A Functional RNA-Origami As Direct Thrombin Inhibitor with Fast-acting and Specific Single-Molecule Reversal Agents in Vivo Model

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 seconds in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulated mainly in the liver over 24 hours and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.