WO2025030108A2 - Compositions de mitsugumine-53 humaine recombinante et leurs procédés d'utilisation - Google Patents

Compositions de mitsugumine-53 humaine recombinante et leurs procédés d'utilisation Download PDF

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WO2025030108A2
WO2025030108A2 PCT/US2024/040744 US2024040744W WO2025030108A2 WO 2025030108 A2 WO2025030108 A2 WO 2025030108A2 US 2024040744 W US2024040744 W US 2024040744W WO 2025030108 A2 WO2025030108 A2 WO 2025030108A2
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rhmg53
lung
injury
transplantation
iri
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WO2025030108A9 (fr
WO2025030108A3 (fr
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Bryan Whitson
Sylvester BLACK
Hua Zhu
Yonggyu Lee
Jung Lye Kim
Doug GOUCHOE
Jianjie Ma
Zhentao ZHANG
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Ohio State Innovation Foundation
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Ohio State Innovation Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to compositions and methods comprising recombinant human Mitsugumin-53 (rhMG53) and uses thereof.
  • IRI ischemia reperfusion injury
  • PWD primary graft dysfunction
  • the endothelial cells are the barrier between the donor graft and the recipient's blood.
  • the IRI leads to injury to the donor graft with the endothelial cells being initially susceptible with injury leading to cell death through apoptosis, necroptosis, and pyroptosis. This initial injury allows for increased injury signaling which promotes recipient leukocyte migration into the donor graft leading to an increased IRI response.
  • composition comprising recombinant human Mitsugumin-53 (rhMG53) protein and its use thereof in treating ischemia reperfusion injury (IRI) and/or primary graft dysfunction (PGD).
  • IRI ischemia reperfusion injury
  • PWD primary graft dysfunction
  • a perfusate composition comprising recombinant human Mitsugumin-53 (rhMG53) protein and a perfusion medium.
  • rhMG53 recombinant human Mitsugumin-53
  • the rhMG53 comprises SEQ ID NO: 1.
  • a preservation solution comprising recombinant human Mitsugumin-53 (rhMG53) protein and a preservation medium.
  • rhMG53 recombinant human Mitsugumin-53
  • the rhMG53 comprises SEQ ID NO: 1.
  • ischemia reperfusion injury IRI
  • rhMG53 recombinant human Mitsugumin-53
  • the rhMG53 protein is administered in a perfusion medium. In some embodiments, the rhMG53 protein is administered in a preservation medium.
  • the rhMG53 comprises SEQ ID NO: 1.
  • the rhMG53 protein is administered to the ex vivo graft before reperfusion of the ex vivo graft. In some embodiments, the rhMG53 protein is administered to the ex vivo graft during reperfusion of the ex vivo graft. In some embodiments, the rhMG53 protein is administered to the ex vivo graft for at least 1 hour.
  • the ex vivo graft is an allograft or a xenograft. In some embodiments, ex vivo graft is a lung, liver, heart, kidney, or intestine graft.
  • a method of treating a subject with primary graft dysfunction comprising administering a recombinant human Mitsugumin-53 (rhMG53) protein to the subject.
  • the rhMG53 protein is administered in a perfusion medium.
  • the rhMG53 comprises SEQ ID NO: 1.
  • the rhMG53 protein is administered is administered intravenously. In some embodiments, the rhMG53 protein is administered subcutaneously.
  • the rhMG53 protein is administered before transplantation of an ex vivo graft. In some embodiments, the rhMG53 protein is administered during transplantation of an ex vivo graft. In some embodiments, the rhMG53 protein is administered after transplantation of an ex vivo graft. In some embodiments, the rhMG53 protein is administered to a donor before recovery. In some embodiments, the rhMG53 protein is administered to a donor during recovery.
  • the ex vivo graft is an allograft or a xenograft. In some embodiments, ex vivo graft is a lung, liver, heart, kidney, or intestine graft.
  • the subject is a human.
  • FIGS. 1A-1B show a method schematic (FIG. 1A) and a computed tomography scan (FIG. IB) of successful mouse lung transplant showing cuffed anastomoses.
  • FIGS. 2A-2D show that bronchoalveolar lavage (BAL) was collected after C57BL/6J mice underwent 1 hour of warm ischemia before lung transplantation and subsequent 5-day survival.
  • Cell count (FIG. 2A), protein levels (FIG. 2B), LDH (FIG. 2C), and HA (FIG. 2D) were measured in the BAL.
  • Significance is denoted by P ⁇ 05.
  • LDH Lactate dehydrogenase
  • HA hyaluronic acid
  • WT wild type
  • KO knockout
  • tPA tissue plasminogen activator.
  • FIGS. 3A-3C show that tissue homogenates were collected after C57BL/6J mice underwent 1 hour of warm ischemia before lung transplantation and subsequent 5-day survival.
  • FIG. 3A shows that when tissues were lysed, ET-1 expression showed no significant difference across all groups.
  • FIGS. 3B and 3C show that when assessing ET-1 and Big ET-1 levels in the BAL, WT- KO had significantly greater levels of ET-1 (FIG. 3B) and Big ET-1 (FIG. 3C) when compared with the other groups.
  • Significance is denoted by P ⁇ 05.
  • ET-1 Endothelin-1
  • BAL bronchoalveolar lavage
  • WT-KO wild-type knockout
  • tPA tissue plasminogen activator.
  • FIGS. 4A-4C show representative hematoxylin and eosin-stained lung sections collected after WI period, subsequent transplant, and survival indicate disrupted architecture and peri bronchial thickening in as shown in FIG. 4A.
  • FIG. 4B shows terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining detects DNA breaks as it occurs in the last phase of apoptosis, there was increased staining in KO recipients compared with WT and tPA recipients.
  • DAPI-stained nuclei are shown in FIG. 4C.
  • WI Warm ischemia
  • KO knockout
  • WT wild type
  • tPA tissue plasminogen activator.
  • FIGS. 5 A-5B show MG53 level in BAL after WI period, subsequent transplant and survival revealed significant increase in WT-tPA compared with WT-KO as shown in FIG. 5A.
  • FIG. 5B shows that when tissue was collected and subsequently lysed, densiometric analysis revealed a significant increase in MG53 levels in tPA recipients compared with all other groups. Significance is denoted by P ⁇ .05. MG53, Mitsugumin-53; BAL, bronchoalveolar lavage; WI, warm ischemia; WT-tPA, wild type-tissue plasminogen activator; WT-KO, wild-type knockout.
  • FIGS. 6A-6B show representative MG53 and DAPI fluorescent staining of lung sections collected following lung transplantation in FIG. 6A.
  • FIG. 6B shows MG53 staining and DAPI staining. Quantification of MG53 in lung tissue showed a significantly greater amount of MG53 in tPA-MG53 mice compared with the mg53-/- mice. Significance is denoted by P ⁇ .05. MG53, Mitsugumin-53.
  • FIG. 7 shows an experimental plan detailing transplantation detail, wt background was used for all donors.
  • Recipients consisted of (1) wt., (2) mg53- or (3) tPA-MG53.
  • MV Wild type; MG53, mitsugumin-53; tPA, tissue plasminogen activator.
  • FIGS. 8A-8C shows that following transplantation and survival, tissue was lysed and evaluated for pro-caspase- 1 and caspase- 1.
  • FIG. 8 A shows western blotting showed more pro- caspase-1 in tPA-MG53 recipients when compared with the other transplant groups.
  • FIG. 8B shows that densiometric analysis revealed significantly greater levels of procaspase-1 in tPA-MG53 recipients as well; however, this was not true for caspase-1 as shown in FIG. 8C.
  • tPA Tissue plasminogen activator
  • MG53 mitsugumin-53
  • WT wild type
  • KO knockout.
  • FIGS. 9A-9C show that following transplantation and survival, BAL was evaluated for TNF-a and IL-lb, whereas tissue was lysed and evaluated for pro-IL-lb. There was no significant difference in TNF-a (FIG. 9A) or IL-lb (FIG. 9B) levels across transplant groups.
  • FIG. 9C shows that when tissues were lysed, and pro-IL-lb levels were measured, there were no significant differences among groups.
  • BAL Bronchoalveolar lavage; TNF-a, tumor necrosis factor-a; IL-lb, interleukin-b; WT, wild type; KO, knockout; tPA, tissue plasminogen activator.
  • FIG. 10 shows that following transplantation and survival, tissue was lysed and evaluated for full-length gasdermin-D and cleaved gasdermin-D.
  • Western blotting as confirmed by densiometric analysis, revealed no significant differences among transplant groups.
  • GSDMD Gasdermin-D
  • WT wild type
  • KO knockout
  • tPA tissue plasminogen activator.
  • FIGS. 11 A-l 1C show injury marker endothelin-1 (ET-1) assessment with in vitro porcine pulmonary artery endothelial cell (PPAEC) cultures. Untreated is vehicle treated and BSA is a protein control.
  • FIG. 11A shows that in supernatant from PPAECs, H/R increased ET-1 release and pre-treatment with 50 pg/mL rhMG53 or post-treatment with 10 or 50 pg/mL rhMG53 mitigated the increase.
  • FIG. 1 IB shows western blot analysis of PPAECs showed similar increases of ET-1 with H/R and decreases with rhMG53 treatment.
  • FIG. 11C shows rapid uptake of fluorescent conjugated Alexa647-rhMG53 into the cytosol.
  • FIGS. 12A-12F show assessment of therapeutic benefit of rhMG53 during rat transplantation model.
  • FIG. 12A shows experimental overview.
  • FIG. 12E shows representative Western blots from tissue.
  • FIGS. 13A-13H show assessment of lung injury indicators in perfusate, and physiological measurements taken during and after ex vivo lung perfusion.
  • LDH Lactate dehydrogenase
  • FIGS. 14A-14C show assessment of morphological and apoptotic changes after EVLP.
  • FIG. 14A shows representative hematoxylin and eosin-stained lung sections collected after the 2h EVLP period indicate disrupted architecture and peribronchial thickening in lungs that underwent Ih of warm ischemia (WI) prior to perfusion.
  • WI warm ischemia
  • lungs that underwent Ih WI and were administered rhMG53 during the perfusion period showed more normal architecture and morphology that was similar to the perfused lungs that had experienced no WI.
  • FIG. 14A shows representative hematoxylin and eosin-stained lung sections collected after the 2h EVLP period indicate disrupted architecture and peribronchial thickening in lungs that underwent Ih of warm ischemia (WI) prior to perfusion.
