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RATIONALE AND OBJECTIVE: Cystic fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators offer significant improvements, but approximately 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimized for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial. METHODS: Air-liquid interface cultures of primary human bronchial epithelial cells (HBEC) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalized human lung epithelial cell line mimicking Class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay (PLA) for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies. RESULTS: rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a Class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence ≤10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalization assay. CONCLUSIONS: The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.
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PURPOSE OF REVIEW: Advances in cystic fibrosis (CF) therapies over the past decade pivotally changed the morbidity and mortality of CF with the advent of cystic fibrosis transmembrane conductance regulator (CFTR) modulators that rescue dysfunctional CFTR protein in individuals with eligible genotypes. However, a significant proportion of the CF population is in need of alternative treatment strategies to address CFTR variants that are ineligible for therapeutic protein correction and/or potentiation. Current drug development efforts of nucleic-acid based therapies (i.e., DNA and RNA based therapies) in CF are informed by historic challenges of CF gene therapy trials, recent FDA guidance informed by non-CF gene therapy trials, and advances in therapeutic applications related to severe acute respiratory syndrome coronavirus 2 vaccine development. These historic and timely developments are of significant relevance for advancing genetic therapies in CF. RECENT FINDINGS: This article reviews the main themes of semi-permanent genetic therapy strategies covering recent literature focused on: adenovirus and adeno-associated virus vector delivery, advances in lentivirus vector use and safety considerations, mRNA delivery and antisense oligonucleotide drug development. SUMMARY: Currently, drug development and clinical trials for genetic therapies in CF are rapidly progressing. This review aims to increase the foundational knowledge of CF genetic therapies.
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COVID-19 , Fibrose Cística , Humanos , Fibrose Cística/genética , Fibrose Cística/terapia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , COVID-19/terapia , Terapia Genética , Genótipo , MutaçãoRESUMO
BACKGROUND: Impaired alveolar fluid clearance, determined in part by alveolar sodium transport, is associated with acute respiratory distress syndrome (ARDS). Nasal sodium transport may reflect alveolar transport. The primary objective of this prospective, observational study was to determine if reduced nasal sodium transport, as measured by nasal potential difference (NPD), was predictive of the development of and outcome from ARDS. METHODS: NPD was measured in 15 healthy controls and in 88 patients: 40 mechanically ventilated patients defined as 'at-risk' for ARDS, 61 mechanically ventilated patients with ARDS (13 who were previously included in the 'at-risk' group) and 8 ARDS survivors on the ward. RESULTS: In at-risk subjects, maximum NPD (mNPD) was greater in those who developed ARDS (difference -8.4 mV; 95% CI -13.8 to -3.7; p=0.005) and increased mNPD predicted the development of ARDS before its onset (area under the curve (AUC) 0.75; 95% CI 0.59 to 0.89). In the ARDS group, mNPD was not significantly different for survivors and non-survivors (p=0.076), and mNPD was a modest predictor of death (AUC 0.60; 95% CI 0.45 to 0.75). mNPD was greater in subjects with ARDS (-30.8 mV) than in at-risk subjects (-24.2 mV) and controls (-19.9 mV) (p<0.001). NPD values were not significantly different for survivors and controls (p=0.18). CONCLUSIONS: Increased NPD predicts the development of ARDS in at-risk subjects but does not predict mortality. NPD increases before ARDS develops, is greater during ARDS, but is not significantly different for controls and survivors. These results may reflect the upregulated sodium transport necessary for alveolar fluid clearance in ARDS. NPD may be useful as a biomarker of endogenous mechanisms to stimulate sodium transport. Larger studies are now needed to confirm these associations and predictive performance.
