Arockiaraj Annie I, Johnson Marie A, Munir Anushe, Ekambaram Prasanna, Lucas Peter C, McAllister-Lucas Linda M, Kemaladewi Dwi U
Department of Human Genetics, School of Public Health, University of Pittsburgh, Pittsburgh, USA.
Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, USA.
bioRxiv. 2023 Mar 7:2023.03.06.531347. doi: 10.1101/2023.03.06.531347.
Merosin-deficient congenital muscular dystrophy (MDC1A) is an autosomal recessive disorder caused by mutations in the gene, resulting in a defective form of the extracellular matrix protein laminin-α2 (LAMA2). Individuals diagnosed with MDC1A exhibit progressive muscle wasting and declining neuromuscular functions. No treatments for this disorder are currently available. We previously showed that postnatal upregulation, achieved through CRISPR activation (CRISPRa), compensates for deficiency and prevents neuromuscular pathophysiology in a mouse model of MDC1A. In this study, we assessed the feasibility of upregulating human as a potential therapeutic strategy for individuals with MDC1A, regardless of their mutations. We hypothesized that CRISPRa-mediated upregulation of human would compensate for the lack of and rescue cellular abnormalities in MDC1A fibroblasts. Global transcriptomic and pathway enrichment analyses of fibroblasts collected from individuals carrying pathogenic mutations, compared with healthy controls, indicated higher expression of transcripts encoding proteins that contribute to wound healing, including Transforming Growth Factor-β (TGF-β) and Fibroblast Growth Factor (FGF). These findings were supported by wound-healing assays indicating that MDC1A fibroblasts migrated significantly more rapidly than the controls. Subsequently, we treated the MDC1A fibroblasts with dCas9-2XVP64 and sgRNAs targeting the promoter. We observed robust expression, which was accompanied by significant decreases in cell migration and expression of β, which are involved in the wound-healing mechanism in MDC1A fibroblasts. Collectively, our data suggest that CRISPRa-mediated upregulation may be a feasible mutation-independent therapeutic approach for MDC1A. This strategy might be adapted to address other neuromuscular diseases and inherited conditions in which strong compensatory mechanisms have been identified.
缺乏层黏连蛋白α2的先天性肌营养不良症（MDC1A）是一种常染色体隐性疾病，由该基因的突变引起，导致细胞外基质蛋白层黏连蛋白α2（LAMA2）出现缺陷形式。被诊断为MDC1A的个体表现出进行性肌肉萎缩和神经肌肉功能衰退。目前尚无针对这种疾病的治疗方法。我们之前表明，通过CRISPR激活（CRISPRa）实现的出生后上调可弥补缺陷，并预防MDC1A小鼠模型中的神经肌肉病理生理过程。在本研究中，我们评估了上调人LAMA2作为MDC1A个体潜在治疗策略的可行性，无论其突变情况如何。我们假设CRISPRa介导的人LAMA2上调将弥补LAMA2的缺乏，并挽救MDC1A成纤维细胞中的细胞异常。与健康对照相比，对携带致病性LAMA2突变个体的成纤维细胞进行的全转录组和通路富集分析表明，编码有助于伤口愈合的蛋白质的转录本表达更高，包括转化生长因子-β（TGF-β）和成纤维细胞生长因子（FGF）。伤口愈合试验支持了这些发现，表明MDC1A成纤维细胞的迁移速度明显快于对照。随后，我们用靶向LAMA2启动子的dCas9-2XVP64和sgRNAs处理MDC1A成纤维细胞。我们观察到LAMA2的强烈表达，同时细胞迁移以及参与MDC1A成纤维细胞伤口愈合机制的β的表达显著降低。总体而言，我们的数据表明CRISPRa介导的LAMA2上调可能是一种针对MDC1A的可行的不依赖突变的治疗方法。该策略可能适用于解决其他已确定有强大补偿机制的神经肌肉疾病和遗传性疾病。
