Ntpdase8 for liver ischemia

Abstract

Methods and compositions for (a) treating or reducing liver ischemia-reperfusion injury, including livers in subjects and livers ready for transplant, or (b) treating sterile inflammatory conditions. The composition includes an NTPDase8 that may be delivered by a lipid nanoparticle or through an mRNA that encodes an NTPDase8.

Description

Docket: 93597 / 7646 (92489-A-PCT)NTPDase8 FOR LIVER ISCHEMIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U. S. Provisional Patent Application Serial No. 63 / 764,731, filed February 28, 2025, and entitled “NTPDase8 FOR LIVER ISCHEMIA” the entire contents of which are hereby incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under HL158519 and GM066189 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] This application incorporates-by-reference nucleotide and / or amino acid sequences which are present in the file named “93597-7646_92489-A-PCT_Sequence_Listing_AWG.xml”, which is 12,646 bytes in size, and which was created on February 23, 2026 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the xml file filed February 26, 2026 as part of this application.BACKGROUND OF THE INVENTION

[0004] The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.

[0005] Liver ischemia-reperfusion (I / R) injury is a serious medical condition, which occurs when the blood supply to the liver is temporarily disrupted and then restored. This condition is frequently encountered in a variety of medical situations, including liver transplantation, substantive liver resections, and after trauma. The pathogenesis of liver I / R injury is complicated, involving a cascade of cellular and molecular events that cause metabolic and oxidative stress, inflammation, and cell death, which all lead to tissue damage1’4.

[0006] NTPDases are a class of ecto-enzymes that hydrolyze nucleotides, such as ATP, ADP, UTP, and UDP, to the respective nucleoside monophosphates and thus participate in nucleotide-14917-4055-5666v.ldependent biological processes. Eight different isoforms of NTPDases have been identified in mammals, each with unique tissue expression, subcellular localization, membrane topology, and substrate specificities. NTPDasel, -2, -3, and -8 have extracellularly oriented catalytic sites. NTPDasel, which is also called CD39, is mostly found on the cell membrane of endothelial cells, immunological cells, and certain cancer cells5'9. CD39 and NTPDase2 are expressed together in blood vessels where CD39 is found primarily on endothelial and smooth muscle cells and NTPDase2 on adventitial cells and fibroblasts7,10.

[0007] In the liver, which has amongst the highest ATPase and ADPase activities of all organs, CD39 is expressed on the vascular endothelium and Kupffer cells whereas NTPDase2 is present on portal fibroblasts and activated hepatic stellate cells11. NTPDase3 is present in multiple organs, including the brain, and pancreas, and has a role in various physiological processes such as metabolic homeostasis, inflammation, neurotransmission and thromboregulation7,8,12 14. NTPDase8, the most recently characterized NTPDase, is primarily expressed in the liver and biliary system with lower amounts of this enzyme have also been found in the jejunum and kidney. The highest levels of NTPD-8 expression in the bile canaliculi and liver indicates that this ectonucleotidase is likely involved in regulating bile secretion and / or nucleoside salvage14'17.

[0008] Since NTPDasel, -2, -3, and -8 have extracellularly oriented catalytic sites these enzymes can control the bioavailability of nucleotides and signaling through P2 receptors in the extracellular space9'11,18‘27. The final products of NTPDase-mediated nucleotide hydrolysis are nucleoside monophosphates, such as AMP, which are further metabolized in the extracellular space by ectoenzymes, such as CD73, to adenosine7. Extracellular nucleotides signal through purinergic ligand-gated ion channel P2X receptors or G-protein coupled P2Y receptors, which often have pro-inflammatory effects28'30. Adenosine signals through Pl or adenosine receptors, the activation of which in general has anti-inflammatory effects7,31'41. Thus, membrane surface NTPDases together with CD73 eventually switch the extracellular milieu microenvironment from pro- to anti-inflammatory4,29,42'48.

[0009] Liver cells release nucleotides into the extracellular space in response to stressful stimuli. These stimuli include partial hepatectomy, which results in the release of ATP into the hepatic vein49, and osmotic stress, which releases ATP from hepatocytes in vitro50. In addition, bile acids induce the release of ATP into the bile in isolated rat liver51. While direct evidence24917-4055-5666v.lindicating fluxes of ATP release during liver I / R injury is not available, indirect evidence demonstrating that CD39 protects the liver from I / R injury indicates that ATP is released and that CD39-mediated degradation is protective4,9’27,52’53.BRIEF SUMMARY OF THE INVENTION

[0010] Herein are disclosed compositions and methods for treating or preventing hepatic ischemia-reperfusion injury.

[0011] In embodiments, the techniques described herein relate to a method of treating or reducing ischemia-reperfusion injury in a subject including administering NTPDase8 effective to be delivered to the subject’s liver in an amount effective to reduce or treat ischemia-reperfusion injury.

[0012] In embodiments, the techniques described herein relate to a method of treating or reducing ischemia-reperfusion injury in liver for transplant into a subject including administering NTPDase8 to the liver in an amount effective to reduce or treat ischemia-reperfusion injury.

[0013] In embodiments, the techniques described herein relate to a method of improving liver surgery including transplanting a liver that has been pretreated with exogenous NTPDase8, wherein the NTPDase8 is effective to reduce or treat ischemia-reperfusion injury, into a subject in need of the transplant.

[0014] In embodiments, the techniques described herein relate to a method of reducing or treating ischemia-reperfusion injury in a subject including administering NTPDase8 to the liver of the subject, wherein the NTPDase8 is effective to prevent or reduce ischemia-reperfusion injury.

[0015] In embodiments, the techniques described herein relate to exogenous NTPDase8 for use in reducing liver damage in a subject, wherein the subject has or is likely to undergo liver ischemia-reperfusion injury.

[0016] In embodiments, the techniques described herein relate to a method of treating or reducing a sterile inflammatory condition in a subject including administering NTPDase8 effective to treat or reduce a sterile inflammatory condition in a subject.34917-4055-5666v.lBRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figs. 1A - IF depict NTPDase2, -3, and -8 regulation of liver injury. Liver injury was determined by measuring ALT (A, C, E) and AST levels (B, D, F) spectrophotometrically. Data are means ± SEM. (n=4-7 / group). **p < 0.01 compared with Entpd8+ / +.

[0018] Figs. 2A-2D depict effects of NTPDase8 on liver I / R. (A) H& E staining (5 X and 40 X) of liver sections. (B) TUNEL staining (10 X confocal tile imaging and 20 X confocal imaging) of liver sections. (C) Mean histological liver injury scores for each parameter. (D) Quantification of TUNEL (+) cells / mm2. Data are mean ± SEM. (n=5-6 / group). *p < 0.05 compared to WT-I / R, **p < 0.01 compared with Entpd8+ +-I / R.

[0019] Figs. 3 A - 3B depict NTPDase8 deficiency in parenchymal cells exacerbates liver injury. Plasma levels of ALT (A) and AST (B) in WT— > WT (WT bone marrow and WT parenchyma), KO— > KO (NTPDase8 KO bone marrow and KO parenchyma) WT^KO (WT bone marrow and NTPDase8 KO parenchyma), and KO^WT (NTPDase8 KO bone marrow and WT parenchyma) mice 6 h after liver injury, n = 5, 6, 6, and 5 mice / group, respectively. Data are presented as mean ± SEM. **p < 0.01 compared to WT— > WT mice.

[0020] Figs. 4A - 4C depict absence of NTPDase8 alters gene expression in the liver of mice. An RNAseq analysis was conducted on the livers of WT or NTPDase8 KO mice that underwent liver I / R with a sample size of 3 mice per group. (A) Heatmap illustrating the results of variancestabilizing transformation (VST) analysis of the expression of the top 50 differentially expressed genes. (B) The volcano map illustrates the primary genes (shown in red) that have been found to have significant differences in expression between the WT and KO groups. These differences were assessed using modified p-values (calculated using the Wald statistic and the Benjamini -Hochberg method) with a threshold of padj < 0.05. The location of Entpd8 in the volcano plot is denoted by yellow. (C) The Stacked bar chart illustrates the proportion of pathways that were upregulated (shown by green) and genes that were downregulated (indicated by red) in KO livers in IPA biological function analysis. The 30 most significant biological pathways were determined using Fisher's exact test, with a threshold of -loglO p-value > 2 (equivalent to a p-value < 0.05). The y axis displays the percentage significance level represented as the logarithm of the p-value.

[0021] Figs. 5 A - 5G depict NTPDase8 protein and gene expressions are downregulated after liver I / R. (A) NTPDase8 gene expression in the liver exposed to sham operation or I / R. (B)44917-4055-5666v.lNTPDase8 protein expression in the liver subjected to sham operation or I / R. Top: immunoblot. Bottom: quantification of the immunoblot, where NTPDase8 normalized to β-actin is shown. (C) (Left) Representative images of NTPDase8 immunohistochemistry in the liver (5 X and 40 X). Dark brown colors indicate NTPDase8 staining. (Right) Quantification of NTPDase8-stained regions as a percentage of the overall tissue area. (D) Confirmation of NTPDase8 downregulation in bulk RNAseq public data of mice subjected to ischemia-reperfusion injury. (E-F) Expression of Entpd8 in Mouse Liver Cells. UMAP plot of single-nucleus RNA sequencing data shows that Entpd8 expression is highly enriched in hepatocytes compared to other liver cell types, including cholangiocytes, mesothelial cells, Kupffer cells, hepatic stellate cells (HSC), dendritic cells (eDCs), endothelial cells, B cells, other myeloid cells, T cells, monocytes, vascular smooth muscle cells (VSMC), NK cells, and plasma cells. (G) NTPDase8 is downregulated 24 hours after initiating normothermic machine perfusion of human livers, where ischemia occurs before placing the livers on normothermic machine perfusion and reperfusion during the perfusion period. Data are mean ± SEM. (n=5-6 / group), **p<0.01 compared with sham (ABD) or pre normothermic machine perfusion (G).

