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Optimizations of in Vitro Mucus and Cell Culture Models to Better Predict in Vivo Gene Transfer in Pathological Lung Respiratory

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Optimizations of in Vitro Mucus and Cell Culture Models to Better Predict in Vivo Gene Transfer in Pathological Lung Respiratory pharmaceutics Review Optimizations of In Vitro Mucus and Cell Culture Models to Better Predict In Vivo Gene Transfer in Pathological Lung Respiratory Airways: Cystic Fibrosis as an Example Rosy Ghanem 1 ,Véronique Laurent 1, Philippe Roquefort 2, Tanguy Haute 1 , Sophie Ramel 3, Tony Le Gall 1 , Thierry Aubry 2 and Tristan Montier 1,4,* 1 Univ Brest, INSERM, EFS, UMR 1078, GGB, F-29200 Brest, France; [email protected] (R.G.); [email protected] (V.L.); [email protected] (T.H.); [email protected] (T.L.G.) 2 IRDL UMR CNRS 6027, Université de Bretagne Occidentale, UFR Sciences et Techniques, 6, Avenue Victor Le Gorgeu CS 93837, CEDEX 3, 29238 Brest, France; [email protected] (P.R.); [email protected] (T.A.) 3 Centre de Ressources et de Compétences de la Mucoviscidose, Fondation Ildys, Presqu’île de Perharidy, 29680 Roscoff, France; [email protected] 4 CHRU de Brest, Service de Génétique Médicale et Biologie de la Reproduction, Centre de Référence des Maladies Rares “Maladies Neuromusculaires”, F-29200 Brest, France * Correspondence: [email protected] Abstract: The respiratory epithelium can be affected by many diseases that could be treated using aerosol gene therapy. Among these, cystic fibrosis (CF) is a lethal inherited disease characterized by airways complications, which determine the life expectancy and the effectiveness of aerosolized treatments. Beside evaluations performed under in vivo settings, cell culture models mimicking in vivo pathophysiological conditions can provide complementary insights into the potential of Citation: Ghanem, R.; Laurent, V.; gene transfer strategies. Such models must consider multiple parameters, following the rationale Roquefort, P.; Haute, T.; Ramel, S.; Le that proper gene transfer evaluations depend on whether they are performed under experimental Gall, T.; Aubry, T.; Montier, T. conditions close to pathophysiological settings. In addition, the mucus layer, which covers the Optimizations of In Vitro Mucus and epithelial cells, constitutes a physical barrier for gene delivery, especially in diseases such as CF. Cell Culture Models to Better Predict Artificial mucus models featuring physical and biological properties similar to CF mucus allow In Vivo Gene Transfer in Pathological determining the ability of gene transfer systems to effectively reach the underlying epithelium. In this Lung Respiratory Airways: Cystic review, we describe mucus and cellular models relevant for CF aerosol gene therapy, with a particular Fibrosis as an Example. Pharmaceutics emphasis on mucus rheology. We strongly believe that combining multiple pathophysiological 2021, 13, 47. https://doi.org/ features in single complex cell culture models could help bridge the gaps between in vitro and 10.3390/pharmaceutics13010047 in vivo settings, as well as viral and non-viral gene delivery strategies. Received: 1 December 2020 Keywords: cystic fibrosis; gene delivery; in vitro model; mucus; airway epithelium Accepted: 28 December 2020 Published: 31 December 2020 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional clai- 1. Introduction ms in published maps and institutio- 1.1. Cystic Fibrosis and Current Protein Treatment nal affiliations. Cystic fibrosis (CF) is one of the most common inherited genetic disorders, affecting over 70,000 people worldwide [1]. CF is induced by numerous mutations listed in a single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) [2]. The CFTR protein is a channel that participates in the exit of chloride ions from the Copyright: © 2020 by the authors. Li- censee MDPI, Basel, Switzerland. cytoplasm. This channel also plays a key role in the regulation of another channel called This article is an open access article ENaC (epithelial sodium channel), involved in the reabsorption of sodium. Altogether, distributed under the terms and con- those channels control the correct hydration of mucus on airway epithelial cells. Alteration ditions of the Creative Commons At- or dysfunction of the CFTR channel leads thus to abnormal ion transport and mucus tribution (CC BY) license (https:// dehydration (Figure1A). Such dehydrated sticky mucus is responsible for the diminution creativecommons.org/licenses/by/ of mucociliary clearance (MCC), leading to a mucosal accumulation. This mucus stagnation 4.0/). provides a propitious environment for bacterial colonization and infections, inducing Pharmaceutics 2021, 13, 47. https://doi.org/10.3390/pharmaceutics13010047 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, x 2 of 25 Pharmaceutics 2021, 13, 47 2 of 25 tion (Figure 