Recent Developments in mRNA-Based Protein Supplementation Therapy to Target Lung Diseases
Authors: Itishri Sahu, A.K.M. Ashiqul Haque, Brian Weidensee, Petra Weinmann, Michael S.D. Kormann
Journal: Molecular Therapy (2019)

Protein supplementation therapy using in vitro-transcribed (IVT) mRNA for genetic diseases contains huge potential as a new class of therapy. From the early ages of synthetic mRNA discovery, a great number of studies showed the versatile use of IVT mRNA as a novel approach to supplement faulty or absent protein and also as a vaccine. Many modifications have been made to produce high expressions of mRNA causing less immunogenicity and more stability. Recent advancements in the in vivo lung delivery of mRNA complexed with various carriers encouraged the whole mRNA community to tackle various genetic lung diseases. This review gives a comprehensive overview of cells associated with various lung diseases and recent advancements in mRNA-based protein replacement therapy. This review also covers a brief summary of developments in mRNA modifications and nanocarriers toward clinical translation. Protein supplementation therapy using in vitro-transcribed (IVT) mRNA for genetic diseases contains huge potential as a new class of therapy. From the early ages of synthetic mRNA discovery, a great number of studies showed the versatile use of IVT mRNA as a novel approach to supplement faulty or absent protein and also as a vaccine. Many modifications have been made to produce high expressions of mRNA causing less immunogenicity and more stability. Recent advancements in the in vivo lung delivery of mRNA complexed with various carriers encouraged the whole mRNA community to tackle various genetic lung diseases. This review gives a comprehensive overview of cells associated with various lung diseases and recent advancements in mRNA-based protein replacement therapy. This review also covers a brief summary of developments in mRNA modifications and nanocarriers toward clinical translation. RNA, a fundamental molecule in the eukaryotic and prokaryotic cells and viruses, came recently into focus for therapeutic approaches:1Robertson M.P. Joyce G.F. The origins of the RNA world.Cold Spring Harb. Perspect. Biol. 2012; 4: a003608Google Scholar, 2Cech T.R. The RNA worlds in context.Cold Spring Harb. Perspect. Biol. 2012; 4: a006742Google Scholar, 3Neveu M. Kim H.J. Benner S.A. The "strong" RNA world hypothesis: fifty years old.Astrobiology. 2013; 13: 391-403Google Scholar tRNAs for nonsense mutation correction, RNA aptamers for binding to a specific target molecule, and RNAi and long noncoding RNAs (lncRNAs) for gene regulation.4Murgola E.J. Prather N.E. Mims B.H. Pagel F.T. Hijazi K.A. Anticodon shift in tRNA: a novel mechanism in missense and nonsense suppression.Proc. Natl. Acad. Sci. USA. 1983; 80: 4936-4939Google Scholar, 5Ellington A.D. Szostak J.W. In vitro selection of RNA molecules that bind specific ligands.Nature. 1990; 346: 818-822Google Scholar, 6Cogoni C. Irelan J.T. Schumacher M. Schmidhauser T.J. Selker E.U. Macino G. Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation.EMBO J. 1996; 15: 3153-3163Google Scholar, 7Chen X. He L. Zhao Y. Li Y. Zhang S. Sun K. So K. Chen F. Zhou L. Lu L. et al.Malat1 regulates myogenic differentiation and muscle regeneration through modulating MyoD transcriptional activity.Cell Discov. 2017; 3: 17002Google Scholar, 8Lundstrom K. Special Issue: Gene Therapy with Emphasis on RNA Interference.Viruses. 