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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：
9810621
负责人：
John Christian Tilton
金额：
$ 69.08万
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/9810621
关键词：
Address Affect Animal Model Animal Testing Animals Antibodies 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 Epithelial Equus caballus Evaluation Family suidae Flow Cytometry Fluorescence Gastrointestinal tract structure Gene Transduction Agent Genes Genetic Diseases Genome engineering Guide RNA Homing Human Image Analysis Imagery 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 Stem cells Subfamily lentivirinae System Technology Testing Tissues Validation Viral base cell type cystic fibrosis mouse cystic fibrosis patients cytotoxicity env Gene Products experimental study flasks gastrointestinal 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 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将基因组编辑复合体体内递送到肺和囊性纤维化(CF)患者的胃肠道、两种组织受累。我们研制出了一种慢病毒- 基于经过合理设计以提高安全性的纳米级蛋白质输送(NanPOD)平台，免疫原性降低，向原代细胞运送货物的效果最佳。在这份提案中，我们将调查细胞- 自由和细胞相关的方法将基因组编辑复合体传输到组织。我们还将生成抗体单链可变区-病毒包膜融合蛋白(scFv-envs)用于特异性靶向肺和肠道细胞，重点是干细胞，以长期纠正基因突变。这些实验将由先进的成像平台指导，从而能够精确地评估动力学和基因组编辑的生物分布，并将在小动物试验中独立验证。最后，这些将与体细胞基因组合作，对大型动物模型进行技术调整和规模调整工程学(SCGE)大型动物实验中心。 UG3阶段(1-3年级) 目的1：优化纳米POD介导的基因组编辑机制在体内向肺和胃肠道组织的转运，在先进的生物发光和低温荧光成像平台的指导下。目标2：制定针对特定细胞类型的纳米POD的策略。目的3：人囊性纤维化(CF)小鼠在肺和胃肠道组织中的测试传递和编辑突变。 UG4阶段(4年级) 目标H1：调整和扩大纳米PODS和“特洛伊木马”细胞的规模，以便将其输送到大型动物肺和胃肠道细胞。目标H2：结合SCGE测试评估大型动物模型中的交付和基因组编辑中锋。