Down Syndrome (DS) is a genetic disorder caused by the triplication of chromosome 21. DS is characterized by defective brain development, manifested by intellectual disability. The molecular mechanisms causing it are not fully understood. Amyloid precursor protein (APP) resides on chromosome 21, thus triplicated in DS. APP plays a role in developmental and post-natal neurogenesis. However, studies examining the role of APP overdose on cortical malformation in DS are scarce. Approximately fifty percent of persons born with DS develop Alzheimer’s Disease (AD) by sixty years of age. Mutations in APP cause familial AD. Increased dosage of APP is predicted to result in enhanced accumulation of APP cleavage products including β-amyloid (Aβ). Aggregated Aβ comprises hallmark amyloid plaques observed in AD brains. Notably, patients with trisomy of chromosome 21 lacking the APP locus do not develop AD. Thus, it would be important to determine whether an additional copy of APP is essential or necessary for the development of AD in DS. Our preliminary studies show that signals critical for neural stem cell proliferation, neuronal differentiation and lamina specification are reduced in DS organoids. This project will test the hypothesis that APP overexpression causes or contributes to deficits in neurogenesis and Alzheimer’s pathology in Down Syndrome. We applied CRISPR-Cas9 technology to eliminate one copy of APP from Down syndrome-derived iPSC [DSAPP]. Using brain organoids developed from DSAPP, DS and isogenic iPSC, experiments in Aim 1 will examine the role of APP in neural stem cell proliferation and neural lineage commitment in DS, Aim 2 will address the role of APP in neuronal differentiation and cortical organization, and Aim 3 will examine whether APP overdose in DS is essential or necessary for the development of AD pathology. These experiments will address critical gaps in understanding DS and AD and identify new therapeutic targets for these disorders.