S C Hyde

Progress and Prospects: The design and production of plasmid vectors

Plasmid DNA (pDNA) expression vectors are fundamental to all forms of non-viral gene transfer. In this review, we discuss principles of pDNA design and production including the impact of bacterially derived sequences on transgene expression and minicircle approaches to minimize their effects. The impact of inclusion of DNA elements such as scaffold matrix attachment regions (S/MARs), transcription factor (TF)-binding sites and tissue-specific promoters are described. The benefits of eliminating CG dinucleotides (CpGs) from the pDNA are also considered.

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.

Sendai virus-mediated CFTR gene transfer to the airway epithelium

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (CftrG551D and Cftrtm1UncTgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Detection of plasmid DNA vectors following gene transfer to the murine airways

Non-viral gene therapy is being considered as a treatment for cystic fibrosis. In clinical studies and in studies using the mouse airways as a model, current formulations result in only transient transgene expression. A number of reasons for this have been proposed including the loss of plasmid DNA from cells. The aim of these studies was to investigate why transgene expression from non-viral vectors is transient in the mouse lung. Plasmid DNA encoding the luciferase reporter gene was complexed with the cationic lipid GL67 and delivered to the mouse airways. The persistence of plasmid DNA in the mouse lungs was investigated using quantitative PCR and Southern hybridization. Results showed that intact plasmid DNA persisted in the mouse lung in the absence of any detectable luciferase activity. The de novo methylation of plasmid DNA in vivo was investigated as a potential cause of this transient gene expression but results suggested that plasmid DNA does not become de novo methylated in the mouse lung. Therefore processes other than the loss of plasmid DNA from the lung or the de novo methylation of plasmid DNA vectors must be responsible for the transient transgene expression.

Long-term persistence of gene expression from adeno-associated virus serotype 5 in the mouse airways.

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) have been used to deliver transgenes to the airways in a variety of pre-clinical and clinical studies. Gene transfer in these studies has been severely restricted by the basolateral localization of rAAV2 receptors. Here, we studied vectors constructed from the AAV5 genome and capsid, which utilize N-linked sialic acid-containing receptors found on the apical surface of airway epithelial cells. We investigated gene transfer efficacy and duration of transgene expression following delivery of rAAV5/5 vectors to the mouse respiratory tract. Robust, dose-dependent transgene expression was observed in the epithelium lining the nose for at least 32 weeks, and for at least 52 weeks in the lung. Importantly, in the lung, transgene expression mediated by rAAV5/5 was 40-fold greater than by rAAV2/2 vectors. A distinct cellular preference for rAAV5/5-mediated transduction was observed, with transgene expression being predominantly restricted to sustentacular cells of the olfactory epithelium in the nose and alveolar type II cells in the lung. Administration of rAAV5/5 vectors to both the nose and lungs led to the rapid development of rAAV5/5-neutralizing antibodies, suggesting that repeated administration may be severely hampered by host immune responses.

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.

Longitudinal assessment of biomarkers for clinical trials of novel therapeutic agents: the run-in study

We will be undertaking a phase IIB clinical trial of repeated application of liposome-based gene therapy over a one year period in approximately 100 CF patients (Multidose Trial). In preparation for this, we sought to address two key questions. Firstly, could we define the optimal set of patients in which the therapy could both be delivered (good access to the airways via nebulisation), and in whom any therapeutic effect was measurable (one or more abnormal measures of lung disease). Secondly, in this set of ‘can deliver—can measure’ patients, which biomarker(s) could be powered to be the primary outcome measure for the trial. To address both questions, we undertook a study (Run-in), cross-sectionally assessing ‘can deliver’ and longitudinally assessing a large set of candidate biomarkers for ‘can measure’.192 patients from age 10 upwards, with FEV1 >40% were enrolled at two clinical centres; 154 of these remained in the study after four visits spaced at approximately 4–5 month intervals. Biomarkers assessed cross-sectionally included radionucleotide deposition scans, CT and mucocilary clearance. Longitudinal biomarkers included a large series of serum, sputum and exhaled breath inflammatory markers, lung physiology, exercise-related assays and quality of life assessment. 12 patients were judged too severe for adequate delivery and were excluded. A shortlist of 4 biomarkers was generated based on a) showing a CF/non-CF difference, b) response to course of intravenous antibiotics, and c) coefficients of variation. These four were matched against the remaining 142 patients, and a further seven patients excluded in whom none of these short listed biomarkers was abnormal. 89 patients (3 or 4 biomarkers abnormal) have been definitely included to progress into the Multidose Trial, and a further 46 (1 or 2 biomarkers abnormal) are awaiting the final primary outcome selection. The Run-in study has, therefore, been able to a) select a cohort of ‘optimal’ patients in which to assess gene therapy and b) provide an indication of which may be the more useful biomarkers to use in phase IIB clinical trials of novel therapeutic agents.

473. The Significance of Academia-Industry Collaboration in Translational Research: A Survey of Over 300 PIs Who Have Received Industry Funding

Due to the high response rate and level of interest in this project, a similar study focusing on the role of collaboration in GT is underway. The initial phase of this study comprises interviews with GT stakeholders from around the world. If you would be willing to participate in this research, please contact natasha.davie@ndcls.ox.ac.uk to arrange a 30-minute interview. No previous experience of collaboration is necessary. Thank you.

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.

P109 Exercise capacity and physical activity in patients with CF: data from the UK CF Gene Therapy Consortium (UKCFGTC) ‘Run-In’ Study

Introduction Exercise capacity is predictive of mortality in CF. Objective measurement of daily physical activity may be related to exercise capacity and both may be useful outcome measures in interventional trials. Participants in this CF study had a field-based estimate of exercise capacity and objective measurement of physical activity at each study visit. Methods Participants wore a pedometer for 7 days prior to each visit. At each visit, London patients performed a standard incremental shuttle-walk test and Edinburgh patients a modified shuttle test (in which running was allowed). Data are expressed as mean (SD). Results Data were analysed from 192 patients over 648 visits. Age at enrolment was 24 (11.9) years (London) and 20.8 (9.9) (Edinburgh) (p=0.052); FEV1 was 67 (17.7)% and 79 (19.5)% for each site respectively (p<0.001). Daily step count at visit 1 was 7491 (2887) in London and 8872 (4089) in Edinburgh (p=0.04) and this difference persisted across subsequent visits. The coefficient of variation (CV) in step counts between visits was 21.3%. Number of shuttles completed in London was 61 (15), and Edinburgh 90 (33) with no trend over the four visits (CV=10 and 16% respectively). In Edinburgh there was a correlation between mean step count and the number of completed shuttles (r=0.46, p<0.001). Step count from both sites, and the number of shuttles completed in Edinburgh, correlated with FEV1 % predicted (r=0.24, p<0.001 and r=0.27, p<0.001 respectively) and with age (r=−0.28, p<0.001 and r=−0.30, p<0.001 respectively). Such correlations were either weaker or not observed in London, however, in this group, number of shuttles correlated with height (r=0.51, p<0.001). Conclusions No changes were detected in exercise capacity or daily activity levels over time. Between site differences were observed in both measures; however, these populations also differ in age and FEV1. The modified shuttle test performed in Edinburgh appeared to better correlate with clinical markers than the standard incremental shuttle test performed in London, and is independent of height. We believe that testing exercise capacity is important in CF and we plan to investigate the other testing methods in the run up to our Multi Dose Gene Therapy Trial.