Methods and systems for imaging or evaluating partial oxygen tension

Abstract

This disclosure describes novel methods for imaging or evaluating partial oxygen tension in a tissue in vitro or in vivo in a subject as well as methods for detecting or evaluating a level of oxygen in a target. A method of identifying an agent capable of modulating partial oxygen tension in a tissue of a subject and a method of evaluating an effect of an agent on partial oxygen tension in a tissue of a subject are also disclosed. The methods involve introducing or expressing in the tissue or in one or more cells therein a luminescent protein capable of oxidizing a substrate with oxygen in the tissue, wherein oxidization of the substrate by the luminescent protein results in emission of a bioluminescence signal, and wherein intensity of the bioluminescence signal is correlated to a level of oxygen in the tissue; contacting the tissue with the substrate under a condition permitting oxidization of the substrate by the luminescent protein and emission of the bioluminescence signal; and imaging or evaluating the partial oxygen tension in the tissue by detecting the bioluminescence signal or determining a level of the bioluminescence signal. Preferably the luminescent protein is a fusion protein comprising a luciferase fused to a fluorescent protein.

Description

[0001] Docket No.: UR 6-24018 / FR 161118.07201

[0002] METHODS AND SYSTEMS FOR IMAGING OR EVALUATING PARTIAL OXYGEN TENSION

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 635,232, filed April 17, 2024. The foregoing application is incorporated by reference herein in its entirety.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The contents of the electronic sequence listing (161118.07201SeqList.xml; Size: 12,389 bytes; and Date of Creation: April 2, 2025) is herein incorporated by reference in its entirety.

[0007] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0008] This invention was made with government support under AT011439, AT012312, and NS128613 awarded by the National Institutes of Health, and W911NF-19-1-0280 awarded by the Army Research Laboratory - Army Research Office. The government has certain rights in the invention.

[0009] FIELD OF THE INVENTION

[0010] This disclosure generally relates to methods and systems for imaging or evaluating partial oxygen tension in a tissue in vitro or in vivo in a subject.

[0011] BACKGROUND OF THE INVENTION

[0012] The human brain uses approximately 20% of total body oxygen consumption at rest. Delivery and demand of oxygen (O2) are so finely balanced that maintaining tissue oxygenation may be the most critical of all brain functions. Yet the understanding of the dynamics of brain tissue oxygen tension (P02) under physiological conditions remains limited, mainly because of the lack of spatially precise measurement techniques for C02 imaging. Currently, tissue Pm can be measured by phosphorescence and by Clark-type electrodes. However, neither approach provides sufficiently high spatiotemporal sensitivity to detect physiological changes in cortical Pm.

[0013] Therefore, there is a critical need for improved methods and systems for evaluating the dynamics of oxygen tension in a tissue.Docket No.: UR 6-24018 / FR 161118.07201

[0014] SUMMARY OF THE INVENTION

[0015] This disclosure addresses the need mentioned above in a number of aspects. In one aspect, this disclosure provides a method of imaging or evaluating partial oxygen tension in a tissue in vitro or in vivo in a subject. In some embodiments, the method comprises: (a) introducing or expressing in the tissue or in one or more cells therein a luminescent protein capable of oxidizing a substrate with oxygen in the tissue, wherein oxidization of the substrate by the luminescent protein results in emission of a bioluminescence signal, and wherein intensity of the bioluminescence signal is correlated to a level of oxygen in the tissue; (b) contacting the tissue with the substrate under a condition permitting oxidization of the substrate by the luminescent protein and emission of the bioluminescence signal; and (c) imaging or evaluating the partial oxygen tension in the tissue by detecting the bioluminescence signal or determining a level of the bioluminescence signal.

[0016] In some embodiments, intensity of the bioluminescence signal is linearly correlated to the level of oxygen in the tissue.

[0017] In some embodiments, the method comprises identifying one or more hypoxic pockets in the tissue. In some embodiments, the method comprises identifying the hypoxic pockets in the tissue based on negative amplitude, Po2(AB / B), where P02 represents the partial oxygen tension, B represents the intensity of the bioluminescence signal, and AB represents a change of the intensity of the bioluminescence signal.

[0018] In some embodiments, the method comprises determining temporal and / or spatial characteristics of the hypoxic pockets. In some embodiments, the method comprises determining a size, an area, a number, a frequency distribution, and / or a change thereof of the hypoxic pockets.

[0019] In some embodiments, the hypoxic pockets constitute a region-of-interest (ROI) of imaging of the partial oxygen tension.

[0020] In some embodiments, the hypoxic pockets are reduced by wakefulness or by locomotion. In some embodiments, the method comprises determining a change of the partial oxygen tension over a period of time. In some embodiments, the period of time is 20, 50, 100, 150, 200, 250, 300, 350, or 400 seconds.

[0021] 168896441.5 2Docket No.: UR 6-24018 / FR 161118.07201

[0022] In some embodiments, the method comprises identifying the hypoxic pockets at a temporal resolution under 225 seconds. In some embodiments, the method comprises identifying the hypoxic pockets at a temporal resolution under 100 seconds. In some embodiments, the method comprises identifying the hypoxic pockets at a temporal resolution under 50 seconds.

[0023] In some embodiments, detecting the bioluminescence signal or determining the level of the bioluminescence signal is insensitive to interference of spectral absorption by hemoglobin.

[0024] In some embodiments, the method comprises introducing a nucleic acid molecule encoding the luminescent protein into the tissue, such that the luminescent protein is expressed in the one or more cells in the tissue. In some embodiments, the method comprises expressing the luminescent protein in the one or more cells in the tissue under control of an astrocytic promoter.

[0025] In some embodiments, the luminescent protein comprises a luciferase subunit derived from a luciferase of Oplophorus Gracilirostris. In some embodiments, the luminescent protein is a fusion protein comprising a luciferase fused to a fluorescent protein. In some embodiments, the luciferase comprises luciferase NanoLuc. In some embodiments, the fluorescent protein comprises fluorescent protein mNeongreen. In some embodiments, the fluorescent protein is GeNL.

[0026] In some embodiments, the fluorescent protein comprises an amino acid sequence of SEQ ID NOs: 1-3 or comprises an amino acid sequence having at least 75% sequence identity with an amino acid sequence of SEQ ID NOs: 1-3.

[0027] In some embodiments, the substrate comprises an imidazopyrazinone substrate. In some embodiments, the imidazopyrazinone substrate comprises furimazine, and wherein the luminescent protein is capable of oxidizing furimazine into furimamide.

[0028] In some embodiments, the tissue is in a brain of a subject. In some embodiments, the tissue is cerebral cortex. In some embodiments, the one or more cells comprise astrocytes.

[0029] In some embodiments, the method further comprises creating a cranial window on the skull of the subject to allow (a) introducing the luminescent protein in the one or more cells in the tissue, (b) contacting the tissue with the substrate, and / or (c) detecting the bioluminescence signal or determining a level of the bioluminescence signal.

[0030] In another aspect, this disclosure provides a method of identifying an agent capable of modulating partial oxygen tension in a tissue of a subject, the method as described herein imagingDocket No.: UR 6-24018 / FR 161118.07201

[0031] or evaluating the partial oxygen tension in the tissue of the subject according to the method as described herein, wherein the subject has been administered with the agent or exposed to the agent; and identifying the agent as a candidate agent if a change of the partial oxygen tension is greater than a reference value.

[0032] In another aspect, this disclosure provides a method of evaluating an effect of an agent on partial oxygen tension in a tissue of a subject. In some embodiments, the method comprises: imaging or evaluating the partial oxygen tension in the tissue of the subj ect according to the method as described herein, wherein the subject has been administered with the agent or exposed to the agent; and determining a change in the partial oxygen tension in the tissue of the subject with respect to a reference value.

