Gerry McLachlan

Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis

In cystic fibrosis (CF), mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in defective transepithelial ion transport, leading to life shortening inflammatory lung disease. Before lung studies, we tested the safety and efficacy of gene delivery to the nasal epithelium of CF patients using pCMV-CFTR–DOTAP cationic liposome complex. A single dose of 400 μg pCMV-CFTR:2.4 mg DOTAP was administered in a randomised, double-blinded fashion to the nasal epithelium of eight CF patients, with a further eight receiving buffer only. Patients were monitored for signs and symptoms for 2 weeks before treatment and 4 weeks after treatment. Inflammatory cells were quantified in a nasal biopsy taken 3 days after treatment. There was no evidence for excess nasal inflammation, circulating inflammatory markers or other adverse events ascribable to active treatment. Gene transfer and expression were assayed by the polymerase chain reaction. Transgene DNA was detected in seven of the eight treated patients up to 28 days after treatment and vector derived CFTR mRNA in two of the seven patients at +3 and +7 days. Transepithelial ion transport was assayed before and after treatment by nasal potential difference during drug perfusion and by SPQ fluorescence halide ion conductance. Partial, sustained correction of CFTR-related functional changes toward normal values were detected in two treated patients. The level of gene transfer and functional correction were comparable to those reported previously using adenoviral vectors or another DNA–liposome complex, but here were sustained and uncompromised by false positives. These results justify further studies with pCMV-CFTR–DOTAP aimed at treating CF lung disease.

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.

Bacterial DNA is implicated in the inflammatory response to delivery of DNA/DOTAP to mouse lungs

Phase 1 clinical trials of liposome-mediated gene therapy for cystic fibrosis have been completed and in all cases the expression level achieved has been low and transient. Clearly, improvements in the efficiency of gene transfer are required. It is now being recognised that delivery of high doses of DNA/liposomes to the mouse airway epithelium can achieve reproducible evidence of transgene, but is often associated with an unacceptable level of inflammation/ toxicity. It has recently been shown that instillation of bacterial DNA causes inflammation in the lower respiratory tract of rodents. The increased number and unmethylated status of CpG motifs, particularly when present in a particular base context, was identified as an important factor in this response. It was suggested that the immune system recognises this molecular pattern as ‘foreign’ thus activating appropriate immune responses. We have found that methylation of DNA decreases the level of several inflammatory cytokines in lavage fluid and surprisingly has a differential effect on expression of the plasmids pCMV CFTR-int6ab and pCMV CAT which only differ in the actual transcription cassette. The severe lung pathology observed did not show a corresponding decrease with methylation suggesting that these cytokines are not the only contributors to the toxicity/inflammation observed.

Regulation of Pulmonary and Systemic Bacterial Lipopolysaccharide Responses in Transgenic Mice Expressing Human Elafin

ABSTRACT The control of lung inflammation is of paramount importance in a variety of acute pathologies, such as pneumonia, the acute respiratory distress syndrome, and sepsis. It is becoming increasingly apparent that local innate immune responses in the lung are negatively influenced by systemic inflammation. This is thought to be due to a local deficit in cytokine responses by alveolar macrophages and neutrophils following systemic bacterial infection and the development of a septic response. Recently, using an adenovirus-based strategy which overexpresses the human elastase inhibitor elafin locally in the lung, we showed that elafin is able to prime lung innate immune responses. In this study, we generated a novel transgenic mouse strain expressing human elafin and studied its response to bacterial lipopolysaccharide (LPS) when the LPS was administered locally in the lungs and systemically. When LPS was delivered to the lungs, we found that mice expressing elafin had lower serum-to-bronchoalveolar lavage ratios of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein 2, and monocyte chemoattractant protein 1, than wild-type mice. There was a concomitant increase in inflammatory cell influx, showing that there was potential priming of innate responses in the lungs. When LPS was given systemically, the mice expressing elafin had reduced levels of serum TNF-α compared to the levels in wild-type mice. These results indicate that elafin may have a dual function, promoting up-regulation of local lung innate immunity while simultaneously down-regulating potentially unwanted systemic inflammatory responses in the circulation.

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.

