Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - GeneREFORM (Genetically Encoded Multicolor Reporter Systems For Multiplexed MRI)
Teaser
In order to fully understand the complexity of biological processes that are reflected by simultaneous occurrences of intra and inter-cellular events, multiplexed imaging platforms are needed. Although optical reporter genes, with their “multicolor†imaging capabilities...
Summary
In order to fully understand the complexity of biological processes that are reflected by simultaneous occurrences of intra and inter-cellular events, multiplexed imaging platforms are needed. Although optical reporter genes, with their “multicolor†imaging capabilities, have revolutionized science their light signal source restricts their use in deep tissues and in large animals (and potentially in humans). The versatility of MRI sensors and reporters may be designed and developed for multiplexed imaging. Indeed, as originally proposed, CEST (Chemical exchange saturation transfer) were used for multiplexed MRI applications exploiting the ability to magnetically label exchangeable protons, which are observable at distinct chemical shifts. Alternatively, another promising option is “multicolor†MRI agents based on heteronuclear non-1H nuclei and 19F-MRI sensors were also designed, developed and considered as additional artificial MRI “colorsâ€. 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. The overall objectives of this projects are to develop “multicolor†MRI sensors that will report on multiple genes expression in an orthogonal manner and will allow multiplexed mapping of reporter genes with unlimited tissue penetration that can be translated in the future to large animals and humans.
Work performed
As originally proposed synthetic nucleoside analogs (specifically, pyrolo-deoxy-cytidine, 5-methyl dihdrothymidine and 8-Azaadenosine), were obtained (purchased or synthetically prepared) and studied for their ability to be used as artificial colors in CEST-MRI experiments. The three nucleosides showed distinct CEST contrast (i.e., 6.0 ppm, 4.9 ppm and 3.0 ppm) and thus are being used as three putative artificial colors for MRI. Moreover, and importantly for this project, the recent purchase of ultrahigh field MRI scanner operating at 15.2 T by the Weizmann Institute provides higher CEST-spectral resolution for better “colors†filtering and expand our capabilities to use natural deoxy-nucleoside such as thymidine (dT) as a CEST agent. The use of such natural nucleoside as CEST MRI agent in addition to other natural nucleosides such as as deoxy-cytidine (dC), and deoxy-adenosine may propose high biological-tolerance to the used imaging agents. Moreover, natural nucleosides could be used for monitoring endogenous nucleoside kinase activities that may be used as reporters for both normal and pathological condition in living organism. A series of mutants Dm-dNK were computationally designed resulting in a mutant-DmdNK that is x10 more active than the wild-type Dm-dNK. The same computational practice is being performed in order to improve HSV1-tk activity. Enzyme activity was studied using fluorescent activated cell sorting (FACS) based assay developed in our lab in the first period of this grant. Such an assay allows us to examine the enzymatic activity multiple mutants of deoxynucleoside kinases (dNK) and compare between different mutants in a high throughput manner. In parallel, we have designed and developed a high throughput screening approach for monitoring dNK activity in bacterial cells using a fully automated and flexible pipetting platform for multichannel liquid handling (CyBio liquid handler, Analytik Jena). Such automated system have optimized for a highly reproducible and accurate experimental protocol for determination of dNK activity based on the accumulation of fluorescent nucleoside and increased fluorescence as a function of dNK activity. This assay allows repetitive screening of multiple 96-well plates providing us with the ability to screen large numbers of mutants simultaneously. 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 demonstrate that small, water-soluble 19Fionic NCs can average out homonuclear dipolar interactions, enabling one to obtain high-resolution 19F NMR signals in solution that reflect the MR properties of F in the crystal core. Decorating 19F-NC surfaces with a biocompatible poly(ethylene glycol) coating maintains colloidal stability in water while preserving the NC high-resolution 19F NMR properties, even after further functionalization. The high content and magnetic equivalence of the fluorides within the NCs enable 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.
Final results
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. Our newly purchased 15.2 T MRI scanner 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. One example is our ability to monitor the natural deoxyribonucleoside thymidine (dT) that, due to the fast exchange rate of its exchangeable proton, could not be used as CEST imaging agents at MRI scanners operating at lower magnetic fields. Such ultrahigh field MRI scanner allows better spectral resolution for monitoring multiple CEST agents with a better separation towards their use in multicolor MRI. Moreover, much higher signal-to-noise ratio is obtained with MRI scanners operating at higher magnetic fields. Such a scanner, obtained by the Weizmann Institute (the Host Institution, HI), is a game changer for multicolor MRI and will be the scanner of choice for the second period of this project.
Fluorine-19 is the second most NMR-sensitive nuclei (after 1H) and therefore it is favorable for MR-based studies (NMR and MRI) covers a wide range of fields from chemistry to structural biology, material sciences and even medicine. 19F-based perfluorocarbon (PFC) nanoemulsions have been proposed as 19F MRI tracers and have been successfully used in a wide range of applications, including in clinical settings. However, PFCs do not have the beneficial properties of inorganic NCs. During the first part of this research we were able to overcome several challenges of obtaining high-resolution 19F-NMR signals from inorganic nanofluorides in water, by sufficient averaging of homonuclear dipolar interactions found in inorganic nanofluorides. This ability allowed us topropose and develop, a novel type of 19F-nanotracers for 19F-MR imaging. The synthesized, purified and fully characterized nanofluorides aimed to combine the advantages of inorganic nanocrystals (e.g., small and controllable sizes, dense fluoride content, monodispersity, colloidal stability, surface modifiability, etc.) with the merit of 19F-MRI. Moreover, and importantly for this research, the use of nanofluorides allow to use them as a platform for multicolor†MRI. Due to the fact that well-defined colloidal inorganic nanoparticles have revolutionized numerous fields in science from basic understanding of materials properties to advanced nanomedicine applications, we believe that the ability to study nanocrystals in solutions with high-resolution NMR could mark the dawn of a new scientific era in material sciences, biology and medicine. Such capability will provide scientists with unique opportunities to study fluoride-based nanorcrystals with high-resolution 19F-NMR and/ or applied them in 19F-MRI studies with a potential to extend it to other NMR-observable nuclei.