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 (4200-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.

The Duchenne and Limb girdle muscular dystrophies (DMD, LGMD) are a heterogeneous group of genetic disorders that affects about 1 in 3500 males each year. Primary mutations in the dystrophin gene result in the absence of the protein in DMD, and mutations in any one of four sarcoglycan (α, β, ∆, γ) genes results in a loss of the entire sarcoglycan complex in LGMD [1] (Fig. 1). In humans these myopathies result in progressive muscle weakness and eventually patient death due to cardiac failure. Despite selective in vitro studies examining the assembly pathway of the dystrophin and sarcoglycan complex in fixed systems, the precise mechanism of how these genes behave in living cells remained unknown. In the present study we have developed and implemented a model system to study dystrophin protein complex (DPC) assembly in living cells. We demonstrate that dystrophin associated proteins (DAP) follow a sequential assembly process which can be observed in real time.Here, we report the construction and characterization of a 4.5kb minidystrophin-EGFP fusion gene, and a 1.9kb α-sarcoglycan -EGFP fusion gene. Live cell microscopy is utilized to show that minidystrophin is deposited at the plasma membrane (PM) (Fig. 2B-E) soon after expression in living muscle cells, early in differentiation. Once at the surface we demonstrate that minidystrophin is firmly anchored to the sarcolemma via interactions with select DPC members that precede the expression of dystrophin and the sarcoglycans ( Fig. 2A). At the same time point early during differentiation, α-sarcoglycan-EGFP does not properly assemble at the PM in living cells (Fig. 3A). DiI lipid dyes and multi-color live cell imaging is used to show that α-sarcoglycan is confined to motile vesicular structures (Fig. 3B-I) that accumulate in large clusters approximately 5 hours after expression, resulting in cell death. In mature mouse muscle containing all normal sarcoglycans, confocal microscopy is used to demonstrate that α-sarcoglycan-EGFP correctly localizes to the sarcolemma of the muscle fiber.Together these data support a model whereby dystrophin expression and localization at the PM precedes and is independent of the sarcoglycans. It is not until later during myotube differentiation that the sarcoglycan proteins assemble and cooperate in a specific fashion to translocate to the PM. These results are the first to demonstrate the systematic and stepwise assembly of the dystrophin protein complex in living cells. Understanding the basic molecular mechanisms that underlie DPC assembly is crucial for the proper development of gene therapies for the muscular dystrophies in humans.