[0033] In another aspect, this disclosure provides a method of evaluating an effect of an activity on partial oxygen tension in a tissue of a subject. In some embodiments, the method comprises: imaging or evaluating the partial oxygen tension in the tissue of the subj ect according to the method as described herein, wherein the subject has performed the activity; and determining a change in the partial oxygen tension in the tissue of the subject with respect to a reference value.

[0034] In yet another aspect, this disclosure provides a method of detecting or evaluating a level of oxygen in a target. In some embodiments, the method comprises: (i) providing a luminescent protein capable of oxidizing a substrate with oxygen, wherein oxidization of the substrate by the luminescent protein results in emission of a bioluminescence signal, and wherein intensity of the bioluminescence signal is correlated to the level of oxygen in the target; (ii) contacting the target or a sample therefrom with the luminescent protein and the substrate under a condition permitting oxidization of the substrate by the luminescent protein and emission of the bioluminescence signal; and (iii) detecting or evaluating a level of the bioluminescence signal, thereby detecting or evaluating the level of oxygen in the target.

[0035] The foregoing summary is not intended to define every aspect of the disclosure, and additional aspects are described in other sections, such as the following detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, or paragraph, or section of this document. Other features and advantages of the invention will become apparent from the following detailedDocket No.: UR 6-24018 / FR 161118.07201

[0036] description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A, IB, 1C, and ID show bioluminescence intensity of GeNL that reports cerebral partial oxygen pressure. FIG. 1A (Top) shows a scheme of the experimental setup. KX-anesthetized mice expressing GeNL under glial fibrillary acidic protein promoter were placed under a macroscope and exposed to different O2 concentrations. An O2-sensitive microelectrode was inserted into the cortex through an acute craniotomy and artificial cerebrospinal fluid supplemented with furimazine (0.25 mM). FIG. 1A (bottom) shows a chemical reaction of furimazine to furimamide catalyzed by GeNL under the presence of O2, leading to light output in the form of bioluminescence. FIG. IB shows a mean trace of the BLI and P02 as measured with the O2 electrode at varying O2 concentrations. The shaded area indicates standard error. FIG. 1C shows BLI intensity [Po2(DB / B)] changes 162% (± 19.13, SEM) when O2 concentration in the tidal air is doubled. A 10% increase in O2 induces a 66% (± 6.70) increase. Under hypoxia, BLI is reduced by 29% (± 2.91). Accordingly, P02 increases by 52.4 mmHg (± 2.4) when O2 concentration is doubled. A 10% increase in O2 increases P02 by 18.6 mmHg (±1.9), a 10% decrease reduces P02 by 9.7 mmHg (±1.6). (Left) One-way repeated measures analysis of variance (ANOVA) F1.272, 10.18 = 82.66, P < 0.0001 main effect of group. Tukey post hoc: 10% versus 30%, P < 0.0001; 10% versus 40%, P < 0.000; 30% versus 40%, P = 0.0006. (Right) One-way repeated measures ANOVA Fi.523,12.18 = 219.7, P < 0.0001 main effect of group. Tukey post hoc: 10% versus 30%, P < 0.0001; 10% versus 40%, P < 0.000; 30% versus 40%, P < 0.0001. FIG. ID shows dependency of change in BLI and corresponding change in PO2 recorded with the O2 electrode in the same region upon changes in O2 concentration of the inhalation air. Data points are fitted with a linear regression with a coefficient of determination, R2, of 0.82. Dots sharing the same color indicate data points from the same mouse.

[0038] FIGS. 2A, 2B, and 2C show characterization of hypoxic pockets in cortex of anesthetized mice. FIG. 2A shows cerebral P02 imaged in somatosensory cortex of KX-anesthetized mice overDocket No.: UR 6-24018 / FR 161118.07201

[0039] 20 min using BLI. FIG. 2B shows a time trace of cerebral P02 from the ROI indicated by inner and outer circles. FIG. 2C shows average distribution of hypoxic pockets.

[0040] FIGS. 3A and 3B show effects of vasodilation and capillary stalling on hypoxic pockets. FIG. 3 A shows cerebral P02 measured in KX-anesthetized mice exposed to 10% CO2 in the inhaled air for 10 min after acute craniotomy. FIG. 3B shows cerebral P02 measured with BLI in KX-anesthetized mice. Capillary stalling was induced by intravascular injection of 4-mm microspheres before the onset of the imaging session.

[0041] FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41, and 4J show increased arousal level suppresses tissue hypoxia. FIG. 4A shows cerebral P02 either measured in KX-anesthetized mice, in awake head -fixed mice during quiet wakefulness in a MAG- 1 mouse holder, or in mobile mice voluntarily running on a Styrofoam sphere. FIG. 4B shows distribution of hypoxic pockets in a single recording session lasting 20 min. FIG. 4C shows frequency distribution of the number of hypoxic pockets. FIG. 4D shows frequency distribution of the area covered by hypoxic pockets in the field of view per second. FIG. 4E shows frequency distribution of duration of hypoxic pockets. FIG. 4F shows frequency distribution of the amplitude of hypoxic pockets. FIG. 4G shows frequency distribution of the size of hypoxic pockets. FIG. 4H shows frequency distribution of number of hypoxic pockets sharing the same region per second. FIG. 41 shows a schematic illustrating the parameters used to calculate the hypoxic burden for each hypoxic pocket (left; x) and for an entire recording / mouse (right; X). FIG. 4J shows a hypoxic burden during KX anesthesia, wakefulness, and mobile wakefulness. N = 9 mice, KX. N = 11 mice, awake. N = 10 mice, mobile. Means ± SEM are shown. Violin plots show median and quartiles. Bars indicating Tukey’s post hoc tests between groups at their edges: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bars, 100 mm.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] This disclosure is provided based at least in part on an unexpected discovery that relative changes in partial oxygen tension can be monitored continuously in tissue using a genetically encoded bioluminescent oxygen indicator. The disclosed methods provide a novel tool to delineate the importance of oxygen tension in physiological processes and neurological diseases.

[0044] Accordingly, this disclosure describes novel methods and systems for imaging or evaluating partial oxygen tension in a tissue zw vitro or in vivo in a subject. In some embodiments,Docket No.: UR 6-24018 / FR 161118.07201

[0045] the method comprises: (a) introducing or expressing in the tissue or in one or more cells therein a luminescent protein capable of oxidizing a substrate with oxygen in the tissue, wherein oxidization of the substrate by the luminescent protein results in emission of a bioluminescence signal, and wherein intensity of the bioluminescence signal is correlated to a level of oxygen in the tissue; (b) contacting the tissue with the substrate under a condition permitting oxidization of the substrate by the luminescent protein and emission of the bioluminescence signal; and (c) imaging or evaluating the partial oxygen tension in the tissue by detecting the bioluminescence signal or determining a level of the bioluminescence signal.

[0046] As used herein, the term “partial oxygen tension” refers to partial pressure of oxygen in a tissue or availability of oxygen for cells in the tissue. As used herein, the term “cortical partial oxygen tension ( 02)” refers to the amount of oxygen dissolved in the fluids of the brain’s cortex. P02 measures the partial pressure of oxygen in the brain tissue, and it indicates the availability of oxygen for the brain’s cells. P02 provides a vital measure of the brain’s oxygen status levels, and it can vary within different regions of the brain.

[0047] As used herein, the term “luminescent protein” refers to proteins that generate light, whether through chemical reactions or by re-emitting absorbed light. An example of luminescent proteins is luciferase that catalyzes reactions with specific substrates, resulting in light emission. Luciferases are a diverse group of enzymes that produce bioluminescence, including firefly luciferase (Photinus pyralis) that uses luciferin as a substrate, along with ATP and magnesium, to produce yellow-green light, renilla luciferase (Renilla reniformis) that uses coelenterazine as a substrate and produces blue light, gaussia Luciferase (Gaussia princeps that uses coelenterazine as a substrate and emits blue light, NanoLuc luciferase - an engineered luciferase derived from the deep-sea shrimp Oplophorus gracilirostris - that uses the substrate furimazine.