[0022] Figs. 6A - 6B depicts ATP levels in the plasma and liver following ER. ATP was measured by a fluorometric assay after ER both in plasma (A) and liver (B). Data are mean ± SEM. (n=5-6 / group). **p <0.01 compared with Entpd8+ / +-V,&p<0.05 compared with Entpd8 -Sham, p<0.0 l compared with EntpdS^' - Sham.

[0023] Figs. 7A - 7B depicts effects of the general P2 receptor agonist suramin on liver injury in NTPDase8+ +and NTPDaseS’ ' mice. Liver injury was determined from plasma levels of ALT (A), and AST (B). Data are presented as mean ± SEM. 'p< 0.01 compared with Entpd8+ +-ER and **p< 0.01 compared with Entpd8~ -I / R. FIG. 8: Determination of intestinal injury after T / HS in lEC-specific A2BR deficient mice. Results with lEC-specific VillinCre-A2BRfl / fl mice and control are shown. Data are mean ± S. D. (n=4 / group). **p<0.05 compared with T / HS-VillinCre-A2BAR+ / +.

[0024] Figs. 8A - 8B depicts NTPDase8 regulation of liver enzymes after T / HS. Alanine aminotransferase (ALT) (A) and aspartate aminotransferase (AST) (B) levels were determined from plasma spectrophotometrically. Data are mean± SEM. (n=4 / group) **p<0.01 compared with T / SS,&&p<0.01 compared with T / HS- and p<0.0 l compared with the corresponding T / SS group.54917-4055-5666v.l

[0025] Fig. 9 depicts a RNA-seq sample variance plot.

[0026] Figs. 10A - 10C depict liver cytokine levels.

[0027] Fig. 11 depicts SDS-PAGE analysis showing NTPDase8 as a ~72 kDa band in the upper panel and B-actin as a ~42 kDa band in the lower panel.

[0028] Figs. 12A - 12E depict NTPDase8 regulation of liver injury. (A) Liver injury was determined by measuring ALT and AST levels spectrophotometrically. (B) Mean histological liver injury scores. (C) Quantification of TUNEL (+) cells / mm2. (D) H& E staining (5× and 40×) of liver sections. (E) TUNEL staining (10x confocal tile imaging and 20x confocal imaging) of liver sections. Data are mean ± SEM. (n = 5-6 / group). *p < 0.05 compared to WT- I / R, **p < 0.01 compared with Entpd8+ +-I / R.

[0029] Figs. 13A - 13E depict NTPDase8 deficiency in parenchymal cells exacerbates liver injury. (A) Plasma levels of ALT and AST in WT — WT (WT bone mar-row and WT parenchyma), KO — KO (NTPDase8 KO bone marrow and KO parenchyma) WT — > KO (WT bone marrow and NTPDase8 KO paren-chyma), and KO —> WT (NTPDase8 KO bone marrow and WT parenchyma) mice 6 h after liver injury. (B) Mean histological liver injury scores. (C) Quantification of TUNEL (+) cells / mm2. (D) H& E staining (5x and 40 ) of liver sections. (E) TUNEL staining (10x confocal tile imaging and 20 confocal imaging) of liver sections, n = 5, 6, 6, and 5 mice / group, respectively. Data are presented as mean ± SEM. **p < 0.01 compared to WT - WT mice.

[0030] Figs. 14A-14E depict effects of the general P2 receptor agonist suramin on liver injury in NTPDase81and NTPDase8 ' mice. (A) Liver injury was deter-mined from plasma levels of ALT and AST. (B) Mean histological liver injury scores. (C) Quantification of TUNEL (+) cells / mm2. (D) H& E staining(5x and 40 ) of liver sections. (E) TUNEL staining (10x confocal tile imaging and 20 confocal imaging) of liver sections. Data are presented as mean ± SEM. p < 0.01 compared with Entpd8+ / +- I / R and **p < 0.01 compared with Entpd8 - I / R.

[0031] Figs. 15A-15E depict NTPDase8 regulation of liver enzymes after T / HS. (A) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined from plasma spectrophotometrically. (B) Mean histological liver injury scores. (C) Quantification of TUNEL (+) cells / mm2(D) H& E staining (5x and 40x) of liver sections. (E) TUNEL staining (10x64917-4055-5666v.lconfocal tile imaging and 20× confocal imaging) of liver sections. Data are mean ± SEM. (n = 4 / group) **p < 0.01 compared with T / SS,&&p < 0.01 compared with T / HS- and##p < 0.01 compared with the corresponding T / SS group.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0032] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0033] In the discussion unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. In some embodiments, about means within a standard deviation using measurements generally acceptable in the art. In some embodiments, about means a range extending to + / - 10% of the specified value. In some embodiments, about includes the specified value. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of and any combination of items it conjoins.

[0034] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described 74917-4055-5666v.lin the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0035] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

[0036] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0037] In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0038] “And / or” as used herein, for example with option A and / or option B, encompasses the separate embodiments of (i) option A, (ii) option B, and (iii) option A plus option B.

[0039] The term “subject” as used in this application means a mammal. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates including humans. Thus, in some embodiments the compositions and / or methods can be used in human medicine or also in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. In some embodiments the compositions and / or methods are particularly desirable for human medical applications, especially in trauma situation. In a preferred embodiment the subject is a human.84917-4055-5666v.l

[0040] The terms “treat”, “treatment” of a disease or condition, and the like refer to slowing down, relieving, ameliorating or alleviating at least one of the symptoms of the condition.

[0041] The terms “therapeutically effective amount” or "amount effective to" encompasses, unless otherwise indicated, an amount sufficient to ameliorate or inhibit a symptom or sign of the medical condition. An effective amount for a particular subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side effects. An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects.General

[0042] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. All combinations of the various elements disclosed herein are within the scope of the invention.NTPDase8

[0043] In some embodiments, the present invention involves the use of NTPDase8.

[0044] In some embodiments, the full length NTPDase8 is human and comprises the sequence: MGLSRKEQVFLALLGASGVSGLTALILLLVEATSVLLPTDIKFGIVFDAGSSHTSLFLYQ WLANKENGTGVVSQALACQVEGPGISSYTSNAAQAGESLQGCLEEALVLIPEAQHRKTP TFLGATAGMRLLSRKNSSQARDIFAAVTQVLGRSPVDFWGAELLAGQAEGAFGWITVN YGLGTLVKYSFTGEWIQPPEEMLVGALDMGGASTQITFVPGGPILDKSTQADFRLYGSD YSVYTHSYLCFGRDQMLSRLLVGLVQSRPAALLRHPCYLSGYQTTLALGPLYESPCVHA TPPLSLPQNLTVEGTGNPGACVSAIRELFNFSSCQGQEDCAFDGVYQPPLRGQFYAFSNF YYTFHFLNLTSRQPLSTVNATIWEFCQRPWKLVEASYPGQDRWLRDYCASGLYILTLLH EGYGF SEETWP SLEFRKQ AGGVDIGWTLGYMLNLTGMIP AD AP AQWRAES YGVW V AK VVFMVLALVAVVGAALVQLFWLQD (SEQ ID NO:1).

[0045] In some embodiments, NTPDase8 has the sequence set forth in GenBank Accession No. AY430414 or AAR04374. In some embodiments, NTPDase8 has the sequence set forth in SEQ ID NO:6. In some embodiments, NTPDase8 is encoded by polynucleotide molecule having the sequence set forth in SEQ ID NO: 7, or SEQ ID NO: 8.94917-4055-5666v.l

[0046] In some embodiments, the present invention involves the use of a functional fragment ofNTPDase8. In some embodiments, the present invention involves the use of a fusion comprising NTPDase8. In some embodiments, a fusion protein comprising NTPDase8, or a functional fraction thereof is administered. Unless otherwise indicated, the methods described herein can be performed using full-length NTPDase8 or a polypeptide derived therefrom, including fusion constructs or fragments that retain the activity relevant to the described method.

[0047] In some embodiments, NTPDase8 is human. In some embodiments, the NTPDase8 is non-human.Administration

[0048] In some embodiments, the NTPDase8 is administered non-genetically (e.g. via mature or soluble protein). In some embodiments, the NTPDase8 is administered genetically (e.g. via mRNA).

[0049] In some embodiments the NTPDase8 is administered extracellularly. In some embodiments, the NTPDase8 is administered intracellularly.Administration location and timing

[0050] In some embodiments, administration can be via transfusion. In some embodiments, administration can be systemic. In some embodiments, administration can be local or direct intraorgan. In some embodiments, administration can be intramuscular or subcutaneous. In some embodiments, administration can be oral or parenteral. In some embodiments, administration is via infusion or injection. In some embodiments, administration is intrathecal or intraventricular. In some embodiments, administration is intravenous.

[0051] In some embodiments, the NTPDase8 is administered to a liver. In some embodiments, the liver is of a subject. In some embodiments, the NTPDase8 is administered via a hepatic portal vein of the subject. In some embodiments, the NTPDase8 is administered via a hepatic artery of the subject.

