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S140 TOWARDS GENE THERAPY FOR CYSTIC FIBROSIS USING A LENTIVIRUS PSEUDOTYPED WITH SENDAI VIRUS ENVELOPES
1U. Griesenbach, 2K. Mitomo, 2M. Inoue, 1L. Somerton, 1C. Meng, 2T. Tabata, 2Y. Ueda, 3G. Frankel, 1R. Farley, 1C. Singh, 1M. Chan, 1F. Munkonge, 1A. Brum, 1S. Xenariou, 1S. Escudero-Garcia, 2M. Hasegawa, 1E. W. F. W. Alton. 1Department of Gene Therapy, Imperial College at the National Heart and Lung Institute and The UK Cysstic Fibrosis Gene Therapy Consortium, London, UK, 2DNAVEC Corporation, Tsukuba, Ibaraki, Japan, 3Division of Cell and Molecular Biology, Imperial College London, London, UK
Gene therapy for cystic fibrosis (CF) is making encouraging progress into clinical trials. However, further improvements in transduction efficiency are desired. To develop a novel gene transfer vector that is improved and truly effective for CF gene therapy, a simian immunodeficiency virus (SIV) was pseudotyped with envelope proteins from Sendai virus (SeV), which is known to transduce unconditioned airway epithelial cells efficiently from the apical side. This novel vector was evaluated in vivo and in vitro directed towards CF gene therapy. Here we show that (1) we can produce relevant titres of an SIV vector pseudotyped with SeV envelope proteins for in vivo use; (2) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF; (3) this can be achieved in a single formulation, and without the need for preconditioning; (4) expression can last for 15 months; (5) readministration is feasible; (6) the vector can transduce human air–liquid interface cultures; and (7) functional CFTR (cystic fibrosis transmembrane conductance regulator) chloride channels can be generated in vitro. Our data suggest that this lentiviral vector may provide a step change in airway transduction efficiency relevant to a clinical programme of gene therapy for CF.
Funding: This work was in part funded by the CF Trust.KM and UG contributed equally to this work.
S141 EVALUATION OF SAFETY AND GENE EXPRESSION WITH A SINGLE DOSE OF PGM169/GL67A ADMINISTERED TO THE NOSE AND LUNG OF INDIVIDUALS WITH CYSTIC FIBROSIS: THE UK CF GENE THERAPY CONSORTIUM “PILOT STUDY”
1J. C. Davies, 1G. Davies, 1N. Voase, 2S. Hyde, 3A. Innes, 4C. Boyd, 4D. Porteous, 1T. Higgins, 1U. Griesenbach, 2D. Gill, 1E. W. F. W. Alton. 1Imperial College, London, UK, 2University of Oxford, Oxford, UK, 3Western General Hospital, Edinburgh, UK, 4University of Edinburgh, Edinburgh, UK
The UK CF Gene Therapy Consortium is working towards a multidose gene therapy study, using the best currently available non-viral gene delivery complex, and whose end point will be to detect clinical benefit rather than proof-of-principle. Based on extensive preclinical testing our selected product is pGM169, a CpG-free human CFTR (cystic fibrosis transmembrane conductance regulator) plasmid with a CpG-free cytomegalovirus (CMV) enhancer and human elongation factor 1α (hCEFI) promoter complexed with GL67A (GL67, DOPE and DMP-PEG5000). We are currently undertaking a single dose study because of a requirement to confirm safety of this “first-in-man” product; however, study design has also been tailored to assess gene expression in vivo in cystic fibrosis (CF) lungs.
A single nebulised dose of 20 ml (53 mg of pGM169 and 286 mg of GL67A) is delivered by an Aeroeclipse II breath-actuated device; a nasal dose (10% of the nebulised volume) is administered on the same occasion using a standard nasal spray device. The latter allows assessment of gene expression without the sampling issues inherent in lower airway assessment, as well as anchoring to our previous clinical trials. Safety measures include physical examination, lung physiology (spirometry, pulse oximetry, lung clearance index), systemic and sputum inflammatory markers, renal and hepatic function and chest CT. We are also measuring antinuclear and antidouble-stranded DNA antibodies, and specific CFTR-related T cell responses. Measurements are made at intervals prior to dosing and during a 28-day follow-up period. Gene expression is assessed by (1) quantitative Taqman RT-PCR for transgene mRNA on nasal and bronchial brushings; (2) anti-CFTR immunohistochemistry; and (3) nasal and lower airway potential difference measurements. Given intersubject variability, paired measurements on individuals will be obtained; bronchoscopies are being performed prior to dosing and at two time points postdosing. Nasal potential difference is measured on serial visits.
