Development of high activity human muscle-specific regulatory cassettes and their

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Project-3 (Hauschka, Stephen D., P.I.) We hypothesize that optimal therapy for DMD and other muscle diseases, whether cell or vector mediated, will require an array of muscle-specific regulatory cassettes for expression of different therapeutic products at different levels. Cassettes meeting some of these goals have been designed & tested, but these contain mouse muscle gene components and all of their testing was done in mouse cell cultures and in mice after systemic viral delivery. Since the sequences of, and spacing between mouse & human muscle gene enhancer control elements differs, and since pilot studies indicate lower activity of mouse cassettes in human muscle cells, it is critical that regulatory cassettes be optimized for expression in mature human muscle fibers. We will design, construct, and test human gene versions of regulatory cassettes based on extensively tested mouse versions of similar cassettes. Individual cassettes will be designed for optimal function in a variety of fast/slow muscle fiber types, as well as in different anatomical muscles. Human cassettes will be built in both "miniature" and large forms to facilitate their packaging with therapeutic cDNAs of different sizes in AAV & Lentiviral (LV) vectors (4.8 & 9 kb packaging limits), as larger cassettes permit including more complex regulatory functions. One cassette type will be designed to produce therapeutic products in response to an externally delivered drug; thus permitting graded synthesis levels of the therapeutic product, depending on the physiological needs of particular patients. Cassette function will be tested in human skeletal muscle cultures, and the best cassettes will be retested in mice and in human muscle xenografts in immunodeficient mice, to mimic the in vivo properties of mature human muscle. Successful DMD gene therapy will likely require stable transduction of patient satellite muscle cells, as well as myofiber nuclei, to ensure continued therapeutic product synthesis following disease-related and natural turnover of muscle fibers. Satellite cell transduction and the maintenance of therapeutic product synthesis will be examined via clonal assays of LV-transduced satellite cells at progressively increased rounds of proliferation; and cassettes will be modified to retain long-term function. RELEVANCE (See instructions): These studies are a necessary prelude to the application of gene- and cell-mediated therapy to the treatment of virtually all human muscle diseases. Additionally, the optimization of muscle regulatory cassette function will permit treating patients as well as ex vivo cultures of donor cells with much lower viral vector doses. This will provide major improvements in patient safety, and the lower vector requirements will save millions of hfialth r.ara dollars.