Down syndrome (DS) is the most common genetic disorder occurring in about 1 in 800 live births, and is characterized by a distinctive facial appearance, intellectual disability, and developmental delays. Gene dosage imbalance in DS patients, primarily caused by an extra copy of chromosome 21 (trisomy 21), is thought to contribute to a broad spectrum of coexisting medical conditions. DS is frequently associated with congenital heart defects (CHDs); approximately 40% of DS patients have some form of CHD, with atrioventricular septal defects (AVSD) being the most prevalent. The swift global spread of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the ongoing pandemic. Intriguingly, DS patients are more vulnerable to SARS-CoV-2 infection: there is a 4-fold increased risk for COVID- 19-related hospitalization and a 10-fold higher risk for COVID-19-related death compared to patients without DS. Mechanisms by which individuals with DS are more susceptible to COVID-19 are largely unknown. Genetically engineered mice made trisomic for homologous chromosome 16 (MMU16) were developed to study genotype- phenotype correlations in DS. However, because authentic SARS-CoV-2 is unable to infect mice due to the inefficient interaction between the viral S-protein and the mouse orthologue of its receptor, human angiotensin converting enzyme 2 (ACE2), it is not ideal to recapitulate SARS-CoV-2 infection in DS patients using current DS mouse models. In this R21 proposal, we aim to bridge this knowledge gap by dissecting the mechanistic causes of the susceptibility of DS patients to COVID-19 using patient-specific induced pluripotent stem cells (iPSCs). Some genes encoded by chromosome 21 (e.g. transmembrane proteinase serine 2, TMPRSS2) are dysregulated in individuals with DS and have been implicated to have a role in SARS-CoV-2 infection. Our central hypothesis is that upregulation of TMPRSS2 in DS leads to enhanced SARS-CoV-2 infection in the lungs, resulting in an enhanced cytokine surge that increases the severity of COVID-19. In Specific Aim 1, we will elucidate the mechanisms underlying the enhanced cytokine surge in Down syndrome in response to SARS- CoV-2 infection using DS iPSC-derived lung organoids. In Specific Aim 2, we will determine the gene-dosage effect of TMPRSS2 on SARS-CoV-2 infection in DS iPSC-derived cardiac and endothelial cells. It is expected that this project will have a major impact on the understanding of susceptibility of DS patients to SARS-CoV-2 infection using clinically relevant and patient-specific cardiac and lung cells.