Novel grafted terpolymers for targeted delivery of CRISPR/Cas9- mediated precise genome editing to the brain

CRISPR/Cas9介导的精确基因组编辑是临床上最有前途的方法之一 管理中枢神经系统(CNS)中的各种人类遗传病。然而， 将这项技术转化为临床应用一直受到几个主要障碍的限制，包括 缺乏安全的方法来同时高效、特异地将Cas9、sgRNA和供体DNA输送到 疾病位置，有限的同源定向修复(HDR)频率由于非主导作用 DNA修复中的同源末端连接(NHEJ)，以及无法穿越血脑屏障(BBB)。至 克服这些挑战，我们建议开发新的、简单的聚合物纳米颗粒，这些纳米颗粒被优化用于输送 通过局部区域和系统管理将精确的基因组编辑传送到大脑。作为初步准备 在这项工作中，我们开发了新的化学，并合成了一组用于基因传递的三聚体。我们建立了 一系列通过对流增强将纳米颗粒(NPs)局部区域输送到大脑的技术 递送(CED)，以及通过自催化将NPs系统递送到大脑的有效方法 脑靶向(ABT)。我们合成了接枝的三聚体纳米粒，可以有效地介导基因的传递 材料，包括CRISPR/CAS9。我们发现了富含亮氨酸重复序列的蛋白31 (LRRC31)优先抑制NHEJ，显著提高CRISPR/CAS9- 中介的精确基因组编辑。在这些进展的基础上，我们建议合成接枝的聚合物纳米颗粒 针对CRISPR/Cas9传递进行优化，识别负责NHEJ抑制的LRRC31基序，以及 评估它们对小鼠的直接、局部区域传递和系统传递精确的基因组编辑 大脑处于UG3开发阶段，并开发扩大聚合物合成和 NPS，并在UH3示范阶段在实验猪身上进行评估。圆满完成 研究将建立一个通用的平台，高效地提供CRISPR/Cas9介导的精确基因组编辑 到大脑，这可能会转化为临床应用。

CRISPR/Cas9- mediated precise genome editing represents one of the most promising approaches to clinical management of a variety of human genetic diseases in the central nervous system (CNS). However, translation of this technology for clinical applications has been limited by several major hurdles, including the lack of safe approaches for efficient, specific delivery of Cas9, sgRNA, and donor DNA simultaneously to the disease location, the limited homology-directed repair (HDR) frequency due to the predominant role of non- homologous end-joining (NHEJ) in DNA repair, and the inability to cross the blood-brain barrier (BBB). To overcome those challenges, we propose to develop novel, simple polymeric NPs that are optimized for delivery of precise genome editing to the brain through both locoregional and systemic administration. As preliminary work, we developed novel chemistry and synthesized a group of terpolymers for gene delivery. We established an array of techniques for locoregional delivery of nanoparticles (NPs) to the brain via convection-enhanced delivery (CED), as well as an effective approach for systemic delivery of NPs to the brain via autocatalytic brain- targeting (ABT). We synthesized grafted terpolymeric NPs that can mediate efficient delivery of genetic materials, including CRISPR/Cas9, to the brain. We discovered leucine-rich repeat-containing protein 31 (LRRC31) that preferentially inhibits NHEJ and significantly enhances the efficiency of CRISPR/Cas9- mediated precise genome editing. Building on those progress, we propose to synthesize grafted polymeric NPs that are optimized for CRISPR/Cas9 delivery, identify LRRC31 motifs responsible for NHEJ inhibition, and evaluate them for direct, locoregional delivery and systemic delivery of precise genome editing to the mouse brain in the UG3 Development Phase, and to develop approaches to scaling up the synthesis of polymers and NPs, and evaluate them in experimental pigs in the UH3 Demonstration Phase. Successful completion of the study will establish a versatile platform for efficient delivery of CRISPR/Cas9- mediated precise genome editing to the brain, which could be potentially translated into clinical applications.