Cas9 RNP delivery to immune cells in vivo via molecular targeting

Cas9 RNP 通过分子靶向递送至体内免疫细胞

基本信息

批准号：
10214471
负责人：
JENNIFER A DOUDNA
金额：
$ 77.63万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-28 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10214471
关键词：
Address Animal Model Animal Testing Antibodies Autologous Transplantation Binding CRISPR/Cas technology Cell membrane Cells Cellular Membrane Clinical Development Disease Endocytosis Endosomes Engineering Ensure Enzymes Fruit Genetic Homing Human Immune Immunoglobulin Fragments Ligand Binding Liver Macaca Macaca mulatta Mediating Methods Mind Molecular Target Mus Organ Peptides Phase Play Preparation Primates Procedures Production Property Proteins RNA Resources Risk Rodent Safety Series Specificity Surface System T-Lymphocyte Technology Testing Therapeutic Therapeutic Uses Validation Viral Vector Virus Work aptamer base cell type clinical development cross reactivity design experimental study genetic manipulation genome editing high throughput screening humanized mouse immunogenic improved in vitro testing in vivo interest intravenous administration lipid nanoparticle new technology nonhuman primate novel pre-clinical prevent protein complex receptor binding recruit scale up screening stem targeted agent targeted delivery therapeutic genome editing uptake

项目摘要

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.

项目概要/摘要 CRISPR-Cas9 已展现出提供临床益处的令人难以置信的潜力，但交付面临挑战目前阻碍了体内基因组编辑的治疗应用。确立了体内基因组编辑的可行性，但这些技术有很大的缺点。载体具有免疫原性，难以制造，并且与脱靶风险增加有关如果靶向肝脏以外的器官，则脂质纳米颗粒不适合全身给药。依赖自体移植的体内疗法在基因操作方面显示出巨大的价值免疫细胞，但该过程仍然存在风险、资源密集型且昂贵得令人望而却步。提供治疗性基因组编辑酶的理想方法是无毒的、与静脉内给药，适合大规模生产，并针对需要的细胞类型考虑到这一切，我们建议以 RNA 蛋白的形式提供 CRISPR-Cas9。 (RNP) 复合物已被证明在局部给药后在体内是安全有效的，并且我们已经制定了一项策略，能够将 Cas9 RNP 与分子结合进行细胞类型特异性递送靶向剂 (MTA)，例如受体结合配体、抗体或适体。我们的建议旨在使用 MTA 束缚的 Cas9 RNP 对体内 T 细胞进行靶向编辑。建立了新型 MTA，以促进 Cas9 RNP 有效且特异性地摄取到 T 细胞中。特征化的抗体 MTA 将指导人类、小鼠和灵长类动物 T 细胞的特异性编辑。 MTA 的筛选将重点关注跨物种反应性，以简化从临床前到临床前的过渡由于Cas9 RNP没有固有的穿过细胞膜的能力，因此其将在临床开发中发挥重要作用。增强了逃离内体的能力，以避免 MTA 诱导后的溶酶体降解我们建立了一种新颖的模块化方法来功能化 Cas9 进行内体逃逸，促进根据需要对特定细胞类型进行重新优化。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) 识别 T 细胞特异性的强大分子靶向剂编辑；(3) 在独立验证后使用靶向 Cas9 RNP 对 T 细胞进行体内基因组编辑。在小鼠中进行编辑后，UH3 阶段将执行以下操作： (1) 扩大靶向 Cas9 RNP 的生产大型动物测试；(2) 验证用于非人类灵长类动物体内基因组编辑的靶向 Cas9 RNP。基于 MTA 的细胞靶向与 Cas9 RNP 的有效内体逃逸的交叉将产生适合静脉注射的多功能基因组编辑平台成功完成。拟议的工作将产生一种工程化的 Cas9 RNP 系统，该系统在体内安全、有效、易于使用制造，并且“即插即用”关于其针对多种感兴趣的细胞类型的分子靶向。