[0052] In some embodiments, the NTPDase8 is administered at least one time during the following time periods: prior to ischemia, after ischemia and prior to reperfusion, and after reperfusion. In some embodiments, the NTPDase8 is administered multiple times throughout the above mentioned time periods. In some embodiments, the NTPDase8 is administered prior to104917-4055-5666v.lischemia in the subject. In some embodiments, the NTPDase8 is administered after ischemia, and prior to reperfusion in the subject. In some embodiments, the NTPDase8 is administered after reperfusion.Non-genetic administration

[0053] In some embodiments, the NTPDase8 is a soluble NTPDase8. In some embodiments, the NTPDase8 comprises a Fc-fusion protein comprising NTPDase8. In some embodiments, the NTPDase8 comprises a PEGylated form of NTPDase8.

[0054] In some embodiments, the NTPDase8 comprises a proteolipid comprising NTPDase8. In some embodiments, the NTPDase8 comprises a lipid nanoparticle comprising NTPDase8. Genetic administration

[0055] In some embodiments, the NTPDase8 is administered via mRNA administration. In some embodiments, the NTPDase8 is administered via viral vector.

[0056] In some embodiments, NTPDase8 is administered via an mRNA encoding NTPDase8. In some embodiments, NTPDase8 is administered via an mRNA encoding a full length NTPDase8. In some embodiments, NTPDase8 is administered via a composition comprising mRNA encoding NTPDase8 and lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise GalNac-lipid nanoparticles.

[0057] In some embodiments, NTPDase8 is administered via an adeno-associated viral vector. In some embodiments, NTPDase8 is administered via an adenovirus. In some embodiments, NTPDase8 is administered via a lentivirus.Compositions

[0058] In some embodiments, the NTPDase8 is contained in pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a soluble NTPDase8 and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprises an mRNA encoding a full length NTPDase8. In some embodiments, a pharmaceutical composition comprises an mRNA encoding full length NTPDase8 and comprises lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise GalNac-lipid nanoparticles.

[0059] In some embodiments, the compositions disclosed herein can be lyophilized and / or freeze dried and are reconstituted for use.114917-4055-5666v.l

[0060] Compositions or pharmaceutical compositions disclosed herein can comprise stabilizers to prevent loss of activity or structural integrity of the enzyme due to the effects of denaturation, oxidation or aggregation over a period of time during storage and transportation prior to use.

[0061] Where a composition or pharmaceutical composition of the present disclosure is used as an injection, infusion, or transfusion, it is desirable to have a pH value in an approximately neutral pH range or human blood pH range, it is also advantageous to minimize surfactant levels to avoid bubbles in the formulation which are detrimental for injection into subjects.

[0062] In some embodiments, the composition or pharmaceutical composition is suitable for intravenous, intramuscular, intraperitoneal, intradermal, intraorgan, and / or subcutaneous injection. In some embodiments, the composition or pharmaceutical composition is in liquid form and has minimized risk of bubble formation and anaphylactoid side effects. In some embodiments, the composition or pharmaceutical composition is isotonic. In an embodiment, the composition or pharmaceutical composition has a pH or 6.8 to 7.4.

[0063] Examples of pharmaceutically acceptable carriers include, but are not limited to, phosphate buffered saline solution, sterile water (including water for injection USP), emulsions such as oil / water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline, for example 0.9% sodium chloride solution, USP. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000, the content of each of which is hereby incorporated in its entirety). In non-limiting examples, the compositions can comprise one or more of dibasic sodium phosphate, potassium chloride, monobasic potassium phosphate, polysorbate 80 (e.g. 2-[2-[3,5-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl (E)-octadec-9-enoate), disodium edetate dehydrate, sucrose, monobasic sodium phosphate monohydrate, and dibasic sodium phosphate dihydrate.Liver conditions

[0064] In some embodiments, the methods comprise the treatment of the liver of a subj ect with a liver condition. In some embodiments, the methods comprise the treatment of a liver of a subject124917-4055-5666v.lundergoing surgery, optionally liver surgery. In some embodiments, the subject has or has undergone shock, heart failure, respiratory failure, hemorrhage, physical trauma, or sepsis.

[0065] In some embodiments, the subject has had a physical trauma-induced hemorrhagic shock.

[0066] In embodiment, the subject is suffering from NASH.

[0067] In some embodiments, the subject has a sterile shock or does not have sepsis, septic shock, or shock associated with an infection.

[0068] In some embodiments, the liver condition is caused by and / or associated with physical trauma-induced hemorrhagic shock, NASH, sterile shock or does not have sepsis, septic shock, or shock associated with an infection.Method of treatment

[0069] In some embodiments, methods of the present invention include a method of treating a liver to reduce or treat ischemia-reperfusion injury comprising administering to the liver a soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8, effective to treat a liver to reduce or treat ischemia-reperfusion injury.

[0070] In some embodiments, the liver is in a subject undergoing or about to undergo a procedure comprising liver surgery, and the soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is administered to the subject.

[0071] In some embodiments, the liver is in a subject undergoing, or who has undergone, a procedure comprising a liver transplant, and the soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is administered to the subject.

[0072] In some embodiments, the liver is in a subject undergoing, or who has undergone, hemorrhagic shock, and the soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is administered to the subject.

[0073] In some embodiments, the liver is to be transplanted into a subject, and the soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is administered to the liver.134917-4055-5666v.l

[0074] In some embodiments, the administering to the liver of a soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is direct administration to the liver. In some embodiments, the administering to the liver is via a hepatic portal vein. In some embodiments, the administering to the liver is via a hepatic artery.

[0075] A method of treating a sterile inflammatory response in a liver comprising administering to the liver a soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8, effective to treat a sterile inflammatory response in a liver.

[0076] A method of treating a liver to reduce or treat ischemia-reperfusion injury comprising administering to the liver a soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8, effective to treat a liver to reduce or treat ischemia-reperfusion injury.

[0077] In embodiments, the liver is in a subject undergoing or about to undergo a procedure comprising liver surgery, and the soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is administered to the subject.

[0078] In embodiments, the liver is in a subj ect undergoing, or who has undergone, a procedure comprising a liver transplant, and the soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is administered to the subject.

[0079] In embodiments, the liver is in a subject undergoing, or who has undergone, hemorrhagic shock, and the soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is administered to the subject.

[0080] In embodiments, the liver is to be transplanted into a subject, and the soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is administered to the liver.

[0081] In embodiments, the soluble NTPDase8 is human.

[0082] In embodiments, the full length NTPDase8 is human.

[0083] In embodiments, the comprising an mRNA encoding full length NTPDase8 comprises lipid nanoparticles. In embodiments, the lipid nanoparticles comprise GalNac-lipid nanoparticles.

[0084] In embodiments, the administering to the liver of a soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8 is direct administration to the liver. In144917-4055-5666v.lembodiments, the administering to the liver is via a hepatic portal vein. In embodiments, the administering to the liver is via a hepatic artery.

[0085] A method of treating a sterile inflammatory response in a liver comprising administering to the liver a soluble NTPDase8 or a composition comprising an mRNA encoding a full length NTPDase8, effective to treat a sterile inflammatory response in a liver.

[0086] In embodiments, the subject has had a physical trauma-induced hemorrhagic shock.

[0087] In embodiments, the subject has a sterile shock or does not have sepsis, septic shock, or shock associated with an infection.

[0088] A pharmaceutical composition comprising a soluble NTPDase8 and a pharmaceutically acceptable carrier.

[0089] A pharmaceutical composition comprising an mRNA encoding a full length NTPDase8. In embodiments, the pharmaceutical composition comprising an mRNA encoding full length NTPDase8 comprises lipid nanoparticles. In embodiments, the lipid nanoparticles comprise GalNac-lipid nanoparticles.

[0090] In embodiments, the techniques described herein relate to a method of treating or reducing ischemia-reperfusion injury in a subject including administering NTPDase8 effective to be delivered to the subject’s liver in an amount effective to reduce or treat ischemia-reperfusion injury.

[0091] In embodiments, the techniques described herein relate to a method of treating or reducing ischemia-reperfusion injury in liver for transplant into a subject including administering NTPDase8 to the liver in an amount effective to reduce or treat ischemia-reperfusion injury.

[0092] In embodiments, the techniques described herein relate to a method, wherein the NTPDase8 is administered prior to ischemia in the subject.

[0093] In embodiments, the techniques described herein relate to a method, wherein the NTPDase8 is administered after ischemia, but prior to reperfusion in the subject.

[0094] In embodiments, the techniques described herein relate to a method, wherein the NTPDase8 is administered after reperfusion.154917-4055-5666v.l

[0095] In embodiments, the techniques described herein relate to a method, wherein the NTPDase8 is administered via a hepatic portal vein of the subject.

[0096] In embodiments, the techniques described herein relate to a method, wherein the NTPDase8 is administered via a hepatic artery of the subject.

[0097] In embodiments, the techniques described herein relate to a method, wherein the NTPDase8 is administered as a composition including a soluble NTPDase8.

[0098] In embodiments, the techniques described herein relate to a method, wherein a composition including an mRNA encoding a full length NTPDase8, so as to permit expression thereof, is administered to the subject.

[0099] In embodiments, the techniques described herein relate to a method, where the composition includes lipid nanoparticles.

[0100] In embodiments, the techniques described herein relate to a method, wherein the lipid nanoparticles include GalNac-lipid nanoparticles.

