Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene

Sakamoto, Miki; Yuasa, Katsutoshi; Yoshimura, M; Yokota, Toshifumi; Ikemoto, Takaaki; Suzuki, Misao; Dickson, George; Miyagoe-Suzuki, Yuko; Takeda, Shin‐ichi

doi:10.1016/s0006-291x(02)00362-5

Cited by 104 publications

(69 citation statements)

References 28 publications

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“…Also, as there are no effective treatments available, 3,4 DMD patients eventually become wheel-chair bound in their early twenties due to progressive muscle weakness, with death resulting from respiratory or cardiac failure. 5 As the genetics of this disease have been well characterized, gene therapy offers hope for treatment. 3 For muscular dystrophies and other muscle degenerative disorders, the use of muscle-specific promoters is highly desirable in the development of gene therapy vectors.…”

Section: Instructionmentioning

confidence: 99%

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

et al. 2008

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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.

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Section: Instructionmentioning

confidence: 99%

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

et al. 2008

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“…3,7,8 To improve the expression efficiency of the LacZ gene in rAAV2-CMVLacZ, a chimeric intron (human b-globin splicing donor and immunoglobulin splicing acceptor, Promega, Madison, WI, USA) is inserted between CMV promoter and the LacZ gene. 8 rAAV2-LacZ-P(À) was generated by deleting the truncated MCK promoter (358 bp) from rAAV2-MCKLacZ, leaving the MCK enhancer intact.…”

Section: Construction and Production Of Raavsmentioning

confidence: 99%

“…[3][4][5][6] We constructed a series of rod-truncated microdystrophin cDNAs, 3 and generated transgenic mdx mice expressing each microdystrophin, and demonstrated that microdystrophin CS1 with four rod repeats and three hinges was a good candidate for therapeutic molecule. 7 We have also showed that the muscle-specific muscle creatine kinase (MCK) promoter in a rAAV drives longer transgene expression than the ubiquitous cytomegalovirus (CMV) promoter in mdx muscle. 8 Therefore, we generated the rAAV2 expressing microdystrophin DCS1 (3.8 kb cDNA) driven by the MCK promoter and introduced it into mdx muscles, and showed that sustained expression of microdystrophin from rAAV significantly ameliorates dystrophic phenotypes of treated mdx mice.…”

Section: Introductionmentioning

confidence: 99%

Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products

et al. 2007

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Using murine models, we have previously demonstrated that recombinant adeno-associated virus (rAAV)-mediated microdystrophin gene transfer is a promising approach to treatment of Duchenne muscular dystrophy (DMD). To examine further therapeutic effects and the safety issue of rAAV-mediated microdystrophin gene transfer using larger animal models, such as dystrophic dog models, we first investigated transduction efficiency of rAAV in wild-type canine muscle cells, and found that rAAV2 encoding b-galactosidase effectively transduces canine primary myotubes in vitro. Subsequent rAAV2 transfer into skeletal muscles of normal dogs, however, resulted in low and transient expression of b-galactosidase together with intense cellular infiltrations in vivo, where cellular and humoral immune responses were remarkably activated.In contrast, rAAV2 expressing no transgene elicited no cellular infiltrations. Co-administration of immunosuppressants, cyclosporine and mycophenolate mofetil could partially improve rAAV2 transduction. Collectively, these results suggest that immune responses against the transgene product caused cellular infiltration and eliminated transduced myofibers in dogs. Furthermore, in vitro interferon-g release assay showed that canine splenocytes respond to immunogens or mitogens more susceptibly than murine ones. Our results emphasize the importance to scrutinize the immune responses to AAV vectors in larger animal models before applying rAAV-mediated gene therapy to DMD patients.

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“…9 Therefore, microdystrophin, in which most of the rod domains of dystrophin are deleted, was developed to fit into rAAV. 10,11 We and others have reported successful improvement of the dystrophic phenotype in skeletal muscles of DMD mice models following introduction of this transgene. 12,13 To achieve transduction of an entire extremity, a limb perfusion by retrograde intravenous administration of the rAAV in larger animal models is being developed.…”

Section: Introductionmentioning

confidence: 99%

Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction

et al. 2011

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Duchenne muscular dystrophy (DMD) is the most common form of the progressive muscular dystrophies characterized by defects of the dystrophin gene. Although primarily characterized by degeneration of the limb muscles, cardiomyopathy is a major cause of death. Therefore, the development of curative modalities such as gene therapy is imperative. We evaluated the cardiomyopathic features of mdx mice to observe improvements in response to intravenous administration of recombinant adeno-associated virus (AAV) type 9 encoding microdystrophin. The myocardium was extensively transduced with microdystrophin to significantly prevent the development of fibrosis, and expression persisted for the duration of the study. Intraventricular conduction patterns, such as the QRS complex duration and S/R ratio in electrocardiography, were also corrected, indicating that the transduced microdystrophin has a protective effect on the dystrophin-deficient myocardium. Furthermore, BNP and ANP levels were reduced to normal, suggesting the absence of cardiac dysfunction. In aged mice, prevention of ectopic beats as well as echocardiographic amelioration was also demonstrated with improved exercise performance. These findings indicate that AAV-mediated cardiac transduction with microdystrophin might be a promising therapeutic strategy for the treatment of dystrophin-deficient cardiomyopathy.

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Micro-dystrophin cDNA ameliorates dystrophic phenotypes when introduced into mdx mice as a transgene

Cited by 104 publications

References 28 publications

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

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

Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products

Improvement of cardiac fibrosis in dystrophic mice by rAAV9-mediated microdystrophin transduction

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