Striated Muscle Gene Transcription and Regulatory Gene Cassettes for Gene Therapy

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DESCRIPTION (provided by applicant): This project combines basic research aimed at understanding how muscle genes are regulated with applied research aimed at improving regulatory cassettes for expressing therapeutic proteins in muscle. Both components focus on the mouse M-creatine kinase gene. MCK is an informative model because it is expressed in skeletal and cardiac muscle, and at varying levels in different muscles & fast/slow muscle fibers. These attributes plus MCK's intrinsically high expression make its components attractive for use in muscle- specific regulatory cassettes. Aims I & 2 continue studies that are identifying control elements & transcription factors associated with four MCK regions containing blocks of highly conserved sequence motifs with unknown functions. Understanding these regions is important because they are involved in muscle gene activation during development, fiber type-specific gene transcription, and response to physiological signals. The overall strategy involves muscle cell culture tests of the transcriptional activity conferred by conserved sequence motifs followed by assays for nuclear factor binding to candidate control elements and quantitative proteomic analysis of differentially-enriched nuclear extracts to identify transcription factor candidates. ChIP analysis is then used to corroborate association of candidate factors with the control elements. Further characterization of control element roles in MCK gene expression in adult muscles & during development is accomplished with systemic viral delivery & transgenic mouse experiments. Information from these studies should be broadly applicable to the regulation of many muscle genes. Aims 3 & 4: Applied aspects of the project use control elements & information from Aims 1 & 2, as well as control regions from other muscle genes, to design optimal regulatory cassettes for gene therapy. Proteins expressed from these cassettes could be used for treating muscle diseases, muscle aging & injury problems, and diseases in which skeletal muscle could be used to secrete therapeutic proteins, e.g., hormone & clotting factor diseases. The cassettes can also be used for many basic research purposes. Aim-3 will optimize the transcriptional activity of cassettes designed to function in different striated muscle types, while also maintaining high muscle specificity to prevent expression in immune system and other non-muscle cells. An additional goal will focus on designing miniature regulatory cassettes that are compatible with packaging large therapeutic cDNAs such as mini- & micro-dystrophins within the limited packaging space in AAV & other viral vectors. The purpose of Aim-4 is to allow therapeutic product levels to be externally regulated by the levels of a non-harmful drug. Cassettes from Aim-3 will be optimized for expressing the DNA-binding & activation domains of previously designed artificial transcription factors (ATFs). Since ATF activity requires dimerization of the 2 domains, and since the amount of dimerization depends on drug concentration, the transcription rate of therapeutic cDNAs linked to unique DNA- binding sites for the ATF can then be regulated by manipulating drug dosage. PUBLIC HEALTH RELEVANCE. Experimental analysis of muscle gene control regions and associated transcription factors is critical for obtaining a full understanding of how muscle genes are regulated during human growth and development and in both healthy and diseased adult muscle. An additional value is that newly discovered control regions can be used to create optimal regulatory cassettes for gene therapy treatments of patients with genetic muscle diseases and with major muscle loss through trauma or geriatric causes. Improved regulatory cassette activity decreases the number of viral vectors needed for patient treatment, thereby increasing safety and decreasing viral production costs.