Short-hairpin RNAs synthesized by T7 phage polymerase do not induce interferon.

Nucleic Acids Research
Takuma GondaiHiroshi Takaku

Abstract

RNA interference (RNAi) mediated by small-interfering RNAs (siRNAs) is a highly effective gene-silencing mechanism with great potential for gene-therapeutic applications. siRNA agents also exert non-target-related biological effects and toxicities, including immune-system stimulation. Specifically, siRNA synthesized from the T7 RNA polymerase system triggers a potent induction of type-I interferon (IFN) in a variety of cells. Single-stranded RNA also stimulates innate cytokine responses in mammals. We found that pppGn (n = 2,3) associated with the 5'-end of the short-hairpin RNA (shRNA) from the T7 RNA polymerase system did not induce detectable amounts of IFN. The residual amount of guanine associated with the 5'-end and hairpin structures of the transcript was proportional to the reduction of the IFN response. Here we describe a T7 pppGn (n = 2,3) shRNA synthesis that does not induce the IFN response, and maintains the full efficacy of siRNA.

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Citations

May 13, 2010·Molecular Therapy : the Journal of the American Society of Gene Therapy·Martin Schlee, Gunther Hartmann
Oct 22, 2009·Nucleic Acids Research·Marta OlejniczakWlodzimierz J Krzyzosiak
Jan 19, 2012·Molecular Cancer Research : MCR·Katherine B ChiappinelliPaul J Goodfellow
Jul 26, 2008·Respiratory Research·Tianbo LiHans G Folkesson
Apr 19, 2012·Nucleic Acid Therapeutics·Tatyana O KabilovaElena L Chernolovskaya
Jan 6, 2009·Immunological Reviews·Martin SchleeGunther Hartmann
Dec 14, 2018·PLoS Pathogens·Zicong XieHongsheng Ouyang

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Methods Mentioned

BETA
transfection
in vitro transcription
electrophoresis
ELISA
enzyme-linked immunosorbent assay

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