Systemic delivery of biotherapeutic RNA to the myocardium transiently modulates cardiac contractility in vivo

Figures

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  Fig. 1.. Observation of cLNP accumulation in the heart. (A) 3H-CHE-cLNP (1 μg of RNA/mouse) tissue distribution in WT vs. Apoe−/− mice 30 min post–IV administration, n ≥ 5. (B) cLNP level in the liver and heart 30 min post-IV measured by LC–MS/MS of ionizable aminolipid. n = 4. (C) Comparison of cLNP uptake in the heart of WT vs. Apoe−/− mice traced by 125I vs. 3H-CHE labels. (D) Autoradiography of the mouse heart 30 min post–IV administration 125I-cLNP (scheme illustrates the approximate position of 2-mm heart slices). (E) Confocal microscopy of the liver, lung, and heart obtained from Apoe−/− mice 30 min post–IV administration of PKH26-labeled cLNP (bar = 25 μm). Alexa Fluor 647–labeled anti-PECAM/CD31 antibody were injected 10 min before killing of mice to denote vascular endothelium. Right panel: Expanded part of the micrograph shows cLNP in the extravascular area of the myocardium. Unless stated otherwise, blue vs. red denote WT vs Apoe−/− mice, respectively. n = 3 to 5 biologically independent animals (A–C and E). Data are shown as M ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001 (t test or two-way ANOVA with Tukey’s post hoc test).

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  Fig. 2.. Pharmacological properties of cardiotropic cLNP. (A and B) 125I-labeled cLNP tissue distribution in WT vs. Apoe−/− mice 30 min post–IV administration. (A) Empty cLNP vs. poly-C RNA-loaded cLNP. (B) 80-nm cLNP vs. 350 nm cLNP. (C) Heart uptake of LNPs formulated with indicated ionizable lipids in comparison with cLNP. (D) Pharmacokinetics of 3H-CHE-cLNP injected in Apoe−/− mice and organs were perfused with 10 mL of PBS and harvested at indicated times. Data are shown as Mean ± SEM, *P < 0.05; **P < 0.01; ***P < 0.001 (t test or two-way ANOVA with Tukey’s post hoc test).

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  Fig. 3.. Cardiac delivery of cLNP/Atp2a2 siRNA. (A) Atp2a2 siRNA-loaded cLNP were IV injected into Apoe−/− mice and SERCA2 level was detected in a 5-d time course. (B) Immunoblot of heart protein isolates 1, 3, or 5 d following 10 μg cLNP/siRNA injection, for SERCA2 relative to GAPDH, with associated densitometry (n = 3, *P < 0.05). (C) Representative CMC cross sections stained for cell membranes with wheat-germ agglutinin (green), and nuclei with DAPI (blue), and (D) associated quantitation of CMC cross-sectional area (N = 3 animals per group, n = 100 CMCs per animal, boxes show the median and 25th and 75th percentiles, *P < 0.01). (E) Heart weight (HW) relative to tibia length (TL) (n = 3, the dashed line represents average HW/TL in uninjected controls; boxes show the median and 25th and 75th percentiles, *P < 0.05).

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  Fig. 4.. cLNP/Atp2a2-siRNA modulates cardiomyocyte contractility. (A) Proposed activity of siRNA-Atp2a2. (B) Measurement of sarcomere shortening in CMCs isolated from mice 3 d after cLNP/siRNA injection. Note reduced fractional shortening, slowed contraction, and relaxation kinetics cLNP/siRNA-treated group (*P < 0.05) Average ± s.e.m. traces of sarcomere shortening, and associated contraction and relaxation parameters averaged over 6 to 10 transients during pacing at 1 Hz (N = 3 animals per group, n = 22 to 49 CMCs per animal). Boxes show the median and 25th and 75th percentiles, *P < 0.05.

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  Fig. 5.. Cre-Lox recombination by LNP/Cre recombinase mRNA indicates significant cLNP access to cardiomyocytes. (A) Schematic picture of Cre-Lox recombination. (B) Plasma apoE depletion by LNP/ApoE-siRNA treatment in WT and mTmG mice. (C) Treatment of mTmG mice with LNP. mTmG mice were pretreated with LNP/ApoE-siRNA and injected with 10 µg of cLNP/Cre-mRNA or SM102/Cre-mRNA in 24, 48, and 72 h. Tissues were harvested at day 8 followed by confocal microscopy analysis. (D) Representative images of heart and liver tissues. Switch of tdTomato to GFP expression indicates recombined cells. Dystrophin staining of heart tissue outlines cardiomyocytes (white borders on heart images) and was used for proper identification. (E) Analysis of Cre-Lox recombination (n = 3, 4 to 5 fields per heart, 220 ±75 CMC/field; analysis was done in a blind fashion). Fractions of GFP-positive cells of total cardiomyocyte number were calculated. The dashed line indicates the basal level in untreated mice. ***P < 0.001 (t test or two-way ANOVA with Tukey’s post hoc test) cLNP vs. SM102-LNP.