[0048] As used herein, the term “bioluminescence signal” refers to the light emitted by cells, tissue, or living organisms as a result of a biochemical reaction, typically involving the enzyme luciferase and a subtract.

[0049] In some embodiments, detecting the bioluminescence signal or determining a level of the bioluminescence signal can be carried out by one or more methods known in the art, including optical detection methods, imaging techniques, spectral analysis, and biochemical assays. Optical detection methods can be performed by one or more sensors, such as photomultiplier tubes (PMTs),Docket No.: UR 6-24018 / FR 161118.07201

[0050] charge-coupled devices (CCDs), electron-multiplying CCDs (EMCCDs), or complementary metal-oxide-semiconductor (CMOS) sensors. Imaging techniques may include long-exposure photography, fluorescence and bioluminescence imaging (BLI), and microscopy. Spectral analysis may include spectrophotometers that measure the intensity and wavelength of bioluminescence and optical filters that help isolate specific wavelengths to distinguish bioluminescence from background light. Biochemical assays may include luciferase reporter assays that measure gene expression or cellular activity and ATP-based bioluminescence assays that detect microbial contamination or metabolic activity.

[0051] In some embodiments, magnetic resonance imaging (MRI) can be used to detect or quantify the bioluminescence signal. For example, MRI-based detection of bioluminescence can be performed according to the method described by Ohlendorf et al. (Nat Biomed Eng. 2024 Jun;8(6):775-786), which is incorporated herein by reference in its entirety. This approach enables the visualization of bioluminescent signals deep within tissues, such as the brain.

[0052] Any suitable method for detecting and measuring bioluminescence signals in tissues may be employed in the context of the methods and systems disclosed herein, including, for example, those described in Bakhsheshian et al. , Proceedings of the National Academy of Sciences 110(51): 20801-06 (2013); Evans et al., Nature Methods 11(4): 393-95 (2014); Fukuchi et al., Molecular Brain 13(1): 122 (2020); Hochgrafe et al. , Molecular Neurobiology 47(3): 868-82 (2013); Iwano et al., Science 359(6378): 935-39 (2018); Liu et al., ACS Chemical Biology 16(12): 2707-18 (2021); Mezzanotte et al., Trends in Biotechnology 35(7): 640-52 (2017); Prescher et al., Current Opinion in Chemical Biology 14(1): 80-89 (2010); Schaub etal., Cancer Research 75(23): 5023-33 (2015); and Su et al., Nature Chemical Biology 19(6): 731-39 (2023), the relevant contents of which are incorporated herein by reference.

[0053] In some embodiments, intensity of the bioluminescence signal is linearly correlated to the level of oxygen in the tissue. In some embodiments, linear correlation refers to the relationship between two variables that can be represented by a straight line. It measures how closely the values of one variable change in relation to another. Positive linear correlation refers to the fact that when one variable increases, the other also increases. Negative linear correlation refers to the fact that when one variable increases, the other decreases.Docket No.: UR 6-24018 / FR 161118.07201

[0054] In some embodiments, the method comprises identifying one or more hypoxic pockets in the tissue. As used herein, “hypoxic pockets” refer to localized areas within a larger environment where oxygen levels are significantly lower than the surrounding regions. These pockets can occur in various ecosystems and biological systems, often with significant ecological, physiological, or medical implications.

[0055] In some embodiments, the method comprises identifying the hypoxic pockets in the tissue based on negative amplitude, T’o2(AB / B), where Pm represents the partial oxygen tension, B represents the intensity of the bioluminescence signal, and AB represents a change of the intensity of the bioluminescence signal.

[0056] In some embodiments, the method comprises determining temporal and / or spatial characteristics of the hypoxic pockets. Temporal characteristics refer to the time-related properties or behavior of hypoxic pockets. These characteristics describe how hypoxic pockets changes, evolves, or behaves over time. Spatial characteristics refer to the properties and attributes of hypoxic pockets in relation to their position, arrangement, and distribution in space. These characteristics help describe how hypoxic pockets are organized and interact within a given area.

[0057] In some embodiments, the method comprises determining a size, an area, a number, a frequency distribution, and / or a change thereof of the hypoxic pockets.

[0058] In some embodiments, the hypoxic pockets constitute a region-of-interest (ROI) of imaging of the partial oxygen tension. A ROI is a specific area within a larger dataset, image, or spatial field that is selected for focused analysis, processing, or measurement. It is commonly used in various fields, such as image processing, medical imaging, remote sensing, and data analysis.

[0059] In some embodiments, the hypoxic pockets are reduced by wakefulness or by locomotion. Wakefulness is a state of consciousness in which an individual is alert, aware, and responsive to their surroundings. It is the opposite of sleep and is regulated by complex neural processes in the brain, particularly in the reticular activating system (RAS). Locomotion is the ability of an organism or object to move from one place to another. It is a fundamental biological function that allows animals, humans, and even some microorganisms to navigate their environment.

[0060] In some embodiments, the method comprises determining a change in the partial oxygen tension over a period of time. In some embodiments, the period of time is 5, 10, 15, 20, 25, 30,Docket No.: UR 6-24018 / FR 161118.07201

[0061] 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400 seconds or any intermediate values therebetween.

[0062] In some embodiments, the method comprises identifying the hypoxic pockets at a temporal resolution under 225 seconds (e. , 225, 220, 215, 210, 205, 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 seconds, or any intermediate values therebetween). In some embodiments, the method comprises identifying the hypoxic pockets at a temporal resolution under 100 seconds (e.g., 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 seconds, or any intermediate values therebetween). In some embodiments, the method comprises identifying the hypoxic pockets at a temporal resolution under 50 seconds (e.g., 50, 45, 40, 35, 30, 25, 20, or 15 seconds, or any intermediate values therebetween).

[0063] In some embodiments, detecting the bioluminescence signal or determining the level of the bioluminescence signal is insensitive (or resistant) to interference of spectral absorption by hemoglobin.

[0064] In some embodiments, the method comprises introducing a nucleic acid molecule encoding the luminescent protein into the tissue, such that the luminescent protein is expressed in the one or more cells in the tissue.

[0065] As used herein, the phrases “nucleic acid,” “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule” are used interchangeably to refer to a polymer of DNA and / or RNA, which can be single-stranded, double-stranded, or multi-stranded, synthesized or obtained (e.g., isolated and / or purified) from natural sources, which can contain natural, non-natural, and / or altered nucleotides, and which can contain natural, non-natural, and / or altered internucleotide linkages including, but not limited to phosphoroamidate linkages and / or phosphorothioate linkages instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.

[0066] In some embodiments, the nucleic acid molecule is a vector. The expression of the luminescent protein can be induced by introducing one or more expression vectors carrying nucleic acids encoding a luminescent protein or fragment thereof. The luminescent protein or fragmentDocket No.: UR 6-24018 / FR 161118.07201

[0067] thereof can be inserted into the proper site of the vector (e.g, operably linked to a promoter). The expression vector is introduced into a selected host cell for amplification and / or polypeptide expression, by well-known methods such as transfection, transduction, infection, electroporation, microinjection, lipofection, the DEAE-dextran method, or other known techniques. These methods and other suitable methods are well known to the skilled artisan.