  • WI warm ischemia
  • TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
  • FIGS. 15A-15C shows assessment of MG53 levels in lung tissue and perfusate during EVLP.
  • FIG. 15A shows Fluorescent microscopy and
  • FIG. 15B shows quantitative analysis of fluorescent micrographs indicates an increase in MG53 in tissue of lungs that underwent Ih warm ischemia (WI) prior to perfusion with rhMG53 (Ih WI vs. Ih WI + rhMG53 P .009).
  • WI Ih warm ischemia
  • C) This increase in tissue MG53 is followed by a decrease of MG53 in the perfusate shows that the exogenous rhMG53 is being used by the tissue during the perfusion period (Ih WI vs.
  • Ih WI + rhMG53 at 30-minutes PO.OOOl, 60-minutes PO.OOOl, 120-minutes PO.OOOl).
  • # significant difference between Oh andlh WI + rhMG53 groups A significant difference between Ih WI andlh WI + rhMG53 groups.
  • *, # , A PO.05; **, ## , AA PO.Ol; ***, ### , p ⁇ Q 001 ' * * * * * * * ###### p ⁇ Q QQ
  • FIGS. 16A-16G show assessment of therapeutic benefit of rhMG53 during porcine transplantation model.
  • FIG. 16A shows the experimental overview.
  • FIG. 16B shows Gross pathology of explanted lungs following reperfusion period. The left lung of the rhMG53 lung showed that the transplanted lobe had less bullae formation, erythema, and injury when treated compared to those who received vehicle.
  • FIG. 16C shows PaCL in the isolated PV of the rhMG53 group (Mean (M): 445.2; Standard Deviation (SD): 55.5) compared to the vehicle group (M:201.9; SD: 182.4) at the end of the reperfusion period (p ⁇ 0.05).
  • M mean
  • SD Standard Deviation
  • FIG. 16D shows systemic PaCCh at the end of the reperfusion period, rhMG53 treated group (M:41.6; SD:4.6) compared to the vehicle group (M: 61.7; SD: 18.7) (p ⁇ 0.05).
  • FIG. 16E shows systemic pH at the end of the reperfusion period, vehicle treated group (M:7.32; SD:0.12) compared to MG53 treated group (M:7.49; SD:0.05) at the end of the reperfusion period (p ⁇ 0.05).
  • FIG. 16F shows ET-1 in the bronchoalveolar lavage (BAL) of the rhMG53 treated pigs as compared to control.
  • FIG. 16G shows ET-1 release was significantly less in the plasma as well (FIG. 17G). Significance denoted by: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001 and **** ⁇ 0.0005.
  • FIGS. 17A-17F show markers of injury suppressed during porcine lung transplantation by rhMG53.
  • TNF-a Tissue necrosis factor-alpha
  • RAGE receptors of advanced glycation end products
  • MCP-1 monocyte chemoattractant factor-1
  • PAI-1 plasminogen activator inhibitor 1
  • IL- 18 interleukin- 18
  • FIG. 17F shows Lactate dehydrogenase (LDH) was assessed in plasma samples and was significantly decreased at the end of the experiment. Significance denoted by: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001 and **** ⁇ 0.0005.
  • FIGS. 18A-18D shows additional assessment of lung injury indicators in perfusate, and physiological measurements taken during and after ex vivo lung perfusion.
  • PA pulmonary artery
  • FIG. 18D shows wet/dry ratio is not significantly changed by warm ischemia (WI), rhMG53 treatment, or perfusion. Dots are individual values with means indicated by lines. Bars represent means ⁇ SD. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****P ⁇ 0.0001.
  • FIG. 19 shows MG53 and CD31 co-localized to damaged endothelium after injury.
  • FIGS. 20A-20C shows m 53 ⁇ ⁇ with ischemic injury for 1 hour and subsequent survival for 24 hours.
  • the upper and lower edges of the box represent the 75th and 25th percentiles, respectively, and the middle line represents the median.
  • the upper and lower whiskers represent maximum and minimum values, and each dot represents an individual animal. Significance denoted by *P ⁇ 05, **P ⁇ 005, ***P ⁇ .0005, ****P ⁇ 0001.
  • LDH Lactate dehydrogenase
  • BAL bronchoalveolar lavage
  • ET-1 endothelin-1
  • HA hyaluronic acid
  • ns nonsignificant
  • OD optical density
  • MG53 mitsugumin- 53.
  • FIGS. 21A-21E shows manual cell differential of BAL samples (n 14 5) fixed onto slides after ischemic injury for 1 hour and subsequent survival for 24 hours.
  • FIG. 21A shows total cell count
  • FIG. 2 IB shows neutrophils per 1 mL
  • FIG. 21C shows macrophages per 1 mL were lower in the exogenous MG53 treatment group but was not significant.
  • neutrophil infiltration was significantly lower compared with macrophages per 1 mL as shown in FIG. 21D; however, this relationship did not hold true for the saline-treated group
  • FIG. 2 IE The upper and lower edges of the box represent the 75th and 25th percentiles, respectively, and the middle line represents the median.
  • the upper and lower whiskers represent maximum and minimum values, and each dot represents an individual animal. Significance denoted by *P ⁇ .05, **P ⁇ 005, ***P ⁇ 0005, ****P ⁇ 0001. ns, Nonsignificant; MG53, mitsugumin-53.
  • FIGS. 22A-22B show TUNEL positive and DAPI staining in nucleus as shown in FIG. 22A.
  • TUNEL staining detects DNA breaks as it occurs in the last phase of apoptosis.
  • the bar graphs represent quantitative results obtained from fluorescent imaging, which show significantly higher amounts of apoptosis in the saline-treated group as shown in FIG. 22B.
  • the upper and lower edges of the box represent the 75th and 25th percentiles, respectively, and the middle line represents the median.
  • the upper and lower whiskers represent maximum and minimum values, and each dot represents an individual animal. Significance denoted by *P ⁇ 05, **P ⁇ 005, ***P ⁇ 0005, ****P ⁇ 0001.
  • IRI Ischemia-reperfusion injury; MG53, mitsugumin-53.
  • FIGS. 23A-23D show mg53 ⁇ ⁇ with ischemic injury for 1 hour and subsequent survival for 24 hours.
  • the upper and lower edges of the box represent the 75th and 25th percentiles, respectively, and the middle line represents the median.
  • the upper and lower whiskers represent maximum and minimum values, and each dot represents an individual animal. Significance denoted by *P ⁇ .05, **P ⁇ .01, ***P ⁇ 001, and **** p ⁇ 0005.
  • IL Interleukin
  • MG53 mitsugumin-53
  • TNF tumor necrosis factor
  • FIGS. 24A-24D show mg53 ⁇ ⁇ with ischemic injury for 1 hour and subsequent survival for 24 hours.
  • Tissue was lysed and evaluated for full-length and cleaved GSDMD: Western blotting in FIG. 24A showed more expression of full-length and cleaved GSDMD within the saline-treated group; densiometric analysis revealed significantly more full-length GSDMD as shown in FIG. 24B and cleaved GSDMD as shown in FIG. 24C expression within the saline-treated group compared with the MG53 -treated group. Additionally, FIG. 24D shows tissue lysate was analyzed for ATP expression, which was significantly higher in the MG53 -treated group.
  • the upper and lower edges of the box represent the 75th and 25th percentiles, respectively, and the middle line represents the median.
  • the upper and lower whiskers represent maximum and minimum values, and each dot represents an individual animal. Significance denoted by *P ⁇ 05, ** P ⁇ 01, *** P ⁇ 001, and **** P ⁇ 0005.
  • MG53 Mitsugumin-53; IRI, ischemia-reperfusion injury; GSDMD, gasdermin-D; ATP, adenosine triphosphate.
  • FIGS. 25A-25B show representative MG53 and CD31 fluorescent staining of lung sections collected after 1-hour ischemic injury and survival for 24 hours as shown in FIG. 25 A. MG53 staining, CD31 staining, and DAPI staining are shown. The merge image of the rhMG53- treated group shows co-localization of MG53 to damaged endothelium after injury. Representative hematoxylin-eosin-stained lung sections collected after ischemia for 1 hour and subsequent survival for 24 hours in the saline and rhMG53 treatment groups, as well as quantification of MG53 in lung sections as shown in FIG. 25B.
  • FIG. 26 shows CLAD/Bronchiolitis obliterans syndrome (BOS)-free patient survival rates of 90-day survivors, grouped by the Grade 3 PGD or not after post-bilateral lung transplantation (BLT).
  • BOS CLAD/Bronchiolitis obliterans syndrome
  • FIG. 27 shows clinical manifestations of allograft dysfunction. This study aims to prevent allograft PGD through preventing vascular endothelial dysfunction, inflammasome progression and pyroptotic injury, and subsequent allograft dysfunction.
  • FIG. 28 shows the study overview and conceptualization of novel and innovative strategy to exploit 1) MG53 cell membrane repair and 2) MG53 modulation of IRI driven inflammation to mitigate PGD inflammatory response to produce a platform for preservation and rescue of lung allograft integrity during lung transplantation.
  • FIGS. 29A-29E show ET-1 elevates in EVLP perfusate (200mL vol.) after Jackpot of warm ischemic injury which is mitigated with addition of 5 pg/mL rhMG53 as shown in FIG. 29A.
  • FIG. 29B shows H4C staining that shows ischemia induces ET-1 levels which are reduced with rhMG53 administration.
  • FIG. 29C shows in the same lung, EVLP delivers rhMG53 which is taken up by the lung.
  • FIG. 29D shows in primary AoEC culture, H/R induces ET-1 elevation that is suppressed with rhMG53.
  • FIG. 29E shows ET-1 levels in the cell culture media are also suppressed with rhMG53 treatment.
  • FIG. 30 shows hypoxia (3hr) and reoxygenation(3hr) causes injury to AoECs as indicated by staining with Annexin V-FITC.
  • Alexa-647 labeling shows culture media supplemented with rhMG53 entering the cells where BSA does not.
  • FIGS. 31A-31C show that CRISPR knocks down VEGFR2 as shown in FIG. 31 A.
  • FIG. 3 IB shows a cell transfected with CRISPR- VEGFR2 does not take up rhMG53, but untransfected cell show uptake of rhMG53.
  • Cells transfected with control CRISP (right panel) display uptake of rhMG53.