To date, 3 patients in an initial non-bronchoscopic cohort have received a half dose of 10 ml and 3 have received the full 20 ml dose. The Data Safety Monitoring Board has granted permission to proceed further with 20 ml dosing, and available data will be presented on safety, tolerability and gene expression.
Funding: UK Cystic Fibrosis Trust.
S142 CERAMIDE IS INCREASED AND ASSOCIATED WITH NEUTROPHILIC INFLAMMATION IN THE LOWER AIRWAY EPITHELIUM OF PEOPLE WITH CYSTIC FIBROSIS
1M. Brodlie, 2M. C. McKean, 3G. E. Johnson, 1A. J. Fisher, 1P. A. Corris, 1J. L. Lordan, 1C. Ward. 1Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK, 2Paediatric Respiratory Unit, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK, 3Cardiopulmonary Transplantation Unit, Freeman Hospital, Newcastle upon Tyne, UK
Introduction and Objectives Cystic fibrosis (CF) results from alterations in the CF transmembrane conductance regulator (CFTR) gene, but the exact pathogenesis of lung disease remains poorly understood. Ceramide is an essential constituent of plasma membranes and regulates many cellular responses. It has recently been shown that CFTR-deficient mice accumulate ceramide in airway epithelial cells, resulting in inflammation and susceptibility to Pseudomonas aeruginosa—two key features of CF lung disease.1 Ceramide accumulation was also demonstrated in nasal epithelial cells from people with CF and qualitatively in a small number of sections of lung.1 The objective of this work was to evaluate ceramide levels quantitatively in the lower airway of people with CF compared with pulmonary hypertension (PH), emphysema and unused donors, and to investigate relationships between epithelial ceramide levels and markers of neutrophilic inflammation and P aeruginosa infection.
Methods Immunohistochemistry was performed on airways from explanted lungs (8 CF, emphysema and PH, respectively) and 8 donor lungs using ceramide, neutrophil elastase (NE) and myeloperoxidase (MPO) antibodies. Ceramide staining was evaluated in the lower airway epithelium and expressed as percentage area. The number of cells staining positive in the airway for NE and MPO/mm basement membrane was also evaluated.
Results Staining for ceramide was significantly increased in the lower airway epithelium of people with CF (median 14.11%) compared with PH (3.03%, p = 0.0009), unused lung donors (3.44%, p = 0.0009) and emphysema (5.06%, p = 0.01) (fig 1). Ceramide staining was increased in emphysematous lungs compared with PH (p = 0.0135) and unused donors (p = 0.0009). The number of NE- and MPO-positive cells in the airway was positively correlated with the percentage of epithelium staining for ceramide (p = 0.001). Ceramide staining was significantly increased in lungs colonised with P aeruginosa (10.1%) compared with those not colonised (3.14% p = 0.0106).
Conclusions Ceramide accumulates in the lower airway epithelium of people with CF, and to a lesser extent in emphysema, and is positively correlated with markers of neutrophilic inflammation and P aeruginosa infection. These data support the hypothesis that ceramide plays a role in the pathogenesis of CF lung disease and may represent a target for pharmacotherapy.
Ceramide staining. *p = 0.0135, **p = 0.01, ***p = 0.009. CF, cystic fibrosis, PH, pulmonary hypertension.
References
S143 INCREASED INCIDENCE OF CYSTIC FIBROSIS-RELATED DIABETES IN PATIENTS INFECTED WITH TRANSMISSIBLE PSEUDOMONAS AERUGINOSA STRAINS
A. Ashish, M. Shaw, T. S. Jordan, H. G. Tan, E. Spencer, A. Collins, P. Dyce, J. Daniels, M. Ledson, M. J. Walshaw. Liverpool Heart annd Chest Hospital, Liverpool, UK
Background Cystic fibrosis-related diabetes (CFRD) is associated with a sixfold increase in mortality. Although its pathophysiology is not well understood, repeated episodes of stress caused by recurrent respiratory infections may exacerbate its development. Furthermore, worsening clinical parameters in CF have also been attributed to chronic infection with Pseudomonas aeruginosa (Psa), but the relationship between infection with transmissible Psa strains (the most prevalent of which is the Liverpool epidemic strain (LES)) and the development of CFRD has not previously been explored. To investigate this further, we compared the incidence of CFRD in two cohorts of our clinic patients, one infected with unique Psa strains and the other with LES.
Methods All 204 patients chronically infected with Psa (139 with LES) were studied. The diagnosis of CFRD was based on World Health Organization criteria. We gathered information on clinical indices of disease severity, including hospital admissions as an index of exacerbation frequency. We carried out a raw univariate analysis on both matched and unmatched cohorts to identify any link between LES status and CFRD. Fisher two-tailed test was used to calculate statistical significance.
Results Using a raw univariate analysis of the entire population, the prevalence of CFRD was greater with LES infection and these patients were older, less well nourished, had worse pulmonary function and required more hospital admissions. However, in two matched cohorts (see table 1) there was still an increased prevalence of CFRD in patients infected with LES, who also had a significantly greater number of respiratory exacerbations.
Conclusions Patients infected with LES have an increased incidence of CFRD, perhaps due to the increased stress this places on their glucose/insulin axis as a result of repeated exacerbations. This study reinforces the need to prevent cross-infection of CF patients with transmissible Psa strains.
S144 BRONCHOALVEOLAR LAVAGE IN CHILDREN WITH CYSTIC FIBROSIS: WHICH LOBE IS BEST?
F. J. Gilchrist, S. Salamat, S. Clayton, J. Peach, J. Alexander, W. Lenney. University Hospital of North Staffordshire, Stoke on Trent, UK
Introduction Bronchoalveolar lavage (BAL) samples are increasingly being used to diagnose lower airway infection in infants and young children with cystic fibrosis (CF). Guidance from the European Respiratory Society (ERS) in 2001 recommended taking a BAL from the most affected lobe or, in diffuse disease, from the right middle lobe (RML). In 2007 an ERS-sponsored workshop recommended that in children with CF two BAL samples should be taken: a triple aliquot from the RML and a single aliquot from the lingula or the most affected lobe. At our unit we have traditionally taken single aliquot BAL samples from multiple lobes. We assessed if we would have missed positive cultures if we had only taken BAL samples from a single lobe (RML) or from two lobes (RML and lingula).
Methods A retrospective case note review was undertaken. It included all paediatric patients with CF who had undergone flexible bronchoscopy between May 2007 and May 2009.
Results We identified 39 bronchoscopies performed on 31 children with CF. BAL samples were sent from all six lobes in 38 bronchoscopies and from four lobes in one. Positive BAL cultures were obtained from 31 bronchoscopies. Had we only used the RML we would have missed 26 positive cultures (14 organisms) in 11 patients. The organisms were: 4 Haemophilus influenzae, 3 Stenotrophomonas maltophilia, 2 Pseudomonas aeruginosa, 2 Sphingomonas paucimobilis and one each of Moraxella catarrhalis, Aspergillus fumigatus and Staphlococcus aureus. Had we used the RML and lingula we would have missed 12 positive cultures (8 organisms) in 7 patients. The organisms were: 3 S maltophilia 2 H influenzae, 2 S paucimobilis and one A fumigatus.
Conclusions This is the first study that has BAL results from multiple lobes in children with CF. These data confirm that the bacterial distribution in CF is different between the two lungs and also between lobes of the same lung. A single lobe BAL is insufficient in assessing patients with CF for lower airway infection. Even when BALs are taken from the RML and lingual, a significant number of infections are missed.