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Síndrome do Desconforto Respiratório , Área Sob a Curva , Humanos , Estudos Prospectivos , Síndrome do Desconforto Respiratório/etiologia , Fatores de RiscoRESUMO
We have shown that a lentiviral vector (rSIV.F/HN) pseudotyped with the F and HN proteins from Sendai virus generates high levels of intracellular proteins after lung transduction. Here, we evaluate the use of rSIV.F/HN for production of secreted proteins. We assessed whether rSIV.F/HN transduction of the lung generates therapeutically relevant levels of secreted proteins in the lung and systemic circulation using human α1-anti-trypsin (hAAT) and factor VIII (hFVIII) as exemplars. Sedated mice were transduced with rSIV.F/HN carrying either the secreted reporter gene Gaussia luciferase or the hAAT or hFVIII cDNAs by nasal sniffing. rSIV.F/HN-hAAT transduction lead to therapeutically relevant hAAT levels (70 µg/ml) in epithelial lining fluid, with stable expression persisting for at least 19 months from a single application. Secreted proteins produced in the lung were released into the circulation and stable expression was detectable in blood. The levels of hFVIII in murine blood approached therapeutically relevant targets. rSIV.F/HN was also able to produce secreted hAAT and hFVIII in transduced human primary airway cells. rSIV.F/HN transduction of the murine lungs leads to long-lasting and therapeutically relevant levels of secreted proteins in the lung and systemic circulation. These data broaden the use of this vector platform for a large range of disease indications.
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Proteína HN/metabolismo , Transfecção/métodos , Proteínas Virais de Fusão/metabolismo , Animais , DNA Complementar/metabolismo , Fator VIII , Técnicas de Transferência de Genes , Genes Reporter , Terapia Genética , Vetores Genéticos , Humanos , Infecções por Lentivirus , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/fisiologia , Camundongos , Sistemas de Translocação de Proteínas/genética , Vírus Sendai/metabolismo , Transdução Genética/métodosRESUMO
We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air-liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and 'benchmarked' against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017.
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Fibrose Cística/genética , Fibrose Cística/terapia , Terapia Genética/métodos , Lentivirus/genética , Animais , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Camundongos , Fator 1 de Elongação de Peptídeos , Regiões Promotoras GenéticasRESUMO
As antibiotic resistance increases, there is a need for new therapies to treat infection, particularly in cystic fibrosis (CF), where Pseudomonas aeruginosa is a ubiquitous pathogen associated with increased morbidity and mortality. Bacteriophages are an attractive alternative treatment, as they are specific to the target bacteria and have no documented side effects. The efficacy of phage cocktails was established in vitro. Two P. aeruginosa strains were taken forward into an acute murine infection model with bacteriophage administered either prophylactically, simultaneously, or postinfection. The infective burden and inflammation in bronchoalveolar lavage fluid (BALF) were assessed at various times. With low infective doses, both control mice and those undergoing simultaneous phage treatment cleared P. aeruginosa infection at 48 h, but there were fewer neutrophils in BALF of phage-treated mice (median, 73.2 × 10(4)/ml [range, 35.2 to 102.1 × 10(4)/ml] versus 174 × 10(4)/ml [112.1 to 266.8 × 10(4)/ml], P < 0.01 for the clinical strain; median, 122.1 × 10(4)/ml [105.4 to 187.4 × 10(4)/ml] versus 206 × 10(4)/ml [160.1 to 331.6 × 10(4)/ml], P < 0.01 for PAO1). With higher infective doses of PAO1, all phage-treated mice cleared P. aeruginosa infection at 24 h, whereas infection persisted in all control mice (median, 1,305 CFU/ml [range, 190 to 4,700 CFU/ml], P < 0.01). Bacteriophage also reduced CFU/ml in BALF when administered postinfection (24 h) and both CFU/ml and inflammatory cells in BALF when administered prophylactically. A reduction in soluble inflammatory cytokine levels in BALF was also demonstrated under different conditions. Bacteriophages are efficacious in reducing both the bacterial load and inflammation in a murine model of P. aeruginosa lung infection. This study provides proof of concept for future clinical trials in patients with CF.
