Volume 24, Issue 1, Pages 66-75 (January 2016) Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA Induces Rapid and Transient Expression  Irene C Turnbull, Ahmed A Eltoukhy, Kenneth M Fish, Mathieu Nonnenmacher, Kiyotake Ishikawa, Jiqiu Chen, Roger J Hajjar, Daniel G Anderson, Kevin D Costa  Molecular Therapy  Volume 24, Issue 1, Pages 66-75 (January 2016) DOI: 10.1038/mt.2015.193 Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Stability of formulated lipidoid nanoparticles tested in vitro on the day of synthesis (day 0) and up to 15 days in storage at 4 °C. Geometric mean fluorescence intensity for cultured HeLa cells was measured 24 hours after transfection with 40 ng eGFPmodRNA (gray), relative to nontreated cells (mean ± SD, n = 3). Particle size (z-average diameter) was determined by dynamic light scattering (black) (mean ± SD, n = 3). Molecular Therapy 2016 24, 66-75DOI: (10.1038/mt.2015.193) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Dose response of intramyocardial injection of formulated lipidoid nanoparticles (FLNP)/eGFPmodRNA in rats. Top panel: Expression of eGFP mRNA measured by real-time polymerase chain reaction in rat myocardium 20 hours after intramyocardial injection of: FLNP with 1, 5, or 10 μg of eGFPmodRNA, and either saline or 10 μg of naked eGFPmodRNA as controls. Values represent mean (±SD) eGFP mRNA expression levels relative to GAPDH, n = 3 rats per group. *P ≤ 0.02 versus naked eGFPmodRNA. Bottom panel: Immunofluorescence micrographs of corresponding frozen tissue sections of rat myocardium for (a) saline-only, (b) 10 μg naked eGFPmodRNA, and (c–e) FLNP/eGFPmodRNA at 1-μg (c), 5-μg (d), or 10-μg (e) doses. Sections incubated with anti-GFP antibody (green), and nuclei stained with DAPI (blue). Upper row shows pseudo-colored images with merged green and blue channels. Lower row shows only the green channel in grayscale. Scale bar = 200 μm for all panels. DAPI, 4',6-diamidino-2-phenylindole. Molecular Therapy 2016 24, 66-75DOI: (10.1038/mt.2015.193) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Expression kinetics after intramyocardial injection of FLNP/eGFPmodRNA in rats. (a) Expression of eGFP mRNA in rat myocardium measured by real-time polymerase chain reaction, from 6 hours to 14 days after intramyocardial injection of formulated lipidoid nanoparticles (FLNP) with 5 μg of eGFPmodRNA (n = 1–5 rats per time point, 19 total). Values represent eGFP mRNA expression levels relative to GAPDH, revealing a nearly exponential decay in mRNA with time postinjection. (b) Representative immunofluorescence microphotographs of frozen tissue sections of rat myocardium collected at selected time points (6 hours to 14 days) from the above study. Sections incubated with anti-GFP antibody (green), and nuclei stained with DAPI (blue). Scale bar = 200 μm for all panels. DAPI, 4',6-diamidino-2-phenylindole. Molecular Therapy 2016 24, 66-75DOI: (10.1038/mt.2015.193) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Biodistribution of eGFP mRNA after intramyocardial injection of FLNP/eGFPmodRNA in rats. Expression of eGFP mRNA measured by real-time PCR in organs harvested 20 hours after intramyocardial injection of formulated lipidoid nanoparticles (FLNP) with 5 μg of eGFPmodRNA. Values represent mean (±SD, n = 3 rats) eGFP mRNA expression levels relative to GAPDH, and normalized by mean expression in the heart. SKM, skeletal muscle. *P < 0.001 versus heart. Inset: Immunofluorescence of frozen tissue section of rat myocardium at 20 hours postinjection shows α-actinin-positive cells with sarcomeres (red) expressing GFP (green). Nuclei stained with DAPI (blue). Scale bar = 10 μm. DAPI, 