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有效地将编辑剂输送到与疾病相关的 组织和细胞。识别以体细胞为靶点的新型递送材料将极大地促进 以编辑为主的治疗策略应用于临床的进展。我们建议筛选一个大型的新型脂类文库 用于RNP(核糖核蛋白)将编辑剂输送到哺乳动物感觉器官内部的纳米颗粒 耳朵。内耳是开发新的递送策略的理想感官器官。它由多个不同的 没有有效投递选择的体细胞类型。主要内耳细胞类型的基因突变一直是 与遗传性听力损失有关，每500名新生儿中就有一人受到影响，目前还没有有效的 治疗。 基于脂质的纳米颗粒载体已经成为最有前途的输送材料之一，并已 已成功应用于临床。我们已经开发了一种组合库方法来 在还原的细胞内环境下合成可降解的类脂纳米粒，并能够 高效低毒地输送生物分子。新型生物可还原脂质纳米粒 (BLNPs)已被用于体内高效低毒的基因组编辑剂的输送。我们 通过阳离子脂质体将基因组编辑的RNP体内输送到哺乳动物内耳，并被拯救 人类遗传性耳聋小鼠模型的听力。开发基于编辑的疗法来治疗不同的 对于多种形式的遗传性听力损失，有必要制定一种针对多种内耳细胞类型的递送策略 同时。哺乳动物的内部具有复杂的结构，细胞类型多样，数量很少， 这使得通过传统的高通量策略来筛选递送技术特别具有挑战性。 缺乏在单个单元格类型级别检测编辑的方法进一步阻碍了我们应用 这项技术在野生型大型动物模型中是必不可少的，这对临床开发这种疗法是必不可少的 申请。通过结合我们的策略来筛选纳米颗粒，以便将编辑材料传递到X-Linked 在体内雄性小鼠内耳中的基因，我们将克服这些障碍，以便有效地传递和编辑 内耳细胞类型多样。对人类内耳组织的体外研究将提供证据 纳米颗粒在人类疾病相关组织中传递的相关性。将我们的工作扩展到大型动物 模型将是这项技术临床应用的重要一步。我们使用纳米颗粒的方法 可应用于其他需要体细胞类型编辑的器官和野生大型动物的研究。

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.