[0068] A wide variety of vectors can be used for the expression of the fusion protein as described. The ability of certain viruses to infect cells or enter cells via receptor-mediated endocytosis, and to integrate into the host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells. Accordingly, in certain embodiments, a viral vector is used to introduce a nucleotide sequence that encodes a luminescent protein or fragment thereof into a host cell for expression. The viral vector will comprise a nucleotide sequence encoding a luminescent protein or fragment thereof operably linked to one or more control sequences, for example, a promoter. Alternatively, the viral vector may not contain a control sequence and will instead rely on a control sequence within the host cell to drive expression of the luminescent protein or fragment thereof. Non-limiting examples of viral vectors that may be used to deliver a nucleic acid molecule include adenoviral vectors, AAV vectors, and retroviral vectors.

[0069] In some embodiments, an adeno-associated virus (AAV) can be used to introduce a nucleotide sequence encoding a luminescent protein or fragment thereof into a host cell for expression. AAV systems have been described previously and are generally well known in the art (Kelleher and Vos, Biotechniques, 17(6): 1110-7, 1994; Cotten et al., Proc Natl Acad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3): 141-64, 1994; Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992). Details concerning the generation and use of rAAV vectors are described, for example, in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein by reference in its entirety for all purposes. In some embodiments, the vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVRh74 (Rhesus macaque-derived AAV), AAVRhlO, or a modified vector thereof.

[0070] In some embodiments, a retroviral expression vector can be used to introduce a nucleotide sequence encoding a luminescent protein or fragment thereof into a host cell for expression. These systems have been described previously and are generally well known in the art (Nicolas andDocket No.: UR 6-24018 / FR 161118.07201

[0071] Rubinstein, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986).

[0072] As used herein, the term “express” refers to the transcription of a polynucleotide or translation of a polypeptide in a cell, such that levels of the molecule are measurably higher in a cell that expresses the molecule than they are in a cell that does not express the molecule. Methods to measure the expression of a molecule are well known to those of ordinary skill in the art, and include without limitation, Northern blotting, RT-PCR, in situ hybridization, Western blotting, and immunostaining.

[0073] In some embodiments, the method comprises expressing the luminescent protein in the one or more cells in the tissue under control of an astrocytic promoter. An astrocytic promoter refers to a specific DNA sequence used to drive or regulate gene expression in astrocytes, which are a type of glial cell in the central nervous system. Astrocytes play key roles in maintaining the bloodbrain barrier, supporting neurons, regulating neurotransmitters, and maintaining homeostasis in the brain. Examples of the astrocytic promoter may include the Glial Fibrillary Acidic Protein (GFAP) promoter. GFAP is a protein that is primarily expressed in astrocytes, and the GFAP promoter can be used to ensure that the target gene or reporter is expressed predominantly in astrocytes.

[0074] Table 1. Representative Sequences

[0075]

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[0077]

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[0079]

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[0081]

[0082] In some embodiments, the astrocytic promoter comprises the nucleotide sequence of SEQ ID NO: 7 or comprises a nucleotide sequence having at least 75% (e.g., 75%, 80%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with the nucleotide sequence of SEQ ID NO: 7.

[0083] In some embodiments, the luminescent protein comprises a luciferase subunit derived from a luciferase of Oplophorus Gracilirostris.

[0084] In some embodiments, the luminescent protein is a fusion protein comprising a luciferase fused to a fluorescent protein. In some embodiments, the luciferase comprises luciferase NanoLuc. NanoLuc is an engineered luciferase derived from the deep-sea shrimp Oplophorus gracilirostris.

[0085] In some embodiments, the fluorescent protein comprises fluorescent protein mNeongreen. mNeonGreen is a highly bright, monomeric green fluorescent protein (GFP) that is widely used in biological research for labeling and tracking specific proteins, cells, or tissues in living organisms. It was derived from a protein found in the shrubby coral (species Discosoma), and has been engineered to have improved properties for use in molecular and cellular biology.Docket No.: UR 6-24018 / FR 161118.07201

[0086] In some embodiments, the fluorescent protein is GeNL or a variant / fragment thereof. GeNL allows real-time monitoring of cellular processes in living organisms due to its bright and stable light emission. GeNL provides a sensitive readout that can be detected in live cells, offering high temporal resolution. The bioluminescent signals emitted by GeNL are non-invasive, which makes it ideal for in vivo studies without the need for external light sources. As part of the Nanoluc family, GeNL is small and efficient, reducing potential interference when fused to other proteins.

[0087] In some embodiments, the fluorescent protein comprises an amino acid sequence of SEQ ID NOs: 1-3 or comprises an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with an amino acid sequence of SEQ ID NOs: 1-3.

[0088] As used herein, “substrate” refers to a substance or compound that is converted or meant to be converted into another compound by the action of an enzyme e.g., luciferase).

[0089] In some embodiments, the substrate comprises an imidazopyrazinone substrate. An imidazopyrazinone substrate is a type of chemical substrate used in biochemical assays, particularly in luciferase-based systems, to produce bioluminescence when catalyzed by a luciferase enzyme. The substrate is typically a small molecule that reacts with the enzyme to generate light, which is often used for detection purposes in biological research. Examples of imidazopyrazinone substrates may include DeepBlueC, coelenterazine, furimazine, and imidazopyrazinone derivatives. DeepBlueC is a bioluminescent substrate designed for use with NanoLuc luciferase, a small and highly efficient luciferase enzyme. DeepBlueC produces a bright blue light upon catalysis by NanoLuc luciferase, making it useful for various bioluminescence assays. Coelenterazine is a bioluminescent substrate used in luciferase assays to generate light. It is a naturally occurring molecule found in several bioluminescent organisms, such as jellyfish, fireflies, and deep-sea shrimp. Furimazine is a bioluminescent substrate specifically designed for use with the NanoLuc luciferase system, which is a highly efficient and small luciferase enzyme derived from the deep-sea shrimp Oplophorus gracilirostris.

[0090] In some embodiments, the imidazopyrazinone substrate comprises furimazine, and wherein the luminescent protein is capable of oxidizing furimazine into furimamide.

[0091] In some embodiments, the tissue is in a brain of a subject. In some embodiments, the tissue is cerebral cortex. In some embodiments, the one or more cells comprise astrocytes.Docket No.: UR 6-24018 / FR 161118.07201

[0092] In some embodiments, the method further comprises creating a cranial window on the skull of the subject to allow (a) introducing the luminescent protein in the one or more cells in the tissue, (b) contacting the tissue with the substrate, and / or (c) detecting the bioluminescence signal or determining a level of the bioluminescence signal.

[0093] In another aspect, this disclosure provides a method of identifying an agent capable of modulating partial oxygen tension in a tissue of a subject. In some embodiments, the method as described herein comprises: imaging or evaluating the partial oxygen tension in the tissue of the subject according to the method as described herein, wherein the subject has been administered with the agent or exposed to the agent; and identifying the agent as a candidate agent if a change of the partial oxygen tension is greater than a reference value.

[0094] The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a “therapeutic agent,” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

[0095] The terms “therapeutic agent,” “therapeutic capable agent,” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.

[0096] As used herein, the term “modulate” is meant to refer to any change in biological state, i.e. increasing, decreasing, and the like.

[0097] The terms “reference level,” “reference value, “control level,” “control value,” “predetermined value,” and “predetermined level” are used interchangeably herein. The terms “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” and “control tissue are used interchangeably herein.Docket No.: UR 6-24018 / FR 161118.07201

[0098] In some embodiments, a reference level or a control level of partial oxygen tension may be determined from a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue that is obtained from a healthy and / or non-diseased part of the body (e.g., tissue or cells) of the same subject or individual, but at different time-points, e.g. , before and after therapy. In some embodiments, a reference level or a control level of partial oxygen tension may be determined from a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue that is obtained from a healthy individual who is not the subject or individual being assessed.

[0099] In another aspect, this disclosure provides a method of evaluating an effect of an agent on partial oxygen tension in a tissue of a subject. In some embodiments, the method comprises: imaging or evaluating the partial oxygen tension in the tissue of the subject according to the method as described herein, wherein the subject has been administered with the agent or exposed to the agent; and determining a change in the partial oxygen tension in the tissue of the subject with respect to a reference value.

