Efficient in Vivo RNP-based Gene Editing in the Sensory Organ Inner Ear Using Bioreducible Lipid Nanoparticles

项目摘要和相关性 基于基因组编辑的治疗的应用需要将编辑剂有效递送到疾病相关的微环境中。 组织和细胞。靶向体细胞的新型递送材料的鉴定将极大地促进靶向体细胞的递送。 将基于编辑的治疗策略推向临床。我们建议筛选一个大型的新脂质库， 用于将编辑剂RNP（核糖核蛋白）递送到哺乳动物感觉器官内的纳米颗粒 耳朵内耳是开发新的传递策略的理想感觉器官。它由多种不同的 体细胞类型没有有效的递送选择。主要内耳细胞类型中的基因突变已经被发现， 与遗传性听力损失有关，500个新生儿中就有一个会受到影响，目前还没有有效的治疗方法。 治疗 基于脂质的纳米颗粒载体已经成为最有前途的递送材料之一， 已成功应用于临床。我们开发了一种组合库方法， 在还原性细胞内环境下合成可降解脂质样纳米颗粒， 以高效率和低毒性递送生物分子。新型生物可还原脂质纳米粒 生物纳米粒（bLNP）已被用于在体内以高效率和低毒性递送基因组编辑剂。我们 通过阳离子脂质体将基因组编辑RNP递送到哺乳动物体内内耳中， 人类遗传性听力损失小鼠模型的听力。开发基于编辑的治疗方法， 对于遗传性听力损失的形式，开发针对多种内耳细胞类型的递送策略至关重要 同步哺乳动物的内部结构复杂，有多种细胞类型， 使得通过常规的高通量策略筛选递送技术特别具有挑战性。 缺乏在单个细胞类型水平上检测编辑的方法进一步阻碍了我们应用 该技术在野生型大型动物模型中的应用对于开发该疗法的临床应用至关重要。 应用程序.通过结合我们的策略来筛选纳米颗粒，以将编辑材料递送到X-连接的 基因在体内雄性小鼠内耳，我们将克服这些障碍， 不同的内耳细胞类型对离体人内耳组织的研究将提供以下证据： 纳米颗粒递送在人类疾病相关组织中的相关性。将我们的工作扩展到大型动物 模型将是这项技术走向临床应用的重要一步。我们的纳米粒子方法 可以应用于需要体细胞类型编辑的其他器官和野生型大型动物的研究。

Project Summary and Relevance Application of genome editing based therapy requires efficient delivery of editing agents into disease-relevant tissues and cells. Identification of novel delivery materials targeting somatic cells will greatly facilitate the advance of editing based therapy strategies to clinic. We propose to screen a large library of novel lipid nanoparticles for RNP (ribonucleoprotein) delivery of editing agents into the mammalian sensory organ inner ear. Inner ear is an ideal sensory organ to develop new delivery strategies. It consists of multiple differentiated somatic cell types without effective delivery options. Gene mutations in the major inner ear cell types have been associated with genetic hearing loss, which affects one in 500 newborns and currently has no effective therapies. Lipid-based nanoparticle carriers have emerged as one of the most promising materials for delivery and have been successfully used in clinical applications. We have developed a combinatorial library approach to synthesize degradable lipid-like nanoparticles under reductive intracellular environments, and capable of delivering biomolecules with high efficiency and low toxicity. The new bioreducible lipid nanoparticles (bLNPs) have been used to deliver genome editing agents with high efficiency and low toxicity in vivo. We have delivered genome editing RNP by cationic liposomes into mammalian inner ear in vivo, and rescued hearing in mouse models of human genetic hearing loss. To develop editing based therapies to treat diverse forms of genetic hearing loss, it is essential to develop a delivery strategy to target multiple inner ear cell types simultaneously. The mammalian inner has a complicated structure with multiple cell types in small numbers, making it particularly challenging to screen a delivery technology by conventional high-throughput strategies. The lack of a method to detect editing at the level of the individual cell type further hinders our ability to apply this technology in wildtype large animal models that are essential for development of this therapy for clinical application. By combining our strategies to screen nanoparticles for delivery of editing materials to X-linked genes in the male mouse inner ears in vivo, we will overcome these hurdles for effective delivery and editing in diverse inner ear cell types. The study of the human inner ear tissues ex vivo will provide evidence of the relevance of nanoparticle delivery in human disease-relevant tissues. Expansion of our work to large animal models will be a major step towards clinical application of this technology. Our approach with nanoparticles can be applied to the study in other organs requiring somatic cell type editing and in wildtype large animals.