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

• 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
• 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

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.

项目总结/摘要 听力损失是世界上最常见的感觉缺陷。残疾性听力损失将影响估计900 到2050年，全球将有200万人死亡，每年的费用为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形式的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 基因或药物作用于模拟人类耳聋的小鼠功能不成熟的新生儿内耳，然后 评估成熟阶段的结构和功能恢复。这项工作利用了 预听哺乳动物内耳以适应水性试剂的显微注射， 影响听觉或前庭功能的获得。然而，迫切需要定义实验性的 模拟成人内耳对治疗性基因操作的反应性的系统。概念 该建议的基础是，将功能沉默的遗传构建体递送至胎儿内耳将使得 在成熟内耳的分化细胞类型中的非创伤性激活。我们假设氨子宫素 将Cre重组酶反应性遗传元件显微注射到携带他莫昔芬的小鼠的耳泡中， 可诱导等位基因将允许控制治疗性基因递送的时间和细胞类型特异性， 危及内耳结构或功能。我们的长期目标是验证内耳的位置和时间 在遗传性和非遗传性模型中，必须调节特定的基因以恢复或保护听觉功能。 遗传性听力损失在目标1中，我们将无创伤地传递一个化学诱导的基因开关， 利用重组腺相关病毒载体（rAAV）将绿色荧光蛋白（GFP）导入胎儿内耳 然后在成年人的内耳中迅速表达。我们假设GFP的表达将 局限于内毛细胞或外毛细胞，Corti器官中的支持细胞亚群，到前庭 嵴和黄斑中的支持细胞以及螺旋神经节神经元，如相关Cre驱动程序所预测的 等位基因。在目标2中，我们将部署遗传开关系统，将成年小鼠的支持细胞重新编程为毛发 通过Pou 4f 3、Gfi 1和Atoh 1转录因子的条件性表达，我们假设 外源性生物活性信号将被有效地传递到成年小鼠内耳中的支持细胞。在Aim中 3，我们将使用可诱导的杂交转录激活系统将支持细胞重编程为毛细胞。 我们假设成年小鼠内源性Pou 4f 3、Gfi 1和Atoh 1的转录激活是通过诱导内源性Pou 4f 3、Gfi 1和Atoh 1的转录激活来实现的。 支持细胞将诱导毛细胞的命运。成功地完成我们的目标可能会建立一个小鼠模型 该系统能够在体内验证可药物化的遗传靶点，这些靶点可以保护听力和平衡， 成熟的内耳