2015; 7: 4482-4487Google Scholar mRNA gives rise to a new therapy for diseases associated with functional protein loss by supplementing the protein with a transcript encoding. The first study of in vitro-transcribed (IVT) mRNA in the late 1980s showed that this mRNA can be directly translated into a functional form immediately after transfection in vitro and in vivo.9Malone R.W. Felgner P.L. Verma I.M. Cationic liposome-mediated RNA transfection.Proc. Natl. Acad. Sci. USA. 1989; 86: 6077-6081Google Scholar, 10Wolff J.A. Malone R.W. Williams P. Chong W. Acsadi G. Jani A. Felgner P.L. Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Google Scholar This was shortly followed by a therapeutic application of mRNA in a temporary reversal of diabetes insipidus, unraveling its therapeutic potential.11Jirikowski G.F. Sanna P.P. Maciejewski-Lenoir D. Bloom F.E. Reversal of diabetes insipidus in Brattleboro rats: intrahypothalamic injection of vasopressin mRNA.Science. 1992; 255: 996-998Scopus (138) Google Scholar Therapeutic applications of mRNA are advantageous due to its unique properties. Cytoplasm being mRNA's functional site requires no transportation across the nuclear membrane. Furthermore, due to the transient nature and biodegradability of mRNA, permanent adverse effects can potentially be avoided.12Newbury S.F. Control of mRNA stability in eukaryotes.Biochem. Soc. Trans. 2006; 34: 30-34Scopus (0) Google Scholar This includes preventing the permanent manipulation of the genome, making mRNA a prevalent molecule for protein supplementation therapy.13Mockey M. Gonçalves C. Dupuy F.P. Lemoine F.M. Pichon C. Midoux P. mRNA transfection of dendritic cells: synergistic effect of ARCA mRNA capping with Poly(A) chains in cis and in trans for a high protein expression level.Biochem. Biophys. Res. Commun. 2006; 340: 1062-1068Google Scholar, 14Holtkamp S. Kreiter S. Selmi A. Simon P. Koslowski M. Huber C. Türeci O. Sahin U. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells.Blood. 2006; 108: 4009-4017Google Scholar, 15Elango N. Elango S. Shivshankar P. Katz M.S. Optimized transfection of mRNA transcribed from a d(A/T)100 tail-containing vector.Biochem. Biophys. Res. Commun. 2005; 330: 958-966Google Scholar Several aspects of mRNA have to be addressed in order to achieve therapeutic benefits: potential immunogenicity mediated by innate immune system reactions (pattern recognition receptor),16Rozenski J. Crain P.F. McCloskey J.A. The RNA Modification Database: 1999 update.Nucleic Acids Res. 1999; 27: 196-197Google Scholar, 17Boccaletto P. Machnicka M.A. Purta E. Piatkowski P. Baginski B. Wirecki T.K. de Crécy-Lagard V. Ross R. Limbach P.A. Kotter A. et al.MODOMICS: a database of RNA modification pathways. 2017 update.Nucleic Acids Res. 2018; 46: D303-D307Google Scholar, 18Ishii K.J. Akira S. TLR ignores methylated RNA?.Immunity. 2005; 23: 111-113Google Scholar, 19Karikó K. Ni H. Capodici J. Lamphier M. Weissman D. mRNA is an endogenous ligand for Toll-like receptor 3.J. Biol. Chem. 2004; 279: 12542-12550Google Scholar degradation of single-stranded mRNA by nucleases, and its negative charge that inhibits the passive crossing of the cell membrane. Concerning these obstacles, solutions such as chemical modifications of nucleosides to reduce immunogenicity and usage of nanocarriers to facilitate crossing the cell membrane are emerging. The recent advancements in the field of nanocarriers suggest the possibility to customize particles for target organs.20Haque A.K.M.A. Dewerth A. Antony J.S. Riethmüller J. Schweizer G.R. Weinmann P. Latifi N. Yasar H. Pedemonte N. Sondo E. et al.Chemically modified hCFTR mRNAs recuperate lung function in a mouse model of cystic fibrosis.Sci. Rep. 2018; 8: 16776Google Scholar, 21Connolly B. Isaacs C. Cheng L. Asrani K.H. Subramanian R.R. SERPINA1 mRNA as a Treatment for Alpha-1 Antitrypsin Deficiency.J. Nucleic Acids. 2018; 2018: 8247935Google Scholar Based on our research interest in lungs, this review focuses on protein replacement therapy of lung disease, especially monogenetic diseases, such as cystic fibrosis (CF) and surfactant protein B (SP-B) deficiency, as well as multifactorial diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. To achieve targeted therapy options, lung structure and methods to reach specific lung cell populations are critical. Therefore, this review gives an overview of lung cell populations and diseases associated with them. Furthermore, a comprehensive summary of mRNA transcript improvements by chemical modification of nucleosides or capping and nanocarriers to target lung cell populations is also featured. The lung has a very unique architecture to enable efficient transfer of oxygen and carbon dioxide required for oxidative metabolism. Inhaled gases travel through the airway tubes via trachea bronchi and bronchioles to the alveoli enriched with blood vessels, the primary site of gas exchange. Inflation and deflation of the lung is a prerequisite for gas exchange at the alveoli. This process requires multiple components like the extracellular matrix, smooth muscle cells, and cartilage for support and flexible collagen and the elastin fiber network for flexibility during inflation and deflation. Precisely regulated surface fluids, electrolytes, and mechanical activity of secretory and ciliated cells determine the mucociliary clearance, whereas on the other hand the epithelium maintains the barrier function. The airway epithelium (tracheal and bronchiolar) consists of goblet cells, brush or tuft cells, ciliated cells, basal cells, neuroendocrine or neuroepithelial bodies, club cells, lineage-negative epithelial progenitor (LNEP) cells, and the newly identified ionocytes.22Plasschaert L.W. Žilionis R. Choo-Wing R. Savova V. Knehr J. Roma G. Klein A.M. Jaffe A.B. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.Nature. 2018; 560: 377-381Google Scholar, 23Montoro D.T. Haber A.L. Biton M. Vinarsky V. Lin B. Birket S.E. Yuan F. Chen S. Leung H.M. Villoria J. et al.A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.Nature. 2018; 560: 319-324Google Scholar The ducts of submucosal glands consist of goblet cells, serous cells, and myoepithelial cells. The alveolar epithelium consists primarily of alveolar type I (ATI) cells (∼95%), macrophages, and alveolar type II (ATII) cells, with close association to capillary endothelial cells of pulmonary microcirculation.24Castranova V. Rabovsky J. Tucker J.H. Miles P.R. The alveolar type II epithelial cell: a multifunctional pneumocyte.Toxicol. Appl. Pharmacol. 1988; 93: 472-483Scopus (126) Google Scholar The terminal airway ducts and alveoli are supported by fibroblasts and myofibroblasts producing extensive elastin-collagen networks, which help with inflation and deflation.25Noguchi A. Reddy R. Kursar J.D. Parks W.C. Mecham R.P. Smooth muscle isoactin and elastin in fetal bovine lung.Exp. Lung Res. 1989; 15: 537-552Google Scholar, 26Morrisey E.E. Hogan B.L. Preparing for the first breath: genetic and cellular mechanisms in lung development.Dev. Cell. 2010; 18: 8-23Google Scholar, 27Boström H. Willetts K. Pekny M. Levéen P. Lindahl P. Hedstrand H. Pekna M. Hellström M. Gebre-Medhin S. Schalling M. et al.PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis.Cell. 1996; 85: 863-873Google Scholar Other important cell populations include stem cells and immune cells, which help in region-specific regeneration and protection against pathogens, respectively. Abnormalities of lung mechanics are observed in various diseases, including CF,28Hilliard T.N. Regamey N. Shute J.K. Nicholson A.G. Alton E.W. Bush A. Davies J.C. Airway remodelling in children with cystic fibrosis.Thorax. 2007; 62: 1074-1080Google Scholar, 29Adam D. Roux-Delrieu J. Luczka E. Bonnomet A. Lesage J. Mérol J.C. Polette M. Abély M. Coraux C. Cystic fibrosis airway epithelium remodelling: involvement of inflammation.J. Pathol. 2015; 235: 408-419Google Scholar asthma,30Elias J.A. Zhu Z. Chupp G. Homer R.J. Airway remodeling in asthma.J. Clin. Invest. 1999; 104: 1001-1006Google Scholar, 31Fehrenbach H. Wagner C. Wegmann M. Airway remodeling in asthma: what really matters.Cell Tissue Res. 2017; 367: 551-569Google Scholar idiopathic pulmonary fibrosis (IPF),32Knudsen L. Ruppert C. Ochs M. Tissue remodelling in pulmonary fibrosis.Cell Tissue Res. 2017; 367: 607-626Google Scholar, 33Wallace W.A. Fitch P.M. Simpson A.J. Howie S.E. Inflammation-associated remodelling and fibrosis in the lung - a process and an end point.Int. J. Exp. Pathol. 2007; 88: 103-110Google Scholar COPD,34Pain M. Bermudez O. Lacoste P. Royer P.J. Botturi K. Tissot A. Brouard S. Eickelberg O. Magnan A. Tissue remodelling in chronic bronchial diseases: from the epithelial to mesenchymal phenotype.Eur. Respir. Rev. 2014; 23: 118-130Google Scholar, 35Salazar L.M. Herrera A.M. Fibrotic response of tissue remodeling in COPD.Hai. 2011; 189: 101-109Google Scholar and bronchiolitis obliterans.34Pain M. Bermudez O. Lacoste P. Royer P.J. Botturi K. Tissot A. Brouard S. Eickelberg O. Magnan A. Tissue remodelling in chronic bronchial diseases: from the epithelial to mesenchymal phenotype.Eur. Respir. Rev. 2014; 23: 118-130Google Scholar, 36Sato M. Keshavjee S. Bronchiolitis obliterans syndrome: alloimmune-dependent and -independent injury with aberrant tissue remodeling.Semin. Thorac. Cardiovasc. Surg. 2008; 20: 173-182Google Scholar These conditions are detrimental in nature and involve multiple factors, such as increased resistance of lung tissue due to fibrosis of the lung; collapsing tubes or thickening of airway walls due to mucus overproduction; and the loss of ciliary function, resulting in airflow obstruction, mucus plugging, chronic infection, and inflammatory damages. In that process, multiple cell populations undergo remodeling, directly contributing to clinical symptoms. Goblet cells, present in the broncho-epithelia and submucosal glands, produce mucins (MUC5B and MUC5AC), and they are differentiated from basal and club cells upon various stimuli (toxic substances, pathogens, particles, and neural and innate immune signals). Goblet cells are not only helping to establish the innate immune system but also key players in pulmonary diseases, as described before.37Ma J. Rubin B.K. Voynow J.A. Mucins, Mucus, and Goblet Cells.Chest. 2018; 154: 169-176Google Scholar These cells produce cytokines and chemokines that recruit and educate innate immune cells, including dendritic cells (DCs), innate lymphoid cells, and eosinophils. This contributes to the CD4+ T helper cell 2 (Th2)-mediated immune response typical of asthma.38Chen G. Korfhagen T.R. Xu Y. Kitzmiller J. Wert S.E. Maeda Y. Gregorieff A. Clevers H. Whitsett J.A. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production.J. Clin. Invest. 2009; 119: 2914-2924Google Scholar, 39Rajavelu P. Chen G. Xu Y. Kitzmiller J.A. Korfhagen T.R. Whitsett J.A. Airway epithelial SPDEF integrates goblet cell differentiation and pulmonary Th2 inflammation.J. Clin. Invest. 2015; 125: 2021-2031Google Scholar Hyper-production of mucus and goblet cell metaplasia are characteristics of Th2-mediated and non-mediated inflammation (pathogen mediated), which leads to complications in CF, COPD, and IPF.40De Rose V. Molloy K. Gohy S. Pilette C. Greene C.M. Airway Epithelium Dysfunction in Cystic Fibrosis and COPD.Mediators Inflamm. 2018; 2018: 1309746Google Scholar, 41Wynn T.A. Cheever A.W. Jankovic D. Poindexter R.W. Caspar P. Lewis F.A. Sher A. An IL-12-based vaccination method for preventing fibrosis induced by schistosome infection.Nature. 1995; 376: 594-596Google Scholar The fluids for hydration are regulated by serous cells that line the acinar region of submucosal glands and are important for the pathology of CF. The mucins are precisely balanced by fluid, and electrolyte transport enables rapid secretion and dispersal of mucus onto the airway surfaces and the movement of the mucus gel up the airway by ciliary activity. In CF, the secretion of chloride and bicarbonate is impaired, disrupting mucociliary clearance due to mutations in CF transmembrane conductance regulator gene (CFTR), which leads to recurrent airway infections, sinusitis, bronchiectasis, and pulmonary tissue remodeling.42Whitsett J.A. Airway Epithelial Differentiation and Mucociliary Clearance.Ann. Am. Thorac. Soc. 2018; 15: S143-S148Google Scholar Brush cells, containing distinctive apical microvilli, are found in multiple organs, e.g., pancreas, intestine, nose, and trachea. Brush cells are known to play an important role in activating the innate immune system in the intestine43Gerbe F. Sidot E. Smyth D.J. Ohmoto M. Matsumoto I. Dardalhon V. Cesses P. Garnier L. Pouzolles M. Brulin B. et al.Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.Nature. 2016; 529: 226-230Scopus (297) Google Scholar and nose (Tas2R receptors),44Tizzano M. Gulbransen B.D. Vandenbeuch A. Clapp T.R. Herman J.P. Sibhatu H.M. Churchill M.E. Silver W.L. Kinnamon S.C. Finger T.E. Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals.Proc. Natl. Acad. Sci. USA. 2010; 107: 3210-3215Google Scholar however, a similar phenomenon in airway trachea requires testing. Recent studies in the trachea have indicated their role as chemosensory for immune surveillance and as respiratory regulators.45Krasteva G. Canning B.J. Hartmann P. Veres T.Z. Papadakis T. Mühlfeld C. Schliecker K. Tallini Y.N. Braun A. Hackstein H. et al.Cholinergic chemosensory cells in the trachea regulate breathing.Proc. Natl. Acad. Sci. USA. 2011; 108: 9478-9483Google Scholar These might be responsible in transducing signals regulating wheezing and coughing during episodes of asthma. However, further studies are required to understand their role.46Gordon E.D. Locksley R.M. Fahy J.V. Cross-Talk between Epithelial Cells and Type 2 Immune Signaling. The Role of IL-25.Am. J. Respir. Crit. Care Med. 2016; 193: 935-936Google Scholar Ciliated cells are characterized by their multiple apical, motile cilia composed of structural proteins and motor proteins (dynein)47Zhang Y.J. O'Neal W.K. Randell S.H. Blackburn K. Moyer M.B. Boucher R.C. Ostrowski L.E. Identification of dynein heavy chain 7 as an inner arm component of human cilia that is synthesized but not assembled in a case of primary ciliary dyskinesia.J. Biol. Chem. 2002; 277: 17906-17915Google Scholar, 48Ostrowski L.E. Blackburn K. Radde K.M. Moyer M.B. Schlatzer D.M. Moseley A. Boucher R.C. A proteomic analysis of human cilia: identification of novel components.Mol. Cell. Proteomics. 2002; 1: 451-465Scopus (346) Google Scholar, 49Ostrowski L.E. Dutcher S.K. Lo C.W. Cilia and models for studying structure and function.Proc. Am. Thorac. Soc. 2011; 8: 423-429Google Scholar that regulate the coordinated bidirectional beating critical for particle and pathogen clearance.50Bustamante-Marin X.M. Ostrowski L.E. Cilia and Mucociliary Clearance.Cold Spring Harb. Perspect. Biol. 2017; 9: a028241Google Scholar, 51Whitsett J.A. Alenghat T. Respiratory epithelial cells orchestrate pulmonary innate immunity.Nat. Immunol. 2015; 16: 27-35Google Scholar Ciliated cells respond to both physical52Sanderson M.J. Dirksen E.R. Mechanosensitivity of cultured ciliated cells from the mammalian respiratory tract: implications for the regulation of mucociliary transport.Proc. Natl. Acad. Sci. USA. 1986; 83: 7302-7306Google Scholar and chemical53Shah A.S. Ben-Shahar Y. Moninger T.O. Kline J.N. Welsh M.J. Motile cilia of human airway epithelia are chemosensory.Science. 2009; 325: 1131-1134Google Scholar stimulation. Mucociliary clearance can be affected by ciliary dysfunction, impaired fluid secretion, disruption of epithelial cell lining, or lack of cough. This impairment can initiate an inflammatory response, damaging the airway epithelium. Disruption in ciliated cell function results in recurrent and persistent infections, morbidity, and mortality in chronic pulmonary disorders.54Horani A. Ferkol T.W. Dutcher S.K. Brody S.L. Genetics and biology of primary ciliary dyskinesia.Paediatr. Respir. Rev. 2016; 18: 18-24Google Scholar In COPD, direct evidence has been provided of suppressed ciliary beating in nasal epithelium55Yaghi A. Zaman A. Cox G. Dolovich M.B. Ciliary beating is depressed in nasal cilia from chronic obstructive pulmonary disease subjects.Respir. Med. 2012; 106: 1139-1147Google Scholar with normal mucus production. Cigarette smoking has been shown to have a detrimental effect on the number and size of cilia in vitro,56Tamashiro E. Xiong G. Anselmo-Lima W.T. Kreindler J.L. Palmer J.N. Cohen N.A. Cigarette smoke exposure impairs respiratory epithelial ciliogenesis.Am. J. Rhinol. Allergy. 2009; 23: 117-122Google Scholar whereas in vivo a slight increase in ciliary beating initially followed by significant loss of cilia over prolonged time was observed.57Simet S.M. Sisson J.H. Pavlik J.A. Devasure J.M. Boyer C. Liu X. Kawasaki S. Sharp J.G. Rennard S.I. Wyatt T.A. Long-term cigarette smoke exposure in a mouse model of ciliated epithelial cell function.Am. J. Respir. Cell Mol. Biol. 2010; 43: 635-640Google Scholar Primary ciliary dyskinesia (PCD) resulting from ciliary dysfunction is caused by recessive mutations in one of multiple genes involved at different points in cilium structure, assembly, and function, which include DNAI,58Guichard C. Harricane M.C. Lafitte J.J. Godard P. Zaegel M. Tack V. Lalau G. Bouvagnet P. Axonemal dynein intermediate-chain gene (DNAI1) mutations result in situs inversus and primary ciliary dyskinesia (Kartagener syndrome).Am. J. Hum. Genet. 2001; 68: 1030-1035Google Scholar DNAH(5,59Olbrich H. Häffner K. Kispert A. Völkel A. Volz A. Sasmaz G. Reinhardt R. Hennig S. Lehrach H. Konietzko N. et al.Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left-right asymmetry.Nat. Genet. 2002; 30: 143-144Google Scholar 1160Pifferi M. Michelucci A. Conidi M.E. Cangiotti A.M. Simi P. Macchia P. Boner A.L. New DNAH11 mutations in primary ciliary dyskinesia with normal axonemal ultrastructure.Eur. Respir. J. 2010; 35: 1413-1416Google Scholar), ARMC4,61Hjeij R. Lindstrand A. Francis R. Zariwala M.A. Liu X. Li Y. Damerla R. Dougherty G.W. Abouhamed M. Olbrich H. et al.ARMC4 mutations cause primary ciliary dyskinesia with randomization of left/right body asymmetry.Am. J. Hum. Genet. 2013; 93: 357-367Google Scholar TXNDC3,62Duriez B. Duquesnoy P. Escudier E. Bridoux A.M. Escalier D. Rayet I. Marcos E. Vojtek A.M. Bercher J.F. Amselem S. A common variant in combination with a nonsense mutation in a member of the thioredoxin family causes primary ciliary dyskinesia.Proc. Natl. Acad. Sci. USA. 2007; 104: 3336-3341Google Scholar HEATR2,63Horani A. Druley T.E. Zariwala M.A. Patel A.C. Levinson B.T. Van Arendonk L.G. Thornton K.C. Giacalone J.C. Albee A.J. Wilson K.S. et al.Whole-exome capture and sequencing identifies HEATR2 mutation as a cause of primary ciliary dyskinesia.Am. J. Hum. Genet. 2012; 91: 685-693Google Scholar HYDIN,64Olbrich H. Schmidts M. Werner C. Onoufriadis A. Loges N.T. Raidt J. Banki N.F. Shoemark A. Burgoyne T. Al Turki S. et al.UK10K ConsortiumRecessive HYDIN mutations cause primary ciliary dyskinesia without randomization of left-right body asymmetry.Am. J. Hum. Genet. 2012; 91: 672-684Google Scholar CDC (39,65Merveille A.C. Davis E.E. Becker-Heck A. Legendre M. Amirav I. Bataille G. Belmont J. Beydon N. Billen F. Clément A. et al.CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs.Nat. Genet. 2011; 43: 72-78Google Scholar 40,66Becker-Heck A. Zohn I.E. Okabe N. Pollock A. Lenhart K.B. Sullivan-Brown J. McSheene J. Loges N.T. Olbrich H. Haeffner K. et al.The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation.Nat. Genet. 2011; 43: 79-84Google Scholar 65,67Horani A. Brody S.L. Ferkol T.W. Shoseyov D. Wasserman M.G. Ta-shma A. Wilson K.S. Bayly P.V. Amirav I. Cohen-Cymberknoh M. et al.CCDC65 mutation causes primary ciliary dyskinesia with normal ultrastructure and hyperkinetic cilia.PLoS ONE. 2013; 8: e72299Google Scholar 103,68Panizzi J.R. Becker-Heck A. Castleman V.H. Al-Mutairi D.A. Liu Y. Loges N.T. Pathak N. Austin-Tse C. Sheridan E. Schmidts M. et al.CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms.Nat. Genet. 2012; 44: 714-719Google Scholar 114,69Knowles M.R. Leigh M.W. Ostrowski L.E. Huang L. Carson J.L. Hazucha M.J. Yin W. Berg J.S. Davis S.D. Dell S.D. et al.Genetic Disorders of Mucociliary Clearance ConsortiumExome sequencing identifies mutations in CCDC114 as a cause of primary ciliary dyskinesia.Am. J. Hum. Genet. 2013; 92: 99-106Google Scholar 15170Hjeij R. Onoufriadis A. Watson C.M. Slagle C.E. Klena N.T. Dougherty G.W. Kurkowiak M. Loges N.T. Diggle C.P. Morante N.F. et al.UK10K ConsortiumCCDC151 mutations cause primary ciliary dyskinesia by disruption of the outer dynein arm docking complex formation.Am. J. Hum. Genet. 2014; 95: 257-274Google Scholar), DNAAF(1,71Loges N.T. Olbrich H. Becker-Heck A. Häffner K. Heer A. Reinhard C. Schmidts M. Kispert A. Zariwala M.A. Leigh M.W. et al.Deletions and point mutations of LRRC50 cause primary ciliary dyskinesia due to dynein arm defects.Am. J. Hum. Genet. 2009; 85: 883-889Google Scholar 2,72Omran H. Kobayashi D. Olbrich H. Tsukahara T. Loges N.T. Hagiwara H. Zhang Q. Leblond G. O'Toole E. Hara C. et al.Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins.Nature. 2008; 456: 611-616Google Scholar 373Mitchison H.M. Schmidts M. Loges N.T. Freshour J. Dritsoula A. Hirst R.A. O'Callaghan C. Blau H. Al Dabbagh M. Olbrich H. et al.Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia.Nat. Genet. 2012; 44: 381-389Google Scholar), RHSP(4A, 9),74Castleman V.H. Romio L. Chodhari R. Hirst R.A. de Castro S.C. Parker K.A. Ybot-Gonzalez P. Emes R.D. Wilson S.W. Wallis C. et al.Mutations in radial spoke head protein genes RSPH9 and RSPH4A cause primary ciliary dyskinesia with central-microtubular-pair abnormalities.Am. J. Hum. Genet. 2009; 84: 197-209Google Scholar DYX1C1,75Tarkar A. Loges N.T. Slagle C.E. Francis R. Dougherty G.W. Tamayo J.V. Shook B. Cantino M. Schwartz D. Jahnke C. et al.UK10KDYX1C1 is required for axonemal dynein assembly and ciliary motility.Nat. Genet. 2013; 45: 995-1003Google Scholar LRRC6,76Horani A. Ferkol T.W. Shoseyov D. Wasserman M.G. Oren Y.S. Kerem B. Amirav I. Cohen-Cymberknoh M. Dutcher S.K. Brody S.L. et al.LRRC6 mutation causes primary ciliary dyskinesia with dynein arm defects.PLoS ONE. 2013; 8: e59436Google Scholar ZMYND10,77Zariwala M.A. Gee H.Y. Kurkowiak M. Al-Mutairi D.A. Leigh M.W. Hurd T.W. Hjeij R. Dell S.D. Chaki M. Dougherty G.W. et al.ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6.Am. J. Hum. Genet. 2013; 93: 336-345Google Scholar CCNO,78Wallmeier J. Al-Mutairi D.A. Chen C.T. Loges N.T. Pennekamp P. Menchen T. Ma L. Shamseldin H.E. Olbrich H. Dougherty G.W. et al.Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia.Nat. Genet. 2014; 46: 646-651Google Scholar and recently identified MCIDAS.79Boon M. Wallmeier J. Ma L. Loges N.T. Jaspers M. Olbrich H. Dougherty G.W. Raidt J. Werner C. Amirav I. et al.MCIDAS mutations result in a mucociliary clearance disorder with reduced generation of multiple motile cilia.Nat. Commun. 2014; 5: 4418Google Scholar Club cells are columnar, secretory cells that express high levels of cytochrome P450-detoxifying enzyme CYP2F, surfactant proteins (SP-A, SP-B, and SP-D), and innate immune proteins, including defensins, lactoferrin, and secretaglobins (SCGB1A1 and SCGB3A1).80Rokicki W. Rokicki M. Wojtacha J. Dżeljijli A. The role and importance of club cells (Clara cells) in the pathogenesis of some respiratory diseases.Kardiochir. Torakochirurgia Pol. 2016; 13: 26-30Google Scholar Upon stimulus or injury, these differentiate into alveolar cells,81Zheng D. Limmon G.V. Yin L. Leung N.H. Yu H. Chow V.T. Chen J. Regeneration of alveolar type I and II cells from Scgb1a1-expressing cells following severe pulmonary damage induced by bleomycin and influenza.PLoS ONE. 2012; 7: e48451Google Scholar, 82Zheng D. Soh B.S. Yin L. Hu G. Chen Q. Choi H. Han J. Chow V.T. Chen J. Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro.Sci. Rep. 2017; 7: 41661Google Scholar goblet cells,38Chen G. Korfhagen T.R. Xu Y. Kitzmiller J. Wert S.E. Maeda Y. Gregorieff A. Clevers H. Whitsett J.A. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production.J. Clin. Invest. 2009; 119: 2914-2924Google Scholar, 83Guseh J.S. Bores S.A. Stanger B.Z. Zhou Q. Anderson W.J. Melton D.A. Rajagopal J. Notch signaling promotes airway mucous metaplasia and inhibits alveolar development.Development. 2009; 136: 1751-1759Google Scholar, 84Roy M.G. Rahmani M. Hernandez J.R. Alexander S.N. Ehre C. Ho S.B. Evans C.M. Mucin production during prenatal and postnatal murine lung development.Am. J. Respir. Cell Mol. Biol. 2011; 44: 755-760Google Scholar or ciliated cells.85Watson J.K. Rulands S. Wilkinson A.C. Wuidart A. Ousset M. Van Keymeulen A. Göttgens B. Blanpain C. Simons B.D. Rawlins E.L. Clonal Dynamics Reveal Two Distinct Populations of Basal Cells in Slow-Turnover Airway Epithelium.Cell Rep. 2015; 12: 90-101Google Scholar However, in humans, these are only localized in terminal bronchioles, and they may play a role in the maintenance of distal bronchioles.86Boers J.E. Ambergen A.W. Thunnissen F.B. Number and proliferation of clara cells in normal human airway epithelium.Am. J. Respir. Crit. Care Med. 1999; 159: 1585-1591Scopus (208) Google Scholar Club cells being progenitor cells also plays an important role in the repair mechanism of the airway epithelium, and, therefore, they are connected to damage responses in CF, COPD, and IPF.80Rokicki W. Rokicki M. Wojtacha J. Dżeljijli A. The role and importance of club cells (Clara cells) in the pathogenesis of some respiratory diseases.Kardiochir.