Cas9 RNP delivery to immune cells in vivo via molecular targeting

项目总结/摘要 CRISPR-Cas9已经证明了提供临床益处的令人难以置信的潜力，但交付的挑战 目前阻碍了体内基因组编辑的治疗用途。病毒载体和脂质纳米颗粒的使用具有 建立了体内基因组编辑的可行性，但这些技术具有实质性的缺点。病毒 载体具有免疫原性，难以制造，并且与脱靶风险增加有关。 编辑.如果脂质纳米颗粒靶向肝脏以外的器官，则不适合全身给药。ex 依赖于自体移植的体内疗法已经显示出在基因操作方面的巨大价值， 免疫细胞，但程序仍然是危险的，资源密集型的，昂贵得令人望而却步。 递送治疗性基因组编辑酶的理想方法将是无毒的，与药物相容的， 静脉内给药，适合大规模生产，并靶向需要的细胞类型， 基因矫正考虑到这一切，我们建议以RNA蛋白的形式递送CRISPR-Cas9。 (RNP)复杂. Cas9 RNP已被证明在局部施用后在体内是安全和有效的，并且 我们已经建立了一种策略，使细胞类型特异性递送的Cas9 RNP拴系到分子， 靶向剂（MTA），如受体结合配体、抗体或适体。 我们的提案旨在使用MTA系留的Cas9 RNP用于体内T细胞的靶向编辑。我们将依靠 已建立的和新的MTA促进Cas9 RNP有效和特异性摄取到T细胞中。好吧-- 这些特征化的抗体MTA将指导人、小鼠和灵长类T细胞中的特异性编辑。新型适体 将重点筛选MTA的跨种属反应性，以简化从临床前到 临床发展。因为Cas9 RNP没有固有的穿过细胞膜的能力，所以它将被称为Cas9 RNP。 在MTA诱导后，随着逃避内体以避免溶酶体降解的能力增强， 内吞作用我们已经建立了一种新的模块化方法来功能化Cas9用于内体逃逸， 便于根据需要对特定细胞类型进行再优化。 在UG3阶段，我们将完成以下三个目标：（1）实现体内兼容的基因组编辑， 使用靶向Cas9 RNP的免疫细胞;（2）鉴定用于T细胞特异性免疫的稳健分子靶向剂。 （3）使用靶向Cas9 RNP进行T细胞的体内基因组编辑。独立验证后， 为了在小鼠中进行基因编辑，UH3阶段将执行以下操作：（1）扩大靶向Cas9 RNP的生产，用于在小鼠中进行基因编辑。 大型动物测试;（2）用于非人灵长类动物体内基因组编辑的靶向Cas9 RNP。 基于MTA的细胞靶向和Cas9 RNP的有效内体逃逸的交叉将产生一种新的靶向。 适用于静脉内给药的通用基因组编辑平台。成功完成 所提出的工作将产生一种工程化的Cas9 RNP系统，其在体内安全、有效、容易实现， 制造，和"即插即用"关于其分子靶向多种感兴趣的细胞类型。

PROJECT SUMMARY / ABSTRACT CRISPR-Cas9 has demonstrated incredible potential to provide clinical benefit, but the challenge of delivery currently hinders therapeutic use of genome editing in vivo. Use of viral vectors and lipid nanoparticles has established the viability of in vivo genome editing, but these technologies have substantial drawbacks. Viral vectors are immunogenic, difficult to manufacture, and have been associated with increased risks of off-target editing. Lipid nanoparticles are unsuitable for systemic administration if targeting organs other than the liver. Ex vivo therapies relying on autologous transplantation have shown the immense value in genetic manipulation of immune cells, but the procedures remain risky, resource-intensive, and prohibitively expensive. An ideal method to deliver therapeutic genome editing enzymes would be non-toxic, compatible with intravenous administration, amenable to large-scale manufacture, and targeted to the cell type in need of genetic correction. With all this in mind, we propose delivery of CRISPR-Cas9 in the form of an RNA-protein (RNP) complex. Cas9 RNP has been shown to be safe and effective in vivo following local administration, and we have established a strategy to enable cell type-specific delivery of Cas9 RNP tethered to a molecular targeting agent (MTA) such as a receptor-binding ligand, antibody, or aptamer. Our proposal aims to use MTA-tethered Cas9 RNP for targeted editing of T cells in vivo. We will rely on established and novel MTAs to promote efficient and specific uptake of Cas9 RNP into T cells. Well- characterized antibody MTAs will direct specific editing in human, mouse, and primate T cells. Novel aptamer MTAs will be screened with a focus on cross-species reactivity to streamline the transition from pre-clinical to clinical development. Because the Cas9 RNP has no inherent ability to cross cellular membranes, it will be augmented with the ability to escape the endosome to avoid lysosomal degradation following MTA-induced endocytosis. We have established a novel modular approach to functionalize Cas9 for endosomal escape, facilitating re-optimization for specific cell types as needed. In the UG3 phase, we will complete the following three aims: (1) Enable in vivo-compatible genome editing of immune cells using targeted Cas9 RNP; (2) Identify robust molecular targeting agents for T cell-specific editing; (3) Use targeted Cas9 RNP for in vivo genome editing of T cells. Following independent validation of editing in mice, the UH3 phase will perform the following: (1) Scale up production of targeted Cas9 RNP for large animal testing; (2) Validate targeted Cas9 RNP for in vivo genome editing in non-human primates. The intersection of MTA-based cell targeting and the efficient endosomal escape of Cas9 RNP will generate a versatile genome editing platform suitable for intravenous administration. Successful completion of the proposed work will result in an engineered Cas9 RNP system that is safe, effective in vivo, readily manufactured, and “plug & play” regarding its molecular targeting to multiple cell types of interest.