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Cited by 196RNA Interference and Gene DeliveryVirus-based gene therapy researchCRISPR and Genetic Engineering

Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver

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Grimm, Dirk, Wang, Lora, Lee, Joyce S., Schürmann, Nina, Gu, Shuo, Börner, Kathleen, Storm, Theresa A., Kay, Mark A.·2010·Journal of Clinical Investigation
DOI 10.1172/jci43565PMC PMC2929739
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Abstract

shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short- and long-term clinical gene silencing in humans.

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  • Factors beyond transcriptional competition, such as saturation of RISC or altered miRNA stability, may contribute to the observed differences in efficacy.

    “This suggests that factors beyond transcriptional competition-such as saturation of the RNA-induced silencing complex (RISC) or altered miRNA stability-may contribute to the observed differences in efficacy 46 .”

    From rAAV-mediated Multi-miRNA Delivery Targeting MAPK Pathway Suppresses Laser-induced Choroidal Neovascularization in Mice (2026) · cites this paper as [46]

    The section “Ago-2 saturation depends on trigger-target sequence homologies” reports Ago-2/RISC saturation and says it occurs at varying efficacies, supporting RISC saturation as a possible contributor to efficacy differences. The Discussion mentions long miRNA half-lives and effects on miRNA biogenesis and maturation, but does not establish that altered miRNA stability contributes to those differences. No page numbers are available in our copy. (LLM, conf=0.83)

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Cited by

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Authors
Grimm, D., Wang, L., Lee, J. S., Schürmann, N., Gu, S., Börner, K., Storm, T. A., & Kay, M. A.
Affiliations
  • Heidelberg University (DE)
  • Stanford Medicine (US)
Corresponding author
Kay, Mark A.
ORCID
Venue
Journal of Clinical Investigation
Publisher
American Society for Clinical Investigation
Published
2010-09-01
Volume
120
Issue
9
Pages
3106-3119
ISSN
0021-9738 (ISSN-L)
DOI
10.1172/jci43565
PMC
PMC2929739
OpenAlex
W2161582292
Cited by
196
References
2
Citations by year
2026: 2 · 2025: 3 · 2024: 9 · 2023: 4 · 2022: 5 · 2021: 8 · 2020: 8 · 2019: 9 · 2018: 6 · 2017: 11 · 2016: 13 · 2015: 18 · 2014: 22 · 2013: 26 · 2012: 22
FWCI
10.17
Type
journal article
Language
en
Open access
Locations
  • Journal of Clinical Investigation (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Chica and Heinz Schaller Foundation
  • National Institutes of Health
  • National Institute of Allergy and Infectious Diseases (AI071068, R01AI071068)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
Argonaute proteins, RNA interference, shRNA, hepatotoxicity, XPO5, Argonaute 2, liver, hepatitis B virus, RNA polymerase III promoter, gene silencing, U6 promoter
MeSH terms
Animals, Female, Genetic Vectors, Hepatitis B virus, Liver, Mice, Transgenic, RNA Polymerase III, Eukaryotic Initiation Factor-2, Transgenes, Gene Silencing, Karyopherins, RNA Interference, Mice, Argonaute Proteins

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