[0101] In embodiments, the techniques described herein relate to a method, wherein the NTPDase8 includes the sequence:MGLSRKEQVFLALLGASGVSGLTALILLLVEATSVLLPTDIKFGIVFDAGSSHTSLFLYQ WLANKENGTGVVSQALACQVEGPGISSYTSNAAQAGESLQGCLEEALVLIPEAQHRKTP TFLGATAGMRLLSRKNSSQARDIFAAVTQVLGRSPVDFWGAELLAGQAEGAFGWITVN YGLGTLVKYSFTGEWIQPPEEMLVGALDMGGASTQITFVPGGPILDKSTQADFRLYGSD YSVYTHSYLCFGRDQMLSRLLVGLVQSRPAALLRHPCYLSGYQTTLALGPLYESPCVHA TPPLSLPQNLTVEGTGNPGACVSAIRELFNFSSCQGQEDCAFDGVYQPPLRGQFYAFSNF YYTFHFLNLTSRQPLSTVNATIWEFCQRPWKLVEASYPGQDRWLRDYCASGLYILTLLH EGYGFSEETWPSLEFRKQAGGVDIGWTLGYMLNLTGMIPADAPAQWRAESYGVWVAK VVFMVLALVAVVGAALVQLFWLQD (SEQ ID NO:1).

[0102] In embodiments, the techniques described herein relate to a method, wherein the NTPDase8 includes the sequence set forth in AY430414 or AAR04374.

[0103] In embodiments, the techniques described herein relate to a method of improving liver surgery including transplanting a liver that has been pretreated with exogenous NTPDase8,164917-4055-5666v.lwherein the NTPDase8 is effective to reduce or treat ischemia-reperfusion injury, into a subject in need of the transplant.

[0104] In embodiments, the techniques described herein relate to a method, further including prior to surgery administering NTPDase8 to the liver in an amount effective to reduce or treat ischemia-reperfusion injury.

[0105] In embodiments, the techniques described herein relate to a method of reducing or treating ischemia-reperfusion injury in a subject including administering NTPDase8 to the liver of the subject, wherein the NTPDase8 is effective to prevent or reduce ischemia-reperfusion injury.

[0106] In embodiments, the techniques described herein relate to exogenous NTPDase8 for use in reducing liver damage in a subject, wherein the subject has or is likely to undergo liver ischemia-reperfusion injury.

[0107] In embodiments, the techniques described herein relate to a method of treating or reducing a sterile inflammatory condition in a subject including administering NTPDase8 effective to treat or reduce a sterile inflammatory condition in a subject.

[0108] In embodiments, the techniques described herein relate to a method, wherein the sterile inflammatory condition is in at least the liver of the subject.174917-4055-5666v.lEXAMPLES

[0110] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.Example 1Materials and Methods

[0111] Ethical statements and animals: All procedures on mice were conducted under the approval of the Columbia University Institutional Animal Care and Use Committee (IACUC), approval number AABL4551 / 2021. Adult male mice (8-12 weeks old, n = 4-6 per group), including Entpd2'1'54, Entpd.3'1'55, and Entpd8''~14mice, and C57BL / 6J wild-type (WT) litter-mate controls, were bred and maintained in a specific pathogen-free facility at Charles River. WT mice were also obtained from the same facility Charles River (Wilmington, MA, USA). All mice were maintained in a pathogen-free environment at Columbia University until use in experiments. They had free access to food and water and were housed in a room with a 12-hour light-dark cycle under non-specific pathogen-free conditions. Ear samples were collected from EntpdZ'1' and Entpd3' ''and genotyping was performed by TransnetYX (Cordova, TN, USA). Entpd8- / - mice were genotyped at Columbia University (Table 1).

[0112] Table 1. shows Primers for qPCR and NTPDase8 genotyping.Target Forward (5’-3’) Reverse(3’-5’)Ms Entpd8vqPCR Qiagen assay #249900Ms Entpd8 TGTAAGGGCCAGAAGGATTG GTACAGACCCGAGGCACAGT genotyping (SEQ ID NO:2) (SEQ IDNO:3)Ms neomycin TCATTCTCAGTATTGTTTTGCC CAGGCATCTAGGATCTGCTC Entpd8 (SEQ ID NO:4) (SEQ ID NO: 5)genotyping

[0113] Study Design and Induction of Liver Ischemia-Reperfusion: Eight-week-old Entpd1', Entpd3"~, and Entpd8'~ mice and their WT controls were anesthetized with isoflurane and exposed184917-4055-5666v.lto 1 h of warm ischemia and 6 h reperfusion injury or sham as described previously56,57. In brief, the shaved abdomen was disinfected with a povidone-iodine solution (7.5%), after which a midline laparotomy was performed. The intestines were gently moved aside with moistened cotton tips to expose the liver and portal vein, and the liver hilum was meticulously dissected using an operating microscope. The median and left lateral lobes were lifted, and the liver hilum was further dissected to separate the quadrate and left lateral lobes. A 10 cm thread was carefully passed around the portal triad to lift it, and a microvascular clamp was applied to occlude the hepatic artery, portal vein, and bile duct to the left and median liver lobes, ensuring immediate blanching of the liver. One hour after occlusion, the clamp was removed, and the muscles and skin were sutured. Blood and tissue samples were collected after a 6-hour reperfusion period. Sham-operated mice were subjected to laparotomy but not vascular clamp56. A fixed-pressure model was used to induce hemorrhagic shock58. Following anesthesia, mice underwent a midline laparotomy of 2 cm. The incision was then sealed using a 4-0 silk suture (034902, Covetrus, USA). The right and left femoral arteries were surgically separated and catheters were inserted to precisely measure blood pressure and extract blood, respectively. For blood withdrawal, a sterile 1 ml syringe containing a 30G needle was connected to PE-10 tubing and filled with 0.2 ml of a 1% heparinized saline solution. Each mouse was administered 1 unit of heparin. The blood pressure was monitored using a continuous blood-pressure monitoring system (Powerlab 8 / 30 AD Instruments Colorado Springs, CO, USA). Following a five-minute baseline recording, mice subjected to shock conditions for a duration of 2.5 hours. By either withdrawing or reinfusing the shed blood, blood pressure was consistently maintained within the range of 28-32 mmHg. Upon completion of the shock phase, the mice were revived with Ringer's Lactate at a concentration three times more than the amount of blood they had shed, for a duration of 15 minutes. Three hours following resuscitation, the mice were euthanized and blood samples were collected. Animals in the T / SS group underwent same procedures except for blood withdrawal.

[0114] RNAseq: The tissue samples were homogenized in the presence of 1 ml Trizol and 5 mm stainless steel bead, using the Qiagen TissueLyser II instrument for 2 min at 25 Hz, twice. Homogenized samples were incubated for 5 min at room temperature after which 200 ml Chloroform was added, mixed well and incubated for 3 min at room temperature. Samples were centrifuged at 10,000 g at 4 °C after which the aqueous layer was transferred to a new tube and mixed with equal volume of 70%. The samples were loaded to Qiagen RNAeasy column, and the194917-4055-5666v.lsamples were processed according to the protocol, including the optional on column DNAse treatment. RNA was eluted in 35 ml of RNAse free water. A Quality Control analysis was performed on the RNA samples using the Agilent Bioanalyzer to assess quality and an RNA Qubit assay was used to determine quantity. Poly(A) RNA enrichment was conducted using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), and the sequencing library was constructed by using the NEBNext Ultra II RNA Library Prep Kit (NEB) (P / N: E7765L, NEB, Ipswich, MA) following the manufacturer’s instructions. End repair, A-tailing, and adapter ligation was performed to generate the final cDNA library. The library quality was assessed using a Bioanalyzer and quantified using a qPCR-based method with the KAPA Library Quantification Kit (P / N: KK4873) and the QuantStudio 12K instrument. Prepared libraries were pooled in equimolar ratios, and the resulting pool was subjected to cluster generation using the NovaSeq X Plus System, following the manufacturer's protocols. 150 bp paired-end sequencing was performed on the NovaSeq XP platform targeting 50M reads per sample. Raw sequencing data (FASTQ files) obtained from the NovaSeq XP was subjected to quality control analysis, including read quality assessment. Real Time Analysis Software (RTA) and NovaSeq X Plus Control Software (1.2.2.48004; Illumina) were used for base calling. MultiQC (vl.7; Illumina) was used for data quality assessments. A single instance of RNA sequencing was conducted, with three biological replicates per group. For RNA-seq analysis, nf-core / rnaseq pipeline is adopted59. Trim Galore was used for read trimming. The reads of each sample were aligned to the mouse reference genome (GRCm38) using the STAR alignment tool. Salmon tool was used to quantify transcript expressions. Heatmap and volcano plots are created in R using was pheatmap and ggplot packages, respectively. DESeq2 is used for differentiallsy expressed gene analysis on quantified transcript expressions. log2 fold change values and standard error (lfcSE) were calculated60'62. The p-values attained by the Wald test and adjusted using the Benjamini-Hochberg adjusted(padj). Statistically different results were considered when padj <0.05. KEGG pathway and Gene Ontology (GO) enrichment analysis for differentially expressed genes are conducted using R cluster Profiler package63'65. The sample variance plot may be accessed in the supplemental material (Fig. 9). Transcriptome data were deposited in NCBI’s Gene Expression Omnibus (GEO) and can be accessible through GEO series accession number: GSE280987.

[0115] Publicly available bulk RNA-sequencing FASTQ files were obtained from NCBI BioProject under accession number PRJNA97313166. Transcript quantification was performed204917-4055-5666v.lusing Kallisto 0.50.0 to estimate transcript abundance. A reference transcriptome for Mus musculus was obtained from Ensembl corresponding to GRCm39. Differential expression analysis was conducted using the DESeq2 R package (v.1.44.0). The count data were normalized using the variance stabilizing transformation method provided by DESeq2.

