Development of new experimental models to understand the genetic basis of allergic bronchopulmonary aspergillosis (ABPA)

Lead Research Organisation: University of Manchester
Department Name: Medical and Human Sciences

Abstract

Asthma is a major respiratory disease characterized by recurrent attacks of breathlessness and wheezing associated with allergen sensitization (e.g. pollen, house dust mite, fungi). In the UK, 5.4 million people have asthma with over 100,000 at risk on a progressive lung disease called allergic bronchopulmonary aspergillosis (ABPA). While the disease is not usually fatal, patients lives can be miserable and the economic impact is very high. ABPA affects people with a defective airway epithelial barrier function such as asthmatics but is further complicated by the growth of the fungal pathogen Aspergillus fumigatus in the airways. We are all exposed to A. fumigatus but <2% of that people gets ABPA, which might be due to a genetically determined deficiency in the patients lungs ability to fend off fungal establishment.
To consider this possibility, the Manchester Fungal Infection Group (MFIG) has sequenced the genome of 242 people with ABPA looking for mutations that might explain the disease. We have found that five mutations are in genes probably involved in avoiding A. fumigatus to be cleared from the respiratory airways. However, the biological function of every single mutation needs to be defined. Classically, transgenic mouse carrying specific mutations have been used to define the biological function of genes. Nonetheless, asthma found in mice and that found in humans are not comparable; therefore a new model to study ABPA is needed. Thus, to study the pathology of ABPA, I propose the development of a human cell culture system comparing cells carrying the five mutations with normal cells. During the last 18 months, I have been working at MFIG developing an in-vitro model using bronchial epithelial cells carrying mutations associated with fungal diseases. Genome edition of cells has been done by using the CRISPR/Cas9 system that allows introducing mutations in cells without changing anything else in the genome. This model allows studying the effect of a single mutation by comparing genome-edited cells with normal ones. By using this model, I have described the role of a mutation in a transcription factor in ABPA patients which modulates A. fumigatus germination. However, there are other associated aspects like the allergic response in ABPA, which cannot be explained only by the transcription-factor mutation.
I would now like to take this work forward with a NC3Rs fellowship by developing an in-vitro model to test the 5 mutated genes discovered in our genetics project described above. I will recreate the mutations in two different cell types, bronchial epithelial cells and macrophages, since they play an essential role in the ABPA immune response. I will challenge the mutated cells with Aspergillus spores and compare them to normal cells so the importance of both, cell type and genetic risk factor in the response against A. fumigatus will be explored. Besides, I will develop an in-vitro model to explore crosstalk interactions between epithelial cells and macrophages carrying the same mutation in order to determine if cell signalling is from epithelial cells to macrophages or the other way around during the initial steps of the immune response to A. fumigatus in ABPA. I will perform this ambitious objective by collaborating with researchers from the University do Minho, Portugal, who have a great deal of experience in studying the importance of genetic risk factors in the development of fungal diseases.
Finally, my results will be confirmed by measuring the response against A. fumigatus in nasal brush and blood samples from ABPA patients with known genetic backgrounds and will then be compared with healthy donors. Samples will be obtained from the Manchester Respiratory and Allergy Biobank. This will allow a better approximation to the real situation in humans.
Biotech companies might have a strong interest in this project as candidate genes for gene therapy or for drug development will be discovered from this job.

Technical Summary

5.4 million people have asthma in the UK with over 100,000 at risk on developing allergic bronchopulmonary aspergillosis (ABPA). ABPA is a progressive lung disease caused by the mayor fungal pathogen Aspergillus fumigatus. Although the disease is not usually fatal, the economic costs are >£10 billion in the UK per year. ABPA affects people with a defective airway epithelial function such as asthmatics. However, the percentage of patients developing ABPA is <2.5%. Genetic factors for the development of disease have not been defined so far. The Manchester Fungal Infection Group, has performed a whole exome sequencing project to investigate genetic variants in ABPA patients that might play a crucial role for the development of this disease. Some of these variants are involved in avoiding A.fumigatus to grow in the respiratory airways. However, functional validation of these at risk factors is need. Transgenic animal models have been used to study genetic predisposing factors in infectious diseases. However, non-animal models will contribute significantly for the replacement of animals in biological research. A cell culture model to study the pathology of ABPA using cells carrying the specific mutations discovered in our genetics project will improve our understanding of this disease and will supply enough information to select genes for the development of new diagnostic test. This training fellowship will define whether in vitro and ex vivo models can contribute to this aim by: (i) Recreating specific gene variants associated to ABPA in monocyte and bronchial epithelial cells by CRISPR/Cas9 system and testing their role in Aspergillus persistence and allergic response. (ii) Studying the interaction between epithelial cells and macrophages to define which cell subset and genetic risk factor orchestrates the ABPA-response.(iii) Validating candidate genetic variants in primary cells from patients with known genetic background.

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