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)：这个小型企业技术转移(STTR)第一阶段项目建议开发自主生物发光的人类干细胞，用于连续、无试剂和实时的生物成像，以满足美国国立卫生研究院对干细胞存活、植入和体内植入后迁移的非侵入性、长期跟踪新技术的要求。干细胞的自我更新能力 分化为其他细胞系已经成为一种有价值的治疗方法，可以从功能上修复以前无法修复的组织和器官。然而，再生医学领域要有效地过渡到转化和临床实践结果，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生物技术公司建议通过创造干细胞系来扩大动物模型的信息能力，这些干细胞系通过表达一种“人性化”的细菌荧光素酶来自我产生生物发光，从而使干细胞能够在其整个生命周期中被连续成像，因为它们在动物宿主中具有生理功能。这与目前市场上依赖于萤火虫的生物发光成像技术有很大不同 荧光素酶基因构建必须提供化学底物才能激活其发光反应，导致细胞功能的单一时间点快照仅提供少量信息，同时重复动物注射会引发未知的和潜在的干扰相互作用，并对动物福利产生不利影响。这项研发工作的具体目标是与田纳西大学医学中心合作，开发iggyBac转座和慢病毒转导方法，以便将生物发光表型简化整合到脂肪来源的间充质干细胞系中，然后在体外3D支架和体内小鼠模型中进行性能评估，以展示其熟练掌握不间断成像和丰富的数据流，远远超过现有的萤火虫荧光素酶方法。在不需要更改仪器或基本生物发光协议的情况下，研究人员可以从萤火虫荧光素酶无缝过渡到490生物技术的人源化细菌荧光素酶技术，以将他们的体内实验研发推进到信息更丰富的终点，所需动物更少。这一创新的成像平台对再生医学领域的贡献将提供更多生理上相关和具有代表性的数据，在临床试验之前预测治疗策略的有效性和安全性。

 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.