Engineered iPSC for therapy of limb girdle muscular dystrophy type 2B

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 is seems to help 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. By transiently introducing several genes that “reprogram” the cells, the patient cells are converted into induced pluripotent stem cells (IPSC), which have the capacity to become any type of cell in the body. The next step is to correct the mutation in dysferlin, so that the cells 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 first year of the project, we have made progress on developing each step of this strategy. We obtained iPSC derived from the cells of a LGMD2B patient. In order to correct the mutation in dysferlin, we applied a new genome editing technology called CRISPR/Cas9. To use this technology, we designed two RNA molecules that would recognize DNA sequences in the vicinity of 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 three molecules into the patient iPSC, the cells used the introduced genetic information to correct precisely the mutation. We verified that the desired correction occurred in the patient iPSC and are isolating individual iPSC clones that carry the correct DNA sequence in place of the mutation. We will verify that the iPSC now make dysferlin protein. To turn the iPSC into muscle precursor cells for transplantation, we applied a cell culture method that included addition of three small molecules that encourage the iPSC to become muscle cells. We verified that this procedure was effective to create muscle precursors that went on to fuse into muscle cells in cell culture within one month. We are testing these cells from various time points in transplantation experiments that involve injecting the cells into mouse muscles. Since we are 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, which are now ready for use, will serve as recipients in our transplantation experiments. We will analyze the transplanted muscles to see whether they contain the cells we transplanted. The transplanted cells can become incorporated into existing muscle fibers by fusion, producing stronger muscle fibers. We will analyze the presence of transplanted cells by staining for the dysferlin protein in the recipient muscles. In addition, we are testing the potential of the transplanted cells to become functional stem cells within the muscle that can give rise to healthy muscle fibers over the long term. These types of muscle stem cells are called satellite cells. To prove that our donor stem cells have become satellite cells in the muscle, we will injure the muscle and try to detect regeneration derived from the donor cells. We will also purify the satellite cells from the recipient muscle and transplant them into another animal, and measure whether donor-derived muscle regeneration occurs in the recipient muscle. If it does, that would indicate that our corrected cells have regenerative ability. In order to evaluate whether our transplanted cells cause an improvement in muscle strength, we are using a sensitive assay to measure the amount of force a particular muscle can generate. We have been collaborating with another lab to apply this method and plan to use it to see if we can detect an improvement in muscle strength that resulted from the transplanted stem cells. If the corrected iPSC show regenerative ability in the recipient muscle and can improve the strength of the muscle, these features would provide proof of principle for an effective stem cell therapy for LGMD2B.

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.