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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Program Projects (P01)
Project #
5P01NS046788-10
Application #
8447008
Study Section
National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
Project Start
Project End
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
10
Fiscal Year
2013
Total Cost
$261,895
Indirect Cost
$94,015
Name
University of Washington
Department
Type
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Parker, Maura H; Tapscott, Stephen J (2013) Expanding donor muscle-derived cells for transplantation. Curr Protoc Stem Cell Biol Chapter 2:Unit 2C.4
Himeda, Charis L; Tai, Phillip W L; Hauschka, Stephen D (2012) Analysis of muscle gene transcription in cultured skeletal muscle cells. Methods Mol Biol 798:425-43
Tai, Phillip W L; Smith, Catherine L; Angello, John C et al. (2012) Analysis of fiber-type differences in reporter gene expression of ?-gal transgenic muscle. Methods Mol Biol 798:445-59
Goncalves, Manuel A F V; Janssen, Josephine M; Nguyen, Quynh G et al. (2011) Transcription factor rational design improves directed differentiation of human mesenchymal stem cells into skeletal myocytes. Mol Ther 19:1331-41
Suga, Tomohiro; Kimura, En; Morioka, Yuka et al. (2011) Muscle fiber type-predominant promoter activity in lentiviral-mediated transgenic mouse. PLoS One 6:e16908
Banks, Glen B; Combs, Ariana C; Chamberlain, Jeffrey S (2010) Sequencing protocols to genotype mdx, mdx(4cv), and mdx(5cv) mice. Muscle Nerve 42:268-70
Kimura, En; Li, Sheng; Gregorevic, Paul et al. (2010) Dystrophin delivery to muscles of mdx mice using lentiviral vectors leads to myogenic progenitor targeting and stable gene expression. Mol Ther 18:206-13
Hall, John K; Banks, Glen B; Chamberlain, Jeffrey S et al. (2010) Prevention of muscle aging by myofiber-associated satellite cell transplantation. Sci Transl Med 2:57ra83
Himeda, Charis L; Ranish, Jeffrey A; Pearson, Richard C M et al. (2010) KLF3 regulates muscle-specific gene expression and synergizes with serum response factor on KLF binding sites. Mol Cell Biol 30:3430-43
Banks, Glen B; Chamberlain, Jeffrey S; Froehner, Stanley C (2009) Truncated dystrophins can influence neuromuscular synapse structure. Mol Cell Neurosci 40:433-41

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