Development of a high-throughput screening assay to identify Nfix-modulating drugs as a novel therapy for muscular dystrophy

Muscular Dystrophies (MDs) are a highly heterogeneous group of genetic muscle wasting disease characterized by progressive degeneration of skeletal muscles that results in impaired mobility, wheel-chair dependency, respiratory complications, cardiac involvement, and, in the most severe case premature death1. In the last years, we reported that dystrophic mice lacking the transcription factor Nfix have delayed cycles of degeneration/regeneration, a metabolic switch towards slow oxidative myofibers and reduced fibrosis, resulting in a morphological and functional amelioration of the disease2,3. Therefore, these findings underscore the potential of Nfix silencing to slow MD progression and preserve muscle mass, highlighting the need for novel approaches to target Nfix in MDs. However, many insights about Nfix are still unknown, making challenging the identification of drugs targeting Nfix. Therefore, we planned to develop a High-Throughput Screening (HTS) assay on a myogenic cell-based system to identify Nfix-modulating drugs. We generated a lentiviral fluorescent, myogenic, and responsive cell-based system, namely pNFIX1-d2EGFP. The pNFIX1-d2EGFP myoblasts have a distinct d2EGFP fluorescent signal, correlated with the Nfix gene expression and they also maintain myogenic abilities, suggesting that the pNFIX1-d2EGFP myoblasts represent an excellent system for HTS purposes in muscular context. Moreover, to overpower the potential underestimation of the number of inhibitor compounds in the HTS assay, we are developing the EpNFIX1-d2EGFP model, a more reliable cell-based system expressing the d2EGFP fluorescent signal based on the entire Nfix endogenous regulation. This approach is extremely ground-breaking and might provide novel alternative drugs to treat MDs, discovering new pathways affecting the Nfix expression.