PROJECT SUMMARY/ABSTRACT The rapid development of genome editing tools has outpaced the ability to deliver these molecules specifically to diseased cells in human patients. A major bottleneck in expanding in vivo applications of genome editing is the lack of targeted, cell-specific delivery vehicles. Current state-of-the-art methods rely on lipid nanoparticles or adeno-associated viruses for delivery. Although these methods can broadly edit tissues in the lung, they lack specificity for diseased cells and pose risks of off-target effects. The safe and effective use of CRISPR-Cas enzymes as treatments for pulmonary and infectious diseases requires the development of cell-type specific delivery vehicles in the lung. In this proposal, we will engineer Enveloped Delivery Vehicles (EDVs) to deliver Cas9 ribonucleoproteins to lung epithelial cells. We will target lung epithelial cells due to their involvement in pulmonary diseases such as surfactant disorders. EDVs are bioengineered virus-like particles derived from lentivirus that package Cas9 proteins. We have previously shown that EDVs can specially edit T-cells in culture and in mice by displaying antibodies on their surface. Building on our laboratory’s expertise with virus-like particles and CRISPR-Cas9 mechanistic biology, we will focus on determining the mechanisms of Cas9 packaging that will enable EDV targeting to lung epithelial cells. Our first aim is to determine how EDVs encapsulate Cas9 and its guide RNA. Preliminary data from experiments using chemical inhibitors and genetic deletions have begun to reveal which viral components of EDVs are necessary to mediate genome editing. Non-essential components will be eliminated to optimize EDV design and enhance editing efficiency. Concurrently, our second aim is to identify antibody fragments that facilitate the entry of EDVs into lung epithelial cells. Antibody fragments displayed on the surface of EDVs will target epitopes commonly utilized by respiratory viruses on epithelial cells. We will evaluate the ability of these antibodies to mediate precise cell entry of EDVs and facilitate genome editing. These two aims are independent but complementary. The outcomes of each will provide valuable insights into the internal and external design elements necessary for targeting and editing lung epithelial cells. While genome editing enzymes hold the potential to treat potentially all genetic diseases, strategies to deliver these components beyond the liver and blood are needed to realize this potential for patients. We anticipate that the mechanistic insights gained from this proposal will guide future efforts to target delivery vehicles to new cell types of interest.