Open-Ended, Dicer-Cleavable Concatemeric siRNA Generated by Rolling Circle Transcription
This invention reduces dosages and improves efficacy of RNA interference drugs including cancer therapeutics.
Researchers
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concatemeric rna molecules, compositions, and methods and uses thereof
United States of America | Granted | 11,230,708
Figures
Design of open-ended periodic shRNA by selective enzymatic digestion of the periodic shRNA hairpin loops, enabling efficient Dicer recognition and processing into siRNAs.
Extended knockdown of GFP by csiRNA (csi25), T1-cleaved csiRNA (T1-csi25), and siRNA (si) with Mirus TranslT-X2® in GFP-expressing HeLa cells, at an effective siRNA concentration of 5 nM.
Luciferase knockdown by luciferase-targeting and scrambled (scr) csiRNA (csi25), T1-cleaved csiRNA (T1-csi25), and siRNA with Mirus TranslT-X2® in luciferase-expressing (a) SKOV3 cells and (b) UCI1O1 cells, with normalization of measured luciferase expression to total protein.
Technology
This designed p-shRNA before cleavage is generated from a dumbbell DNA template consisting of a 21 to 29 base pair region encoding a siRNA sequence, with one single stranded loop containing one or more guanosine residues, and the other loop containing no guanosine residues. The p-shRNA is then treated with RNase T1 which specifically cleaves single stranded RNA at the 3’ ends of guanosine residues, to produce siRNA repeats connected by single stranded linkers at one end, with the other end accessible for efficient Dicer cleavage. The inventors have shown that this Dicer-cleavable p-shRNA demonstrates much greater silencing activity than unmodified p-shRNA in GFP-expressing HeLa cells, as well as significant reporter gene knockdown in SKOV3 and UCI101 ovarian cancer cells. This p-shRNA’s greater molecular weight and flexibility compared to siRNA can potentially allow more stable complexation using less delivery materials making it ideal for optimizing gene delivery vehicles.
Problem Addressed
Short interfering RNAs (siRNAs) have great therapeutic potential but clinical translation has been hindered by poor in vivo stability, low loading efficiency, and dose-limiting toxicity. The generation of periodic short hairpin RNA (concatemeric siRNA) via rolling circle transcription (RCT) of a dumbbell shaped DNA template, which may be processed by Dicer proteins into siRNAs, has been reported as an alternative source of RNA interference. However, due to a lack of open ends, Dicer cleavage of periodic short hairpin RNA (p-shRNA) is inefficient. This invention is a more stable, cleavable p-shRNA containing open ends for efficient Dicer processing enabling high silencing efficacy with smaller amounts of delivery materials.
Advantages
- Improves efficacy of RNA inference drugs while allowing for reduced dosage amounts and frequencies
Publications
Wu, C., Li, J., Wang, W., & Hammond, P. T. (2018). Rationally Designed Polycationic Carriers for Potent Polymeric siRNA-Mediated Gene Silencing. ACS Nano, 12(7), 6504-6514. doi: 10.1021/acsnano.7b08777.
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