Cas9 RNP delivery to immune cells in vivo via molecular targeting

项目摘要/摘要 CRISPR-CAS9已经证明了提供临床益处的难以置信的潜力，但交付的挑战 目前阻碍了体内基因组编辑的治疗应用。病毒载体和脂质纳米粒的使用已经 建立了体内基因组编辑的可行性，但这些技术有很大的缺陷。病毒式传播 载体是免疫原性的，难以制造，并且与偏离目标的风险增加有关。 正在编辑。如果靶向肝脏以外的器官，脂质纳米粒不适合全身给药。例如 依靠自体移植的活体疗法在基因操作方面显示出巨大的价值。 免疫细胞，但这一过程仍然存在风险、资源密集型和昂贵得令人望而却步。 理想的提供治疗性基因组编辑酶的方法是无毒的，与 静脉给药，适合大规模生产，针对需要的细胞类型 基因修正。考虑到所有这些，我们建议以RNA-蛋白质的形式递送CRISPR-Cas9 (RNP)复合体。Cas9 RNP在体内局部给药后被证明是安全和有效的，并且 我们已经建立了一种策略，使特定细胞类型的Cas9 RNP能够与分子捆绑在一起 靶向剂(MTA)，如受体结合配体、抗体或适配子。 我们的建议旨在使用MTA拴系的Cas9 RNP在体内对T细胞进行靶向编辑。我们将依靠 已建立的和新的MTA，以促进T细胞高效和特异性地摄取Cas9 RNP。好的- 特化的抗体MTA将指导人类、小鼠和灵长类T细胞的特异性编辑。新型适配子 将对MTA进行筛选，重点放在跨物种的反应性上，以简化从临床前到 临床发展。因为Cas9 RNP没有穿过细胞膜的固有能力，所以它将是 增强了逃脱内体以避免MTA诱导的溶酶体降解的能力 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以实现内体逃逸， 便于根据需要对特定单元类型进行重新优化。 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)确定针对T细胞特异性的强大分子靶向试剂 编辑；(3)使用靶向Cas9 RNP进行体内T细胞基因组编辑。在独立验证之后 在MICE中编辑，UH3阶段将执行以下操作：(1)扩大靶向Cas9 RNP的生产 大型动物试验；(2)验证靶向Cas9 RNP在非人类灵长类动物体内基因组编辑的有效性。 基于MTA的细胞靶向和Cas9 RNP的有效内体逃逸的交集将产生 适合静脉给药的通用基因组编辑平台。圆满完成 拟议的工作将导致一个工程的Cas9 RNP系统，它是安全的，在体内有效，容易 制造，并“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.