Abstract 256: mRNA-based Nanoparticles Are Highly Effective Agents For Cardiac Gene Therapy

Abstract

Gene therapy holds promise for repair and regeneration of damaged myocardium after infarction. Viral vectors used in most studies, while highly effective, carry concerns of safety and immunogenicity. Nanoparticle (NP)-enabled transfection using polymeric and lipidoid reagents mitigates some of the concerns related to the use of viral vectors. However, these agents have been less effective in transfecting cardiac myocytes, which are primarily post-mitotic and only weakly facilitate DNA entry to the nucleus. Because translation occurs in the cytosol, transfection with RNA obviates the need for nuclear entry. Recent work has shown that synthetic alterations to messenger RNA (mRNA) can prolong its half-life in the cytosol and diminish the host’s immune response. We hypothesized that transfection of cardiac myocytes with lipidoid NPs formed using modified mRNA would lead to high levels of protein expression. Neonatal rat (NRCM), adult rat (ARCM) and human embryonic stem cell-derived (hES-CM) cardiac myocytes were transfected with modified GFP-mRNA NPs using a lipidoid vehicle (Stemfect TM , Stemgent, Cambridge, MA) or Lipofectamine2000 (LF, Invitrogen) and GFP expression was assessed after 24 hours. Average transfection efficiency in NRCM was significantly higher using Stemfect versus LF as measured by flow cytometry (48.5% vs. 6.6% at 20ng mRNA; 47.4% vs. 11.8% at 40ng mRNA, 48.1% vs. 18.6% at 80ng mRNA; 48.1% vs. 28% at 160ng mRNA; n=3, p<0.01 for each pair). In hES-CM transfection was also more robust with Stemfect NPs versus LF (91.6% vs. 62.2%, p<0.05 at 40ng mRNA; 96.1 vs. 87.8%, p<0.01 at 160ng mRNA; n=3). GFP expression was noted by fluorescence microscopy to be higher in ARCMs transfected with Stemfect NPs versus LF. In conclusion, we have shown that difficult-to-transfect cardiac myocytes are readily transfected with mRNA-based NPs in vitro and with significantly higher efficiency when using Stemfect rather than Lipofectamine. Gene therapy with mRNA represents a novel approach to cardiac repair and may be preferable to DNA because of the transient window of protein expression and lower side effect profile.