  • FIGS. 32A-32C show EaHy926 endothelial cells and THP-1 macrophages (black box) and EaHy926 with THP-1 with A7G53- - (grey box) co-culture with H/R demonstrating their interaction resulting in increased expression of IL-ip as shown in FIG. 32A, TNF-a as shown in FIG. 32B, and lactate dehydrogenase (LDH) as shown in FIG. 32C that is significantly reduced with exogenous rhMG53 treatment.
  • FIGS. 32A-32C show EaHy926 endothelial cells and THP-1 macrophages (black box) and EaHy926 with THP-1 with A7G53- - (grey box) co-culture with H/R demonstrating their interaction resulting in increased expression of IL-ip as shown in FIG. 32A, TNF-a as shown in FIG. 32B, and lactate dehydrogenase (LDH) as shown in FIG. 32
  • FIGS. 34A-34B show IF staining in mouse lung slides.
  • FIGS. 34A and 34B show staining of GsdmD-N, MPO, and CD68 in mouse lung slides.
  • FIG. 35 shows murine lung transplantation experiment.
  • WT lungs were transplanted to either WT (WT-WT) or mg53-/- (WT-KO) recipients.
  • WT-WT WT-WT
  • WT-KO mg53-/-
  • FIGS. 36A-36B show IF staining and western blot images wherein LPS+Ni treatment leads to translocation of RFP-MG53 to plasma membrane as shown in FIG. 36A.
  • FIG. 36B shows rhMG53 treatment does not change activation of caspase- 1 in THP-1 cells.
  • FIGS. 37A-37B show western blotting images.
  • FIG. 37A shows MG53 co-IP with GsdmD.
  • FIG. 37B shows the effect of MG53 on caspase 1 mediated GsdmD and Sumo-GsdmD cleavage.
  • Sumo-GsdmD a 6xHis tag and a sumo domain joined to the N-terminus of GsdmD.
  • FIGS. 38A-38B show the measurement of pyroptosis activity.
  • FIG. 38A shows a schematic representation of the fluorescence liposome leakage assay.
  • FIG. 38B shows a dose response curve of MG53 as represented by rate of leakage (pyroptosis activity).
  • FIG. 39B shows the mg53ko recipient pairings demonstrated much higher levels of cleaved GSDM-D
  • FIG. 39C shows lower levels of pro-caspase 1.
  • FIG. 39D shows that under histology, there was a reduction in TUNEL staining in the tPA recipients and increase in mg53ko.
  • FIG. 40 shows scRNA-seq, new lung atlas nomenclature (ref 63), demonstrating neutrophil enriched populations are the main cell type undergoing pyroptosis (upper right panel).
  • the apoptotic pathway demonstrated minimal change and observed was in pneumocytes, not neutrophils.
  • FIGS. 41A-41F s h ow th at the etiology and mechanism of this inflammatory protection and the impact of neutrophil pyroptosis is the basis of these investigations.
  • Murine lung transplant as shown in FIGS. 41A-41B, and survival as shown in FIG. 41C.
  • WT donor mice lung transplantation (n 6/group) into WT (WT-WT) and mg53-/- (WT-KO) recipient mice was conducted.
  • F IG S . 4 1 D - 4 1 F s h ow th at the mg53-/- recipient demonstrates an exuberant inflammatory response.
  • FIGS. 42A-42C show rhMG53 delivered via EVLP to a porcine warm ischemic injury model preserves structure as shown in FIG. 42A and decreases circulating IL-6 as shown in FIG. 42B and LDH levels as shown in FIG. 42C.
  • FIGS. 44A-44D show images and graphs wherein FIG. 44 A shows EVLP of human donor lungs and FIG. 44B shows TUNEL staining of normal human lung and representative samples of those unable to be rescued with EVLP demonstrating higher degree of apoptosis as shown in FIG. 44C and Caspase 3/7 activity as shown in FIG. 44D in the non-rescuable lungs over the perfusion duration of 4 hours in human EVLP validating the selected small animal biomarkers.
  • FIG. 45 shows serum lactate dehydrogenase levels in pigs at baseline and through the transplant and 4-hour repression period where saline vehicle control or rhMG53 was administered through central venous catheter.
  • FIGS. 46A-46B show tissue level analysis of explanted pig lung of the non-transplanted donor right lung (ischemic injury only control) and the transplanted left lung in the recipient (injured, repercussion lung post-transplant) and the recipient animal native right lung (which was not transplanted or ischemic) where saline vehicle control or rhMG53 were administered through central venous catheter.
  • Adenosine triphosphate (ATP) FIG. 46A
  • tissue necrosis factor alpha TNF-a
  • FIGS. 47A-47B show plasminogen activator inhibitor-1 (PAI-1) (FIG. 47 A) measured in bronchioalveolar lavage fluid (BAL) (FIG. 47B) and in tissue for pig lung of the non-transplanted donor right lung (ischemic injury only control) and the transplanted left lung in the recipient (injured, repercussion lung post-transplant) and the recipient animal native right lung (which was not transplanted or ischemic) where saline vehicle control or rhMG53 were administered through central venous catheter.
  • PAI-1 plasminogen activator inhibitor-1
  • FIGS. 48A-48C show endothelin-1 (ET-1) levels in bronchioalveolar lavage fluid (BAL) (FIG. 48A), tissue (FIG. 48B), and plasma (FIG. 48C) for pig lung of the non-transplanted donor right lung (ischemic injury only control) and the transplanted left lung in the recipient (injured, repercussion lung post-transplant) and the recipient animal native right lung (which was not transplanted or ischemic) where saline vehicle control or rhMG53 were administered through central venous catheter.
  • FIGS. 49A-49B show MCP-1 levels in BAL as shown in FIG. 49A and lung tissues as seen in FIG. 49B, were measured in vehicle and MG53 treated groups.
  • FIG. 50 shows AGER levels in BAL were measured in vehicle and MG53 treated groups.
  • FIGS. 51A-51B shows IL-18 levels in BAL as shown in FIG. 51A and lung tissues as shown as FIG. 5 IB, were measured in vehicle and MG53 treated groups.
  • FIGS. 52A-52B show CXCL2 levels in BAL as shown in FIG. 52A and lung tissues as shown in FIG. 52B, were measured in vehicle and MG53 treated groups.
  • FIGS. 53A-53B show H&E staining of lung tissues from indicated groups Pl, P3.1, P3.2 and P3.3 as shown in FIG. 53A and groups P2, P4.1, P4.2 and P4.3 as shown in FIG. 53B.
  • FIGS. 54A-54B show H&E staining of lung tissues from indicated groups P5.1, P6.1, P6.2, P6.3, and P6.4 as shown in FIG. 54A and groups P7.1, P9.1, P9.2, P9.3, and P9.4 as shown in FIG. 54B.
  • FIGS. 55A-55B show H&E staining of lung tissues from indicated groups P13.1, P15.1, P15.2, P15.3, and Pl 5.4 as shown in FIG. 55 A and groups P8.1, P10.1, Pl 0.2, Pl 0.3, and Pl 0.4 as shown in FIG. 55B.
  • FIGS. 56A-56B show H&E staining of lung tissues from indicated groups P17.1, P18.1, P18.2, P18.3, and P18.4 as shown in FIG. 56A and groups PI L I, P12.1, P12.2, P12.3, and P12.4 as shown in FIG. 56B.
  • FIGS. 57A-57B show H&E staining of lung tissues from indicated groups P20.1, P22.1, P22.2, P22.3, and P22.4 as shown in FIG. 57A and groups P19.1, P21.1, P21.2, P21.3, and P21.4 as shown in FIG. 57B.
  • FIGS. 58A-58D show PV PaO2 levels in transplanted lung as shown in FIG. 58A, systemic arterial PaCO2 levels as shown in FIG. 58B, and systemic arterial pH levels as shown in FIG. 58C, and LDH release in plasma as shown in FIC. 58D.
  • FIG. 59 shows experimental plan/model for rhMG53 administration to before/during transplant and ex vivo organ perfusion to determine the optimum administration time/duration.
  • data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • administering to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, or via a transdermal patch, and the like. Administration includes self-administration and the administration by another.
  • beneficial agent and “active agent” are used interchangeably herein to refer to a chemical compound or composition that has a beneficial biological effect.
  • beneficial biological effects include both therapeutic effects, i.e., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., prevention of a disorder or other undesirable physiological condition.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like.
  • buffer refers to a solution consisting of a mixture of acid and its conjugate base, or vice versa.
  • the solution is used as a means of keeping the pH at a nearly constant range to be used in a wide variety of chemical and biological applications.
  • compositions, methods, etc. include the recited elements, but do not exclude others.
  • Consisting essentially of' when used to define compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • control is an alternative subject or sample used in an experiment for comparison purposes.
  • a control can be "positive” or “negative.”
  • composition refers to any agent that has a beneficial biological effect.
  • beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., Primary Graft Dysfunction).
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • composition when used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • the composition disclosed herein comprises rhMG53.
  • detect or “detecting” refers to an output signal released for the purpose of sensing of physical phenomenon. An event or change in environment is sensed and signal output released in the form of light.
  • diagnosis refers to the act of process of identifying the nature of an illness, disease, disorder, or condition in a subject by examination or monitoring of symptoms.
  • Effective amount of an agent refers to a sufficient amount of an agent to provide a desired effect.
  • the amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • “enhance”, “enhanced”, “enhancement”, “enhancing”, and any grammatical variations thereof as used herein, refers to an act of intensifying, increasing, or further improving the quality, value, or extent of a biological function, composition, compound, cell, or tissue.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA occurs.
  • a polynucleotide such as a gene, a cDNA, or an mRNA
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see,
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the compliment of a test sequence.
  • the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
  • sequence comparisons typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence algorithm program parameters Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
  • Ischemia-reperfusion refers to an occurrence when cellular dysfunction and death follow restoration of blood flow to a tissue or organ that previously experienced ischemia.
  • ischemia refers to an inadequate blood supply to an organ or part of the body. Ischemia can occur to any tissue or organ that requires or has an established blood supply, including but not limited to the heart, liver, kidneys, brain, and muscles.
  • “increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level so long as the increase is statistically significant.
  • the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur.
  • the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
  • the term “preventing” a disease, a disorder, or unwanted physiological event in a subject refers to the prevention of a disease, a disorder, or unwanted physiological event or prevention of a symptom of a disease, a disorder, or unwanted physiological event
  • “Pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
  • the term When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
  • “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • carrier encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
  • polypeptide refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.
