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.