Project Summary / Abstract The development of site specific nucleases for precise gene engineering has advanced basic understanding of genes and their connection to phenotype-causing mutations and physiologically relevant endpoints and treatment strategies to cure human diseases and medical disorders. It is critically important to mention that the safe and efficient delivery approaches ensure the utilization of these programmable nucleases, thereby improving the therapeutic potential of gene therapy. There are some terrific delivery systems and vesicles that include viruses and viral captives that take advantages of their ability to target particular cell types. Nanoparticles such as lipid nanoparticles, peptide nanoparticles, and gold nanoparticles can allow encapsulation of the molecules for the delivery. However, still clever ways to try to address challenges in delivery of molecules are required. We propose a revolutionary approach to realize controlled delivery of Cas9-ribonucleoprotein (RNP). Our long-term goal is to develop a platform for delivering various types of macromolecules efficiently, effectively, and safely for cell engineering in vitro and in vivo. The goal of this project is to develop a gas vesicle (GV) and ultrasound based delivery approach (GVUS) to improve cell viability and cargo delivery efficiency by conjugating GVs and purified RNP and monomeric streptavidin fusion protein (RNP-mSA-bioGV) to form protein clouds for precise delivery using optimized ultrasound excitations after the investigation of ultrasound parameters to induce stable oscillations and cavitation of GVs. GVs are biocompatible and intact for a long time due to their stability while immediate clearance is available upon brief sonication. Stable oscillations and cavitation of GVs under ultrasound excitation will be used for controlled disruption of cell plasma membrane for intracellular delivery. In specific aim 1, we will investigate GV dynamics under different ultrasound excitations and find optimized ultrasound parameters to generate stable oscillations and cavitation of GVs. We will test various delivery modes using optimized parameters. We will develop RNP-mSA-bioGV protein clouds for controlled delivery of RNP for gene editing, followed by the characterization of the protein clouds in specific aim 2. Primary mouse T cells will be engineered using developed protein clouds delivered by GVUS to study gene editing precision and in vitro anti-tumor activities of engineered T cells to assess the feasibility of the proposed approach in specific aim 3. An innovative approach to use protein clouds controlled by ultrasound will revolutionize the current delivery techniques to engineer cells for research in laboratories and clinical applications. Although many cell therapies have significant challenges in manufacturing and cost, we expect that GVUS approach combined with protein clouds will contribute to realize simpler and cheaper patient-specific and cell-base...