  • nucleic acid as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides.
  • nucleobase refers to the part of a nucleotide that bears the Watson/Crick basepairing functionality.
  • the most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.
  • ribonucleic acid and “RNA” as used herein mean a polymer composed of ribonucleotides.
  • deoxyribonucleic acid and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.
  • polynucleotide refers to a single or double stranded polymer composed of nucleotide monomers.
  • recombinant refers to a human manipulated nucleic acid (e.g., polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g., polynucleotide), or if in reference to a protein (i.e., a “recombinant protein”), a protein encoded by a recombinant nucleic acid (e.g., polynucleotide).
  • a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g., polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g.
  • a recombinant expression cassette may comprise nucleic acids (e.g., polynucleotides) combined in such a way that the nucleic acids (e.g., polynucleotides) are extremely unlikely to be found in nature.
  • human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second nucleic acid (e.g., polynucleotide).
  • nucleic acids e.g., polynucleotides
  • nucleic acids can be manipulated in many ways and are not limited to the examples above.
  • reduced generally means a decrease by a statistically significant amount.
  • reduced means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10- 100% as compared to a reference level so long as the decrease is statistically significant.
  • a “subject” (or a “host”) is meant an individual.
  • the "subject” can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal.
  • the subject can be a mammal such as a primate or a human.
  • Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject.
  • “Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result.
  • Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.
  • the term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect.
  • a desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
  • a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
  • “Therapeutic agent” refers to any composition that has a beneficial biological effect.
  • Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition.
  • the terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • therapeutic agent when used, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • treating or “treatment” of a subject includes the administration of a drug to a subject with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder.
  • the terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, and improvement or remediation of damage.
  • A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B- F, and C-E would be considered disclosed.
  • This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
  • the present disclosure discloses a composition comprising recombinant human Mitsugumin-53 (rhMG53) protein and its use thereof in treating ischemia reperfusion injury (IRI) and/or primary graft dysfunction (PGD).
  • rhMG53 recombinant human Mitsugumin-53
  • a perfusate composition comprising recombinant human Mitsugumin-53 (rhMG53) protein and a perfusion medium.
  • rhMG53 recombinant human Mitsugumin-53
  • the rhMG53 comprises SEQ ID NO: 1, or a fragment of variant thereof.
  • Mitsugumin-53 is a protein that functions as an essential component of plasma membrane repair.
  • MG53 belongs to the tripartite motifcontaining (TRIM) protein family and is primarily found in skeletal muscle and alveolar epithelial cells.
  • TAM tripartite motifcontaining
  • MG53 when cell membranes are damaged, MG53 is released into the bloodstream, it detects the oxidized external environment and attaches to phosphatidylserine on membrane vesicles to facilitate membrane repair. MG53 then guides these vesicles to mend the compromised membrane using a "plug and patch" approach.
  • MG53 has been shown to have therapeutic benefit in decreased ischemia-reperfusion associated injury in both hilar clamp and transplantation models.
  • a perfusion medium is any fluid, such as blood or saline, perfused through the blood vessels of an organ, ensuring that it receives adequate oxygen and nutrients for its proper functioning.
  • the perfusion medium is circulated through the organ using a perfusion circuit.
  • the perfusion medium is blood.
  • the perfusion medium is saline, in some embodiments, the perfusion medium is a cryo-preservant,
  • the perfusion medium is rich in cell nutrients (such as, for example, vitamins, minerals, fatty acids and/or amino acids (such as, for example, L. Glutamine)).
  • the perfusate composition comprises rhMG53 and a perfusion medium, wherein the rhMG53 comprises SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises a nucleotide sequence.
  • rhMG53 comprises SED ID NO: 2, wherein SEQ ID NO: 2 comprises an amino acid sequence.
  • rhMG53 comprises sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or of higher identity) compared to SEQ ID NO: 1.
  • rhMG53 comprises sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or of higher identity) compared to SEQ ID NO: 2.
  • a preservation solution comprising recombinant human Mitsugumin-53 (rhMG53) protein and a preservation medium.
  • rhMG53 recombinant human Mitsugumin-53
  • the rhMG53 comprises SEQ ID NO: 1.
  • a preservation medium is a fluid that prevents injury to organs/grafts for transplantation during transport of the organ/graft from a donor to a recipient.
  • the preservation medium is a fluid, such as, for example, the UW solution, histidine- tryptophan-ketoglutarate (HTK), and Celsior.
  • the preservation medium is a potassium-containing and hyperosmolar solution.
  • the preservation solution comprises lactobionate, raffinose, and hydroxyethyl starch as osmotic agents and other components including glutathione, adenosine, and the free-radical scavenger allopurinol.
  • the glutathione content serves to facilitate the regeneration of cellular adenosine triphosphate (ATP) and maintain membrane integrity and adenosine provides the substrate for ATP regeneration during reperfusion.
  • the preservation medium comprises tryptophan and/or ketoglutarate, wherein tryptophan serves as a membrane stabilizer and antioxidant, whereas ketoglutarate acts as a substrate for anaerobic metabolism during preservation.
  • the preservation solution comprises relatively low-potassium and/or a strong histidine buffer.
  • the rhMG53 is administered as a recombinant protein. In some embodiments, the rhMG53 is administered through the delivery of a nucleic acid.
  • the rhMG53 is administered through the delivery of an mRNA encoding rhMG53.
  • the mRNA can include modified nucleotides.
  • the rhMG53 is administered through the delivery of a DNA sequence encoding rhMG53.
  • the rhMG53 is administered through the delivery of a nanoparticle comprising rhMG53.
  • the preservation solution comprises rhMG53 and a preservation medium, wherein the rhMG53 comprises SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises a nucleotide sequence.
  • rhMG53 comprises SED ID NO: 2, wherein SEQ ID NO: 2 comprises an amino acid sequence.
  • rhMG53 comprises sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or of higher identity) compared to SEQ ID NO: 1.
  • rhMG53 comprises sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or of higher identity) compared to SEQ ID NO: 2.
  • the perfusate composition or the preservative solution can comprise a vector (such as, for example, a plasmid, a cosmid, or an artificial chromosome) encoding rhMG53.
  • a vector such as, for example, a plasmid, a cosmid, or an artificial chromosome
  • the vector encoding rhMG53 is a viral vector.
  • Viral vectors are molecular tools used to deliver genetic material into cells that can be performed inside a living organism (in vivo), or in cell culture (in vitro).
  • the viral vector includes, but is not limited to adeno-associated viral (AAV) vectors, adenoviral vectors, retroviral vectors, lentiviral vectors, and hybrid viral vectors.
  • AAV adeno-associated viral
  • a method of treating or preventing ischemia reperfusion injury (IRI) in an ex vivo graft comprising administering a recombinant human Mitsugumin-53 (rhMG53) protein to the ex vivo graft.
  • rhMG53 recombinant human Mitsugumin-53
  • the rhMG53 protein is administered in a perfusion medium of any of the preceding aspects.
  • the rhMG53 protein is administered in a preservation medium of any of the preceding aspects.
  • the preservation solution comprises rhMG53 and a preservation medium, wherein the rhMG53 comprises SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises a nucleotide sequence.
  • rhMG53 comprises SED ID NO: 2, wherein SEQ ID NO: 2 comprises an amino acid sequence.
  • rhMG53 comprises sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or of higher identity) compared to SEQ ID NO: 1.
  • rhMG53 comprises sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or of higher identity) compared to SEQ ID NO:
  • the rhMG53 protein is administered to the ex vivo graft before reperfusion of the ex vivo graft. In some embodiments, the rhMG53 protein is administered to the ex vivo graft during reperfusion of the ex vivo graft.
  • reperfusion is the restoration of blood flow to an organ or tissue after having been blocked due to explantation or reperfusion injury.
  • the rhMG53 protein is administered to the ex vivo graft for at least 1 hour. In some embodiments, the rhMG53 is administered to the ex vivo graft for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes or more. In some embodiments, the rhMG53 is administered to the ex vivo graft for 2,
  • a graft is an organ or piece of tissue taken from a donor and transplanted into a recipient.
  • a graft can be an allograft or a xenograft.
  • the allograft is a graft taken from one subject and transplanted in a non-identical subject of the same species.
  • a xenograft is a graft taken from one subject and transplanted to another subject belonging to another species, e.g., animal to human.
  • the ex vivo graft is an allograft or a xenograft.
  • ex vivo graft is a lung, liver, heart, kidney, or intestine graft. In some embodiments, ex vivo graft is a lung. In some embodiments, ex vivo graft is a liver. In some embodiments, ex vivo graft is a heart. In some embodiments, ex vivo graft is a kidney. In some embodiments, ex vivo graft is an intestine graft.
  • the method suppresses the ischemic reperfusion injury in an ex vivo graft relative to an untreated ex vivo graft. In some embodiments, the method suppresses the ischemic reperfusion injury in an ex vivo graft by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more relative to an untreated ex vivo graft.
  • a method of treating a subject with primary graft dysfunction comprising administering a recombinant human Mitsugumin-53 (rhMG53) protein to the subject.
  • the rhMG53 protein is administered in a perfusion medium of any of the preceding aspects. In some embodiments, the rhMG53 protein is administered in a preservation medium of any of the preceding aspects.
  • the preservation solution comprises rhMG53 and a preservation medium
  • the rhMG53 comprises SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises a nucleotide sequence.
  • rhMG53 comprises SED ID NO: 2, wherein SEQ ID NO: 2 comprises an amino acid sequence.
  • rhMG53 comprises sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or of higher identity) compared to SEQ ID NO: 1.
  • rhMG53 comprises sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or of higher identity) compared to SEQ ID NO: 2.
  • the rhMG53 protein is administered to the subject before transplantation of the ex vivo graft. In some embodiments, the rhMG53 protein is administered to the subject during transplantation of the ex vivo graft. In some embodiments, the rhMG53 protein is administered to the subject after transplantation of the ex vivo graft.
  • the rhMG53 protein is administered to a donor before recovery. In some embodiments, the rhMG53 protein is administered to a donor during recovery.
  • the rhMG53 protein is administered to the subject for at least 1 hour. In some embodiments, the rhMG53 is administered to the subject for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes or more. In some embodiments, the rhMG53 is administered to the ex vivo graft for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 hours or more.
  • the ex vivo graft is an allograft or a xenograft.
