NOVEL MARKERS FOR IMAGING GENE EXPRESSION IN VIVO USING MRI

非侵入性成像的能力，在体内的基因表达的持续时间和位置是重要的生物医学研究和分子医学的未来。本申请的目的是开发可用于各种分子成像应用的通用MRI报告基因盒。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递送到活小鼠脑中可以在MR图像中引起强有力的对比。当细胞从生物体中螯合内源性铁时，报告蛋白被制成超顺磁性的。我们的方法是独特的，因为细胞通过遗传手段原位构建MRI造影剂。不需要大体积金属络合物，从而简化了细胞内递送。我们的建议包括三个具体目标：（1）我们将设计第二代MRI报告基因，以提高灵敏度和最小报告基因序列长度。MRI报告基因将被整合到复制缺陷型病毒载体中，包括腺病毒、单纯疱疹病毒和水疱性口炎病毒，用于随后的细胞培养和体内研究;（2）使用病毒载体，我们将在已建立的细胞系中对报告基因的化学、生物化学、生物物理和毒理学性质进行广泛的体外分析;（3）我们将通过在小鼠CNS中纵向成像MRI报告子来证明体内原理验证。我们将通过局部注射和使用组织特异性启动子靶向其表达来定量评估它们在不同组织中的对比效应。最后，我们将使用一系列神经病理学标记物寻找转导神经元的脑组织病理学变化的证据。总体而言，该平台技术可适用于检查许多组织类型中的基因表达，因此存在大量潜在的体内应用。实例包括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，与NIH的使命高度相关。 使人们能够可视化活生物体内基因表达的工具对医学和生物医学研究的未来至关重要。新兴的遗传医学领域需要非侵入性成像方法，可以指示治疗基因是否已被递送到正确的细胞以及所需的蛋白质是否已被制造。 为了实现这一目标，我们的项目将开发新技术，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.