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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/8981338
• 关键词: Address; Adipose tissue; Animal Experiments; Animal Model; Animal Welfare; Animals; Bacterial Luciferases; Biological Models; Bioluminescence; Businesses; Cell Line; Cell Lineage; Cell Therapy; Cell physiology; Cells; Chemicals; Clinical Trials; Color; Complement; Data; Data Collection; Dependence; Discrimination; Disease; Electroporation; Engineering; Engraftment; Evaluation; Fireflies; Firefly Luciferases; Fluorescence; Gene Delivery; Gene Transfer; Genes; Genome; Green Fluorescent Proteins; Healed; Health; Human; Image; Imaging technology; Implant; In Vitro; Injection of therapeutic agent; Legal patent; Life; Light; Longevity; Luc Gene; Luciferases; Marketing; Measurement; Mediating; Medical center; Mesenchymal Stem Cells; Methods; Monitor; Morphologic artifacts; Noise; Organ; Osteogenesis; Outcome; Output; Performance; Phase; Phenotype; Physiological; Play; Procedures; Production; Protocols documentation; Reagent; Regenerative Medicine; Renilla; Reporter; Reporter Genes; Research; Research Personnel; Role; Safety; Signal Transduction; Small Business Technology Transfer Research; Stem cells; Subfamily lentivirinae; Surface; Techniques; Technology; Technology Transfer; Tennessee; Therapeutic; Therapeutic Effect; Time; Tissue Engineering; Tissues; Transduction Gene; Transfection; Transplantation; Transposase; United States National Institutes of Health; Universities; Variant; base; bioimaging; clinical practice; consumer demand; data acquisition; expression vector; healing; human stem cells; imaging platform; implantation; improved; in vivo; in vivo imaging; innovation; instrumentation; light emission; matrigel; migration; mouse model; novel; public health relevance; research and development; response; scaffold; self-renewal; stem cell fate; stem cell therapy; treatment strategy; two-dimensional

 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（由应用程序提供）：这一小型企业技术转移（STTR）I期项目提案，旨在开发自动发光的人类干细胞，以持续，无试剂和实时的生物影像图，以解决国家卫生研究院的新技术请求，以寻求无创的，长期的长期跟踪干细胞生存，雕刻，雕刻，迁移，迁移，以及In vivo Inclantation in vivo Intherantions。干细胞自我更新的能力 并分化为其他细胞谱系已成为一种有价值的治疗方法，可以在功能上治愈以前无法弥补的时机和器官。但是，对于再生医学领域，要有效地向翻译和临床实践结果过渡，将需要对动物模型的强烈依赖，以充分了解干细胞的能力和复杂性。 490 Biotech提案通过创建干细胞系来扩大动物模型的信息能力，从而通过表达“人源化”细菌荧光素酶来自我产生生物发光光，从而使干细胞在其动物宿主中的物理作用时一生都可以在整个生命周期中连续成像。这与依赖萤火虫的生物发光成像技术的当前市场有很大不同 荧光素酶基因构建体必须用化学基材提供以激活其光发射响应，从而与重复的动物注射串联的细胞功能的单个时间点快照只能带有重复的动物注射，从而引起未知的相互作用并潜在相互作用的相互作用并不利地影响动物福利。与田纳西州医学中心的大学合作，这项研发的特定目标是开发PiggyBac的换位和慢病毒翻译方法，用于简化生物发光表型的整合到脂肪衍生的间质干细胞线上，并在adipose衍生的间质干细胞系中进行性能评估，并在体外评估范围内，以表现出体外3D的范围，以表现出不合时宜的范围。现有的萤火虫荧光素酶方法。由于必要的仪器或基本生物发光方案没有变化，研究人员可以无缝地从萤火虫荧光素酶过渡到490 Biotech的人源性细菌荧光素酶技术，以推进其体内实验性研发，再到更有用的终点，而所需的动物则更少。这个创新成像平台对再生医学领域的贡献将为预测治疗策略在临床试验之前的有效性和安全性至关重要。