Self-Complementary Recombinant Adeno-Associated Viral Vectors: Packaging Capacity And The Role of Rep Proteins in Vector Purity
Abstract
Self-complementary adeno-associated viral (scAAV) vectors bypass the requirement for viral second-strand DNA synthesis, but the packaging capacity of these vectors ( approximately 2.4 kb) is significantly smaller than that of conventional AAV vectors ( approximately 4.8 kb). We constructed human recombinant green fluorescent protein (hrGFP) expression cassettes ranging from 2.3 to 4.1 kb. Each vector was biologically active, but the transduction efficiency of vectors containing <3.3-kb genomes was significantly higher than those containing 3.5-kb genomes or larger. However, scAAV vectors containing up to approximately 3.3-kb genomes also contained single-stranded genomes, and 3.5-kb and larger genomes were packaged only as single-stranded DNA. These data suggest that the maximum packaging capacity of scAAV vectors is approximately 3.3 kb. The production of single-stranded genomes was not due to repair of the terminal resolution site (trs) in the inverted terminal repeats in the AAV genome, but rather was partly due to the use of AAV helper plasmid, known to lead to higher levels of expression of Rep proteins. The use of a helper plasmid known to lead to reduced levels of Rep proteins led to the generation of scAAV vectors that contained approximately 90% of the viral genomes in double-stranded forms. These studies demonstrate the feasibility of achieving encapsidation of larger genomes into scAAV vectors than was suggested originally, but underscore the need to exercise cauti…
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The increased efficiency of scAAV is compensated for by the loss of half the packaging capacity (2.4 kb of scAAV vs. 4.8 kb of ssAAV).
“The increased efficiency is compensated for the loss of half the packaging capacity (2.4 kb of scAAV vs. 4.8 kb of ssAAV) [12].”
From Designing and optimizing AAV-mediated gene therapy for neurodegenerative diseases: from bench to bedside (2024) · cites this paper as [12]
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- Authors
- Wu, J., Zhao, W., Zhong, L., Han, Z., Li, B., Ma, W., Weigel-Kelley, K. A., Warrington, K. H., & Srivastava, A.
- Affiliations
- University of Florida Health (US)
- Division of Cellular & Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, U.S.A.
- Venue
- Human Gene Therapy
- Publisher
- SAGE Publications
- Published
- 2007-02
- Volume
- 18
- Issue
- 2
- Pages
- 171-182
- ISSN
- 1043-0342 (ISSN-L)
- 1557-7422
- OpenAlex
- W2036846364
- Cited by
- 108
- References
- 5
- Citations by year
- 2026: 5 · 2025: 5 · 2024: 5 · 2023: 2 · 2022: 3 · 2021: 5 · 2020: 2 · 2019: 4 · 2018: 6 · 2017: 5 · 2016: 5 · 2015: 9 · 2014: 8 · 2013: 3 · 2012: 5
- FWCI
- 5.35
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Human Gene Therapy (publishedVersion)
- PubMed (publishedVersion)
- Funders
- U.S. Public Health Service
- National Heart, Lung, and Blood Institute (R01HL076901, R01HL065570, R01HL-65570, R01 HL-76901)
- National Institute of Diabetes and Digestive and Kidney Diseases (P01DK058327)
- National Institute of Biomedical Imaging and Bioengineering (R01EB002073)
- 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
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- self-complementary AAV vectors, adeno-associated virus, packaging capacity, Rep protein, single-stranded DNA, double-stranded DNA, helper plasmid, inverted terminal repeats, transduction efficiency, gene therapy vectors
- MeSH terms
- Dependovirus, DNA-Binding Proteins, DNA, Recombinant, DNA, Viral, Genetic Vectors, HeLa Cells, Humans, Transduction, Genetic, Viral Proteins, Virus Replication, Genome, Viral, Sequence Deletion, DNA, Complementary, Virus Assembly
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