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，由于衰老或暴露于耳毒剂以及病毒或细菌病原体而可能会丢失。失去的毛细胞在禽类中自发再生，而不是哺乳动物的耳蜗。因此，毛细胞的损失导致人类永久性的听力缺陷。缺乏针对毛细胞损失的有效治疗方法以及许多其他形式的获得和遗传的听力障碍，这引发了人们对基因转移技术潜在应用以恢复正常人耳蜗功能的兴趣。最近的基因转移研究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，基本的螺旋 - 环螺旋转录激活剂MATH1既需要且足以诱导成熟的耳蜗非感官上皮细胞，以区分毛细胞。尽管非常令人兴奋和令人鼓舞，但在基因治疗方法治疗毛细胞损失的方法可以成功地应用于人类之前，仍有许多问题要解决。例如，自然毛细胞在高度定义的细胞结构中，由一行内毛细胞和三排外毛细胞组成。此外，毛细胞的立体膜必须与直接接触的tecorial膜接触。内毛细胞与支撑细胞的不同亚群有关，并与螺旋神经神经元进行不同类型的突触接触。内部和外毛细胞在成熟的耳蜗中也执行不同的功能。内毛细胞充当主要的机械感觉细胞，而外毛细胞具有更多的扩增能力。因此，我们假设新生的毛细胞需要位于感觉上皮脊的内部和外毛细胞的天然行中，以便与支撑细胞，tectorial膜和神经元进行适当的接触才能实现。迄今为止发表的毛细胞再生研究依靠腺病毒载体来传递，并且CMV启动子驱动Math1基因的本构表达。这些条件主要导致位于正常感觉上皮脊外的异位毛发细胞的发展。我们最近证明，与腺相关的病毒（AAV）可以有效地转导围绕成熟耳蜗中毛细胞的支撑细胞。我们还确定，当表达由神经胶质原纤维酸性蛋白（GFAP）启动子驱动时，转基因表达可以局限于这些支持细胞群。我们进一步证明，通过将Math1蛋白与雌激素受体蛋白融合来使MATH1活性（在他莫昔芬存在下）。因此，我们建议使用AAV传递GFAP-MATH1/ER构建体并驱动融合蛋白在支持细胞中的表达。提出的研究将使用体外和体内模型同时进行，以研究瞬时Math1活性在特定支持细胞群体对新生毛细胞的定位和发育中的影响。