[0116] The mouse single-nucleus RNA sequencing (snRNA-seq) data were obtained from the Gene Expression Omnibus (GEO) under accession number GSE256398. The data from floxed untreated mice were used for analysis. Human liver data were retrieved from published data67from the GEO database with accession GSE263614. Differential expression analysis was performed between pre-reperfusion liver at each time point during normothermic machine perfusion (NMP) and between transplanted and non-transplanted liver at the same NMP time points. The analysis followed the previously published DESeq2 pipeline68. Technical artifacts such as ambient background RNA and empty droplets were removed using the remove-background function in CellBender (v.0.2.0) (fpr = 0.1). The resulting gene expression matrices were imported into R and analyzed using the Seurat package (v.5.1.0). Low-quality cells or outlier cells were filtered using a standard of “nFeatureRNA < 200 or nFeatureRNA > 7500 or nCount_RNA > 60000 or percent.mt > 20”. Data were analyzed using the NormalizeData, FindVariableFeatures (nFeatures = 3000), ScaleData and RunPCA (npcs = 50), RunUMAP, FindNeighbors and FindClusters functions with default parameters otherwise indicated. The main cell types were identified manually by checking the expressions of marker genes as described69. Cellular distributions and cluster identities were visualized using DimPlot. A specific gene expression was visualized using FeaturePlot.

[0117] Generation of NTPDase8 bone marrow chimeric mice: Bone marrow chimeric mice were created using a previously described method48. In summary, 8-10 weeks old WT or Entpd8' ‘ mice were euthanized, and bone marrow cells were collected from the femur and tibia under sterile conditions by flushing with saline solution. The collected bone marrow cells were centrifuged at 400 Xgfor 5 minutes, resuspended, and counted. Bone marrow recipient mice were irradiated with a total dose of 12 Gray from SnAgCu (MultiRad 350, CT, USA) and administered in two fractions. Each recipient mouse received a tail injection of ~107bone marrow cells in 0.2 ml sterile saline solution. The chimeric mice were housed for at least 8 weeks before experimentation, with tetracycline (100 μg / ml) added to their drinking water for the first 2 weeks post-transplantation. These chimeric mice later underwent liver I / R injury as previously described.214917-4055-5666v.l

[0118] Measurement of hepatic injury: To measure hepatic injury, plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were assessed. Plasma samples were collected 6 hours after the sham surgery or liver I / R induction. ALT (MAK052, Sigma, CT, USA) and AST (MAK055, Sigma, CT, USA) levels were measured using commercial kits70. Sections of the liver were cut at 5 μm thickness, stained with the hematoxylin-eosin method, and scanned with a Leica AT2 slide scanner (Leica AT2, USA). Stained sections were scored for I / R injury by assessment of interstitial edema (0-4), sinusoid congestion (0-4), hepatocellular necrosis (0-4), hepatocyte vacuolation (0-4) and leukocyte infiltration (0-4 )71.

[0119] Immunohistochemistry and TUNEL staining: Five μm thickness liver sections were incubated for 30 min at room temperature in 5% bovine serum albumin (BSA) diluted in PBS and then incubated overnight at 4 °C with primary antibody (Polyclonal guinea pig anti-mouse NTPDase8; ectonucleotidases-ab.com) diluted in 1% BSA. Sections were washed twice with PBS-Tween (0.1%) and incubated with 0.3% hydrogen peroxide in PBS for 10 min. Sections were incubated with an Avidin / Biotin blocking kit (Vector Laboratories) for 15 min at room temperature followed by 2 washes in PBS-Tween and then incubated with a biotin-labeled secondary antibody (Biotinylated goat anti-guinea pig IgG, Vector Laboratories, BA-7000), which was diluted 1000 times in PBS, for 1 h at room temperature, after which samples were washed 2 more times. Complex avidin / biotinylated horseradish peroxidase (Vector laboratories) was then added for 30 min at room temperature to optimize the reaction. After washing twice with PBS-Tween, peroxidase activity was revealed with the substrate DAB for 2 to 3 min. After washing with distilled water, tissues were counterstained with aqueous hematoxylin as recommended and mounted14. To assess DNA fragmentation, liver sections were fixed for 10 minutes at 4 °C with 4% paraformaldehyde in 0.1 mol / L PBS. Next, terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL) labeling (in situ cell death detection kit, Roche) was carried out. In brief, sections were stained with streptavidin-FITC following labeling with terminal deoxynucleotidyl transferase mix, which contained 12.5 mg / mL terminal deoxynucleotidyl transferase and 25 mg / mL biotinylated dUTP (Roche). DNA fragmented cells were assessed by tile imaging with a confocal microscope (Zeiss LSM 900, Germany) by counting TUNEL-positive cells in specified arrays of all sections72.

[0120] Western blot analysis: For western blot analysis, liver tissue samples were homogenized, sonicated, and lysed using RIPA buffer (Sigma 20-108, USA) containing a protease 224917-4055-5666v.linhibitor cocktail. Total protein content was determined from each sample by Qubit 4.0 (Invitrogen, Life Technologies, USA). 50 μg of protein were size-fractionated using 4-20% Mini-Protean TGX Stain Free (4568023, Bio-Rad, Life Sciences Research) electrophoresis gel and then transferred to a PVDF membrane (1620174, Bio-Rad, Life Sciences Research). After transfer, membranes were rinsed in TBS-tween and blocked for 1 hour at room temperature with 5% nonfat dry milk in TBS-tween followed by incubating with NTPDase8 polyclonal primary antibody at a 1:800 dilution (Ptlab, 21010-1-AP, USA) for 2 hours at 37 °C. Thereafter, membranes were incubated with horseradish peroxidase-conjugated goat-anti rabbit (ab97051; Abeam, USA) secondary antibody, diluted 1:2000 in the blocking solution for 2 h at 37 °C. The membranes were stripped and then re-probed using an HRP-conjugated anti P-actin antibody (ab20272, Abeam, USA) to ensure constant protein loading. The Clarity Western ECL Substrate Kit (1708280, BioRad, Life Sciences Research) was then used to develop the membranes70.

[0121] RT-qPCR: All reagents utilized in this procedure were sourced from Thermo Fisher -Applied Biosystems, USA. Snap-frozen samples were homogenized using TRIzol reagent, and RNA was extracted following the manufacturer's guidelines. Subsequently, reverse transcription was performed to generate cDNA. The RT-qPCR reactions, utilizing 20-100 ng of cDNA, were conducted on an Applied Biosystems QuantStudio 3 PCR system with Master Mix PowerUp and specific primers. Data analysis was carried out using the 2 − Δ / ΔCT method described previously, with normalization to the appropriate housekeeping gene17.

[0122] Determination of plasma and liver ATP levels: Plasma and liver ATP levels were measured by a fluorometric assay after sample deproteinization according to the manufacturer’s instruction (ab83355, Abeam, USA)70.

[0123] ELISA: TNF-a, IL-6, and MCP-1 in liver were determined using commercially available kits (Duoset R& D Systems, DY410, DY406, DY479 Minneapolis, USA), according to the instructions.

[0124] Statistics: The DESeq2 algorithm was utilized for the study of differentially expressed genes in the RNAseq data processing. The log2 fold change values were obtained, the standard error (IfcSE) was established, and the Wald statistic was used to generate the p-value. The displayed data represents the Benjamini -Hochberg corrected p-values (padj). The volcano graphic illustrates the FDR-corrected p-values. The 2-tailed unpaired Student t test was utilized for234917-4055-5666v.lcomparing two groups. One-way ANOVA was employed to compare three or more groups. The software utilized was GraphPad Prism version 8. Results were deemed statistically significant when the p-value was less than or equal to 0.05.Results

[0125] NTPDases differentially regulate liver I / R injury: Plasma ALT and AST levels were comparable between WT and Entpd2~'~ or Entpd3'~ mice after I / R injury (Figure 1A-D). However, after I / R injury, in Entpd8'' mice, plasma ALT and AST levels were elevated (Figure 1 E-F) compared to WT mice, indicating increased injury. Consequently, in the following experiments, the role of NTPDase8 in regulating liver injury was further analyzed.

[0126] After liver I / R, H& E and TUNEL staining were performed to determine I / R-associated histological changes and apoptosis, respectively in WT and Entpd8''' mice. H& E staining demonstrated that the absence of NTPDase8 exacerbates ER-induced histological alterations (Figure 2A-C). Along with this, NTPDase8 deficiency resulted in increased TUNEL staining, indicating increased apoptosis (Figure 2B-D).

[0127] NTPDase8 deficiency in parenchymal cells exacerbates liver injury: the increased liver injury in Entpd8'' mice was due to a deficiency of NTPDase8 in hematopoietic cells using bone marrow chimeric mice was investigated. WT mice receiving syngeneic EntpdS'1' bone marrow showed no increase in liver injury when compared to WT mice transplanted with WT bone marrow. In addition, Entpd8'' mice receiving Entpd8" bone marrow and Entpd8~~ mice transferred with WT bone marrow both had similarly increased liver injury compared with WT mice transferred with WT bone marrow. These results suggest that NTPDase8 on non-myeloid parenchymal cells protects against liver I / R injury (Figure 3 A and ).

