Project Summary Osteoporosis is a disease of skeletal fragility that causes fractures in 50% of women and 25% of men over age 50. The most commonly prescribed anti-resorptive osteoporosis medications cannot cure osteoporosis, while currently available bone-building anabolic osteoporosis medications are limited by waning efficacy. There is still a great unmet need for safe and sustained approaches to increasing bone formation. We have shown that 2.3-kb rat type I collagen promoter-driven green fluorescent protein (Col2.3GFP) is highly expressed in mature osteoblasts. We isolated Col2.3GFP(hi) osteoblasts from bones, cultured bone chips, and directed differentiation of embryonic stem cells, and by RNA-sequencing identified 593 genes that are enriched in mature osteoblasts. Gene ontology (GO) analysis identified ER-to-Golgi vesicle trafficking as the most highly enriched GO term. Our preliminary data reveal that transient knockdown in MC3T3 osteoblasts of several vesicle trafficking genes results in increased mineralized nodule formation and accelerated osteoblast marker expression. Our central hypothesis is that disruption of vesicle trafficking impairs bone formation due to collagen overmodification and hypermineralization. We will leverage several innovative methods: direct reprogramming of fibroblasts to derive induced osteoblasts, CRISPR/Cas9 gene editing to delete vesicle trafficking genes in induced osteoblasts, Col2.3GFP as a cell-autonomous osteoblast reporter, and subcutaneous transplantation of gene-edited osteoblasts to assess in vivo bone formation. We screened vesicle trafficking genes with transient siRNA knockdown, and selected 9 genes (Preb, Stx5a, Rab2a, Gosr2, Bet1, Ramp1, Arf4, Cog6, Pacs1) whose knockdown increased mineralized nodule formation, osteoblast marker expression, and ER stress. In Aim 1 we will determine whether disruption of vesicle trafficking increases mineralization due to collagen overmodification. We will perform permanent knockdown by CRISPR/Cas9 gene editing of each gene in mouse and human induced osteoblasts with the Col2.3GFP osteoblast reporter and assess osteoblast marker expression and mineralized nodule formation. We will measure collagen production and post-translational modification by prolyl hydroxylation, and determine whether inhibition of collagen overmodification can restore osteoblast function. In Aim 2 we will determine whether disruption of vesicle trafficking impairs bone formation in vivo. We will examine bone formation in vivo by subcutaneous transplantation of gene-edited mouse and human iOBs. We will determine whether inhibition of collagen overmodification restores bone formation in vivo. Successful completion of these aims will identify novel genes involved in osteogenesis as potential therapeutic targets for the treatment of osteoporosis, and will provide mechanistic insights into the role of vesicle trafficking machinery in osteoblast function.