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Note: This covers both the year 1 Postnatal modeling of FSHD in mice and year 2 Testing of DUX4 RNAi therapy in a new cell model of FSHD grants (See 2011 and 2012 grants).
The research goals of 2011-13 were to 1) create a model of FSHD that displayed features of the disease in muscles of laboratory mice through production of human DUX4 protein and 2) to test a therapeutic approach to block production of DUX4 protein in patient cell lines grown in tissue culture dishes in the laboratory.
In 2013 a mouse model was created that shows toxicity from expression of a full size DUX4 protein as proposed in research goal 1. We injected a DNA shuttle vector in a normal mouse muscle that carries the DUX4 gene for muscle cell production of DUX4 protein. Features of the mouse muscle tissue, while not completely unique to FSHD, are consistent with changes seen in FSHD patient tissues. We observe fiber splitting and immune cell infiltrate that appears targeted to individual muscle fibers. This is the first example of disease effects that appear to mimic the muscle changes in patients with both low and high DUX4 expression. With our gene delivery method we are able to compare muscle cell changes at high and low doses of our shuttle vector that are proportional to DUX4 protein levels measured in the mouse muscle. Our future challenge is to characterize cellular pathways of muscle cell death in the DUX4 mice and relate them to cell death in FSHD muscle for validation of the model. The DUX4 expressing mice provide a powerful system for testing therapeutic approaches as a complex biological system and will be used to evaluate the RNA interference therapeutic approach we have developed. Plans are underway to facilitate the use of this model to evaluate additional potential therapies to maximize the usefulness of this test platform and speed therapy development.
In the past year we worked to reduce DUX4 protein in FSHD cells grown in the laboratory (collaboration with Dr. Dan Miller) to test the RNA interference (RNAi) therapy we have developed. RNAi involves expression of specific cellular molecules that will interact specifically with a messenger RNA to destroy it, so that DUX4 protein cannot be made. Testing a variety of RNAi target sequences in different shuttles for delivery to cells grown in the laboratory has provided information regarding effective DUX4 target regions with this method. We are able to decrease the amount of DUX4 protein and are optimizing our shuttles and RNAi sequences to maximize DUX4 protein reduction in the FSHD cells. In addition, we are continuing to test the DUX4 RNAi sequences in the D4Z4-2.5 transgenic mouse model of FSHD and will soon begin testing in our new DUX4 expressing mouse model described above. Since cell and animal models each have the ability to inform us about the potential for a therapy to be successful in clinical trials, we are gathering data from both types of models to identify promising candidate DUX4 RNAi therapeutics for clinical trials.