Associate Professor Boston University, United States
Introduction:: The overall goal of this project is to create a non-invasive, easily administered, and simply monitored diagnostic method for heart transplant rejection. Our approach to achieving this is to develop a sensitive and specific cell-based sensor that unites modular targeting receptors and signal amplifying genetic circuits for expression of a reporter molecule that is detectable in blood. If successful, this research will demonstrate the feasibility of an innovation strategy for diagnosis of heart transplant rejection and may enable more regular testing and earlier detection for improved patient outcomes in the clinic.
Materials and Methods:: Cell Lines: The key cell lines that we are using are HEK293T cells, NK92, CTLL-2, 2H-11, and HUVEC. NK92 and CTLL-2 will be routinely verified by cell surface staining. Furthermore, since NK92 and CTLL-2 are used as chassis to house our genetic circuits for detection of transplant rejection, its exact identity will have little impact to the conclusion of the project. The expression of p-selectin and MHC class I on target cells 2H-11 and HUVEC will be verified almost everytime an experiment is done with them using antibody staining. We will test for mycoplasma contamination periodically and whenever a new lines of cells are thawed. DNA constructs: During the course of these projects, many DNA constructs will be generated. All DNA constructs will be sequence-verified. Every DNA constructs that will be used in publications will be stored separately in a dedicated space in the freezer. The constructs will again be sequence-verified before publication. Mouse models: Animal research involves utilizing 6-8 week old female mice purchased from the Jackson laboratory (cat#: 001303). Female and male mice will be intravenously inoculated with 5-10x106 cells from cancer cell lines engineered to express different levels of the antigens. Xenograft tumors will be allowed to develop for 5-7 days post-inoculation when engineered NK cells will be delivered via iv or ip injection. Tumor burden will be measured using luciferase and IVIS imaging.
Results, Conclusions, and Discussions:: I have collected in vivo data that shows anti-CD19 synNotch Jurkat T cells (7.5x106) can detect CD19+ Nalm6 leukemia cells (5x106) and produce Gluc at 26 fold over no Nalm6 condition. This highly encouraging data demonstrates the feasibility of using SynNotch immune cells as diagnostics, even without the circuits and optimization proposed in this work. Additionally, I have preliminary data showing that Jurkat T cells transduced with constitutive Gluc reporters can produce high levels of Gluc after 48 hours without irradiation (fold change of 982x). My lab has also shown that we can use FlpO to control the similarly high level of Gluc production in HEK293T cells. Using our shRNA design, we have collected preliminary data demonstrating that this cFFL can, in transiently transfected Human Embryonic Kidney (HEK) cells, improve the SNR of doxycycline-inducible expression of FlpO, when compared to a system without shRNA production. We have also evaluated our system with a synNotch input in HEK cells. Transiently transfected αCD19 synNotch has low inducibility and high basal activity in HEK cells. Therefore, we integrated the synNotch into the HEK cells and selected the cells with the lowest basal activity. However, this caused the maximum activity to also decrease significantly. We posit that the Notch receptor activity is not optimal in HEK cells because synNotch has a much higher dynamic range in immune cells. To combat this loss in activity, we show that the DE circuit significantly improves and digitizes the αCD-19 synNotch receptor signaling output. These promising results will be a foundation for developing our DE circuits using synNotch as the tumor antigen sensor in NK92 cells. Finally, our lab has shown effective design and implementation of universal CAR for targeting of Her2+ cells which suggests the feasibility of engineering universal synNotch receptors in future work.
