CMV-Induced Embryonic Cochlear Pathogenesis

描述(申请人提供)：先天性巨细胞病毒(CMV)感染是儿童感音神经性耳聋(SNHL)最常见的非遗传原因，占所有SNHL的20%至60%。据估计，每年在美国出生的至少8000名婴儿将患有CMV引起的先天性SNHL，其中相当大一部分儿童在新生期后表现出起病延迟和听力进行性恶化。目前，对巨细胞病毒引起的出生缺陷的机制知之甚少。由于宫内CMV感染引起SNHL，而出生后感染不会引起SNHL，我们推测CMV诱导的耳蜗畸形是由病毒诱导的多个宿主(胚胎)细胞信号通路的失调引起的，而这些信号通路对于正常的耳蜗形态发生至关重要，而且畸形的严重程度与时间有关。我们最近建立了一种新的小鼠胚胎器官培养模型，证明了CMV在体外活动性感染小鼠胚胎耳蜗会导致严重的耳蜗表型异常和迷路炎。我们的初步结果表明，这种新的小鼠胚胎耳蜗培养模型模拟了先天性巨细胞病毒感染儿童的病理。我们的目的是利用我们建立的CMV诱导的耳蜗器畸形的体外胚胎小鼠器官培养模型来描述CMV诱导的耳蜗病的分子病理学。从长远来看，我们的目标是确定CMV诱导的耳蜗畸形发生的分子机制，并确定关键的细胞靶点，以针对新的耳蜗特异性出生后治疗。我们设计了一系列相互关联的实验，以解决三个具体目标。目的1：研究MCMV对体外培养的胚胎耳蜗组织形态、组织分化和细胞增殖的影响。目的2：确定MCMV诱导的一组“耳蜗病相关”信号分子基因表达的差异。目的3：确定MCMV诱导的耳蜗病的分子病理学的时间依赖性差异。我们的初步研究结果将提供CMV诱导的耳蜗病理学的详细分子表型。将出现一个知情和合理的框架，在此框架内，可能会计划和提出未来的机制研究，以确定哪些细胞信号通路在CMV诱导的耳蜗病发病中是功能必不可少的，并可能出现针对新疗法的假定靶点。我们的体外模型还具有额外的翻译价值，为所有类别的新候选、耳蜗靶向、出生后抗CMV药物(包括抗病毒药物)提供了一个初步筛选系统。这项研究具有重要的临床意义，因为CMV引起的SNHL的发病率相当高，目前的抗病毒治疗本身就是致畸的，受感染的儿童长期使用现有的抗病毒药物存在严重的安全问题。 公共卫生相关性：先天性巨细胞病毒(CMV)感染是儿童感音神经性耳聋(SNHL)最常见的非遗传原因，占所有SNHL的20%至60%。据估计，每年在美国出生的至少8000名婴儿将患有先天性巨细胞病毒引起的SNHL。由于目前对CMV诱导的耳蜗肌异常的机制知之甚少，本项目的目标是开始描述CMV诱导的耳蜗病的分子病理学。本研究的最终目的是确定CMV诱导的耳蜗畸形发生的分子机制，并确定细胞靶点，针对这些细胞靶点，可以针对新的耳蜗特异性、出生后治疗来改善由于先天性CMV感染引起的SNHL。此外，我们的体外模型具有额外的翻译价值，为所有类别的新组织靶向药物的初步筛选提供了一个有效的模型系统，因为它们具有阻止CMV诱导的病理的能力。这在临床上很重要，因为CMV引起的SNHL的患病率相当高，目前的抗病毒治疗本身就是致畸的，受感染的儿童长期使用现有的抗病毒药物存在严重的安全问题。

DESCRIPTION (provided by applicant): Congenital cytomegalovirus (CMV) infection is the most common non-genetic cause of sensorineural hearing loss (SNHL) in children, accounting for between 20 and 60% of all SNHL. It is estimated that at least 8000 infants born annually in the US will have congenital CMV-induced SNHL, with a significant proportion of these children exhibiting delayed onset and progressive deterioration of hearing after the newborn period. Currently, little is known about the mechanism underlying CMV-induced birth defects. Since in utero CMV infection causes SNHL whereas postnatal infection does not, we postulate that CMV-induced cochlear malformations are caused by viral-induced dysregulation of multiple host (embryonic) cell signaling pathways essential for normal cochlear morphogenesis and that the severity of dysmorphogenesis is time-dependent. We have recently developed a novel mouse embryonic organ culture model of CMV-induced cochlear malformations and demonstrate that active CMV infection of embryonic mouse cochlears in vitro induces severely abnormal cochlear phenotypes and labyrinthitis. Our preliminary results indicate that this novel embryonic mouse cochlear culture model mimics the pathology seen in children with congenital CMV infection. Our objective is to initiate delineation of the molecular pathology of the CMV-induced cochlear abnormalities using our novel in vitro embryonic mouse organ culture model of CMV-induced cochlear malformation. Long-term, our goal is to identify the molecular mechanisms underlying CMV-induced cochlear dysmorphogenesis and identify key cellular targets against which new cochlear- specific, postnatal therapies can be directed. We have designed a series of interrelated experiments to address three Specific Aims. Aim 1: Characterize mCMV-induced changes in embryonic cochlear morphology, histodifferentiation and cell proliferation in vitro. Aim 2: Determine mCMV-induced differences in the gene expression of a focused set of "cochlea-related" signaling molecules. Aim 3: Determine time-dependent differences in the molecular pathology of mCMV-induced cochlear abnormalities. The results of our initial studies will provide a detailed molecular phenotype of CMV-induced cochlear pathology. An informed and rational framework will emerge within which future mechanistic studies may be planned and proposed to identify which cellular signaling pathways are functionally essential for CMV-induced cochlear pathogenesis, and likely putative targets against which new therapies can be directed will emerge. Our in vitro model also has the added translational value of providing a system for initial screening of all classes of new candidate, cochlear-targeted, postnatal anti-CMV drugs, including antivirals. This line of investigation is clinically critical because the prevalence of CMV-induced SNHL is considerable, present antiviral therapies are teratogenic in themselves, and long-term use of current antiviral drugs by infected children present serious safety concerns. PUBLIC HEALTH RELEVANCE: Congenital cytomegalovirus (CMV) infection is the most common non-genetic cause of sensorineural hearing loss (SNHL) in children, accounting for between 20 and 60% of all SNHL. It is estimated that at least 8000 infants born annually in the US will have congenital CMV-induced SNHL. Since little is presently known about the mechanisms underlying CMV-induced abnormalitites, the objective of this project is to initiate delineation of the molecular pathology of the CMV-induced cochlear abnormalities. The ultimate goal of this research is to identify the molecular mechanisms underlying CMV-induced cochlear dysmorphogenesis and identify cellular targets against which new cochlear-specific, postnatal therapies can be directed to ameliorate SNHL due to congenital CMV infection. In addition, our in vitro model has the added translational value of providing an efficient model system for initial screening of all classes of new tissue-targeted drugs for their ability to preclude CMV-induced pathology. This is clinically important because the prevalence of CMV-induced SNHL is considerable, present antiviral therapies are teratogenic in themselves, and long-term use of current antiviral drugs by infected children present serious safety concerns.