A functional RNA-origami as direct thrombin inhibitor with fast-acting and specific single-molecule reversal agents in vivo model

Abhichart Krissanaprasit; Emily Mihalko; Katherine Meinhold; Aryssa Simpson; Jennifer Sollinger; Sanika Pandit; Daniel M Dupont; Jørgen Kjems; Ashley C Brown; Thomas H LaBean

doi:10.1016/j.ymthe.2024.05.002

A functional RNA-origami as direct thrombin inhibitor with fast-acting and specific single-molecule reversal agents in vivo model

Mol Ther. 2024 May 7:S1525-0016(24)00302-2. doi: 10.1016/j.ymthe.2024.05.002. Online ahead of print.

Affiliations

¹ Department of Materials Science and Engineering, College of Engineering, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: akrissa@ncsu.edu.
² Joint Department of Biomedical Engineering, College of Engineering, North Carolina State University and University of North Carolina-Chapel Hill, Raleigh, NC 27695, USA.
³ Department of Materials Science and Engineering, College of Engineering, North Carolina State University, Raleigh, NC 27695, USA.
⁴ Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, 8000 Aarhus, Denmark.
⁵ Joint Department of Biomedical Engineering, College of Engineering, North Carolina State University and University of North Carolina-Chapel Hill, Raleigh, NC 27695, USA; Comparative Medicine Institute, North Carolina State University and University of North Carolina, Chapel Hill, NC 27695, USA.
⁶ Department of Materials Science and Engineering, College of Engineering, North Carolina State University, Raleigh, NC 27695, USA; Comparative Medicine Institute, North Carolina State University and University of North Carolina, Chapel Hill, NC 27695, USA. Electronic address: thlabean@ncsu.edu.

PMID: 38720458
DOI: 10.1016/j.ymthe.2024.05.002

Abstract

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 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h 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.

Keywords: RNA nanotechnology; RNA origami; anticoagulant; aptamer; direct thrombin inhibitor; in vivo; murine model; nanomedicine; nucleic acid nanostructure; reversal agent.