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代表了临床上最有前途的方法之一。 管理中枢神经系统（CNS）中的各种人类遗传性疾病。然而，在这方面， 将该技术转化为临床应用受到几个主要障碍的限制，包括 缺乏将Cas9、sgRNA和供体DNA同时有效、特异性递送至受试者的安全方法。 疾病的位置，有限的同源性定向修复（HDR）的频率，由于占主导地位的作用， DNA修复中的同源末端连接（NHEJ），以及不能穿过血脑屏障（BBB）。到 克服这些挑战，我们提出开发新的，简单的聚合物纳米粒子，优化交付 通过局部和全身给药对大脑进行精确的基因组编辑。作为初步 工作中，我们开发了新的化学和合成了一组用于基因传递的三元共聚物。我们建立 一系列通过对流增强将纳米颗粒（NPs）局部递送到大脑的技术 递送（CED），以及通过自催化将NP全身递送至脑的有效方法。 脑靶向（ABT）。我们合成了接枝的三元聚合物纳米颗粒，可以介导基因的有效传递， 包括CRISPR/Cas9在内的所有材料。我们发现了富含亮氨酸重复序列的蛋白质31 （LRRC31），其优先抑制NHEJ并显著增强CRISPR/Cas9 - 1的效率。 介导的精确基因组编辑。在此基础上，我们提出了合成接枝聚合物纳米粒子的方法 针对CRISPR/Cas9递送进行优化，鉴定负责NHEJ抑制的LRRC31基序，以及 评估它们对小鼠进行精确基因组编辑的直接、局部递送和系统递送 在UG3开发阶段的大脑，并开发方法来扩大聚合物的合成， NP，并在UH3示范阶段的实验猪中对其进行评估。成功完成 研究将建立一个多功能平台，用于有效递送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.