Full-Length Dystrophin Reconstitution with Adeno-Associated Viral Vectors
Open accessAbstract
Duchenne muscular dystrophy (DMD) is the most common lethal muscle disorder in children. It is caused by mutations of the dystrophin gene. Adeno-associated virus (AAV)-mediated gene replacement therapy has been actively pursued to treat DMD. However, this promising therapeutic modality has been challenged by the small packaging capacity of the AAV vector. The size of the full-length dystrophin cDNA is >11 kb, while an AAV virus can carry only a 5 kb genome. Innovative high-capacity AAV vectors may offer an opportunity to express the full-length dystrophin coding sequence. Here we describe several sets of tri-AAV vectors for full-length human dystrophin delivery. In each set, the full-length human dystrophin cDNA was split into three fragments and independently packaged into separate recombinant AAV vectors. Each vector was engineered with unique recombination signals for directional recombination. Tri-AAV vectors were coinjected into the tibialis anterior muscle of dystrophin-deficient mdx4cv mice. Thirty-five days after injection, dystrophin expression was examined by immunofluorescence staining. Despite low reconstitution efficiency, full-length human dystrophin was successfully expressed from the tri-AAV vectors. Our results suggest that AAV can be engineered to express an extra-large (up to 15 kb) gene that is approximately three times the size of the wild-type AAV genome. Further optimization of the trivector strategy may expand the utility of AAV for human gene therapy.
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Triple AAV vectors can increase the genome capacity to up to 14 kb, thus expanding the possibilities for full-length reconstitution of large transgenes.
“However, triple AAV vectors can increase the genome capacity to up to 14 kb, thus expanding the possibilities for full-length reconstitution of large transgenes [28].”
From Circumventing the packaging limit of AAV-mediated gene replacement therapy for neurological disorders (2022) · cites this paper as [28]
The Discussion says tri-AAV vectors may expand total carrying capacity and enable delivery of large genes such as full-length dystrophin. It gives the capacity as up to 15 kb, however, not the cited 14 kb. (LLM, conf=0.87)
Experimental — accuracy not yet measuredDual/triple vector approaches split the transgene into two or three separate sequences delivered by independent AAVs and allow reassembly to produce the full-length transgene upon co-infection.
“In the so-called 'dual/triple vector approaches,' the transgene is split into either two or three separate sequences delivered by independent AAVs and allowed to reassemble to produce the full-length transgene upon co-infection [28].”
From Circumventing the packaging limit of AAV-mediated gene replacement therapy for neurological disorders (2022) · cites this paper as [28]
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Cites
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Research works printed but not linked: 3
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Trapani, I., Toriello, E., de Simone, S., Colella, P., Iodice, C., Polishchuk, E. V., Sommella, A., Colecchi, L., Rossi, S., Simonelli, F., et al. (2015) · Human Molecular Genetics · doi:10.1093/hmg/ddv386 · Cited by 94 · see citing works
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- Authors
- Lostal, W., Kodippili, K., Yue, Y., & Duan, D.
- Affiliations
- University of Missouri (US)
- Corresponding author
- Duan, Dongsheng
- Venue
- Human Gene Therapy
- Publisher
- SAGE Publications
- Published
- 2014-06
- Volume
- 25
- Issue
- 6
- Pages
- 552-562
- ISSN
- 1043-0342 (ISSN-L)
- 1557-7422
- PMC
- PMC4064726
- OpenAlex
- W2083455105
- Cited by
- 77
- References
- 6
- Citations by year
- 2026: 5 · 2025: 2 · 2024: 4 · 2023: 3 · 2022: 6 · 2021: 6 · 2020: 3 · 2019: 6 · 2018: 6 · 2017: 10 · 2016: 8 · 2015: 13 · 2014: 5
- FWCI
- 7.88
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Human Gene Therapy (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Muscular Dystrophy Association
- University of Washington
- 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
- Health Sciences › Medicine › Genetics › Neurogenetic and Muscular Disorders Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- Duchenne muscular dystrophy, dystrophin, adeno-associated virus vectors, gene replacement therapy, gene therapy
- MeSH terms
- Dependovirus, Animals, Cloning, Molecular, Genetic Vectors, Humans, Male, Recombinant Proteins, Transduction, Genetic, Genetic Therapy, Gene Expression, Dystrophin, Mice, Inbred mdx, Muscle, Skeletal, Muscular Dystrophy, Duchenne, Cell Line, Tumor
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