[0100] In another aspect, this disclosure provides a method of evaluating an effect of an activity on partial oxygen tension in a tissue of a subject. In some embodiments, the method comprises: imaging or evaluating the partial oxygen tension in the tissue of the subj ect according to the method as described herein, wherein the subject has performed the activity; and determining a change in the partial oxygen tension in the tissue of the subject with respect to a reference value.

[0101] In yet another aspect, this disclosure provides a method of detecting or evaluating a level of oxygen in a target. In some embodiments, the method comprises: (i) providing a luminescent protein capable of oxidizing a substrate with oxygen, wherein oxidization of the substrate by the luminescent protein results in emission of a bioluminescence signal, and wherein intensity of the bioluminescence signal is correlated to the level of oxygen in the target; (ii) contacting the target or a sample therefrom with the luminescent protein and the substrate under a condition permitting oxidization of the substrate by the luminescent protein and emission of the bioluminescence signal; and (iii) detecting or evaluating a level of the bioluminescence signal, thereby detecting or evaluating the level of oxygen in the target.

[0102] Additional DefinitionsDocket No.: UR 6-24018 / FR 161118.07201

[0103] To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0104] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0105] Unless specifically stated otherwise, as apparent from the above discussion, it is appreciated that throughout the description, discussions utilizing terms such as “processing,” “performing,” “receiving,” “computing,” “calculating,” “determining,” “identifying,” “displaying,” “providing,” “merging,” “combining,” “running,” “transmitting,” or the like, refer to the action and processes of a computer system, or similar electronic computing device, that manipulates and transforms data represented as physical (or electronic) quantities within the computer system memories or registers or other such information storage, transmission or display devices.

[0106] “Contacting” refers to the process of bringing into contact at least two distinct species or substances such that they can react with one another or interact such that one has an effect on the other.

[0107] As used herein, the “amount” or “level” of a biomarker is a detectable level or amount in a sample. These can be measured by methods known to one skilled in the art. These terms encompass a quantitative amount or level e.g., weight or moles), a semi -quantitative amount or level, a relative amount or level (e.g., weight % or mole % within class), a concentration, and the like. Thus, these terms encompass absolute or relative amounts or levels or concentrations of a biomarker in a sample. The expression level or amount of biomarker assessed can be used to determine the response to treatment.Docket No.: UR 6-24018 / FR 161118.07201

[0108] The terms “patient,” “subject,” “host,” and “individual” are used interchangeably herein and refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and / or rhesus monkeys ( Macaca mulatta )) and baboon ( Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri ) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees ( Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish.

[0109] It is noted here that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0110] As used herein, “plurality” means two or more. As used herein, a “set” of items may include one or more of such items.

[0111] As used herein, “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0112] As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like do not necessarily refer to the same embodiment, but may unless the context dictates otherwise.

[0113] As used herein, the terms “and / or” or “ / ” means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0114] As used herein, the term “substantially” does not exclude “completely,” e.g., a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the present disclosure.Docket No.: UR 6-24018 / FR 161118.07201

[0115] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

[0116] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0117] As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the present disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure.

[0118] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the present disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[0119] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps ofDocket No.: UR 6-24018 / FR 161118.07201

[0120] the method occur sequentially, the steps may occur in any order, unless noted otherwise. In cases in which a method may include a combination of steps, each and every combination or subcombination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0121] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present disclosure. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0122] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0123] Examples

[0124] EXAMPLE 1

[0125] This disclosure presents a methodology to measure relative changes in P02 using the oxygen-dependent reaction of a luminescent substrate guided by its enzyme expressed in astrocytes. This method has a superior signal-to-noise ratio due to its bioluminescence origin and a spatiotemporal resolution that can visualize the dynamics of cortical P02 in awake-behaving mice. Green enhanced Nano-lantern (GeNL) is a luminescent fusion protein consisting of the luciferase NanoLuc (M. P. Hall et al., ACS Chem. Biol. 7, 1848-1857 (2012)) and the fluorescent protein mNeongreen (K. Suzuki etal., Nat. Commun. 7, 13718 (2016)). During the enzymatic conversion of its luminescent substrate furimazine to furimamide, energy is emitted in the form of light. The fluorescent fusion protein acts as a fluorescence amplifier, increasing the quantum yield through Forster resonance energy transfer (FRET). The enzymatic reaction of GeNL with furimazine depends on O2 (FIG. 1A), and the intensity of the bioluminescence signal is linearly correlated to the availability of O2 when O2 is the rate-limiting factor in the enzymatic reaction.Docket No.: UR 6-24018 / FR 161118.07201

[0126] Green enhanced Nano-lantern can detect oxygen in mouse cortex

[0127] To assess whether the oxygen dependency can be used in vivo to visualize spontaneous Pm dynamics in the brain, GeNL was expressed in cortical astrocytes of wild-type mice and measured bioluminescence intensity (BLI) after topical administration of the substrate furimazine through a cranial window (FIG. 1A). BLI followed cerebral Pm when O2 concentration of the breathing air was changed stepwise from 10% to 40% under ketamine-xylazine (KX) anesthesia. The baseline level of air O2 was kept at 20% and then changed for I -min periods followed by a I -min recovery phase at baseline levels. Changing the O2 concentration from 20% to 40% increased BLI by approximately 200%, whereas a reduction of O2 concentration in the inhaled air to 10% decreased BLI by approximately % from baseline (FIG. IB). To record the absolute 02 in the BLI imaging field, an Ch-sensitive Clark-type microelectrode was placed in the field of view during the calibration protocol. Cerebral as placed increased by 20 mmHg at 30% O2 and by 50 mmHg at 40% O2 and reduced by 10 mmHg from baseline at 10% O2 (FIGs. IB and 1C). The reaction time and slope of BLI occurred in parallel with the electrode recordings, indicating similar reaction times with no time lag between change in O2 and change in BLI. BLI peaked faster than electrode recordings upon increase of O2 concentration but declined at a similar rate when O2 was lowered, perhaps indicating substrate availability limitations under conditions with unphysiologically high O2 concentrations. ABLI is correlated AP02 in a linear manner (FIG. ID), although availability of free O2 might limit the sensitivity at low O2 supply. GeNL BLI is thus an accurate measure of relative Pm .

[0128] Increased metabolism leads to increased carbon dioxide (CO2) concentrations as a byproduct of cellular respiration. CO2 is a known vasodilator and alters pH. The pH change was thus investigated in brain tissue upon O2 calibration to exclude the possibility of pH alterations causing the effect on BLI described above. Mice expressing the genetically encoded fluorescent pH sensor pHuji (Y. Shen, M. Rosendale, R. E. Campbell, D. Perrais, J. Cell Biol. 207, 419-432 (2014)) in the extracellular space were implanted with a fiber photometry probe and exposed to the same protocol for O2 calibration under KX anesthesia. Manipulating O2 did not alter pH during O2 calibration.

[0129] Activation of the whisker barrel cortex by contralateral whisker stimulation leads to an increase in local blood flow and an increase in P02. The whisker barrel cortex was next imaged inDocket No.: UR 6-24018 / FR 161118.07201

[0130] awake behaving mice while stimulating the whiskers with air puffs to test whether BLI can detect the sensory -induced cortical P02 change. Mice were exposed to a series of 10 brief whisker stimulations (5 Hz, 50-ms duration for 10 s, 50-s interstimulation interval). During whisker stimulation, BLI increased in the field of view, closely following the individual series of whisker stimulation trials. Moreover, KX-anesthetized mice with simultaneous 02 microelectrode recording showed similar consistent correlation between BLI and DP02. Notably, the amplitude and the timing of BLI response differed between awake and KX-anesthetized mice, supporting the notion that anesthesia dampens tissue P02 and functional hyperemia dynamics.

