Development of a "Cell Splicing" Technology Platform

Project Summary: The general scientific community already separates different layers or subcellular fractions (i.e., membrane vs. cytoplasm vs. nucleus) as well as subcomponent organelles/machinery (such as mitochondria, lysosomes, etc.) in order to study and better understand cell function. This Trailblazer research proposal seeks to discover how

we might repurpose such components, with major emphasis presently on nuclear transfer or exchange as part of a new synthetic biology approach in creating cell-based therapies. Such efforts will lead to the development of a massively expanded toolbox of interventional therapies with a wide array of potential downstream

applications in biomedicine (i.e., treatments for cancer, genetic and infectious disease, autoimmunity, and tissue injury and repair). This will be achieved through the following: 1) Generate methods to efficiently isolate nuclei from macrophage and T cells for fusion into enucleated red blood cells and platelets. Methods for nuclear isolation will first be optimized using drug and

density centrifugation-induced cellular blebbing and fractionation to isolate nuclei- vs. cytoplasmic component- containing vesicles, called karyoplasts and cytoplasts, respectfully. Karyoplasts will be derived from innate immune macrophage and adaptive immune T cells, and then fused (with PEG) into naturally enucleated RBCs

and platelets, and derived cell constructs will be monitored for viability and function over time. 2) Develop storage, freezing, and thawing requirements to maintain viability of cell-derived cytoplasts and karyoplasts, and fusion constructs. This will be done by exploring different freezing media types, constituent chemical concentrations, or altered protocol temperature kinetics to both store (short vs. long-

term) as well as thaw cells or their components with preserved structure and function (Figure 1, middle). 3) Characterize macrophage- & T cell-derived cytoplasts, as well as new variant cells following nuclear exchange between enucleated macrophage and T cell bodies. Prior enucleation studies show modified cell behavior, therefore it is not only of interest to investigate nuclear exchange but also what happens

to enucleated cells. In addition, nuclear exchange will be attempted with both fresh as well as frozen karyoplast and cytoplast components, with all fusion constructs tested for morphology/viability, proliferation, cytokine expression, and behaviors either derived or distinct from donor cells. This approach will also allow us to

determine how constructs may be tunable as part of a larger plug and play system. 4) Test new constructs in functional assays in vitro and in a therapeutic cancer model in vivo. This strategy will provide a platform to create new cell behaviors related to functional activities like macrophage- related adherence and phagocytosis, as well as T cell-mediated perforin/granzyme cytolysis. Therefore,

constructs will be tested in vitro in adhesion, migration, and co-culture (cytolysis) assays as well as for anti-tumor activity in vivo in mice.