1A). Such dehydrated sticky mucus is responsible for the diminution of mu- cociliary clearance (MCC), leading to a mucosal accumulation. This mucus stagnation pro- vides a propitious environment for bacterial colonization and infections, inducing related chronicrelated inflammation. chronic inflammation. In the CF In context, the CF poly context,nuclear polynuclear neutrophils neutrophils accumulation accumulation in the mu- in custhe is mucus responsible is responsible for the forproduction the production and th ande excretion the excretion of lysosomal of lysosomal enzymes enzymes which which di- rectlydirectly contribute contribute to totissue tissue remodeling remodeling [3,4]. [3,4 ].The The remodeling remodeling of of secretory secretory cells cells induces induces an an overproductionoverproduction of of mucin, mucin, the the main main constituen constituentt of of mucus, mucus, leading leading to to severe severe consequences consequences forfor the the respiratory respiratory exchanges exchanges [5]. [5]. Figure 1. Schematic representation of the respiratory system and the use of an aerosol to target the pulmonary epithelium. Figure 1. Schematic representation of the respiratory system and the use of an aerosol to target the pulmonary epithelium. (A) The upper and lower respiratory tract with an emphasis on a bronchial section constituted by mucous glands and smooth (A) The upper and lower respiratory tract with an emphasis on a bronchial section constituted by mucous glands and muscles. In the case of cystic fibrosis (CF), sticky mucus is overproduced and creates a bronchial plug. (B) Pseudostratified smooth muscles. In the case of cystic fibrosis (CF), sticky mucus is overproduced and creates a bronchial plug. (B) Pseu- dostratifiedairway epithelium airway epithelium composed composed of goblet, of ciliated, goblet, andciliated, basal and cells. basal The cells. mucosa The mucosa is covered is covered with airways with airways surface surface liquid liquid(ASL) (ASL) which which contains contains two layers, two layers, a periciliary a periciliary liquid liquid layer directlylayer directly in contact in contact with the with cilia the and cilia mucus and mucus layer. CFlayer. mucus CF mucusis colonized is colonized with opportunistic with opportunistic bacteria bacteria such as suchStaphylococcus as Staphylococcus aureus aureusand Pseudomonas and Pseudomonas aeruginosa aeruginosa. These. These bacteria bacteria form a formbiofilm a biofilm participating participating in the in viscous the viscous anddense and dense aspect aspect of the of CF the mucus. CF mucus. When When nanoparticles nanoparticles (NPs) (NPs) such such as lipoplexesas lipoplexes are areaerosolized, aerosolized, they they have have to crossto cross the the mucus mucus barrier barrier before before reaching reaching the the epithelial epithelial cells cells to deliver to deliver therapeutic therapeutic compounds compounds such suchas acids as acids nucleic nucleic construction. construction. Today,Today, pulmonary pulmonary complications complications are are the the pr primaryimary cause cause of of CF-related CF-related morbidity morbidity and and mortality.mortality. Similar Similar manifestations manifestations exist exist in inthe the gastrointestinal gastrointestinal and and genital genital tracts tracts but butare, are,at present,at present, dealt dealt with with by bysymptomatic symptomatic treatmen treatmentsts such such as as supplementary supplementary pancreatic pancreatic en- en- zymeszymes [6]. [6]. During During the the last last decade, decade, new new dr drugsugs have have been been developed developed regarding regarding the the CFTR CFTR mutations.mutations. Ivacaftor Ivacaftor (Kalydeco (Kalydeco®®),), a aCFTR CFTR potentiator potentiator channel, channel, is is indicated indicated for for the the G551D G551D mutation.mutation. This This potentiator cancan be be associated associated with with correctors correctors such such as lumacaftor as lumacaftor (Orkambi (Or-®) kambior tezacaftor®) or tezacaftor (Symdeko (Symdeko®) for the®) homozygote for the homozygote F508del mutation.F508del mutation. More recently, More arecently, tritherapy, a tritherapy,namely, elexacaftor-tezacaftor-ivacaftor namely, elexacaftor-tezacaftor-ivacaftor (Trikafta® ),(Trikafta showed®), an showed FEV1 (forced an FEV1 expiratory (forced expiratoryvolume) higher volume) than higher 10% benefit than 10% for patientsbenefit withfor patients a single with F508del a single allele F508del [7]. Unfortunately, allele [7]. Unfortunately,these molecules these are mutation-dependentmolecules are mutation and-dependent are sometimes and are responsible sometimes for responsible several side foreffects several (such side as effects hepatitis, (such abdominal as hepatitis, pain) abdominal [8]. Even pain) though [8]. Even pharmacology though pharmacology
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