Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways.

INTRODUCTION: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19. METHODS: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging. RESULTS: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73-97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control. CONCLUSION: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.

Hedgehog signaling in the airway epithelium of patients with chronic obstructive pulmonary disease.

Genome-wide association studies have linked gene variants of the receptor patched homolog 1 (PTCH1) with chronic obstructive pulmonary disease (COPD). However, its biological role in the disease is unclear. Our objective was to determine the expression pattern and biological role of PTCH1 in the lungs of patients with COPD. Airway epithelial-specific PTCH1 protein expression and epithelial morphology were assessed in lung tissues of control and COPD patients. PTCH1 mRNA expression was measured in bronchial epithelial cells obtained from individuals with and without COPD. The effects of PTCH1 siRNA knockdown on epithelial repair and mucous expression were evaluated using human epithelial cell lines. Ptch1+/- mice were used to assess the effect of decreased PTCH1 on mucous expression and airway epithelial phenotypes. Airway epithelial-specific PTCH1 protein expression was significantly increased in subjects with COPD compared to controls, and its expression was associated with total airway epithelial cell count and thickness. PTCH1 knockdown attenuated wound closure and mucous expression in airway epithelial cell lines. Ptch1+/- mice had reduced mucous expression compared to wildtype mice following mucous induction. PTCH1 protein is up-regulated in COPD airway epithelium and may upregulate mucous expression. PTCH1 provides a novel target to reduce chronic bronchitis in COPD patients.

Sialyl Lewis X modification of the epidermal growth factor receptor regulates receptor function during airway epithelial wound repair.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a major regulator of airway epithelial cell (AEC) functions such as migration, proliferation and differentiation, which play an essential role in epithelial repair. EGFR is a glycoprotein with 12 potential N-glycosylation sites in its extracellular domain. Glycosylation of EGFR has been shown to modulate its function. Previously, our laboratory demonstrated an important role of the carbohydrate structure sialyl Lewis x (sLe(x)) in airway epithelial repair. OBJECTIVE: To examine whether an sLe(x) decoration of EGFR can modulate receptor function during AEC repair. METHODS: Primary normal human bronchial epithelial (NHBE) cells were cultured in vitro. Co-localization of sLe(x) and EGFR was examined using confocal microscopy. Expressions of RNA and protein were analysed using RT-PCR and Western blotting. The final step in the synthesis of sLe(x) was catalysed by a specific alpha-1,3-fucosyltransferase (FucT-IV). To evaluate the role of sLe(x) in EGFR activation, a knockdown of the FucT-IV gene with small interfering RNA (siRNA) and an inhibitory anti-sLe(x) antibody (KM-93) was used. RESULTS: We demonstrated a co-localization of sLe(x) with EGFR on NHBE cells using confocal microscopy. Using a blocking antibody for sLe(x) after a mechanical injury, we observed a reduction in EGFR phosphorylation and epithelial repair following injury. FucT-IV demonstrates a temporal expression coordinate with epithelial repair. Down-regulation of FucT-IV expression in NHBE by specific siRNA suppressed sLe(x) expression. The use of FucT-IV siRNA significantly reduced phosphorylation of EGFR and prevented epithelial repair. An immunohistochemical analysis of human normal and asthmatic airways showed a significant reduction in sLe(x) and tyrosine-phosphorylated EGFR (pY(845)-EGFR) in the epithelium of asthmatic subjects compared with that of normal subjects. CONCLUSION: The present data demonstrate that sLe(x), in association with EGFR, in NHBE is coordinate with repair. This glycosylation is important in modulating EGFR activity to affect the repair of normal primary AEC.

Erythropoietin inhibits respiratory epithelial cell apoptosis in a model of acute lung injury.

Fas-mediated apoptosis of the alveolar epithelium is important in the pathogenesis of acute respiratory distress syndrome. Erythropoietin (EPO) has cytoprotective properties in other organ systems, and is relatively deficient in critical illness. This study investigates a potential role for EPO in reducing apoptosis in a model of acute lung injury. Apoptosis was induced in human alveolar epithelial (A549) cells or normal human bronchial epithelial (NHBE) cells by Fas activation with CH-11 Fas-crosslinking antibody or by co-culture with polymorphonuclear neutrophils in a transwell system. The effect of recombinant human (rh)EPO on apoptosis was measured by poly(ADP-ribose) polymerase cleavage and cell death detection assay. The specific EPO-EPO receptor (EPOR)-mediated effect was determined using an EPO-blocking antibody or EPOR small interfering RNA. Expression of EPOR was demonstrated in A549, NHBE and normal human alveolar epithelium. Fas- and neutrophil-mediated apoptosis of A549 and NHBE cells was inhibited by rhEPO by a specific EPO-EPOR-mediated mechanism. This anti-apoptotic effect was associated with induction of a pro-apoptotic Bcl-xL/Bax ratio. EPO has cytoprotective properties in respiratory epithelium in an in vitro model, which may indicate a potential therapeutic role in acute lung injury.