In vivo delivery of CRISPR Cas9-guide RNA nucleoprotein complexes using the nanoPOD platform

使用 nanoPOD 平台体内递送 CRISPR Cas9 引导 RNA 核蛋白复合物

基本信息

批准号：
10004163
负责人：
John Christian Tilton
金额：
$ 69.43万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
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：
2019-09-01 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10004163
关键词：
Address Affect Animal Model Animal Testing Animals Antibodies Bar Codes Binding Biodistribution Biological Bioluminescence Biomedical Engineering CRISPR/Cas technology Cell Line Cells Chimeric Proteins Clinical Trials Collaborations Complex Cystic Fibrosis DNA DNA Repair DNA Sequence Alteration Development Disease Distal Engineering Ensure Enterobacteria phage P1 Cre recombinase Equus caballus Evaluation Family suidae Flow Cytometry Fluorescence Gastrointestinal tract structure Gene Transduction Agent Genes Genetic Diseases Genome engineering Guide RNA Homing Human Immune Kinetics Libraries Luciferases Luminescent Proteins Lung Macaca mulatta Mediating Membrane Methods Modeling Molecular Monitor Mus Mutation Nucleic Acids Nucleoproteins Organoids Patients Phase Phenotype Proteins RNA Receptor Cell Regenerative Medicine Reporter Ribonucleoproteins Safety Somatic Cell Subfamily lentivirinae System Technology Testing Tissues Validation Viral Visualization base cell type cystic fibrosis mouse cystic fibrosis patients cytotoxicity env Gene Products epithelial stem cell experimental study flasks gastrointestinal gastrointestinal epithelium genome editing imaging platform imaging system immunogenicity improved in vivo interest nanoparticle delivery nanoscale novel novel strategies novel therapeutics rare genetic disorder receptor repaired scale up somatic cell gene editing stem cells targeted delivery tool trafficking whole animal imaging

项目摘要

Project Abstract This project aims to optimize technologies for the in vivo delivery of genome editing complexes to the lung and gastrointestinal tract, two tissues affected in cystic fibrosis (CF) patients. We have developed a lentivirus- based nanoscale protein delivery (nanoPOD) platform that has been rationally engineered for improved safety, reduced immunogenicity and optimal cargo delivery to primary cells. In this proposal, we will investigate cell- free and cell-associated methods transport of genome editing complexes to tissues. We will also generate antibody single chain variable fragment (scFV)–viral Env fusion proteins (scFv-Envs) for specific targeting of lung and gut cells with an emphasis on stem cells for long-lasting correction of genetic mutations. These experiments will be guided by advanced imaging platforms enabling precise evaluation of kinetics and biodistribution of genome editing and will be independently validated in small animal testing. Finally, these technologies will be adapted and scaled for large animal models in collaboration with the Somatic Cell Genome Engineering (SCGE) large animal testing centers. UG3 Phase (Years 1-3) Aim 1: Optimize nanoPOD mediated transport of genome editing machinery to lung and GI tissues in vivo, guided by advanced bioluminescence and cryofluorescence imaging platforms. Aim 2: Develop strategies to target nanoPODs to specific cell types. Aim 3: Test delivery and editing in lung and GI tissues using reporter mice with human cystic fibrosis (CF) mutations. UG4 Phase (Year 4) Aim H1: Adapt and scale nanoPODs and the “Trojan Horse” cells for delivery to large animal lung and GI cells. Aim H2: Assess delivery and genome editing in a large animal model in collaboration with the SCGE testing centers.

项目摘要该项目旨在优化用于体内基因组编辑复合物向肺部和胃肠道，两种组织在囊性纤维化（CF）患者中。我们已经开发了慢病毒 - 基于纳米级蛋白（Nanopod）平台，该平台经过合理设计，以提高安全性，降低了免疫原性和最佳货物递送到原代细胞。在此提案中，我们将研究细胞 - 基因组编辑复合物传输到组织的游离和细胞相关方法。我们还将生成抗体单链可变片段（SCFV） - 病毒融合蛋白（SCFV-ENVS）用于特定靶向的特定靶向肺部和肠道细胞重点是干细胞，以恢复遗传突变的持久校正。这些实验将以高级成像平台为指导，以实现动力学和动力学的精确评估基因组编辑的生物分布，将在小动物测试中独立验证。最后，这些技术将与体细胞基因组合作，对大型动物模型进行调整和缩放工程（SCGE）大型动物测试中心。 UG3阶段（1-3年） AIM 1：优化纳米动物介导的基因组编辑机械传输到体内肺和GI组织的运输，在晚期生物发光和冷荧光成像平台的指导下。目标2：制定策略将纳米动物靶向特定的细胞类型。 AIM 3：使用人囊性纤维化（CF）的记者小鼠在肺和GI组织中进行测试和编辑突变。 UG4阶段（第4年） AIM H1：适应和缩放纳米动物和“特洛伊木马”细胞，用于传递到大型动物肺和胃肠道细胞。 AIM H2：与SCGE测试合作评估大型动物模型中的传递和基因组编辑中心。