Stanford Technology Accelerating Medicines Partnership Center

 描述(由申请人提供)：斯坦福技术加速药物伙伴关系(STAMP)中心的广泛、长期目标是在RA/SLE AMP网络中为AMP研究开发和实施多路机械分析方面的领导者。虽然我们建议专注于SLE，但我们的方法将用于RA的初步研究，并可应用于任何自身免疫性疾病。参与AMP计划的研究人员和斯坦福大学的其他合作者一直在高通量基因组学和蛋白质组学技术的开发方面引领创新者，用于研究癌症和自身免疫。项目1(Steve Quake和Howard Chang)将使用从组织样本和血液中分离的单个细胞的RNA-Seq进行转录谱分析，然后使用基于Fluidigm的微流体设备(C1)或FACS将其分离到单独的孔中。Chang实验室将确定转录因子的结合以打开染色质，抑制和/或激活涉及NFAT、NFB、RORS、IRF、STATS和其他转录因子的转录程序。项目2(Garry Nolan)将使用CyTOF研究从血液和组织中获得的细胞，表征离散细胞亚群中的细胞表面分子、信号分子和磷酸特异性表位。来自项目1的上调转录本将在UH3资助期的迭代过程中通知细胞周期蛋白抗体的选择。项目3(Bill Robinson、Mark Davis和PJ Utz)将使用自身抗体分析、流式细胞仪、曲线谱测序和酵母展示来表征血液和组织中分离的抗原特异性B和T细胞。罗宾逊和戴维斯实验室将通过对FACS分类的浆母细胞、FACS或四聚体分类的T细胞以及从组织中分离的细胞的重链和轻链基因进行测序，来表征B和T细胞受体的谱系。将克隆、表达和纯化单抗以用于抗原鉴定。将使用蛋白质阵列发现抗原靶标(Utz和Robinson)，这将成为所有AMP网络中心的核心分析。还提出了试点项目，包括血浆病毒测序和开发和实施多路离子束成像(MIBI)的两个项目。最后，为了确保血液、肾脏、皮肤和滑膜的可用性，我们将从斯坦福大学和旧金山湾区(SLE)、加州大学洛杉矶分校(UCLA)和雪松-西奈(SLE)、加州大学洛杉矶分校(UCSD)(RA)以及其他AMP中心招募受试者。在一个AMP中心集成所有分析将有助于发现途径、分子和新的药物靶点。

 DESCRIPTION (provided by applicant): The broad, long term objective of the Stanford Technology Accelerating Medicines Partnership (STAMP) Center is to serve as the leader within the RA/SLE AMP Network in the development and implementation of multiplexed mechanistic assays for AMP studies. Although we propose to focus on SLE, our methods will be used in pilot studies for RA and can be applied to any autoimmune disease. Investigators participating in this AMP proposal, and other collaborators at Stanford, have been leading innovators in the development of high-throughput genomics and proteomics technologies for studying cancer and autoimmunity. Project 1 (Steve Quake and Howard Chang) will perform transcript profiling using RNA-Seq of single cells isolated from tissue samples and blood, then separating into individual wells using Fluidigm-based microfluidics devices (C1) or FACS. The Chang lab will determine binding of transcription factors to open chromatin, repressing and/or activating transcriptional programs involving NFAT, NF¿B, RORs, IRFs, STATs, and other transcription factors. Project 2 (Garry Nolan) will employ CyTOF to study cells obtained from blood and tissue, characterizing cell surface molecules, signaling molecules, and phospho-specific epitopes in discrete cellular subsets. Upregulated transcripts from Project 1 will inform selection of CyTOF antibodies in an iterative process during the UH3 funding period. Project 3 (Bill Robinson, Mark Davis and PJ Utz) will use autoantibody profiling, FACS, repertoire-sequencing and yeast display to characterize antigen- specific B and T cells in blood and isolated from tissues. The Robinson and Davis labs will characterize the B and T cell receptor repertoire by sequencing heavy and light chain genes from FACS sorted plasmablasts, and FACS or tetramer sorted T cells, as well as cells isolated from tissue. Monoclonal antibodies will be cloned, expressed, and purified for antigen identification. Antigen targets will be discovered (Utz and Robinson) using protein arrays, which will serve as a core assay for all AMP Network Centers. Pilot projects are also proposed including plasma virome sequencing and two projects to develop and implement multiplexed ion beam imaging (MIBI). Finally, to insure availability of blood, kidney, skin, and synovium for our studies, subjects will be recruited from Stanford and the Bay Area (SLE); UCLA and Cedars- Sinai (SLE); and UCSD (RA), as well as from other AMP Centers. Integration of all assays at one AMP Center will facilitate discovery of pathways, molecules, and new drug targets.