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Carga Bacteriana/efeitos dos fármacos , Bacteriófagos/crescimento & desenvolvimento , Pulmão/microbiologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/virologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/microbiologia , Modelos Animais de Doenças , Feminino , Inflamação/tratamento farmacológico , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologiaRESUMO
Since cloning of the CFTR gene more than 20 years ago a large number of pre-clinical and clinical CF gene therapy studies have been performed and a vast amount of information and know-how has been generated. Here, we will review key studies with a particular emphasis on clinical findings. We have learnt that the lung is a more difficult target than originally anticipated, and we describe the strength and weaknesses of the most commonly used airway gene transfer agents (GTAs). In our view, one of the most significant developments in recent years is the generation of lentiviral vectors, which efficiently transduce lung tissue. However, focused and co-ordinated efforts assessing lentiviral vector safety and scaling up of production will be required to move this vector into clinical lung gene therapy studies.
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Fibrose Cística/terapia , Terapia Genética/métodos , Vetores Genéticos , Lentivirus/genética , Pulmão , Animais , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Técnicas de Transferência de Genes , Humanos , Pulmão/patologia , Pulmão/virologia , CamundongosRESUMO
We have previously reported cyanide at concentrations of up to 150 µM in the sputum of cystic fibrosis patients infected with Pseudomonas aeruginosa and a negative correlation with lung function. Our aim was to investigate possible mechanisms for this association, focusing on the effect of pathophysiologically relevant cyanide levels on human respiratory cell function. Ciliary beat frequency measurements were performed on nasal brushings and nasal air-liquid interface (ALI) cultures obtained from healthy volunteers and cystic fibrosis patients. Potassium cyanide decreased ciliary beat frequency in healthy nasal brushings (n = 6) after 60 min (150 µM: 47% fall, p<0.0012; 75 µM: 32% fall, p<0.0001). Samples from cystic fibrosis patients (n = 3) showed similar results (150 µM: 55% fall, p = 0.001). Ciliary beat frequency inhibition was not due to loss of cell viability and was reversible. The inhibitory mechanism was independent of ATP levels. KCN also significantly inhibited ciliary beat frequency in ALI cultures, albeit to a lesser extent. Ciliary beat frequency measurements on ALI cultures treated with culture supernatants from P. aeruginosa mutants defective in virulence factor production implicated cyanide as a key component inhibiting the ciliary beat frequency. If cyanide production similarly impairs mucocilliary clearance in vivo, it could explain the link with increased disease severity observed in cystic fibrosis patients with detectable cyanide in their airway.
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Cílios/metabolismo , Cianetos/química , Fibrose Cística/metabolismo , Mucosa Nasal/metabolismo , Escarro/metabolismo , Trifosfato de Adenosina/química , Sobrevivência Celular , Células Cultivadas/efeitos dos fármacos , Fibrose Cística/microbiologia , Voluntários Saudáveis , Humanos , Muco/fisiologia , Mucosa Nasal/microbiologia , Cianeto de Potássio/química , Prognóstico , Infecções por Pseudomonas/fisiopatologia , Pseudomonas aeruginosa , Testes de Função Respiratória , Sistema Respiratório/fisiopatologiaRESUMO
RATIONALE: Lung clearance index (LCI) is a more sensitive measure of lung function than spirometry in cystic fibrosis (CF) and correlates well with abnormalities in high-resolution computed tomography (HRCT) scanning. We hypothesized LCI would be equally sensitive to lung disease in primary ciliary dyskinesia (PCD). OBJECTIVES: To test the relationships between LCI, spirometry, and HRCT in PCD and to compare them to the established relationships in CF. METHODS: Cross-sectional study of 127 patients with CF and 33 patients with PCD, all of whom had spirometry and LCI, of which a subset of 21 of each had HRCT performed. HRCT was scored for individual features and these features compared with physiological parameters. MEASUREMENTS AND MAIN RESULTS: Unlike in CF, and contrary to our hypothesis, there was no correlation between spirometry and LCI in PCD and no correlation between HRCT features and LCI or spirometry in PCD. CONCLUSIONS: We show for the first time that HRCT, spirometry, and LCI have different relationships in different airway diseases and that LCI does not appear to be a sensitive test of airway disease in advanced PCD. We hypothesize that this results from dissimilarities between the components of large and small airway disease in CF and PCD. These differences may in part lead to the different prognosis in these two neutrophilic airway diseases.
