J. Alastair Innes

Sputum Proteomics in Inflammatory and Suppurative Respiratory Diseases

RATIONALE Markers of inflammatory activity are important for assessment and management of many respiratory diseases. Markers that are currently unrecognized may be more valuable than those presently believed to be useful. OBJECTIVES To identify potential biomarkers of suppurative and inflammatory lung disease in induced sputum samples. METHODS Induced sputum was collected from 20 healthy control subjects, 24 patients with asthma, 24 with chronic obstructive pulmonary disease, 28 with cystic fibrosis (CF), and 19 with bronchiectasis. Twelve patients with CF had sputum sampled before and after antibiotic therapy for an infective exacerbation. The fluid phase of induced sputum was analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy on three protein array surfaces. Some protein markers were selected for identification, and relevant ELISA assays sought. For 12 patients with CF, both SELDI-TOF and ELISA monitored changes in inflammatory responses during infective exacerbations. MEASUREMENTS AND MAIN RESULTS SELDI-TOF identified potential biomarkers that differentiated each of the disease groups from healthy control subjects: at a significance of P < 0.01, there were 105 for asthma, 113 for chronic obstructive pulmonary disease, 381 for CF, and 377 for bronchiectasis. Peaks selected for protein identification yielded calgranulin A, calgranulin B, calgranulin C, Clara cell secretory protein, lysosyme c, proline rich salivary peptide, cystatin s, and hemoglobin alpha. On treatment of an infective CF exacerbation, SELDI-TOF determined falls in levels of calgranulin A and calgranulin B that were mirrored by ELISA-measured falls in calprotectin (heterodimer of calgranulins A and B). CONCLUSIONS Proteomic screening of sputum yields potential biomarkers of inflammation. The early development of a clinically relevant assay from such data is demonstrated.

Changes in physiological, functional and structural markers of cystic fibrosis lung disease with treatment of a pulmonary exacerbation

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.

A randomised, double-blind, placebo-controlled phase IIB clinical trial of repeated application of gene therapy in patients with cystic fibrosis

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.

Sputum Trace Metals Are Biomarkers of Inflammatory and Suppurative Lung Disease

BACKGROUND Induced sputum cytology and protein biomarkers can be used to assess airways inflammation. Increases in sputum iron have been described in inflammatory lung disease. We hypothesized that other sputum metals may be affected by airways inflammation and investigated their potential value as biomarkers. METHODS Sputum was obtained from 20 healthy control subjects and from patients with inflammatory pulmonary diseases (23 with cystic fibrosis [CF], 16 with bronchiectasis, 17 with asthma, and 23 with COPD), and iron, zinc, manganese, and copper were measured. Fourteen patients with CF were also studied through an exacerbation cycle. RESULTS Sputum zinc and iron were elevated in CF and non-CF bronchiectasis vs controls (P < .001, zinc; P < .01 iron). Manganese was elevated in asthma (P < .01) and bronchiectasis (P < .05) vs controls. Copper was elevated in CF vs controls (P < .05). Zinc decreased (P < .01) following treatment of CF exacerbation. In subjects with CF zinc levels correlated with other biomarkers. CONCLUSIONS These results suggest a relationship of high concentrations of total zinc and iron with airways inflammation in CF and non-CF bronchiectasis, with longitudinal changes being observed in CF. Further work is required to elucidate potential inflammatory mechanisms related to these observations.

Assessment of F/HN-Pseudotyped Lentivirus as a Clinically Relevant Vector for Lung Gene Therapy

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.

Effects of cystic fibrosis lung disease on gas mixing indices derived from alveolar slope analysis.

S(cond) and S(acin) are derived from analysis of concentration-normalized phase III slopes (Sn(III)) of a multiple breath inert gas washout. Studies in healthy and COPD subjects suggest these reflect ventilation heterogeneity in conducting and acinar airway zones respectively, but similar studies in cystic fibrosis (CF) are lacking. S(cond), S(acin) and lung clearance index (LCI, a measure of overall gas mixing efficiency) were measured in 22 adults and 18 children with CF and 17 adult and 29 child controls. Plethysmography and gas transfer measurements were performed in adults, and spirometry in all subjects. S(cond) was elevated in almost all CF patients, including children with mild disease and normal LCI. However, S(cond) did not correlate with other measurements and appeared to reach a maximum; further increase in ventilation heterogeneity being restricted to S(acin). The nature and/or severity of CF lung disease may invalidate assumptions underlying the ability to separate phase III slope analysis of ventilation heterogeneity into proximal and peripheral components, and LCI may be a better indicator of gas mixing in this population.

Biomarkers for cystic fibrosis lung disease: application of SELDI-TOF mass spectrometry to BAL fluid.