  • ex vivo graft is a lung, liver, heart, kidney, or intestine graft.
  • composition of any of the preceding aspects is administered in combination with an inhibitor selected from a group comprising, a white blood cell inhibitor, a protease inhibitor, such as, for example, CD38 inhibitor, reactive oxygen species inhibitor, vasodilators (i.e., nitric oxide) or antioxidants, such as, for example, catalase, superoxide dismutase,
  • an inhibitor selected from a group comprising, a white blood cell inhibitor, a protease inhibitor, such as, for example, CD38 inhibitor, reactive oxygen species inhibitor, vasodilators (i.e., nitric oxide) or antioxidants, such as, for example, catalase, superoxide dismutase,
  • the method suppresses an immune response in the subject relative to an untreated subject with graft transplantation. In some embodiments, the method suppresses the immune response in the subject by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or more relative to an untreated subject with a graft transplantation.
  • the organ/graft transplant is a lung, a liver, a heart, a kidney, or an intestine graft. In some embodiments, the organ/graft transplant is a lung.
  • Example 1 MG53 mitigates warm ischemic lung injury in a murine model of transplantation.
  • IRI ischemia-reperfusion injury
  • PGD primary graft dysfunction
  • Mitsugumin-53 is a protein that functions as an essential component of plasma membrane repair. Previous studies have shown that MG53 can reduce ventilator-associated lung injury as well as IRI in transplant relevant models. Modulation or administration of MG53 and its related cell membrane repair pathway is potentially a novel therapeutic for the treatment of IRI-related PGD during lung transplantation.
  • MG53 -related repair of the cell membrane is an integral component of protection against IRI, and therefore when wild-type lungs are transplanted into genetically absent or overexpressing recipients of MG53, the severity of injury is most pronounced in the genetically absent recipient.
  • wild-type (wf) lung donor allografts were transplanted into a wl background, a MG53 knockout (mg53-/-), or a constitutively overexpressed MG53 (tissue plasminogen activator-MG53) recipient mouse after 1 hour of warm ischemic injury. Mice survived for 5 days after transplantation. Bronchi oalveol ar lavage, serum, and tissue were collected at sacrifice. Bronchi oalveolar lavage, serum, and tissue markers of apoptosis and a biometric profile of lung health were analyzed.
  • mice For transplant experiments, wt donor mice, all with 1 hour of warm ischemic injury, were randomly assigned to 1 of 3 recipient groups with the following backgrounds ( Figure 7): (1) wt background (control) (n 14 6/group); (2) mg53-/- background (n 14 4/group); (3) tPA-MG53 background (n 14 6/group). Protocols for a donor lung harvest and subsequent transplantation of the donor graft into the recipient have been described earlier. To summarize, mice were administered ketamine and xylazine (Henry Schein). When a surgical plane of anesthesia was met, a tracheostomy tube was placed and a thoracoabdominal incision was made.
  • mice were then sacrificed by cutting the inferior vena cava (IVC). While the lungs were still ventilating, the right and left ventricles were cut, and the lungs were gravity flushed with preservation solution (Perfadex; XVIVO) with an angiocatheter that was introduced directly into the pulmonary artery (PA). The ventilator was then disconnected from the endotracheal tube inflated with a predetermined volume. A clamp was placed on the trachea to keep the lungs inflated.
  • IVC inferior vena cava
  • PA pulmonary artery
  • mice were then prepped and anesthetized as described earlier.
  • a midline abdominal laparotomy was performed, and 0.5 mL of blood was collected from the IVC, for further biochemical analysis.
  • the mice were subsequently humanely killed by cutting the IVC.
  • the diaphragm was then dissected along the thoracic arch and the thoracic cavity was exposed by median sternotomy.
  • a bronchoalveolar lavage (BAL) was performed by filling a syringe with 0.8 mL of sterile saline, injecting it into the endotracheal tube, and withdrawing twice.
  • the right and left ventricles were cut, and the lungs were flushed by gravity through the pulmonary artery using an angiocatheter with 5 mL of prechilled phosphate-buffered saline solution (GIBCO).
  • GEBCO prechilled phosphate-buffered saline solution
  • the superior lobe and the postcaval lobe were snap frozen for protein and RNA expression analysis, the middle lobe was preserved in 10% neutral buffered formalin for histology and the inferior lobe was used for wet to dry weight ratio.
  • the left lung of the recipient was divided into 3 parts: the upper region was collected for snap freeze, the middle region was used for histology, and the lower region was used for wet-to-dry weight ratio.
  • Lactate dehydrogenase (LDH) release assay LDH release was measured in perfusate using the LDH cytotoxicity detection assay kit (Clontech Laboratories) according to the manufacturer’s instructions. The optical density values were analyzed at 490 or 492 nm by subtracting the reference value at 620 nm.
  • Membranes were incubated with primary mouse antibody against ET-1, gasdermin D (Abeam) IL-lb, and cleaved caspase-1 (Cell Signaling Technology). The membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) for 1 hour at room temperature. Protein levels on the membrane were determined using Western Lightning Plus-ECL (PerkinElmer), and autoradiography film (Denville Scientific). Incubation with monoclonal mouse b-actin antibody (Sigma-Aldrich) was conducted for comparative control. Quantitative results were obtained using Image J software (National Institutes of Health).
  • MG53 Staining For the immunohistochemical analysis, paraffin-embedded, formalin- fixed sections of murine lung were cut from tissue blocks at 4- to 6-mm thickness and mounted on slides. Mounted tissue sections were heated at 60°C for 30 minutes and washed 4 times in xylene each for 10 minutes to remove the paraffin wax, followed by rehydration through an ethanol series, before washing and storing in dH2O until use. Antigen retrieval was conducted via a 30- minute incubation in 100°C sodium citrate buffer followed by preincubation in 1% fetal bovine serum/glycine blocking solution. A specific primary antibody against MG53 (1 :200, rabbit, homemade) incubated with the tissue section overnight.
  • mg53-l- mice had significantly greater levels of markers of overall cell lysis and endothelial cell injury. Overexpression of MG53 resulted in a signature similar to that of wt controls. At the time of explant, tissue plasminogen activator-MG53 recipient tissue expressed significantly greater levels ofMG53, measured by immunohistochemistry, compared with mg53-l-, demonstrating up- take of endogenous overexpressed MG53 into donor tissue.
  • pro-caspase- 1, caspase-1, TNF-a, IL- 1b, gasdermin D activity, as a biochemical indicators of programmed cell death, were also measured.
  • pro-caspase-1 and caspase 1 levels were analyzed via Western blotting (Figure 8).
  • Pro-caspase- 1 was greater in the tPA-MG53 recipient lobe as compared to the both the wt donor lobe and mg53-/- recipient lobe (Figure 8B).
  • Caspase- 1 levels were not significantly different among the groups (Figure 8C).
  • the transplantation groups there were no significant differences in TNF-a levels in the BAL or IL-lb levels in BAL or tissue ( Figure 9).
  • Gasdermin-D and cleaved gasdermin-D levels were not significantly different amongst groups either ( Figure 10).
  • MG53 is an integral part of the cell membrane repair machinery and can be used for the use of new therapeutics in lung transplantation.
  • MG53 is initially involved in cell membrane repair and machinery. To summarize, when cell membrane damage occurs, MG53 senses the oxidized environment outside the cell and binds to phospholipids on membrane vesicles within the cytoplasm. MG53 then brings these vesicles to the damaged membrane and facilitates their repair through a “plug-and-patch” mechanism. Protection of the endothelium is paramount, as damage from IRI disrupts this barrier, leading to migration of immune cells into the alveolar space causing further edema, injury, and eventually can manifest clinically as PGD. After 1 hour of warm ischemia, subsequent transplantation, and a 5-day survival, circulating MG53 was still found in quantifiable levels within donor lung tissue.
  • MG53 staining confirmed the protein was still visualized within the lung parenchyma as well and further confirmed that it was congregated around the alveoli and presumably laying within the endothelium, i.e., the site of injury. This confirms previous studies and shows a finding of prolonged presence of MG53 in donor tissues. This confirms that MG53 not only has the ability to migrate to the affected endothelial cells during injury but also can be present for a sustained period of time contributing to further repair and regeneration.
  • IRI is a known contributor to PGD and is mediated by inflammatory signaling cascades that lead to impaired cell function, resulting in endothelial cell death. During these cascades, markers of inflammation and cell damage are released. In this study, markers of acute lung injury, cellular integrity, and death were specifically looked at.
  • ET-1 is a marker of endothelial cell integrity during lung injury, and previously high levels of ET-1 have been shown to correlate with the development of PGD. ET-1 has also been studied at several time points throughout transplantation: before organ procurement, intraoperatively, and immediately postoperatively.
  • ET-1 and Big ET-1 amino acid from which ET-1 is cleaved were found at significantly greater levels in BAL of the MG53 knockout transplantation group compared with MG53 overexpressers and control.
  • LDH is a widely recognized marker of cellular inflammation and damage, and specifically in transplantation models, it has been found at substantially lower levels of perfusate in the lungs that are considered suitable for transplantation post-ex vivo lung perfusion. Post transplantation, acute rejection is often accompanied by an increase in serum LDH. Endogenous MG53 was able to mitigate cellular inflammation in both M and tPA-MG53 recipients.
  • Apoptosis is a major type of cell loss and contributes to IRI during lung transplantation. Quantification of cell death (i.e., apoptosis) was evaluated by TUNEL staining, which showed an increase in intensity in mg53-/- recipients compared with wt and tPA-MG53 recipients.
  • endogenous circulating MG53 can be delivered effectively to the lung transplanted after warm ischemic injury and due to the endogenous nature of the recipient animal, the effect persists after injury and preserve cellular integrity, decrease cellular inflammation, and mitigate apoptosis.
  • all recipients received wt lung allografts, which have the ability to produce endogenous MG53 from their endothelium.
  • these cells can only produce finite quantities of MG53, which fall well below normal circulating levels in mice, thus limiting the ability to prevent injury when transplanted into mg53-/- recipients as evident by this study.
  • Example 2 MG53 preserves endothelia integrity to protect against ischemia reperfusion injury during lung transplantation .
  • Lung transplantation is hampered by a lack of suitable donors, which is highlighted by the fact that only around 20% of eligible donor allografts end up being successfully transplanted.
  • extended criteria donors are being used with increasing frequency, however, have been shown to have worse peri-operative outcomes and decreased survival.