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Síndrome de Kartagener/fisiopatologia , Pulmão/fisiopatologia , Testes de Função Respiratória/métodos , Estudos Transversais , Fibrose Cística/diagnóstico por imagem , Fibrose Cística/fisiopatologia , Volume Expiratório Forçado , Humanos , Síndrome de Kartagener/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Sensibilidade e Especificidade , Espirometria , Tomografia Computadorizada por Raios XAssuntos
Antibacterianos/imunologia , Antibacterianos/uso terapêutico , Fibrose Cística/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/efeitos dos fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Fibrose Cística/complicações , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Infecções por Pseudomonas/etiologia , Adulto JovemRESUMO
The UK Cystic Fibrosis Gene Therapy Consortium has been working towards clinical gene therapy for patients with cystic fibrosis for several years. We have recently embarked on a large, multi-dose clinical trial of a non-viral, liposome-based formulation powered for the first time to detect clinical benefit. The article describes the details of the protocol.
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Regulador de Condutância Transmembrana em Fibrose Cística/uso terapêutico , Fibrose Cística/terapia , Terapia Genética/métodos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Método Duplo-Cego , Seguimentos , Humanos , Estudos Prospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Clinical trials in cystic fibrosis (CF) have been hindered by the paucity of well characterised and clinically relevant outcome measures. AIM: To evaluate a range of conventional and novel biomarkers of CF lung disease in a multicentre setting as a contributing study in selecting outcome assays for a clinical trial of CFTR gene therapy. METHODS: A multicentre observational study of adult and paediatric patients with CF (>10 years) treated for a physician-defined exacerbation of CF pulmonary symptoms. Measurements were performed at commencement and immediately after a course of intravenous antibiotics. Disease activity was assessed using 46 assays across five key domains: symptoms, lung physiology, structural changes on CT, pulmonary and systemic inflammatory markers. RESULTS: Statistically significant improvements were seen in forced expiratory volume in 1 s (p<0.001, n=32), lung clearance index (p<0.01, n=32), symptoms (p<0.0001, n=37), CT scores for airway wall thickness (p<0.01, n=31), air trapping (p<0.01, n=30) and large mucus plugs (p=0.0001, n=31), serum C-reactive protein (p<0.0001, n=34), serum interleukin-6 (p<0.0001, n=33) and serum calprotectin (p<0.0001, n=31). DISCUSSION: We identify the key biomarkers of inflammation, imaging and physiology that alter alongside symptomatic improvement following treatment of an acute CF exacerbation. These data, in parallel with our study of biomarkers in patients with stable CF, provide important guidance in choosing optimal biomarkers for novel therapies. Further, they highlight that such acute therapy predominantly improves large airway parameters and systemic inflammation, but has less effect on airway inflammation.
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Antibacterianos/administração & dosagem , Fibrose Cística/tratamento farmacológico , Volume Expiratório Forçado/fisiologia , Pneumopatias/tratamento farmacológico , Pulmão/fisiopatologia , Tomografia Computadorizada por Raios X , Adolescente , Adulto , Antibacterianos/uso terapêutico , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Criança , Fibrose Cística/diagnóstico , Fibrose Cística/fisiopatologia , Feminino , Humanos , Injeções Intravenosas , Interleucina-6/sangue , Complexo Antígeno L1 Leucocitário/sangue , Pulmão/diagnóstico por imagem , Pneumopatias/diagnóstico , Pneumopatias/fisiopatologia , Masculino , Recidiva , Resultado do Tratamento , Adulto JovemRESUMO
Several studies suggest that sex may affect cystic fibrosis (CF) disease severity, with females with CF being more severely affected. In this context, it has been suggested that sex hormones may influence the CF phenotype. A large proportion of females with CF regularly use oral contraceptives (OCs), but the effect of their use on disease severity is unclear. Here, we retrospectively assessed the effects of OCs on clinical outcomes in females with CF. Data from 681 females were available, of whom 42% had taken OCs for varying periods of time. We first performed an inter-patient analysis comparing annual change in % predicted forced expiratory volume in 1 s, body mass index and total days of intravenous antibiotic use over a 5-yr study period in 57 females exposed to and 57 females not exposed to OCs. There were no differences between the two groups. We next performed an intra-patient analysis of the same outcomes over a 3-yr period of OC exposure and a 3-yr period of no OC exposure in the same patient (exposure followed by non-exposure, n=27; non-exposure followed by exposure, n=23), but again did not detect any differences in any of the clinical outcomes. Our data suggests that the use of OCs does not affect CF disease severity.