[0128] Role of NTPDase8 in regulating gene expression levels after liver I / R injury: To study possible mechanisms by which NTPDase8 is protective and how this differs from the role of CD39, bulk RNAseq analysis of livers from WT and Entpd8'' mice that had liver I / R injury was performed. Heat map visualization of the data confirmed that NTPDase8 (Figure 4A) expression was highly downregulated in EntpdS'' livers, which was also confirmed in the volcano plot (Figure 4B). The most regulated signaling pathways were identified by Ingenuity Pathway Analysis (Qiagen IPA software) and these pathways included those associated with B lymphocyte signaling, such as macrophage activation, and cytokine signaling (Figure 4C). Of the 50 most differentially244917-4055-5666v.lregulated genes, 6 were downregulated and 44 up-regulated in Entpd^'1' vs. WT livers (Figure 4A and B). Upregulated genes include many members of the immunoglobulin family, checkpoint genes, such as Pdcdl and Ctla4, and the anti-inflammatory cytokine IL10. One notable downregulation was A2m, which is normally expressed at high levels in the liver and is involved in the regulation of inflammation (Figure 4B).

[0129] NTPDase8 expression is downregulated in the ischemic mouse liver: the NTPDase8 gene and protein expression using quantitative real-time PCR, western blot, and 1HC in WT sham and I / R mice was analyzed. It was found that transcripts of NTPDase8 were downregulated in the I / R versus sham liver (Figure 5A). Additionally, NTPDase8 protein expression was suppressed in the ischemic mouse liver (Figure 5B and C).

[0130] It was confirmed that I / R causes NTPDase8 downregulation by analyzing RNAseq data from a public dataset (PRJNA973131) (Figure 5D)66. Moreover, analysis of the collected scRNAseq data from the non-ischemic mouse liver indicated that NTPDase8 is expressed exclusively in hepatocytes (Figure 5E and F).

[0131] Role of NTPDase8 in regulating extracellular ATP levels: Since NTPDase8 degrades ATP, it was hypothesized that Entpd8- / - mice would have increased ATP levels. First, the effect of liver I / R on ATP levels in both plasma and tissues was determined and it was noted that ATP was increased in plasma. In line with the hypothesis, it was further found that Entpd8- / - mice have increased ATP levels in both the liver and plasma compared to those in WT mice (Figure 6A and B).

[0132] P2 receptor blockade rescues the increased liver injury of Entpd1' mice: To study the role of ATP signaling in mediating the increased injury of Entpd8''' mice, it was then investigated whether blocking P2 receptors could mitigate liver injury in these mice. It was observed that the non-selective P2 receptor antagonist suramin decreased ALT and AST levels in both Entpd8- / -and WT mice, indicating that ATP signaling through P2 receptors contributes to injury in both Entpd8- / - and WT mice (Figure 7A and B).

[0133] NTPDase8 deficiency fails to affect the host’s inflammatory response to liver injury: To investigate the function of NTPDase8 in inflammatory responses, the cytokine responses of WT and Entpd8 ~ mice to liver injury were examined. No significant differences were observed in liver cytokine levels (Fig. 10 A-C.254917-4055-5666v.l

[0134] NTPDase8 deficiency exacerbates liver injury after T / HS: Finally, the role of NTPDase8 in another ischemia-reperfusion model was tested, where I / R was induced by trauma hemorrhagic shock and resuscitation. In this model Entpd8 ~ mice had increased liver injury compared to EntpdS mice, confirming the protective effect of NTPDase8 in liver I / R (Figure 8A andB).Discussion

[0135] Disclosed herein is the expression and function of members of the extracellular NTPDase family within the context of liver I / R injury. The results demonstrated a protective role of NTPDase8, which was comparable to that seen for Cd39 / NTPDasel, as NTPDase2 and -3 did not appear to regulate liver I / R injury.

[0136] NTPDase2 is expressed in multiple tissues, including the liver, heart, and vascular adventitia. In the liver, the role of NTPDase2 has been studied in several chronic and acute injury models. After chronic CCI4 intoxication, Entpd2' / ' mice exhibit more severe liver fibrosis than WT mice26. In addition, NTPDase2 protected against acetaminophen-induced acute liver injury73. NTPDase2 is expressed primarily on periportal fibroblasts within the liver, which is different from that of NTPDase8. In addition, while NTPDase2 causes a more sustained elevation of ADP accumulation than NTPDase8 in the presence of extracellular ATP, these enzymes can differentially modulate ATP vs ADP-mediated P2 purinergic signaling. Thus, the differential expression and function of NTPDase2 and 8 may underpin altered roles in liver I / R injury and possibly in other liver injury models.

[0137] Studies have shown NTPDase3 is expressed in pancreatic islet, central nervous system neurons, peripheral nerves, and various epithelial cells in the digestive, reproductive, renal, and respiratory systems74'76. In the pancreas, relative NTPDase3 expression is found to be much lower than that of NTPDase8 in the liver11,14, which may explain why NTPDase3 failed to affect the host's response to liver I / R injury in these studies.

[0138] As indicated above, NTPDase8 modulates the concentration of endogenous purinergic agonists at the cell surface as it degrades the generally proinflammatory and injurious ATP and ADP77. It was tested whether in the absence of NTPDase8, extracellular ATP concentrations would increase, which would in turn aggravate parenchymal injury through increased P2 receptor signaling.264917-4055-5666v.l

[0139] Elevated ATP levels in the extracellular space (plasma) and P2 receptor blockade ameliorated liver injury in NTPDase8 deficient mice subjected to liver ER were detected. However, other mechanisms may also be involved in the protective role of NTPDase8, inclusive of adenosinergic responses.

[0140] The liver is thought to be the main source of purines for tissues unable to synthesize purines de novo, and the presence of NTPDase8 in the canalicular portion of hepatocytes in parallel with ecto-5 '-nucleotidases and nucleoside transporters supports roles for NTPDase8 in nucleoside salvage in the liver7879. Thus, NTPDase8 may suppress liver injury by salvaging purines and therefore promoting the energetically beneficial tissue environment. This is in line with the finding that liver I / R injury causes decreased intracellular ATP levels80, leading to a potentially energetically catastrophic situation and cell death4.

[0141] It was shown that NTPDase8 expression is significantly reduced in leukocytes isolated from septic patients and NTPDase8 expression is also decreased in livers of septic vs. control mice14,17. Moreover, cytokine treatment of hepatocytes suppresses NTPDase8 expression of hepatocytes in vitro14’7. Thus, it appears that various types of stressful stimuli down-regulate the expression of NTPDase8. While the scRNAseq data indicate that at least in non-ischemic liver, NTPDase8 is exclusively present in hepatocytes, the cellular and molecular mechanisms of NTPDase8 down-regulation after I / R have yet to be studied.

[0142] In conclusion, these studies demonstrate that NTPDase8 is important in the degrading extracellular ATP in the liver and that by this action, NTPDase8 protects against severe liver injury following I / R injury by limiting deleterious P2 receptor signaling.

