Year 2

The goal of this project is to develop a stem cell therapy that will improve the medical condition of patients with limb girdle muscular dystrophy type 2B. In this disease, patients have a mutation in each copy of their gene encoding dysferlin. Dysferlin is a protein that is primarily expressed in muscle, where it has an important role in the repair of muscle cells. When the protein is missing, the muscle fibers are weaker and gradually break down. Patients lose muscle strength and become dependent on a wheelchair for mobility. In our strategy, we start with cells from the patient, so they will be immunologically matched. The cells are “reprogrammed” into induced pluripotent stem cells (iPSC), which have the capacity to become any type of cell. The next step is to correct the mutation in dysferlin, so that the iPSC will make dysferlin protein. Then the cells are differentiated into muscle precursor cells and transplanted into the body, where they can repair the defective muscle tissue. During the second year of the project, we have made progress on developing each step of this strategy. We previously obtained iPSC derived from the cells of a LGMD2B patient. In order to correct the mutation in dysferlin, we applied a genome editing technology called CRISPR/Cas9. To use this technology, we designed an RNA molecule that recognized DNA sequences near the mutation. We also designed a DNA sequence that matched the area of the mutation, but contained the correct DNA sequence in place of the mutated sequence. After introducing these molecules into the patient iPSC, the cells used the introduced genetic information to correct precisely the mutation. We isolated iPSC clones that carried the correct DNA sequence in place of the mutation. Using two different techniques that measure the amount of dysferlin made by the cells, we verified that after differentiation into muscle precursors, the corrected iPSC now made dysferlin protein in amounts similar to normal cells. To turn the iPSC into muscle precursor cells for transplantation, we applied a cell culture method that included addition of three small molecules that encouraged the iPSC to become muscle cells. We verified that this procedure was effective to create muscle precursor cells. The method caused the iPSC to start expressing several proteins that are characteristic of muscle stem cells. These proteins included PAX7 and CD56. By 21 days of differentiation, the majority of the cells expressed these markers. The differentiated cells also went on to fuse into muscle cells in cell culture within one month. We tested these cells in transplantation experiments that involved injecting the cells into mouse muscles. Since we were placing human cells into a mouse, in order to avoid immune rejection of the cells, we first created a new mouse strain by crossing a mouse model of LGMD2B with severely immune-deficient mice. The resulting mice serve as recipients in our transplantation experiments. The transplanted cells can become incorporated into existing muscle fibers by fusion, producing stronger muscle fibers. We analyzed the transplanted muscles to see whether they contained the cells we injected. We detected the presence of transplanted cells by staining for specific human proteins in the recipient muscles that identified the cells as being derived from the donor cells we injected. In order to distribute corrected muscle cells widely in the large muscles of human patients, we developed methods whereby we could inject the cells into an artery and allow them to become distributed into the muscles through the bloodstream. To perform this assay, we prepared muscle stem cells then injected them into the femoral artery of a mouse model. Because mice are small, this procedure has to be done under a low-power microscope. The donor cells were labeled with various markers so that we could track them in recipient animals. By analyzing for the presence of these markers in the muscles of the recipient animals, we showed that our donor cells had engrafted into all the leg muscles that we examined. We believe that this type of method, based on vascular injection, will be valuable for delivery of therapeutic cells to the muscles. In order to evaluate whether our transplanted cells cause an improvement in muscle strength, we are developing assays to measure the difference between normal mice and our mouse models of limb girdle muscular dystrophy 2B. The assays we are developing include a treadmill assay to measure how long the mice can run, as a measure of muscle strength. We are also measuring differences inside the muscles that are indicators of whether the muscle is healthy or diseased. If the corrected iPSC can improve the strength and health of the muscles in the mice, these features would provide proof of principle for an effective stem cell therapy for LGMD2B.