A C Boyd

CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression

Pulmonary delivery of plasmid DNA (pDNA)/cationic liposome complexes is associated with an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response and brief duration of transgene expression. We demonstrate that retention of even a single CpG in pDNA is sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors do not. Using a CpG-free pDNA expression vector, we achieved sustained (≥56 d) in vivo transgene expression in the absence of lung inflammation.

Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung

We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to assess three non-viral GTAs: 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n=8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1–10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients.

Sputum and serum calprotectin are useful biomarkers during CF exacerbation

BACKGROUND Adequate monitoring of cystic fibrosis lung disease is difficult. CF exacerbation offers a unique setting to test the utility of biomarkers in the assessment of changing airways inflammation. We hypothesised that levels of calprotectin in sputum (and serum) would change informatively following treatment of an exacerbation. METHODS 27 patients with CF were recruited at onset of pulmonary exacerbation. Sputum and serum were collected at the start and end of anti-biotic therapy. Sputum calprotectin, interleukin-8 (IL8), and myeloperoxidase (MPO) were measured, as were serum calprotectin, CRP and vascular endothelial growth factor (VEGF). RESULTS Sputum calprotectin decreased following treatment of an exacerbation (p<0.05), and was superior to other sputum markers. Serum calprotectin, CRP, and VEGF also decreased significantly (p=0.002, p=0.002, p=0.013 respectively). Serum calprotectin level following treatment had predictive value for time to next exacerbation (p=0.032). CONCLUSIONS This study demonstrates the superiority of calprotectin (in sputum and serum) as a biomarker of CF exacerbation over better-established markers.

Toxicology study assessing efficacy and safety of repeated administration of lipid/DNA complexes to mouse lung

For gene therapy to improve lung function in cystic fibrosis (CF) subjects, repeated administration of the gene transfer agent over the lifetime of patients is likely to be necessary. This requirement limits the utility of adenoviral and adeno-associated viral vectors (both previously evaluated in CF gene therapy trials) because of induced adaptive immune responses that render repeated dosing ineffective. For CF gene therapy trials, non-viral vectors are currently the only viable option. We previously showed that the cationic lipid formulation GL67A is the most efficient of several non-viral vectors analysed for airway gene transfer. Here, we assessed the efficacy and safety of administering 12 inhaled doses of GL67A complexed with pGM169, a CpG-free plasmid encoding human CFTR complementary DNA, into mice. We show that repeated administration of pGM169/GL67A to murine lungs is feasible, safe and achieves reproducible, dose-related and persistent gene expression (>140 days after each dose) using an aerosol generated by a clinically relevant nebuliser. This study supports progression into the first non-viral multidose lung trial in CF patients.

DEVELOPMENT OF AN OPTIMAL F/HN PSEUDOTYPED SIV VECTOR FOR CF GENE THERAPY

We are developing lung gene transfer vectors to treat acquired and inherited lung disorders such as cystic fibrosis, and have identified two platforms for efficient respiratory gene delivery: one non-viral system based on CpG-free plasmid DNA combined with cationic lipids (pDNA/GL67A), which has recently completed evaluation in a Phase IIb clinical study; and one novel viral system based on a recombinant simian immunodeficiency virus pseudotyped with the F/HN proteins of Sendai virus (rSIV. F/HN) to promote airway cell uptake. Here we report on the development of a “third generation” rSIV. F/HN vector suitable for use in the clinic. The vector is manufactured by transient transfection of cultured human cells using five producer plasmids, all of which have been engineered to be pharmacopoeia compliant. A variety of vector configurations, including a range of enhancers/promoters and transgenes, were evaluated in a panel of airway models. rSIV. F/HN vectors directed high-level, robust gene expression in fully differentiated human airway cells, human nasal brushings and human and sheep lung slices. In the mouse nose and lung, the preferred configuration directed up to x500-fold higher transgene expression than the non-viral platform, for the lifetime of the animal. Transgene expression was observed in 14.1% of lung epithelial cells (p < 0.0001 compared to controls). Repeated monthly administration (3X) was possible without loss of expression or significant histological inflammatory reactivity. Reassuringly, insertion site profiling from transduced cell lines and mouse nose/lung samples reveals a pattern of integration comparable to those reported for other lentiviral vectors in clinical development, with no evidence for enrichment of insertion at undesirable loci. The stability of rSIV. F/HN vectors was evaluated in two bronchoscope catheters and two aerosol generation devices. Encouragingly for clinical translation, no significant loss of transduction activity was noted with any of these clinically relevant delivery devices (p = 0.64). Delivery of rSIV. F/HN expressing CFTR to sheep lung resulted in CFTR mRNA at ∼30% the levels of endogenous ovine CFTR (p < 0.0001 compared to non-treated lobes), exceeding presumed therapeutic levels. With the majority of translational hurdles addressed, we are now entering toxicology studies and the final stages of pharmaceutical development prior to entering clinical trials.

