Abstract:Near-IR fluorescence (NIRF) imaging is a new technology using IR fluorescent protein (iRFP) gene labelling and is potentially useful for in vivo applications. In the present study, we expressed iRFP and the TNF-related apoptosis inducing ligand gene in mesenchymal stem cells (MSCs) using adeno-associated virus (AAV) and showed that iRFP-labelled MSCs can be detected by fluorescence microscopy. We injected mice with MSCs labelled with AAV-iRFP, which we were then able to detect by whole-animal NIRF imaging. Our… Show more
“… 27 Novel OI genes are constantly being developed, such as infrared fluorescence proteins or the very small endogenously fluorescent protein UnaG, which can be used to monitor gene transfer in AAV vectors, which have limited genetic capacity. 123 , 124 , 125 Figure 4 Mechanisms of optical imaging modalities and examples from oncolytic virotherapy and gene therapy studies (A) Fluorescence: Incident excitation wavelength (Ex. λ) excites the fluorophore within the intracellular GFP and causes an emission wavelength (Em.…”
Reporter gene imaging (RGI) can accelerate development timelines for gene and viral therapies by facilitating rapid and noninvasive
in vivo
studies to determine the biodistribution, magnitude, and durability of viral gene expression and/or virus infection. Functional molecular imaging systems used for this purpose can be divided broadly into deep-tissue and optical modalities. Deep-tissue modalities, which can be used in animals of any size as well as in human subjects, encompass single photon emission computed tomography (SPECT), positron emission tomography (PET), and functional/molecular magnetic resonance imaging (f/mMRI). Optical modalities encompass fluorescence, bioluminescence, Cerenkov luminescence, and photoacoustic imaging and are suitable only for small animal imaging. Here we discuss the mechanisms of action and relative merits of currently available reporter gene systems, highlighting the strengths and weaknesses of deep tissue versus optical imaging systems and the hardware/reagents that are used for data capture and processing. In light of recent technological advances, falling costs of imaging instruments, better availability of novel radioactive and optical tracers, and a growing realization that RGI can give invaluable insights across the entire
in vivo
translational spectrum, the approach is becoming increasingly essential to facilitate the competitive development of new virus- and gene-based drugs.
“… 27 Novel OI genes are constantly being developed, such as infrared fluorescence proteins or the very small endogenously fluorescent protein UnaG, which can be used to monitor gene transfer in AAV vectors, which have limited genetic capacity. 123 , 124 , 125 Figure 4 Mechanisms of optical imaging modalities and examples from oncolytic virotherapy and gene therapy studies (A) Fluorescence: Incident excitation wavelength (Ex. λ) excites the fluorophore within the intracellular GFP and causes an emission wavelength (Em.…”
Reporter gene imaging (RGI) can accelerate development timelines for gene and viral therapies by facilitating rapid and noninvasive
in vivo
studies to determine the biodistribution, magnitude, and durability of viral gene expression and/or virus infection. Functional molecular imaging systems used for this purpose can be divided broadly into deep-tissue and optical modalities. Deep-tissue modalities, which can be used in animals of any size as well as in human subjects, encompass single photon emission computed tomography (SPECT), positron emission tomography (PET), and functional/molecular magnetic resonance imaging (f/mMRI). Optical modalities encompass fluorescence, bioluminescence, Cerenkov luminescence, and photoacoustic imaging and are suitable only for small animal imaging. Here we discuss the mechanisms of action and relative merits of currently available reporter gene systems, highlighting the strengths and weaknesses of deep tissue versus optical imaging systems and the hardware/reagents that are used for data capture and processing. In light of recent technological advances, falling costs of imaging instruments, better availability of novel radioactive and optical tracers, and a growing realization that RGI can give invaluable insights across the entire
in vivo
translational spectrum, the approach is becoming increasingly essential to facilitate the competitive development of new virus- and gene-based drugs.
“…Mesenchymal stromal cells (MSCs), which have already shown therapeutic promise in inflammatory and degenerative diseases, have recently emerged as cell-based (drug) carriers [ 100 , 101 ]. MSCs are multipotent cells that exhibit fibroblast-like morphology and can differentiate into adipocytic, myogenic, osteogenic, and chondrogenic lineages [ 101 , 102 ]. The minimal criteria for the definition of human MSCs, as defined by the International Society for Cell therapy, are very broad, leaving room for different interpretations and, consequently, heterogeneous reports of cell types [ 100 , 103 ].…”
Surgeons rely almost completely on their own vision and palpation to recognize affected tissues during surgery. Consequently, they are often unable to distinguish between different cells and tissue types. This makes accurate and complete resection cumbersome. Targeted image-guided surgery (IGS) provides a solution by enabling real-time tissue recognition. Most current targeting agents (tracers) consist of antibodies or peptides equipped with a radiolabel for Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT), magnetic resonance imaging (MRI) labels, or a near-infrared fluorescent (NIRF) dye. These tracers are preoperatively administered to patients, home in on targeted cells or tissues, and are visualized in the operating room via dedicated imaging systems. Instead of using these ‘passive’ tracers, there are other, more ‘active’ approaches of probe delivery conceivable by using living cells (macrophages/monocytes, neutrophils, T cells, mesenchymal stromal cells), cell(-derived) fragments (platelets, extracellular vesicles (exosomes)), and microorganisms (bacteria, viruses) or, alternatively, ‘humanized’ nanoparticles. Compared with current tracers, these active contrast agents might be more efficient for the specific targeting of tumors or other pathological tissues (e.g., atherosclerotic plaques). This review provides an overview of the arsenal of possibilities applicable for the concept of cell-based tracers for IGS.
“…iRFP720 was used to study metastasis of prostate cancer in bones of live mice [97]. Proteins from the iRFP series have been successfully used for visualization of tumors of internal organs of live mice [11,12,68,79,98]. The use of photoactivated PAiRFP proteins allowed to significantly increase the image contrast owing to the sig nal registration from the two states of PAiRFP during visualization of tumors at the early stages of their devel opment in mice [23].…”
Section: Application Of Nir Fluorescent Biomarkers Reporters and Bimentioning
High transparency, low light scattering, and low autofluorescence of mammalian tissues in the near infrared (NIR) spectral range (~650 900 nm) open a possibility for in vivo imaging of biological processes at the micro and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes-natural NIR light absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP like fluores cent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that com bine fluorescence and bioluminescence methods with photoacoustic tomography.
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