PROJECT SUMMARY Since 2017, six CAR-T cell therapies and four hematopoietic stem cell (HSC) therapies have been approved in the U.S. and E.U. to treat cancers, hemophilia, ALD, MLD, and ADA-SCID. These therapies use a lentivirus (LV) to efficiently deliver and integrate a therapeutic gene into the host cell genome, but LVs are expensive to produce and their semi-random genomic integration patterns have been shown to induce cancer in some patients (e.g., 3/67 patients treated with SkysonaTM). Therefore, while LV is a highly efficient gene delivery vehicle, its disadvantages have motivated the search for alternative non-viral gene delivery (NVGD) methods. The goal of this collaborative multi-PI proposal is to develop NVGD methods that are less expensive and safer than LV, while achieving comparably high levels of transgene expression. In Aim 1, the Bracaglia lab will develop novel polymeric gene delivery vehicles that maximize the delivery of siRNAs (or other cargoes) to T cells and HSCs. In Aim 2, Drs. Elmer and Huang will collaborate to discover and test novel drugs that temporarily inhibit host cell proteins that repress foreign genes. Finally, the Elmer lab will optimize the non-viral integration of transgenes in Aim 3 by identifying the safest and most effective sites for transgene integration in the T cell and HSC genomes. Altogether, these efforts will result in the development of novel NVGD methods that significantly improve a wide range of current and future gene therapies and provide a holistic research experience in cellular engineering for at least 18 undergraduate students that will work on this project as part of our Senior Project Studio (SPS), in which students will work with the PIs on the proposed projects in Aims 1-3 for course credit. In addition, the PIs will also involve dozens of additional students in the project by designing relevant hands-on research projects that will be incorporated into six lab and lecture courses that are taught by the PIs.