A cell-specific inducible model of hearing loss

描述(申请人提供)：耳聋模型的制作方法多种多样，但小鼠模型存在的问题阻碍了其在听觉生理学研究中的应用。许多方法依赖于对耳朵以外的器官细胞造成损害的药物，因此导致不可接受的高死亡率。现有的方法也会对耳蜗内的多个结构造成损害。我们的目标是开发一种遗传模型，在该模型中，我们可以诱导耳蜗中特定类型的细胞死亡，包括毛细胞和螺旋神经节神经元，这两种细胞类型是导致听力损失的关键细胞类型。这样的模型将加快细胞功能和发育以及内耳再生的研究。如果一种高度可重复性的模型能够在不损害死亡细胞周围组织的情况下导致细胞的凋亡性死亡，将加快通过细胞移植或通过使用基因操作或刺激内源性细胞来替代丢失的细胞类型的治疗手段进行细胞替换的过程。我们在目标1中的目标是使含有用于细胞靶向的基因的小鼠被置于CMV启动子之后，以实现转基因的强表达。该构建体包含一个停止序列，该序列阻止在lox位点之间克隆的死亡诱导caspase基因的表达，以允许重组酶将其移除。在这个构建中，人caspase-3基因与FK506结合位点融合，增加了另一层对照。转基因的活性将在AIM 1a中进行体外和体内测试。我们的目标是获得提供部分或完全消融的小鼠系，这将通过目标1b的转基因方法和目标1c的敲入方法来实现。我们在目标2中的目标是测试两个品系的小鼠的耳蜗靶向细胞消融。在第一组小鼠中，我们将通过将转基因基因与专用于毛细胞的Cre小鼠杂交来靶向内耳毛细胞，而在第二行小鼠中，我们将Caspase转基因小鼠与Cre小鼠杂交，这将允许靶向螺旋神经节神经元。这种方法，使用与Cre小鼠的杂交来靶向选择的细胞类型，并用FK-506类似物治疗通过caspase-3的二聚化来诱导细胞死亡，使我们能够控制组织类型的指定和细胞死亡的时间。 公共卫生相关性：在这笔赠款中，我们将开发一种用于耳聋研究的小鼠模型。这一模型将对内耳疾病治疗的发展至关重要，在内耳疾病的治疗中，毛细胞或神经元的替换将得到评估。由于耳蜗细胞的丧失与感觉神经性听力损失之间存在明显的相关性，因此，我们再生这些细胞的能力的提高将直接导致听力损失的新治疗方法。

DESCRIPTION (provided by applicant): Deafness models have been made by a variety of approaches but problems with models in the mouse have hampered their use for studies of auditory physiology. Many methods rely on drugs that cause damage to cells in organs other than the ear and therefore lead to unacceptably high rates of mortality. The available methods also result in damage to multiple structures in the cochlea. Our objective is to develop a genetic model in which we can induce the death of specific cell types in the cochlea including hair cells and spiral ganglion neurons, two critical cell types for hearing loss. Such a model would expedite studies on cell function and development as well as inner ear regeneration. A model that was highly reproducible and that resulted in apoptotic cell death without damage to tissues that surround the dead cells would expedite procedures for cell replacement by cell transplantation or by use of genetic manipulation or therapeutic means of stimulating endogenous cells to replace the lost cell types. Our objective in Aim 1 is to make mice that contain a gene for cell targeting placed behind a CMV promoter for strong expression of the transgene. The construct contains a STOP sequence that prevents expression of a death-inducing caspase gene cloned between lox sites to permit its removal by a recombinase. In this construct the human caspase-3 gene is fused with FK506 binding sites, adding a further layer of control. Activity of the transgene will be tested in vitro and in vivo in Aim 1a. Our objective is to have lines of mice that provide partial or complete ablation, which is to be achieved by a transgenic approach in Aim 1b and a knock-in approach in Aim 1c. Our objective in Aim 2 is to test two lines of mice for targeted cell ablation in the cochlea. In the first line of mice we will target inner ear hair cells by crossing the transgenic to a Cre mouse specific for hair cells, while in the second line of mice we will cross the caspase transgenic with a Cre mouse that will allow targeting of spiral ganglion neurons. The approach, using a cross to a Cre mouse to target the cell type of choice, and treatment with an FK-506 analogue to induce cell death by dimerization of caspase-3, permits us to control both specification of tissue type and timing of cell death. PUBLIC HEALTH RELEVANCE: In this grant we will develop a mouse model to be used for the study of deafness. This model will be critical for the development of treatments for inner ear disorders in which replacement of hair cells or neurons will be evaluated. Because of the clear correlation between loss of cochlear cells and sensorineural hearing loss, improvements in our ability to regenerate these cells will lead directly to new treatments for loss of hearing.