Fetal gene therapy for congenital deafness and imbalance

针对先天性耳聋和失衡的胎儿基因治疗

• 批准号: 10475412
• 负责人: JOHN Vincent BRIGANDE
• 金额: $ 2.66万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10475412
• 关键词: Abdomen; Acoustics; Acute; Affect; Age; Alleles; Assisted Reproductive Technology; Auditory Brainstem Responses; Biological Models; Birth; Breeding; CRISPR/Cas technology; Capsid; Child; Clinic; DNA Sequence Alteration; Detection; Development; Embryo; Equilibrium; Evaluation; Exons; Exposure to; Fetal Development; Fetal Hair; Fetus; Frequencies; Functional disorder; Future; Gene Transduction Agent; Genes; Genetic; Gestational Age; Goals; Green Fluorescent Proteins; Hair Cells; Head; Head Movements; Health Sciences; Hearing; Human; Immunofluorescence Immunologic; Induced Mutation; Intervention; Knowledge; Laboratories; Labyrinth; Live Birth; Location; Macaca mulatta; Mediating; Medicine; Methodology; Modeling; Molecular Virology; Movement; Mus; Mutation; Neonatal; Observational Study; Operative Surgical Procedures; Oregon; Partner in relationship; Patients; Pharmacotherapy; Positioning Attribute; Preclinical Testing; Pregnancy; Preparation; Primates; Recombinant adeno-associated virus (rAAV); Reflex action; Research; Resources; Retinitis Pigmentosa; Rhesus; Safety; Scaffolding Protein; Second Pregnancy Trimester; Sensory; Sensory Hair; Startle Reaction; Stimulus; Synaptic Transmission; Technology; Testing; Therapeutic; Time; Toxic effect; Translating; Treatment Efficacy; Ultrasonics; Ultrasonography; United States; Universities; Usher Syndrome Type 1C; Uterus; Viral; Virus; adeno-associated viral vector; arm; arm movement; base; comparative; congenital deafness; congenital hearing loss; deaf; deafness; early adolescence; early onset; experience; fetal; gene therapy; genetic manipulation; genome editing; hearing impairment; hearing restoration; human disease; human model; in vivo; inner ear diseases; membranous labyrinth; mouse model; mutant; neonatal mice; neonate; nonhuman primate; novel therapeutic intervention; postnatal; prenatal; pressure; programs; protein expression; ribbon synapse; safety testing; sound; surgical service; therapeutic gene; transduction efficiency; vector

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.

项目摘要/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于基因的治疗策略。鼠标是主要的模型系统 由于自然和诱导突变的可用性，新生儿内耳的可及性，以及其 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，病毒介导的基因 针对出生后第0天(P0)至P5的小鼠内耳最佳产量的治疗和药物治疗 恢复听力和平衡。此后的干预大大减少或完全消除了治疗性 福利。关键的是，P0-P5小鼠内耳功能不成熟，在P12辅音为 声学惊吓反射的出现。在人类中，声音惊吓出现在怀孕19周期间 妊娠中期，提示小鼠从P0-P5开始的治疗效果窗口可能 预测人类胎儿的产前疗效窗口。这一提议的概念基础是，早期的 新生小鼠内耳功能模拟胎儿期人类内耳。辨别基因疗法是否被定义为 在早期新生小鼠内耳可以安全有效地移植到临床，建立高等脊椎动物模型 以胎儿听力早熟为特征的系统是必要的。我们的长期目标是建立 恒河猴模型系统测试胎儿和新生儿基因疗法治疗先天性耳聋和 不平衡。在目标1中，我们将定义恒河猴胎儿听力的开始。纯音在100,250， 500、1000或3000赫兹将以越来越大的强度通过产妇的腹部传播。超声波 对头部、手臂或躯干剧烈运动的评估将显示惊恐。我们预测，这一数字将会下降 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所做的那样。我们进一步假设 治疗干预的最佳时间将在听力开始年龄之前。在目标2中，我们将定义 进入内耳的胎儿存活手术。一种编码绿色荧光的腺相关病毒载体 蛋白质(GFP)将被显微注射到膜迷路中。病毒的转导效率将被估计 采用整体免疫荧光法检测GFP。我们假设一个基于AAV2的载体伪类型化 通过人工合成或自然产生的衣壳，将有力地转导体内大多数未成熟的毛细胞 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 具有Hardin双等位基因突变的胚胎。我们假设正确的目标定位将产生一个模型 Usher综合征1C型，以先天性耳聋和严重前庭功能障碍为特征。成功 拟议研究的完成将确定启动恒河猴胎儿基因治疗的最佳孕龄； 鉴定能够向胎儿感觉毛细胞输送和谐素的AAV载体；并创建灵长类动物模型 先天性内耳疾病。这些资源将在未来的研究中部署，以测试安全性和有效性 对胎儿和新生儿进行基因治疗以挽救听力和平衡。