Cell-based therapies hold tremendous potential to treat cancer, inflammatory conditions, and degenerative diseases. However, despite a few notable successes, cell-based therapies face key clinical barriers, including cell source heterogeneity, inefficient directed migration (homing) to target tissues, cell dosing or controllability, inability to affect tissue microenvironments, and patient safety. Bioengineering may potentially enhance cell capabilities, but raises safety concerns regarding the control of genetically engineered cells in vivo. Therefore, bioengineering methods

to improve cell-based therapy efficacy must prioritize predictable behavior and safety.

To address these issues, the Klemke Laboratory developed a platform technology that couples cell biongineering with cell enucleation (mass removal of nuclei) to enhance cellular function and controllability. By adapting methods developed in the 1970s, mesenchymal stromal cells (MSCs) were enucleated by ultracentrifugation in discontinuous Ficoll gradients containing cytochalasin B. Combining cell bioengineering with enucleation both decreases the risk of engraftment and oncogenesis, and provides opportunities for customized pre- and/or post-enucleation bioengineering. We named these bioengineered, enucleated cells “CargocytesTM”.

Cargocytes were characterized by absent nuclei, viability for 2-3 days, functional protein translational machinery, retention of receptor expression, and directional migration, and then further investigated via bioengineering with therapeutically-relevant properties. For in vitro studies, Cargocytes were bioengineered and then examined for secretion of functional cytokines and expression of functional chemokine receptors. Next, Cargocytes in vivo abilities were examined in two independent mouse models. First, bioengineered Cargocytes injected intratumorally in a mouse breast cancer model produced functional antitumor cytokine that slowed tumor progression and increased animal survival. Second, bioengineered Cargocytes injected intravenously in a mouse ear inflammation model both homed to the site of inflammation and produced functional anti-inflammatory cytokine that reduced inflammation. This suggests that bioengineered, enucleated cells are more efficacious than non-engineered cells, and lacking nuclei uniquely contributes to safety and controllability in vivo.

To my knowledge, therapeutic bioengineering of enucleated cells has not been previously described, and Cargocytes represent a unique entity in the cell-based therapy field as either a stand-alone or adjuvant therapy. By retaining biologically important cell-like functions yet with advantages like small size and defined lifespan, Cargocytes potentially resolve key barriers currently limiting cell-based therapies.

Many mesenchymal stem cell (MSC) based therapies have the common issue ofcells becoming trapped in the lung vasculature. Our novel enucleated cell therapy, named “Cargocytes,” also encounter this issue, though to a lesser degree due to their smaller size. Lung trapping can be especially dangerous in a therapeutic because it can cause embolisms in the lung vasculature. We therefore designed and optimized a series of protocols to further reduce the cell size of these Cargocytes and obtain minimal lung trapping after intravenous injection. By using a spinner flask for 3D culture, we show that the size of the MSCs and Cargocytes can be significantly reduced after 48 hours of culturing, as compared to cells cultured in a 2D environment. After optimizing the spinner flask culture, we then proceeded to optimize the enucleation process for 3D cells such that we could get a sufficient yield at an acceptable efficiency. We next compared the cell surface markers of 2D MSCs and 3D MSCs. Markers for MSCs remained constant even after culturing in 3D for 48 hours. Because Cargocytes are a novel platform technology, some diagnostic assays have yet to be conducted on these new cell carriers. We conducted a series of tests to look at markers of apoptosis, such as an Annexin V and multi-caspase assay. We found the profiles to be comparable to that of the parental MSCs. Finally, we engineered hTERT-MSCs with exogenous CDK4 to potentially allow cells to grow under 3D culture conditions. Although the CDK4 engineering did not promote growth of MSCs in 3D conditions, the cells were able to survive much longer. A cell cycle analysis also revealed that these newly engineered cells have slightly more cells in the G2 phase compared to parental hTERT-MSCs.