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。再生医学领域进展的重大障碍是鉴定具有真正治疗潜力的基因和创造可靠的策略来有效地调节它们的表达和功能。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病毒载体和体内电穿孔的功能获得研究。在目前的提案中，我们试图定义一个快速，成本有效，技术上简化的实验范式，使通过体内蛋白转导调节基因表达在耳蜗前体。几乎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用处。然而，两项新技术已经出现，显示出巨大的潜力：蛋白质的表面重塑和病毒样颗粒。通过替换非保守残基，蛋白质的表面重塑促进了内吞作用，部分是通过最大化与糖萼中硫酸化蛋白聚糖的生产相互作用。下一代病毒样颗粒来自禽类病毒载体，并有效地向受感染细胞传递蛋白质而不是核酸有效载荷。在目的1中，我们建议通过体外微量注射生物活性Cre重组酶，利用表面重塑和病毒样颗粒形式，启动耳蜗前体的体细胞重组。在子目标A中，我们使用荧光报告基因的一个固定等位基因测试了这两种格式，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听力和平衡获得的潜在影响。在subaim B中，我们将通过cre介导的对固定的Atoh1基因的重组，生成无调性同源物1 （Atoh1）表达的内耳马赛克。我们预测，取消Atoh1的表达将减少感觉毛细胞的形成数量，并允许我们验证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，同时保持其蛋白质转导特性。在目的2中，我们提出通过体外微量注射表面重塑蛋白/核酸复合物，用表达质粒或小干扰RNA （siRNA）转染生物前体。在子目标A中，我们将定义高效表达质粒转染的参数，并测试预测会诱导额外毛细胞形成的Atoh1构建体的生物活性。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，以降低基因表达并扰乱毛细胞命运规范。上述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。

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.