To deliver muscle cells from a healthy donor (containing a healthy gene) to Duchenne muscles to compensate for lost muscle tissue and to allow normal dystrophin production by the donor cells.
Muscle consists of muscle fibers, which do not divide and muscle stem cells that lay on top of the fiber (Figure 4). When the muscle fiber is damaged, these muscle stem cells (also called satellite cells or myoblasts) will start dividing and will migrate to the location of the damage and fuse with the damaged muscle to repair it. These stem cells can be isolated from a muscle biopsy and expanded in the lab and then transplanted into Duchenne muscle.
Image of Muscle Stem Cells
Muscle stem cells are unable to travel from the bloodstream into muscle.
Local injection into affected muscles
Even muscles injected directly into muscles do not migrate beyond 1-2 mm from the needle tract.
A clinical trial where muscle stem cells were injected with 100 injections in a small area of muscle (0.25-1 cm2) has been completed in Canada (Tremblay and Skuk). The treatment was safe and dystrophin positive fibers could be detected in a biopsy taken from the treated area. A new trial for local myoblast transplantation in a forearm muscle of patients of 16 years and older is ongoing in Canada.
Nevertheless, it is unfeasible to use this form of treatment to deliver muscle cells to all muscles in the body.
There are other stem cells present in blood, blood vessel walls and fat tissue that can also participate in muscle formation. These cells can be isolated and expanded in the lab. An advantage is that these cells are probably able to travel from the bloodstream into muscles, thus allowing body wide treatment.
Although these cells are able to participate in muscle formation the efficiency is at the moment very low (<1% of the transplanted cells ends up in muscle).
A trial where CD133+ cells were obtained from Duchenne patients (isolated from blood), expanded in the lab and then transplanted back into hand muscles of patients has been completed in Italy (Torrente).
Ways to increase the efficiency of this approach are currently under investigation. Promising results have been obtained in mouse and dog models with ‘mesangioblasts’ (group of Giulio Cossu) and ‘CD133+’ cells (group of Ivan Torrente).
A trial to assess the safety of transplantation of mesangioblasts (obtained from unaffected brothers) into Duchenne patients has been performed in Italy (Cossu). Five patients were injected intra-arterially with mesangioblasts. This was a safety trial and no improvement of muscle function was expected or detected. Further work is ongoing to improve the transplantation protocol for potential future studies.
It is difficult to obtain sufficient amounts of stem cells for transplantation, because these stem cells have only a limited capacity to divide.
It is now possible to reprogram adult cells so they acquire stem cell characteristics (induced pluripotent stem cells, or iPS cells). These cells have a huge proliferative potential and can differentiate into all possible cell types. However, the trick is to have them differentiate only into muscle progenitor cells. Work is currently ongoing to generate clinical grade iPS cells and to optimize the protocol to obtain muscle progenitor cells in a safe and efficient and standardized way
Transplantation of donor muscles will elicit an immune response (like the transplantation of any tissue into another person).
Administration of drugs that suppress the immune system. This is standard treatment for individuals receiving donor tissue. Unfortunately, chronic treatment with these drugs is not without side effects (e.g. one is more prone to infections).
Isolate muscle cells from the patients, expand them in the lab and treat them (e.g. with gene therapy) in the lab. Then transplant the patient’s own cells back (autologous transplantation). Gene therapy is much more efficient in cells (in the lab) than in tissue (in a person). In addition, because the patient’s own cells are transplanted, immune suppression may not be necessary.
For this to work, ways to efficiently deliver muscle cells or other stem cells to muscle still have to be optimized (see challenge 1-4). Furthermore, it is possible the immune system will still respond to the transplanted cells even though they are from the patient: due to the manipulation in the lab, the cells are likely changed and the immune system may pick up on this.
In the lab, it is now possible to make minor changes in the DNA of a cell without having to add a gene (using DNA ‘scissors’ – different types have been developed: ZNF, TALEN and RGN (also called CRISPR/Cas9)). These DNA scissors work at a low efficiency. In cultured cells, the cell in which the scissor was successful, has to be identified (usually only ~1 in 1000) and then expanded to be transplanted in mouse models.
Generally, the genetic mistake in the dystrophin is quite large and the DNA scissors cannot repair large mistakes. However, it is possible to introduce a mistake to hide an exon permanently (see exon skipping section). This work is in an early stage and a lot of work will be needed to assess whether this method is safe and whether it is applicable to humans. In 2016 three publications in the journal Science showed proof-of-concept of this approach in the mdx mouse model and in 2018, a paper showed proof-of-concept in a dog model. While this is encouraging, one should bear in mind that the DNA scissors will need to be delivered to the majority of muscle cells. As such it faces the challenges of gene therapy or cell therapy with respect to the translation step from mouse and larger animals and humans. Furthermore, more research is need to assess how specific the scissors are and whether they are safe to use in human beings. For more information see this blog.
Another way to use stem cells:
Heart stem cells may have the potential to temporarily slow down heart pathology in DMD patients by producing protective growth factors. This effect is transient, because the stem cells will die. Capricor has conducted a safety trial in DMD patients with heart disease to assess how well local injection of donor heart stem cells into the heart is tolerated. The procedure was well tolerated and there was a small decrease in fibrosis observable in hearts from patients in the treated group. Furthermore, there was a trend for improved upper limb function suggesting also a protective effect of the stem cells on skeletal muscle. Capricor is has completed a phase 2 trial where ambulant and non ambulant patients were treated every 3 months with an intravenous delivery of 150 million stem cells (CAP-1002) or placebo. This trial was temporarily placed on hold after two patients developed an acute immune response requiring hospitalization. Consequently, patients are pretreated with corticosteroids and anti-histamines to reduce the risk of a severe immune response. Preliminary analysis again suggests improved upper limb function compared to placebo, although this is based on a small number of patients (8 vs 12). Patients who were involved in the clinical trial can continue treatment or start treatment (if they were in the placebo-group) in an open-label study. A phase 3 placebo-controlled trial to assess safety and efficacy of treatment with these stem cells is currently ongoing. The primary endpoint will be the performance upper limb and the trial will be in both ambulatory and non ambulatory patients.