Duchenne Muscular Dystrophy (DMD) is an early onset, progressive, and inevitably fatal disease affecting 1/5000 males, caused by mutations in the DMD gene (X-chromosome) that prevent expression of dystrophin (Dys). There is no cure for DMD, and effective therapeutics are desperately needed. The overall goal of the proposed research is to develop an in vitro assay system (the ?Stem Cell-derived Muscle Function Assay? [SCMFA]) that will enable high throughput testing of chemical compounds for potential therapeutic effects against DMD. The SCMFA will feature skeletal muscle (SkM) differentiated from human pluripotent stem cells (hPSCs, either induced pluripotent [hiPSCs], or NIH-approved human embryonic stem cell [hESC] lines) representing subjects that are unaffected, DMD, or Becker Muscular Dystrophy (which is closely related to DMD but milder). The project will be a collaboration between Vala Sciences Inc that specializes in development of cell-based assays using automated microscopy, and Genea Biocells US, a company that specializes in human stem cells and skeletal muscle diseases.
The Specific Aims for Phase I are: 1) Develop hiPSC lines for the project (while we already have certain hESC lines, the assay will ultimately feature only hiPSC lines) and methods to seed and differentiate the cells to SkM on patterned substrates and flexible membranes (all in 96-well dishes); 2) Development of Kinetic Image Cytometry methods to quantify muscle function (calcium and voltage transients, contractile motion, and generation of reactive oxygen species [ROS]) and Structured Illumination Microsopy (SIM ? similar to confocal) quantify the contractile apparatus and biomarker expression; and 3) Development of methods to stress the cells relevant to DMD (pacing of contraction, and stretch of the cells). Experiments will also be conducted to test the hypothesis that DMD SkM is more susceptible to stress-induced activation ROS formation and activation of TRPC (stretch) channels. In Phase II, additional hiPSC lines will be developed, the assay will be multiplexed and miniaturized to 384 well dishes, and pilot chemical libraries will be screened. Vala Sciences Inc and Genea Biocells US will market the SCMFA to pharmaceutical companies, world-wide, interested in developing therapeutics for DMD. The methods developed will also be applicable to other inherited muscular dystrophies and related afflictions.
Duchenne Muscular Dystrophy (DMD) is an inherited disease affecting 1 out of 5000 boys. The boys become weak because their muscle degenerates, losing their ability to walk by the time they are a teenager, and die before the age of 30 because they can?t breath anymore. DMD is always fatal, and there is no cure (certain medications prolong life, but not by very long). We are proposing to develop a new way to test drugs for their ability to improve the health of muscle of DMD patients. We will start with human stem cells that represent subjects with DMD (we will use stem cells that are approved for research use by the US government). We will then differentiate the stem cells to muscle cells which we will put into multi-well dishes (96- or 384-wells per dish), so that we can test many drugs on them in the same experiment. We will use automated digital microscopes to determine if any of the drugs increase the strength and health of the muscle. Once these test methods are developed, we will be able to test hundreds of thousands of different chemicals to identify drugs that will help boys with DMD.
