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一个完全自主的、无试剂的生物发光（lux）报告基因系统提供一个新的平台技术，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，由于高背景、细胞毒性或外源试剂添加要求等因素，每种报告系统都有内在的局限性。我们最近的研究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）在人类细胞系中的表达。这些发展为克服其他报告系统的局限性提供了一条清晰的途径，并为在哺乳动物系统中早期诊断、治疗效果和疾病复发中成像体内基因表达提供了一种新的能力。这种能力建立在lux报告基因系统的基础上，该系统提供了所有生物化学底物的内源性合成和循环，这些底物是完全自催化光生产所需的基因表达反应。在我们迄今为止的努力中，我们使用生物信息学分析和递归PCR方法重新设计了光habdus luminesens的细菌荧光素酶luxAB基因，以优化密码子在HEK293细胞中的表达。这项工作证明了在无细胞实验中添加正癸醛后，体内产生的转录物和成熟蛋白具有高水平的生物发光。本研究的具体目标是通过表达完整lux操纵子（luxCDABE）构建一种稳定的能够自主生物发光的哺乳动物细胞系，并通过本构自主生物发光成像在结直肠癌细胞模型中阐明该报告基因的价值。本研究的工作假设是，人类癌细胞系（HCT-116）可以被设计成有效地表达完整的细菌生物发光反应，并在体内被监测。这项研究的最终目标是为一个完全自主的、无试剂的生物发光（lux）报告基因系统提供一个新的平台技术，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的高通量筛选。该技术将为全动物模型的早期诊断、治疗效果和疾病复发提供体内基因表达成像能力。

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.