BACKGROUND For cystic fibrosis (CF) patients there is a lack of good assays of disease activity and response to new therapeutic interventions, including gene therapy. Current measures of airways inflammation severity are insensitive or non-specific. METHODS Bronchoalveolar lavage fluid from 39 CF children and 38 respiratory disease controls was obtained at bronchoscopy and analysed by surface enhanced laser desorption ionisation time of flight (SELDI-TOF) mass spectrometry. Recognized proteins were assessed for CF disease specificity. Individual protein identification of specific peaks was performed. RESULTS 1277 proteins/peptides, >4 kDa, were detected using 12 different surfaces and binding conditions. 202 proteins/peptides were differentially expressed in the CF samples (p<0.001), 167 up-regulated and 35 down-regulated. The most discriminatory biomarker had a mass of 5.163 kDa. The most abundant, with a mass of 10.6 kDa, was identified as s100 A8 (calgranulin A). CONCLUSIONS The application of SELDI-TOF mass spectrometry allows evaluation of proteins in BAL fluid avoiding the limitations of only analysing predetermined proteins and potentially identifying proteins not previously appreciated as biomarkers. Its application to cystic fibrosis should enable appropriate evaluation of evolving illness, of gene therapy and other new therapies.

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis.

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.

A Phase I/IIa Safety and Efficacy Study of Nebulized Liposome-mediated Gene Therapy for Cystic Fibrosis Supports a Multidose Trial