4',6-diamidino-2-phenylindole. Molecular Therapy 2016 24, 66-75DOI: (10.1038/mt.2015.193) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Intracoronary delivery of FLNP/eGFPmodRNA via left ventricle injection with temporary aortic cross-clamping in the rat. Bar graph displays the biodistribution of eGFP mRNA in organs harvested 20 hours (black bars, n = 4) and 14 days (gray bars, n = 2) after intracoronary delivery of formulated lipidoid nanoparticles (FLNP) with 10 μg of eGFPmodRNA. Values represent mean (±SD) expression levels of eGFP mRNA measured by real-time PCR relative to GAPDH. SKM, skeletal muscle. *P < 0.005 relative to heart at 20 hours; no statistical analysis was done for the 14-week samples. Inset: GFP expression in rat myocardium collected at 20 hours postdelivery, demonstrated by immunofluorescence of frozen tissue section incubated with anti-GFP antibody (green), and DAPI (blue). Scale bar = 200 μm. DAPI, 4',6-diamidino-2-phenylindole. Molecular Therapy 2016 24, 66-75DOI: (10.1038/mt.2015.193) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Direct intramyocardial delivery of FLNP/eGFPmodRNA in a pilot large animal study in healthy and diseased pigs. (a) Expression of eGFP mRNA in healthy pig myocardium 20 hours after intramyocardial injection of formulated lipidoid nanoparticles (FLNP) with 72-μg dose of eGFPmodRNA, compared to untreated sham animal. Biodistribution showed lower GFP expression in off-target organs than in the heart. Values represent mean (±SD) eGFP mRNA expression levels relative to GAPDH. (b) Expression of eGFP mRNA 20 minutes after intramyocardial injection of FLNP/eGFPmodRNA (36-μg dose) at two LV sites in a pig with heart failure at 12 weeks post-MI, compared to non-injection remote LV site and untreated sham animal. Insets: Immunofluorescence microscopy of frozen tissue sections of pig LV myocardium from subendocardium of injection site 20 hours postinjection in healthy pig (top inset) and from border zone (BZ) injection site at 20 minutes postinjection in MI pig (bottom inset) show expression of GFP (red). Nuclei stained with DAPI (blue). Scale bars = 50 μm. (c–e) Colocalization studies show GFP expression (red) in α-actinin-positive cardiomyocytes with sarcomeres (c), in vimentin-positive cardiac fibroblasts (d), and in macrophages (MΦ) in the spleen (e, arrows). Nuclei stained with DAPI (blue). Scale bars = 10, 25, and 25 μm as indicated. DAPI, 4',6-diamidino-2-phenylindole. Molecular Therapy 2016 24, 66-75DOI: (10.1038/mt.2015.193) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Intracoronary delivery of FLNP/eGFPmodRNA in a pilot large animal study in healthy and diseased pigs. (a) Expression of eGFP mRNA in healthy and diseased (48 hours post-MI) pig myocardium 20 hours after intracoronary injection of FLNP with 500 μg dose of eGFPmodRNA, comparing expression levels in different regions of the heart. LA, left atrium; LV, left ventricle; RV, right ventricle. Insets: Immunofluorescence of posterior LV frozen tissue sections from healthy (left inset) and postinfarct (right inset) hearts show GFP expression (red), and nuclei stained with DAPI (blue). Scale bars = 200 μm. (b) Immunofluorescent microscopy studies show GFP expression (red) colocalizes with α-smooth muscle actin-positive smooth muscle cells in the media (m) and also with cells in the intima (i) and adventitia (a) layers of the coronary vessel wall, which is surrounded by GFP-positive myocardial tissue. Scale bars = 100 and 25 μm, as shown. DAPI, 4',6-diamidino-2-phenylindole. Molecular Therapy 2016 24, 66-75DOI: (10.1038/mt.2015.193) Copyright © 2016 American Society of Gene & Cell Therapy Terms and Conditions