Developing a CEST Reporter Gene (RMI)

描述（由申请人提供）： 细胞迁移、运输和归巢的非侵入性成像是一个新兴的新领域，可以为我们提供对细胞-组织相互作用动力学的更深入了解，并为使用干细胞和祖细胞的新型细胞疗法的开发提供指导。与其他成像模态（即，PET、SPECT和生物发光成像），MR成像具有最高的空间分辨率，并且可以提供解剖学和功能信息，但是其遭受用于使用合适的标记物/示踪剂检测细胞的极高的信噪比阈值。在一定程度上，这种限制已经解决了使用细胞内的内体标记与超顺磁性纳米粒子。然而，以这种方式诱导的基于磁化率的T2（*）对比度具有显著的缺点，包括产生低强度“黑洞”（模糊组织形态）、难以区分活细胞和死细胞、低强度成像伪影的存在、长期金属毒性的不确定性，以及最重要的是，细胞增殖后标记物的稀释。 我们提出了一种新的方法，使用CEST（化学交换饱和转移）报告基因的标记细胞的MR检测。该方法基于酰胺富集的人工蛋白质的表达，即，富赖氨酸蛋白（LRP）和富银蛋白（阿普），可以通过CEST成像在纳摩尔范围内检测。使用这些分子探针的优点是：1）基因产物可以直接可视化而不需要底物（不需要组织穿透）; 2）检测灵敏度不受细胞增殖的限制; 3）只有活细胞应该提供CEST对比度; 4）对比度可以重复地“打开”和“关闭”;和5）可以采用双细胞或三细胞标记策略。我们有初步的数据表明，CEST报告基因可以克隆，在转染细胞中表达，并通过MR CEST成像在幻影中特异性检测，而不影响细胞活力或增殖。我们假设这种检测在体内也是可能的。为了实现这一目标，我们的目标是合成新的，更有效的CEST报告基因，并检测双标记的LRP/阿普转染胶质瘤细胞和神经干细胞分别在活的动物。

DESCRIPTION (provided by applicant): Non-invasive imaging of cell migration, trafficking, and homing is an emerging new field that can provide us with a deeper insight into the dynamics of cell-tissue interactions, as well as provide guidance to the development of novel cell therapies using stem cells and progenitors. Compared to other imaging modalities (i.e., PET, SPECT, and bioluminescent imaging), MR imaging has the highest spatial resolution and can provide both anatomical and functional information, but it suffers from a severely high signal-to noise threshold for the detection of cells using suitable labels/tracers. To a certain degree, this limitation has been resolved using intracellular endosomal tagging with super-paramagnetic nanoparticles. However, the magnetic susceptibility-based T2 (*) contrast induced this way has significant drawbacks, including the creation of hypointense "black holes" (obscuring tissue morphology), difficult differentiation between live and dead cells, the presence of hypointense imaging artifacts, uncertainty about long-term metal toxicity, and, most important, dilution of label following cell proliferation. We are proposing a new approach for the MR detection of labeled cells using a CEST (Chemical Exchange Saturation Transfer) reporter gene. The method is based on the expression of amide-enriched artificial proteins, i.e., lysine-rich protein (LRP) and argenine-rich protein (ARP) that can be detected by CEST imaging in the nanomolar range. The advantages of using these molecular probes are: 1) the gene product can be visualized directly without the need of a substrate (no tissue penetration needed); 2) detection sensitivity is not limited by cell proliferation; 3) only live cells should provide CEST contrast; 4) the contrast can be "switched-on" and "switched-off" repeatedly; and 5) double- or triple-cell labeling strategies may be pursued. We have initial data showing that a CEST reporter gene can be cloned, expressed in transfected cells, and specifically detected by MR CEST imaging in phantoms, without affecting cell viability or proliferation. We hypothesize that this detection is also possible in vivo. To achieve this goal, our aim is to synthesize novel, more efficient CEST reporter genes, and to detect double-labeled LRP/ARP transfected glioma cells and neural stem cells individually in live animals.