Development of Noninvasive Bioluminescence Imaging for Cancer Diagnosis and Thera

描述（由申请人提供）：本研究的最终目标是为完全自主的无试剂生物发光（lux）报告基因系统提供一种新的平台技术，用于癌症诊断、体内成像、基于组织的生物传感和哺乳动物细胞中的高通量筛选。近年来，利用荧光蛋白（如GFP）、萤火虫荧光素酶（luc）或水蛋白进行体外和体内成像的报告基因技术取得了巨大进展。然而，对于更广泛的成像和生物传感应用，这些报告系统中的每一个都具有固有的局限性，这些局限性可归因于诸如高背景、细胞毒性或对外源试剂添加的要求等因素。我们最近的发展已经证明了细菌生物发光系统（luxCDABE）在低等真核生物中的完全表达以及“人源化”细菌荧光素酶（luxAB）在人细胞系中的表达。这些发展提供了一个明确的路径，克服其他报告系统的局限性，并创造了一个新的能力，在体内基因表达成像在早期诊断，治疗效果和疾病复发的哺乳动物系统。这种能力建立在lux报告基因系统的基础上，该系统提供了作为基因表达响应的完全自催化光产生所需的所有生物化学底物的内源合成和再循环。在我们迄今为止的努力中，使用生物信息学分析和递归PCR方法来重新设计发光杆菌的细菌荧光素酶luxAB基因，以在HEK 293细胞中进行密码子优化表达。这项工作证明了在添加正癸醛的无细胞测定中证明的具有高水平生物发光的转录物和成熟蛋白的体内产生。这项拟议的研究的具体目标是构建一个稳定的哺乳动物细胞系，能够自主生物发光的完整的勒克斯操纵子（luxCDABE）的表达，并阐明本报告的组成性自主生物发光成像在结直肠癌细胞模型的价值。这项研究的工作假设是，人类癌细胞系（HCT-116）可以被工程化，以有效地表达完整的细菌生物发光反应，并在体内监测。本研究的最终目标是提供一种新的平台技术，用于癌症诊断，体内成像，基于组织的生物传感和哺乳动物细胞中的高通量筛选的完全自主的，无试剂的生物发光（lux）报告基因系统。该技术将提供在整个动物模型中对早期诊断、治疗功效和疾病复发中的体内基因表达进行成像的能力。

DESCRIPTION (provided by applicant): The ultimate goal for this research is to provide a new platform technology for a fully autonomous, reagentless bioluminescent (lux) reporter gene system for cancer diagnostics, in vivo imaging, tissue-based biosensing, and high throughput screening in mammalian cells. Reporter gene technology for in vitro and in vivo imaging employing fluorescent proteins (such as GFP), firefly luciferase (luc), or aqueorin has made tremendous advances in recent years. However, for even broader imaging and biosensing applications, each of these reporter systems has intrinsic limitations attributable to factors such as high background, cytotoxicity, or requirements for exogenous reagent additions. Our recent developments have demonstrated complete expression of the bacterial bioluminescence system (luxCDABE) in lower eukaryotes as well as expression of "humanized" bacterial luciferase (luxAB) in human cell lines. These developments provide a clear path for overcoming limitations of other reporter systems and the creation of a new capability for imaging in vivo gene expression in early diagnosis, therapeutic efficacy and disease re-occurrence in mammalian systems. This capability builds upon the lux reporter gene system which provides endogenous synthesis and recycling of all biochemical substrates required for fully auto-catalytic light production as a gene expression response. In our efforts to date, a bioinformatics analysis and recursive PCR approach were used to re-engineer the bacterial luciferase, luxAB genes, of Photorhabdus luminescens for codon optimized expression in HEK293 cells. This work demonstrated in vivo production of transcript and mature protein with high levels of bioluminescence demonstrated in a cell-free assay with added n-decanal. The specific goal of this proposed research is to construct a stable mammalian cell line capable of autonomous bioluminescence from expression of the complete lux operon (luxCDABE) and to elucidate the value of this reporter by constitutive autonomous bioluminescence imaging in a colorectal cancer cell model. The working hypothesis of this research is that a human cancer cell line (HCT-116) can be engineered to efficiently express the complete bacterial bioluminescence reaction and be monitored in vivo. The ultimate goal for this research is to provide a new platform technology for a fully autonomous, reagentless bioluminescent (lux) reporter gene system for cancer diagnostics, in vivo imaging, tissue-based biosensing, and high throughput screening in mammalian cells. This technology will provide the capability for imaging in vivo gene expression in early diagnosis, therapeutic efficacy and disease re-occurrence in whole animal models.