[0131] Oxygen dynamics in the cerebral cortex

[0132] Continuous imaging of BLI showed that LO2 under resting conditions was highly dynamic, exhibiting local transient dips in P02 (FIG. 2A). These local hypoxic events were spatially constricted, lasting several seconds up to minutes, and typically showed a sharp onset and offset in relative tissue P02 (FIG. 2B). Given their negative relative amplitude in P02 and the characteristic sharply defined border, they are called “hypoxic pockets.”

[0133] Hypoxic pockets were identified based on their negative amplitude [7?o2(AB / B)], typical sharp onset and offset, clear edges, and long duration. Hypoxic pockets were observed throughout 20-minute-long recordings (FIG. 2C). In KX-anesthetized mice, 200 ± 22 hypoxic pockets (mean ± SEM) were detected during this time. Within a single frame recorded at 1 Hz, 8.12 ± 0.04 hypoxic pockets were observed per square millimeter, covering 2.43 ± 0.02% of the field of view. Hypoxic pockets covered an area of 1823 ± 27 mm2while lasting 48.2 ± 1.0 s. o2(AB / 5) decreased by 27.2 ± 4.5% from the baseline before each hypoxic pocket. Hypoxic pockets had an average diameter of 45.29 ± 0.31 mm and an almost circular shape. Hypoxic pockets often occurred repeatedly in the same area. Within a 20-min recording, on average, 73.2 ± 5.1 of those regions of interest (ROIs) were detected with on average 2.8 ± 0.1 hypoxic pockets. Each minute, 0.15 ± 0.01 hypoxic pockets occurred within a given ROI, meaning that every ~7 min, a hypoxic pocket occurred at the same place.

[0134] To quantify whether P02 during hypoxic pockets indeed reaches the hypoxic threshold in the cortex (<18 mmHg), the Po2( B / B) decrease in hypoxic pockets was compared with those during hypoxia (10% inhaled O2), where P02 reached 11 mmHg (FIG. IB). During hypoxia, o2(AB / B) was reduced by 29.2 ± 2.9%, which is similar to the decrease observed in hypoxicDocket No.: UR 6-24018 / FR 161118.07201

[0135] pockets. Using the electrode and BLI correlation, it was determined that a 20.6% i\B!R reduction corresponds to the hypoxic threshold.

[0136] Spectral absorption by hemoglobin is a common cause of artifacts in imaging fluorescent biosensors in vivo. To exclude the possible signal interference by hemoglobin as the underlying cause of the hypoxic pockets, the mNeonGreen fluorescence of GeNL was recorded instead. Hemoglobin absorption should also affect the mNeonGreen fluorescence, which would be subject to hemoglobin absorption, but does not depend on O2. However, no events with similar spatiotemporal characteristics as hypoxic pockets were observed in the mNeonGreen fluorescence traces. O2 tension in and around venules is lower than in and around arterioles. The distance of hypoxic pockets from arterioles and venules was thus measured. Hypoxic pockets were closer to venules than to arterioles, with an average distance of 28.1 ± 0.7 and 48.0 ± 1.2 mm, respectively.

[0137] Tissue oxygenation is closely linked to the availability of O2 and, therefore, to capillary circulation of red blood cells. It was thus hypothesized that hypoxic pockets result from hemodynamic changes in the microcirculation. Intrinsic optical spectroscopy imaging (IOSI) was used to monitor hemoglobin dynamics in the brain of resting awake mice. Hemoglobin dynamics were recorded at the isosbestic point for total hemoglobin concentration ([HbT]). The analysis identified areas of low [HbT] that shared their characteristic onset and offset dynamics with hypoxic pockets measured with BLI. On average 0.9 ± 0.01 events of low [HbT] were detected per square millimeter per second in IOSI measurements, which lasted 16.5 ± 0.4 s covering 7344 ± 402 mm2. During these events, [HbT] decreased by 1.9 ± 0.1 mmHg. Whereas bioluminescence allows only the measurement of tissue hypoxia, IOSI reflects blood volume and thus transient localized decreases in hemoglobin concentration with similar temporal and spatial properties as shown for hypoxic pockets.

[0138] Effects of vasodilation and capillary stalling on hypoxic pockets

[0139] Brain activity is accompanied by transient increases in blood flow due to vasodilation, a phenomenon called neurovascular coupling or functional hyperemia. Whether hyperemia suppresses the number of hypoxic pockets and induces vasodilation by increasing CO2 in air (hypercapnia) was investigated (Fig. 3A). The elevation of CO2 in the inhaled air increased tissue P02 reversibly to 118.4 ± 0.3% concurrently with a sharp decrease in the number of hypoxic pockets per square millimeter per second in a reversible manner, from 3.9 ± 0.1 to 2.3 ± 0.1 andDocket No.: UR 6-24018 / FR 161118.07201

[0140] back to 4.7 ± 0.1, a reduction of 41% when CO2 was lowered. Area covered by hypoxic pockets changed by 0.54%, from 1.01 ± 0.03% to 0.47 ± 0.02%, a reduction of 53%. 73o2(A8 / B) decrease within hypoxic pockets during hypercapnia was reduced by 4% and stayed low after hypercapnia during the time of recording. Furthermore, the duration of hypoxic pockets was reduced by 17 s, and their spatial expansion was reduced by 65% (FIG. 3B). After resolution of hypercapnia, hypoxic pocket duration and size recovered (FIG. 3B). This observation is consistent with the finding that hypercapnia decreases the portion of poorly perfused capillaries in the rat brain cortex. To assess whether increased acidification during hypercapnia is involved, extracellular pH was measured in the brain parenchyma during 10% CO2 as described above. A 5-min period of 10% CO2 decreased pHuji intensity by 6.4% in a reversible manner. Typically, fluorescent proteins are quenched in acidic environments, yet an increase in BLI during hypercapnia was observed, excluding the possibility that the observation was an artificial effect of pH on the sensor’ s intensity.

[0141] Isoflurane is another potent vasodilator that increases blood volume but not tissue pH. Mice before and during isoflurane anesthesia were compared, and a decrease in the number of hypoxic pockets similar to that seen for CO2 was observed. During isoflurane anesthesia, the number of hypoxic pockets per second was effectively decreased from 6.2 ± 0.01 to 1.9 ± 0.03, a reduction of 69%. The area covered by hypoxic pockets was reduced by 0.83%, a decrease of 13%. The P BIB') decrease within hypoxic pockets was 21% less in isofl urane-anesthetized mice than in awake mice, and duration decreased by 15 s. The size of hypoxic pockets was reduced by 13%.

[0142] A direct consequence of hypercapnia and isoflurane anesthesia-induced vasodilation is an increase in tissue Pm. Increased tissue Pm might compensate for low-oxygenated areas by increasing O2 diffusion and thus reducing the prevalence of hypoxic pockets (25). To uncouple vasodilation and tissue oxygenation, the O2 in the inhalation air was increased from 20% to 30%, inducing hyperoxia. This led to a 55% increase in Pm during hyperoxia, which is substantially higher than the increase measured under hypercapnia. During hyperoxia, the number of hypoxic pockets per second dropped from 3.5 ± 0.0.04 to 3.1 ± 0.03, a decrease of 11%, which is far less compared with the isoflurane and hypercapnia conditions, where hypoxic pockets were reduced by 69 and 41%, respectively. During hyperoxia, Po2(AB / J8) in hypoxic pockets decreased 15.9% less than during normoxia, which might be explained by the accompanying increase in P02 leading to an overall increased tissue P02, reducing the amplitude of the hypoxic pockets. Furthermore, theDocket No.: UR 6-24018 / FR 161118.07201

[0143] duration of hypoxic pockets was reduced by 13 s (41.5 ± 1 versus 28.5 ± 1 s), and their size increased slightly by 13%. These observations show that vasodilation more potently controls the hypoxic pockets than does blood oxygenation.

