The development of reporter genes for different imaging modalities has revolutionized the way many current biomedical studies are designed, examined, and implemented. MRI reporter genes allow the longitudinal study of the same subject with unlimited tissue penetration, which may be useful for studying dynamic biological processes, such as the survival of transplanted therapeutic cells, their long-term fate, and their functionality. Moreover, MRI reporters have the potential for clinical translation. This proposal aims to develop, optimize, and implement a Chemical Exchange Saturation Transfer (CEST)-MRI genetically engineered reporter system. We have recently used a synthetically designed nucleoside as a CEST-based agent for the in vivo imaging of the herpes simplex virus type-1 thymidine kinase (HSV1-tk) reporter gene expression with MRI. Capitalizing on the capability of the HSV1-tk to phosphorylate synthetic nucleoside analogs, as do other enzymes of the deoxyribonucleoside kinase (dNK) family, the compound 5-methyl-5,6-dihydrothymidine (5-MDHT) has been used as a CEST reporter probe for imaging the HSV1-tk reporter gene. The relatively large ? (5 ppm) of the imino exchangeable proton of 5-MDHT makes it suitable for diamagnetic CEST imaging with minimal contributions of endogenous contrast and limited effects from direct water saturation. The drosophila melanogaster dNK (Dm-dNK) phosphorylates a wider range of nucleoside analogs compared to HSV1-tk, including fluorescent nucleosides, and therefore, could be considered as an alternative MRI reporter gene. This proposal aims to develop, optimize, and implement a CEST-MRI genetic reporter system that couples a dNK reporter gene with an appropriate CEST reporter probe. As a first step, a series of potential CEST probes will be examined to characterize their imaging capabilities, such as ?, kex, and contrast enhancement, which are critical for the design of CEST agents. Then, their activity with a recombinant Dm-dNK enzyme will be studied to determine the optimal probe (i.e., substrate). We will compare the Dm-dNK reporter system with our previous reporter system based on the HSV1-tk reporter gene through in vitro assessments of the imaging capabilities. This will include quantitative sensitivity determination by studying te obtained CEST contrast per number of live cells expressing the transgene. Moreover, the cytotoxicity effect of both reporters will be tested. As an example of a potential application, we will capitalize on the ample experience of our research group in pancreatic islet cell engraftment into animal models of diabetes. We will engineer pancreatic islet cells to express the preferred dNK reporter gene, and transplant these islet cells into diabetic mice. The long-term survival of the engrafted cells will be monitored, longitudinally and non-invasively, using CEST-MRI. The CEST contrast that reports on the viability of the cells will be correlated with their therapeutic effect, i.e., insulin secretion and blood glucose levels. Upon completion of this study, we anticipate establishing a new approach for the non-invasive imaging of gene expression with MRI for a broad spectrum of biomedical applications.
Engraftment of therapeutic cells is a new treatment strategy for many diseases; including type I diabetes; where pancreatic islet cells are transplanted to provide physiological insulin regulation. We propose to develop improved CEST-MRI reporter genes for real-time monitoring of the fate of the engrafted cells; longitudinally and noninvasively This approach will help to optimize and improve cell-therapy-based applications in animal models of Type 1 diabetes in particular; and in other potential cell-based treatments in general.
