COVID-19 Expansion for AIRe Program

Background: Under the DoD-funded Anticipating Influenza Resistance and Evolution (AIRE) Program, we performed single-cell RNA-Sequencing (scRNA-Seq) on nasal samples from healthy subjects and patients with influenza. These studies revealed: (1) what cells were infected with influenza (a surprisingly large number of different cell types were infected), (2) what cells were significant producers of virus (only ciliated epithelial cells), and (3) what cells were major producers of host cytokines. We also found that influenza enhanced expression of angiotensin-converting enzyme 2 (ACE2)-expressing epithelial cells-likely targets of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19). We will expand these studies by performing scRNA-Seq on both nasal wash and tracheal aspirates from patients with COVID-19 to define what cells are highly productive for virus in both the upper and lower respiratory tracts. To define targets for drugs or gene modulation, we will perform a CRISPRa/i screen using cells derived from the human airway and compare candidates to our current CRISPRa/i screen of influenza host factors. Using Cas9-expressing embryonic stem (ES) cells, we will produce organ cultures of human lung cells. Lung cells will be infected with SARS-CoV-2, and we will test the findings obtained in the CRISPR screen by knocking out selected genes and examining the effect of their deletion on virus expression. Relevance to Focus Area(s): Our proposal for the expansion award addresses the following points of the topic area, Respiratory Health: • By defining specific virus-infected human airway cells in patients with COVID-19, we can develop a strategy to minimize infection and replication. • Using scRNA-Seq, we will identify host factors to target to prevent severe COVID-19, including manifestations such as acute respiratory distress syndrome (ARDS). • By determining the function of host factors essential for SARS-CoV-2 infection, we will identify therapeutic targets for reducing the incidence and/or severity of COVID-19 and other respiratory viral pathogens. Objectives: To use a collaborative, multi-disciplinary approach to determine the host response to COVID-19 in patient samples and identify the critical factors involved in disease pathogenesis that can be targeted for effective preventive and therapeutic measures. Specific Aims: Aim 1: Identify the cell types and host responses involved in COVID-19 pathogenesis using scRNA-Seq on patient samples. Aim 2: Perform whole-genome CRISPR activating and inactivating screens to identify host factors that can be modulated to target SARS-CoV-2. Aim 3: Test the function of candidates important for SARS-CoV-2 pathogenesis using human embryonic stem cells differentiated into various cell types and organoids. Impact: Short-Term Impact: The immediate impact of successful completion of the proposed work is that we will determine the cell types infected and the host response to enable targeted therapeutic and prevention strategies; provide a better understanding of the disease pathogenesis; understand how drugs targeting SARS-CoV-2 work and help to better establish the timing of treatments. Long-Term Impact: Results from our work can help direct future therapeutic and prevention strategies; define what cells to target with drugs; what genes and host defenses might be modulated to prevent or treat infection; and provide CRISPR screening and human ES cell-based technologies that can be used to address other emerging viral threats. Relevance to Military Health: Respiratory viruses pose a great risk to the military due to the potential for rapid spread in a confined environment, where crowding, a strenuous work environment, and physiological stress occur. The COVID-19 pandemic continues to impact military readiness with disruptions to basic training and deployment. Our military is also at the frontlines helping first responders during this pandemic, and valuable resources are being diverted to address the pandemic. In addition, the disease results in higher morbidity and mortality to older individuals, making our older Veterans particularly susceptible. Less