Eric Alton

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. Proof of principle for low-level airway gene transfer was established in most, but not all, trials. It is currently unclear whether current gene transfer efficiency will lead to improvements in clinically relevant endpoints such as inflammation or infection. In addition to addressing this important question, we and others are further improving airway gene transfer, by modifying existing and developing new gene transfer agents. Here, we describe pre-clinical methods related to assessing correction of the CF chloride transport defect.

Cystic Fibrosis Gene Therapy – Not Low-hanging Fruit

T he last 25 years have shown that it has been comparatively slow and difficult to develop cystic fibrosis (CF) gene therapy; the lung is a complex target organ. However, research has steadily progressed and recently it was shown that non-viral gene therapy can stabilise CF lung disease. These data, in addition to the development of potent lentiviral vectors, have renewed interest in CF gene therapy within academia and industry.

Molecular Therapies for Cystic Fibrosis

Abstract This chapter describes the therapeutic strategies for cystic fibrosis which are based on targeting cystic fibrosis transmembrane conductance regulator (CFTR), either at the gene or protein level. We provide updates on small molecule CFTR modulators and gene therapy, focusing on clinical development and evaluation. The field has seen significant progress over recent years, particularly with the CFTR potentiator, ivacaftor, in patients with class III mutations. Increased understanding of the abnormalities in the structure and function of CFTR protein will help optimize the approaches required for normalizing function and, in doing so, aid the rational design of clinical trials—both in terms of the development of more efficacious drugs and the selection of appropriate patient populations. While progress with gene therapy remains some way behind, potential benefits (including being mutation agnostic and a nonsystemic route of delivery) remain significant. It may be that future optimal approaches will harness the benefits of more than one of these approaches and lead to considerable synergy. The ultimate goal for molecular and advanced therapies in cystic fibrosis is to find drugs or combinations of drugs capable of restoring CFTR function, applicable to patients with any genetic mutation.

Gene Therapy for Lung Diseases

Gene therapy is under development for a variety of lung disease, both those caused by single gene defects, such as cystic fibrosis and α1-antitrypsin deficiency, and multifactorial diseases such as cancer, asthma, lung fibrosis, and ARDS. Both viral and nonviral approaches have been explored, the major limitation to the former being the inability to repeatedly administer, which renders this approach perhaps more applicable to conditions requiring single administration, such as cancer. Progress in development and clinical trials in each of these diseases is reviewed, together with some potential newer approaches for the future.

496. Recombinant Sendai Virus Vector to Evaluate Vector Distribution in a Large Animal Model of Lung Gene Transfer

We have described gene delivery in sheep as a large animal model for airway gene transfer. This model allows comparison of different GTAs in terms of efficacy and safety using doses, delivery methods and assays, relevant to human studies. Proof-of-principle for non-viral gene transfer has been established however, levels of transgene expression are too low on a per cell basis to characterise which cells are targeted. Our preferred method for delivery is by aerosol however, not all GTAs are stable following aerosolisation in conventional jet or ultrasonic nebulisers and the cost of producing GTAs in sufficient quantities for whole lung aerosol studies can be prohibitive. These issues were behind our initial studies with delivery by instillation to individual segments. Unfortunately this approach is associated with increased toxicity as a result of pooling of material in the terminal bronchiole/alveolar duct regions. The Trudell AeroProbe* catheter develops an aerosol at the tip and can be passed through a bronchoscope. This allows localised delivery of GTAs, reducing the amount of material required. A further advantage of the AeroProbe* is that it is a single-pass aerosol device and subjects the GTA to less shear stress. An important question to be addressed when comparing delivery with different devices, is how their respective distribution patterns compare. Here we used a recombinant Sendai virus (DF/SeV-LacZ) vector, shown to be highly efficient at transducing airway epithelial cells in a variety of species, to investigate distribution patterns of delivery methods. SeV was delivered either by instillation to single lung segments via a polyethylene catheter (PEC), by AeroProbe* directed at segments or by AeroProbe* directed at the whole lung. In terms of stability, 100% and 49 +/- 3% of infectious virus particles survived the PEC and AeroProbe* respectively (n=3/group). In vivo studies show the variation in distribution of the different delivery methods. Instillation of 5ml SeV (3e9 CIU) to the same segment of different animals (n=3) results in varied distribution. Data from these animals confirm the absence of spillover as determined by histological assessment of adjacent segments. Segmentally-directed delivery of 5ml SeV (1e10 CIU) via AeroProbe* gives a different distribution. We observed areas of expression at airway bifurcations from impaction of aerosol particles at these sites and expression around sub-mucosal gland openings and within the glands themselves. Spillover to adjacent segments was observed in this case. Delivery of 24ml SeV (4e10 CIU) to the whole lung via the AeroProbe* gave a different type of expression pattern again. SeV was distributed throughout the lung. An even pattern of distribution all the way to the peripheral alveolar regions can be observed in several segments although the extent varies. These distribution patterns will have implications for sampling. It is clear from these studies that the properties of SeV make it an ideal vector to study the distribution pattern of different devices in our ovine model.