Expanding AAV Packaging Capacity with Trans-splicing or Overlapping Vectors: A Quantitative Comparison
Open accessAbstract
Recombinant adeno-associated (rAAV) viral vectors hold great therapeutic potential for human diseases. However, a relatively small packaging capacity (less than 5 kb) has limited the application of rAAV for certain diseases such as cystic fibrosis and Duchenne muscular dystrophy. Here we compared two mechanistically distinct approaches to overcome packaging restraints with rAAV vectors. The trans-splicing approach reconstitutes gene expression from two independent rAAV vectors, each encoding unique, nonoverlapping halves of a transgene. This process requires intermolecular concatamerization and subsequent splicing between independent vectors. A distinct overlapping vector approach uses homologous recombination between overlapping regions in two independent vectors. Using the beta-galactosidase gene as template, trans-splicing approaches were threefold (in primary fibroblasts) and 12-fold (in muscle tissue) more effective in generating full-length transgene products than the overlapping vector approach. Nevertheless, the efficiency of trans-splicing remained moderate at approximately 4.3% (for muscle) and 7% (for fibroblasts) of that seen with a single vector encoding the full-length transgene. The efficiency of trans-splicing was augmented 1185-fold by adenoviral E4, but not E2a, gene products. This augmentation was much less pronounced with the overlapping vectoring approach (12-fold). Trans-splicing and overlapping vector approaches are two viable alternatives to expand rAA…
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Split vector systems exploiting head-to-tail concatamerization have been developed to circumvent the small packaging capacity of AAV genomes.
“Recently, split vector systems, exploiting head-to-tail concatamerization formation, have been developed to circumvent the small packaging capacity of AAV genomes (13-16, 41, 52).”
From Packaging Capacity of Adeno-Associated Virus Serotypes: Impact of Larger Genomes on Infectivity and Postentry Steps (2005) · cites this paper as [14]
The trans-splicing method to expand the size of ORFs that can be successfully expressed using AAV vectors was originally developed in the laboratory of John Engelhardt and has since been utilized in several studies with varying success.
“The trans-splicing method to expand the size of ORFs that can be successfully expressed using AAV vectors was originally developed in the laboratory of John Engelhardt 52,53 and has since been utilized in several studies with varying success (e.g., refs. 53-57 ).”
From Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids (2016) · cites this paper as [55]
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Self-citation
- Authors
- Duan, D., Yue, Y., & Engelhardt, J. F.
- Affiliations
- University of Iowa (US)
- Corresponding author
- Duan, Dongsheng
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2001-10
- Volume
- 4
- Issue
- 4
- Pages
- 383-391
- ISSN
- 1525-0016 (ISSN-L)
- OpenAlex
- W2084711412
- Cited by
- 262
- References
- 5
- Citations by year
- 2026: 6 · 2025: 17 · 2024: 5 · 2023: 7 · 2022: 7 · 2021: 11 · 2020: 15 · 2019: 15 · 2018: 6 · 2017: 10 · 2016: 9 · 2015: 10 · 2014: 14 · 2013: 10 · 2012: 9
- FWCI
- 6.55
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Funders
- National Heart, Lung, and Blood Institute (HL 58340, R01HL058340)
- National Institute of Diabetes and Digestive and Kidney Diseases (P30 DK 54759, P30DK054759)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Health Sciences › Medicine › Genetics › Neurogenetic and Muscular Disorders Research
- Keywords
- recombinant adeno-associated virus vectors, trans-splicing, homologous recombination, transgene expression efficiency, lacZ reporter, muscle cells, fibroblasts, cystic fibrosis, Duchenne muscular dystrophy
- MeSH terms
- Dependovirus, Animals, beta-Galactosidase, Fibroblasts, Genetic Vectors, Mice, Inbred C57BL, Muscles, Proviruses, Recombination, Genetic, Sequence Homology, Nucleic Acid, Genetic Therapy, Adenovirus E2 Proteins, Adenovirus E4 Proteins, Gene Transfer Techniques, Virus Assembly, Transgenes, Trans-Splicing, Mice
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