Programmable therapeutic macromolecule delivery via engineered trogocytosis - PROJECT SUMMARY/ABSTRACT Efficient cell-specific delivery is the key bottleneck of applying macromolecule therapies to various diseases. Traditional delivery vehicles each have their own limitations. For instance, viral vectors and nanoparticles are limited by poor specificity, small cargo capacity, and low programmability to different tissue/cell types. As cell- based therapies gain clinical momentum, we propose a paradigm shift by using autologous or allogeneic cells as vehicles to deliver therapeutic biomolecules, enabling precise, context-dependent delivery to various cells. Inspired by the natural phenomenon of trogocytosis, where one cell acquires a patch of membrane and associated molecules from another cell through direct contact, we engineered donor cells with customized receptors and a pH-sensitive fusion protein (termed fusogen) to deliver large cargos such as CRISPR-Cas9 and base editors into recipient cells. Upon trogocytosis, the fusogen facilitates cargo endosomal escape, while a release domain preferentially cleaves at the acidic pH frees the cargo within the recipient’s cytosol or nucleus for downstream function (e.g., gene editing in the nucleus or triggering apoptosis in the cytosol). We termed this cell-based delivery system “Trogocytosis-based tRANSfer and Functional Effector Release” (TRANSFER). In the proposed studies, we aim to develop TRANSFER as a therapeutically relevant system and demonstrate its applications for disease treatment in vivo. In Aim 1, we will improve TRANSFER using primary cells as the donors (e.g., T cells and retinal pigment epithelial cells [RPE]) for cell-type-specific delivery to various primary recipient cells by refining key components, including transmembrane domains, fusogens, and release domains, developing targeting strategies for primary recipient cells, and expanding the range of cargos delivered. In Aim 2, leveraging our expertise on retinal biology and diseases, we will evaluate TRANSFER in simultaneous prime editor and neurotrophic factor delivery in a preclinical retinal degeneration model, providing a proof-of-concept that could extend beyond neurodegenerative diseases. Overall, this project lays the groundwork for a novel, powerful, patient-friendly cell-based system that addresses major hurdles in delivering macromolecular therapeutics (including editing enzymes and biologics). By synergizing the safety and targeted potential of cell-based therapies with the precision of engineered release mechanisms, TRANSFER could significantly expand treatment options for neurodegenerative, ocular, and other diseases where localized, large-cargo delivery is critical. If successful, it can greatly expand the methods for efficient therapeutic cargo delivery in vivo, fueling the development of next generation cell therapy and gene therapy toward disease such as retina disorder, muscular disease, and neurological diseases.
