NOVEL MARKERS FOR IMAGING GENE EXPRESSION IN VIVO USING MRI

能够非侵入性地成像基因在体内表达的持续时间和位置对于生物医学研究和分子医学的未来是重要的。这项应用的目标是开发一种通用的MRI报告基因盒，可用于各种分子成像应用。我们实验室的最新结果表明，将编码金属蛋白报告基因的载体输送到活着的小鼠大脑中，可以在磁共振图像中产生强烈的对比度。当细胞将内源性铁从生物体中隔离出来时，报告蛋白就会变得超顺磁性。我们的方法是独特的，因为细胞通过遗传手段在原位构建MRI造影剂。不需要大块的金属络合物，从而简化了细胞内的传递。我们的建议包括三个具体目标：(1)我们将设计第二代MRI报告程序，以提高灵敏度和最小报告序列长度。我们将把核磁共振记者整合到腺病毒、单纯疱疹病毒和水泡性口炎病毒等复制缺陷病毒载体中，用于后续的细胞培养和体内研究；(2)利用这些病毒载体，我们将在已建立的细胞系中对核磁共振记者的化学、生化、生物物理和毒理学特性进行广泛的体外分析；(3)我们将通过对小鼠中枢神经系统的纵向成像来证明其在体内的原理。我们将通过焦点注射和使用组织特异性启动子靶向表达来定量评估它们在不同组织中的对比效果。最后，我们将使用一系列神经病理标记物在报告转导的脑组织中寻找病理变化的证据。总体而言，这一平台技术可以适用于检测多种组织类型的基因表达，因此在体内有大量潜在的应用。例如，基因疗法的临床前测试，例如用于治疗转移性疾病，以及用于药物开发的转基因模型。因此，这与美国国立卫生研究院的使命高度相关。层层概述--使人们能够在活体内可视化基因表达的工具对于医学和生物医学研究的未来是至关重要的。基因医学的新兴领域需要非侵入性的成像方法，以表明治疗基因是否已被传递到正确的细胞，以及所需的蛋白质是否已被制造出来。为了实现这一目标，我们的项目将开发新的技术，使用传统的磁共振成像(MRI)扫描仪在体内可视化基因表达。

The ability to non-invasively image the duration and location of gene expression in vivo is important for the future of biomedical research and molecular medicine. The goal of this application is to develop a general purpose MRI reporter gene cassette that can be used in a variety of molecular imaging applications. Recent results from our laboratory demonstrate that vectors encoding metalloprotein reporters delivered into the living mouse brain can elicit potent contrast in MR images. The reporter protein is made superparamagnetic as the cell sequesters endogenous iron from the organism. Our approach is unique because the cell constructs the MRI contrast agent in situ by genetic means. No bulky-metal complex is required, thereby simplifying intracellular delivery. Our proposal consists of three Specific Aims: (1) we will design second generation MRI reporters for improved sensitivity and minimal reporter sequence length. MRI reporters will be incorporated into replication-defective viral vectors including adenovirus, herpes simplex virus, and vesicular stomatitis virus, for subsequent cell culture and in vivo studies; (2) using the viral vectors we will perform extensive in vitro analyses of the chemical, biochemical, biophysical and toxicological properties of the reporters in established cell lines; (3) we will demonstrate proof-of-principle in vivo by longitudinally imaging the MRI reporters in the CNS of mice. We will quantitatively evaluate their contrasting effects in different tissues via focal injections and by targeting their expression using tissue-specific promoters. Finally, we will look for evidence of pathologic changes in reporter-transduced brain tissues using an array of neuropathological markers. Overall, this platform technology can be adapted to examine gene expression in many tissue types, and thus there are a large number of potential in vivo applications. Examples include preclinical testing of gene therapeutics, such as for the treatment of metastatic disease, and use in transgenic models for drug development. Thus, there is high relevance to the mission of the NIH. Lay Summary - Tools that enable one to visualize gene expression within the living organism are of fundamental importance to the future of medicine and biomedical research. The emerging field of genetic medicine requires non-invasive imaging methods that can indicate if the therapeutic genes have been delivered to the correct cells and if the desired proteins have been made. Towards this goal, our project will develop new technologies to visualize gene expression in vivo using conventional magnetic resonance imaging (MRI) scanners.