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：
10664098
负责人：
JENNIFER A DOUDNA
金额：
$ 125.74万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-23 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10664098
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.

项目摘要 /摘要 CRISPR-CAS9表现出了提供临床益处的难以置信的潜力，但是交付的挑战目前，在体内阻碍了基因组编辑的治疗用途。使用病毒载体和脂质纳米颗粒具有建立了体内基因组编辑的生存能力，但是这些技术具有很大的缺点。病毒性的向量具有免疫原性，难以生产，并且与脱靶的风险增加有关编辑。如果针对肝脏以外的其他器官，则脂质纳米颗粒不适合全身给药。前任依靠自体移植的体内疗法表明，免疫细胞，但这些程序仍然存在风险，资源密集并且非常昂贵。传递热基因组编辑酶的理想方法是无毒的，与静脉内给药，适合大规模制造，针对需要的细胞类型考虑到所有这些，我们建议以RNA-蛋白质的形式提供CRISPR-CAS9 （RNP）复合物。 CAS9 RNP在当地管理后的体内被证明是安全有效的，并且我们已经建立了一种策略，以启用特定于细胞类型的cas9 rnp的递送到分子靶向剂（MTA），例如接收器结合配体，抗体或apatamer。我们的建议旨在使用MTA系的CAS9 RNP进行体内T细胞的靶向编辑。我们将依靠建立且新颖的MTA，以促进CAS9 RNP对T细胞的有效且特定的摄取。出色地- 表征的抗体MTA将指导人，小鼠和灵长类动物T细胞中的特定编辑。新颖的apatmer MTA将筛选，重点是跨物种的反应性，以简化从临床前的过渡到临床发展。因为CAS9 RNP没有跨细胞膜的遗传性，所以它将是增强了逃脱内体的能力，以避免MTA引起的溶酶体降解内吞作用。我们已经建立了一种新型的模块化方法，可以使Cas9官能化以进行内体逃生，根据需要促进对特定细胞类型的重新选择。在UG3阶段，我们将完成以下三个目标：（1）启用体内兼容的基因组编辑使用靶向CAS9 RNP的免疫细胞；（2）确定T细胞特异性的强大分子靶向剂编辑；（3）使用靶向CAS9 RNP进行T细胞的体内基因组编辑。在独立验证之后在小鼠中进行编辑，UH3阶段将执行以下操作：（1）扩大目标CAS9 RNP的生产大动物测试；（2）在非人类隐私中验证靶向的CAS9 RNP进行体内基因组编辑。基于MTA的细胞靶向与CAS9 RNP的有效内体逃逸的交集将产生多功能基因组编辑平台适合静脉内给药。成功完成拟议的工作将导致设计的CAS9 RNP系统，该系统很容易，在体内有效，很容易关于其分子靶向多种细胞类型感兴趣的分子靶向，制造的和“插件”。