[0143] The foregoing merely illustrates the principles of the disclosure. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. It will thus be appreciated that those skilled in the art will be able to devise numerous systems, arrangements, and procedures which, although not explicitly shown or described herein, embody the principles of the disclosure and can be thus within the spirit and scope of the disclosure. Various different exemplary embodiments can be used together with one another, as well as interchangeably therewith, as should be understood by those having ordinary skill in the art. In addition, certain terms used in the present disclosure, including the specification, drawings and claims thereof, can be used synonymously in certain instances, including, but not274917-4055-5666v.llimited to, for example, data and information. It should be understood that, while these words, and / or other words that can be synonymous to one another, can be used synonymously herein, that there can be instances when such words can be intended to not be used synonymously. Further, to the extent that the prior art knowledge has not been explicitly incorporated by reference herein above, it is explicitly incorporated herein in its entirety. All publications referenced are incorporated herein by reference in their entireties.REFERENCES1. Walsh KB, Toledo AH, Rivera-Chavez FA, Lopez-Neblina F, Toledo-Pereyra LH.Inflammatory mediators of liver ischemia-reperfusion injury. Exp Clin Transplant. Jun 2009;7(2):78-93.2. Pacher P, Hasko G. Endocannabinoids and cannabinoid receptors in ischaemiareperfusion injury and preconditioning. Br J Pharmacol. Jan 2008;153(2):252-62.doi: 10.1038 / sj.bjp.07075823. Cao Z, Mulvihill MM, Mukhopadhyay P, et al. Monoacylglycerol lipase controls endocannabinoid and eicosanoid signaling and hepatic injury in mice. Gastroenterology. Apr 2013; 144(4):808-817 el5. doi: 10.1053 / j.gastro.2012.12.0284. Sun X, Imai M, Nowak-Machen M, et al. Liver damage and systemic inflammatory responses are exacerbated by the genetic deletion of CD39 in total hepatic ischemia. Purinergic Signal. Dec 2011;7(4):427-34. doi: 10.1007 / sl 1302-011-9239-6.5. Giuliani AL, Sarti AC, Di Virgilio F. Extracellular nucleotides and nucleosides as signalling molecules. Immunol Lett. Jan 2019;205:16-24. doi:10.1016 / j.imlet.2018.11.006.6. Mizumoto N, Kumamoto T, Robson SC, et al. CD39 is the dominant Langerhans cell associated ecto-NTPDase:: Modulatory roles in inflammation and immune responsiveness. Nat Med. Apr 2002;8(4):358-365. doi: DOI 10.1038 / nm0402-358.7. Robson SC, Sevigny J, Zimmermann H. The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance. Purinergic Signal. Jun 2006;2(2):409-30. doi: 10.1007 / sl 1302-006-9003-5.284917-4055-5666v.l8. Yegutkin GG. Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade. Bba-Mol Cell Res. May 2008;1783(5):673-694. doi:10.1016 / j.bbamcr.2008.01.024.9. Kaczmarek E, Koziak K, Sevigny J, et al. Identification and characterization of CD39 / vascular ATP diphosphohydrolase. J Biol Chem. Dec 20 1996;271(51):33116-22. doi:10.1074 / jbc.271.51.3311610. Sevigny J, Sundberg C, Braun N, et al. Differential catalytic properties and vascular topography of murine nucleoside triphosphate diphosphohydrolase 1 (NTPDasel) and NTPDase2 have implications for thromboregulation. Blood. Apr 152002;99(8):2801-2809. doi: DOI 10.1182 / blood. V99.8.280111. Fausther M, Lecka J, Kukulski F, et al. Cloning, purification, and identification of the liver canalicular ecto-ATPase as NTPDase8. Am J Physiol Gastrointest Liver Physiol. Mar 2007;292(3): G785-95. doi: 10.1152 / ajpgi.00293.200612. Belcher SM, Zsamovszky A, Crawford PA, Hemani H, Spurting L, Kirley TL.Immunolocalization of ecto-nucleoside triphosphate diphosphohydrolase 3 in rat brain:Implications for modulation of multiple homeostatic systems including feeding and sleep-wake behaviors. Neuroscience. 2006; 137(4): 1331-1346. doi: 10.1016 / j. neuroscience.2005.08.086 13. Haas CB, Lovaszi M, Braganhol E, Pacher P, Hasko G. Ectonucleotidases in Inflammation, Immunity, and Cancer. J Immunol. May 1 2021;206(9): 1983-1990.doi:10.4049 / jimmunol.200134214. Haas CB, Lovaszi M, Pacher P, et al. Extracellular ectonucleotidases are differentially regulated in murine tissues and human polymorphonuclear leukocytes during sepsis and inflammation. Purinergic Signal. Dec 2021;17(4):713-724. doi:10.1007 / sl 1302-021-09819-1 15. Bigonnesse F, Levesque SA, Kukulski F, et al. Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-8. Biochemistry-Us. May 11 2004;43(18):5511-5519. doi:10.1021 / bi0362222294917-4055-5666v.l16. Fausther M, Lecka J, Kukulski F, et al. Cloning, purification, and identification of the liver canalicular ecto-ATPase as NTPDase8. Am J Physiol-Gastr L. Mar 2007;292(3): G785-G795. doi: 10.1152 / ajpgi.00293.200617. Lovaszi M, Nemeth ZH, Kelestemur T, et al. Evaluation of Components of the Extracellular Purinergic Signaling System in Human Sepsis. Shock. Apr 1 2024;61(4):527-540. doi: 10.1097 / SHK.000000000000223018. Chadwick BP, Frischauf AM. The CD39-like gene family: identification of three new human members (CD39L2, CD39L3, and CD39L4), their murine homologues, and a member of the gene family from Drosophila melanogaster. Genomics. Jun 15 1998;50(3):357-67. doi: 10.1006 / geno.1998.531719. Wang TF, Rosenberg PA, Guidotti G. Characterization of brain ecto-apyrase: evidence for only one ecto-apyrase (CD39) gene. Brain Res Mol Brain Res. Jul 1997;47(l-2):295-302. doi: 10.1016 / sO 169-328x(97)00066- 120. Lemmens R, Vanduffel L, Kittel A, Beaudoin AR, Benrezzak O, Sevigny J. Distribution, cloning, and characterization of porcine nucleoside triphosphate diphosphohydrolase-1. Eur J Biochem. Jul 2000;267(13):4106-14. doi:10.1046 / j.1432-1327.2000.01462.x21. Mateo J, Harden TK, Boyer JL. Functional expression of a cDNA encoding a human ecto-ATPase. Br J Pharmacol. Sep 1999;128(2):396-402. doi: 10.1038 / sj.bjp.070280522. Smith TM, Kirley TL. Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPases and CD39 ecto-apyrasesl. Comparative Study Research Support, Non-U. S. Gov't. Biochimica et biophysica acta. Jul 28 1998; 1386(l):65-78.23. Vorhoff T, Zimmermann H, Pelletier J, Sevigny J, Braun N. Cloning and characterization of the ecto-nucleotidase NTPDase3 from rat brain: Predicted secondary structure and relation to other members of the E-NTPDase family and actin. Purinergic Signal. Sep 2005;l(3):259-70. doi: 10.1007 / s 11302-005-6314-x24. Lavoie EG, Kukulski F, Levesque SA, Lecka J, Sevigny J. Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3. Biochem Pharmacol. May 15 2004;67(10):1917-26. doi:10.1016 / j.bcp.2004.02.012304917-4055-5666v.l25. Bigonnesse F, Levesque SA, Kukulski F, et al. Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-8. Biochemistry-Us. May 11 2004;43(18):5511-9. doi:10.1021 / bi036222226. Feldbrugge L, Jiang ZG, Csizmadia E, et al. Distinct roles of ecto-nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) in liver regeneration and fibrosis. Purinergic Signal. Mar 2018;14(1):37-46. doi: 10.1007 / sl 1302-017-9590-327. Sandhu B, Perez-Matos MC, Tran S, et al. Global deletion of NTPDase3 protects against diet-induced obesity by increasing basal energy metabolism. Metabolism. May2021;118: 154731. doi: 10.1016 / j.metabol.2021.15473128. Lovaszi M, Branco Haas C, Antonioli L, Pacher P, Hasko G. The role of P2Y receptors in regulating immunity and metabolism. Biochem Pharmacol. May 2021;187: 114419. doi: 10.1016 / j.bcp.2021.11441929. Antonioli L, Blandizzi C, Pacher P, Hasko G. The Purinergic System as a Pharmacological Target for the Treatment of Immune-Mediated Inflammatory Diseases.Pharmacol Rev. Jul 2019;71 (3):345-382. doi: 10.1124 / pr.117.01487830. Di Virgilio F, Ferrari D, Falzoni S, et al. P2 purinoceptors in the immune system.Research Support, Non-U. S. Gov't Review. Ciba Found Symp. 1996;198:290-302; discussion 302-5.31. Hasko G, Szabo C, Nemeth ZH, Kvetan V, Pastores SM, Vizi ES. Adenosine receptor agonists differentially regulate IL-10, TNF-alpha, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J Immunol. Nov 15 1996;157(10):4634-40.32. Hasko G, Kuhel DG, Chen JF, et al. Adenosine inhibits IL-12 and TNF-[alpha] production via adenosine A2a receptor-dependent and independent mechanisms. Faseb J. Oct 2000;14(13):2065-74.33. Csoka B, Himer L, Selmeczy Z, et al. Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function. FASEB J. Oct 2008;22(10):3491-9. doi:fj.08-107458 [pii] 10.1096 / fj.08-107458314917-4055-5666v.l34. Csoka B, Nemeth ZH, Virag L, et al. A2A adenosine receptors and C / EBPbeta are crucially required for IL- 10 production by macrophages exposed to Escherichia coli. Blood. Oct 1 2007; 110(7):2685-95. doi:blood-2007-01-065870 [pii] 10.1182 / blood-2007-01-065870 35. Csoka B, Selmeczy Z, Koscso B, et al. Adenosine promotes alternative macrophage activation via A2A and A2B receptors. Comparative Study FASEB journal: official publication of the Federation of American Societies for Experimental Biology. Jan 2012;26(1):376-86. doi: 10.1096 / fj.11-19093436. Nemeth ZH, Csoka B, Wilmanski J, et al. Adenosine A2A receptor inactivation increases survival in polymicrobial sepsis. J Immunol. May 1 2006;176(9):5616-26. doi: 176 / 9 / 5616 [pii] 37. Csoka B, Koscso B, Toro G, et al. A2B Adenosine Receptors Prevent Insulin Resistance by Inhibiting Adipose Tissue Inflammation via Maintaining Alternative Macrophage Activation. Diabetes. Mar 2014;63(3):850-66. doi: 10.2337 / db 13-057338. Csoka B, Nemeth ZH, Rosenberger P, et al. A2B adenosine receptors protect against sepsis-induced mortality by dampening excessive inflammation. J Immunol. Jul 1 2010;185(l):542-50. doi:jimmunol.0901295 [pii] 10.4049 / jimmunol.090129539. Koscso B, Csoka B, Selmeczy Z, et al. Adenosine augments IL-10 production by microglial cells through an A2B adenosine receptor-mediated process. Research Support, N. I. H., Extramural Research Support, N. I. H., Intramural Research Support, U. S. Gov't, Non-P. H. S. Journal of immunology. Jan 1 2012; 188(l):445-53. doi: 10.4049 / jimmunol.110122440. Hasko G, Kuhel DG, Salzman AL, Szabo C. ATP suppression of interleukin- 12 and tumour necrosis factor-alpha release from macrophages. Br J Pharmacol. Mar 2000;129(5):909-14. doi:10.1038 / sj.bjp.070313441. Hasko G, Szabo C. IL-12 as a therapeutic target for pharmacological modulation in immune-mediated and inflammatory diseases: regulation of T helper 1 / T helper 2 responses. Br J Pharmacol. Jul 1999; 127(6): 1295-304. doi:10.1038 / sj.bjp.070268942. Antonioli L, Blandizzi C, Csoka B, Pacher P, Hasko G. Adenosine signalling in diabetes mellitus-pathophysiology and therapeutic considerations. Nat Rev Endocrinol. Apr 2015;11(4):228-241. doi:10.1038 / nrendo.2015.10324917-4055-5666v.l43. Antonioli L, Blandizzi C, Fornai M, Pacher P, Lee HT, Hasko G. P2X4 receptors, immunity, and sepsis. Curr Opin Pharmacol. Aug 2019;47:65-74.doi: 10.1016 / j. coph.2019.02.01144. Antonioli L, Blandizzi C, Pacher P, Hasko G. Immunity, inflammation and cancer: a leading role for adenosine. Research Support, N. I. H., Extramural Research Support, N. I. H., Intramural Research Support, Non-U. S. Gov't Review. Nature reviews Cancer. Dec2013; 13(12):842-57. doi:10.1038 / nrc361345. Antonioli L, Pacher P, Vizi ES, Hasko G. CD39 and CD73 in immunity and inflammation. Research Support, N. I. H., Extramural Research Support, U. S. Gov't, Non-P. H. S. Trends Mol Med. Jun 2013;19(6):355-67. doi:10.1016 / j.molmed.2013.03.00546. Csoka B, Nemeth ZH, Toro G, et al. Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing. FASEB J. Sep 2015;29(9):3626-37. doi:10.1096 / fj.15-27245047. Hasko G, Csoka B, Koscso B, et al. Ecto-5'-nucleotidase (CD73) decreases mortality and organ injury in sepsis. J Immunol. Oct 152011;187(8):4256-67. doi:jimmunol.1003379 [pii]10.4049 / jimmunol.100337948. Csoka B, Nemeth ZH, Toro G, et al. CD39 improves survival in microbial sepsis by attenuating systemic inflammation. FASEB J. Jan 2015;29(1):25-36. doi: 10.1096 / fj.14-253567 49. Gonzales E, Julien B, Serriere-Lanneau V, et al. ATP release after partial hepatectomy regulates liver regeneration in the rat. J Hepatol. Jan 2010;52(1):54-62.doi: 10.1016 / j.j hep.2009.10.00550. Schlosser SF, Burgstahler AD, Nathanson MH. Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides. Proc Natl Acad Sci U S A. Sep 3 1996;93(18):9948-53. doi: 10.1073 / pnas.93.18.994851. Nathanson MH, Burgstahler AD, Masyuk A, Larusso NF. Stimulation of ATP secretion in the liver by therapeutic bile acids. Biochem J. Aug 152001;358(Pt 1): 1-5. doi:10.1042 / 0264-6021:3580001334917-4055-5666v.l52. Hart ML, Gorzolla IC, Schittenhelm J, Robson SC, Eltzschig HK. SP1-dependent induction of CD39 facilitates hepatic ischemic preconditioning. J Immunol. Apr 12011;184(7):4017-24. doi:jimmunol.0901851 [pii]10.4049 / jimmunol.090185153. Dranoff JA, Kruglov EA, Robson SC, Braun N, Zimmermann H, Sevigny J. The ectonucleoside triphosphate diphosphohydrolase NTPDase2 / CD39Ll is expressed in a novel functional compartment within the liver. Hepatology. Nov 2002;36(5):1135-44.doi: 10.1053 / jhep.2002.3682354. Vandenbeuch A, Anderson CB, Parnes J, et al. Role of the ectonucleotidase NTPDase2 in taste bud function. Proc Natl Acad Sci U S A. Sep 3 2013;110(36):14789-94.doi: 10.1073 / pnas.130946811055. Feldbrugge L, Moss AC, Yee EU, et al. Expression of Ecto-nucleoside Triphosphate Diphosphohydrolases-2 and -3 in the Enteric Nervous System Affects Inflammation in Experimental Colitis and Crohn's Disease. J Crohns Colitis. Sep 1 2017; 11(9): 1113-1123. doi: 10.1093 / ecco-j cc / jj x05856. Yazdani HO, Tohme S. Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury. J Vis Exp. Aug 302019;(150)doi: 10.3791 / 5974857. Pacher P, Vaslin A, Benko R, et al. A new, potent poly(ADP -ribose) polymerase inhibitor improves cardiac and vascular dysfunction associated with advanced aging. J Pharmacol Exp Ther. Nov 2004;311(2):485-91. doi: 10.1124 / jpet.104.06965858. Kelestemur T, Nemeth ZH, Pacher P, Antonioli L, Hasko G. A 2a Adenosine Receptors Regulate Multiple Organ Failure after Hemorrhagic Shock in Mice. Shock. Oct 1 2022;58(4):321-331. doi:10.1097 / SHK.000000000000198559. Ewels PA, Peltzer A, Fillinger S, et al. The nf-core framework for community-curated bioinformatics pipelines. Nat Biotechnol. Mar 2020;38(3):276-278. doi:10.1038 / s41587-020-0439-x60. Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. Jan 1 2013;29( 1): 15-21. doi:10.1093 / bioinformatics / bts635344917-4055-5666v.l61. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. doi:10.1186 / sl3059-014-0550-8 62. Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. Apr 2017; 14(4):417-419. doi:10.1038 / nmeth.419763. Ashbumer M, Ball CA, Blake JA, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. May 2000;25(l):25-9. doi: 10.1038 / 75556 64. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. Jan 1 2000;28(l):27-30. doi:10.1093 / nar / 28.1.2765. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. May 2012;16(5):284-7. doi:10.1089 / omi.2011.0118 66. Huang K, Wang C, Mei B, et al. Bile acids attenuate hepatic inflammation during ischemia / reperfusion injury. JHEP Rep. Aug 2024;6(8):101101. doi:10.1016 / j.jhepr.2024.10110167. Hautz T, Hackl H, Gottschling H, et al. Transcriptomic signatures during normothermic liver machine perfusion correspond with graft quality and predict the early graft function.EBioMedicine. Oct 2024; 108: 105330. doi: 10.1016 / j.ebiom.2024.105330354917-4055-5666v.l

