DMD remains a devastating and incurable muscle-wasting disease caused by DNA mutations in the gene that codes for dystrophin, a protein that plays a key role in muscle cell health. Most DNA mutations cause a frameshift in the RNA, and result in no expression of functional dystrophin, the protein encoded by the DMD gene. When dystrophin is completely missing from the muscle, the muscle cells are susceptible to repeated cycles of damage, and lead to progressive weakness everywhere in the body including the heart and lungs. Exon-skipping is a promising therapy that aims to repair the expression of the dystrophin protein by repairing the reading frame of the mRNA. This strategy has been demonstrated to successfully restore a small fraction of the normal amount of dystrophin, and is likely to be therapeutically beneficial. However, increasing the amount of dystrophin produced in this strategy is very likely to improve the impact of exon skipping on those affected by DMD. We have identified a combination therapy that improves the effectiveness of exon-skipping therapy in mouse muscle and in human DMD patient stem cell derived muscle cells in culture. In the second year of this project, we have expanded the set of cell lines from DMD patient skin biopsies. These biopsies have been grown in culture and induced into muscle cells using direct reprogramming. We have tested the effectiveness of exon skipping enhancement in these human cell lines to determine if multiple independent mutations are relevant for this potential therapy. We have shown that different cell lines can be repaired by exon skipping strategies, and that the compound can enhance skipping in a dish. At 24 months in the project, we have also completed a 6 month trial of over 180 mice that are in eight different treatment groups including with or without compound (Dantrolene) and either without antisense oligo or with antisense oligo at three different doses administered weekly by IV in mdx mice. Mdx mice are the main mouse model of Duchenne, and we can readily assess dystrophin protein expression and DMD RNA repair in mouse tissues. The experiment is recently completed, but all of the data are blinded. The mouse muscles are being assessed and will be correlated with functional measurements in the 8 different mouse groups. The combination therapy appears to have been well tolerated over the 6 month time frame, and analysis of the muscle specimens to quantitatively assess dystrophin restoration and DMD exon skipping are underway. Additional tissues including blood, kidney, liver and heart are being assessed for potential toxicity related to the combination therapy.