Peter Tennant

Enhanced lung gene expression after aerosol delivery of concentrated pDNA/PEI complexes.

A major limitation of many self-assembling nonviral gene transfer formulations is that they are commonly prepared at relatively low component concentrations. While this typically has little impact on their use in cell culture, it can severely limit the progress of in vivo studies. In order to overcome this, we have developed a simple, scalable, pharmaceutically acceptable concentration method that has allowed us to increase the concentration of a commonly used pDNA/PEI formulation from 0.2 to >8 mg/ml plasmid DNA (pDNA). Crucially, the concentration method was found to have only minimal impact on the electrostatic properties or size of the pDNA/PEI particles. When delivered as an aerosol to the mouse lung, the concentrated pDNA/PEI formulations resulted in a 15-fold increase in lung reporter gene expression, with minimal impact in terms of inflammation or toxicity. Importantly, this performance advantage was replicated after aerosol administration to sheep lungs, with reporter gene expression being similarly approximately 15-fold higher than with the conventional pDNA/PEI formulation, and lung inflammation falling to background levels. These findings demonstrate that concentrated pDNA/PEI formulations offer increased aerosol gene transfer with decreased inflammatory sequelae, and represent a promising advance in the field of nonviral lung gene transfer. It seems likely that similar benefits might be achievable with alternative delivery routes and with other nonviral formulations.

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.

Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung

Existing methods of non‐viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep.

Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer

The cationic lipid GL67A is one of the more efficient non-viral gene transfer agents (GTAs) for the lungs, and is currently being evaluated in an extensive clinical trial programme for cystic fibrosis gene therapy. Despite conferring significant expression of vector-specific mRNA following transfection of differentiated human airway cells cultured on air liquid interfaces (ALI) cultures and nebulisation into sheep lung in vivo we were unable to detect robust levels of the standard reporter gene Firefly luciferase (FLuc). Recently a novel secreted luciferase isolated from Gaussia princeps (GLuc) has been described. Here, we show that (1) GLuc is a more sensitive reporter gene and offers significant advantages over the traditionally used FLuc in pre-clinical models for lung gene transfer that are difficult to transfect, (2) GL67A-mediated gene transfection leads to significant production of recombinant protein in these models, (3) promoter activity in ALI cultures mimics published in vivo data and these cultures may, therefore, be suitable to characterise promoter activity in a human ex vivo airway model and (4) detection of GLuc in large animal broncho-alveolar lavage fluid and serum facilitates assessment of duration of gene expression after gene transfer to the lungs. In summary, we have shown here that GLuc is a sensitive reporter gene and is particularly useful for monitoring gene transfer in difficult to transfect models of the airway and lung. This has allowed us to validate that GL67A, which is currently in clinical use, can generate significant amounts of recombinant protein in fully differentiated human air liquid interface cultures and the ovine lung in vivo.

The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep.

Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.

Validation of recombinant Sendai virus in a non-natural host model

We have previously shown that recombinant Sendai virus (SeV) vector, derived from murine parainfluenza virus, is one of the most efficient vectors for airway gene transfer. We have also shown that SeV-mediated transfection on second administration, although reduced by 60% when compared with levels achieved after a single dose, is still high because of the efficient transfection achieved by SeV vector in murine airways. Here, we show that these levels further decrease on subsequent doses. In addition, we validated SeV vector repeat administration in a non-natural host model, the sheep. As part of these studies we first assessed viral stability in a Pari LC Plus nebuliser, a polyethylene catheter (PEC) and the Trudell AeroProbe. We also compared the distribution of gene expression after PEC and Trudell AeroProbe administration and quantified virus shedding after sheep transduction. In addition, we show that bronchial brushings and biopsies, collected in anaesthetized sheep, can be used to assess SeV-mediated gene expression over time. Similar to mice, gene expression in sheep was transient and had returned to baseline values by day 14. In conclusion, the SeV vector should be strongly considered for lung-related applications requiring a single administration of the vector even though it might not be suitable for diseases requiring repeat administration.

Variability of the Sheep Lung Microbiota

ABSTRACT Sequencing technologies have recently facilitated the characterization of bacterial communities present in lungs during health and disease. However, there is currently a dearth of information concerning the variability of such data in health both between and within subjects. This study seeks to examine such variability using healthy adult sheep as our model system. Protected specimen brush samples were collected from three spatially disparate segmental bronchi of six adult sheep (age, 20 months) on three occasions (day 0, 1 month, and 3 months). To further explore the spatial variability of the microbiotas, more-extensive brushing samples (n = 16) and a throat swab were taken from a separate sheep. The V2 and V3 hypervariable regions of the bacterial 16S rRNA genes were amplified and sequenced via Illumina MiSeq. DNA sequences were analyzed using the mothur software package. Quantitative PCR was performed to quantify total bacterial DNA. Some sheep lungs contained dramatically different bacterial communities at different sampling sites, whereas in others, airway microbiotas appeared similar across the lung. In our spatial variability study, we observed clustering related to the depth within the lung from which samples were taken. Lung depth refers to increasing distance from the glottis, progressing in a caudal direction. We conclude that both host influence and local factors have impacts on the composition of the sheep lung microbiota. IMPORTANCE Until recently, it was assumed that the lungs were a sterile environment which was colonized by microbes only during disease. However, recent studies using sequencing technologies have found that there is a small population of bacteria which exists in the lung during health, referred to as the “lung microbiota.” In this study, we characterize the variability of the lung microbiotas of healthy sheep. Sheep not only are economically important animals but also are often used as large animal models of human respiratory disease. We conclude that, while host influence does play a role in dictating the types of microbes which colonize the airways, it is clear that local factors also play an important role in this regard. Understanding the nature and influence of these factors will be key to understanding the variability in, and functional relevance of, the lung microbiota.