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Anticoncepcionais Orais/efeitos adversos , Fibrose Cística , Adolescente , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Índice de Gravidade de Doença , Adulto JovemRESUMO
RATIONALE: Ongoing efforts to improve pulmonary gene transfer thereby enabling gene therapy for the treatment of lung diseases, such as cystic fibrosis (CF), has led to the assessment of a lentiviral vector (simian immunodeficiency virus [SIV]) pseudotyped with the Sendai virus envelope proteins F and HN. OBJECTIVES: To place this vector onto a translational pathway to the clinic by addressing some key milestones that have to be achieved. METHODS: F/HN-SIV transduction efficiency, duration of expression, and toxicity were assessed in mice. In addition, F/HN-SIV was assessed in differentiated human air-liquid interface cultures, primary human nasal epithelial cells, and human and sheep lung slices. MEASUREMENTS AND MAIN RESULTS: A single dose produces lung expression for the lifetime of the mouse (~2 yr). Only brief contact time is needed to achieve transduction. Repeated daily administration leads to a dose-related increase in gene expression. Repeated monthly administration to mouse lower airways is feasible without loss of gene expression. There is no evidence of chronic toxicity during a 2-year study period. F/HN-SIV leads to persistent gene expression in human differentiated airway cultures and human lung slices and transduces freshly obtained primary human airway epithelial cells. CONCLUSIONS: The data support F/HN-pseudotyped SIV as a promising vector for pulmonary gene therapy for several diseases including CF. We are now undertaking the necessary refinements to progress this vector into clinical trials.
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Fibrose Cística/genética , Terapia Genética/métodos , Vetores Genéticos , Lentivirus/genética , Análise de Variância , Animais , Fibrose Cística/terapia , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Imunodeficiência SímiaRESUMO
Bacteriophages (phages) are antimicrobials with resurgent interest that are being investigated for the treatment of antibiotic refractory infection, including for Pseudomonas aeruginosa (Pa) lung infection in cystic fibrosis (CF). In vitro work supports the use of this therapy in planktonic and biofilm culture models; however, consistent data are lacking for efficacy across different clinical Pa strains, culture models, and in combination with antibiotics in clinical use. We first examined the efficacy of a 4-phage cocktail as an adjunct to our CF centre's first-line systemic combination antibiotic therapy (ceftazidime + tobramycin) for 16 different clinical Pa strains and then determined subinhibitory interactions for a subset of these strains with each antibiotic in planktonic and biofilm culture. When a 4-phage cocktail (4 × 108 PFU/mL) was added to a ceftazidime-tobramycin combination (ceftazidime 16 mg/mL + tobramycin 8 mg/mL), we observed a 1.7-fold and 1.3-fold reduction in biofilm biomass and cell viability, respectively. The four most antibiotic resistant strains in biofilm were very susceptible to phage treatment. When subinhibitory concentrations of antibiotics and phages were investigated, we observed additivity/synergy as well as antagonism/inhibition of effect that varied across the clinical strains and culture model. In general, more additivity was seen with the phage-ceftazidime combination than with phage-tobramycin, particularly in biofilm culture, where no instances of additivity were seen when phages were combined with tobramycin. The fact that different bacterial strains were susceptible to phage treatment when compared to standard antibiotics is promising and these results may be relevant to ongoing clinical trials exploring the use of phages, in particular in the selection of subjects for clinical trials.
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BACKGROUND: Handheld spirometry allows monitoring of lung function at home, of particular importance during the COVID-19 pandemic. Pediatric studies are unclear on whether values are interchangeable with traditional, clinic-based spirometry. We aimed to assess differences between contemporaneous, home (unsupervised) and clinic (supervised) spirometry and the variability of the former. The accuracy of the commercially available spirometer used in the study was also tested. METHODS: Data from participants in the Clinical Monitoring and Biomarkers to stratify severity and predict outcomes in children with cystic fibrosisc (CLIMB-CF) Study aged ≥ 6 years who had paired (±1 day) clinic and home forced expiratory volume in 1 s (FEV1 ) readings were analyzed. Variability during clinical stability over 6-months was assessed. Four devices from Vitalograph were tested using 1 and 3 L calibration syringes. RESULTS: Sixty-seven participants (median [interquartile range] age 10.7 [7.6-13.9] years) provided home and clinic FEV1 data pairs. The mean (SD) FEV1 % bias was 6.5% [±8.2%]) with wide limits of agreement (-9.6% to +22.7%); 76.2% of participants recorded lower results at home. Coefficient of variation of home FEV1 % during stable periods was 9.9%. Data from the testing of the handheld device used in CLIMB-CF showed a potential underread. CONCLUSION: In children and adolescents, home spirometry using hand-held equipment cannot be used interchangeably with clinic spirometry. Home spirometry is moderately variable during clinical stability. New handheld devices underread, particularly at lower volumes of potential clinical significance for smaller patients; this suggests that supervision does not account fully for the discrepancy. Opportunities should be taken to obtain dual device measurements in clinic, so that trend data from home can be utilized more accurately.
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COVID-19 , Fibrose Cística , Adolescente , Humanos , Criança , Fibrose Cística/diagnóstico , Pandemias , COVID-19/diagnóstico , Espirometria , Volume Expiratório ForçadoRESUMO
BACKGROUND: Studies in cystic fibrosis (CF) generally focus on inflammation present in the airway lumen. Little is known about inflammation occurring in the airway wall, the site ultimately destroyed in end-stage disease. OBJECTIVE: To test the hypothesis that inflammatory patterns in the lumen do not reflect those in the airway wall of children with CF. METHODS: Bronchoalveolar lavage (BAL) fluid and endobronchial biopsies were obtained from 46 children with CF and 16 disease-free controls. BAL cell differential was assessed using May-Gruenwald-stained cytospins. Area profile counts of bronchial tissue immunopositive inflammatory cells were determined. RESULTS: BAL fluid from children with CF had a predominance of neutrophils compared with controls (median 810×10(3)/ml vs 1×10(3)/ml, p<0.0001). In contrast, subepithelial bronchial tissue from children with CF was characterised by a predominance of lymphocytes (median 961 vs 717 cells/mm(2), p=0.014), of which 82% were (CD3) T lymphocytes. In chest exacerbations, BAL fluid from children with CF had more inflammatory cells of all types compared with those with stable disease whereas, in biopsies, only the numbers of lymphocytes and macrophages, but not of neutrophils, were higher. A positive culture of Pseudomonas aeruginosa was associated with higher numbers of T lymphocytes in subepithelial bronchial tissue (median 1174 vs 714 cells/mm(2), p=0.029), but no changes were seen in BAL fluid. Cell counts in BAL fluid and biopsies were positively correlated with age but were unrelated to each other. CONCLUSION: The inflammatory response in the CF airway is compartmentalised. In contrast to the neutrophil-dominated inflammation present in the airway lumen, the bronchial mucosa is characterised by the recruitment and accumulation of lymphocytes.
