Using a systems approach to identify the mechanism of action of correctors

Tese de doutoramento, Biologia (Biologia de Sistemas), Universidade de Lisboa, Faculdade de Ciências, 2018Cystic Fibrosis (CF), the most common life-shortening genetic disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, an ion channel expressed at the apical membrane of epithelial cells. High-throughput screens (HTS) identified several novel molecules potentially targeting the underlying CFTR defect but only for some patients: potentiator VX-770 (Ivacaftor/Kalydeco), for subjects bearing G551D and other gating mutations, the combination corrector/potentiator VX-809 (Lumacaftor)/VX-770 (Orkambi) for F508del-homozygous patients and another similar combination VX-661 (Tezacaftor)/VX-770 is under approval. The main objective of this PhD work was to study new compounds that correct the basic CF defect, by rescuing CFTR protein traffic and function, focusing both on individual responses of CF patients with different CFTR mutations to these new drugs, and their mechanism of action. Chapter 1 focusses on the measurement of functional responses on human bronchial epithelial cells (HBE’s) derived from CF lung explants bearing different CFTR mutations to VX-809 namely: A561E, N1303K, G542X and Y1092X. Our data showed a positive response of A561E/A561E to VX-809 and F508del/Y1092X but not F508del/G542X. In Chapter 2, we evaluated the efficacy of CFTR modulators (correctors/potentiators) in physiologically relevant tissues, namely rectal biopsies, intestinal organoids, (HBE’s) and human nasal epithelial cells (HNE’s), from CF patients with rare CFTR mutations. Data obtained here showed that neither R560S nor H1079P-could not be rescued by any of the CFTR modulators, but 3849+10kbC>T and R334W and c.120del23-CFTR were rescued by VX-770 alone or with VX-809. In Chapter 3 we evaluated the efficacy of two novel CFTR correctors (B9, E12) in primary HBE cells, and three novel compounds E-act mimics (C2, C5, and C7) as enhancers of alternative Cl- channel TMEM16A in human intestinal organoids. In Chapter 4 (final) we assessed the effect of CFTR modulators and their possible additivity with F508del-CFTR genetic revertants 4RK, R1070W, and G550E to understand the mechanism of action of small molecule correctors and another variant diacidic ER exit code DD/AA in CFBE mCherry cells expressing these varinats by Ussing chamber analysis with or without CFTR modulators. Our data show that C18 and VX-661 and low temperature (But not VX-809) rescued DD/AA to the cell surface and genetic revertants restore the channel function without any CFTR modulator. Altogether, results from this work bring new insights into how the CFTR genotype may influence CFTR function and response to CFTR modulators and how each patient should be assessed individually for the responsiveness to the CFTR modulators towards personalized therapeutics

Herbo Glow: Formulation and Evaluation of a Botanical Face Pack

Aim/Objectives: The main goal of this research was to create and test a herbal face pack that would improve the health and appearance of the skin by utilising all-natural components. The goal of the research is to create a face pack out of all-natural herbs as a substitute for manufactured cosmetics. Materials: As for the contents, we formulated it using a blend of popular herbal herbs that are well recognised for their beneficial effects on the skin. The skin-nourishing, antibacterial, and brightening characteristics of turmeric, neem, sandalwood, orange peel, and rose petals were among them. Multani mitti, also known as Fuller\u27s Earth, was one of them. Methods: A fine face pack formulation was created by drying, powdering, and blending the chosen herbs in the suitable amounts. Testing for organoleptic properties, particle size, pH, smoothness, spreadability, grittiness, and stability were all part of the physicochemical assessment of the finished product. Furthermore, patch testing was used to assess irritancy in the participants. Results: The physical properties of the herbal face pack were found to be desirable, including a fine texture, an appropriate pH, decent spreadability, and the absence of grittiness. After 30 days, the product\u27s physical and chemical properties had not changed. While testing on volunteers, we did not find any indications of skin irritation or negative consequences. Discussions: The findings indicate that the herbal face pack offers the intended cosmetic advantages, such nourishing the skin, enhancing its radiance, and washing it, without the hazards of synthetic ingredients. Using only pure herbal powders guarantees that the composition is safe, biocompatible, and effective. Conclusion: In conclusion, the results show that it is possible to create and test a cosmetic face pack that is completely herbal. To keep skin healthy and radiant, the mixture provides an alternative to synthetic cosmetic face packs that is safe, natural, and effective

Using Drugs to Probe the Variability of Trans-Epithelial Airway Resistance

BACKGROUND:Precision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial 'set-point' may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the 'reference range of normality' remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport. METHODOLOGY/PRINCIPAL FINDINGS:Brushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm2), trans-epithelial voltage (-62.3/-1.8 mV) and calculated equivalent current (-125.0/-3.2 μA/cm2; all non-normal, P<0.001). A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport. CONCLUSIONS/SIGNIFICANCE:Healthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium transport. Our data may guide the choice of the background set point in subjects with airway diseases and frame the reference range for the future delivery of precision airway medicine

Formulation and Evaluation of Fast Disintegrating Tablets of Atenolol Using Natural and Synthetic Superdisintegrants

