In utero protein transduction to interrogate inner ear sensory patch formation

子宫内蛋白质转导研究内耳感觉斑的形成

基本信息

批准号：
8586482
负责人：
JOHN Vincent BRIGANDE
金额：
$ 11.55万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-12-01 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8586482
关键词：
Address Adopted Auditory BHLH Protein Birds Cell Differentiation process Cell Line Cell surface Cells Characteristics Complex Data Dependence Development Developmental Gene Electroporation Electrostatics Embryo Embryonic Development Endocytosis Epithelium Equilibrium Ganglia Gene Expression Gene Transfer Techniques Genes Genetic Genetic Recombination Glycocalyx Goals Hair Cells Hearing Homologous Gene Human In Vitro Injection of therapeutic agent Inorganic Sulfates Internal Ribosome Entry Site Intervention Knockout Mice Knowledge Laboratories Labyrinth LoxP-flanked allele Mediating Microinjections Modality Mus Nucleic Acids Organ of Corti Otic Placodes Pathway interactions Pattern Phenocopy Plasmids Property Proteins Proteoglycan RNA Reagent Recombinants Regenerative Medicine Regulation Reporter Research Retina Sensory Sensory Hair Small Interfering RNA Specific qualifier value Surface Surface Properties Testing Therapeutic Time Transfection Unspecified or Sulfate Ion Sulfates Viral Vector Virus Virus-like particle Wild Type Mouse base cell fate specification cell type cellular transduction cost effective gain of function gene function gene therapy in utero in vivo inner ear diseases interest knock-down loss of function membranous labyrinth mouse model mutant mouse model nerve supply new technology next generation novel otoconia postnatal programs protein complex protein expression public health relevance recombinase regenerative research study tool transgene expression

项目摘要

DESCRIPTION (provided by applicant): A long term goal of our laboratory is to define gene-based strategies that restore auditory and vestibular function in the diseased or damaged inner ear. Significant barriers to progress in the field of regenerative medicine are the identification f genes that have authentic therapeutic potential and the creation of reliable strategies to efficaciously modulate their expression and function. To begin to address these barriers, we have devised in utero gene transfers techniques that permit gain-of-function studies in the developing mouse inner ear that rely on viral vectors and in vivo electroporation. In the present proposal, we seek to define a rapid, cost effective, and technically simplified experimental paradigm that enables modulation of gene expression in otic precursors by in vivo protein transduction. Virtually all proteins do not spontaneously enter cells which restricts their usefulness as research tools. However, two new technologies have emerged that show enormous potential: surface remodeling of proteins and virus-like particles. Surface remodeling of proteins by replacement of nonconserved residues facilitates endocytosis in part by maximizing productive interactions with sulfated proteoglycans in the glycocalyx. Next generation virus-like particles are derived from an avian viral vector and effectively deliver a protein rather than a nucleic acid payload to the infected cell. In Aim 1, we propose to initiate somatic recombination in otic precursors by transuterine microinjection of bioactive Cre recombinase using the surface remodeling and virus-like particle formats. In subaim A, we test both formats using a floxed allele of a fluorescent reporter to define the time course of recombination, the type and distribution of recombined cells, and the potential impact of these reagents on postnatal acquisition of hearing and balance. In subaim B, we will generate inner ears mosaic for atonal homolog 1 (Atoh1) expression by Cre-mediated recombination of the floxed Atoh1 gene. We predict that abrogation of Atoh1 expression will reduce the number of sensory hair cells formed and allow us to test the hypothesis that Atoh1 positive cells can instruct the formation of Atoh1 negative hair cells. An additional property of surface remodeled proteins is their ability to reversibly complex with nucleic acids while retaining their protein transduction characteristics. In Aim 2, we propose to transfect otic precursors with expression plasmid or small interfering RNA (siRNA) by transuterine microinjection of surface remodeled protein/nucleic acid complexes. In subaim A, we will define the parameters for efficient expression plasmid transfection and test the bioactivity of an Atoh1 construct which is predicted to induce the formation of extra hair cells. In subaim B, we will define the parameters for efficiet siRNA transfection and test the bioactivity of siRNAs directed against Atoh1 to knock down gene expression and perturb hair cell fate specification. Successful completion of the proposed studies will establish a gain- and loss-of-function experimental platform to discern genes that have therapeutic potential and will introduce in vivo protein transduction as a potential therapeutic strategy for regenerative interventions in the diseased inner ear.

描述（由申请人提供）：我们实验室的长期目标是定义基于基因的策略，以恢复患病或受损内耳的听觉和前庭功能。再生医学领域进展的重大障碍是识别具有真正治疗潜力的基因以及创建有效调节其表达和功能的可靠策略。为了开始解决这些障碍，我们设计了子宫内基因转移技术，允许在发育中的小鼠内耳中进行依赖于病毒载体和体内电穿孔的功能获得研究。在本提案中，我们寻求定义一种快速、具有成本效益且技术上简化的实验范式，该范式能够通过体内蛋白质转导来调节耳前体中的基因表达。事实上，所有蛋白质都不会自发进入细胞，这限制了它们作为研究工具的用途。然而，两项新技术的出现显示出巨大的潜力：蛋白质和病毒样颗粒的表面重塑。通过替换非保守残基对蛋白质进行表面重塑，部分通过最大化与糖萼中的硫酸化蛋白多糖的生产性相互作用来促进内吞作用。下一代病毒样颗粒源自禽类病毒载体，并有效地将蛋白质而不是核酸有效负载传递到受感染的细胞。在目标 1 中，我们建议通过使用表面重塑和病毒样颗粒形式经子宫显微注射生物活性 Cre 重组酶来启动耳前体中的体细胞重组。在 subaim A 中，我们使用荧光报告基因的 floxed 等位基因测试这两种格式，以确定重组的时间过程、重组细胞的类型和分布，以及这些试剂对出生后获得听力和平衡的潜在影响。在 subaim B 中，我们将通过 Cre 介导的 floxed Atoh1 基因重组来生成无调性同源物 1 (Atoh1) 表达的内耳镶嵌。我们预测 Atoh1 表达的废除将减少形成的感觉毛细胞的数量，并允许我们检验 Atoh1 阳性细胞可以指导 Atoh1 阴性毛细胞形成的假设。表面重塑蛋白的另一个特性是它们能够与核酸可逆地复合，同时保留其蛋白转导特性。在目标 2 中，我们建议通过表面重塑蛋白/核酸复合物的子宫显微注射，用表达质粒或小干扰 RNA (siRNA) 转染耳前体。在子目标 A 中，我们将定义有效表达质粒转染的参数，并测试 Atoh1 构建体的生物活性，预计该构建体会诱导额外毛细胞的形成。在子目标 B 中，我们将定义有效 siRNA 转染的参数，并测试针对 Atoh1 的 siRNA 的生物活性，以敲低基因表达并扰乱毛细胞命运规范。成功完成拟议的研究将建立一个功能获得和丧失的实验平台，以识别具有治疗潜力的基因，并将引入体内蛋白质转导作为患病内耳再生干预的潜在治疗策略。