Year 2

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 develop into 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 as starting material.

The first milestones for 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 to do the same for human cells (2B).
Our project took 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. Our laboratory 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 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. 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.

In the Milestone 1B 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 several weeks, while the stem cells are grown in plastic dishes in an incubator. The cells are grown in culture fluids that contain substances that allow the cells to differentiate from generalized stem cells into cells that are committed to produce muscle. We analyzed the cells at different time points to see if they had the characteristics of muscle precursor cells. 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 and also demonstrated that they contained messenger RNA that encoded muscle proteins. We also verified that the cells expressed the dystrophin gene that we inserted into them and produced normal dystrophin protein. 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 20 – 50% 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 Milestone 2A, we introduced these corrected and differentiated mouse stem cells into DMD-model mice to repair muscle damage. We injected the cells into a leg muscle, and three weeks later, we detected engrafted cells by staining for dystrophin. In the coming year, we will carry out the final experiments, Milestone 3, in which human cells that have been reprogrammed and corrected are engrafted into disease model mice.