7. PROJECT SUMMARY/ABSTRACT Many emerging therapeutic strategies employ macromolecules, like proteins or RNAs, to manipulate intracellular contents and processes, thus mitigating disease. The success of these therapeutic strategies requires the ability to safely and efficiently deliver macromolecules into cells within the body, which poses a significant challenge. To overcome the biological barriers to macromolecular delivery, researchers have developed various delivery vehicles, such as viral vectors and nanoparticles, that can package and deliver macromolecular cargos into desired target cells. Recently, cell-derived bioparticles, including virus-like particles and extracellular vesicles, have emerged as promising delivery vehicles that combine key benefits of both viral- and nanoparticle-based delivery methods. However, a major challenge associated with these new methods is their poor manufacturability, which severely limits their prospects for clinical translation. The proposed work seeks to unlock the full potential of cell-derived bioparticles for therapeutic macromolecule delivery by developing new strategies for highly efficient bioparticle production. Initial efforts will focus on identifying, understanding, and manipulating genes in bioparticle producer cells that influence bioparticle production efficiency. To determine genes that regulate bioparticle production, we will assess the effects of thousands of different genetic perturbations on bioparticle production by performing pooled genome- wide knockdown and activation screens in producer cells. These screens will utilize a unique design in which sequencing a given bioparticle’s contents will identify the perturbation in the cell that produced that bioparticle. We will perform separate screens to interrogate (1) the production of virus-like particles that package ribonucleoprotein cargos and (2) the production of extracellular vesicles that package messenger RNA cargos, yielding insights into two distinct types of cell-derived bioparticles and two important classes of therapeutically relevant macromolecular cargos. These results will not only enhance our understanding of the mechanisms of cell-derived bioparticle formation but also reveal new ways to manipulate producer cells to improve particle production. In parallel, we will establish a new paradigm for directly transferring therapeutic bioparticles from producer cells into target cells via cell-to-cell contact, a strategy that has the potential to substantially improve delivery potency relative to existing methods. This investigation will lay a foundation for potentially transformative therapeutic approaches in which patient-derived cells are engineered into bioparticle producer cells and transplanted into the body, where they would subsequently engage desired target cells to deliver therapeutics via locally released bioparticles. Collectively, these studies will dramatically expand the utility of cell-derived bioparticles as delivery vehicles for macromolecular therapeutics.

8. PROJECT NARRATIVE The ability to deliver macromolecules into cells within the body is required for many emerging therapeutic modalities, including genome editing therapies, but there are few available methods for safe and efficient macromolecular delivery. The proposed work will explore the use of cell-derived bioparticles for therapeutic macromolecular delivery, with a focus on (1) understanding cellular factors that influence bioparticle formation and (2) developing new strategies for highly efficient bioparticle production. These advances would overcome the key limitations that currently prevent the widespread use of cell-derived bioparticles, unlocking the potential of these bioparticles to serve as next-generation therapeutic delivery vehicles to treat a wide range of diseases.