权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Vesicle Trafficking and Osteoblast Function

囊泡运输和成骨细胞功能

基本信息

批准号：
10709486
负责人：
JOY Y WU
金额：
$ 17.12万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-23 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10709486
关键词：
Acceleration Adult Affect Age Biological Assay Biology CRISPR/Cas technology Cells Collagen Collagen Type I Data Fibroblasts Fracture Future Gene Deletion Gene Expression Gene Mutation Genes Golgi Apparatus Green Fluorescent Proteins Human Hydroxylation Impairment Knockout Mice Measures Methods Modification Monitor Mus Nodule Ontology Osteoblasts Osteogenesis Osteoporosis Pharmaceutical Preparations Pluripotent Stem Cells Post-Translational Protein Processing Production Protocols documentation Qualifying Rattus Reporter Role Small Interfering RNA Source Techniques Vesicle Woman bone conditional knockout directed differentiation embryonic stem cell endoplasmic reticulum stress gene transplantation for gene therapy in vitro Assay in vivo innovation insight interest knock-down men mineralization new therapeutic target novel osteoblast differentiation promoter skeletal disorder small hairpin RNA subcutaneous therapeutic target trafficking transcriptome sequencing

项目摘要

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.

项目总结