Merosin-deficient congenital muscular dystrophy (MDC1A) is caused by mutations in the LAMA2 gene, resulting in defective extracellular matrix protein LAMA2. Individuals affected by MDC1A have progressive muscle wasting and declining neuromuscular functions, for which no treatment is currently available. We previously showed that postnatal Lama1 upregulation, achieved using CRISPR activation (CRISPRa), compensates for the lack of Lama2 and improves neuromuscular pathophysiology in MDC1A mice. In this project, we aim to assess the feasibility of upregulating human LAMA1 as a therapeutic potential for MDC1A patients, irrespective of their mutations. We hypothesize that CRISPRa-mediated upregulation of human LAMA1 can compensate for the lack of LAMA2 and rescue cellular abnormalities in MDC1A fibroblasts.
First, we performed global transcriptomic and pathway enrichment analyses on three MDC1A fibroblasts carrying different pathogenic LAMA2 mutations. We identified key players in the wound healing mechanism, such as TGF-beta and FGF signaling pathways, as overactive compared to healthy control fibroblasts. Furthermore, the MDC1A fibroblasts migrate significantly faster in an automated wound-healing assay coupled with real-time imaging, which provides crucial functional validation of the transcriptomic data. Subsequently, we treated the MDC1A fibroblasts with dCas9-2XVP64 and sgRNAs targeting the human LAMA1 promoter and observed robust LAMA1 expression. Importantly, the upregulation of LAMA1 significantly reduces the overactive migration and the expression of genes belonging to the TGF-beta and FGF signaling pathways in MDC1A fibroblasts.
Collectively, our data provide strong molecular and functional evidence of the feasibility of CRISPRa-mediated LAMA1 upregulation as a mutation-independent therapeutic approach for MDC1A, thereby bridging the translation of this strategy from mouse to patient-relevant models. We envision the adaptability of the approach and technology for other neuromuscular diseases and inherited conditions in which strong compensatory genes exist.