  • IRI ischemia reperfusion injury
  • the result of this injury is the breakdown of the endothelial barrier, while also for innate immune cells to populate the alveolar space and cause further damage to the lung parenchyma. Clinically, this results in poor allograft function and primary graft dysfunction (PGD) following transplantation.
  • PGD is a significant adverse event that occurs after lung transplantation, and unfortunately can affect up to 30% of patients leading to unnecessary morbidity and mortality. Thus, protecting the endothelium during transplantation is critical to preventing the sequala of IRI and thus mitigating PGD.
  • MG53 Mitsugumin-53
  • TAM tripartite motif-containing
  • MG53 detects the oxidized external environment and attaches to phosphatidylserine on membrane vesicles. It then guides these vesicles to mend the compromised membrane using a "plug and patch" approach.
  • MG53-mediated repair constitutes an integral component of lung protection through preservation of endothelial cell integrity during allograft transplantation and can rescue marginal allografts by protecting against IRI during lung transplantation.
  • two well-established and complimentary models of IRI were employed, further validated rhMG53 administration in small animal transplantation which culminated in successful porcine lung transplantation.
  • Endothelial cell primary culture Primary porcine pulmonary artery endothelial cells (PPAEC) were harvested as previously described. Briefly, fresh pulmonary artery (PA; 10-14 cm), was sourced from heparinized and euthanized pigs, and PPAEC were isolated by suturing and clamping off the branches and ends of the vessels, and filling the PA with collagenase (Sigma, St. Louis, MO) for 15 minutes. The endothelium was then denuded with gentle scraping and cells were suspended in 20% FBS (Lonza, Walkersville, MD) before centrifuging at 200 G for 5 minutes.
  • FBS Longza, Walkersville, MD
  • Cells were cultured in MEM containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin (Lonza) with bovine brain extract at 37°C in a humidified atmosphere of 5% CO2 in air until confluent.
  • hypoxia conditions by filling an Atmospheric Control Unit (BGM LAB TECH, Ortenberg, DE) with N2 gas (hypoxia).
  • BGM LAB TECH, Ortenberg, DE Atmospheric Control Unit
  • rhMG53 10 or 50 ug/ml
  • hypoxic conditions For the post-treatment group, after 3h of hypoxia, cells were washed and treated with rhMG35 (10 or 50 ug/ml) and then incubated in normoxic conditions (reoxygenation).
  • the control perfusate consisted of 4% human serum albumin (CLS Behring, King of Prussia, PA), Williams’ Medium (Sigma), and heparin (0.01 units/mL).
  • rhMG53 perfusate consisted of the control perfusate with 50ug/mL of rhMG53.
  • Ventilation parameters were then set with a flow speed initially 5-10% predicted cardiac output and increased to 20% of predicted cardiac output no later than 15 minutes after beginning perfusion.
  • Physiologic lung parameters were measured continuously using Power Lab software (Adlnstruments, Sydney, AU). The perfusate was sampled immediately before entering the PA or after exiting the LA via sample ports at Oh, Ih, and 2h and analyzed by a blood gas analyzer (ABL- 90 Flex Plus, Radiometer American Inc., Brea, CA). Additionally, an aliquot was frozen for further analysis. Tissue was harvested and processed immediately following perfusion. The right upper lobe was used for wet-to-dry weight ratio. The middle right lobe was fixed in 10% neutral buffered formalin. The lower right lobe, upper left lobe, and lower left lobe were snap frozen.
  • Donor lung allografts were procured and preserved in the standard fashion. When explanted, the lungs were filled with room air (3 mL/kg) covered in moistened gauze and left on a warming surgery board (37°C) for Ih to induce ischemia. After Ih of warm ischemia, a left thoracotomy was then performed on the recipient rat and prepared for transplantation in the standard fashion. The donor PA and PV were flushed with 0.2 mL of heparinized saline (1 U/mL) prior to anastomosis, and allograft was then transplanted into the recipient.
  • the clamp around the hilum was removed, allowing for reperfusion and ventilation of the donor lung.
  • the thoracotomy and laparotomy incisions were then closed using nylon suture.
  • the rats then survived for 3h under ventilation and anesthesia.
  • After 3h hours of reperfusion blood was collected from the recipients for further analysis.
  • the recipient was subsequently euthanized, and tissue was harvested and processed immediately.
  • the recipient’s left and right lungs were divided into three parts: the upper region was collected for snap-frozen, the middle region was used for histology, and the lower region was used for wet-to-dry weight ratio.
  • Perfadex pre-chilled preservation solution
  • the recipient was anesthetized, lined up for invasive monitoring, prepped, and draped in standard sterile fashion.
  • the donor allograft was prepared on the back table, and a BAL sample was taken from the right donor lung for a baseline injury profile.
  • a recipient left pneumonectomy was performed.
  • a left lung transplant was performed, and prior to completion of anastomosis, methyl-prednisone and either rhMG53 or control was administered systemically via the jugular vein.
  • the chest was left open, and the pig survived under anesthesia for 4 hours.
  • blood samples were obtained via central access for analysis (ABL-90 Flex Pl, Radiometer American Inc., Brea, CA).
  • tissue and BAL samples were taken from the right native lung and left donor allograft. To avoid compounding injury from dependent atelectasis, the lower lobes of the animal were not used for sample collection. The right and left middle lobes (or middle lobe equivalent) were used for BAL collection, while the left and right upper lobes were used for snap-frozen, histology, and wet-to-dry weight ratio.
  • Biochemical Assays Colorimetry and fluorescence for all assays were measured using the POLARstar Omega multi-mode microplate reader (BMG LABTECH, Stafford, TX). ET-1 (Enzo Life Sciences, Farmingdale, NY), Big ET-1 (Enzo Life Sciences), and HA (R&D Systems, Minneapolis, MN) levels were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions. LDH release was measured using the LDH cytotoxicity detection assay kit (Clontech Laboratories, Mountain View, CA) according to the manufacturer’s instructions. The optical density values were analyzed at 490 or 492 nm by subtracting the reference value at 620 nm. Caspase-3/7 activity was detected in lung tissue extracts with the Apo-ONE Homogeneous Caspase-3/7 kit (Promega, Madison, WI) according to the manufacturer’s instructions.
  • Western blot analysis Western blot analysis was performed using culture media and cell lysates to evaluate ET-1 (Abeam, Cambridge, UK) and P-actin (Sigma, St. Louis, MO) as a comparative control. Equal amounts of supernatants or proteins were electrophoresed on 6-12% SDS-PAGE gels and transferred onto a nitrocellulose membrane. Membranes were blocked in a Tris-buffered saline/0.1% Tween 20 and 5% nonfat dry milk for 3h, incubated with primary antibodies for 2h at room temperature or overnight at 4°C, and incubated with horseradish peroxidase-conjugated secondary antibodies for Ih at room temperature. Western blots were imaged using Western Lightning Plus-ECL (PerkinElmer, Waltham, MA) and autoradiography film (Denville Scientific, Metuchen, NJ), and quantified by using ImageJ (NIH, Bethesda, MD).
  • ET-1 Abeam, Cambridge, UK
  • P-actin Sigma, St
  • TUNEL staining Apoptotic cells in lung tissue section were determined using the In Situ Cell Death Detection kit (Roche, Indianapolis, IN), which is a TUNEL assay. Tissues were also stained with 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei. The TUNEL and DAPI images were merged to allow for visual identification and quantification of apoptotic cells by ImageJ (National Institute of Health) analysis.
  • Histological Analysis Histologic specimens were stored in 10% formalin, transferred to ethanol for paraffin embedding, and 5-pm sections were cut and processed. Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining was performed using standard methods.
  • paraffin-embedded formalin-fixed sections of murine lung were cut from tissue blocks at 4-6pm thickness and mounted on slides. Mounted tissue sections were heated at 60°C for 30 min and washed four times in xylene each for 10 min to remove the paraffin wax, followed by rehydration through an ethanol (EtOH) series, before washing and storing in dH2O until use.
  • EtOH ethanol
  • Antigen retrieval was conducted via 30 min incubation in 100°C sodium citrate buffer followed by pre-incubation in 1% FBS/glycine blocking solution.
  • a specific primary antibody against MG53 (1 :200, Rabbit, Homemade) incubated with the tissue section overnight. The details for creation of the homemade MG53 primary antibody have been described in detail before.
  • tissue sections were stained with Alexa Fluor 647 (Rabbit) and Alexa Fluor 555 (Rat) second antibodies (1 :500, A32733, Invitrogen; 1 :500, A21434, Invitrogen).
  • the stained tissue sections were mounted in DAPI Fluoromount-G (0100-20, Southern Biotech). Images were taken using the Nikon A1R-MP Laser Scanning Confocal Microscope.
  • rhMG53 Modulates Injury Marker Endothelin-1 In Primary Endothelial Cell Cultures: Using porcine pulmonary artery endothelial cells (PPAECs), pre-treatment (50ug/mL) and post-treatment with rhMG53 (10 and 50ug/mL) reduced ET-1 production in supernatant as compared to untreated control ( Figure 11 A). When cells were lysed, Western blot analysis showed that treatment before H/R (pre-treatment) with 50ug/mL of rhMG53, but not treatment after hypoxia and before reoxygenation (post-treatment), resulted in significantly decreased ET-1 production within cells (Figure 1 IB).
  • MG53 Administration During Rodent Transplantation Prevents Endothelial Injury With the goal of providing evidence that administration of exogenous human recombinant MG53 (rhMG53) is efficacious in mitigating damage to the endothelium in marginal donor allografts, rodent single lung transplantation after a period of 1 hour of warm ischemia was conducted. Prior to reperfusion of the transplanted allograft, the recipients were treated systemically with either rhMG53 (1 mg/kg) or saline (Figure 12A).
  • Perfusate was collected during EVLP at 0-, 30-, 60-, and 120-minutes and was analyzed for markers of endothelial, and cell membrane damage.
  • Composite ET-1+ Big ET-1 was significantly increased in lung allografts which underwent ischemic injury prior to reperfusion, which was mitigated by addition of rhMG53 to the perfusate (Figure 18). Big ET-1 release was significantly mitigated by the addition of rhMG53 during all time points (Figure 13B), while the active ET-1 release was significantly decreased at 60- and 120-minutes (Figure 13C).
  • LDH lactate dehydrogenase
  • Figure 13D lactate dehydrogenase
  • HA hyaluronic acid
  • PaO2 was significantly decreased in both the no injury and injury without treatment group, while this difference was not seen in the rhMG53 -treated group ( Figure 13F).
