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.

Increased expression of angiotensinconverting enzyme 2 (ACE2), TMPRSS2 and furin in idiopathic pulmonary fibrosis (IPF) and lymphangioleiomyomatosis (LAM) patients' lung tissue: Implications for COVID -19

Background and Aims: Patients with chronic lung disease are highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to COVID-19. ACE2 is the main receptor for SARS-CoV-2 attachment. Our previous study reported higher ACE2 levels in smokers and COPD patients. Current study investigates if patients with interstitial lung diseases (ILDs) such as IPF and LAM have elevated levels of ACE2, transmembrane peptidase serine 2 (TMPRSS2) and Furin, increasing their risk for SARS-CoV-2 infection. Methods: Surgically resected lung tissue from IPF, LAM patients and normal controls (NC) was immunostained for ACE2, TMPRSS2 and Furin. Percentage ACE2 expression was measured in small airway (SA) epithelium and alveolar areas. Analysis was done using computer-assisted Image- ProPlus 7.0 software. Results: Compared to NC, the percentage ACE2 expression significantly increased in the SA epithelium of IPF (p<0.01), LAM (p<0.001) and in alveolar areas of IPF (p<0.001), LAM (p<0.001). We also observed elevated TMPRSS2 and Furin expression in the same lung tissue areas of IPF and LAM against NC. There was significant positive correlation between smoking history and ACE2 expression in the IPF for SA epithelium (r=0.81, p<0.05) and alveolar areas (r=0.94, p<0.01). Conclusions: This study has investigated the ACE2, TMPRSS2, and Furin in resected lung tissue of IPF and LAM, which suggests that people with ILDs are at higher risk of developing severe COVID-19 infection. To further understand and provide potential therapeutic targets for ILDs, we need to explore other cell types such as type II pneumocytes, alveolar macrophages and endothelial cells.

Effect of therapeutics on the modulation of ACE2 expression in airway epithelium: Implications for covid-19

RATIONALE: Coronavirus disease 2019 (COVID-19) is a global pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV2). SARS-CoV-2 uses the receptor angiotensin-converting enzyme 2 (ACE2) to gain entry to host cells in the respiratory tract epithelium. Clinical features post viral infection include symptoms of viral pneumonia, fever, cough, chest discomfort, and in severe cases dyspnea and bilateral lung infiltration. Adults with any age are at risk of getting COVID-19, however co-morbidities like hypertension, COPD, smoking, type 2 diabetes, asthma, obesity etc. increase infection susceptibility. Modern day therapeutics to control aforementioned health conditions include angiotensin II receptor blockers (ARBs), ACE inhibitors (ACEi), and anti-inflammatory drugs such as dexamethasone and hydroxychloroquine. Since binding of viral spike protein with receptor ACE2 initiates viral entry in the host, hence ACE2 has been one of the main candidates to understand the mechanism of viral infection. In this study our aim was to investigate if ACE2 expression levels can be modulated with therapeutic interventions. Methods: In an in vitro model of monolayer cultures, human airway epithelial (1HAEo-) cell lines were treated with losartan and telmisartan (ARB), captopril (ACEi), and hydroxychloroquine at half-log concentrations from 1 to 100 μM, and fluticasone, ciclesonide, and dexamethasone (steroids) at half-log concentrations from 0.3 to 10 μM. Whole cell lysates were obtained at 24hr post-treatment to quantify ACE2 expression via western blotting. Statistical analysis was performed using one-way ANOVA and Dunnett's multiple comparison test. Results: The in vitro experiments have shown that total ACE2 protein expression in 1HAEo-cells is modulated in response to certain drugs. Compared to untreated control cells, losartan treatment showed ∼45 % significant increase in ACE2 expression at both 3 μM and 10 μM concentrations (p< 0.01), whereas dexamethasone showed ∼ 40% significant increase at 10 μM dose (p< 0.001). Captopril treatment showed ∼35% decrease at 30 and 100 μM (p<0.01), whereas ciclesonide treatment demonstrated statistically significant decrease in ACE2 expression with all tested concentrations. No difference in ACE2 expression was observed with telmisartan and hydroxychloroquine treatments. Conclusion: Our findings suggest that daily medications like losartan, captopril, dexamethasone and ciclesonide for certain pre-existing conditions may modulate ACE2 protein levels in airway epithelium hence possibly priming for COVID19 infections if exposed. This affect susceptibility for COVID19 infection and severity of illness in vulnerable populations. Hence basic understanding of mechanism of action for daily standard of care prophylactic therapies can reduce or prevent SAR-CoV-2 infection in at-risk individuals.