The dystrophin gene is the largest gene in the body containing 79 exons, which are the portions of the DNA that contain the instructions for your cell to create a protein. Due to the small size of vectors used to deliver transgenes to cells, and the large size of the dystrophin gene, it is not currently possible to deliver the entire dystrophin gene to a cell. Researchers have therefore designed microdystrophin transgenes that maintain key pieces of genetic information to make a functional shortened protein.
This shortening of the protein does create new challenges. For instance many of the exons that are a part of the dystrophin gene have to be excluded from these microdystrophins. The dystrophin protein has many tasks within the body, including the very important role of stabilizing the muscle cell membrane. To stabilize the muscle cell membrane, the dystrophin protein interacts with several other beneficial proteins on the muscle cell membrane. Researchers try to be selective in which parts of the dystrophin gene are included in a microdystrophin transgene, but ultimately some of those regions that interact with other proteins will be missing. This means that a microdystrophin protein won’t be as effective as having the full length dystrophin protein, but it is still likely to provide some benefit compared to no dystrophin at all.
Another unknown with microdystrophin gene replacement is the durability of the transgene, in other words, how long will the muscle cells continue to produce the microdystrophin. The transgene does not become a part of a person’s DNA. While animal studies have been conducted, we will need to evaluate the durability in humans over time to understand how long the transgene lasts in cells. There are multiple dynamics that could impact durability. For instance, there could be loss of transgene through dilution. As an individual grows their body will develop more muscle than when they were a child. It is possible that as new muscle cells form, they won’t have a copy of the microdystrophin transgene or protein and would therefore act like typical dystrophic muscle. Another potential limitation around durability is muscle damage. It is possible that as muscles are used or damaged through physical activity that those cells containing a microdystrophin transgene will be lost. Monitoring of patients who received microdystrophin gene replacement will be critical to our understanding of durability and identifying if and when redosing is required.
It is also important to note that only living muscle cells carrying the transgene would produce a microdystrophin. This means that any muscle tissue which has been replaced by fat or fibrosis will not benefit from the microdystrophin transgene. As more individuals at different stages of the disease are exposed to microdystophin gene therapy, we will learn if there is an ideal stage to deliver this particular gene replacement strategy.
As discussed in the previous section, the dystrophin gene is the largest gene in the human body. Because the vectors used in current gene therapy are so small it creates a limit on how much genetic information can be packaged to create a micro-dystrophin protein.
The dystrophin protein is large and has many different protein domains. While the main role of the dystrophin protein is to stabilize the muscle membrane, it does not do this alone. The dystrophin protein interacts with a number of other proteins in the cell.
Researchers have tried to rationally design micro-dystrophins to keep key portions of the dystrophin protein, but do recognize that many important regions will be missing; therefore the micro-dystrophins may be missing domains that interact with other muscle cell proteins and may not be as functional as larger dystrophin proteins.
This means that while the micro-dystrophins won’t be as effective as full-length dystrophin they are still likely to provide more benefit than no dystrophin.
One major challenge that will take time to truly understand is the length of time the micro-dystrophin transgene will remain in the cells. There have been animal studies that can help to predict how long the micro-dystrophin transgene will remain in the cells and produce dystrophin, but these studies suggest that the production of micro-dystrophin will not be permanent. Multiple factors could impact how long the micro-dystrophin transgenes remain in the muscle cells.
It will be important for researchers and clinicians to monitor the changes in micro-dystrophin expression in muscle over time.
Finally, we expect benefit to only come from delivery of micro-dystrophin to muscle tissue. In Duchenne, over time, muscle tissue is replaced by fat and fibrosis. We don’t expect any benefit from delivery of micro-dystrophin into fat and fibrotic tissues. This is why age and stage of disease when delivering a gene therapy may be relevant for how much benefit is seen in a given individual.
