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。

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.