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

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.

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.

The use of laboratory animals remains central to study the pathology of infectious diseases and the identification of new biological pathways that can be crucial to the process of drug discovery. Currently, these studies uses tens-of-thousands of mice per infectious disease, while the use of an in vitro model could significantly replace this requirement, mainly when genetic susceptibility factors for the development of an infectious disease will be studied. There is increasing data which suggests in vitro systems are valid and in some instances preferable experimental platforms for addressing clinically pertinent questions of genetic susceptibility factors to disease, for example in cystic fibrosis, hepatitis or tuberculosis infections.
The aim of this study is to replace the use of animal models in interrogating genetic susceptibility factors for the development of the most frequent form of fungal allergy, allergic bronchopulmonary aspergillosis (ABPA). Globally, more than 500,000 mice have been used in studying genetic susceptibility factors to respiratory infectious disease since 2000 according to the recorded data in pubmed. This project, will directly contribute to use a minimum of 200 fewer mice and a minimum of £20k will be saved.
The current proposal will expand the body of data in which important insights into human susceptibility factors for fungal diseases can be derived directly from in vitro and ex vivo models, and thereby reinforce the validity of this approach. There are a number of additional points in this proposal which are directly relevant to the 3Rs strategy: (i) disease in animals differs from human, (ii) some of the genes associated to disease in humans are not present in mice, (iii) there is no good animal model to study fungal allergy, (iv) endpoint to test fungal allergy is not death. Thus, the development of alternative models using cell lines or primary cells to avoid the severity of the procedures in mouse models to study fungal allergy and widely any other infectious diseases, is directly compatible with the mission and philosophy of the 3Rs.
The major benefit of this research is the demonstration of a method by which data acquired using an in vitro an ex vivo experimental model can be used to make inferences regarding genetic susceptibility factors to fungal diseases. This model will have a clear impact at the Manchester Fungal Infection Group in the University of Manchester where the animal models used to study other fungal diseases such as chronic pulmonary aspergillosis an invasive aspergillosis might be considerable replaced in more than 50%. The proposed study will have therefore, a clear impact on scientists pursuing the advancement in the discovery of genetic risk factors underlining other infectious diseases and in this way to have a higher impact in the 3Rs mission by decreasing the high number of animals used in biological research.
Finally, results derived from this project will impact on people suffering from disease, clinicians and biotech companies. More than 4 million individuals suffer from asthma in the UK with >100,000 cases of ABPA. Fungal complications of asthma are uncommon but give rise to disproportionate severity accounting for a large proportion of hospital admission and healthcare costs. Knowledge of pathways underpinning fungal complications of asthma is likely to inform much needed development of biomarkers for diagnosis in this area. Furthermore, patients identified as having ABPA face lifelong treatment with expensive antifungal drugs, thus novel ABPA therapies are also required.