To the Editor: The vast majority of treatments for cystic fibrosis (CF) target the downstream consequences of the disease and are incompletely effective. The success of the CF transmembrane conductance regulator (CFTR) potentiator ivacaftor has illustrated the clinical benefits arising from restoration of CFTR protein function (1). This agent is applicable as a monotherapy for a minority of patients with specific, rare mutations. CFTR gene therapy, a mutation-independent alternative, has demonstrated proof of principle for gene transfer in animal models and human trials, but only one study (using a viral vector) has unsuccessfully assessed whether clinical outcomes can be improved (2). In preparation for a phase IIb clinical trial of repeatedly administered, nonviral, liposome-mediated CFTR gene transfer assessing clinically meaningful outcomes (3), the UK CF Gene Therapy Consortium (www.cfgenetherapy.org.uk) undertook a single-application safety and dose-ranging study (NCT00789867). Some of the results of these studies have been previously reported in the form of abstracts (4, 5). The chosen plasmid DNA expresses CFTR under the control of the human cytomegalovirus enhancer/elongation factor 1α sequence (6), a modified EF1a promoter aiming for extended duration of expression (7), and was rendered CpG-free to minimize a host inflammatory response (6). The cationic lipid, GL67A, was chosen on the basis of extensive preclinical testing (8). After informed consent, adult patients with CF received a single nebulized +/− nasal dose of pGM169/GL67A. Reconstitution and preparation of pGM169/GL67A was undertaken on the day, and doses were delivered in sealed negative-pressure cubicles after pretreatment with inhaled salbutamol (albuterol). Preplanned adjunctive therapies including ibuprofen, prednisolone, or paracetamol were administered to some patients. The primary outcome of the clinical study was safety; assessment included examination, standard hematology/biochemistry, adverse events, spirometry, lung clearance index, chest computed tomography scan, gas transfer, bronchial biopsy histology, and immune markers. pGM169-specific DNA and mRNA were measured on nasal and lower airway brushings, with potential difference also measured bronchoscopically and nasally. For the latter, “responders” were defined as demonstrating chloride secretion 5 mV or more greater than their mean predose value, and greater than any of their predose responses. A total of 35 subjects (Tables 1 and ​and2)2) received a nebulized dose (5 ml, n = 8; 10 ml, n = 10; 20 ml, n = 17) via an AeroEclipse II (Trudell Medical International, London, ON, Canada) breath-actuated nebulizer (9). Three subjects undertook slow delivery (∼75 vs. 25 min for each 5 ml). Standard spray devices were used for nasal delivery (2 ml, n = 21). According to pre-/post-device weighing, a mean (SD) of 88.7% (2.9%) of expected nebulized dose and 94.5% (15.0%) of expected nasal dose was delivered. There were two serious adverse events: one occurred after the predosing bronchoscopy (swelling of the uvula related to intubation) and led to observation overnight in hospital, and the other was an episode of pancreatitis occurring around Day 10 after dosing (10 ml nebulized cohort). The subject was exocrine pancreatic sufficient and had likely experienced previous similar, but undiagnosed, episodes. Table 1. Baseline Demographics Table 2. Postdosing Responses Overall, in the trial, 94.3% of subjects experienced at least one adverse event, the majority of which were mild to moderate in severity and resolved spontaneously or with standard antipyretics. The most common occurred on the day of dosing and largely resolved within 24–48 hours (Tables 1 and ​and2):2): Typically, within the first few hours after dosing, a mild, self-limiting influenza-like systemic response was seen, most frequently in the 20-ml patients. This was not affected by slow delivery or coadministration of ibuprofen or prednisolone but was clearly dose-related and reduced by paracetamol. Symptoms of headache and/or tiredness were reported by 82, 70, and 13%, and raised serum inflammatory markers were recorded in 100, 60, and 63% of the 20-, 10-, and 5-ml groups respectively, with dose-related trends in maximal values. No patient dosed with 5 ml had a temperature higher than 38°C (Table 2). A relatively asymptomatic, dose-related, restrictive drop in spirometry was also observed, with no change in respiratory rate or oxygen saturation. No patient dosed with 5 ml showed a more than 20% relative fall in FEV1 (Table 2). The 20-ml group showed a small, significant (P < 0.05) mean (SD) drop in gas transfer (transfer factor for carbon monoxide corrected for alveolar volume and hemoglobin concentration) on Day 2 of 4.5% (6.0%), which returned to baseline values by Day 14. No changes were seen in the other cohorts. Two of the 20-ml patients had small areas of ground glass opacity reported on their Day 2 chest computed tomography scans, which resolved by Day 14. No significant changes were seen in endobronchial histology (20 ml; n = 10). Consistent with the proposed excretion route for lipids, small but significant serum creatinine rises within the normal range could be detected 8 hours after dosing in the 20- and 10-ml groups, but not the 5-ml cohort; there were no other biochemical changes. Bilirubin rose on Day 1 in all dosing groups, as with creatinine, remaining within the normal range, and normalized by Day 2. There was no evidence of immune responses based on double-stranded DNA antibodies or human CFTR-specific T cells. Lung clearance index, a sensitive marker of pulmonary dysfunction (10), was included as a safety assay. Fourteen 20-ml patients with paired predosing and 28-day postdosing values showed a small but significant increase (i.e., a deterioration; Figure 1A). In contrast, and unexpectedly, on post hoc analysis, 11 of 14 patients in the lower-dosing groups (5 and 10 ml) showed a small but significant improvement (Figure 1A). Figure 1. (A) Lung clearance index (LCI) increased (worsened) by a mean (SEM) of 0.75 (0.3) units in the 20-ml group (P = 0.03), whereas it decreased (improved) in the 5-/10-ml patients by 0.32 (0.1) (P = 0.04). The difference in ... With respect to bronchial samples, 10 patients (all 20 ml) had paired pre- and postdosing bronchoscopies. pGM169-specific DNA was detected in all bronchial brushing samples at levels ∼1000-fold higher than in the nasal samples. pGM169-specific mRNA was detected in 2 of 10 postdosing samples. Paired bronchoscopic potential difference measurements were interpretable for 8 of 10 patients. There was a trend toward an increase in chloride secretion (Figure 1B) but no changes in sodium-related parameters. With respect to nasal samples, pGM169-specific DNA was detected in all 15 brushing samples taken between Day 2 and Day 14 after dosing and in two of six samples at Day 28. pGM169-specific mRNA was detected in 3 of 21 postdosing samples, with all positive samples being observed at either Day 14 (n = 2) or Day 28 (n = 1). In keeping with previous published data, there were no changes in sodium parameters on nasal potential difference. In contrast, 6 of 16 subjects (37.5%) demonstrated a “response” in terms of chloride secretory capacity. Responses were seen most commonly in the zero chloride perfusion phase and at the 14-day point; they were of sustained duration in one subject (Figure 1C). These data were important in informing the design of the phase IIb trial. Thus, based on these findings, 5 ml was selected as the optimal dose, with paracetamol being used as an adjuvant to minimize the risk of unblinding. Although well-tolerated, the adverse effects of the 20-ml doses were considered prohibitive for use in a repeated administration trial. We consider that the efficient delivery of large volumes of viscous fluid into the airways led acutely to both the influenza-like and restrictive responses, analogous to those seen after bronchoalveolar lavage, and masking the effect of plasmid DNA CpG depletion. At lower volumes, the latter effect was “revealed,” allowing safe dosing of 5 ml. The unexpected improvement in lung clearance index after only one administration at the lower doses was intriguing; larger numbers and longer follow-up are needed to confirm or refute this finding. The variable responses both in molecular and CFTR functional terms underscore the technical challenges inherent in these assays and the limited sensitivity to low levels of gene expression (11). The clean safety profile and encouraging improvements in a sensitive measure of airway health lend support to progression to a phase IIb multidose trial designed to detect clinical improvements after prolonged administration.

The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep.

Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.