22 Scientific Article The Israel Chemist and Chemical Engineer Issue 9 · January 2023 · Tevet 5783 the mapping of transgene expression in a pseudomulti-color fashion, noninvasively [34]. Summary and outlook Using synthetic chemistry, supramolecular chemistry, nanofabrication, and protein engineering approaches to generate various types of novel molecular formulations as MRI sensors with unique features, we demonstrated several strategies with which to obtain artificial MRI colors. Having demonstrated the potential of small-sized inorganic nanofluorides to be used as imaging agents for 19F-MRI applications, and considering the extensive use of inorganic NCs for in vivo imaging, we envision that further uses of nanofluorides in additional scenarios are just a matter of time. Moreover, the extensive demonstrations of nanofluorides as ideal matrices for NCs with upconversion fluorescent properties and demonstrations of the use of nanofluorides for CT imaging show the potential to further develop our fluoride-based NCs as materials for multimodal-imaging, beyond multicolor 19F-MRI. The innovat ive GEST and paraGEST approaches that we have proposed in which hostguest chemistry is combined with 19F-NMR, CEST, and MRI should be further developed to obtain novel molecular systems for molecular and cellular MRI. While the main challenge in applying GEST-MRI in vivo is the need to deliver both the host and the guest to the imaging region, relying on brain-deliverable molecular guests (i.e., fluorinated anesthetics), the potential to modify molecular hosts to recognize biomarkers of neuropathologies (e.g., amyloid-beta plaques in Alzheimer’s disease) create opportunities to develop GEST-MRI as a diagnostic tool. Finally, the successful demonstration of the ability to map transgene expression with GeneREFORM, in a pseudo-multicolor fashion, noninvasively, could transform MRI into a powerful tool with capabilities and possibilities that have, to date, been unattainable. GeneREFORM should be further developed to provide additional MRI “colors” and should be applied in developing fields, such as cell-based and genebased therapies. In conclusion, there is no doubt that luminescent colors have changed research in the life sciences from the ground up, and, although they are still the obvious choice to illuminate the complexity of biological systems, MRI-based “colors” can extend the available “multicolor” toolbox for scenarios that are not amenable to the use of light. Moreover, and different from other imaging modalities, the information obtained by MRI “colors” can be correlated with other anatomical and physiological parameters that are routinely obtained in MRI studies. to convert 5-MDHT; and the variant HSV1-TK_7B was confirmed to be a specific dNK for 5-MDHT and not reactive with pdC. Obtaining two highly active and highly orthogonal dN/dNK pairs (pdC/Dm-DNK_7C, Figure 7c and 5-MDHT/ HSV1-TK_7B, Figure 7d), the molecular components of GeneREFORM were established. Using these components, we demonstrated the ability to use GeneREFORM to map the simultaneous expression of two different transgenes with MRI in two different animal models. In the first model, tumor cells expressing either of the two transgenes, HSV1-TK_7B or Dm-dNK_7C, were inoculated intracranially into the two brain hemispheres of immunodeficient mice. Seven days later, after two contralateral tumors were developed in the brains of this group of mice, a mixture of pdC and 5-MDHT was injected intravenously into the studied subjects. The CEST maps obtained at ∆w = 5 and ∆w = 6 ppm revealed the accumulation of 5-MDHT or pdC in HSV1-TK_7B- or Dm-dNK_7C-expressing cells, respectively, as manifested by the pseudo-colored CEST map display (Figure 8a). A similar observation was obtained when the expression of the orthogonal transgenes was mediated by adeno-associated virus (AAV) vectors (Figure 8b). Overall, we showed, for the first time, the development and implementation of a genetically encoded reporter system, GeneREFORM, that is not based on luminescence and enables a b In vivo (MRI) Ex vivo (Fluorescence) Figure 8. In vivo MRI of GeneREFORM. (a) Schematic illustration of the performed in vivo study of intracranial injection of transgene-expressing cells, and the CEST maps on an anatomical MR image of the mouse brain. The fluorescent images of a brain cryo-section show the expression of the transgenes. (b) Schematic illustrationof the invivo studyof intracranial injection of AAV-Dm-dNK_7C and AAV-HSV1-TK_7B in two sets of representative mice showing the CEST maps on an anatomical MR image and the corresponding fluorescent images of a brain cryo-section showing the transgene expression. Modified with permission from Reference 34.
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