The behaviour of the cystic fibrosis respiratory epithelium and its response to multidose CFTR gene therapy

Abstract

Cystic fibrosis (CF) is a clinical syndrome resulting from inherited mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) protein, whose absent or reduced function results in abnormal epithelial ion transport and an abnormal transepithelial potential difference (PD), leading to downstream epithelial dysfunction and a pathognomonic clinical phenotype. Most treatments to date manage the disease sequelae of airway mucus and infection, but correction of the underlying defect for all patients is the ultimate aim. Gene therapy offers the potential as a universal treatment to ameliorate CFTR function, leading to clinical benefit.

This thesis will centre on the recently completed Multidose Trial (MDT) – the repeated application of non-viral CFTR gene therapy in patients with cystic fibrosis. The trial was undertaken during the entirely of this PhD, and recruited 136 patients (aged ≥12) with CF, and randomised to receive 12, monthly nebulised doses of a cationic-lipid (pGM169/GL67A), or placebo. The hypothesis of the study was that repeated administration of non-viral gene therapy would produce vector-specific epithelial CFTR, leading to demonstrable improvements in lung function and measureable de novo chloride ion transport in the upper (nasal) and lower (bronchial) airway. The study reports stability in lung function with gene therapy (n=62) at 48 weeks compared to a decline in the placebo group (n=54), concluding a significant treatment effect of a relative improvement of 3.7% (p=0.046) in percent-predicted forced expiratory volume in 1 second (FEV1) at follow-up; other clinical parameters further support a treatment benefit, however a reduction in the frequency of pulmonary exacerbations was not detected. A post hoc analysis identified a significant treatment benefit of 6.4% in patients with more severe airways disease (ppFEV1 50-70%), however failed to detect a treatment effect in patients with less severe disease (ppFEV1 70-90%). Individual patients demonstrated de novo chloride transport in the nasal and lower airway, as measured by epithelial potential difference, with a significant difference being measured in the lower airway in response to gene therapy (-4.4 mV, p=0.03); no difference was identified in the upper airway in response to active treatment.

The thesis will next explore the relationships of epithelial PD between the nasal and lower airway with measurements of lung function at baseline, and in response to treatment with gene therapy. At baseline, trends between sodium transport in the nasal epithelium and FEV1 and a lung clearance index (LCI) (p=0.01) were identified; no relationship was identified between chloride indices, or with any measurement from the lower airway. No relationship between the electrophysiology of the upper and lower airway epithelium was detected.

The author will present a novel method used to maintain blinding whilst performing nasal PD (NPD) measurements during the MDT, using a 2-operator technique. This technique is reported as not inferior to the standard NPD method and supports its validity of its use for future studies, but provides caution that this method may take longer to perform and that more data may be excluded owing to poor NPD trace quality.

The thesis concludes by describing two studies designed to further understand the performance and interpretation of PD measurements, and discussing the overall usefulness of airway PD as a clinical trial outcome. The first study investigates the amount of total chloride secretion (in healthy (non-CF) volunteers (n=18)) in the nasal epithelium by perfusing ‘standard nasal’ (with amiloride) and ‘lower airway’ (sans amiloride) solutions, reporting that more (approximately 50%) chloride is secreted in the presence of amiloride-containing solutions, and when the duration of perfusion was extended.

The final study aimed to define the minimum period for performing sequential NPDs in the same (CF) patient, reporting that 4/8 (50%) CF patients basal PD had returned after 30 min, and that basal PD had returned by 60 min in all patients, concluding that repeated measurements could be made over a short timeframe for clinical and research studies.