Stephen N. Smith

Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebo-controlled trial

BACKGROUND We and others have previously reported significant changes in chloride transport after cationic-lipid-mediated transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to the nasal epithelium of patients with cystic fibrosis. We studied the safety and efficacy of this gene transfer to the lungs and nose of patients with cystic fibrosis in a double-blind placebo-controlled trial. METHODS Eight patients with cystic fibrosis were randomly assigned DNA-lipid complex (active) by nebulisation into the lungs followed 1 week later by administration to the nose. Eight control patients followed the same protocol but with the lipid alone (placebo). Safety was assessed clinically, by radiography, by pulmonary function, by induced sputum, and by histological analysis. Efficacy was assessed by analysis of vector-specific CFTR DNA and mRNA, in-vivo potential difference, epifluorescence assay of chloride efflux, and bacterial adherence. FINDINGS Seven of the eight patients receiving the active complex reported mild influenza-like symptoms that resolved within 36 h. Six of eight patients in both the active and placebo groups reported mild airway symptoms over a period of 12 h following pulmonary administration. No specific treatment was required for either event. Pulmonary administration resulted in a significant (p<0.05) degree of correction of the chloride abnormality in the patients receiving active treatment but not in those on placebo when assessed by in-vivo potential difference and chloride efflux. Bacterial adherence was also reduced. We detected no alterations in the sodium transport abnormality. A similar pattern occurred following nasal administration. INTERPRETATION Cationic-lipid-mediated CFTR gene transfer can significantly influence the underlying chloride defect in the lungs of patients with cystic fibrosis.

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype phenotype correlations

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one‐step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with ‘null’ mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR‐related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement (‘null’) and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild‐type CFTR activity. The long‐term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype‐phenotype correlations.

Validation of Nasal Potential Difference Measurements in Gut-Corrected CF Knockout Mice

Attempts at correcting the nasal potential difference (PD) in cystic fibrosis (CF) mice have long been used in preclinical gene and small molecule therapy development. However, in general, CF mice suffer from intestinal disease, are runted, and have high mortality rates; they are therefore difficult to work with, especially if large numbers are required. Because of this, large-scale PD studies in CF mice have not been performed. Working with CF mice has become substantially easier after the generation of the gut-corrected CF-knockout mouse. Fatty acid-binding promoter (FABp)-mediated expression of CFTR in the gut, but not the airways, prevents the intestinal disease of the CF knockout mouse. This model has given us the unique opportunity to systematically study PDs in large numbers of CF mice. The nose, but not the lungs, of these animals mimic the bioelectric defect seen in humans. We have therefore assessed the bioelectrics of the respiratory epithelium comparing FABp-CF and wild-type mice. The large body of data gathered in CF and wild-type mice allowed us, for the first time, to establish power calculations that should inform sample sizes required in gene and small molecule therapy development. In addition, we address the important issues of intra-animal variability as well as intra- and inter-operator variability for scoring the traces, and the effect of age and sex on nasal PD in CF mice. These data should allow a more informed use of CF animals in future studies.

Effect of IBMX and alkaline phosphatase inhibitors on Cl− secretion in G551D cystic fibrosis mutant mice

Some cystic fibrosis transmembrane conductance regulator (CFTR) mutations, such as G551D, result in a correctly localized Cl- channel at the cell apical membrane, albeit with markedly reduced function. Patch-clamp studies have indicated that both phosphatase inhibitors and 3-isobutyl-1-methylxanthine (IBMX) can induce Cl- secretion through the G551D mutant protein. We have now assessed whether these agents can induce Cl- secretion in cftrG551D mutant mice. No induction of Cl-secretion was seen with the alkaline phosphatase inhibitors bromotetramisole or levamisole in either the respiratory or intestinal tracts of wild-type or cftrG551D mice. In contrast, in G551D intestinal tissues, IBMX was able to produce a small CFTR-related secretory response [means ± SE: jejunum, 1.8 ± 0.9 μA/cm2, n = 7; cecum, 3.7 ± 0.8 μA/cm2, n = 7; rectum (in vivo), 1.9 ± 0.9 mV, n = 5]. This was approximately one order of magnitude less than the wild-type response to this agent and, in the cecum, was significantly greater than that seen in null mice ( cftrUNC ). In the trachea, IBMX produced a transient Cl- secretory response (37.3 ± 14.7 μA/cm2, n = 6) of a magnitude similar to that seen in wild-type mice (33.7 ± 4.7 μA/cm2, n = 9). This response was also present in null mice and therefore is likely to be independent of CFTR. No effect of IBMX on Cl-secretion was seen in the nasal epithelium of cftrG551D mice. We conclude that IBMX is able to induce detectable levels of CFTR-related Cl- secretion in the intestinal tract but not the respiratory tract through the G551D mutant protein.

Limitations of the murine nose in the development of nonviral airway gene transfer.

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter. In a study of approximately 400 mice with CF, vector-specific CF transmembrane conductance regulator (CFTR) mRNA was detected in nasal epithelial cells of 82% of mice treated with a cytomegalovirus-plasmid (pCF1-CFTR), and 62% of mice treated with an ubiquitin C-plasmid. We then assessed whether CFTR gene transfer corrected a panel of CFTR-specific endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex vivo bacterial adherence. Importantly, even with the comparatively large number of animals assessed, the CFTR function studies were only powered to detect changes of more than 50% toward wild-type values. Within this limitation, no significant correction of the CF phenotype was detected. At the current levels of gene transfer efficiency achievable with nonviral vectors, the murine nose is of limited value as a stepping stone to human trials.