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

项目摘要 该项目旨在优化将基因组编辑复合物体内递送至肺部的技术， 胃肠道，囊性纤维化（CF）患者的两种组织受影响。我们发明了一种慢病毒- 基于纳米级蛋白质递送（nanoPOD）平台，该平台已被合理设计以提高安全性， 降低免疫原性和向原代细胞的最佳货物递送。在这份提案中，我们将调查细胞- 游离和细胞相关方法将基因组编辑复合物转运至组织。我们还将产生 抗体单链可变片段（scFV）-病毒Env融合蛋白（scFv-Envs），用于特异性靶向 肺和肠细胞，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，能够精确评估动力学， 基因组编辑的生物分布，并将在小动物测试中独立验证。最后这些 将与体细胞基因组合作，对这些技术进行调整，并将其用于大型动物模型 工程（SCGE）大型动物试验中心。 UG 3阶段（1-3年） 目的1：优化nanoPOD介导的基因组编辑机器向体内肺和胃肠道组织的转运， 由先进的生物发光和冷冻荧光成像平台引导。 目标2：开发将nanoPOD靶向特定细胞类型的策略。 目的3：使用患有人类囊性纤维化（CF）的报告小鼠测试肺和GI组织中的递送和编辑 突变。 UG 4阶段（第4年） 目标H1：调整和扩展纳米POD和“特洛伊木马”细胞，以递送到大型动物肺和GI细胞。 目标H2：与SCGE测试合作，评估大型动物模型中的递送和基因组编辑 中心.

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.