Delivery of Preassembled Double-Stranded Short Interference RNA-Argonaute Protein Complexes for Enhanced RNAi-Based Therapeutics
Increasing the efficiency of knockdown using siRNA or miRNA has the potential to boost RNAi technology’s performance in the clinic.
Researchers
-
argonaute protein-double stranded rna complexes and uses
United States of America | Granted | 10,994,025
Figures
Codelivering double stranded (ds) siRNA with recombinant Ago2 results in superior silencing of GFP expression. 20nM ds siRNA against GFP (siGFP) was transfected alone or along with Ago2 at 1:0.5 and 1:1 molar ratios in (A) HEK293T and (B) NIH3T3 cells which were modified to stably express GFP. Relative GFP levels were quantified by flow cytometry at 24, 48 and 72 hr post transfection and normalized to nontransfected GFP-expressing cells.
Codelivering miRNA34a with recombinant Ago2 results in superior silencing of miRNA34a targets. (A) We first engineered a GFP with a mi34a binding site at 3’ untranslated microregion (UTR). (B), 20nMRNA34a was transfected alone or along with Ago2 at 1:0.5 and 1:1 molar ratios in HEK293T cells which were modified to express GFP-miRNA34a binding site. Relative GFP levels were quantified by flow cytometry at 24, 48, 72 and 96 hr post transfection.
(C) To test if co-delivery of Ago2 could improve the silencing of endogenous microRNA34a targets, 20nM microRNA34a was transfected alone or along with Ago2 at 1:0.5 into ovarian cancer cells, OVCAR8. Co-delivery of Ago2 results in ~3 fold silencing of the microRNA34a target, CDK4 compared to traditional delivery of microRNA34a alone.
Technology
Ago2 is a core component of RISC complex that executes the actual silencing step of RNAi. The current invention preassembles siRNA or miRNA with Ago2 and demonstrates that transfection of preassembled complexes has a higher knockdown efficiency at the same concentration compared to siRNA or miRNA alone. This effect is as high as 3 fold and increases as the molar ratio of Ago2 to siRNA/miRNA gets closer to 1:1.
Problem Addressed
Delivering sufficient amount of siRNA or miRNA to achieve efficient knockdown is a challenge in RNAi therapy. Thus, there is a need to increase the efficiency of knockdown per molecule delivered. The current invention delivers preassembled siRNA or miRNA with argonaute protein (Ago2) to enhance knockdown efficiency.
Advantages
- Preassembled siRNA or miRNA with Ago2 protein increases knockdown efficiency by as much as 3 fold
License this technology
Interested in this technology? Connect with our experienced licensing team to initiate the process.
Sign up for technology updates
Sign up now to receive the latest updates on cutting-edge technologies and innovations.