Year 1

The goal of this project is to develop a new approach for therapy of Duchenne muscular dystrophy (DMD). In our strategy, we use skin cells from patients as the starting material and convert these cells into stem cells by adding “reprogramming” genes. We then add the therapeutic dystrophin gene to the stem cells to correct the mutation that causes DMD. The corrected stem cells are grown in a manner so that they will become muscle precursor cells. This process is called differentiation. The differentiated muscle precursor cells are injected into diseased muscles to restore healthy muscle fibers. The overall goal of the project is to demonstrate the entire strategy in a mouse model of DMD, using both mouse and human cells.

The milestones for the first year of the project were 1A) to demonstrate the complete strategy for making stem cells from the mouse model and adding a correct dystrophin gene at a precise location in the chromosomes, and 1B) to demonstrate differentiation of mouse and human stem cells into muscle precursor cells that will later be used for engraftment. We achieved both of these milestones.

Milestone 1A. Our project takes advantage of the recent discovery that ordinary skin cells can be “reprogrammed” into stem cells that are similar in their properties to embryonic cells. The reprogramming process is carried out by introducing four genes into skin cells that can change the pattern of expression of genes in the cells to that of embryonic cells. The reprogramming genes are usually introduced into cells by putting them into viruses that can incorporate, or integrate, themselves into the chromosomes. This process is effective, but leaves behind viruses embedded in the chromosomes, which can activate genes that cause cancer. My laboratory has developed a safer method for reprogramming, in which no viruses are used. Instead, we utilize an enzyme that can place a single copy of the reprogramming genes into a safe place in the chromosomes.

In our method, the reprogramming genes are present on small circles of DNA that are easily made from bacteria grown in the laboratory. The circles of DNA, along with DNA that codes for the integration enzyme, are introduced into patient skin cells. The enzyme causes the reprogramming genes to incorporate into a chromosome at a single, safe location. After the cells are reprogrammed, the reprogramming genes, which are no longer needed, are precisely removed from the chromosomes by using another enzyme. These cells appear to be safe to use in the clinic.

In addition, we developed a method to add the therapeutic dystrophin gene to the reprogrammed cells at a precise location. By adding a correct copy of the dystrophin gene, the stem cells now have the potential to make healthy muscle. These corrected stem cells were used to create muscle precursor cells in Milestone 1B.

Milestone 1B. In these experiments, we demonstrated that cells reprogrammed and corrected by our methods can be grown in such a way that they differentiate into muscle precursor cells that have the capacity to become healthy muscle fibers. This process of differentiation takes place over a period of about two to three weeks, while the stem cells are grown in plastic dishes in an incubator. The cells are grown in culture medium that contains substances that allow the cells to differentiate from generalized stem cells into cells that are committed to produce muscle.

We followed two procedures published in the literature for differentiation of the cells. We analyzed the cells at different time points to see if they had the characteristics of muscle precursor cells. First, we observed the cells under the microscope and saw that they fused together into long fibers, which is characteristic of muscle cells. Moreover, the fibers began to contract and twitch, which is typical of muscle fibers.

To analyze the cells at the molecular level, we stained them with antibodies that recognize proteins that are made in muscle precursor cells. We were able to detect staining in some of the cells in the culture, indicating that they were becoming muscle precursor cells. This was demonstrated for both human and mouse stem cells.

To measure what fraction of the cells had become muscle precursors, we mixed the culture containing differentiated mouse stem cells with an antibody that binds to the surface of muscle precursor cells. The cells were analyzed with an instrument that can measure how many cells in the culture bind the antibody. We found that 5 – 10% of the cells stained with the antibody. This result indicated that a significant fraction of the cells had become muscle precursors cells, with the potential to be engrafted.

In the coming year, these corrected and differentiated mouse stem cells will be introduced into DMD mice to repair muscle damage. We will also apply our reprogramming and correction methods to human cells from DMD patients.