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Brônquios/patologia , Fibrose Cística/imunologia , Pneumonia/complicações , Adolescente , Fatores Etários , Remodelação das Vias Aéreas/fisiologia , Biópsia , Líquido da Lavagem Broncoalveolar/citologia , Criança , Pré-Escolar , Fibrose Cística/complicações , Fibrose Cística/patologia , Fibrose Cística/fisiopatologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Volume Expiratório Forçado/fisiologia , Humanos , Lactente , Subpopulações de Linfócitos/imunologia , Masculino , Infecções Oportunistas/complicações , Infecções Oportunistas/imunologia , Infecções Oportunistas/patologia , Infecções Oportunistas/fisiopatologia , Pneumonia/imunologia , Pneumonia/patologia , Pneumonia/fisiopatologia , Testes de Função Respiratória , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Capacidade Vital/fisiologiaRESUMO
BACKGROUND: The nuclear membrane of differentiated airway epithelial cells is a significant barrier for nonviral vectors. Trans-cyclohexane-1,2-diol (TCHD) is an amphipathic alcohol that has been shown to collapse nuclear pore cores and allow the uptake of macromolecules that would otherwise be too large for nuclear entry. Previous studies have shown that TCHD can increase lipid-mediated transfection in vitro. METHODS: We aimed to reproduce these in vitro studies using the cationic lipid GL67A, which we are currently assessing in cystic fibrosis trials and, more importantly, we assessed the effects of TCHD on transfection efficiency in differentiated airway epithelium ex vivo and in mouse lung in vivo using three different drug delivery protocols (nebulisation and bolus administration of TCHD to the mouse lung, as well as perfusion of TCHD to the nasal epithelium, which prolongs contact time between the airway epithelium and drug). RESULTS: TCHD (0.5-2%) dose-dependently increased Lipofectamine 2000 and GL67A-mediated transfection of 293T cells by up to 2 logs. Encouragingly, exposure to 8% TCHD (but not 0.5% or 2.0%) increased gene expression in fully differentiated human air liquid interface cultures by approximately 20-fold, although this was accompanied by significant cell damage. However, none of the TCHD treated mice in any of the three protocols had higher gene expression compared to no TCHD controls. CONCLUSIONS: Although TCHD significantly increases gene transfer in cell lines and differentiated airway epithelium ex vivo, this effect is lost in vivo and further highlights that promising in vitro findings often cannot be translated into in vivo applications.
Assuntos
Cicloexanos/farmacologia , Cicloexanóis/farmacologia , Técnicas de Transferência de Genes , Poro Nuclear/efeitos dos fármacos , Sistema Respiratório/efeitos dos fármacos , Animais , Células Cultivadas , Cicloexanos/administração & dosagem , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Epitélio/efeitos dos fármacos , Feminino , Terapia Genética , Vetores Genéticos , Humanos , Lipídeos/farmacologia , Pulmão/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/efeitos dos fármacos , TransfecçãoRESUMO
The prospect of gene therapy for inherited and acquired respiratory disease has energized the research community since the 1980s, with cystic fibrosis, as a monogenic disorder, driving early efforts to develop effective strategies. The fact that there are still no approved gene therapy products for the lung, despite many early phase clinical trials, illustrates the scale of the challenge: In the 1990s, first-generation non-viral and viral vector systems demonstrated proof-of-concept but low efficacy. Since then, there has been steady progress toward improved vectors with the capacity to overcome at least some of the formidable barriers presented by the lung. In addition, the inclusion of features such as codon optimization and promoters providing long-term expression have improved the expression characteristics of therapeutic transgenes. Early approaches were based on gene addition, where a new DNA copy of a gene is introduced to complement a genetic mutation: however, the advent of RNA-based products that can directly express a therapeutic protein or manipulate gene expression, together with the expanding range of tools for gene editing, has stimulated the development of alternative approaches. This review discusses the range of vector systems being evaluated for lung delivery; the variety of cargoes they deliver, including DNA, antisense oligonucleotides, messenger RNA (mRNA), small interfering RNA (siRNA), and peptide nucleic acids; and exemplifies progress in selected respiratory disease indications.