Brown, Thomas Iain

2019-02-15T14:32:36Z

2019-02-15T14:32:36Z

2005

Previous work has demonstrated the importance of neutrophil derived proteolytic enzymes in the pathogenesis of acute pulmonary inflammation. The protease/anti-protease balance theory of lung disease relies on the presence of substances within the lung which inhibit these enzymes. These include alpha-1-antiprotease, secretory leukoprotease inhibitor (SLPI) and elafin. Indeed these inhibitors have been used to modulate the inflammatory response both in vitro and in vivo. Whilst only limited data currently exists in relation to the ovine orthologs of these inhibitors the potential of this species as an intermediate model for lung-directed gene therapy has recently been highlighted and warrants further applied research. This thesis is directed towards this aim.

The first section details the cloning and characterisation of the ovine forms of SLPI and elafin. The ovine orthologs show many characteristics in common with the human forms of these proteins including distributions at mucosal sites and activity against neutrophil elastase in vitro. The identification of the sequences of the genes encoding ovine SLPI and elafin has expanded the current knowledge based on members of the whey acidic protein (WAP) and Trappin families of protein.

The ovine elafin sequence facilitated the construction of a replication-deficient adenovirus (Ad-oelafin) for use as an efficient vector in lung-directed gene therapy in the context of lipopolysaccharide (LPS)-mediated direct lung injury. The data demonstrates that local lung administration of Ad-o-elafin up-regulates the innate immune response to intra-pulmonary LPS both within areas of the lung directly exposed to the virus and also in spatially disparate lung segments not receiving adenovirus. Additionally, the endobronchial administration of Ad-o-elafin abrogates the circulating leukocytosis seen as a result of intra-pulmonary LPS instillation when compared to control adenovirus. This suggests a role for local elafin up-regulation in the stimulation of the innate immune response with a concomitant decrease in the systemic response.

These data expand a relatively narrow knowledge base in relation to lung-directed adenoviralmediated gene therapy in large animal models. In particular the extent of influence of a local gene delivery has been highlighted and is potentially of direct relevance to the design of applied protocols aimed at modulating the inflammatory response in the lung.

http://hdl.handle.net/1842/34908

The University of Edinburgh

Annexe Thesis Digitisation Project 2019 Block 22

Anti-protease gene therapy in the lung

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy