PSMA-based Gene Reporter-Probe System

基于 PSMA 的基因报告探针系统

• 批准号: 7140356
• 负责人: MARTIN G POMPER
• 金额: $ 35.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140356
• 关键词: NIH Roadmap Initiative tag; SCID mouse; bioimaging /biomedical imaging; carboxypeptidase; cell line; chemical cleavage; confocal scanning microscopy; enzyme activity; enzyme substrate; fluorescent dye /probe; molecular /cellular imaging; molecular probes; peptide chemical synthesis; plasmids; positron emission tomography; protein localization; reporter genes; surface antigens; technology /technique development; transfection /expression vector

DESCRIPTION (provided by applicant): For cellular and molecular imaging to gain clinical utility beyond the standard probes used in nuclear medicine and the few experimental trials that employ super-paramagnetic nanoparticles, a practical, biocompatible system that employs signal amplification to improve sensitivity is needed. Use of gene reporter/probe systems is becoming recognized as the best way not only to track the movement or activation of cells but also to study protein-protein interactions and other aspects of signal transduction non-invasively. A survey of the field shows that the gene reporter/probe systems that have been developed to date lack either bio-compatibility due to immunogenicity, suffer from relatively low sensitivity, are already widely expressed throughout normal tissues endogenously or are incapable of reliably sequestering the imaging agent within the cell in a high-affinity interaction. For those reasons we have chosen to embark upon a focused, interdisciplinary program to build and test a sensitive, multimodality gene reporter/probe system based on the prostate specific membrane antigen (PSMA). PSMA is ideally suited to becoming a gene reporter/probe system because it is a human, transmembrane protein that has a highly restricted expression pattern, which will limit background signal, and because the corresponding optical, magnetic resonance and nuclear imaging probes are remarkably simple to synthesize. Most germane to this application, however, is the fact that PSMA possesses enzymatic and transporter activities, and we intend to exploit those features to develop a high-sensitivity reporter/probe system. Specifically, we will 1) construct a dual enzymatic plasmid that will produce firefly luciferase concurrently with PSMA, 2) synthesize select, fluorescent, high-affinity probes to PSMA, 3) prepare enzymatic PSMA-based reporter substrates that will be both fluorescent and radioactive and 4) validate this system in vitro and in vivo. Because the high-affinity probes do not serve as substrates, we will be able to compare their ability to those of the enzymatic probes for detection of cellular events, essentially producing a variety of probes with different pharmacokinetics and sensitivities for different indications. We believe that the PSMA gene reporter/probe system will represent a clinically translatable alternative to those that currently exist and that because of the enzymatic and transporter capabilities of PSMA, the system will also be of significantly enhanced sensitivity.

描述（由申请人提供）： 为了使细胞和分子成像在核医学中使用的标准探针和少数使用超顺磁性纳米粒子的实验试验之外获得临床实用性，需要一种实用的生物相容性系统，该系统利用信号放大来提高灵敏度。基因报告/探针系统的使用逐渐被认为是跟踪细胞运动或激活的最佳方法，也是非侵入性研究蛋白质-蛋白质相互作用和信号转导其他方面的最佳方法。该领域的调查表明，迄今为止开发的基因报告/探针系统要么由于免疫原性而缺乏生物相容性，要么灵敏度相对较低，要么已经在正常组织中广泛内源表达，要么无法可靠地隔离成像细胞内的药物以高亲和力相互作用。出于这些原因，我们选择开展一项重点突出的跨学科计划，以构建和测试基于前列腺特异性膜抗原 (PSMA) 的敏感、多模态基因报告基因/探针系统。 PSMA 非常适合成为基因报告器/探针系统，因为它是一种人类跨膜蛋白，具有高度受限的表达模式，这将限制背景信号，并且因为相应的光学、磁共振和核成像探针非常容易使用合成。然而，与该应用最密切相关的是 PSMA 具有酶活性和转运蛋白活性，我们打算利用这些特征来开发高灵敏度报告/探针系统。具体来说，我们将 1) 构建双酶质粒，该质粒将与 PSMA 同时产生萤火虫荧光素酶，2) 合成针对 PSMA 的选择性荧光高亲和力探针，3) 制备基于酶 PSMA 的报告底物，该底物将具有荧光和放射性4）在体外和体内验证该系统。由于高亲和力探针不充当底物，因此我们将能够将它们与酶探针检测细胞事件的能力进行比较，从而基本上生产出针对不同适应症具有不同药代动力学和敏感性的各种探针。我们相信，PSMA 基因报告/探针系统将代表现有系统的临床可转化替代方案，并且由于 PSMA 的酶促和转运功能，该系统的灵敏度也将显着增强。