Periodic Reporting for period 4 - GeneREFORM (Genetically Encoded Multicolor Reporter Systems For Multiplexed MRI)
Reporting period: 2020-11-01 to 2021-04-30
The dynamicity and complexity of biological processes are crucial for the function and communication of live cells, and the proper regulation or inappropriate occurrences associated with these biological processes can involve in any aspect of health or disease. Researchers and clinicians from diverse fields of science—from physics through chemistry, engineering, and biology to medicine—are challenged by the complexity of intra- and inter-cellular processes. A major challenge is to monitor, characterize, quantify, and better understand elusive biological events and to shed light on their multiplexity. Although optical reporter genes, with their “multicolor” imaging capabilities, have revolutionized science their light (e.g. luminescence, fluorescence) signal source restricts their use in deep tissues and in large animals (and potentially in humans). The versatility of MRI sensors and reporters allows their design and development with color-like features for multiplex imaging. The overall goal of this project is to further expand our knowledge and gain a better understanding of the multifaceted processes in complex organisms, multiplexed imaging setups are desperately needed. To this end, inspired by the multicolor capabilities of optical reporters, this project aims to develop, optimize, and implement MRI reporters (sensors) with artificial “multicolor” characteristics. Capitalizing on (i) the frequency encoding, color-like features of several types of MRI contrast mechanisms, among them are CEST-MRI and 19F-MRI and on (ii) enzyme engineering procedures that allow the optimization of enzymatic activity for a desired substrate, “multicolor” MRI reporter systems were developed. The impact of the proposed imaging platform on many fields in biomedicine will grow rapidly with advances in developing fields, such as cell-based therapies, as well as personalized and/ or regenerative medicine that mandate creative, multiplexed monitoring abilities. For many biological processes that are still illusive, and for others that cannot yet be monitored in the deep tissues, the proposed platform, which aims to allow in vivo imaging of multiple reporter genes expression, may be the light at the end of the tunnel and perform (upon successful creation) beyond the state-of-the-art multicolor imaging reporters.
By combining the frequency-encodability of CEST-MRI that allows virtual magnetic tagging of biomolecules to generate artificial MRI-based colors, with orthogonal evolution of promiscuous enzymes of the deoxyribonucleoside kinase (dNK) family a platform for colored MRI mapping of genes expression was developed. By applying an automated evolution-guided protein design approach on two promiscuous dNKs, namely HSV1-TK and Dm-dNK, followed by a single-site mutagenesis resulted in two orthogonal dNKs (“reporter genes”) that specifically convert two, MRI-detectable, synthetic deoxyribonucleosides (“reporter probes”). Systemically administrated “reporter probes”, which exclusively accumulate in cells expressing the mutated-dNK transgenes upon their specific phosphorylation, were virtually tagged and displayed as pseudo-colored MRI maps for non-invasive, in vivo visualization of transgenes expression based on a proton exchange contrast mechanism. As alternative to “multicolor” proton CEST agents, where pronounced and complex endogenous magnetization transfer processes occur and might contribute to endogenous CEST, MTC and NOE, complicating data interpretation, we propose to use fluorinated MRI sensors for “artificial” multicolor imaging. We demonstrated that small, water-soluble 19F-ionic NCs can average out homonuclear dipolar interactions, enabling one to obtain high-resolution 19F NMR signals in solution. Decorating 19F-NC surfaces with a biocompatible coating enabled their use as imaging tracers for in vivo 19F MRI by facilitating a “hot-spot” display of their distribution. We also demonstrated that MRI signals of different nanofluorides (i.e. CaF2 and SrF2) can be color-encoded based on the difference between their 19F NMR chemical shifts and displayed in a multiplexed manner. Furthermore, we combined CEST-MRI and 19F-MRI as a novel approach for multicolor MRI. Specifically, capitalizing on reversible host-guest binding dynamics and using CEST in 19F-MRI framework, pseudo-colored maps of complexed arrays were demonstrated ashowing the ability to generate MRI-based polychromatic palette, as an advanced strategy for light-free, multicolor-mapping.
The proposed research aims to mimic the multicolor capabilities of optical reporters that are, currently, the state-of-the-art imaging tool in basic sciences. The ability to monitor multiplexed biological systems, non-invasively and present them in a multicolor fashion offers imaging capabilities that are not exist today. The 15.2 T MRI scanner used for this project provides us with an imaging platform that position us even further in regard to imaging performances and allow us with capabilities to obtain more artificial colors for CEST MRI applications. Moreover, the demonstration of the applicability diverse nanoifluorides (e.g. CaF2 or SrF2) to provide “color-like” features of 19F-MRI for multiplexed mapping, showing the potentiality of the approach to monitor multiple targets, simultaneously, at the same imaging frame. Finally, our demonstration of a novel approach for non-invasive mapping of multiple targets by MRI based on the combination of CEST MRI and 19F-MRI with capability to amplify MR signals of micromolar concentrations of thermally-polarized nuclear spins could be further developed and applied for a variety of applications.