CRISPRa-induced upregulation of human compensates for -deficiency in Merosin-deficient congenital muscular dystrophy.

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.