Novel Mini–Dystrophin Gene Dual Adeno-Associated Virus Vectors Restore Neuronal Nitric Oxide Synthase Expression at the Sarcolemma
Open accessAbstract
Six- to 8-kb mini-dystrophin genes are promising candidates for Duchenne muscular dystrophy (DMD) gene therapy. Several dual adeno-associated virus (AAV) mini-dystrophin vectors have been tested in dystrophin-deficient mice. Despite the encouraging preclinical results, none of the existing dual AAV vectors can restore sarcolemmal neuronal nitric oxide synthase (nNOS) expression. Localization of nNOS to the sarcolemma may greatly improve the therapeutic outcome in DMD (Lai, Y., Thomas, G.D., Yue, Y., et al. [2009]. J. Clin. Invest. 119, 624-635). In this study, we developed a series of dual AAV expression vectors to express a synthetic minigene that carries the nNOS localization domain. To help validate dual vector reconstitution, we also included a FLAG tag and a GFP reporter at different ends of the minigene. These dual AAV vectors were packaged in Y445F tyrosine mutant AAV-6 and tested in dystrophin-null mdx4cv mice by direct muscle injection. All dual vectors expressed GFP/FLAG-tagged mini-dystrophin and restored sarcolemmal nNOS. However, the reconstitution efficiency was significantly different among different sets. The dual vector set YZ27/YZ22 yielded the highest transduction efficiency (∼90%). Further development of this set dual vector may lead to more effective DMD gene therapy.
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Overlapping vectors work particularly well in muscle tissue, ameliorating phenotypes linked to Duchenne muscular dystrophy and dysferlinopathies.
“This strategy was shown to work particularly well in muscle tissue, ameliorating phenotypes linked to Duchenne muscular dystrophy (DMD) [43][44][45] and dysferlinopathies [46][47][48].”
From Circumventing the packaging limit of AAV-mediated gene replacement therapy for neurological disorders (2022) · cites this paper as [45]
The Results section (‘Dual vector set IV resulted in superior transduction in mdx4cv muscle’; page unavailable) reports that the overlapping-vector set had the highest transduction efficiency in muscle, and the Discussion reports restored mini-dystrophin and sarcolemmal nNOS in dystrophin-deficient mice. This supports the DMD-related muscle result, but the paper does not show that treatment ameliorated a disease phenotype such as muscle function. The dysferlinopathy clause is attached to different citations, not this source. (LLM, conf=0.84)
Experimental — accuracy not yet measuredThe choice of the best dual-vector design can depend significantly on the nature of the therapeutic protein.
“These results provide further evidence that the choice of the best dual-vector design can depend significantly on the nature of the therapeutic protein.”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [68]
The Discussion reports that the best-performing dual-vector set for mini-dystrophin depended on the gene-splitting site and the sequence used for recombination; the Results also show significantly higher transduction with set IV. But the study compares designs for one therapeutic protein, not different proteins, so it does not directly establish that the preferred design depends on the nature of the protein. (LLM, conf=0.82)
Experimental — accuracy not yet measuredIn the mdx4cv mouse model of Duchenne muscular dystrophy, muscle transduction was significantly greater when the dual overlapping vector system was used, with an expression level approximately three times higher than with vectors based on the hybrid approach.
“Interestingly, they found that muscle transduction in the mdx4cv mouse model of Duchenne muscular dystrophy was significantly greater when the dual overlapping vector system was used-with an expression level approximately three times higher than with vectors based on the hybrid approach.”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [68]
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Experimental — accuracy not yet measuredA simple, dual overlap vector system outperformed three independent hybrid systems.
“For instance, Zhang and Duan demonstrated that a simple, dual overlap vector system outperformed three independent hybrid systems, 68 and Pryadkina et al. obtained similar results when they analyzed the efficiency of dysferlin overexpression obtainable with dual overlapping vector or hybrid vector systems. 45”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [68]
Hybrid systems are not always the most efficient vector systems available.
“It needs to be pointed out, however, that hybrid systems are not always the most efficient vector systems available.”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [68]
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References 31 · Cited by 95
Research works printed but not linked: 3
- Duan, D., Yan, Z., and Engelhardt, J.F. (2006). Expanding the capacity of AAV vectors. In Parvoviruses . M.E. Bloom, S.F. Cotmore, R.M. Linden, C.R. Parrish, and J.R. Kerr, eds. (Hodder Arnold; distributed in the United States by Oxford University Press, London, New York) pp. 525-532.
- Thomas, G.D., Sander, M., Lau, K.S., et al. (1998). Impaired metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle. Proc. Natl. Acad. Sci. U. S. A. 95, 15090-15095.
- Zhong, L., Li, B., Mah, C.S., et al. (2008). Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc. Natl. Acad. Sci. U. S. A. 105, 7827-7832.
- Authors
- Zhang, Y., & Duan, D.
- Affiliations
- University of Missouri (US)
- Corresponding author
- Duan, Dongsheng
- ORCID
- Duan, Dongsheng
- Venue
- Human Gene Therapy
- Publisher
- SAGE Publications
- Published
- 2012-01
- Volume
- 23
- Issue
- 1
- Pages
- 98-103
- ISSN
- 1043-0342 (ISSN-L)
- 1557-7422
- PMC
- PMC3260444
- OpenAlex
- W2002153634
- Cited by
- 95
- References
- 31
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 4 · 2023: 4 · 2022: 1 · 2021: 6 · 2020: 3 · 2019: 6 · 2018: 10 · 2017: 8 · 2016: 6 · 2015: 13 · 2014: 12 · 2013: 8 · 2012: 8
- FWCI
- 5.59
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Human Gene Therapy (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Muscular Dystrophy Association
- National Institutes of Health
- National Heart, Lung, and Blood Institute (R01HL091883, HL-91883)
- National Institute of Neurological Disorders and Stroke (R21NS062934, NS-62934)
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR49419, R01AR049419)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
- Health Sciences › Medicine › Genetics › Neurogenetic and Muscular Disorders Research
- Keywords
- Duchenne muscular dystrophy, mini-dystrophin, neuronal nitric oxide synthase, gene therapy, dystrophin-deficient mice
- MeSH terms
- Dependovirus, Animals, Genetic Vectors, Humans, Injections, Intramuscular, Male, Muscular Dystrophy, Animal, Plasmids, Sarcolemma, Blotting, Western, Genetic Therapy, Dystrophin, Gene Transfer Techniques, Mice, Inbred mdx, Muscle, Skeletal, Green Fluorescent Proteins, Mice, Nitric Oxide Synthase Type I
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