In vivo assessment of luciferase expression

All in vivo experiments were conducted after review and approval by the Institutional Animal Care and Use Committee (IACUC) at Lawrence Livermore National Laboratory. Female BALB/c mice (Harlan), aged 5-8 weeks and weighing about 20 g, were used for all in vivo imaging studies. Mice were bilaterally injected intramuscularly at the midline of each hindlimb quadricep with 50 μL of unformulated RNA or NLP formulated RNA complexes. All test materials were formulated in PBS and filter-sterilized. Four days after injection, luciferase expression levels were assessed using an IVIS Spectrum instrument (Perkin Elmer) at the University of California, Davis, Center for Molecular and Genomic Imaging (CMGI). Five minutes before imaging, mice were injected intraperitoneally with 0.15 mg/g of luciferin solution (Caliper Lifesciences). Mice were subsequently anesthetized [2% (vol/vol) isoflurane in oxygen] and positioned in the IVIS instrument to image from both dorsal and ventral positions. Relative luciferase expression was based on quantifying the total flux (photons per second) in a pre-defined area (region of interest was consistent for all analyses). The expression level of luciferase for each mouse is reported as the highest total flux value from one of the limbs and positions. All acquisition and imaging parameters were kept constant across experiments (e.g. exposure time, binning, field-of-view). Error bars represent SD. Statistical analyses was performed using GraphPad PRISM 7 software. Significance analyses for in vivo luciferase expression was performed using ANOVA followed by Kruskal-Wallis or Tukey’s post hoc test for multiple comparisons.