Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis.

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models.

Transgene sequences free of CG dinucleotides lead to high level, long-term expression in the lung independent of plasmid backbone design.

Non-viral aerosol gene therapy offers great potential for treating chronic lung diseases of the airways such as cystic fibrosis (CF). Early clinical trials showed that transgene expression in the airways was transient whereas maximal duration of transgene expression is essential in order to minimise the frequency of aerosol treatments. Improved vector design, such as careful selection of the promoter/enhancer, can lead to more persistent levels of transgene expression, but multiple factors affect expression in vivo.

Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial.

Background: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis.

Methods: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK.

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.

Pharmacological Characterization of a Novel ENaCα siRNA (GSK2225745) With Potential for the Treatment of Cystic Fibrosis.

Lung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC(50) (half maximal effective concentration) = 0.

CpG-free plasmid expression cassettes for cystic fibrosis gene therapy.

Clinical studies are underway for the aerosol delivery of plasmid DNA complexed with Genzyme Lipid GL67A to the lungs of patients with cystic fibrosis (CF). Plasmid vectors contain several functional elements all of which play a role in determining the efficacy of the final clinical product. To optimise the final plasmid, variations of CpG-free 5' enhancer elements and 3'UTR regions were inserted into a common CpG-free, plasmid backbone containing Luciferase or CFTR transgenes.

The use of CpG-free plasmids to mediate persistent gene expression following repeated aerosol delivery of pDNA/PEI complexes.

Aerosol gene therapy offers great potential for treating acquired and inherited lung diseases. For treatment of chronic lung diseases such as cystic fibrosis, asthma and emphysema, non-viral gene therapy will likely require repeated administration to maintain transgene expression in slowly dividing, or terminally differentiated, lung epithelial cells. When complexed with plasmid DNA (pDNA), the synthetic polymer, 25 kDa branched Polyethylenimine (PEI), can be formulated for aerosol delivery to the lungs.

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.

The use of carboxymethylcellulose gel to increase non-viral gene transfer in mouse airways.

We have assessed whether viscoelastic gels known to inhibit mucociliary clearance can increase lipid-mediated gene transfer. Methylcellulose or carboxymethylcellulose (0.25-1.

Limitations of the murine nose in the development of nonviral airway gene transfer.

A clinical program to assess whether lipid GL67A-mediated gene transfer can ameliorate cystic fibrosis (CF) lung disease is currently being undertaken by the UK CF Gene Therapy Consortium. We have evaluated GL67A gene transfer to the murine nasal epithelium of wild-type and CF knockout mice to assess this tissue as a test site for gene transfer agents. The plasmids used were regulated by either (1) the commonly used short-acting cytomegalovirus promoter/enhancer or (2) the ubiquitin C promoter.

An immunocytochemical assay to detect human CFTR expression following gene transfer.

Background: To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery.

Methods: We characterised an antibody (G449) which satisfied the criteria for use in clinical trials.

Assessment of CFTR function after gene transfer in vitro and in vivo.

Cystic fibrosis (CF) a monogenic lethal disease and, therefore, ideally suited for the development of gene therapy. The first clinical trials were carried out shortly after cloning the CF gene in 1989. Since then, 25 trials have been carried out.

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.

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 (>or=56 d) in vivo transgene expression in the absence of lung inflammation.

Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice.

Gene therapy is being investigated in the treatment of lung-related aspects of the genetic disease, Cystic fibrosis (CF). Clinical studies have demonstrated CF transmembrane conductance regulator (CFTR) expression in the airways of adults with CF using a variety of gene transfer agents. In utero gene therapy is an alternative approach that facilitates vector transduction of rapidly expanding populations of target cells while avoiding immune recognition of the vector.

Coating of adeno-associated virus with reactive polymers can ablate virus tropism, enable retargeting and provide resistance to neutralising antisera.

Background: Copolymers based on poly-[N-(2-hydroxypropyl) methacrylamide] (HPMA) have been used previously to enable targeted delivery of adenovirus. Here we demonstrate polymer-coating techniques can also be used to modify and retarget adeno-associated virus (AAV) types 5 and 8.

Methods: Three strategies for modifying transductional targeting of AAV were employed.

Lack of repeat transduction by recombinant adeno-associated virus type 5/5 vectors in the mouse airway.

While recombinant adeno-associated virus (rAAV) vectors promote long-term transgene expression in the lungs and other organs, the goal of correcting chronic inherited lung diseases such as cystic fibrosis with this type of viral gene transfer vector is limited by the requirement of achieving stable potent transgene expression, potentially requiring vector readministration. Here we evaluated the abilities of rAAV type 5/5 (rAAV5/5) vectors based on the genome and capsid of AAV5 to efficiently transduce the lungs and nasal epithelium of mice after repeated administration. Transduction efficiency as judged by reporter gene expression was markedly reduced on a second rAAV5/5 administration and effectively abolished on a third.

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

Background: 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.