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

基本信息

批准号：
9807781
负责人：
JOHN Vincent BRIGANDE
金额：
$ 20.81万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9807781
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 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 neonate nonhuman primate novel therapeutics postnatal prenatal pressure programs protein expression ribbon synapse safety testing sound surgical service transduction efficiency vector

项目摘要

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遗传突变的广泛了解已导致基于基因的治疗策略旨在挽救感觉功能。鼠标是主要模型系统由于自然和诱发突变的可用性，新生儿内耳的可及性及其对遗传操作的响应。这些研究的惊人观察是病毒介导的基因针对产后第0天（P0）通过P5小鼠内耳产量最佳的疗法和药物治疗拯救听力和平衡。此后干预大大减少或完全消除治疗性好处。至关重要的是，P0-P5小鼠的内耳在功能上不成熟，在p12辅音的听力发作与声学惊吓反射的出现。在人类中，声音惊吓是在第19周出现的怀孕的第二个三个月，表明小鼠P0-P5的治疗功效窗口可能鉴定人类胎儿的产前疗效窗口。该提议的概念基础是早期新生小鼠内耳在功能上对产前人体内耳进行建模。辨别基因疗法是否定义在新生小鼠早期，内耳可以安全有效地转化为诊所，脊椎动物模型较高需要以早熟的胎儿听力出现为特征的系统。我们的长期目标是建立测试胎儿与新生儿基因疗法的恒河猴猕猴模型系统，以治疗先天性耳聋和不平衡。在AIM 1中，我们将定义恒河猴中胎儿听力的发作。 100、250的纯音， 500、1000或3,000 Hz将随着强度的增加而在孕产妇腹部进行传播。超声波评估急性头部，手臂或躯干运动将表明惊吓。我们预测惊吓会降低频率刺激将在发育过程中像在人类胎儿中一样首先出现。我们进一步假设干预治疗的最佳时间将在听力发作的年龄之前。在AIM 2中，我们将定义胎儿存活手术以进入内耳。编码绿色荧光的腺相关病毒载体蛋白质（GFP）将被微注射到膜迷宫中。病毒转导效率将估计整个固定免疫荧光以检测GFP。我们假设一个基于AAV2的矢量为伪型使用合成或天然的衣壳将牢固地转导大部分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胚胎中有双性突变的胚胎。我们假设正确的目标将产生一个模型 Usher综合征1C的特征是先天性耳聋和深刻的前庭功能障碍。成功的拟议的研究的完成将定义恒河猴启动胎儿基因治疗的最佳胎龄。确定能够将和声传递到胎儿感觉毛细胞的AAV载体；并创建一个灵长类动物模型先天性内耳病。这些资源将在以后的研究中部署以测试安全性和功效胎儿与新生儿基因疗法以挽救听力和平衡。