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: 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
• 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

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.

项目概要/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 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 由于自然突变和诱导突变的存在、新生儿内耳的可及性及其 对基因操纵的反应。这些研究的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 的小鼠内耳产量最佳的疗法和药物疗法 拯救听力和平衡。此后的干预大大减少或完全消除治疗 好处。重要的是，P0-P5 小鼠内耳功能不成熟，听力开始于 P12 与 声惊跳反射的出现。人类在妊娠第 19 周时会出现听觉惊吓。 妊娠中期，表明小鼠 P0-P5 的治疗功效窗口可能 预测对人类胎儿的产前疗效窗口。该提案的概念基础是早期 新生小鼠内耳的功能模拟了产前人类内耳。辨别基因疗法是否被定义 在早期新生小鼠内耳中可以安全有效地转化为临床的高等脊椎动物模型 需要以胎儿听力早熟为特征的系统。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 100、250、 500、1000 或 3,000 Hz 将以越来越强的强度穿过孕妇腹部。超声波 对头部、手臂或躯干的剧烈运动的评估将表明惊吓。我们预测惊喜会降低 频率刺激将在发育过程中首先出现，就像在人类胎儿中一样。我们进一步假设 治疗干预的最佳时间将早于听力出现的年龄。在目标 2 中，我们将定义 进入内耳的胎儿生存手术。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。将估计病毒转导效率 通过整体免疫荧光检测 GFP。我们假设基于 AAV2 的向量假型化 具有合成或天然存在的衣壳将有力地转导毛细胞中的大多数未成熟毛细胞。 胎儿内耳。目标 3 将采用基于 CRISPR/Cas9 的基因组编辑技术来培育恒河猴 Harmonin 具有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 1C 型 Usher 综合征的特征是先天性耳聋和严重的前庭功能障碍。成功的 拟议研究的完成将确定在恒河猴中启动胎儿基因治疗的最佳胎龄； 鉴定能够将 Harmonin 传递至胎儿感觉毛细胞的 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 胎儿与新生儿基​​因治疗以挽救听力和平衡。