Support cell specific expression of regulatable Math-1

支持可调节 Math-1 的细胞特异性表达

• 批准号: 7140495
• 负责人: DAVID J POULSEN
• 金额: $ 16.82万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140495
• 关键词: adeno associated virus group; cochlea; ear hair cell; gene delivery system; gene expression; gene therapy; genetic regulation; guinea pigs; hearing disorders; immunocytochemistry; in situ hybridization; laboratory mouse; nerve /myelin protein; organ of Corti; otoacoustic emission; tamoxifen; transcription factor; transfection /expression vector

DESCRIPTION (provided by applicant): Hair cells are the mechanosensory cells of the cochlea and may be lost as a consequence of aging or exposure to ototoxic agents and viral or bacterial pathogens. Lost hair cells spontaneously regenerate within the avian but not the mammalian cochlea. Thus, hair cell loss leads to permanent hearing deficits in humans. The lack of effective treatments for hair cell loss and many other forms of acquired and inherited hearing disorders has prompted interest in the potential application of gene transfer techniques to restore normal cochlear function. Recent gene transfer studies have examined the potential of driving hair differentiation in mature mammalian cochlea. It has been determined that the basic helix-loop-helix transcriptional activator Math1, is both required and sufficient to induce mature cochlear non-sensory epithelial cells to differentiate into hair cells. While extremely exciting and encouraging, a number of issues remain to be worked out before gene therapy approaches to treating hair cell loss can be successfully applied in humans. For example, natural hair cells are arrayed within a highly defined cytoarchitecture composed of a single row of inner hair cells and three rows of outer hair cells. In addition, the stereocilia of hair cells must come into contact with the tectorial membrane which come into direct contact. The inner and outer hair cells are associated with different subpopulations of support cells and make different types of synaptic contacts with spiral ganglion neurons. The inner and outer hair cells also perform different functions within the mature cochlea. Inner hair cells serve as the primary mechanosensory cells while outer hair cells serve more of an amplification capacity. Therefore, we hypothesize that nascent hair cells will need to be localized within the natural rows of inner and outer hair cells of the sensory epithelial ridge in order to make the appropriate contacts with support cells, the tectorial membrane and neurons to be functional. Hair cell regeneration studies published to date have relied on Adenoviral vectors to deliver, and the CMV promoter to drive constitutive expression of the Math1 gene. These conditions predominantly resulted in the development of ectopic hairs cells located outside of the normal sensory epithelial ridge. We have recently demonstrated that Adeno-associated virus (AAV) can efficiently transduce support cells immediately surrounding hair cells in the mature cochlea. We have also established that transgene expression can be limited to these support cell populations when expression is driven by the glial fibrillarv acidic protein (GFAP) promoter. We have further demonstrated that Math1 activity can be made inducible (in the presence of tamoxifen) by fusing the Math1 protein with the estrogen receptor protein. Therefore, we propose to use AAV to deliver a GFAP-Math1/ER construct and drive expression of the fusion protein specifically in support cells. Proposed studies will be carried out using both in vitro and in vivo models to study the effects of transient Math1 activity in specific support cell populations on the localization and development of nascent hair cells.

描述（由申请人提供）：毛细胞是耳蜗的机械感觉细胞，可能由于老化或暴露于耳毒性剂和病毒或细菌病原体而丢失。失去的毛细胞在鸟类而不是哺乳动物的耳蜗内自发再生。因此，毛细胞损失导致人类永久性听力缺陷。由于缺乏有效的治疗方法来治疗毛细胞损失和许多其他形式的获得性和遗传性听力障碍，因此人们对基因转移技术在恢复正常耳蜗功能方面的潜在应用产生了兴趣。最近的基因转移研究已经检查了驱动成熟哺乳动物耳蜗中毛发分化的潜力。已经确定碱性螺旋-环-螺旋转录激活因子Math 1是诱导成熟耳蜗非感觉上皮细胞分化为毛细胞所必需的并且是足够的。虽然非常令人兴奋和鼓舞，但在基因治疗方法成功应用于人类之前，仍有许多问题有待解决。例如，天然毛细胞排列在由一排内毛细胞和三排外毛细胞组成的高度限定的细胞结构中。此外，毛细胞的静纤毛必须与直接接触的盖膜接触。内毛细胞和外毛细胞与不同的支持细胞亚群相关，并与螺旋神经节神经元形成不同类型的突触接触。内毛细胞和外毛细胞在成熟的耳蜗内也执行不同的功能。内毛细胞作为主要的机械感觉细胞，而外毛细胞则具有更多的放大能力。因此，我们假设，新生的毛细胞将需要定位在感觉上皮嵴的内毛细胞和外毛细胞的自然行内，以便与支持细胞、盖膜和神经元进行适当的接触。迄今为止发表的毛细胞再生研究依赖于腺病毒载体来递送，并且CMV启动子驱动Math 1基因的组成型表达。这些条件主要导致异位毛细胞位于正常感觉上皮嵴外的发展。我们最近已经证明，腺相关病毒（AAV）可以有效地支持细胞直接周围的毛细胞在成熟的耳蜗。我们还建立了转基因表达可以限制在这些支持细胞群体时，表达是由胶质纤维酸性蛋白（GFAP）启动子驱动。我们已经进一步证明，Math 1的活性可以诱导（在他莫昔芬的存在下）通过融合Math 1蛋白与雌激素受体蛋白。因此，我们建议使用AAV来递送GFAP-Math 1/ER构建体并驱动融合蛋白在支持细胞中特异性表达。将使用体外和体内模型进行拟议的研究，以研究特定支持细胞群中的瞬时Math 1活性对新生毛细胞的定位和发育的影响。