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Construction and analysis of compact muscle-specific promoters for AAV vectors

Gene Therapy volume 15, pages 1489–1499 (2008)Cite this article

Abstract

Adeno-associated viral (AAV) vectors have been broadly used for gene transfer in vivo for various applications. However, AAV precludes the use of most of the original large-sized tissue-specific promoters for expression of transgenes. Efforts are made to develop highly compact, active and yet tissue-specific promoters for use in AAV vectors. In this study, we further abbreviated the muscle creatine kinase (MCK) promoter by ligating a double or triple tandem of MCK enhancer (206-bp) to its 87-bp basal promoter, generating the dMCK (509-bp) and tMCK (720-bp) promoters. The dMCK promoter is shorter but stronger than some previously developed MCK-based promoters such as the enh358MCK (584-bp) and CK6 (589-bp) in vitro in C2C12 myotubes and in vivo in skeletal muscles. The tMCK promoter is the strongest that we tested here, more active than the promiscuous cytomegalovirus (CMV) promoter. Furthermore, both the dMCK and tMCK promoters are essentially inactive in nonmuscle cell lines as well as in the mouse liver (>200-fold weaker than the CMV promoter). The dMCK promoter was further tested in a few lines of transgenic mice. Expression of LacZ or minidystrophin gene was detected in skeletal muscles throughout the body, but was weak in the diaphragm, and undetectable in the heart and other tissues. Similar to other miniature MCK promoters, the dMCK promoter also shows preference for fast-twitch myofibers. As a result, we further examined a short, synthetic muscle promoter C5-12 (312-bp). It is active in both skeletal and cardiac muscles but lacks apparent preference on myofiber types. Combination of a MCK enhancer to promoter C5-12 has increased its strength in muscle by two- to threefold. The above-mentioned compact muscle-specific promoters are well suited for AAV vectors in muscle-directed gene therapy studies.

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Acknowledgements

This work was supported by grants to Xiao Xiao from the National Institutes of Health (NIH) and the Muscular Dystrophy Association (MDA), and the scholarships to Bing Wang from the Duchenne Muscular Dystrophin Research Center (DMDRC) and Center for Cell Therapeutics (CCT), University of Pittsburgh. We would like to thank Jim Cummins and Melessa Salay for editorial assistance.

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Authors and Affiliations

  1. Department of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA

    B Wang, F H Fu & L Zhou

  2. Division of Molecular Pharmaceutics, UNC School of Pharmacy, Chapel Hill, NC, USA

    J Li, C Chen & X Xiao

  3. Cardiovascular Institute, University of Pittsburgh, Pittsburgh, PA, USA

    X Zhu

  4. Department of Anatomy and Molecular Cell Biology, The State University of New York, Brooklyn, NY, USA

    X Jiang

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Correspondence to B Wang.

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Wang, B., Li, J., Fu, F. et al. Construction and analysis of compact muscle-specific promoters for AAV vectors. Gene Ther 15, 1489–1499 (2008). https://doi.org/10.1038/gt.2008.104

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