Machine learning-enabled Comparative Transcriptomic Profiling to Validate NanoScript-induced Inner Ear Hair Cells

项目总结 儿童早期听力损失显著影响学习和社交技能的获得，而听力 成年人的失落往往会导致社交孤立，无法执行许多日常的社交功能。一个 感觉神经性听力损失的主要原因是内耳感觉毛细胞的丧失。终生接触 估计15%的美国成年人(约3600万)会有一些 听力损失的形式。一种很有前途的减轻听力损失和耳聋的方法是细胞替代疗法 通过将支持细胞转化为毛细胞。不幸的是，目前的转分化方法 依赖病毒传递对于临床翻译来说可能是不安全和不切实际的。因此，迫切需要 开发替代平台，以调节基因表达和诱导转分化，在一种高效的，非 适合恢复听力的病毒方式。 为此，我们的长期目标是开发纳米脚本，这是一种创新的、基于纳米颗粒的人工 转录因子平台能够以非病毒的方式有效地调节基因表达。vbl.使用 NanoScript，我们将把支持细胞转化为有功能的毛细胞。NanoScript由一个 具有特定小分子和多肽的纳米颗粒，这些小分子和多肽旨在模仿个体 天然转录因子(TF)蛋白的结构域。转铁蛋白是一种内源性多域蛋白，可协调 许多细胞功能，包括分化。由于NanoScrip是转铁蛋白的功能复制品，它可以 将病毒传递的TF替换为基于再生医学的应用。这项提案的总体目标是 是设计三种纳米脚本，模仿毛细胞分化所必需的三种转录因子(Gfi1、Pou4f3和Atoh1； 平均绩点)。我们将测试GPA-NanoScript是否与相同的DNA序列结合并激活基因表达 体外培养。接下来，我们将确定向GPA-NanoScrip添加表观遗传调节子是否会与相同的 以转铁蛋白为靶标，局部改变染色质结构，增强基因表达。最后，我们会 使用耳蜗外植体确定GPA-NanoScript是否促进支持细胞的转分化 通过单细胞转录组分析转化为毛细胞。使用体外模型产生新生毛细胞将 作为测试再生医学纳米脚本技术的跳板。它还将建立NanoScrip 作为研究人员通过直接重编程产生功能细胞的有效和非病毒工具。

PROJECT SUMMARY Hearing loss during early childhood significantly affects learning and acquisition of social skills, while hearing loss in adults can often result in social isolation and the inability to perform many routine social functions. A leading cause of sensorineural hearing loss is the loss of sensory hair cells of the inner ear. A lifetime exposure to aminoglycoside and loud sounds will result in an estimated 15% of adult Americans (~36 million) having some form of hearing loss. A promising approach to mitigate hearing loss and deafness is a cell replacement therapy by transdifferentiating supporting cells into hair cells. Unfortunately, current approaches for transdifferentiation rely on viral delivery may be unsafe and impractical for clinical translation. Therefore, there is a critical need to develop alternative platforms for regulating gene expression and inducing transdifferentiation in an efficient, non- viral manner that is suitable for restoration of hearing. To this end, our long-term goal is to develop NanoScript, an innovative, tunable nanoparticle-based artificial transcription factor platform capable of effectively regulating gene expression in a non-viral manner. Using NanoScript, we will transdifferentiate supporting cells into functional hair cells. NanoScript consists of a nanoparticle functionalized with specific small molecules and peptides that are designed to mimic the individual domains of natural transcription factor (TF) proteins. TFs are endogenous, multi-domain proteins that orchestrate many cellular functions, including differentiation. Since NanoScript is a functional replica of TF proteins, it can replace virally-delivered TFs for regenerative medicine-based applications. The overall objective of this proposal is to design three NanoScripts that mimic three TFs essential for hair cell differentiation (Gfi1, Pou4f3, and Atoh1; GPA). We will test whether GPA-NanoScript binds to the same DNA sequence and activate gene expression in vitro. Next, we will determine if the addition of epigenetic modulators to GPA-NanoScript will bind to the same targets as the TF proteins, locally alter the chromatin structure and enhance gene expression. Finally, we will use cochlear explants to determine whether GPA-NanoScript promotes transdifferentiation of supporting cells into hair cells by single-cell transcriptome analysis. Generation of nascent hair cells using an ex vivo model will serve as a springboard to test NanoScript technology for regenerative medicine. It will also establish NanoScript as an effective and non-viral tool for researchers to generate functional cells via direct reprogramming.