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Media type:
E-Article
Title:
A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry
Contributor:
Kwak, Seo Young;
Han, Hee Dong;
Ahn, Hyung Jun
Published:
Springer Science and Business Media LLC, 2019
Published in:
Scientific Reports, 9 (2019) 1
Language:
English
DOI:
10.1038/s41598-019-39407-8
ISSN:
2045-2322
Origination:
Footnote:
Description:
AbstractThe transient silencing effects currently demonstrated by nonviral siRNA delivery systems limit the therapeutic utility of RNAi, but it remains a technical challenge to prolong duration of gene silencing. We have developed a T7 autogene-based hybrid mRNA/DNA system to enable long-term expression of shRNA in cytoplasm in vitro and in vivo. This hybrid mRNA/DNA system consists of T7 polymerase (T7pol) mRNA, pT7/shRNA-encoding DNA fragment and T7 autogene plasmid, and it can generate higher levels of T7pol proteins, compared to pCMV-triggering T7 autogene system, especially without the need of nuclear entry of any gene. A large amount of T7pol proteins produced are used to induce pT7-driven expression of shRNA in cytoplasm, and through cellular processing of RNA hairpins, mature siRNAs are generated for more than 13 days. We here demonstrate that a single liposomal delivery of this hybrid system leads to the long-term silencing effects in vitro and in vivo, in contrast to the conventional siRNA methods relying on the repeated administrations every 2 or 3 days. These sustainable shRNA expression properties in cytoplasm can provide an efficient strategy to address the limitations caused by shRNA-encoding plasmid DNA systems such as low nuclear entry efficiency and short-term silencing effect. The development of long-term shRNA expression system in vivo could scale down administration frequency of RNAi therapeutics in the treatment of chronic diseases, thereby increasing its clinical utility.