Project 467258

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) is a gene-editing technology that can be used to treat disease. One impactful potential application is to use this technology to engineer a patients own immune cells to fight cancer, but the success of this application is presently limited by the methods that are used to deliver the CRISPR/Cas9 system to cells. Relevant modes of delivery are currently extremely inefficient, too harsh for fragile immune cells leading to cell death, or unable to deliver agents as large as CRISPR/Cas9. The proposed research aims to optimize microbubble (MB) mediated sonoporation as a safe method to address these challenges. Sonoporation involves the use of ultrasound to generate an acoustic field in which MBs can interact with cells by creating temporary pores in them, thus allowing for cargo such as CRISPR/Cas9 to be delivered. By designing MBs with properties to encourage pore formation in cells, gentler ultrasound parameters can be used, thus improving both delivery efficiency and cell survival. Sonoporation with these customized MBs will be carried out on immune cells and subsequently assessed for their ability to deliver the cargo into a large percentage of cells while causing minimal cell death. The delivery of a smaller cargo, selected based upon existing sonoporation studies with commercial MBs, will be tested first as an easier task and a proof of concept, followed by the delivery of the CRISPR/Cas9 system which will be compared to other leading delivery methods. By developing this technique of customized MB mediated sonoporation, CRISPR/Cas9-based technologies will continue to pioneer new potential applications in immune cells and other fragile cell types for disease treatment.