Lipid nanoparticle delivery of a microRNA-145 inhibitor improves experimental pulmonary hypertension

Abstract

Therapies that exploit RNA interference (RNAi) hold great potential for improving disease outcomes. However, there are several challenges that limit the application of RNAi therapeutics. One of the most important challenges is effective delivery of oligonucleotides to target cells and reduced delivery to non-target cells. We have previously developed a functionalized cationic lipopolyamine (Star:Star-mPEG-550) for in vivo delivery of siRNA to pulmonary vascular cells. This optimized lipid formulation enhances the retention of siRNA in mouse lungs and achieves significant knockdown of target gene expression for at least 10days following a single intravenous injection. Although this suggests great potential for developing lung-directed RNAi-based therapies, the application of Star:Star-mPEG mediated delivery of RNAi based therapies for pulmonary vascular diseases such as pulmonary arterial hypertension (PAH) remains unknown. We identified differential expression of several microRNAs known to regulate cell proliferation, cell survival and cell fate that are associated with development of PAH, including increased expression of microRNA-145 (miR-145). Here we test the hypothesis that Star:Star-mPEG mediated delivery of an antisense oligonucleotide against miR-145 (antimiR-145) will improve established PAH in rats. We performed a series of experiments testing the in vivo distribution, toxicity, and efficacy of Star:Star-mPEG mediated delivery of antimiR-145 in rats with Sugen-5416/hypoxia induced PAH. We showed that after subchronic therapy of three intravenous injections over 5weeks at 2mg/kg, antimiR-145 accumulated in rat lung tissue and reduced expression of endogenous miR-145. Using a novel in situ hybridization approach, we demonstrated substantial distribution of antimiR-145 in the lungs as well as the liver, kidney, and spleen. We assessed toxic effects of Star:Star-mPEG/antimiR-145 with serial complete blood counts of leukocytes and serum metabolic panels, gross pathology, and histopathology and did not detect significant off-target effects. AntimiR-145 reduced the degree of pulmonary arteriopathy, reduced the severity of pulmonary hypertension, and reduced the degree of cardiac dysfunction. The results establish effective and low toxicity of lung delivery of a miRNA-145 inhibitor using functionalized cationic lipopolyamine nanoparticles to repair pulmonary arteriopathy and improve cardiac function in rats with severe PAH.

Keywords: Antisense oligonucleotide; Lipid nanoparticle; Lung delivery; MicroRNA-145; Pulmonary hypertension; Sugen5416/hypoxia.

Figure 1. Experimental Approach

On day 0, Adult male Sprague Dawley rats were injected with Sugen-5416 (20 mg/kg, SC), exposed to normobaric hypoxia (10 % O₂) for three weeks, and returned to room air an additional 10 weeks to induce PAH. Animals were randomized to treatments and received intravenous injections at week 8, 10, and 12. Evaluation of therapeutic effects occurred at week 13 and tissues were collected for molecular analysis.

Figure 2. Staramine lipopolyamine mediated delivery of antimiR-145 delivers active inhibitor to lung tissue

(A) Quantitative measurement of antimiR-145 in 13-week Sugen5416/hypoxia PAH rat lung and heart (right ventricle, RV and left ventricle plus septum, LV). (B) Measurement of endogenous miR-145 levels in 13-week Sugen5416/hypoxia PAH rat lungs. The Y-axis represents expression of miR-145 levels relative to U6 small nuclear RNA. Data are presented as Mean±SEM and analyzed with 1 way ANOVA followed by Dunnett’s Multiple Comparison Test (*P<0.05 compared to PAH, N=6). A. In situ hybridization of FFPE lung sections using an antimiR-145 sequence specific probe. Sections are from 13-week Sugen5416/hypoxia rats treated with three intravenous dose of 2 mg/kg antimiR-145 formulated in Star:Star-mPEG (a, b),or 13-week Sugen5416/hypoxia rats treated with placebo (5% dextrose) (c, d). Blue speckles represent the antimiR-145 (arrowheads), and tissue is counterstained with nuclear fast red.

Figure 3. Biodistribution of antimiR-145 in extrapulmonary organs

In situ hybridization of FFPE sections from 13-week Sugen5416/hypoxia rats treated with three intravenous dose of 2 mg/kg antimiR-145 formulated in Star:Star-mPEG. Serial sections from liver (left), spleen (center), and kidney (right) were stained using a scramble sequence probe (top) or an antimiR-145 sequence specific probe (middle). Blue signals represent antimiR-145 positive areas and tissue is counterstained with nuclear fast red. Lower panels are sections from 13-week Sugen5416/hypoxia rats treated with placebo (5% dextrose) showing no positive signal.

Figure 4. Lack of effect of antimiR-145 on leucocyte counts, electrolytes, metabolites and marker proteins assessed by comprehensive metabolic panels of rats with PAH

A. Lack of effect of antimiR-145 or nonsilencing control oligonucleotide on body weight and spleen weight. Blood was collected at week 12 after two doses of antimiR-145 (2 mg/kg) or nonsilencing control oligonucleotide (2 mg/kg) were administered at weeks 8 and 10. B. Assessment of leukocyte count with differential, serum electrolytes and metabolites and marker proteins. Data are presented as Mean±SEM and analyzed with 1 way ANOVA followed by Dunnett’s Multiple Comparison Test, N = 4–7.

Figure 5. AntimiR-145 therapy repairs pulmonary arteriopathy

A. Hematoxylin and Eosin stain of FFPE rat lung sections showing improvement of arteriopathy after antimiR-145 treatment at 13 Wk Sugen5416/hypoxia PAH rats in comparison to normal age-matched control rats and 13 Wk Sugen5416/hypoxia rats. B. Morphological measurements of pulmonary arteries in lung tissue from 13 weeks Sugen5416/hypoxia PAH rats showing increased arterial wall thickness in vessels sized 50–200 µm. Data are presented as frequency distributions analyzed by One-way nonparametric ANOVA followed by Dunn’s Test (*P<0.05 compared to PAH). The nonsilencing control had no significant effect on wall thicknesses compared to untreated PAH animals and is not shown for the sake of clarity.

Figure 6. Effect of antimiR-145 on RV function

Hemodynamic measurement of: (A) right ventricular systolic pressure (RVSP) and (B) mean systemic arterial pressure (MSAP). (C) Assessment of RV hypertrophy by measurement of RV/LV + septum (S) weight ratio, Data are presented as Mean±SEM and analyzed with 1 way ANOVA followed by Dunnett’s Multiple Comparison Test (*P<0.05 compared to PAH, N=8–18). Representative echocardiographic images of (D) parasternal short axis views and (E) Pulsed wave Doppler traces from the right ventricular outflow tract.