[0144] Local cerebral microcirculation is mainly regulated by changes in vascular resistance. Reversible adhesion of circulating leukocytes can effectively halt capillary blood flow, a phenomenon called capillary stalling. To directly test whether capillary stalling elicits hypoxic pockets, microspheres (diameter: 4 mm) were delivered intravascularly (FIG. 3B) to occlude capillaries. O2 dynamics and microspheres were recorded in the same field of view simultaneously at 1 Hz using a hybrid BLI-fluorescence microscope that uses the readout time of the BLI camera to record microsphere fluorescence with a second camera. After injection of microspheres, the number of hypoxic pockets decreased from 8.2 ± 0.07 to 5.9 ± 0.06. In contrast, the total area covered by hypoxic pockets per second increased from 2.4% to 5.9%, an increase of 146%. Whereas Pm( B / B) only marginally changed, the duration of microsphere-induced hypoxic pockets was Il s shorter. Microsphere-induced hypoxic pockets were 445% larger than in the control group. Taken together, upon microsphere injection, the area of hypoxic tissue was increased, as reflected by an increase in size but not in the number of pockets. A plausible explanation for this observation is that upon microsphere injection, hypoxic pockets near each other may have fused together, thus decreasing the number of individually detected events. When interrogating the dynamics of individual spheres and local tissue O2 dynamics, it was clear that influx of a microsphere quickly triggered a local decrease in PO2 with a similar temporal dynamic as the spontaneously occurring hypoxic pockets.

[0145] Hypoxic pockets are reduced by wakefulness and further suppressed by locomotion To detect O2 dynamics in awake animals and assess the effect of locomotion, mice were trained to tolerate head restraint. One group of awake mice was recorded on a stationary platform, whereas a second group of mice was placed on a polystyrene ball, allowing head-fixed locomotion (FIG. 4A). The number of hypoxic pockets in behaving mice was reduced by 17% compared to KX-anesthetized mice (FIGs. 4B, and 4C). Yet the pockets increased in surface area by 41% (Fig.

[0146] 4G). Hypoxic pockets lasted, on average, 8 s less in awake mice, whereas the amplitude was unaffected (FIGs. 4E and 4F). Taken together, the P02 maintains the same characteristics but is more dynamic in the awake state than in the anesthetized state, possibly reflecting the higher levelDocket No.: UR 6-24018 / FR 161118.07201

[0147] of neuronal activity and thereby blood flow in wakefulness compared with anesthesia. It was also sought to determine whether the number of hypoxic pockets correlates with mice actively running, where increased blood flow and tissue Pm is expected. Twenty-four hours after surgery, mice were placed on an air-supported polystyrene ball, and Pm dynamics were recorded (FIG. 4A). Compared with immobilized awake mice, the number of hypoxic pockets was reduced by 35%, from 6.7 ± 0.05 to 4.3 ± 0.03, and the percentage of area covered by hypoxic pockets at any given time was reduced by 33% (FIG. 4D). On average, hypoxic pockets lasted 7 s less in mice that could freely run (FIG. 4E).

[0148] Further, the amplitude of hypoxic pockets was dampened by 10.4% compared with immobilized mice (FIG. 4F). The spatial coverage of the hypoxic pockets was further reduced by 26% in running mice compared with immobile mice (FIG. 4G), whereas the number of ROIs of hypoxic pockets did not differ between KX-anesthetized, mobile, and immobile mice (FIG. 4H). It is unlikely that the decrease of hypoxic pockets during active locomotion is due to a general increase in blood flow, as their highly structured spatial characterization is preserved, which is in contrast to the expected general linear increase in blood flow. The burden of hypoxic pockets on the brain was characterized as a measure of the effect of area, duration, and amplitude (FIG. 41). The analysis showed that the hypoxic burden is reduced by 52% in running mice compared with quiet awake mice (FIG. 4J). Earlier studies have shown that sensory stimulation can reduce prevalence of capillary stalls (§. E. Erdener et al., J. Cereb. Blood Flow Metab. 39, 886-900 (2019)). To test whether a similar paradigm suppresses the occurrence of hypoxic pockets, a series of 10 whisker stimulations were delivered to anesthetized mice. The stimulation resulted in a 35% decrease in the number of hypoxic pockets. Thus, functional hyperemia suppressed the occurrence of hypoxic pockets, whether initiated by sensory stimulation in anesthetized mice or by active running in awake behaving mice.

[0149] Discussion

[0150] This study shows that relative changes in Pm can be monitored continuously in wide cortical regions of awake behaving mice by a genetically encoded bioluminescent oxygen indicator. By monitoring the bioluminescence signal of GeNL expressed in astrocytes, it was found that cortical Pm constantly fluctuates during physiological conditions, giving rise to spatially and temporally defined hypoxic pockets. Manipulations that either increased or blocked capillary flowDocket No.: UR 6-24018 / FR 161118.07201

[0151] showed that local interruption of the microcirculation is responsible for the occurrence of hypoxic pockets. This conclusion was supported by imaging (i.e., IOSI) of cortical hemoglobin absorption. Monitoring the local hemoglobin concentration identified a transient local reduction in hemoglobin, which shared characteristic onset and offset dynamics with hypoxic pockets, thus providing an alternative approach to validate the occurrence of spatially restricted P02 fluctuations during physiological conditions.

[0152] Two-photon imaging of cortical capillaries has documented that the velocity of red blood cells can vary by more than a factor of 10. A characteristic trait of the mammalian cortex is the unbalanced prevalence of arterioles and venules. A modeling study predicted that low-flow regions in the capillary bed are inevitable owing to the many sources and sinks of blood flow and that they tend to form around whichever vessel is more numerous. In contrast to humans, where the ratio is 2.5:1 (arterioles: venules), in mice, venules outnumber arterioles by a factor of 2.6, which is expected to increase the low-flow regions preferentially around venules. Indeed, the analysis supported this notion by demonstrating that hypoxic pockets tend to appear closer to venules than to arterioles in the mouse cortex.

[0153] GeNL was expressed under an astrocytic promoter to take advantage of the fact that the fine astrocytic processes infiltrate all parts of the neuropil. However, it was not expected that the astrocyte-selective expression of GeNL had substantial impact on the observations. Oxygen diffuses freely across the neuropil with no restrictions imposed by either plasma membranes or by specific cell types.

[0154] The methods as disclosed are unexpectedly capable of detecting hypoxic pockets that cannot be detected by existing methods. Recording P02 concentration with a temporal resolution of 225 to 400 s per frame found hypoxic micropockets in the cortex of awake old mice using the phosphorescence probe PtP-C343. However, the more frequent hypoxic events reported lasted only ~50 s and could not be detected when imaging with a slow, minute-lasting temporal resolution. Recently, 1 / f-like fluctuations in P02 have been observed using Clark electrodes and linked to red blood cell spacing heterogeneity, which is in line with the observations in this study. However, P02 declines with age, and it would be of interest to use the P02 bioluminescence imaging introduced here to assess whether aging is linked to a progressive increase in the duration and / or spatial expansion of hypoxic pockets. Another open question is whether the transient hypoxicDocket No.: UR 6-24018 / FR 161118.07201

[0155] pockets contribute to the noise observed in the functional magnetic resonance imaging (fMRI) blood oxygen level-dependent (BOLD) signal. The hypoxic pockets are below the spatial resolution of fMRI but are likely contributing to the considerable noise during rest. It is in this regard that the analysis showed that the burden of the hypoxic pockets decreased when neuronal activity was increased, indicating that the increase in BOLD signal may, in part, reflect a drop in occurrence of hypoxic pockets. This conclusion is supported by the finding that increased capillary blood flow reduces the relative portion of capillary stalls.

