Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver
Dirk Grimm, … , Theresa A. Storm, Mark A. Kay
Dirk Grimm, … , Theresa A. Storm, Mark A. Kay
Published August 9, 2010
Citation Information: J Clin Invest. 2010;120(9):3106-3119. https://doi.org/10.1172/JCI43565.
View: Text | PDF
Research Article Genetics

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

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.

Authors

Dirk Grimm, Lora Wang, Joyce S. Lee, Nina Schürmann, Shuo Gu, Kathleen Börner, Theresa A. Storm, Mark A. Kay

×

Figure 3

Strategies to improve in vivo RNAi efficacy, toxicity, and persistence.

Options: View larger image (or click on image) Download as PowerPoint
Strategies to improve in vivo RNAi efficacy, toxicity, and persistence. ...
(A and C) Adult hAAT-transgenic mice (n = 3 to 5) were infused with the shown vectors, following the injection regime in B. Arrows in B indicate periods of measurable hAAT knockdown. C-X, CMV–Xpo-5; S1/8, serotypes 1/8; T-A, TTR–Ago-2; T-X, TTR–Xpo-5. (D) Measurements of serum ALT levels in mice (each bar represents 1 mouse) 12 days after injection of a low (L) or high (H) dose (8 × 1010 or 3 × 1011) of H25 shRNA-encoding sdsAAV-8, together with the indicated Ago-2/Xpo-5 AAV expression vectors (or none as control). (E) In vivo hAAT knockdown in hAAT-transgenic mice (n = 3–5) infused with sdsAAV-8 vectors expressing hAAT-19 shRNA (a shorter and less toxic form of hAAT-25; ref. 3) under the U6, H1, or 7SK pol III promoter. Note the much more persistent long-term hAAT silencing with the H1 and 7SK constructs, which correlated well with their prolonged persistence in the livers (Southern blots in F: each lane represents 1 mouse; bottom panel, DNA copy standard) and is most likely due to their inherently weaker shRNA expression (Northern blots in G). Also note the consistent drops in vector genomes and shRNA levels with the stronger and thus toxic U6 promoter constructs. (H and I) Validation in the HBV transgenic mouse model, showing delayed but ultimately safe and potent long-term in vivo shRNA expression and target suppression with the H1 and 7SK promoters, but not the more toxic U6 vectors. (I) Relative HBV surface antigen levels 13 months after infusion (P < 0.01, H1 versus U6; P < 0.05, 7SK versus U6).