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

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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

DOI 10.1128/jvi.70.5.3227-3234.1996PMC PMC190186

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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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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
DOI: 10.1128/jvi.70.5.3227-3234.1996
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
Licence: journals.asm.org/non-commercial-tdm-license (tdm)
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

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

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