Engineered Stem Cells for Inner Ear Pharmacotherapy

描述（由申请人提供）：该申请代表了一项探索性和发展性研究提案，旨在开发新的工程成人脂肪来源干细胞ASCs系，从龙猫中收获（产生chASCs），可用于自体移植，将营养物质直接输送到内耳。有相当多的证据表明，成人内耳的内在康复能力有限，但在许多情况下，这种能力将是非常有价值的。这些包括耳保护病例，在这些病例中，其他系统性危及生命的疾病需要使用同样具有耳毒性的积极治疗。还有很多其他的情况会受益于斯卡帕氏或螺旋神经节神经元的再生能力。同时，很明显，内耳保护和神经康复是通过神经营养因子的应用而增强的，其中脑源性神经营养因子（BDNF）是其中的重要成员。因此，长期提供BDNF的策略的发展将在提供耳保护和刺激内耳康复方面具有重大益处。本申请提出了一项研究计划，其中我们将测试使用chASCs作为基于细胞的递送系统向内耳提供BDNF的有效性。这项工作利用了长期准备的栗鼠模型，其中直接进入淋巴周围空间已经发展。实验将通过慢病毒转染来收获、表征和工程chASCs，以表达增强的绿色荧光蛋白（GFP）和GFP标记的BDNF。将实施生产稳定细胞系的策略。然后将这两种细胞系直接移植到龙猫的淋巴周围空间。利用这些细胞系可以监测移植的chASCs分泌BDNF的内在能力，以及转基因表达导致的BDNF分泌增强。chASCs的这种旁分泌功能将通过ELISA方法进行评估。两种转基因系统中GFP的表达将使我们能够通过受体颞骨的组织学分析来批判性地评估这些细胞在体内的整合和存活。通过记录听脑干反应和单神经元电生理学来评估外周听觉和前庭系统的内耳功能，以确定移植对内耳功能的潜在影响。总之，这些实验代表了内耳成体工程干细胞旁分泌功能的直接测试。虽然模型系统包含神经营养因子的分泌，但它将作为无数其他耳活性剂的概念证明。这些实验的成功结果可以立即在PI实验室开发的栗鼠内耳神经康复模型中进行测试。

DESCRIPTION (provided by applicant): This application represents an exploratory and development research proposal to develop new lines of engineered adult adipose-derived stem cells ASCs, harvested from chinchillas (resulting in chASCs), that can be used for autologous transplantation for the delivery of trophins directly to the inner ear. There is considerable body of evidence demonstrating the limited intrinsic rehabilitative capabilities of the adult inner ear, yet there are numerous conditions in which such capabilities would be extremely valuable. These include cases of otoprotection, in which other systemic life-threatening conditions warrant the use of aggressive therapies that are also ototoxic. Still other conditions abound that would benefit from regenerative capabilities of Scarpa's or spiral ganglion neurons. At the same time, it is clear that inner ear protection and neurorehabilitation is enhanced through the application of neurotrophins, of which brain-derived neurotrophic factor (BDNF) is an important member. Therefore, the development of a strategy to provide BDNF for prolonged periods would be of significant benefit in providing otoprotection and stimulating inner ear rehabilitation. The present application presents a research plan in which we will test the efficacy of using chASCs as a cell-based delivery system to provide BDNF to the inner ear. This work takes advantage of a chronically-prepared chinchilla model in which direct access to the perilymphatic space has been developed. Experiments will be conducted to harvest, characterize, and engineer chASCs via lentiviral transfection to express enhanced green fluorescent protein (GFP) and GFP-tagged BDNF. Strategies will be implemented to produce stable cell lines. These two cell lines will then be transplanted directly to the perilymphatic space in chinchillas. The use of these lines enable the capability to monitor the intrinsic capabilities of transplanted chASCs to secrete BDNF, as well as the enhanced secretion of BDNF resulting from transgene expression. This paracrine function of chASCs will be evaluated through ELISA methodologies. The GFP expression in both transgene systems will enable us to critically evaluate the integration and survival of these cells in vivo through histologic analysis of the recipient temporal bones. Inner ear function of both the peripheral auditory and vestibular systems will be assessed by recording the auditory brainstem response and through single neuron electrophysiology to identify the potential influence of the transplantation on inner ear function. In summary, these experiments represent a direct test of paracrine function of adult engineered stem cells in the inner ear. While the model system incorporates the secretion of neurotrophin, it will serve as a proof-of- concept of a myriad of other otoactive agents. The successful outcome of these experiments can be immediately tested in a model of inner ear neurorehabilitation in the chinchilla developed in the laboratory of the PI. PUBLIC HEALTH RELEVANCE: The research to be conducted under this exploratory and development proposal will test the efficacy of utilizing adult adipose-derived stem cells as a means to deliver trophin therapies directly to the inner ear. We will test the intrinsic paracrine capabilities of these cells as a source of brain-derived neurotrophic factor (BDNF), as well as BDNF resulting from cells that have been engineered via viral transfection. Such a cell-based therapy system takes advantage of the intrinsic and engineered capabilities of stem cells, and if successful will be extremely advantageous in cases requiring neurorehabilitation or neuroprotection of the inner ear.