Fetal gene therapy for congenital deafness and imbalance

项目总结/摘要 对导致先天性听力损失和失衡的基因突变的广泛了解导致了 旨在挽救感觉功能的基因治疗策略。小鼠是主导模型系统 由于天然和诱导突变的可用性，新生儿内耳的可及性，及其 对基因操纵的反应。这些研究中一个引人注目的观察结果是， 针对出生后第0天（P0）至P5小鼠内耳的治疗和药物治疗 拯救听力和平衡。此后的干预大大减少或完全消除了治疗 效益重要的是，P0-P5小鼠内耳功能不成熟，在P12时听力开始， 声音惊吓反射的出现在人类中，声音惊吓在妊娠第19周出现， 妊娠中期，这表明小鼠P0-P5的治疗效果窗口可能 断言在人类胎儿中的产前有效窗口。这一建议的概念基础是， 新生小鼠内耳在功能上模拟了产前人内耳。为了辨别基因疗法是否定义了 在早期新生小鼠内耳可以安全有效地转化为临床，更高的脊椎动物模型 需要一种以胎儿听力的早熟出现为特征的系统。我们的长期目标是建立 恒河猴模型系统，用于测试胎儿与新生儿基因疗法以治疗先天性耳聋， 不平衡在目标1中，我们将定义恒河猴胎儿听力的开始。纯音100，250， 500、1000或3，000 Hz将以增加的强度通过母体腹部传输。超声 急性头部、手臂或躯干运动的评估将指示惊吓。我们预测， 频率刺激将在发育过程中首先出现，就像它们在人类胎儿中一样。我们进一步假设 进行治疗干预的最佳时间应早于听力开始的年龄。在目标2中，我们定义 胎儿存活手术进入内耳一种编码绿色荧光蛋白的腺相关病毒载体， 蛋白（GFP）将被显微注射到膜迷路中。将估计病毒转导效率 通过整体包埋免疫荧光检测GFP。我们假设一个基于AAV 2的载体假型化 用合成的或天然存在的衣壳将有力地覆盖毛细胞中的大多数未成熟毛细胞。 胎儿内耳在Aim 3中，将部署基于CRISPR/Cas9的基因组编辑技术来创建恒河猴 harmonin双等位基因突变的胚胎。我们假设，正确的目标将产生一个模型， 以先天性耳聋和严重前庭功能障碍为特征的1C型Usher综合征。成功 完成拟议的研究将确定在恒河猴中开始胎儿基因治疗的最佳胎龄; 鉴定能够将harmonin递送至胎儿感觉毛细胞的AAV载体;以及 先天性内耳疾病这些资源将用于未来的研究，以测试安全性和有效性。 胎儿与新生儿基因治疗的对比，以挽救听力和平衡。

PROJECT SUMMARY/ABSTRACT Extensive knowledge of the genetic mutations responsible for congenital hearing loss and imbalance has led to gene-based therapeutic strategies aimed at rescuing sensory function. The mouse is the dominant model system because of the availability of natural and induced mutations, the accessibility of the neonatal inner ear, and its responsiveness to genetic manipulation. A striking observation from these studies is that virus-mediated gene therapies and pharmacotherapies targeted to the postnatal day 0 (P0) through P5 mouse inner ear yield optimal rescue of hearing and balance. Intervention thereafter dramatically lessens or entirely eliminates therapeutic benefits. Critically, the P0-P5 mouse inner ear is functionally immature with hearing onset at P12 consonant with the emergence of the acoustic startle reflex. In humans, acoustic startle arises at gestational week 19 during the second trimester of pregnancy, suggesting that the window of therapeutic efficacy from P0-P5 in the mouse may predicate a prenatal window of efficacy in the human fetus. The conceptual basis of this proposal is that the early neonatal mouse inner ear functionally models the prenatal human inner ear. To discern if gene therapies defined in the early neonatal mouse inner ear may safely and effectively translate to the clinic, a higher vertebrate model system characterized by the precocious emergence of fetal hearing is needed. Our long-term goal is to establish a rhesus macaque model system to test fetal versus neonatal gene therapy to treat congenital deafness and imbalance. In Aim 1, we will define the onset of fetal hearing in the rhesus macaque. Pure tones at 100, 250, 500, 1000, or 3,000 Hz will be transmitted across the maternal abdomen with increasing intensities. Ultrasonic assessment of acute head, arm, or torso movements will indicate startle. We predict that startle to lower frequency stimuli will emerge first during development as they do in the human fetus. We further hypothesize that the optimal time to intervene therapeutically will precede the age of hearing onset. In Aim 2, we will define a fetal survival surgery to access the inner ear. An adeno-associated viral vector encoding green fluorescent protein (GFP) will be microinjected into membranous labyrinth. The viral transduction efficiency will be estimated by whole mount immunofluorescence to detect GFP. We hypothesize that an AAV2-based vector pseudotyped with a synthetic or naturally occurring capsid will robustly transduce the majority of immature hair cells in the fetal inner ear. In Aim 3, a CRISPR/Cas9-based genome editing technology will be deployed to create rhesus embryos with bi-allelic mutations in harmonin. We hypothesize that correct targeting will produce a model of Usher syndrome type 1C characterized by congenital deafness and profound vestibular dysfunction. Successful completion of the proposed studies will define the optimal gestational age to initiate fetal gene therapy in rhesus; identify an AAV vector capable of delivering harmonin to fetal sensory hair cells; and create a primate model of congenital inner ear disease. These resources will be deployed in future studies to test the safety and efficacy of fetal versus neonatal gene therapy to rescue hearing and balance.