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

Funding Type: 
Early Translational IV
Grant Number: 
Award Value: 
Disease Focus: 
Muscular Dystrophy
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 
Limb girdle muscular dystrophy type 2B (LGMD 2B) is a form of muscular dystrophy that leads to muscle degeneration and disability. In LGMD 2B, a vital muscle protein is mutated, and its absence leads to progressive degeneration of muscles in the body that are needed for mobility. To create a therapy, we will provide a new supply of stem cells that carry the missing protein that is lacking. These cells will be delivered to the body in such a way that they will engraft into the muscles and produce new, healthy muscle tissue on an ongoing basis. We now possess methods to create stem cells that can become muscle cells out of adult skin cells by a process known as “reprogramming”. By reprogramming adult cells, together with addition to them of a correct copy of the gene that is mutated in LGMD 2B, we will create stem cells that have the ability to create new, healthy muscle cells in the body of a patient. This is the type of strategy that we are developing in this proposal. The corrected muscle stem cells will be transplanted into mice with LGMD 2B, and the ability of the cells to generate healthy new muscle tissue and increased muscle strength will be evaluated. This project could lead to a new stem cell therapy that could improve the clinical condition of LGMD 2B patients. If we are successful with this disease, similar methods could be used to treat other degenerative disorders, and perhaps even some of the degeneration that occurs during muscle injury and normal aging.
Statement of Benefit to California: 
The proposed research could lead to a stem cell therapy for limb girdle muscular dystrophy type 2B (LGMD 2B). This outcome would deliver a variety of benefits to the state of California. There would be a profound personal benefit to the Californians affected directly or indirectly by LGMD 2B. Progress toward a cure for LGMD 2B is also likely to accelerate the development of treatments for other degenerative disorders. The most obvious targets would be other forms of muscular dystrophy and neuromuscular disorders. Muscle injury, and even some of the normal processes of muscle aging, may be treatable by a similar strategy. An effective stem cell therapy for LGMD 2B would also bring economic benefits to the state by reducing the huge burden of costs associated with the care of patients with long-term degenerative disorders. Many of these patients would be more able to contribute to the workforce and pay taxes. Another benefit is the effect of novel, cutting-edge technologies developed in California on the business economy of the state. Such technologies can have a profound effect on the competitiveness of California through the formation of new manufacturing and health care delivery facilities that would employ California citizens and bring new sources of revenue to the state. Therefore, this project has the potential to bring health and economic benefits to California that are highly desirable for the state.
Progress Report: 
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.