Dystrophin gene mutations lead to Duchenne muscular dystrophy (DMD), a severe muscle disease that affected nearly all muscles in the body. A cure for DMD requires body-wide therapy. Adeno-associated virus (AAV) is currently the only viral vector that can efficiently transduce whole body muscle. Thus AAV is the vector-of-choice for DMD gene therapy. Despite great promise, AAV gene therapy is challenged by the small viral packaging capacity (5 kb maximal). The 11.5 kb full-length dystrophin coding sequence cannot be delivered by a single AAV vector. To overcome this obstacle, investigators have developed abbreviated micro/mini-dystrophin genes. Microgenes can fit into a single AAV but it cannot restore muscle force to the normal level. A 6 kb H2-R19 minigene fully recovers muscle force. My first and only R01 was to develop dual AAV vectors to express the 6 kb minigene in a single muscle in mdx mice, a mild mouse model for DMD. We have successfully accomplished and surpassed this goal. In this renewal, we will further advance dual AAV gene therapy for DMD. Despite the fact that the 6 kb minigene can fully restore muscle force, this minigene cannot restore neuronal nitric oxide synthase (nNOS) to the sarcolemma. The loss of sarcolemmal nNOS leads to functional ischemia in mdx mice and DMD patients. We recently developed a novel 7 kb minigene that recruits nNOS to the sarcolemma. In this renewal, we will dissect out the structure motif(s) responsible for nNOS recruiting in the dystrophin gene. Furthermore, we will establish the therapeutic advantage(s) of the 7 kb minigene in transgenic mice by measuring muscle force and blood perfusion. Most importantly, we will develop novel dual AAV vectors to express the 7 kb minigene. We have previously demonstrated an efficient mini-dystrophin gene therapy with a pair of the trans-splicing AAV (tsAAV, a dual vector approach). This proof-of-principle study is performed in mdx mice by local injection. In this renewal, we will test the hypotheses that (1) systemic whole body dual AAV vector gene transfer can be achieved in a symptomatic dystrophin/utrophin double knockout (dko) mouse DMD model;(2) furthermore, systemic minigene therapy with dual AAV vectors can ameliorate muscle pathology, restore muscle force and blood perfusion in dko mice. Our long-term goal is to develop an effective minigene therapy for DMD patients. Establishing systemic gene transfer in a large animal model is a logical next step. We have recently characterized a Corgi dog model for DMD. However canine muscle has been notoriously difficult to transduce with any vector. We have now overcome this hurdle and achieved systemic single AAV transduction in wild type newborn dogs. This is the first demonstration of a whole body gene transfer in a large animal model. In this renewal, we will test the hypotheses that (1) systemic dual AAV vector transduction can be achieved in newborn dogs;(2) systemic dual AAV minigene therapy is feasible in neonatal dystrophic dogs. In summary, our study will establish the foundation for dual AAV minigene gene therapy in DMD patients in the future.

Public Health Relevance

. Duchenne muscular dystrophy is a life threatening disease affecting a fairly large population (more than one in 3,500 newborns). It is caused by dystrophin gene mutations. Dual AAV-mediated minigene therapy hold great promise to cure the disease. Our work will advance current dual vector therapy by providing a functionally superior minigene, by targeting all body muscles in a symptomatic mouse model, and by systemic approach in a dog model. Our findings will pave the way to eventually move dual AAV gene therapy to human trials in the future.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR049419-09
Application #
8136552
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Nuckolls, Glen H
Project Start
2008-06-15
Project End
2013-05-31
Budget Start
2011-06-01
Budget End
2012-05-31
Support Year
9
Fiscal Year
2011
Total Cost
$391,167
Indirect Cost
Name
University of Missouri-Columbia
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
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Duan, Dongsheng; Hakim, Chady H; Ambrosio, Carlos E et al. (2015) Early loss of ambulation is not a representative clinical feature in Duchenne muscular dystrophy dogs: remarks on the article of Barthélémy et al. Dis Model Mech 8:193-4
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Wang, Y; Zhang, K; Wasala, N B et al. (2015) Optical polarization tractography revealed significant fiber disarray in skeletal muscles of a mouse model for Duchenne muscular dystrophy. Biomed Opt Express 6:347-52
McGreevy, Joe W; Hakim, Chady H; McIntosh, Mark A et al. (2015) Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy. Dis Model Mech 8:195-213
Duan, Dongsheng (2015) Duchenne muscular dystrophy gene therapy in the canine model. Hum Gene Ther Clin Dev 26:57-69
Wasala, Nalinda B; Zhang, Keqing; Wasala, Lakmini P et al. (2015) The FVB Background Does Not Dramatically Alter the Dystrophic Phenotype of Mdx Mice. PLoS Curr 7:
Kodippili, Kasun; Vince, Lauren; Shin, Jin-Hong et al. (2014) Characterization of 65 epitope-specific dystrophin monoclonal antibodies in canine and murine models of duchenne muscular dystrophy by immunostaining and western blot. PLoS One 9:e88280
Hakim, Chady H; Yue, Yongping; Shin, Jin-Hong et al. (2014) Systemic gene transfer reveals distinctive muscle transduction profile of tyrosine mutant AAV-1, -6, and -9 in neonatal dogs. Mol Ther Methods Clin Dev 1:14002
Lostal, William; Kodippili, Kasun; Yue, Yongping et al. (2014) Full-length dystrophin reconstitution with adeno-associated viral vectors. Hum Gene Ther 25:552-62

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