In utero protein transduction to interrogate inner ear sensory patch formation

描述（由申请人提供）：我们实验室的长期目标是确定基于基因的策略，以恢复患病或受损内耳的听觉和前庭功能。在再生医学领域取得进展的重大障碍是鉴定具有真实治疗潜力的基因，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，我们试图定义一个快速，成本效益高，技术上简化的实验范式，使耳前体的基因表达的调制在体内蛋白转导。事实上，所有的蛋白质都不能自发地进入细胞，这限制了它们作为研究工具的有用性。然而，两种新技术已经出现，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的蛋白质的表面重塑部分地通过最大化与糖萼中的硫酸化蛋白聚糖的生产性相互作用来促进内吞作用。下一代病毒样颗粒来源于禽类病毒载体，并有效地将蛋白质而不是核酸有效载荷递送至受感染的细胞。在目的1中，我们提出通过经子宫显微注射生物活性Cre重组酶，使用表面重塑和病毒样颗粒形式在耳前体中启动体细胞重组。在子目标A中，我们使用荧光报告基因的floxed等位基因来测试两种形式，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。在子目标B中，我们将通过Cre介导的floxed Atoh 1基因的重组产生用于无调同源物1（Atoh 1）表达的内耳嵌合体。我们预测，废除Atoh 1表达将减少形成的感觉毛细胞的数量，并允许我们测试的假设，Atoh 1阳性细胞可以指示Atoh 1阴性毛细胞的形成。表面重塑蛋白质的另一个特性是它们与核酸可逆复合的能力，同时保留它们的蛋白质转导特性。目的2：通过经子宫显微注射重组蛋白质/核酸复合物，用表达质粒或小干扰RNA（siRNA）阻断耳前体细胞。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Atoh 1构建体的生物活性，该构建体被预测为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Atoh 1的siRNA敲低基因表达和扰乱B毛细胞命运特化的生物活性。拟议研究的成功完成将建立一个获得和丧失功能的实验平台，以识别具有治疗潜力的基因，并将引入体内蛋白转导作为患病内耳再生干预的潜在治疗策略。

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.