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.

Integrative genomic analysis highlights potential genetic risk factors for COVID-19

RATIONALE: The genes that influence the pathophysiology of COVID-19 have yet to be identified. Association analysis has found genetic loci for COVID-191. We used integrative genomics (IG) to combine gene expression and proteomic information with COVID-19 susceptibility loci in order to identify candidate genes for this disease. METHODS: For these analyses we used the COVID-19 Host Genetics Initiative genome-wide association (GWA) meta-analysis version 4 (COVID-19 positive versus COVID-19 negative), the Lung eQTL study2 (n=1,038), eQTLGen3 study (n=31,784) and the INTERVAL4 study (n=3,301). We conducted two IG methods (Bayesian Colocalization [coloc] and Summary Based Mendelian Randomization) to link gene and protein expression in lung and blood tissues with COVID-19 susceptibility loci. We identified the most consistently colocalized gene and conducted a Mendelian Randomization (MR) to assess the causal association of its protein ('exposure') with COVID-19 susceptibility ('outcomes'). Significant MR was set as P<0.05. RESULTS: The expression of 6 genes in lung and 12 in blood colocalized with COVID-19 susceptibility loci. SMR results demonstrated that the expression levels of 6 genes in lung tissue and 5 in blood were associated with COVID-19. Out of the candidate genes identified, two (ABO and SLC6A20) were within previously identified loci (Figure 1). Based on the SMR we found that the expression of SLC6A20 in lung was associated with a higher risk of COVID-19. Novel discovered associations included ERMP1, FCER1G, and CA11, genes which have been previously linked with respiratory diseases (i.e.: asthma) and host immune responses (i.e.: neutrophil and eosinophil counts). COVID-19 susceptibility also colocalized with plasma protein levels of ABO. Based on MR, ABO demonstrated a significant causal association (P = 2.10 × 10-5) with the risk of COVID-19 with increased levels of this protein in plasma associated with an increased risk of COVID-19. The top variant in the MR test (rs505922) was in complete linkage disequilibrium with the variant responsible for the blood O genotype, conferring reduced risk. CONCLUSIONS: This multi-omics approach led to the discovery of novel genes associated with COVID-19. We found that the ABO protein is a causal risk factor for COVID-19, with blood group O being protective against COVID-19. REFERENCES: 1. Ellinghaus, D. et al. N. Engl. J. Med. (2020). 2. Hao, K. et al. PLoS Genet. (2012). 3. Ṽsa, U. et al. bioRxiv. (2018). 4. Sun, B. B. et al. Nature. (2018) .

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.