Abstract 19616: M3-RNA Encapsulation Technology Enables Rapid and Temporally Restricted Protein Expression
Introduction: Targeted induction of protein synthesis by exogenous delivery of coding synthetic mRNA would provide a unique new resource for intervention. Here, an M3-RNA (Microencapsulated Modified Messenger RNA) approach was evaluated. This technology will enable us to attain rapid protein expression as mRNA is translated within the cytoplasm.
Hypothesis: Using M3-RNA technology we can achieve targeted delivery of mRNA with temporally restricted protein expression.
Methods: In vitro and in vivo transfection transfection using modified messenger RNA (mmRNA) was assessed utilizing nanoparticle sized liposomes.
Results: In vitro protein expression was analyzed upon transfection with mCherry mRNA in multiple cell lines (HEK293, HUVEC, Human Dermal and Cardiac Fibroblasts, HL-1 Cardiomyocyte cell line). Rapid expression of reporter genes within the cells was seen in as early as 2-4 hours and lasted for as long as 7 days and transfection efficiency was assessed using flow cytometry. We found almost identical action potential and calcium transients within transfected and untransfected cells confirming that overexpression does not alter the HL-1 cardiomyocytes functionality. In addition, structural integrity of transfected cardiomyocyte was confirmed using α-actinin and scarcomeric myosin antibody. We utilized M3-RNA technology to achieve in vivo transfection by microencapsulating mmRNA in nanoparticle-sized liposomes (70-100nm). Targeted and temporally restricted expression of FLuc mmRNA was assessed in using subcutaneous, intramuscular, intrahepatic, intrarenal and intraocular routes. Rapid expression of FLuc was observed in 2-4 hours that lasted for almost 72 hours except in highly metabolizing organs like liver and kidney. Similar expression of M3-mCherry RNA was also observed. Intracardiac FLuc expression by injecting M3-RNA in left ventricle of heart in mice revealed protein expression within the mouse heart that sustained for 72 hours.
Conclusions: M3-RNA approach enabled us to attain rapid protein expression in multiple cell lines and in targeted organs in mice. This novel technological platform is capable of inducing robust and transient protein expression; a new approach for targeted, temporally restrained gene therapy.
Author Disclosures: R.D. Singh: None. T.A. Witt: None. C. Livia: None. M.L. Hillestad: None. V.M. Dhivya: None. R.J. Crespo-diaz: None. P.G. Stalboerger: None. A. Terzic: None. A. Behfar: None.
- © 2016 by American Heart Association, Inc.