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

哮喘是一种主要的呼吸系统疾病，其特征是与过敏原(如花粉、屋尘螨、真菌)相关的反复发作的呼吸困难和喘息。在英国，有540万人患有哮喘，其中超过10万人面临一种名为过敏性支气管肺曲霉菌病(ABPA)的进行性肺部疾病的风险。虽然这种疾病通常不会致命，但患者的生活可能会很悲惨，经济影响非常大。ABPA影响呼吸道上皮屏障功能缺陷的人，如哮喘患者，但由于真菌病原体烟曲霉在呼吸道中的生长，ABPA进一步复杂化。我们都接触过烟曲霉，但其中2%的人患有ABPA，这可能是因为患者肺部抵御真菌建立的能力存在遗传缺陷。为了考虑这种可能性，曼彻斯特真菌感染小组(MFIG)对242名ABPA携带者的基因组进行了测序，以寻找可能解释这种疾病的突变。我们已经发现，五个基因突变可能与避免烟曲霉菌从呼吸道中清除有关。然而，每个单一突变的生物学功能都需要定义。传统上，携带特定突变的转基因小鼠被用来定义基因的生物学功能。尽管如此，在老鼠身上发现的哮喘和在人类中发现的哮喘是不能比较的；因此需要一个新的模型来研究ABPA。因此，为了研究ABPA的病理学，我建议开发一种人类细胞培养系统，将携带这五种突变的细胞与正常细胞进行比较。在过去的18个月里，我一直在MFIG工作，利用携带与真菌疾病相关的突变的支气管上皮细胞开发一个体外模型。细胞的基因组编辑是通过使用CRISPR/Cas9系统完成的，该系统允许在细胞中引入突变，而不会改变基因组中的任何其他东西。这个模型允许通过比较基因组编辑的细胞和正常细胞来研究单一突变的影响。通过使用这个模型，我已经描述了ABPA患者中转录因子突变对烟曲霉菌萌发的调节作用。然而，还有其他相关方面，如ABPA的过敏反应，这不能仅用转录因子突变来解释。现在，我想与NC3R奖学金一起推进这项工作，开发一个体外模型来测试在我们上述遗传学项目中发现的5个突变基因。我将重建两种不同细胞类型的突变，即支气管上皮细胞和巨噬细胞，因为它们在ABPA免疫反应中起着至关重要的作用。我将用曲霉孢子挑战突变细胞，并将它们与正常细胞进行比较，以探索两者、细胞类型和遗传风险因素在对抗烟曲霉菌反应中的重要性。此外，我将开发一个体外模型来探索携带相同突变的上皮细胞和巨噬细胞之间的串扰相互作用，以确定在ABPA对烟曲霉的免疫反应的初始步骤中，细胞信号是从上皮细胞到巨噬细胞还是反过来。我将通过与葡萄牙明尼奥大学的研究人员合作来实现这一雄心勃勃的目标，他们在研究遗传风险因素在真菌疾病发展中的重要性方面拥有丰富的经验。最后，我的结果将通过测量鼻刷和已知遗传背景的ABPA患者的血液样本中对烟曲霉菌的反应来证实，然后与健康的捐赠者进行比较。样本将从曼彻斯特呼吸和过敏生物库获得。这将允许更好地接近人类的真实情况。生物技术公司可能会对这个项目产生浓厚的兴趣，因为基因治疗或药物开发的候选基因将从这项工作中被发现。

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.