Transfection efficiency and toxicity following delivery of naked plasmid DNA and cationic lipid–DNA complexes to ovine lung segments

We defined, using a novel large animal model system, the acute pathologic response to localized pulmonary administration of either naked plasmid DNA (pDNA) or cationic lipid-pDNA complexes (pDNA:GL67) and related such responses to concomitant indicators of transfection efficiency, namely levels of chloramphenicol acetyl transferase (CAT) protein and mRNA in specific lung tissue compartments. We instilled doses of 0.2, 1, and 5 mg pDNA to spatially distinct lung segments in six anesthetized sheep and doses of 0.2, 1, and 5 mg pDNA:GL67 to a further six sheep. Twenty-four hours after gene delivery the sheep were euthanized and necropsy examination with sampling of relevant tissues was carried out. Levels of plasmid-derived CAT-specific mRNA and CAT protein in samples derived from segments treated with either pDNA or pDNA:GL67 increased in relation to the administered dose. Levels of mRNA and protein expression were greater for pDNA:GL67 than for pDNA alone. A significant correlation was observed between mRNA and protein expression in samples derived from airways treated with pDNA:GL67. Histopathological changes following administration of both pDNA and pDNA:GL67 were characterized by a neutrophilic inflammation predominantly oriented on airways. The severity of the inflammatory response appeared to correlate with the administered dose of DNA and was generally more severe for pDNA:GL67.

Chemokine and cytokine expression in murine intestinal epithelium following Nippostrongylus brasiliensis infection

Infection of mice with the nematode parasite Nippostrongylus brasiliensis results in a well characterized intestinal mastocytosis with intraepithelial migration of mucosal mast cells (MMC). The molecules mediating this response are unknown. We examined expression of several putative mast cell chemoattractants in intestinal epithelium following N. brasiliensis infection. Expression of the chemokines monocyte chemoattractant protein‐1 (MCP‐1), macrophage inflammatory protein‐1α(MIP‐1α), RANTES (regulated on activation normal T‐cell expressed and secreted), fractalkine, and thymocyte expressed chemokine (TECK); and the cytokines stem cell factor (SCF) and transforming growth factor β1 (TGFβ1), was constitutive and no alteration was detected following infection. MCP‐1 expression was also constitutive but at much lower levels and increased expression was detected on days 7 and 14 postinfection. Expression of MCP‐1 in whole jejunum was at much higher levels than in epithelium. Constitutive expression of MCP‐1, MIP‐1α and TGFβ1 was also detected in cultured bone marrow‐derived homologues of MMC. In an intestinal epithelial cell line (CMT‐93), there was constitutive expression of SCF, TGFα1, fractalkine and MCP‐1. The results show that, in vivo, epithelium is a potentially important source of mast cell chemoattractants.

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.

Aerosol delivery of DNA/liposomes to the lung for cystic fibrosis gene therapy

Abstract Lung gene therapy is being evaluated for a range of acute and chronic diseases, including cystic fibrosis (CF). As these therapies approach clinical realization, it is becoming increasingly clear that the ability to efficiently deliver gene transfer agents (GTAs) to target cell populations within the lung may prove just as critical as the gene therapy formulation itself in terms of generating positive clinical outcomes. Key to the success of any aerosol gene therapy is the interaction between the GTA and nebulization device. We evaluated the effects of aerosolization on our preferred formulation, plasmid DNA (pDNA) complexed with the cationic liposome GL67A (pDNA/GL67A) using commercially available nebulizer devices. The relatively high viscosity (6.3±0.1 cP) and particulate nature of pDNA/GL67A formulations hindered stable aerosol generation in ultrasonic and vibrating mesh nebulizers but was not problematic in the jet nebulizers tested. Aerosol size characteristics varied significantly between devices, but the AeroEclipse II nebulizer operating at 50 psi generated stable pDNA/GL67A aerosols suitable for delivery to the CF lung (mass median aerodynamic diameter 3.4±0.1 μm). Importantly, biological function of pDNA/GL67A formulations was retained after nebulization, and although aerosol delivery rate was lower than that of other devices (0.17±0.01 ml/min), the breath-actuated AeroEclipse II nebulizer generated aerosol only during the inspiratory phase and as such was more efficient than other devices with 83±3% of generated aerosol available for patient inhalation. On the basis of these results, we have selected the AeroEclipse II nebulizer for the delivery of pDNA/GL67A formulations to the lungs of CF patients as part of phase IIa/b clinical studies.