PROJECT SUMMARY Congenital lung diseases, such as inherited surfactant protein syndromes, cystic fibrosis, and alpha-1 antitrypsin deficiency, are a significant source of pediatric morbidity and mortality. Treatment options for neonatal patients with lung disorders that present with respiratory failure are limited to palliative care or pediatric lung transplant. As such, there is a clear clinical demand for new therapies that allow for early correction of congenital lung diseases to reduce pediatric morbidity and mortality. Recent advances in gene editing technologies, such as CRISPR-Cas9 systems, have unlocked the potential to correct pathogenic mutations and thereby treat congenital disorders at their source. Performing gene editing in utero offers the added benefits of reversing genetic abnormalities prior to the transition to postnatal life, when pulmonary function becomes essential, and harnessing normal developmental properties of the fetus for more efficient correction. Traditionally, viral vectors have been used to study in utero gene therapy in animal models. Although these studies are encouraging, discovery of alternative, potentially safer, delivery vehicles will advance the field toward clinical translation. Thus, this proposal aims to investigate the potential of ionizable lipid nanoparticles (LNPs), a promising non-viral delivery platform, for nucleic acid delivery to the mouse fetal lung. Fetal lung optimized lipid nanoparticles (FLO-LNPs) will be generated through a multi-stage optimization scheme. In Aim 1, a diverse library of 24 ionizable lipid structures will be screened to identify the ionizable lipid that best delivers mRNA to the fetal lung. In Aim 2, LNP formulations will be optimized using a Design of Experiments scheme for minimal toxicity and maximal delivery of a CRISPR-Cas9 systems in mouse precision cut lung slices. In Aim 3, the optimized LNP formulation will be modified via antibody conjugation and tested for cell-specific targeting in the fetal mouse lung. Ultimately, this proposal – conducted as an interdisciplinary project between sponsors in the Department of Bioengineering, Perelman School of Medicine, and Children’s Hospital of Philadelphia at the University of Pennsylvania – will allow for the development of a novel LNP delivery platform that can be applied in subsequent work to deliver in utero gene therapies for congenital lung disease.