Gene Transfer to the Inner Ear

基因转移至内耳

基本信息

批准号：
6640390
负责人：
ANNE E LUEBKE
金额：
$ 19.69万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-08-01 至 2005-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The proposed study will determine the efficacy of in vivo gene delivery to the adult mouse cochlea using a modified 2nd generation adenovirus [El-, E2b-, E3-] and test cochlear function, as assessed by distortion product to acoustic emissions (DPOAEs) and auditory brainstem responses (ABRs). The magnitude of DPOAEs are reliable indicators of functioning outer hair cells, while ABR threshold measures are an indicator of functioning inner hair cells. Our initial findings support the ability of adenovirus to deliver transgenes to guinea pig cochlear hair cells in vivo, but it has not yet been determined if such gene-transter techniques can be used to change endogenous levels of genes present in hair cells, and if gene-transfer can be used to affect functional changes in cochlear physiology. The first Specific Aim is to deliver a modified 2nd generation adenovirus to the adult mouse cochlea. We will also determine if there is any toxicity associated with introducing and expressing a foreign transgene by comparing serum levels of transaminase and liver pathology from animals that have had equal titers of modified 2nd generation adenovirus containing a foreign transgene (LacZ) to that of modified 2nd generation adenovirus infection without a foreign transgene, Throughout the experiment, DPOAEs and ABRs will be monitored to determine if there is any loss of cochlear function due to viral infection and possible immune clearance of hair cells. The second Specific Aim will determine if protein expression in the cochlea can be modulated using virally-mediated gene transfer using the KCN04 channel as a model. We will overexpress functional KCN04 channels and dominant-negative mutant (G285S) KCN04 channels. We will then use gene transfer to determine if over-expression of normal and dominant-negative mutant KCN04 channels can be used to change the ability of adult hair cells to transduce auditory signals. In both Aims we will use molecular biological cloning, virus production, immunohistochemical, Western blotting, DPOAE and ABR measurements, and aseptic surgical techniques. KCNQ4 mutations are found in the human autosomal dominant non-syndromic deafness disorder, DFNA2, and families with this mutation exhibit a significant hearing loss. Once we can mimic human disease using gene transfer, future studies can employ gene transfer techniques to rescue mutant phenotypes. Information gained from these studies will aid development of theraupeutic strategies targeted at genes involved in deafness.

描述（由申请人提供）：拟议的研究将使用修改后的第二代腺病毒[EL-，E2B-，E3-]和测试耳蜗功能，确定体内基因递送到成年小鼠耳蜗的功效，该功能是通过畸变产物评估的，对声学发射（DPOAES）和听觉脑系统响应（ARREDITY BRAINTACE RESSES）。 DPOAE的大小是功能性外毛细胞的可靠指标，而ABR阈值度量是内部毛细胞运行的指标。我们的初始发现支持腺病毒在体内向豚鼠的毛毛细胞传递转基因的能力，但是尚未确定是否可以使用这种基因 - 传递技术来改变毛细胞中存在的内源性基因水平，以及是否可以使用基因转移来影响人口研究的功能变化。第一个具体目的是向成年小鼠耳蜗提供改良的第二代腺病毒。我们还将确定是否存在与引入和表达外国转基因相关的毒性，通过将跨激酶和肝脏病理的血清水平和肝脏病理学比较，来自具有相等的经过修改的第二代腺病毒的动物的血清和肝脏病理学水平，这些腺病毒含有外元病毒（LACZ），该腺病毒（LACZ）与未经改性的adenovirus Insement and Extery Insement and Ins Extery and Ins Extere and Ins Extere and Ins Extere and Ins Ins Ins iS iS dpo A.由于病毒感染和毛细胞的可能免疫清除而导致的耳蜗功能。第二个特定目的将确定使用使用KCN04通道作为模型的病毒介导的基因转移来调制耳蜗中的蛋白质表达。我们将过表达功能性KCN04通道和显性阴性突变体（G285S）KCN04通道。然后，我们将使用基因转移来确定是否可以使用正常和显性阴性突变体KCN04通道的过表达来改变成年毛细胞传递听觉信号的能力。在这两个目标中，我们都将使用分子生物克隆，病毒产生，免疫组织化学，蛋白质印迹，DPOAE和ABR测量以及无菌外科手术技术。在人类常染色体显性非共生聋哑疾病，DFNA2和这种突变的家族中发现KCNQ4突变显示出明显的听力损失。一旦我们可以使用基因转移模仿人类疾病，未来的研究就可以采用基因转移技术来挽救突变体表型。从这些研究中获得的信息将有助于开发针对涉及聋哑基因的theraupteutic策略。