Cited by 761Virus-based gene therapy researchRNA Interference and Gene DeliveryBacteriophages and microbial interactions

Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors

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Ferrari, F K, Samulski, T, Shenk, T, Samulski, R J·1996·Journal of Virology

Abstract

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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Registry

Crossref · 10.1128/jvi.70.5.3227-3234.1996

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45 of 45 references verified0 citation errors · 0 not checkable

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  • Second strand synthesis after capsid uncoating in nucleus is long-recognized as the rate-limiting step of rAAV transduction.

    “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.”

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Authors
Ferrari, F. K., Samulski, T., Shenk, T., & Samulski, R. J.
Affiliations
University of North Carolina at Chapel Hill (US)
Venue
Journal of Virology
Published
1996-05
Volume
70
Issue
5
Pages
3227-3234
ISSN
  • 0022-538X (ISSN-L)
  • 1098-5514
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
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
  • Physical Sciences › Environmental Science › Ecology › 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

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