Recombinase-Mediated Reprogramming and Dystrophin Gene Addition in mdx Mouse Induced Pluripotent Stem Cells.

This paper demonstrates a complete strategy for a stem cell therapy for muscular dystrophy. In this study, we modeled our strategy in mice. We used cells from mice that have a mutation in the same gene, dystrophin, that is mutated in patients that have Duchenne muscular dystrophy. We started with skin cells from the mouse and used a new method to turn the skin cells into stem cells. This procedure, called "reprogramming", was done without using any viruses, and we did not place genes into the chromosomes at random. Instead, we placed the genes needed for reprogramming into a single, safe location in the mouse chromosomes. This was done by positioning the genes in the chromosome with a recombinase enzyme that recognizes specific DNA sequences. We used the recombinase in combination with a circle of DNA called a plasmid that carried the reprogramming genes. We verified the position by DNA sequencing and made sure that no genes were disrupted during the reprogramming process. Then we added a correct copy of the coding sequence for mouse dystrophin. To do this, we used a second recombinase enzyme that recognizes a specific DNA sequence that was present on the original reprogramming plasmid. We verified that the dystrophin gene was now present in the cells. The corrected cells were then turned into muscle precursor cells by growing the cells in a specific way in culture dishes. After about two weeks in cell culture, the cell population was sorted on an instrument that allowed us to purify cells that were making a protein typical of muscle precursor cells. We took the positive cells and injected them into a leg muscle in the disease model mice. We showed that these cells became incorporated into the muscle and expressed the dystrophin gene. Thus, this study provides a model for our strategy for a muscular dystrophy therapy. We are now doing similar experiments in human cells, starting with skin cells from muscular dystrophy patients. By using a strategy similar to the one we used in this mouse study, we hope to develop a therapy for human muscular dystrophies.