Year 3 + NCE
This project has led to great progress in the development of a stem cell therapy for Duchenne muscular dystrophy. During the project period, we went from a conceptual strategy to making all parts of the strategy work, while at the same time discovering improvements in all aspects. The studies began with developing a new and potentially safer way to reprogram mouse cells. We started with skin cells from mdx disease model mice and introduced a plasmid, or circle of DNA, that encoded four genes that could reprogram the skin cells back into embryonic-like cells. We used an enzyme from bacteria called a “recombinase” to paste the reprogramming genes into a safe place in the mouse chromosomes. The next step was to use a second recombinase enzyme to place a correct copy of the dystrophin gene, the gene that is mutated in this form of muscular dystrophy, into a precise position next to the reprogramming genes. Once this was accomplished, we used a third recombinase to delete the portions of inserted DNA that were no longer needed, including the reprogramming genes. These steps left us with “induced pluripotent stem cells”, or iPSC, that were corrected for the disease-causing mutation. In the next step, we used methods to grow the iPSC that induced them to become muscle precursor cells. We measured these changes by monitoring several proteins that are typical of muscle cells. These muscle proteins began to appear in the iPSC as they were undergoing the differentiation process. Once the cells were differentiated, we injected them into the leg muscles of living mice that had muscular dystrophy. We showed that the cells we injected were able to engraft into the muscle, where they could repair and replace damaged muscle fibers. Having successfully carried out the complete stem cell strategy using mouse cells, we published our findings in a scientific journal and sought to develop a similar strategy using human cells. We found that the reprogramming strategy that we had used in mouse cells did not work well in human cells. Therefore, we turned to a reprogramming method that was recently reported by two labs, in which plasmids based on Epstein-Barr virus are used to carry the reprogramming genes into human cells. The long-lasting plasmids provided a sufficient dose of the reprogramming genes, such that the human cells became iPSC. In order to supply a correct copy of the mutated gene, we developed a new method of genome engineering called DICE, for dual integrase cassette exchange. In this method, a short DNA sequence called a “landing pad” was positioned in a special place in the chromosomes called H11. This location has features that make it favorable as a spot to place introduced genes. The landing pad contains recognition sequences for two different recombinase enzymes. When a piece of DNA carrying the genes we want to insert is flanked by recognition sequences for the two enzymes, the landing pad is replaced by the gene we would like to insert. By using this method, we generated iPSC that had a new gene inserted precisely at the H11 location. The next step is to differentiate the cells into muscle precursor cells. The procedure that had worked in mouse cells was not effective for the human cells. We tried two new methods, and both generated human muscle precursor cells at good efficiency. We transplanted the differentiated muscle precursor cells into leg muscles of immune-deficient mice. The mice needed to be immune-deficient in order to accept grafts of human cells without rejecting the cells. We obtained evidence that the human cells successfully engrafted into the muscle. Until now, we had been introducing the stem cells by injecting them directly into a muscle with a needle. This procedure works well in the small muscles of a mouse, but would not work well in the much larger muscles of a human. Therefore, we also began developing a new stem cell delivery method in which the stem cells are introduced into an artery, where they can access muscle tissue by passing through the blood vessel wall and into the muscle tissue. We generated preliminary results suggesting that this arterial delivery system might be a successful means to distribute healthy stem cells to diseased muscles throughout the body. We intend to continue developing this stem cell strategy so that it can be used to help repair the muscles in patients with muscular dystrophy.