Claims

CLAIMSWhat is claimed is:

1. A method of treating or reducing ischemia-reperfusion injury in a subject comprising administering NTPDase8 effective to be delivered to the subject’s liver in an amount effective to reduce or treat ischemia-reperfusion injury.

2. A method of treating or reducing ischemia-reperfusion injury in liver for transplant into a subject comprising administering NTPDase8 to the liver in an amount effective to reduce or treat ischemia-reperfusion injury.

3. The method of claim 1, wherein the NTPDase8 is administered prior to ischemia in the subject.

4. The method of claim 1, wherein the NTPDase8 is administered after ischemia, and prior to reperfusion in the subject.

5. The method of claim 1, wherein the NTPDase8 is administered after reperfusion.

6. The method of claim 1, wherein the NTPDase8 is administered via a hepatic portal vein or a hepatic artery of the subject.

7. The method of any of claims 1 or 3-6, wherein the ischemia-reperfusion injury is in a liver of the subject.

8. The method of any of claims 1-7, wherein the NTPDase8 is administered as a composition comprising a soluble NTPDase8.

9. The method of claim 1, wherein a composition comprising an mRNA encoding a full length NTPDase8, so as to permit expression thereof, is administered to the subject.

10. The method of claim 9, where the composition comprises lipid nanoparticles.

11. The method of claim 10, wherein the lipid nanoparticles comprise GalNac-lipid nanoparticles.364917-4055-5666v.l12. The method of any of claims 1-11, wherein the NTPDase8 comprises the sequence: MGLSRKEQVFLALLGASGVSGLTALILLLVEATSVLLPTDIKFGIVFDAGSSHTSL FLYQWLANKENGTGVVSQALACQVEGPGISSYTSNAAQAGESLQGCLEEALVLI PEAQHRKTPTFLGATAGMRLLSRKNSSQARDIFAAVTQVLGRSPVDFWGAELLA GQAEGAFGWITVNYGLGTLVKYSFTGEWIQPPEEMLVGALDMGGASTQITFVPG GPILDKSTQADFRLYGSDYSVYTHSYLCFGRDQMLSRLLVGLVQSRPAALLRHP CYLSGYQTTLALGPLYESPCVHATPPLSLPQNLTVEGTGNPGACVSAIRELFNFSS CQGQEDCAFDGVYQPPLRGQFYAFSNFYYTFHFLNLTSRQPLSTVNATIWEFCQR PWKLVEASYPGQDRWLRDYCASGLYILTLLHEGYGFSEETWPSLEFRKQAGGVD IGWTLGYMLNLTGMIPADAPAQWRAESYGVWVAKVVFMVLALVAVVGAALV QLFWLQD (SEQ ID NO: 1).

13. The method of any of claims 1-11, wherein the NTPDase8 comprises the sequence set forth in AY430414 or AAR04374.

14. A method of improving liver surgery comprising transplanting a liver that has been pretreated with exogenous NTPDase8, wherein the NTPDase8 is effective to reduce or treat ischemia-reperfusion injury, into a subject in need of the transplant.

15. The method of claim 14, further comprising prior to surgery administering NTPDase8 to the liver in an amount effective to reduce or treat ischemia-reperfusion injury.

16. A method of reducing or treating ischemia-reperfusion injury in a subject comprising administering NTPDase8 to the liver of the subject, wherein the NTPDase8 is effective to prevent or reduce ischemia-reperfusion injury.

17. Exogenous NTPDase8 for use in reducing liver damage in a subject, wherein the subject has or is likely to undergo liver ischemia-reperfusion injury.

18. A method of treating or reducing a sterile inflammatory condition in a subject comprising administering NTPDase8 effective to treat or reduce a sterile inflammatory condition in a subject.374917-4055-5666v.l19. The method of Claim 18, wherein the sterile inflammatory condition is in at least the liver of the subject.

20. The method of any of claims 1-19, wherein the subject is human.384917-4055-5666v.l

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