PROJECT SUMMARY As asthma worsens, occlusion of airways with mucus significantly contributes to airflow obstruction. In addition, individuals suffering with asthma are often susceptible to viral respiratory infections which may lead to severe worsening of disease symptoms. Recent evidence has suggested mucus obtained from individuals with severe asthma possesses altered mucin composition. However, how these changes alter the functional properties of the mucus gel is not yet fully understood. The overall objectives in this application are to (i) establish new models where mucus composition can be precisely controlled for mechanistic assessment of its function and (ii) understand the role of mucus composition in viral infection-induced exacerbation of disease. The central hypothesis is airway mucus with an imbalanced concentration of the 2 primary gel-forming airway mucins, MUC5B and MUC5AC, possesses significantly altered biophysical properties that leads to mucus overaccumulation and impaired function of mucus as a barrier to respiratory viruses in asthma. The central hypothesis will be tested through development of genetically engineered human lung tissue culture models capable of producing mucus with a composition representative of healthy and asthmatic airways. Our approach will enable for a mechanistic understanding of conditions under which the mucus gel becomes immobile, thus reducing its ability to be effectively cleared and increasing the likelihood for mucus plug formation in the lungs of individuals with asthma. In addition, we will explore the impact of mucin composition on the barrier function of mucus towards influenza virus. Based on these mechanistic studies, we will develop a new mucin-targeted therapeutic approach to reverse disease symptoms and prevent severe infection which will be tested using a mouse model of allergic asthma. The research proposed in this application is innovative as it employs tools from molecular biology, biophysics, and engineering to develop a novel means to manipulate and assess mucus composition by closely mimicking its properties in healthy and asthmatic airways. If successful, the results of this work will be significant as our approach may provide new insights into the biological function of mucus in asthma, also with relevance to other related muco-obstructive lung diseases. This work aligns with the goals of the Stephen I. Katz mechanism as it represents a significant shift in research direction for the PI using unique approaches and new techniques to address long-standing questions on the emergent properties of MUC5B and MUC5AC in asthma.