A Lung Segmental Model of Chronic Pseudomonas Infection in Sheep

Background Chronic lung infection with Pseudomonas aeruginosa is a major contributor to morbidity, mortality and premature death in cystic fibrosis. A new paradigm for managing such infections is needed, as are relevant and translatable animal models to identify and test concepts. We sought to improve on limitations associated with existing models of infection in small animals through developing a lung segmental model of chronic Pseudomonas infection in sheep. Methodology/Principal Findings Using local lung instillation of P. aeruginosa suspended in agar beads we were able to demonstrate that such infection led to the development of a suppurative, necrotising and pyogranulomatous pneumonia centred on the instilled beads. No overt evidence of organ or systemic compromise was apparent in any animal during the course of infection. Infection persisted in the lungs of individual animals for as long as 66 days after initial instillation. Quantitative microbiology applied to bronchoalveolar lavage fluid derived from infected segments proved an insensitive index of the presence of significant infection in lung tissue (>104 cfu/g). Conclusions/Significance The agar bead model of chronic P. aeruginosa lung infection in sheep is a relevant platform to investigate both the pathobiology of such infections as well as novel approaches to their diagnosis and therapy. Particular ethical benefits relate to the model in terms of refining existing approaches by compromising a smaller proportion of the lung with infection and facilitating longitudinal assessment by bronchoscopy, and also potentially reducing animal numbers through facilitating within-animal comparisons of differential therapeutic approaches.

Lung Microbiota Changes Associated with Chronic Pseudomonas aeruginosa Lung Infection and the Impact of Intravenous Colistimethate Sodium

Background Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density. This potentially suggests a role for the latter in the pathogenesis of exacerbations. However, other data suggesting that changes in P. aeruginosa sputum culture status may not reliably predict an improvement in clinical status, and data indicating no significant changes in either total bacterial counts or in P. aeruginosa numbers in sputum samples collected prior to pulmonary exacerbation sheds doubt on this assumption. We used our recently developed lung segmental model of chronic Pseudomonas infection in sheep to investigate the lung microbiota changes associated with chronic P. aeruginosa lung infection and the impact of systemic therapy with colistimethate sodium (CMS). Methodology/Principal Findings We collected protected specimen brush (PSB) samples from sheep (n = 8) both prior to and 14 days after establishment of chronic local lung infection with P aeruginosa. Samples were taken from both directly infected lung segments (direct) and segments spatially remote to such sites (remote). Four sheep were treated with daily intravenous injections of CMS between days 7 and 14, and four were treated with a placebo. Necropsy examination at d14 confirmed the presence of chronic local lung infection and lung pathology in every direct lung segment. The predominant orders in lung microbiota communities before infection were Bacillales, Actinomycetales and Clostridiales. While lung microbiota samples were more likely to share similarities with other samples derived from the same lung, considerable within- and between-animal heterogeneity could be appreciated. Pseudomonadales joined the aforementioned list of predominant orders in lung microbiota communities after infection. Whilst treatment with CMS appeared to have little impact on microbial community composition after infection, or the change undergone by communities in reaching that state, when Gram negative organisms (excluding Pseudomonadales) were considered together as a group there was a significant decrease in their relative proportion that was only observed in the sheep treated with CMS. With only one exception the reduction was seen in both direct and remote lung segments. This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment. Conclusions/Significance The proportional increase in Pseudomonadales relative to other Gram negative bacteria in the lungs of sheep treated with systemic CMS highlights the potential for such therapies to inadvertently select or create a niche for bacteria seeding from a persistent source of chronic infection.

Analysis of airway epithelial regeneration and repair following endobronchial brush biopsy in sheep

ABSTRACT Understanding the fundamental processes involved in repairing the airway wall following injury is fundamental to understanding the way in which these processes are perturbed during disease pathology. Indeed complex diseases such as asthma and chronic obstructive pulmonary disease (COPD) have at their core evidence of airway wall remodeling processes that play a crucial functional role in these diseases. The authors sought to understand the dynamic cellular events that occur during bronchial airway epithelial repair in sheep. The injury was induced by endobronchial brush biopsy (BBr), a process that causes epithelial débridement and induces a consequential repair process. In addition, the current experimental protocol allowed for the time-dependent changes in airway wall morphology to be studied both within and between animals. The initial débridement was followed by evidence of dedifferentiation in the intact epithelium at the wound margins, followed by proliferation of cells both within the epithelium and in the deeper wall structures, notably in association with the submucosal glands and smooth muscle bundles. Seven days after injury, although the airway wall was thickened at the site of damage, the epithelial layer was intact, with evidence of redifferentiation. These studies, in demonstrating broad agreement with previous studies in small animals, indicate the wider relevance of this system as a comparative model and should provide a solid basis upon which to further characterize the critical cellular and molecular interactions that underlie both effective restitution and pathological repair.