Genetically-encoded molecular imaging of genome-edited immune cells - Project Summary Adoptive cell transfers constitute a new paradigm in cell-based therapeutics with wide-ranging applications from neurodegenerative, cardiovascular, autoimmune disorders as well as cancer. Autologous lymphocytes from the patient are genetically engineered to express receptors to specific antigens on the cell surface of target cells and reinfused back into the patient for therapeutic action. However, once administered the fate of cells remains uncertain. In order to answer important questions regarding distribution, turnover and eventual survival of the cells, techniques to non-invasively monitor these genome-edited cells is necessary. Molecular imaging, particularly Positron emission tomography is advantageous over traditional diagnostic and imaging tools because it enables diagnosis and tracking of radiotracers in real-time, is non-invasive and has the highest sensitivity among clinical imaging modalities. We propose to develop analogues of Green fluorescent protein/luciferase for nuclear imaging, thus combining the highly desirable elements from both these powerful modalities towards a chemogenetic nuclear imaging modality. The metallophore-transporter complex found in bacteria is genetically encoded in the prokaryotic DNA and offers the ideal chemical-biological pair that can be expressed on mammalian cells to enable nuclear imaging and tracking of these cells. By engineering mammalian cells to ectopically express the bacterial transporters, we will be able to selectively target and image the genome -edited cells in vivo using metallophore-radionuclide probes. Bacterial metallophores have evolved to serve as metal chelators for a wide variety of metals with the majority showing a high binding affinity for iron. However, several pathogenic bacteria secrete metallophores with the highest binding affinity for copper(II) (Cu). We, therefore, propose to use 64Cu, a popular radionuclide in Positron Emission Tomography (PET) imaging to generate metallophore/64Cu complexes as contrast agents. We have identified metallophores that are able to evade the innate immune system and avoid imminent sequestration. Combined with simple and one-step processing techniques, they are attractive agents for depth-independent real-time imaging, tracking and identifying genome-edited cells. Because the native or wild type cells do not express these transporters, we expect minimal uptake in normal mammalian cells including native bacterial “commensal” flora, as they remain exclusive to pathogenic bacteria. We hypothesize that through a combination of facile coordinate complexation chemistry, high selectivity, immune evasiveness and non-endogenous nature, bacterial metallophore/64Cu will serve as ideal nuclear imaging probes to identify and accurately detect GECs in vivo. In the first phase we will develop and evaluate the bacteria transporter protein mammalian expression vector and subsequently will be able to incorporate the vectors into CAR-T-Cells for in vivo reporting. In the next phase we will determine the in vivo pharmacokinetics, biodistribution and PET imaging of the probes and genome-edited cells in animal models. In summary this project we will use metallophores as dual role compounds - as a chelator as well as a targeting ligand for imaging, which is novel and innovative. If successful, this strategy will enable precise labeling of individual genome-edited cells non-invasively in vivo and potentially avoiding the shortfall of previous PET reporters such as immunogenicity and low selectivity. Due to the broad impact and transformative potential of this project we envision a quick and clear path to clinical trials beyond project phase.
