Gene Transfer to the Inner Ear

基因转移至内耳

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/6640390
• 关键词: Adenoviridae; audiometry; biological signal transduction; biotechnology; cochlea; cochlear microphonic potentials; deafness; ear hair cell; gene delivery system; gene expression; gene mutation; gene therapy; immunocytochemistry; laboratory mouse; molecular cloning; otoacoustic emission; ototoxin; potassium channel; transfection /expression vector; western blottings

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）：所提出的研究将确定使用经修饰的第二代腺病毒[El-，E2 b-，E3-]向成年小鼠耳蜗进行体内基因递送的功效，并测试耳蜗功能，如通过声发射失真产物（DPOAE）和听觉脑干反应（ABR）评估的。DPOAE的幅度是外毛细胞功能的可靠指标，而ABR阈值测量是内毛细胞功能的指标。我们的初步研究结果支持腺病毒在体内将转基因传递到豚鼠耳蜗毛细胞的能力，但尚未确定这种基因转移技术是否可用于改变毛细胞中存在的基因的内源性水平，以及基因转移是否可用于影响耳蜗生理学的功能变化。第一个具体目标是将修饰的第二代腺病毒递送至成年小鼠耳蜗。我们还将通过比较来自具有相同滴度的含有外源转基因的经修饰的第二代腺病毒（LacZ）与没有外源转基因的经修饰的第二代腺病毒感染的动物的转氨酶和肝脏病理学的血清水平来确定是否存在与引入和表达外源转基因相关的任何毒性。在整个实验中，将监测DPOAE和ABR，以确定是否存在由于病毒感染和毛细胞可能的免疫清除导致的耳蜗功能丧失。第二个特定目标将确定是否可以使用KCN 04通道作为模型，使用病毒介导的基因转移来调节耳蜗中的蛋白质表达。我们将过表达功能性KCN 04通道和显性阴性突变体（G285 S）KCN 04通道。然后，我们将使用基因转移，以确定是否正常和显性负突变KCN 04通道的过度表达可以用来改变成年毛细胞的能力，以消除听觉信号。在这两个目标中，我们将使用分子生物学克隆，病毒生产，免疫组织化学，蛋白质印迹，DPOAE和ABR测量，和无菌手术技术。KCNQ 4突变在人类常染色体显性遗传性非综合征性耳聋疾病DFNA 2中发现，具有该突变的家族表现出显著的听力损失。一旦我们能够利用基因转移来模拟人类疾病，未来的研究就可以利用基因转移技术来拯救突变的表型。从这些研究中获得的信息将有助于开发针对耳聋相关基因的治疗策略。