Expressing humanized bacterial luciferase in stem cells: Moving beyond firefly luciferase to expand the informational capacity of animal models for regenerative medicine

在干细胞中表达人源化细菌荧光素酶：超越萤火虫荧光素酶，扩大再生医学动物模型的信息能力

基本信息

批准号：
8981338
负责人：
Dan Morrison
金额：
$ 15万
依托单位：
490 BIOTECH, INC.
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2017-02-28
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): This Small Business Technology Transfer (STTR) Phase I project proposes to develop autonomously bioluminescent human stem cells for continuous, reagent-free, and real-time bioimaging to address the National Institutes of Health's request for new techniques for non-invasive, long-term tracking of stem cell survivability, engraftment, and migration following in vivo implantation. The ability of stem cells to self- renew and differentiate into other cell lineages has emerged as a valuable therapeutic approach to functionally heal previously irreparable tissues and organs. However, for the regenerative medicine field to effectively transition toward translational and clinical practice outcomes, a strong dependence on animal models will be required to fully understand the capabilities and complexities of stem cells. 490 BioTech proposes to expand the informational capacity of animal models by creating stem cell lines that self- generate bioluminescent light via expression of a 'humanized' bacterial luciferase, thereby enabling stem cells to be continuously imaged throughout their lifetime as they physiologically function within their animal host. This differs significantly from the current market of bioluminescent imaging technologies that rely on a firefly luciferase gene construct that must be provided with a chemical substrate to activate its light emission response, resulting in only marginally informative single time point snapshots of cell function in tandem with repetitive animal injections that invoke unknown and potentially interfering interactions and adversely effects animal welfare. In partnership with the University o Tennessee Medical Center, the specific objectives of this R&D effort are to develop piggyBac transposition and lentiviral transduction methods for streamlined integration of the bioluminescent phenotype into adipose-derived mesenchymal stem cell lines followed by performance evaluation in in vitro 3D scaffolds and in vivo mouse models to demonstrate proficiency toward uninterrupted imaging and enriched data flows that far exceed that of existing firefly luciferase methods. With no change in instrumentation or fundamental bioluminescent protocols necessary, researchers can seamlessly transition from firefly luciferase to 490 BioTech's humanized bacterial luciferase technology to advance their in vivo experimental R&D to more informative endpoints with fewer animals required. The contribution of this innovative imaging platform to the field of regenerative medicine will provide more physiologically relevant and representative data critical to predicting the efficacy and safety of treatment strategies as they precede to clinical trials.

描述（由申请人提供）：该小型企业技术转让 (STTR) 第一阶段项目提议开发自主生物发光人类干细胞，用于连续、无试剂和实时生物成像，以满足美国国立卫生研究院对新技术的要求。无创、长期跟踪体内植入后干细胞的存活率、植入和迁移能力。并分化成其他细胞谱系已成为一种有价值的治疗方法，可以功能性地治愈以前无法修复的组织和器官。然而，为了使再生医学领域有效地向转化和临床实践结果过渡，需要充分依赖动物模型。了解干细胞的能力和复杂性。490 BioTech 提出通过创建干细胞系来扩展动物模型的信息能力，这些干细胞系通过表达“人源化”细菌荧光素酶来自我产生生物发光，从而使干细胞能够随着萤火虫在动物宿主体内的生理功能的变化，它们的整个生命周期都会被持续成像，这与目前依赖萤火虫的生物发光成像技术市场有很大不同。荧光素酶基因构建体必须提供化学底物才能激活其光发射反应，导致细胞功能的单一时间点快照与重复的动物注射相结合，从而引起未知和潜在干扰的相互作用并对动物福利产生不利影响。与田纳西大学医学中心合作，这项研发工作的具体目标是开发piggyBac转座和慢病毒转导方法，以简化将生物发光表型整合到脂肪来源的间充质干中细胞系，然后进行体外 3D 支架和体内小鼠模型的性能评估，以证明其对不间断成像和丰富数据流的熟练程度，远远超过现有的萤火虫荧光素酶方法，无需改变仪器或基本生物发光方案，研究人员可以无缝过渡。从萤火虫荧光素酶到 490 BioTech 的人源化细菌荧光素酶技术，推动他们的体内实验研发，以更少的动物获得更多信息。再生医学领域的创新成像平台将提供更多的生理相关性和代表性数据，对于预测临床试验前治疗策略的有效性和安全性至关重要。