Enhanced MR reporter for immuno-imaging.

TR&D 2摘要 BTRC的TR& D2将开发新的合成非金属报告基因， 交换饱和转移（CEST）MRI用于精确可视化和精确定位生物过程， 活的有机体。使用富含赖氨酸的蛋白质（LRP）作为这样的原型基因，我们以前已经证明了 1）我们可以对快速分裂的肿瘤细胞进行成像，而不受目前标记稀释效应的限制， 2）我们可以对启动子驱动的特异性基因表达进行成像; 3)we can image图像oncolytic溶瘤virotherapy病毒疗法.在本TR&D中，我们的目标是大幅提高CEST对比度， LRP的生物相容性，以进一步传播到科学界，并创造一个途径， 最终的临床翻译。利用先进的、基于合理设计的分子遗传工程方法， 我们将首先开发所谓的“增强型”LRP或eLRP（Aim 1a）。增强的定义是转录 和翻译效率、蛋白质重折叠、最佳质子交换率和最强CEST对比度。为 后者，定制设计的高通量筛选方法将用于确定最佳肽 序列配置。接下来，我们将使用一系列免疫学方法“人源化”eLRP以创建heLRP。 分析（目标1b）。我们将使用已建立的算法来鉴定诱导T细胞和/或体液免疫应答的表位。 相反的反应。重新工程化的LRP将经历重复的筛选过程，直到所有免疫原性 在不影响CEST对比度的情况下被消除。或者，我们将使用人鱼精蛋白-1 （hPRM 1）作为起始模板以通过DNA改组产生嵌合LRP/hPRM 1构建体。没有 来自heLRP免疫的兔的血清多克隆抗体将用作TR&D 2的最终关键标准 传播。在这个免疫学筛选过程中，我们将同时鉴定 HeLRP，即，“免疫原性”LRP或iLRP。在体内转染后，该iLRP将用作新的免疫抑制剂。 同时诱导抗肿瘤免疫应答和可视化随后肿瘤细胞治疗诊断载体 回归（目标2）。最后，我们旨在演示如何使用eLRP提供独特的动态洞察力 转化为生物过程，并由细胞间相互作用定义。我们选择了树突状细胞（DC） 以免疫疗法为例。验证研究证实组成型表达的eLRP DC 在接种后迁移至淋巴结时可在体内检测到（目标3a），我们将研究何时 并且其中DC活化在将抗原呈递给CD 4+细胞时发生。我们的目标是使用IL-12来实现这一目标。 启动子驱动的特异性表达（Aim 3b）。同时，我们将评估时间进程和生物分布 在表达Ova的黑色素瘤小鼠模型中使用BLI的活化的Ova特异性CD 4+转基因细胞。我们 LRP报告基因在基础细胞生物学和细胞功能障碍的研究中具有广泛的应用。 各种疾病模型，因为它们可以从头设计和计算机模拟，因此具有无限的潜力， 操作和微调所需的精确可视化的生物过程的问题。

SUMMARY for TR&D 2 TR&D 2 of the BTRC will develop novel synthetic, non-metallic reporter genes that can be detected with chemical exchange saturation transfer (CEST) MRI for precise visualization and pinpointing of biological processes in living organisms. Using a lysine-rich protein (LRP) as such a prototype gene, we have previously demonstrated that 1) We can image rapidly dividing tumor cells without the limitation of a label dilution effect that currently exists with conventional MR contrast agents; 2) We can image promoter-driven specific gene expression; and 3) we can image oncolytic virotherapy. In this TR&D, we aim to dramatically improve the CEST contrast and biocompatibility of LRP for further dissemination to the scientific community, and to create a pathway towards eventual clinical translation. Using advanced, rational design-based molecular-genetic engineering approaches, we will first develop a so-called “enhanced” LRP, or eLRP (Aim 1a). Enhancement is defined by transcription and translation efficiency, protein refolding, optimal proton exchange rate, and strongest CEST contrast. For the latter, a custom-designed high-throughput screening methodology will be used to determine optimal peptide sequence configurations. Next, we will “humanize” eLRP to create heLRP, using an array of immunological assays (Aim 1b). We will use established algorithms to identify epitopes that induce a T cell and/or humoral response in reverse. Re-engineered LRPs will undergo reiterated screening processes until all immunogenicity has been eliminated without compromising CEST contrast. Alternatively, we will use human protamine-1 (hPRM1) as a starting template to create chimeric LRP/hPRM1 constructs through DNA shuffling. The absence of serum polyclonal antibodies from heLRP-immunized rabbits will be used as a final key criteria for TR&D 2 dissemination. During this immunological screening process, we will simultaneously identify the counterpart of heLRP, i.e., an “immunogenic” LRP or iLRP. Following in vivo transfection, this iLRP will be used as a new theranostic vector to simultaneously induce an anti-tumor immune response and visualize subsequent tumor cell regression (Aim 2). Finally, we aim to demonstrate how eLRP can be used to provide a unique dynamic insight into biological processes and as defined by cell-cell interactions. We have chosen dendritic cell (DC) immunotherapy as an example. Following a validation study to confirm that constitutively expressed eLRP DCs can be detected in vivo when migrating to lymph nodes following vaccination (Aim 3a), we will investigate when and where DC activation occurs upon presenting antigen to CD4+ cells. We aim to accomplish this using IL-12 promoter-driven specific expression (Aim 3b). Concurrently, we will assess the time course and biodistribution of activated, Ova-specific CD4+ transgenic cells using BLI in an Ova-expressing melanoma mouse model. Our LRP reporters will have many applications in the study of basic cell biology and cell malfunctioning in a wide variety of disease models, as they can be designed de novo and in silico, and hence have unlimited potential for manipulation and fine-tuning as needed for the precise visualization of the biological process in question.