Therapeutic cellular reprogramming in the adult mammalian inner ear by fetal gene transfer

通过胎儿基因转移对成年哺乳动物内耳进行治疗性细胞重编程

基本信息

批准号：
10063987
负责人：
JOHN Vincent BRIGANDE
金额：
$ 19.25万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10063987
关键词：
Ablation Actins Action Potentials Adopted Adoption Adult Affect Alleles Astrocytes Auditory Biological Models Brain Cell Differentiation process Cells Chemicals Chick Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Code Complementary DNA Confocal Microscopy Cre driver Crista ampullaris Enterobacteria phage P1 Cre recombinase Epithelial Equilibrium Event Excision Functional disorder GFI1 gene Gene Delivery Gene Expression Gene Transfer Genes Genetic Genetic Recombination Genetic Transcription Goals Green Fluorescent Proteins HSF1 Hair Cells Hearing Hybrids Individual Inherited Inner Hair Cells LGR5 gene Labyrinth Location Mediating Mediator of activation protein Microinjections Modeling Mus Neonatal Neurons Organ of Corti Otic Vesicle Outer Hair Cells Parvalbumins Pharmaceutical Preparations Pharmacology Pharmacotherapy Phenotype Pillar Cell Proteins Reagent Recombinants Recovery of Function Reporter SLC17A8 gene Sensory Signal Transduction Site Specificity Structure Supporting Cell System Tamoxifen Testing Therapeutic Transcription Coactivator Transcription Initiation Site Transcriptional Activation Transgenic Organisms Validation Work World Health Organization adeno-associated viral vector aqueous beta Actin cell type cost deafness design diphtheria toxin receptor drug discovery fetal genetic element genetic manipulation hearing impairment hearing preservation human model in vivo inner ear diseases interest macula mouse model new technology novel therapeutic intervention p65 postnatal postsynaptic preservation promoter protein expression response socioeconomics spiral ganglion targeted treatment therapeutic candidate therapeutic gene transcription factor transduction efficiency transgene expression vector

项目摘要

PROJECT SUMMARY/ABSTRACT Hearing loss is the most common sensory deficit worldwide. Disabling hearing loss will affect an estimated 900 million individuals globally by 2050 at an annual cost of US$ 750 billion. There is compelling socioeconomic rationale to devise novel therapeutic strategies to treat hereditary and non-hereditary forms of inner ear disease. Mouse models of deafness and vestibular dysfunction are most commonly exploited to test gene and pharmacotherapeutics designed to rescue sensory function. A widespread experimental approach is to deliver genes or drugs to the functionally immature neonatal inner ears of mice that model human deafness and then assess structural and functional recovery at mature stages. This work takes advantage of the plasticity of the pre-hearing mammalian inner ear to accommodate microinjection of aqueous reagents without significantly affecting acquisition of auditory or vestibular function. However, a pressing need is to define experimental systems that model the responsivity of the adult inner ear to therapeutic genetic manipulation. The conceptual basis of this proposal is that delivery of functionally silent genetic constructs to the fetal inner ear will enable atraumatic activation in differentiated cell types of mature inner ear. We hypothesize that transuterine microinjection of Cre recombinase-responsive genetic elements into the otic vesicle of mice harboring tamoxifen- inducible alleles will permit control of the timing and cell type-specificity of therapeutic gene delivery without compromising inner ear structure or function. Our long term goal is to verify where in the inner ear and when specific genes must be modulated to restore or protect auditory function in models of hereditary and non- hereditary hearing loss. In Aim 1, we will atraumatically deliver a chemically inducible genetic switch flanking green fluorescent protein (GFP) to the fetal inner ear using a recombinant adeno-associated viral vector (rAAV) and then pharmacologically trigger expression in the adult inner ear. We hypothesize that GFP expression will be constrained to inner or outer hair cells, subsets of supporting cells in the organ of Corti, to vestibular supporting cells in the cristae and maculae, and spiral ganglion neurons as predicated by relevant Cre driver alleles. In Aim 2, we will deploy the genetic switch system to reprogram adult mouse supporting cells into hair cells by conditional expression of the Pou4f3, Gfi1, and Atoh1 transcription factors. We hypothesize that exogenous bioactive signals will be efficiently transmitted to supporting cells in the adult mouse inner ear. In Aim 3, we will use an inducible hybrid transcriptional activation system to reprogram supporting cells into hair cells. We hypothesize that forced transcriptional activation of endogenous Pou4f3, Gfi1, and Atoh1 in adult mouse supporting cells will induce a hair cell fate. Successful completion of our aims may establish a mouse model system that enables in vivo validation of druggable genetic targets that can preserve hearing and balance in the mature inner ear.

项目概要/摘要听力损失是全世界最常见的感觉缺陷。失能性听力损失将影响大约 900 人到 2050 年，全球将有 100 万人口，每年损失 7,500 亿美元。有令人信服的社会经济设计新的治疗策略来治疗遗传性和非遗传性内耳疾病的基本原理。耳聋和前庭功能障碍的小鼠模型最常用于测试基因和旨在挽救感觉功能的药物治疗。一种广泛的实验方法是提供将基因或药物植入功能未成熟的小鼠新生内耳中，模拟人类耳聋，然后评估成熟阶段的结构和功能恢复。这项工作利用了可塑性预听哺乳动物内耳可适应水性试剂的显微注射而不会显着影响听觉或前庭功能的获得。然而，迫切需要定义实验模拟成人内耳对治疗性基因操作的反应性的系统。概念性的该提议的基础是，向胎儿内耳传递功能沉默的基因构建体将能够成熟内耳分化细胞类型的无创伤激活。我们假设宫内将 Cre 重组酶反应性遗传元件显微注射到含有他莫昔芬的小鼠耳囊中诱导等位基因将允许控制治疗基因传递的时间和细胞类型特异性，而无需损害内耳结构或功能。我们的长期目标是验证内耳的位置和时间必须调节特定基因以恢复或保护遗传性和非遗传性疾病模型中的听觉功能遗传性听力损失。在目标 1 中，我们将无创地在侧翼传递化学诱导的基因开关使用重组腺相关病毒载体 (rAAV) 将绿色荧光蛋白 (GFP) 注入胎儿内耳然后通过药理学触发成人内耳的表达。我们假设 GFP 表达将仅限于内毛细胞或外毛细胞、柯蒂氏器中支持细胞的子集、前庭根据相关 Cre 驱动程序的预测，嵴和黄斑以及螺旋神经节神经元中的支持细胞等位基因。在目标 2 中，我们将部署基因开关系统，将成年小鼠支持细胞重新编程为毛发细胞通过 Pou4f3、Gfi1 和 Atoh1 转录因子的条件表达来实现。我们假设外源生物活性信号将有效传输至成年小鼠内耳的支持细胞。瞄准 3，我们将使用诱导型混合转录激活系统将支持细胞重新编程为毛细胞。我们假设成年小鼠中内源性 Pou4f3、Gfi1 和 Atoh1 的强制转录激活支持细胞将诱导毛细胞命运。成功完成我们的目标可能会建立小鼠模型系统能够体内验证可药物遗传靶标，从而保护听力和平衡成熟的内耳。