[0156] Monitoring P02 with BLI is limited to detection of short-term fluctuations, excelling at measuring relative changes over periods of minutes rather than providing accurate baseline P02 comparisons over extended durations or across groups. It is important to note that although BLI is effective for assessing relative P02, it does not offer absolute quantification. Capillary blood flow is essential for supplying the brain with O2 and glucose, which are needed to support the high metabolic demand associated with normal brain function. Numerous studies have shown a link between reduced cerebral blood flow and cognitive decline, including changes in microvasculature structure and flow. The existence of non-perfused capillaries was discovered decades ago. More recently, transient disruptions of flow caused by neutrophil adhesion at the single capillary level were identified as a potential mechanism that contributes to cerebral blood flow changes driving neurological deficits. Increased capillary stalling has been observed in models of Alzheimer’s disease, raising questions about the long-term impact of capillary stalling and its potential role in long-term neuronal viability. Hypoxia-induced increase in expression of hypoxia inducible factor la (HIFla) impairs plasticity by disrupting synaptic physiology and spatial memory. This study predicts that physical inactivity has direct effects on tissue P02 by favoring capillary occlusions and increasing the number of hypoxic pockets. Conversely, simply increasing sensory input or locomotion rapidly suppresses the occurrence of hypoxic pockets, perhaps explaining the linkage between a sedentary lifestyle and an increased risk of dementia.

[0157] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variationsDocket No.: UR 6-24018 / FR 161118.07201

[0158] are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the claims or disclosure. All references cited herein are incorporated by reference in their entireties.

Claims

Docket No.: UR 6-24018 / FR 161118.07201CLAIMSWhat is claimed is:

1. A method of imaging or evaluating partial oxygen tension in a tissue in vitro or in vivo in a subject, comprising:introducing or expressing in the tissue or in one or more cells therein a luminescent protein capable of oxidizing a substrate with oxygen in the tissue, wherein oxidization of the substrate by the luminescent protein results in emission of a bioluminescence signal, and wherein intensity of the bioluminescence signal is correlated to a level of oxygen in the tissue;contacting the tissue with the substrate under a condition permitting oxidization of the substrate by the luminescent protein and emission of the bioluminescence signal; and imaging or evaluating the partial oxygen tension in the tissue by detecting the bioluminescence signal or determining a level of the bioluminescence signal.

2. The method of claim 1, wherein intensity of the bioluminescence signal is linearly correlated to the level of oxygen in the tissue.

3. The method of any one of the preceding claims, comprising identifying one or more hypoxic pockets in the tissue.

4. The method of claim 3, comprising identifying the hypoxic pockets in the tissue based on negative amplitude, Po2(AB / B), where P02 represents the partial oxygen tension, B represents the intensity of the bioluminescence signal, and AB represents a change of the intensity of the bioluminescence signal.

5. The method of any one of claims 3-4, comprising determining temporal and / or spatial characteristics of the hypoxic pockets.

6. The method of any one of claims 3-5, comprising determining a size, an area, a number, a frequency distribution, and / or a change thereof of the hypoxic pockets.Docket No.: UR 6-24018 / FR 161118.072017. The method of any one of claims 3-6, wherein the hypoxic pockets constitute a region-of-interest (ROI) of imaging of the partial oxygen tension.

8. The method of any one of claims 3-7, wherein the hypoxic pockets are reduced by wakefulness or by locomotion.

9. The method of any one of claims 3-8, comprising determining a change of the partial oxygen tension over a period of time.

10. The method of claim 9, wherein the period of time is 20, 50, 100, 150, 200, 250, 300, 350, or 400 seconds.

11. The method of any one of claims 3-10, comprising identifying the hypoxic pockets at a temporal resolution under 225 seconds.

12. The method of any one of claims 3-11, comprising identifying the hypoxic pockets at a temporal resolution under 100 seconds.

13. The method of any one of claims 3-12, comprising identifying the hypoxic pockets at a temporal resolution under 50 seconds.

14. The method of any one of the preceding claims, wherein detecting the bioluminescence signal or determining the level of the bioluminescence signal is insensitive to interference of spectral absorption by hemoglobin.

15. The method of any one of the preceding claims, comprising introducing a nucleic acid molecule encoding the luminescent protein into the tissue, such that the luminescent protein is expressed in the one or more cells in the tissue.

16. The method of any one of the preceding claims, comprising expressing the luminescent protein in the one or more cells in the tissue under control of an astrocytic promoter.Docket No.: UR 6-24018 / FR 161118.0720117. The method of any one of the preceding claims, wherein the luminescent protein comprises a luciferase subunit derived from a luciferase of Oplophorus Gracilirostris .

18. The method of any of the proceeding claims, wherein the luminescent protein is a fusion protein comprising a luciferase fused to a fluorescent protein.

19. The method of claim 18, wherein the luciferase comprises luciferase NanoLuc.

20. The method of claim 18, wherein the fluorescent protein comprises fluorescent protein mNeongreen.

21. The method of any one of the preceding claims, wherein the fluorescent protein comprises an amino acid sequence of SEQ ID NOs: 1-3 or comprises an amino acid sequence having at least 75% sequence identity with an amino acid sequence of SEQ ID NOs: 1-3.

22. The method of any one of the preceding claims, wherein the fluorescent protein is GeNL.

23. The method of any one of the preceding claims, wherein the substrate comprises an imidazopyrazinone substrate.

24. The method of claim 23, wherein the imidazopyrazinone substrate comprises furimazine, and wherein the luminescent protein is capable of oxidizing furimazine into furimamide.

25. The method of any one of the preceding claims, wherein the tissue is in a brain of a subject.

26. The method of any one of the preceding claims, wherein the tissue is cerebral cortex.

27. The method of any of the proceeding claims, wherein the one or more cells comprise astrocytes.

28. The method of any one of the preceding claims, wherein the method further comprises creating a cranial window on the skull of the subject to allow (a) introducing the luminescentDocket No.: UR 6-24018 / FR 161118.07201protein in the one or more cells in the tissue, (b) contacting the tissue with the substrate, and / or (c) detecting the bioluminescence signal or determining a level of the bioluminescence signal.

29. A method of identifying an agent capable of modulating partial oxygen tension in a tissue of a subject, comprising:imaging or evaluating the partial oxygen tension in the tissue of the subject according to the method of any one of the preceding claims, wherein the subject has been administered with the agent or exposed to the agent; andidentifying the agent as a candidate agent if a change of the partial oxygen tension is greater than a reference value.

30. A method of evaluating an effect of an agent on partial oxygen tension in a tissue of a subject, comprising:imaging or evaluating the partial oxygen tension in the tissue of the subject according to the method of claims 1-28, wherein the subject has been administered with the agent or exposed to the agent; anddetermining a change in the partial oxygen tension in the tissue of the subject with respect to a reference value.

31. A method of evaluating an effect of an activity on partial oxygen tension in a tissue of a subject, comprising:imaging or evaluating the partial oxygen tension in the tissue of the subject according to the method of claims 1-28, wherein the subject has performed the activity; anddetermining a change in the partial oxygen tension in the tissue of the subject with respect to a reference value.

32. A method of detecting or evaluating a level of oxygen in a target, comprising:providing a luminescent protein capable of oxidizing a substrate with oxygen, wherein oxidization of the substrate by the luminescent protein results in emission of a bioluminescence signal, and wherein intensity of the bioluminescence signal is correlated to the level of oxygen in the target;Docket No.: UR 6-24018 / FR 161118.07201contacting the target or a sample therefrom with the luminescent protein and the substrate under a condition permitting oxidization of the substrate by the luminescent protein and emission of the bioluminescence signal; anddetecting or evaluating a level of the bioluminescence signal, thereby detecting or evaluating the level of oxygen in the target.

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