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Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors
Open accessAbstract
The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100- and 1,000-fold. The increase in transduction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV life cycle. Chemical and physical agents that have been shown to induce helper-free replication of wild-type AAV were also able to stimulate recombinant AAV transduction, suggesting that the phenomenon might affect AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis of a second strand on the single-stranded genomic AAV DNA. These data suggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have important implications for the use of AAV vectors in gene therapy protocols.
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“Second strand synthesis after capsid uncoating in nucleus is long-recognized as the rate-limiting step of rAAV transduction (89); an improvement in such efficacy could allow rAAV administration at lower dose.”
From Immunomodulation in Administration of RAAV: Preclinical and Clinical Adjuvant Pharmacotherapies (2021) · cites this paper as [89]
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Metadata
- Authors
- Ferrari, F. K., Samulski, T., Shenk, T., & Samulski, R. J.
- Affiliations
- University of North Carolina at Chapel Hill (US)
- ORCID
- Shenk, T
- Venue
- Journal of Virology
- Publisher
- American Society for Microbiology
- Published
- 1996-05
- Volume
- 70
- Issue
- 5
- Pages
- 3227-3234
- ISSN
- 0022-538X (ISSN-L)
- 1098-5514
- PMC
- PMC190186
- OpenAlex
- W2165815741
- Cited by
- 761
- References
- 46
- Citations by year
- 2026: 13 · 2025: 16 · 2024: 16 · 2023: 17 · 2022: 13 · 2021: 15 · 2020: 25 · 2019: 17 · 2018: 15 · 2017: 10 · 2016: 22 · 2015: 27 · 2014: 21 · 2013: 17 · 2012: 27
- FWCI
- 24.99
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Virology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (5r01dk042701-06)
- National Heart, Lung, and Blood Institute (HL48327, P01HL051818, HL51818)
- National Institute of Diabetes and Digestive and Kidney Diseases (DK 42701)
- 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 › Immunology and Microbiology › Virology › Bacteriophages and microbial interactions
- Keywords
- recombinant adeno-associated virus vectors, viral uncoating, gene therapy vectors
- MeSH terms
- Dependovirus, Adenoviridae, beta-Galactosidase, Cell Line, DNA, Viral, Genetic Vectors, HeLa Cells, Humans, Kinetics, Plasmids, Recombinant Proteins, Recombination, Genetic, Transduction, Genetic, Transfection, Ultraviolet Rays, X-Rays, Gene Expression, Mutagenesis, Open Reading Frames
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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