An in vivo genetic screen of lung function candidate genes using tissue specific RNAi in Drosophila melanogaster

Background: Chronic Obstructive Pulmonary Disease (COPD) is a major cause of worldwide mortality and morbidity with current strategies in management dependent on accurate diagnosis, followed by appropriate treatment. However, there are no current treatment-modifying approaches in COPD apart from smoking cessation. Genome-wide association studies (GWAS) are providing an unprecedented insight into the genetic basis of human disease including respiratory diseases such as COPD. We have recently completed a large GWAS of lung function including data from 144,318 individuals and identified 68 high priority genes for COPD for further study. However, translation of GWAS findings to new biological understanding and therapeutic opportunities remains a challenge with a heavy reliance on animal studies and slow progress as individual genes are studied one by one. There is a need to rethink this approach.

The hypotheses underlying this studentship proposal is that the use of non-mammalian systems such as Drosophila melanogaster have significant potential to accelerate translation of genetic findings to new understanding in lung disease and shift the reliance on animal models with associated benefits for the replacement, reduction and refinement of animal work.

Aims: This studentship has four specific aims, (1) Complete an RNAi screen of 61 lung function gene orthologues (representing 52/68 priority genes) in the fly dorsal thorax, (2) Complete a secondary screen in the fly tracheal system, using genes that were found to affect epithelial integrity in the fly dorsal thorax, (3) Characterise hits from Aim (2). Determine the molecular basis of phenotypes observed and (4) Translate findings into human systems.

Experimental Plan: We will express RNAi hairpin vectors in clones (using Flp/FRT, MARCM and Pannier-Gal4) to individually knock down 61 candidate genes in the dorsal thorax, followed by imaging of living pupae at 18-20h after puparium formation to observe cell and tissue morphology and behaviour in high resolution. Two independent RNAi transgenes will be used to limit false positive/negative results. Genes that are found to affect epithelial morphology and or integrity in the dorsal thorax will be tested in the larval tracheal system (ppk4-Gal4 driven RNAi expression). 3rd instar larvae will be dissected by ventral filleting and effects on epithelial cell morphology, epithelial integrity, and/or airway branching identified. Candidate genes prioritised from the tracheal screen will be investigated mechanistically including; using different GFP reporters to label subcellular structures, such as ECad:GFP to label the adherens junction or Viking:GFP to label the basement membrane. Tracheal function will also be assessed, e.g. through a behavioural assay in response to hypoxia. Finally, lead genes will be investigated to provide initial human translation including; expression profiling in lung tissue from COPD patients and control subjects, expression profiling in human lung development and investigation of the role of genes in human bronchial epithelial cells function, e.g. tight junction formation.

Impact: Overall this studentship has potential to make a paradigm shift away from the established route of generating a knockout mouse for each candidate gene one at a time to investigate gene function in the airway biology. This proof of concept study primarily addresses the replacement and reduction of animal work in respiratory research, however through the use of Drosophila genetic techniques, this project has scope to provide initial mechanistic understanding with associated refinement of animal research in the future. If all 3Rs are realised, a 50% decrease in mouse experiments represents ~16,000 mice per year. This studentship will lead to rapid advances in our understanding of genes implicated in COPD and potentially identify new therapeutic opportunities.