Full length dystrophin transcription by dCas9 in fibroblasts and Cell Cycle Evaluation

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that cause it to be prematurely terminated or not fully transcribed. A lack of dystrophin in the muscle sarcolemma leads to progressive muscle degeneration. Definitive treatment of DMD will require dystrophin to be restored in all affected skeletal muscles, using cell therapy, gene therapy, or a combination of the two. Interestingly, a very small fraction of muscle fibers are able to produce dystrophin-positive fibers, called revertant fibers through natural exon skipping, indicating that increasing the number of the revertant fibers, may be a possible therapeutic approach for DMD. Using viral expression vectors carrying an inactivated Crispr/Cas9 system, called dCas9, fused to different transcriptional activators, we were able to cause 10T½ fibroblasts to induce transcription of the endogenous full-length dystrophin. We also used this model to investigate what effect transcribing extremely large genes like dystrophin has on the cell cycle of these cells. These findings open the possibility of using this technology to increase transcription of full-length dystrophin in dystrophic cells through natural exon skiping and ultimately increasing the amount of these revertant fibers for DMD therapy, as well understanding these cells on a molecular level.