Volume 19, Issue 6, Pages 1116-1122 (June 2011) Systemic Delivery of Tumor Suppressor microRNA Mimics Using a Neutral Lipid Emulsion Inhibits Lung Tumors in Mice  Phong Trang, Jason F Wiggins, Christopher L Daige, Chris Cho, Michael Omotola, David Brown, Joanne B Weidhaas, Andreas G Bader, Frank J Slack  Molecular Therapy  Volume 19, Issue 6, Pages 1116-1122 (June 2011) DOI: 10.1038/mt.2011.48 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Biodistribution of systemically delivered microRNAs (miRNA) mimics. (a) A group of six mice was injected each with 20 µg neutral lipid emulsion-formulated miR-124. After killing, three mice were used to isolate total RNA from liver, kidney, and lung (nonperfused, NP). Organs from the remaining three mice were perfused with 0.9% saline solution before subjected to RNA isolation (P). Perfused and nonperfused organs from nontreated mice served as negative controls for endogenous miR-124 expression levels in these tissues under these conditions. miR-124 copy numbers per 10 ng RNA were determined by quantitative reverse transcriptase PCR using a miR-124 standard curve. Standard deviations and data values are shown in the graph. (b) miR-124 copy numbers per 10 ng RNA of whole blood from nontreated mice (n = 6) or mice injected intravenously with the miR-124 formulation (n = 6) are shown. Molecular Therapy 2011 19, 1116-1122DOI: (10.1038/mt.2011.48) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Systemic delivery of luciferase siRNA (si-luc) to orthotopic H460-luc lung tumors in mice. (a) IVIS images of mice carrying luciferase-expressing lung tumors were taken right before and 48 hours after intravenous injection of si-luc (animals number 3–4) or negative control (animals number 1–2) formulated with neutral lipid emulsion. (b) Quantitative analysis of data shown in a. The data are presented as percent (%) luminescence 48 hours post-treatment relative to the luminescence at time of injection (100%). Molecular Therapy 2011 19, 1116-1122DOI: (10.1038/mt.2011.48) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Systemic delivery of let-7b mimic reduces lung tumor burden in a Kras activated non-small cell lung cancer model. Whole lungs and tumor histologies (H&E) are shown. (a) Mice treated with let-7b display a significant reduction in lung lesions (arrows) in four out of five treated animals compared to mice treated with (b) miR-NC. (c) immunohistochemistry stainings directed against Ki-67, and (TUNEL) assay of apoptotic bodies in K-ras G12D mice treated with let-7b (left panel) and miR-NC (right panel), respectively. Immunohistochemistry stainings at a ∼100-fold magnification are shown. (d) Quantitative analysis of tumor burden in LSL-K-ras G12D animals treated with Ad-Cre and let-7b (n = 4) versus LSL-K-ras G12D animals treated with Ad-Cre and miR-NC (n = 5). The ratios of tumor area versus normal lung area are presented as a box-and-whisker plot. The two-tailed P value is indicated. (e) let-7b expression in mouse lungs (n = 5 for each group) 48 hours after last treatment. Boxes represent interquartile ranges (between the 25th and 75th quartiles) and the two-tailed P value is indicated. The total range, mean (open diamond), and median (blank bar) are shown. H&E, hematoxylin and eosin; miR-NC, negative control microRNA; TUNEL, TdT-mediated dUTP nick end labeling. Molecular Therapy 2011 19, 1116-1122DOI: (10.1038/mt.2011.48) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 miR-34a mimics reduce lung tumor burden in an autochthonous non-small cell lung cancer model. Whole lungs and tumor histologies (H&E) are shown. (a) LSL-K-ras G12D mice tumors were allowed to develop for 10 weeks. Then, synthetic miR-34a or miR-NC conjugated with neutral lipid emulsion were intravenously delivered by tail vein injections. Mice treated with miR-34a (second row) display a significant reduction in lung lesions, hyperplasias, and adenomas (arrows) compared to mice treated with miR-NC (first row). (b) Immunohistochemistry stainings directed against Ki-67, and (TUNEL) assay of apoptotic bodies in K-ras G12D mice treated with miR-34a (left panel) and miR-NC (right panel), respectively. Immunohistochemistry stainings at a ∼100-fold magnification are shown. Insets show Ki-67-specific staining at a 400-fold magnification. (c) Quantitative analysis of tumor burden in LSL-K-ras G12D animals treated with Ad-Cre and miR-34a (n = 5) versus LSL-K-ras G12D animals treated with Ad-Cre and miR-NC (n = 5). The ratios of tumor area versus normal lung area are presented as a box-and-whisker plot. The two-tailed P value is indicated. (d) miR-34a expression in mouse lungs (n = 5 for each group) 48 hours after last treatment. Boxes represent interquartile ranges (between the 25th and 75th quartiles) and the two-tailed P value is indicated. The total range, mean (open diamond), and median (blank bar) are shown. H&E, hematoxylin and eosin; miR-NC, negative control microRNA; TUNEL, TdT-mediated dUTP nick end labeling. Molecular Therapy 2011 19, 1116-1122DOI: (10.1038/mt.2011.48) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions