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：
8425964
负责人：
JOHN Vincent BRIGANDE
金额：
$ 30.8万
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 2014-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8425964
关键词：
Address Adopted Alleles 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 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

Address Adopted Alleles 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 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

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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，以恢复患病或损坏的内耳或损害的听觉和前庭功能。再生医学领域进步的重大障碍是具有真实治疗潜力的识别基因，也是创建可靠的策略以有效调节其表达和功能。为了开始解决这些障碍，我们已经在子宫基因转移技术中设计了，这些技术允许在发育中的小鼠内耳中获得功能性研究，该研究依赖于病毒载体和体内电穿孔。在本提案中，我们试图定义快速，具有成本效益和技术简化的实验范式，该范式可以通过体内蛋白质转导来调节眼膜前体中的基因表达。几乎所有蛋白质都不会自发进入细胞，从而限制其作为研究工具的实用性。然而，已经出现了两种新技术，这些技术显示出巨大的潜力：蛋白质和病毒样颗粒的表面重塑。通过替换未经保存的残基对蛋白质进行表面重塑，部分通过与糖脂中硫酸化蛋白聚糖的生产性相互作用最大化，部分促进了内吞作用。下一代病毒样颗粒源自禽病毒载体，并有效地传递蛋白质，而不是核酸有效载荷给感染的细胞。在AIM 1中，我们建议使用表面重塑和类似病毒的颗粒格式对生物活性Cre重组酶进行非生物活性CRE重组酶的微注射来启动细胞前体的体细胞重组。在Subaim A中，我们使用荧光报告基因的floxed等位基因来测试两种格式，以定义重组的时间过程，重组细胞的类型和分布以及这些试剂对听力后听到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基因的重组来生成内耳的镶嵌物1（ATOH1）表达。我们预测，ATOH1表达的废除将减少形成的感觉毛细胞的数量，并使我们能够检验AtOH1阳性细胞可以指导ATOH1阴性毛细胞的形成的假设。表面重塑蛋白的另一个特性是它们与核酸逆转复杂的能力，同时保留其蛋白质转导特性。在AIM 2中，我们建议通过表面重塑的蛋白/核酸复合物对表达质粒或小干扰RNA（siRNA）转染眼膜前体。在Subaim 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，该构建体预测会诱导多余的毛细胞的形成。在Subaim B中，我们将定义效率siRNA转染的参数，并测试针对ATOH1的siRNA的生物活性，以击倒基因表达和扰动毛发细胞命运的规范。成功完成拟议的研究将建立一个功能和功能丧失的实验平台，以识别具有治疗潜力的基因，并将在体内蛋白质转导中引入患病内耳疾病再生干预措施的潜在治疗策略。