  • Delta PaO2 i.e., the change in oxygenation from the pulmonary artery canula compared to the left atrium canula, a significant decrease was seen in the injury group, however not noted in the rhMG53 group or no injury group ( Figure 13G).
  • Pulmonary vascular resistance (PVR) was significantly decreased in all groups at 120-minutes (Figure 13H), while pulmonary artery (PA) pressure was significantly increased in all groups at the end of the perfusion period ( Figure 18). Wet-to-dry ratio showed no significant differences showing that any edema caused by the 120-minutes perfusion did not differ among groups ( Figure 18).
  • rhMG53 Mitigates Endothelial Injury and Preserves Lung Function in a Large Animal Transplantation Model After demonstrating cellular protection and preserved physiologic function after administration of rhMG53 in small animal models, the hypothesis was tested in a large animal model. Pigs were assigned to treatment group or vehicle control, and after a period of 24-hours of cold static storage (CSS), a single left-lung transplant was performed.
  • CCS cold static storage
  • Pigs were treated with rhMG53 (1 mg/kg) or control (saline) by central access prior to reperfusion of the transplanted lobe (Figure 16A). All pigs that were administered rhMG53 were able to survive the full reperfusion period (4 hours), while 1 pig died at ⁇ 1 hour into reperfusion in the control group.
  • rhMG53 s ability to mitigate release of known markers of endothelial injury is shown, and furthermore preserve lung function during transplantation with exogenous administration.
  • This investigation builds on the scientific premise that targeting the elemental process of cell membrane repair is a means to preserve the quality of the donor lung, thereby improving the outcome of lung transplantation.
  • the data generated by this study supports the notion that rhMG53 has therapeutic benefit in protecting endothelial cells exposed to IRI and protect allograft lungs from further injury during EVLP and employed in lung transplantation to mitigate IRI improving early outcomes.
  • rhMG53 administered during the perfusion period mitigated release of known markers of endothelial and cell membrane integrity, as well as apoptotic response, demonstrating protection of this barrier. Furthermore, oxygenation was preserved throughout the perfusion period by the administration of rhMG53, demonstrating preserved physiologic function. Finally, analysis of rhMG53 in the perfusate showed accumulation of rhMG53 in the lung tissue, and simultaneous reduction of rhMG53 in the perfusate over time, which further supports that administration of rhMG53 is readily used by lung tissue to prevent further injury. Finally, the porcine transplantation model showed that rhMG53 administration decreased inflammation while preserving oxygenation and mitigating a respiratory acidosis.
  • ET-1 is a potent vasoconstrictor and is released in response to hypoxia and inflammation. When the endothelium is injured, ET-1 is released which exerts its own effects on pulmonary vasculature and the alveoli. ET-1 increases capillary permeability through the upregulation of vascular endothelial growth factor, as well as increases capillary hydrostatic pressure.
  • ET-1 When the endothelium is injured, ET-1 is released which exerts its own effects on the alveoli. ET-1 mediates nitric oxide release from endothelial cells, which signals epithelial cells in the alveoli to down regulate Na + /K + adenosine 5 '-triphosphatase which disrupts fluid clearance causing edema within the parenchyma. Additionally, the inhibition of ET-1 has been shown to decrease hypoxia induced apoptosis. All of these effects of ET-1 were readily mitigated throughout the experiments (as evident by directly decreasing edema or preserving oxygenation through presumably less alveolar edema), as well as downstream products of ET-1 to include: MCP-1, PAI-1, and IL-18.
  • ET-1 expression can be regulated by a variety of factors, including RAGE.
  • RAGE has been shown to mediate interference of membrane resealing of endothelial cells and promotes endothelial cell death and further damage to this membrane.
  • NF-KB nuclear factor-kappa beta
  • MG53 The action of MG53 is multifactorial and confers protection through a variety of anti-pyroptotic and necroptotic pathways, in which case MG53’s ability to decrease ET- 1 release involves the RAGE-NF -KB -TNF-a axis. The interference of this axis is evident throughout this study.
  • Example 3 Mitsugumin-53 mitigation of ischemia-reperfusion injury in a mouse model.
  • IRI ischemia- reperfusion injury
  • Mitsugumin 53 is a tripartite motif-containing family protein predominately expressed in skeletal muscle and alveolar epithelial cells. Upon tissue injury, it is released into the circulation to facilitate cell membrane repair. MG53’s cell membrane repair capabilities are well described. In brief, when plasma membranes are damaged, MG53 senses the oxidized extracellular environment and binds to phosphatidylserine on membrane vesicles. It then traffics these vesicles to seal the damage membrane through a “plug and patch” method. 7 Previous studies have shown MG53 can reduce acute lung injury, as well as offer cardioprotection to cardiac myocytes damaged by IRI.
  • rhMG53 Modulation or administration of an exogenously produced recombinant human MG53 (rhMG53) and its related cell membrane repair is a potential novel therapeutic for the treatment of IRI and prevention of PGD associated with lung transplantation. It was hypothesized that exogenous administration of rhMG53 would confer endothelial protection and mitigate lung injury in a transplant-relevant in vivo model of IRI.
  • mice C57BL/6J mice were subjected to 1 hour of ischemia (via left lung hilar clamp), followed by 24 hours of reperfusion.
  • m 53 ⁇ ⁇ mice were administered exogenous recombinant human mitsugumin-53 or saline before reperfusion.
  • Tissue, bronchoalveolar lavage, and blood samples were collected at death and used to quantify the extent of lung injury via histology and biochemical assays.
  • mice All in vivo experiments used C57BL/6J (The Jackson Laboratory) MG53 knockout (/i/i U ) mice weighing 20 to 30 g.
  • a transgenic mouse line with genetically absent ability to secrete MG53 was generated by interrupting the MG53 DNA sequence as previously described.
  • the founder mice were confirmed with absent levels of MG53 in blood circulation by immunoblotting. All animals were housed under standard conditions (humidity: 45%-70%, temperature: 21 °C ⁇ 3 °C, 12-hour light-dark cycle) and provided ad libitum access to water and standard diet. Animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee (Protocol #2012A00000135). All experiments were performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978).
  • mice were then kept alive for 24 hours under standard conditions. At death, the mice were prepped, anesthetized, and intubated as described earlier. A midline abdominal laparotomy was performed, and 0.5 mL of blood was collected from the inferior vena cava. The mice were killed by cutting the inferior vena cava, and the thoracic cavity was exposed. A bronchoalveolar lavage (BAL) was performed by filling a syringe with 0.8 mL of sterile saline, injecting it into the endotracheal tube and withdrawing twice.
  • BAL bronchoalveolar lavage
  • the right and left auricles were cut, and the lungs were flushed by gravity through the pulmonary artery using an angiocatheter with 5 mL of prechilled phosphate-buffered saline solution (GIBCO).
  • GEBCO prechilled phosphate-buffered saline solution
  • the heart-lung block was then removed.
  • the left lung was divided into 2 parts: The upper region was collected for snap-freezing, and the lower region was used for histology. Blood and tissue were analyzed for a previously described biometric profile of lung health.
  • Biochemical Assays Colorimetry and fluorescence for all biochemical assays were measured using the POLARstar Omega multi-mode microplate reader (BMG LABTECH).
  • Enzyme-Linked Immunosorbent Assay Tissue homogenates were collected from the lungs. The levels of endothelin-1 (ET-1) (Enzo Life Sciences), hyaluronic acid (HA), tumornecrosis factor alpha (TNF-a), and interleukin (IL)-lb (R&D Systems) were measured by enzyme-linked immunosorbent assay according to the manufacturer’s instructions.
  • E-1 Endothelin-1
  • HA hyaluronic acid
  • TNF-a tumornecrosis factor alpha
  • IL interleukin
  • Lactate Dehydrogenase Release Assay Lactate dehydrogenase release was measured in perfusate using the LDH cytotoxicity detection assay kit (Clontech Laboratories) according to the manufacturer’s instructions. The optical density values were analyzed at 492 nm and normalized relative to plate reference absorbance at 620 nm and background LDH in control wells.
  • Adenosine Triphosphate Assay Adenosine triphosphate (ATP) level in tissue was measured using the ATP Colorimetric/Fluorometric Assay Kit (Biovision), according to the manufacturer’s instructions. Absorbance was measured (OD 570 nm) in a micro-plate reader.
  • the membrane was incubated with horseradish peroxidase as a secondary antibody (Jackson ImmunoResearch Laboratories) for 1 hour at room temperature. Protein levels on the membrane were determined using Western Lightning Plus-ECL (PerkinElmer) and autoradiography film (Denville Scientific). Incubation with monoclonal mouse b-actin antibody (Sigma- Aldrich) was conducted for comparative control. Quantitative results were obtained using Image J software (NIH).
  • TUNEL Staining Apoptotic cells in the lung tissue section were determined using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer’s instructions. The assay uses terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), which identifies apoptotic cells by labeling DNA fragmentation sites. The tissue was also counterstained with DAPI to permit visualization of cell nuclei. After TUNEL staining, samples were cover-slipped using Prolong Diamond (P36966; Thermo Fisher Scientific) mounting medium. Tissues were imaged using a Leica Thunder 3D imaging system equipped with a Leica K5 fluorescence imaging camera.
  • TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling
  • TUNEL and DAPI images were merged to allow visual identification of apoptotic cells using LasX V2.3.0 and ImageJ analysis software.
  • Immunohistochemistry Staining Paraffin-embedded formalin-fixed sections of murine lung were cut from tissue blocks at 4- to 6-mm thickness and mounted on slides. Mounted tissue sections were heated at 60 °C for 30 minutes and washed 4 times in xylene each for 10 minutes to remove the paraffin wax, followed by rehydration through an ethanol series, before washing and storing in dH2O until use.
  • Antigen retrieval was conducted via 30 minutes of incubation in 100 °C sodium citrate buffer followed by preincubation in 1% fetal bovine serum/glycine blocking solution.
  • MG53 A specific primary antibody against MG53 (1 :200, Rabbit, Homemade) and CD31(1 :200, Rat, 550,274, BD Pharmingen) was incubated with the tissue section overnight.
  • the details for creation of homemade MG53 primary antibody have been described in detail.
  • tissue sections were stained with Alexa Fluor 647 (Rabbit) and Alexa Fluor 555 (Rat) second antibodies (1 :500, A32733, Invitrogen; 1 :500, A21434, Invitrogen).
