This strategy applies to dysferlin genes containing a stop codon mutation. Imagine that you are traveling on the road that makes dysferlin protein. In order to obtain a full length dysferlin protein, you need to get to the end of the road. However, somewhere along the road there is a washed-out bridge that stops you from going further. A stop codon mutation is like a washed-out bridge that prevents the cell from manufacturing a full-length dysferlin protein. If cells stop making the dysferlin protein before it is completed, the truncated portion of dysferlin that has been made will be too short to function, and will eventually break down and disappear. Therefore, you need to find another way to cross the washed-out bridge. Reading through a stop codon (the effect of PTC124/Ataluren) is like a new bridge that allows cells to skip past the washed-out bridge and finish the journey to get a functional full-length dysferlin protein.
What approaches may lead to a cure for dysferlinopathy?
Click on the strategies below to see non-technical, metaphor-based descriptions of the potential scientific approaches to treating LGMD2B/Miyoshi/dysferlinopathy. For more technical descriptions, please visit the funded projects section of the website.
Click on the strategies below to learn more.
1. Strategies based on restoring functional dysferlin protein
Think of a mutated dysferlin gene as an electrical wire that has a fault somewhere in the middle. The idea behind exon skipping is to remove the fault by cutting the wire on either side of the fault and then rejoining the two ends of the wire so that the fault is removed. The resulting wire is shorter than the original one, but can potentially still do the same job. A mutated dysferlin gene can also be thought of as a scratched CD. When you get to the damaged section of the CD, the music cannot proceed, but exon skipping is like skipping past the track where the scratched section is, so you can listen to the rest of the album.
Gene editing involves repairing the mutation in the gene. Think of the dysferlin gene as a string of holiday lights. If there is a defect in one of the bulbs, the whole string of lights will not work. The way gene editing works is like replacing the faulty bulb, so that the whole string of lights will work.
Gene therapy involves a variety of methods to put a functional copy of the dysferlin gene back into muscle cells. No change is made to the mutant gene, but now an additional viable copy of the gene is present. Imagine you are a supervisor and have an employee who is not doing his job. The job needs to get done, but you are not able to fire the ineffective employee because he is the boss's son. So you bring in a new person who can do the job. Therefore, even though the ineffective employee is still there, the new employee does enough good work and the job gets done.
Proteins normally fold into predefined structures and when this folding gets messed up the protein cannot function. The way the protein folds is carefully controlled so that it does not form the wrong shape and lose its specific function. There are specific proteins in cells whose job it is to make sure that other proteins fold properly — like molecular teachers instructing the young proteins how to behave. If the protein does not fold appropriately, there are other proteins that get rid of it — much like an errant student flunking out of school. This may explain why most of the dysferlin mutations result in decreased protein levels. So, not only can mutations in a gene affect the function of the encoded protein, they can also affect how much of it there is. However, an alternative to getting rid of the student is to try REALLY hard to get them to learn — offer tutoring and mentoring and perhaps some guidance. Similarly, some compounds may act as chaperones inside a cell and help the young proteins fold, potentially enough to keep them from being tossed out.
2. Strategies based on preventing muscle cell damage/death even in the absence of dysferlin
Dysferlin has a specific role in muscle cells, and when it is mutated, that job does not get done. However, one solution may be to find other proteins or small molecules that can do something similar enough to get the task completed. Think of dysferlin as part of the "tool kit" that cells use to repair damage, just like a hammer in a carpenter's box of tools when doing a repair job. If the hammer is not there, the carpenter can’t do the repair the way he would normally do it. However, the carpenter could improvise and use something else as a hammer. While this tool might not be ideal for the job, it is better than nothing. There are other proteins similar to dysferlin in muscle cells that might be able to do the job of the missing dysferlin protein, such as other protein family members like myoferlin
Outside of every cell is a membrane that holds all the cell parts together. If the membrane is weak, then it will be susceptible to external damage. Think of the cell membrane of muscle cells as a dam, which is normally strong but becomes weak and unstable when dysferlin is mutated. When severe storms come, the unstable dam will break. Membrane stabilization is a strategy based on finding ways to shore up or reinforce the dam to make it stronger, so that it holds when the storms come.
Too much calcium inside a cell can be toxic, and calcium leaks into muscle cells when they are damaged. Imagine that there is a leak in the floor and water is slowly seeping in, thereby damaging the furniture in the room. Similarly, calcium enters through a crack in the cell membrane and causes damage in the cell. If we were to install a pump that pumps out this water (or calcium), then we could limit the amount of damage caused. The idea here is to regulate the amount of calcium inside the cell, without necessarily fixing the original crack in the floor (i.e., the cell membrane).
All cells possess the capacity to self-terminate or be killed by external factors. If we can regulate the process by which a cell dies, we might be able to give the cell's repair mechanisms more time to act. Think of muscle cells as bubbles in a sink. We want to keep the sink full of bubbles, but without dysferlin the muscle cell bubbles rupture easily, leaving the sink with very few bubbles. But imagine that the bubbles go through different phases before they burst. If we could prevent factors, such as wind, from causing early rupture of the bubbles, then the bubbles will last longer. Similarly, muscle cells go through different stages before they degenerate completely, and if we can stop muscle cells from dying at an early stage, these cells may have a chance to repair themselves, stay alive and be functional.
There is pervasive invasion of immune system cells into degenerating muscle tissue in dysferlinopathy. Some parts of the immune system are designed to eliminate problems, but this can have deleterious effects upon the adjoining cells that are healthy. Think of the immune system as the fire department. Sometimes, if the immune system does not work correctly, the fire department shows up when there is a fire in the neighborhood, but instead of dousing only the house that is on fire, they also drench all the surrounding houses, causing more damage than the original fire did. The fire department needs the right instructions, so only the house on fire gets doused with water. In another words, we need to control the immune system by giving it the right chemical signals, so that it only targets the degenerated muscle cells, and does not cause damage to other healthy muscle cells.
3. Strategies based on replacing damaged/dead muscle tissue
In muscular dystrophies, the muscle tissue breaks down more rapidly than it can rebuild. However, one possible way to push the balance toward healthy muscle is to speed up the regenerative process. Think of muscle cells as bubbles in a sink. We want to keep the sink full of bubbles, but without dysferlin the muscle cell bubbles rupture easily, leaving the sink with very few bubbles. We can solve the problem by quickly making new bubbles and adding them to the sink, so the sink will continue to be filled with bubbles even if the bubbles keep bursting. In the case of muscle that is lacking dysferlin, if we can increase the number of new muscle cells that the body makes (i.e. muscle regeneration), then dysferlin deficient patients can keep getting new muscles, even if their muscle cells do not live very long.
Another strategy to restore muscle is to add new cells that can become muscle. Think of muscle cells as bubbles in a sink. We want to keep the sink full of bubbles, but without dysferlin the muscle cell bubbles rupture easily, leaving the sink with very few bubbles. We can solve the problem by adding new bubbles to the sink, and it is even better if the new bubbles are made from better detergents so that they last longer than the original bubbles. In the case of dysferlin deficiency, if we can introduce stem cells that have a healthy dysferlin gene into muscle, then these stem cells will form new muscle cells that will not die quickly like the ones with a defective dysferlin gene.