U Griesenbach

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.

Gene Therapy Progress and Prospects: Cystic fibrosis

Our first review on progress and prospects in cystic fibrosis (CF) gene therapy was published in this series in October 2002. We now summarize the progress made since then and comment on the prospects for CF gene therapy over the next couple of years. Three clinical trials have been carried out, further supporting the proof-of-principle that gene transfer to the airway epithelium is feasible. Developments in viral and non-viral vectors, as well as recent alternative strategies such as gene repair, trans-splicing and stem cell therapy will be reviewed.

Gene transfer to the lung: Lessons learned from more than 2 decades of CF gene therapy ☆

Gene therapy is currently being developed for a wide range of acute and chronic lung diseases. The target cells, and to a degree the extra and intra-cellular barriers, are disease-specific and over the past decade the gene therapy community has recognized that no one vector is good for all applications, but that the gene transfer agent (GTA) has to be carefully matched to the specific disease target. Gene therapy is particularly attractive for diseases that currently do not have satisfactory treatment options and probably easier for monogenic disorders than for complex diseases. Cystic fibrosis (CF) fulfils these criteria and is, therefore, a good candidate for gene therapy-based treatment. This review will focus on CF as an example for lung gene therapy, but lessons learned may be applicable to other target diseases.

Comparison between intratracheal and intravenous administration of liposome-DNA complexes for cystic fibrosis lung gene therapy

Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA–vector formulations are two strategies to obtain gene transfer to the lung. It is still uncertain, however, which of these two modes of delivery will be more effective in the treatment of cystic fibrosis and other lung diseases. In this study, we attempted to optimize formulations of the cationic liposome DODAC:DOPE (dioleoyldimethylammonium- chloride:dioleoylphosphatidylethanolamine) complexed to plasmids encoding chloramphenicol acetyltransferase for i.t. and i.v. injection into CD-1 mice and compared the two methods. Our results showed that both methods conferred reporter gene expression in the lung that was significantly higher relative to injection of plasmid DNA alone. Expression using either mode of administration was maximal 24 h after injection and declined to around 10% of day 1 levels 2 weeks after injection. For i.v. delivery of DODAC: DOPE–DNA complexes multilamellar vesicles were more effective than large unilamellar vesicles in all organs investigated. Recombinant DNA could be detected in the distal lung region following either route of administration. However, i.t. administration predominantly led to DNA deposition in epithelial cells lining the bronchioles, eg in clara cells, whereas i.v. administration resulted in DNA deposition in the alveolar region of the lung including type II alveolar epithelial cells.

Changes in physiological, functional and structural markers of cystic fibrosis lung disease with treatment of a pulmonary exacerbation

Background Clinical trials in cystic fibrosis (CF) have been hindered by the paucity of well characterised and clinically relevant outcome measures. Aim To evaluate a range of conventional and novel biomarkers of CF lung disease in a multicentre setting as a contributing study in selecting outcome assays for a clinical trial of CFTR gene therapy. Methods A multicentre observational study of adult and paediatric patients with CF (>10 years) treated for a physician-defined exacerbation of CF pulmonary symptoms. Measurements were performed at commencement and immediately after a course of intravenous antibiotics. Disease activity was assessed using 46 assays across five key domains: symptoms, lung physiology, structural changes on CT, pulmonary and systemic inflammatory markers. Results Statistically significant improvements were seen in forced expiratory volume in 1 s (p<0.001, n=32), lung clearance index (p<0.01, n=32), symptoms (p<0.0001, n=37), CT scores for airway wall thickness (p<0.01, n=31), air trapping (p<0.01, n=30) and large mucus plugs (p=0.0001, n=31), serum C-reactive protein (p<0.0001, n=34), serum interleukin-6 (p<0.0001, n=33) and serum calprotectin (p<0.0001, n=31). Discussion We identify the key biomarkers of inflammation, imaging and physiology that alter alongside symptomatic improvement following treatment of an acute CF exacerbation. These data, in parallel with our study of biomarkers in patients with stable CF, provide important guidance in choosing optimal biomarkers for novel therapies. Further, they highlight that such acute therapy predominantly improves large airway parameters and systemic inflammation, but has less effect on airway inflammation.

Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo.

We have assessed whether magnetic forces (magnetofection) can enhance non-viral gene transfer to the airways. TransMAGPEI, a superparamagnetic particle was coupled to Lipofectamine 2000 or cationic lipid 67 (GL67)/plasmid DNA (pDNA) liposome complexes. In vitro transfection with these formulations resulted in approximately 300- and 30-fold increase in reporter gene expression, respectively, after exposure to a magnetic field, but only at suboptimal pDNA concentrations. Because GL67 has been formulated for in vivo use, we next assessed TransMAGPEI in the murine nasal epithelium in vivo, and compared this to naked pDNA. At the concentrations required for in vivo experiments, precipitation of magnetic complexes was seen. After extensive optimization, addition of non-precipitated magnetic particles resulted in approximately seven- and 90-fold decrease in gene expression for naked pDNA and GL67/pDNA liposome complexes, respectively, compared to non-magnetic particles. Thus, whereas exposure to a magnetic field improved in vitro transfection efficiency, translation to the in vivo setting remains difficult.

Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes

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 efficiently transduce unconditioned airway epithelial cells from the apical side. This novel vector was evaluated in mice in vivo and in vitro directed toward CF gene therapy. Here, we show that (i) we can produce relevant titers of an SIV vector pseudotyped with SeV envelope proteins for in vivo use, (ii) this vector can transduce the respiratory epithelium of the murine nose in vivo at levels that may be relevant for clinical benefit in CF, (iii) this can be achieved in a single formulation, and without the need for preconditioning, (iv) expression can last for 15 months, (v) readministration is feasible, (vi) the vector can transduce human air-liquid interface (ALI) cultures, and (vii) functional CF transmembrane conductance regulator (CFTR) 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.

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.

Emerging significance of plasmid DNA nuclear import in gene therapy

The signal-mediated import of plasmid DNA (pDNA) into nondividing mammalian cell nuclei is one of the key biological obstacles to nonviral therapeutic pDNA delivery. Overcoming this barrier to pDNA transfer is thus an important fundamental objective in gene therapy. Here, we outline the rationale behind current and future strategies for signal-mediated pDNA nuclear import. Results obtained from studies of the nuclear delivery of pDNA coupled to experimentally defined nuclear localisation signal (NLS) peptides, in conjunction with detergent-permeabilised reconstitution cell assays, direct intracellular microinjection, cell-based transfection, and a limited number of in vivo experiments are discussed.

Use of ultrasound to enhance nonviral lung gene transfer in vivo.

We have assessed if high-frequency ultrasound (US) can enhance nonviral gene transfer to the mouse lung. Cationic lipid GL67/pDNA, polyethylenimine (PEI)/pDNA and naked plasmid DNA (pDNA) were delivered via intranasal instillation, mixed with Optison microbubbles, and the animals were then exposed to 1 MHz US. Addition of Optison alone significantly reduced the transfection efficiency of all three gene transfer agents. US exposure did not increase GL67/pDNA or PEI/pDNA gene transfer compared to Optison-treated animals. However, it increased naked pDNA transfection efficiency by approximately 15-fold compared to Optison-treated animals, suggesting that despite ultrasound being attenuated by air in the lung, sufficient energy penetrates the tissue to increase gene transfer. US-induced lung haemorrhage, assessed histologically, increased with prolonged US exposure. The left lung was more affected than the right and this was mirrored by a lesser increase in naked pDNA gene transfer, in the left lung. The positive effect of US was dependent on Optison, as in its absence US did not increase naked pDNA transfection efficiency. We have thus established proof of principle that US can increase nonviral gene transfer, in the air-filled murine lung.