Mentoring Patient Oriented Research in sensory disorders

摘要： 平衡功能需要感觉毛细胞。前庭毛细胞退化导致平衡 功能障碍表现为头晕、眩晕。在小鼠中，自发再生程度有限 发生在椭圆囊中，这是一种检测线性加速度的前庭器官。此外，添加 转录因子Atoh1强健增强了小鼠椭圆囊中毛细胞的再生，但是 再生毛细胞相对于天然毛细胞仅部分成熟。在初步实验中，我们 表征了一种新型 AAV-ATOH1 结构，可导致更成熟的毛细胞再生 小鼠椭圆囊。以再生人体毛细胞恢复平衡功能为长期目标， 仍然存在两个主要障碍：1）我们对分子特征的了解不完整 人类毛细胞、支持细胞和毛细胞前体，2) 成人内耳组织 不易用于测试在动物模型中发现的有前途的疗法。 为了克服这些障碍，我们设计了一种手术方法来获取活的椭圆囊 通常具有正常听觉和前庭功能的已故器官捐献者。我们已经开始了 收集他们的医疗记录、单细胞转录组和椭圆囊的组织学切片。在 与此同时，我们还从接受手术切除的前庭神经鞘瘤患者那里获取了椭圆囊。 在此，我们建议增加器官捐献者和前庭神经鞘瘤患者的招募 描述并验证毛细胞、支持细胞和毛细胞前体的转录组 这两个队列中的成人椭圆囊（目标 1）。此外，我们将确定我们的小说是否 AAV-ATOH1 增强培养的人类椭圆囊中的毛发再生和成熟（目标 2）。 该奖项的另一个目标是促进我作为患者研究人员和导师的职业生涯—— 定向研究。为了建立在我之前在实验室研究和指导方面的经验的基础上，这个奖项 旨在保护我的时间并帮助我获得知识和技能以 1) 研究人类毛细胞 再生；2）指导其他人进行以患者为导向的研究。 总之，我们将应用最先进的技术（单细胞 RNA 测序、基因 疗法、生物信息学策略）来研究活体人类椭圆囊中的前庭毛细胞再生。我们 组建了一支由合作者和经验丰富的导师组成的团队，从事以患者为导向的研究。在 在这个 5 年提案结束时，我们将获得 1) 人类毛细胞的转录组，支持 细胞和毛细胞前体，2) 揭示了新型 AAV-ATOH1 是否能增强再生和 人类前庭毛细胞的成熟，以及 3) 增强了我在以下方面执行和指导他人的能力 以患者为导向的感觉障碍研究。

Abstract: Sensory hair cells are required for balance function. Vestibular hair cell degeneration causes balance dysfunction manifested as dizziness and vertigo. In mice, a limited degree of spontaneous regeneration occurs in the utricle, a vestibular organ detecting linear acceleration. Moreover, addition of the transcription factor Atoh1 robust enhances regeneration of hair cells in the mouse utricle, but regenerated hair cells mature only partially relative to native hair cells. In preliminary experiments, we have characterized a novel AAV-ATOH1 construct leading to regeneration of more mature hair cells in the mouse utricle. With the long-term goal of regenerating human hair cells to restore balance function, two major obstacles remain: 1) we have an incomplete understanding of the molecular signatures of human hair cells, supporting cells and hair cell precursors, 2) adult human inner ear tissues are not readily available to test promising therapeutics discovered in animal models. To overcome these obstacles, we have designed a surgical method to procure live utricles from deceased organ donors who typically have normal auditory and vestibular function. We have begun assembling their medical records, single-cell transcriptomes, and histologic sections of utricles. In parallel, we have procured utricles from vestibular schwannoma patients undergoing surgical resection. Here, we propose to increase the recruitment of organ donors and vestibular schwannoma patients and delineate and validate the transcriptomes of hair cells, supporting cells, and hair cell precursors in adult human utricles in these two cohorts (Aim 1). Furthermore, we will determine whether our novel AAV-ATOH1 enhances hair regeneration and maturation in cultured human utricles (Aim 2). Another goal of this award is to further my career as an investigator and mentor in patient- oriented research. To build on my previous experience in bench research and mentoring, this award is designed to protect my time and help me gain knowledge and skills to 1) study human hair cell regeneration and 2) mentor others pursuing patient-oriented research. In summary, we will apply state-of-the art technologies (single-cell RNA-sequencing, gene therapy, bioinformatic strategies) to study vestibular hair cell regeneration in live human utricles. We have assembled a team of collaborators and experienced mentors in patient-oriented research. At the end of this 5-year proposal, we will have 1) delineated transcriptomes of human hair cells, supporting cells, and hair cell precursors, 2) revealed whether novel AAV-ATOH1 enhances regeneration and maturation of human vestibular hair cells, and 3) enhanced my ability to perform and mentor others in patient-oriented research in sensory disorders.