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

• 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
• 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

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(申请人提供)：我们实验室的一个长期目标是确定基于基因的策略，恢复患病或受损内耳的听觉和前庭功能。在再生医学领域取得进展的主要障碍是识别具有真正治疗潜力的基因，以及创建可靠的策略来有效地调节其表达和功能。为了开始解决这些障碍，我们设计了宫内基因转移技术，允许在依赖病毒载体和体内电穿孔的发育中的小鼠内耳进行功能获得研究。在目前的提案中，我们试图定义一种快速、经济、技术简化的实验范式，使之能够通过体内蛋白质转导来调节耳科前体的基因表达。几乎所有的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有效性。然而，已经出现了两项显示出巨大潜力的新技术：蛋白质表面重塑和病毒样颗粒。通过替换非保守残基的蛋白质表面重塑促进内吞作用，部分是通过最大限度地与糖萼中硫酸蛋白多糖的有效相互作用来实现的。下一代类病毒颗粒来自禽类病毒载体，有效地将蛋白质而不是核酸有效地运送到受感染的细胞。在目标1中，我们建议通过经子宫内膜微量注射具有生物活性的Cre重组酶，利用表面重塑和病毒样颗粒形式启动耳聋前体的体细胞重组。在次级目标A中，我们使用荧光报告器的一种流动等位基因来测试这两种形式，以确定重组的时间进程、重组细胞的类型和分布，以及这些试剂对出生后听力和平衡获得的潜在影响。在子目标B中，我们将通过Cre介导的小花Atoh1基因的重组来产生无调性同源1(Atoh1)表达的内耳镶嵌。我们预测，Atoh1表达的取消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阴性毛细胞形成的假设。表面修饰蛋白质的另一个特性是它们能够与核酸可逆地络合，同时保持它们的蛋白质转导特性。在目的2中，我们建议通过经子宫内膜微量注射表面修饰的蛋白质/核酸复合体的方法，将表达载体或小干扰RNA(SiRNA)导入耳科前体细胞。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结构的生物活性，该结构预计可诱导额外毛细胞的形成。在子目标B中，我们将确定高效siRNA转染的参数，并测试针对Atoh1的siRNAs的生物活性，以下调基因表达并扰乱毛细胞命运。拟议研究的成功完成将建立一个功能获得和功能丧失的实验平台，以识别具有治疗潜力的基因，并将在体内引入蛋白质转导作为一种潜在的治疗策略，用于疾病内耳的再生干预。