Oral disintegrating tablet (ODT) is defined as “A solid dosage form containing medical substances or active ingredient which disintegrates rapidly usually within a matter of seconds when placed upon the tongue”. The aim of the present research is to formulate Atenolol fast disintegrating tablets. Atenolol is β1- cardio selective adrenergic receptor blocker, widely used in the treatment of hypertension, angina pectoris, arrhythmias and myocardial infarction. It works by slowing down the heart and reducing the work load of the heart. The conventional tablets of atenolol are reported to exhibit fluctuations in the plasma drug levels after administration. Atenolol fast disintegrating tablets were prepared by using direct compression method using Synthetic as wel as Natural superdisintegrants like sodium starch glycolate, Cross carmellose sodium and Miriabilis jalapa starch. The prepared tablets were characterized for their hardness, weight variation, disintegration time, wetting time, water absorption ratio friability, and in vitro dissolution studies. The ability of the tablet to release the drug faster depends on the concentration and type of superdisintegrants. In this study the fast disintegrating tablets containing Cross carmellose sodium, Sodium starch glycolate and Miriabilis jalapa starch as the super disintegrant in the ratio of 1:2:3 Shows better release of drug. About 97.92% of the drug was released from the tablets in 10mins. Therefore, based on the physico chemical properties, in vitro drug release profile F9 formulation containing Miriabilis jalapa starch is optimized as the best formulation. Keywords: Fast Disintegrating Tablets, Superdisintegrants, Atenolol, Miriabilis jalapa starch, In vitro evaluation

Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells.

Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. pBECs collected from healthy donors (n = 5) were CR using g-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or bronchial epithelial growth medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza)-(AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral RNA was assessed by RT-qPCR and anti-viral proteins quantified by LEGENDplex following Rhinovirus-A1b infection. CRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency compared to BEGM media. PneumaCult media cultures exhibited increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. There are distinct structural and functional differences in pBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing CRpBECs ALI experiments for specific research questions

A novel treatment of cystic fibrosis acting on-target:cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts ‘on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment

The Complex Association between COPD and COVID-19.

Chronic obstructive pulmonary disease (COPD) is significant cause of morbidity and mortality worldwide. There is mounting evidence suggesting that COPD patients are at increased risk of severe COVID-19 outcomes; however, it remains unclear whether they are more susceptible to acquiring SARS-CoV-2 infection. In this comprehensive review, we aim to provide an up-to-date perspective of the intricate relationship between COPD and COVID-19. We conducted a thorough review of the literature to examine the evidence regarding the susceptibility of COPD patients to COVID-19 infection and the severity of their disease outcomes. While most studies have found that pre-existing COPD is associated with worse COVID-19 outcomes, some have yielded conflicting results. We also discuss confounding factors such as cigarette smoking, inhaled corticosteroids, and socioeconomic and genetic factors that may influence this association. Furthermore, we review acute COVID-19 management, treatment, rehabilitation, and recovery in COPD patients and how public health measures impact their care. In conclusion, while the association between COPD and COVID-19 is complex and requires further investigation, this review highlights the need for careful management of COPD patients during the pandemic to minimize the risk of severe COVID-19 outcomes

Inflammation-induced loss of CFTR-expressing airway ionocytes in non-eosinophilic asthma.

BACKGROUND AND OBJECTIVE: Severe asthma is a heterogeneous disease with subtype classification according to dominant airway infiltrates, including eosinophilic (Type 2 high), or non-eosinophilic asthma. Non-eosinophilic asthma is further divided into paucigranulocytic or neutrophilic asthma characterized by elevated neutrophils, and mixed Type 1 and Type 17 cytokines in the airways. Severe non-eosinophilic asthma has few effective treatments and many patients do not qualify for biologic therapies. The cystic fibrosis transmembrane conductance regulator (CFTR) is dysregulated in multiple respiratory diseases including cystic fibrosis and chronic obstructive pulmonary disease and has proven a valuable therapeutic target. We hypothesized that the CFTR may also play a role in non-eosinophilic asthma. METHODS: Patient-derived human bronchial epithelial cells (hBECs) were isolated and differentiated at the air-liquid interface. Single cell RNA-sequencing (scRNAseq) was used to identify epithelial cell subtypes and transcriptional activity. Ion transport was investigated with Ussing chambers and immunofluorescent quantification of ionocyte abundance in human airway epithelial cells and murine models of asthma. RESULTS: We identified that hBECs from patients with non-eosinophilic asthma had reduced CFTR function, and did not differentiate into CFTR-expressing ionocytes compared to those from eosinophilic asthma or healthy donors. Similarly, ionocytes were also diminished in the airways of a murine model of neutrophilic-dominant but not eosinophilic asthma. Treatment of hBECs from healthy donors with a neutrophilic asthma-like inflammatory cytokine mixture led to a reduction in ionocytes. CONCLUSION: Inflammation-induced loss of CFTR-expressing ionocytes in airway cells from non-eosinophilic asthma may represent a key feature of disease pathogenesis and a novel drug target

Human Primary Epithelial Cell Models: Promising Tools in the Era of Cystic Fibrosis Personalized Medicine

Cystic fibrosis (CF) is an inherited disorder where individual disease etiology and response to therapeutic intervention is impacted by CF transmembrane regulator (CFTR) mutations and other genetic modifiers. CFTR regulates multiple mechanisms in a diverse range of epithelial tissues. In this Review, we consolidate the latest updates in the development of primary epithelial cellular model systems relevant for CF. We discuss conventional two-dimensional (2-D) airway epithelial cell cultures, the backbone of in vitro cellular models to date, as well as improved expansion protocols to overcome finite supply of the cellular source. We highlight a range of strategies for establishment of three dimensional (3-D) airway and intestinal organoid models and evaluate the limitations and potential improvements in each system, focusing on their application in CF. The in vitro CFTR functional assays in patient-derived organoids allow for preclinical pharmacotherapy screening to identify responsive patients. It is likely that organoids will be an invaluable preclinical tool to unravel disease mechanisms, design novel treatments, and enable clinicians to provide personalized management for patients with CF

Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing.

Funder: Fondazione Fibrosi Cistica - FFC#1/2017Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272-26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272-26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases