RESUMEN
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with F508del being the most prevalent mutation. The combination of CFTR modulators (potentiator and correctors) has provided benefit to CF patients carrying the F508del mutation; however, the safety and effectiveness of in utero combination modulator therapy remains unclear. We created a F508del ferret model to test whether ivacaftor/lumacaftor (VX-770/VX-809) therapy can rescue in utero and postnatal pathologies associated with CF. Using primary intestinal organoids and air-liquid interface cultures of airway epithelia, we demonstrate that the F508del mutation in ferret CFTR results in a severe folding and trafficking defect, which can be partially restored by treatment with CFTR modulators. In utero treatment of pregnant jills with ivacaftor/lumacaftor prevented meconium ileus at birth in F508del kits and sustained postnatal treatment of CF offspring improved survival and partially protected from pancreatic insufficiency. Withdrawal of ivacaftor/lumacaftor treatment from juvenile CF ferrets reestablished pancreatic and lung diseases, with altered pulmonary mechanics. These findings suggest that in utero intervention with a combination of CFTR modulators may provide therapeutic benefits to individuals with F508del. This CFTR-F508del ferret model may be useful for testing therapies using clinically translatable endpoints.
Asunto(s)
Aminofenoles , Aminopiridinas , Benzodioxoles , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Hurones , Quinolonas , Animales , Femenino , Embarazo , Aminofenoles/uso terapéutico , Aminofenoles/farmacología , Aminopiridinas/farmacología , Aminopiridinas/uso terapéutico , Benzodioxoles/uso terapéutico , Benzodioxoles/farmacología , Agonistas de los Canales de Cloruro/uso terapéutico , Agonistas de los Canales de Cloruro/farmacología , Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Modelos Animales de Enfermedad , Combinación de Medicamentos , Mutación , Quinolonas/farmacología , Quinolonas/uso terapéuticoRESUMEN
Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.
Asunto(s)
Fibrosis Quística , Modelos Animales de Enfermedad , Hurones , Pulmón , Transgenes , Animales , Humanos , Animales Modificados Genéticamente , Linaje de la Célula , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Hurones/genética , Hurones/fisiología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Pulmón/citología , Pulmón/metabolismo , Pulmón/patología , Tráquea/citología , Transgenes/genéticaRESUMEN
Alpha-1 antitrypsin deficiency (AATD) is the most common genetic cause and risk factor for chronic obstructive pulmonary disease, but the field lacks a large-animal model that allows for longitudinal assessment of pulmonary function. We hypothesized that ferrets would model human AATD-related lung and hepatic disease. AAT-knockout (AAT-KO) and PiZZ (E342K, the most common mutation in humans) ferrets were generated and compared with matched controls using custom-designed flexiVent modules to perform pulmonary function tests, quantitative computed tomography (QCT), bronchoalveolar lavage (BAL) proteomics, and alveolar morphometry. Complete loss of AAT (AAT-KO) led to increased pulmonary compliance and expiratory airflow limitation, consistent with obstructive lung disease. QCT and morphometry confirmed emphysema and airspace enlargement, respectively. Pathway analysis of BAL proteomics data revealed inflammatory lung disease and impaired cellular migration. The PiZ mutation resulted in altered AAT protein folding in the liver, hepatic injury, and reduced plasma concentrations of AAT, and PiZZ ferrets developed obstructive lung disease. In summary, AAT-KO and PiZZ ferrets model the progressive obstructive pulmonary disease seen in AAT-deficient patients and may serve as a platform for preclinical testing of therapeutics including gene therapy.
Asunto(s)
Enfermedad Pulmonar Obstructiva Crónica , Enfisema Pulmonar , Deficiencia de alfa 1-Antitripsina , Animales , Hurones , Humanos , Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/terapiaRESUMEN
Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.
Asunto(s)
Fibrosis Quística/tratamiento farmacológico , Glucosamina/análogos & derivados , Mucina 5B/metabolismo , Depuración Mucociliar/efectos de los fármacos , Moco/efectos de los fármacos , Polímeros/farmacología , Animales , Fibrosis Quística/genética , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Modelos Animales de Enfermedad , Hurones , Glucosamina/farmacología , Glucosamina/uso terapéutico , Humanos , Ratones , Ratones Endogámicos CFTR , Mucina 5B/química , Moco/metabolismo , Polímeros/uso terapéutico , Estructura Cuaternaria de Proteína/efectos de los fármacos , Ratas , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/patología , Viscosidad/efectos de los fármacosRESUMEN
Cystic fibrosis (CF) is a multiorgan disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). In patients with CF, abnormalities initiate in several organs before birth. However, the long-term impact of these in utero pathologies on disease pathophysiology is unclear. To address this issue, we generated ferrets harboring a VX-770 (ivacaftor)-responsive CFTR G551D mutation. In utero VX-770 administration provided partial protection from developmental pathologies in the pancreas, intestine, and male reproductive tract. Homozygous CFTR G551D/G551D animals showed the greatest VX-770-mediated protection from these pathologies. Sustained postnatal VX-770 administration led to improved pancreatic exocrine function, glucose tolerance, growth and survival, and to reduced mucus accumulation and bacterial infections in the lung. VX-770 withdrawal at any age reestablished disease, with the most rapid onset of morbidity occurring when withdrawal was initiated during the first 2 weeks after birth. The results suggest that CFTR is important for establishing organ function early in life. Moreover, this ferret model provides proof of concept for in utero pharmacologic correction of genetic disease and offers opportunities for understanding CF pathogenesis and improving treatment.
Asunto(s)
Aminofenoles/administración & dosificación , Agonistas de los Canales de Cloruro/administración & dosificación , Fibrosis Quística/tratamiento farmacológico , Quinolonas/administración & dosificación , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Glucemia/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Hurones , Técnicas de Sustitución del Gen , Genitales Masculinos/anomalías , Genitales Masculinos/efectos de los fármacos , Edad Gestacional , Humanos , Masculino , Mutación , Páncreas Exocrino/efectos de los fármacos , Páncreas Exocrino/patología , Páncreas Exocrino/fisiopatología , Embarazo , Infecciones del Sistema Respiratorio/etiología , Infecciones del Sistema Respiratorio/prevención & control , Investigación Biomédica TraslacionalRESUMEN
RATIONALE: Classical interpretation of cystic fibrosis (CF) lung disease pathogenesis suggests that infection initiates disease progression, leading to an exuberant inflammatory response, excessive mucus, and ultimately bronchiectasis. Although symptomatic antibiotic treatment controls lung infections early in disease, lifelong bacterial residence typically ensues. Processes that control the establishment of persistent bacteria in the CF lung, and the contribution of noninfectious components to disease pathogenesis, are poorly understood. OBJECTIVES: To evaluate whether continuous antibiotic therapy protects the CF lung from disease using a ferret model that rapidly acquires lethal bacterial lung infections in the absence of antibiotics. METHODS: CFTR (cystic fibrosis transmembrane conductance regulator)-knockout ferrets were treated with three antibiotics from birth to several years of age and lung disease was followed by quantitative computed tomography, BAL, and histopathology. Lung disease was compared with CFTR-knockout ferrets treated symptomatically with antibiotics. MEASUREMENTS AND MAIN RESULTS: Bronchiectasis was quantified from computed tomography images. BAL was evaluated for cellular differential and features of inflammatory cellular activation, bacteria, fungi, and quantitative proteomics. Semiquantitative histopathology was compared across experimental groups. We demonstrate that lifelong antibiotics can protect the CF ferret lung from infections for several years. Surprisingly, CF animals still developed hallmarks of structural bronchiectasis, neutrophil-mediated inflammation, and mucus accumulation, despite the lack of infection. Quantitative proteomics of BAL from CF and non-CF pairs demonstrated a mucoinflammatory signature in the CF lung dominated by Muc5B and neutrophil chemoattractants and products. CONCLUSIONS: These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Infecciones/microbiología , Inflamación/microbiología , Enfermedades Pulmonares/microbiología , Pulmón/microbiología , Pulmón/fisiopatología , Infecciones del Sistema Respiratorio/microbiología , Animales , Modelos Animales de Enfermedad , Hurones/microbiología , Infecciones/fisiopatología , Inflamación/fisiopatología , Enfermedades Pulmonares/fisiopatología , Infecciones del Sistema Respiratorio/fisiopatologíaRESUMEN
The cystic fibrosis (CF) field is the beneficiary of five species of animal models that lack functional cystic fibrosis transmembrane conductance regulator (CFTR) channel. These models are rapidly informing mechanisms of disease pathogenesis and CFTR function regardless of how faithfully a given organ reproduces the human CF phenotype. New approaches of genetic engineering with RNA-guided nucleases are rapidly expanding both the potential types of models available and the approaches to correct the CFTR defect. The application of new CRISPR/Cas9 genome editing techniques are similarly increasing capabilities for in vitro modeling of CFTR functions in cell lines and primary cells using air-liquid interface cultures and organoids. Gene editing of CFTR mutations in somatic stem cells and induced pluripotent stem cells is also transforming gene therapy approaches for CF. This short review evaluates several areas that are key to building animal and cell systems capable of modeling CF disease and testing potential treatments.