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.

项目摘要/摘要听力损失是全球最常见的感觉赤字。禁用听力损失将影响估计的900 到2050年，全球百万个人的年成本为7500亿美元。有令人信服的社会经济制定新的治疗策略来治疗内耳疾病的遗传性和非遗传形式的理由。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药。一种广泛的实验方法是交付对人的耳聋的功能不成熟的新生儿内耳的基因或药物，然后评估成熟阶段的结构和功能恢复。这项工作利用了预见的哺乳动物内耳，可容纳水试剂的显着注射影响听觉或前庭功能的获取。但是，紧迫的需求是定义实验模拟成人内耳对治疗性遗传操作的反应性的系统。概念该提议的基础是，将功能无声的遗传结构传递到胎儿内耳将启用成熟内耳的分化细胞类型中的漫画激活。我们假设伸缩剂将CRE重组酶反应性遗传元素的显微注射到具有他莫昔芬的小鼠的耳囊中诱导等位基因将允许控制治疗基因递送的时间和细胞类型特异性损害内耳的结构或功能。我们的长期目标是验证内耳的位置和何时必须调节特定基因以恢复或保护遗传和非 - 遗传听力损失。在AIM 1中，我们将进行化学诱导的遗传开关侧翼绿色荧光蛋白（GFP）使用重组腺相关病毒载体（RAAV）到胎儿内耳。然后在成人内耳中触发药理触发表达。我们假设GFP表达将受约束内毛细胞或外毛细胞，在Corti器官中支撑细胞的子集，前庭由相关的CRE驱动器依据等位基因。在AIM 2中，我们将部署遗传开关系统以将成年小鼠支撑细胞重新编程为头发通过POU4F3，GFI1和ATOH1转录因子的条件表达的细胞。我们假设这一点外源生物活性信号将有效地传播到成年小鼠内耳中的支撑细胞。目标 3，我们将使用诱导的杂交转录激活系统将支撑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的强迫转录激活支持细胞会诱导毛细胞命运。成功完成我们的目标可能会建立鼠标模型可以在体内验证可毒品的遗传靶标的系统成熟的内耳。