P209 Standardisation Of Lung Clearance Index In A Multicentre Clinical Trial

Introduction Lung clearance index (LCI) is a sensitive and repeatable non-invasive measure of ventilation inhomogeneity derived from the multiple breath washout (MBW) technique. It is more sensitive to early lung disease than traditional lung function measurements. Before it can be adopted as a primary endpoint in multicentre trials, it must be demonstrated that it can be applied with minimal inter-operator variability. LCI is a major secondary outcome in our gene therapy multidose trial. Aim To assess LCI achievability and intra- and inter-site agreement. Method 136 CF patients at two sites with FEV1 50–90% predicted were randomly allocated on a 1:1 basis to receive 12 monthly nebulised doses of active gene therapy product or placebo. LCI was performed in triplicate on seven occasions for each subject using a MBW technique completed on an InnocorTM device using 0.2% SF6. Stringent quality control criteria have been developed, including offset calculations and minimal acceptable differences between tests. LCI was calculated using customised offline analysis software (SimpleWashout, Igor Pro) by a single operator at each site. To test inter-operator agreement, every seventh MBW from each timepoint was randomly selected, without subject duplication, and used to calculate LCI values by both operators separately. Results A total of 854 LCIs were performed during the trial, and technically acceptable measurements were achieved in 95.9% and 94.2% of tests at the two sites (mean 94.8%). 118 (13.8%) of LCIs were analysed independently by two operators, with a full range LCI values represented (range 7.24–19.21). The 95% limits of agreement (LoA) for LCI values were -0.04 to 0.04 (mean difference 0.00) and for FRC values were -0.01 to 0.01 (mean difference 0.00). Conclusions Our results demonstrate that LCI is an achievable outcome measure in a multicentre trial in 94.8% of attempts. Separate offline analysis completed by two operators, with appropriate training and knowledge of the test, produces mean LCI and FRC inter-site differences of 0.00. LCI is feasible and appropriate for use as a surrogate endpoint in multicentre clinical trials using stringent methodology. Abstract P209 Figure 1 Bland-Altman plot of LCI inter-operator difference

25 SELDI-TOF-MS ProteinChip profiling of serum and nasal cells from Cystic Fibrosis patients

Surface enhanced laser desorption/ionisation-time of flight (SELDI-TOF) mass spectrometry (MS) is an array based proteomic technology. It combines a variety of chromatographic surfaces that bind proteins from biological mixtures according to physicochemical properties with MS analysis. The relative expression of proteins at specific molecular weights can then be compared among different samples. The aim of this work is to establish protein profiles in Cystic Fibrosis (CF) serum and airway cell lysate for biomarker identification. These proteins may serve as diagnostic/prognostic markers or even as new targets for CF therapy. Serum and nasal epithelial cells (collected by brushing) were obtained from CF patients (n 13/15) and controls (n 11/9). Protein extracts were applied to a range ProteinChip surfaces (anionic exchange, cationic exchange and metal affinity) and analysed by SELDI-TOF-MS. The mass spectral data demonstrate a larger group of proteins with differential expression in serum compared to nasal cells lysate. 45 peaks (p < 0.05) differentiated CF serum from control. 9 peaks (p < 0.05) differentiated CF nasal cell lysate from control. Further work is required to validate these results in a larger cohort of CF patients and to identify candidate biomarkers with MS protein characterisation. Work supported by FCT/FEDER research grants (POCTI/SAU-MMO/56163/2004). PA is a recipient of FCT doctoral fellowship. CB and DP collaborated equally to supervision of the experiments. 2. CFTR Cell biology~Physiology