Gene Silencing Using 4'-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response

Our plain-English summary of Gene Silencing Using 4'-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response, by N. Tarashima, H. Ando, T. Kojima, et al (Molecular Therapy. Nucleic Acids, 2016).

The study

• Authors: N. Tarashima, H. Ando, T. Kojima, N. Kinjo, Y. Hashimoto, K. Furukawa, T. Ishida, N. Minakawa
• Journal: Molecular Therapy. Nucleic Acids
• Published: 2016;5(1):e274
• Cited by: 14 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4'-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4'-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4'-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response.

In plain English

This laboratory study developed an "intelligent shRNA expression device" (iRed): a PCR-amplified 4'-thioDNA, about 362 base pairs, encoding only the U6 promoter and a short hairpin RNA, compared with the 6,503 base pair parental plasmid DNA. Four versions were made, each substituting one nucleotide type (dSA, dSG, ST or dSC). In HeLa cells transfected with luciferase reporters, pDNA suppressed pGL2 firefly luciferase expression by 95% from 24 to 72 hours at 1.0 nmol/l; the dSG and dSC iReds silenced luciferase more potently than synthetic shRNA, with dSC iRed the most potent, and shRNA transcription from the iReds increased up to 72 hours. In BALB/c nu/nu mice given intrapleural MSTO-211H-Luc human pleural mesothelioma cells, dSC iRed and the natural device markedly reduced tumour bioluminescence, although the authors state no statistically significant difference from controls. In BALB/c mice injected intravenously with 20 micrograms of DNA in PEGylated lipoplexes, pDNA strongly raised TNF-alpha, IL-6, INF-gamma and INF-alpha, while dSC and ST iReds induced lower levels.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory experimental study combining in vitro cell-culture transfection experiments with mouse in vivo experiments (orthotopic mesothelioma model and cytokine assays)
• Who was studied: HeLa cells in culture; BALB/c nu/nu male mice (5 weeks old) bearing MSTO-211H-Luc human pleural mesothelioma cells in the left pleural cavity, four mice per group; and BALB/c male mice (5 weeks old) for cytokine measurement; work done at Tokushima University, Japan
• Exposure: Transfection/injection of 4'-thioDNA iReds (dSA, dSG, dSC, ST) encoding U6 promoter plus shRNA, compared with shRNA-expressing plasmid DNA, synthetic shRNA, natural dsDNA device and negative control dsDNA; in vivo doses 1 mg/kg (devices) or 0.077 mg/kg (shRNA) intrapleurally, or 20 micrograms intravenously in PEGylated lipoplexes
• Main results:
  • The iRed with 4'-thiomodification was 362 base pairs, versus 6,503 base pairs for the parental plasmid DNA; dSA iRed incorporated 209 and ST iRed 212 4'-thio residues, dSG iRed 113 and dSC iRed 118
  • In HeLa cells, pDNA suppressed pGL2 firefly luciferase expression by 95% from 24 to 72 hours post-transfection at 1.0 nmol/l
  • In HeLa cells, dSG iRed and dSC iRed silenced luciferase more potently than synthetic shRNA, with dSC iRed the most potent
  • iReds targeting pGL2 luciferase did not significantly suppress pGL3 luciferase activity at 1.0 nmol/l, indicating target specificity
  • In the orthotopic mesothelioma mice, dSC iRed and the natural device markedly suppressed tumour luciferase activity, but no statistically significant difference from the control group was recognised; no significant body-weight loss occurred in any group
  • Four hours after intravenous injection of lipoplexes, pDNA strongly induced TNF-alpha, IL-6, INF-gamma and INF-alpha in the serum
  • dSC iRed and ST iRed induced lower levels of these cytokines than pDNA
• The authors' conclusion: The authors concluded that the 4'-thioDNA iRed acted as a bioisostere of natural DNA template for DNA polymerase and RNA polymerase III to synthesise shRNA with potent gene-silencing activity, and that its lack of immunogenicity indicates potential as an RNAi therapeutic
• Limitations the authors state:
  • The authors state that iReds containing more than two types of 4'-thio units could not be prepared, so only one type of 2'-deoxy-4'-thionucleoside unit was used
  • The authors state that further work is needed on which dSNTP is superior and on PCR conditions with more than two dSNTP types
  • The authors note the in vivo silencing difference did not reach statistical significance because of the high standard deviation in the control group

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma.

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