed amelioration of disease phenotypes in a LAMA2-CMD mouse model. However, there are more than 600 pathogenic mutations identified in the patient populations, which significantly hampers future translation of any LAMA2 mutation correction strategy to patients. In contrast, the attenuation of disease pathogenicity by targeted modulation of the expression of disease modifier genes would be beneficial to all individuals affected with LAMA2-CMD. We were the first to develop a CRISPR activation-based approach to postnatally upregulate a disease modifier gene Lama1, which is structurally and functionally similar to Lama2, in the mouse model. These findings led to the hypothesis that targeted LAMA1 gene activation can serve as a mutation-independent therapeutic approach for LAMA2-CMD. We will test this hypothesis by performing a dedicated preclinical evaluation of efficacy and safety profiles of Lama1 upregulation in a severe LAMA2-CMD mouse model. In addition, we will bridge the translation of the strategy from a mouse-to patient-relevant models and evaluate the effect of human LAMA1 upregulation on cellular impairments, including mitochondrial respiration, migration, and myelination. Successful completion of these experiments will pave the way towards the development of mutation-independent therapeutic interventions for LAMA2-CMD and potentially other muscular dystrophies.