Programmable In Vivo Delivery and Monitoring of Proteins by Extracellular Vesicles

Genetically modified cells have revolutionized disease treatments by harnessing the power of the immune system. However, these approaches present challenges, including limited control over cell interactions, significant safety concerns, and the need to genetically engineer cells ex vivo. In PROVIDE, I propose a conceptually novel approach to engineer cells in vivo by delivering specific membrane proteins to target cells using extracellular vesicles (EVs). EVs are natural mediators of intercellular communication and can serve as biocompatible delivery vehicles of molecules. Furthermore, EVs are capable of displaying specialized proteins through genetic engineering of parental cells. However, the use of EVs as treatments faces challenges including limited targeting to and fusion with target cells, poor pharmacokinetics, and inefficient technologies for loading EVs with therapeutic or labeling agents. I hypothesize that rationally programming EVs to display specialized proteins can systematically address each of these issues. PROVIDE will develop a modular library of surface proteins displayed on the surface of EVs to enhance targeting, pharmacokinetics, and membrane fusion for efficient in vivo delivery of therapeutic proteins directly into the membrane of target cells. Moreover, ion transporters capable of shuttling a wide range of substrates across the membrane, including multiple specialized MRI agents with different ""colors"", will be expressed in the membrane of EVs. This will enable in vivo monitoring of multiplex target delivery and tracking of modified cells using advanced heteronuclear MRI techniques. As a demonstration, I will simultaneously deliver a receptor-ligand pair to tumor and T cells, respectively, and monitor the recruitment of engineered T cells to engineered tumor cells in vivo.PROVIDE addresses key limitations of current cell engineering strategies and will develop a cell-free platform for precise image guided treatments""