Gene Transfer to the Inner Ear

描述（由申请人提供）：拟议的研究将确定使用修饰的第二代腺病毒[El-， E2b-， E3-]在体内将基因递送到成年小鼠耳蜗的有效性，并通过声发射失真产物（dpoae）和听觉脑干反应（ABRs）来评估耳蜗功能。dpoae的大小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内源性基因水平，以及基因转移是否可用于影响耳蜗生理学的功能变化。第一个特异性目标是将修饰的第二代腺病毒传递到成年小鼠耳蜗。我们还将通过比较感染含有外源转基因修饰的第二代腺病毒（LacZ）和不含外源转基因修饰的第二代腺病毒感染的动物的血清转氨酶水平和肝脏病理，来确定引入和表达外源转基因是否有任何毒性。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而导致的耳蜗功能丧失。第二个特异性目标将确定耳蜗中的蛋白质表达是否可以通过病毒介导的基因转移（以KCN04通道为模型）来调节。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正常和显性阴性突变KCN04通道的过度表达可以用来改变成人毛细胞转导听觉信号的能力。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、Western blotting、DPOAE和ABR测量以及无菌手术技术。KCNQ4突变在人类常染色体显性非综合征性耳聋疾病DFNA2中发现，具有这种突变的家庭表现出严重的听力损失。一旦我们可以通过基因转移模拟人类疾病，未来的研究可以利用基因转移技术来拯救突变表型。从这些研究中获得的信息将有助于开发针对与耳聋有关的基因的治疗策略。

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.