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

 描述（由申请人提供）：本小企业技术转让（STTR）I期项目拟开发用于连续、无试剂和实时生物成像的自主生物发光人类干细胞，以满足美国国立卫生研究院对体内植入后干细胞存活性、植入和迁移的非侵入性、长期跟踪新技术的要求。干细胞自我更新的能力 并分化成其它细胞谱系已经成为一种有价值的治疗方法，用于功能性地治愈先前不可修复的组织和器官。然而，为了使再生医学领域有效地向转化和临床实践成果过渡，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提出通过创建干细胞系来扩展动物模型的信息容量，所述干细胞系经由“人源化”细菌荧光素酶的表达而自我产生生物发光，从而使得干细胞能够在其整个生命周期中随着其在其动物宿主内的生理功能而连续成像。这与目前市场上依赖萤火虫的生物发光成像技术有很大不同 荧光素酶基因构建体，其必须提供有化学底物以激活其光发射响应，导致与引起未知和潜在干扰相互作用并不利地影响动物福利的重复动物注射串联的细胞功能的仅少量信息的单个时间点快照。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，用于将生物发光表型流线型整合到脂肪中，衍生的间充质干细胞系，然后在体外3D支架和体内小鼠模型中进行性能评估，以证明对不间断成像和丰富数据流的熟练程度，现有的萤火虫荧光素酶方法。在仪器或基本生物发光协议没有必要改变的情况下，研究人员可以从萤火虫荧光素酶无缝过渡到490 BioTech的人源化细菌荧光素酶技术，以推进他们的体内实验研发，以更少的动物为所需的更多信息的终点。这个创新的成像平台对再生医学领域的贡献将提供更多的生理相关和代表性数据，这些数据对于预测临床试验之前治疗策略的有效性和安全性至关重要。

 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.