  • the stained tissue sections were mounted in DAPI Fluoromount-G (0100-20, Southern Biotech). Images were taken using the Nikon A1R-MP Laser Scanning Confocal Microscope.
  • Histological Analysis Histological samples were stored in 10% formalin and transferred to ethanol for paraffin embedding, and 5-mm sections were cut and processed. Hematoxylin-eosin and IHC staining were performed using standard methods.
  • BAL fluid was centrifuged at 800g for 10 minutes, and the supernatant was removed. The cells were then resuspended in the appropriate buffer and automatic cell count was performed using Countess 3 (Invitrogen). The resuspended cells were diluted to an appropriate concentration and loaded into a cytospin funnel, and again centrifuged at 800g for 10 minutes. The slides were then dried overnight at room temperature and stained with Differential Quick Stain Kit (Electron Microscopy Sciences) and analyzed with a light microscope for manual cell differential.
  • recombinant human mitsugumin-53 showed a significant decrease in an established biometric profile of lung injury as measured by lactate dehydrogenase and endothelin- 1 in the bronchoalveolar lavage and plasma. Biochemical markers of apoptosis and pyroptosis (interleukin- lb and tumor necrosis factor-a) were also significantly mitigated, overall demonstrating recombinant human mitsugumin-53 ’s ability to decrease the inflammatory response of ischemiareperfusion injury. Exogenous recombinant human mitsugumin-53 administration showed a trend toward decreasing overall cellular infiltrate and neutrophil response. Fluorescent colocalization imaging revealed recombinant human mitsugumin-53 was effectively delivered to the endothelium.
  • TUNEL staining of lung sections indicated that apoptosis was also increased by IRI and mitigated by rhMG53 administration ( Figure 22A).
  • the degree of apoptosis in TUNEL staining was further quantified using Gene 5 software (Agilent Technologies). This also showed a significantly higher amount of apoptosis in saline-treated mice compared with rhMG53 treatment ( Figure 22B).
  • IL-lb was significantly decreased in the treatment group when compared with control ( Figure 23 A), as well as the BAL fluid ( Figure 23B).
  • TNF-a showed a similar pattern and was significantly delivered to lung tissue, as demonstrated by the MG53 staining.
  • co-localization of MG53- and CD31- stained cells showed that rhMG53 effectively delivered the endothelium, that is, the site of injury.
  • Imaging demonstrated that m 53 ⁇ ⁇ mice treated with vehicle had no background MG53 expression.
  • MG53 and CD31 co-localization in the saline-treated group demonstrated no overlap of MG53 and CD 31.
  • rhMG53 was effectively agent in lung transplantation.
  • IRI in lung tissue results in endothelial dysfunction and injury, manifesting as a recruitment of immune cells into the injured parenchyma and airway and resultant alveolar edema.
  • the classic cell type associated with early IRI-induced PGD is the neutrophil. Neutrophils invade the parenchyma and alveolar space early during IRI leading to exacerbated lung injury and inflammation. In addition to causing direct oxidative tissue, neutrophils amplify the proinflammatory response through interactions with a multitude of immune cells. This neutrophilic inflammation is particularly damaging in the transplantation setting due to enhancement of the adaptive immune response increasing the potential for graft rejection.
  • the benefit of blocking neutrophil activation during reperfusion has been shown to be protective against IRI in lung allografts. The ability to hamper neutrophil recruitment and response after reperfusion is a way to prevent worsening IRI. rhMG53 administration attenuated the cellular response in the alveolar fluid and mitigated neutrophil migration from IRI in alveolar fluid.
  • IRI is a complex process mediated by a multitude of damage-associated molecular patterns (DAMPs) and inflammatory signaling cascades that lead to impaired cell function, resulting in endothelial cell death. During this process, markers of inflammation and cell damage are released and can be quantified in the systemic circulation.
  • DAMPs damage-associated molecular patterns
  • TNF-a is common proinflammatory cytokine that is upregulated downstream by DAMPS.
  • ET-1 is a marker of endothelial cell integrity during lung injury and has both mitogenic and proinflammatory effects that are implicated in the pathophysiology of IRI.
  • Clinically ET-1 is a prognostic biomarker, and elevations of serum ET-1 have been shown to correlate with the development of PGD.
  • ET-1 release was significantly mitigated by administration of MG53 compared with control in the BAL and serum of mice subjected to IRI.
  • cellular release of LDH is a widely recognized marker of cell membrane compromise, oxidative stress, and damage.
  • LDH has been found at substantially lower levels in perfusate of lungs that are considered suitable for transplantation after ex vivo lung perfusion. Furthermore, post-transplantation elevation of serum LDH is an early clinical indicator of acute rejection.
  • Administration of exogenous MG53 was able to mitigate oxidative stress in mice with IRI as well.
  • HA is a marker of acute lung injury, and during transplantation can be seen elevated in patients with acute rej ection. In this study, there was a nonsignificant trend in reducing HA release in both the BAL and plasma through the administration of rhMG53.
  • Apoptosis is a form of programmed cell death that is triggered in response to hypoxia during IRI. Cells induce death via proteolysis from caspases. This form of injury contributes to IRI during the early phases of allograft reperfusion. Apoptosis is most severe after reperfusion, and prior studies have noted induction of apoptosis as early as 30 minutes after reperfusion. Quantification of apoptosis was evaluated by TUNEL staining, which demonstrates a decreased intensity in animals that were treated with rhMG53. In this work, apoptotic cell death was observed as long as 24 hours after IRI.
  • Pyroptosis is another form of programmed cell death and is more often associated with IRI than apoptosis. It differs from apoptosis in that cell death arises from plasma membrane disruption, organelle swelling, and mitochondria dysfunction.
  • Damage and formation of DAMPs are recognized by NOD-like receptors, which results in the formation of inflammasomes. Inflammasomes recruit and subsequently turn pro-caspase 1 into its active form.
  • Activated caspase 1 splits full-length GSDMD to cleaved GSDMD and activate IL-lb, initiating pryoptosis.
  • cleaved GSDMD interacts with cell-membrane lipids to form transmembrane pores, which allows IL-lb, additional cytokines, and signals to be released and promote further inflammation and cell death.
  • This step in the IRI model was significantly suppressed by the administration of rhMG53 and was further confirmed by the mitigation of IL-lb release.
  • ATP was measured in tissue homogenate. When pretreated with MG53, ATP was significantly higher in tissue when compared with control. These cells with a high amount of ATP indicate functional respiration and intact mitochondrial function.
  • MG53 is involved in cell membrane repair and machinery. Protection of the endothelium is paramount in lung transplantation because damage to this barrier leads to further edema, injury, and eventual failure of the allograft. The delivery of therapeutics to the endothelium is vital to any candidate treatment intended to mitigate IRI, because the endothelium is the barrier between the recipient blood and the donor organ. IHC imaging after IRI and subsequent 24-hour survival demonstrated a co-localization of MG53 and CD31.
  • CD31 is a transmembrane immunoglobin-type inhibitory receptor that is expressed on endothelial cells.
  • Example 4 Mitsugumin-53 regulates pyroptosis and cell integrity during lung transplantation.
  • Lung transplantation was the only definitive therapy for end-stage lung disease. While the waiting list of patients eligible for transplant grows, the number of suitable donor organs falls far short of the demand. Less than 20% of offered lungs are successfully transplanted due to a limited ischemic time, and poor donor lung quality, manifested by pulmonary edema, hypoxia, or trauma. To address the critical unmet demand for suitable donor allografts, attention is directed to donation after circulatory death (DCD) and marginal or extended criteria donors. Unfortunately, ischemiareperfusion injury (IRI), which happens universally in transplantation, is especially pronounced in these allografts.
  • DCD circulatory death
  • IRI ischemiareperfusion injury
  • IRI happens universally in transplantation, triggering inflammatory responses and cell death which leads to PGD, organ rejection, and increased mortality;
  • IRI contributes to pyroptosis and release of inflammatory cytokines;
  • maintaining endothelial cellular integrity prevents IRI;
  • MG53 is a novel factor to regulate pyroptosis, preserving cell integrity.
  • lung IRI induced endothelial cell disruption and immune cell pyroptosis are important events leading to lung transplant failure where MG53 plays dual roles in promoting lung tissue integrity and inhibiting post-transplant inflammation to improve success of lung transplantation.
  • the mechanism of MG53 in preservation of lung endothelial cell integrity in transplant related injury is used to derive a mechanism for how circulating MG53 preserves the endothelial integrity, improves cell survival and reduces inflammation during IRI. Mechanisms are determined by which pulmonary vascular endothelial cells contribute to leukocyte signaling and inflammation. Furthermore, precision cut lung specimens allow modeling of endothelial cell-to-tissue level interaction and potential benefit of these therapies.
  • This step focuses on the role of MG53 in regulating transplantation induced pyroptosis.
  • the therapeutic benefits of MG53 to repair cell membrane damage induced by GsdmD- N mediated membrane pore formation are evaluated.
  • mg53- tPA-MG53, GsdmD-/- and WT mice are used to determine the role of MG53 in post-IRI induced neutrophilic pyroptosis.
  • the effective dose and time window of rhMG53 to protect neutrophils against pyroptotic death is determined.
  • lung transplantation experiments are performed in neutrophil specific GsdmD-/- mice to determine the role of pyroptosis in an in vivo model.
  • EVLP large animal and human lung ex vivo lung perfusion
  • rhMG53 to preserve and rescue lung allograft integrity.
  • the therapeutic benefit of rhMG53 supplementation for pulmonary applications is established.
  • Expertise in EVLP and transplantation is used to test the efficacy of rhMG53 in rescuing the injured allograft procured from pigs and non- transplantable human donor lungs.
  • Ischemia reperfusion injury drives PGD, whose hallmark is inflammation, leucocyte infiltration, and the activation of cellular immune responses that impact long term survival ( Figure 26).
  • the lung transplant community discards -80% of potential donor allografts out of fear of PGD.
  • PGD leads to pulmonary vascular endothelial cell injury ( Figure 27) and increased vascular permeability, which results in increased extravascular water/edema and respiratory failure.
  • agents providing effective mitigation of inflammation and PGD are a novel and of therapeutic benefit.
  • Pyroptosis is critical for the inflammatory response to IRI: Pyroptosis, a lytic form of cell death, is a key pathway triggering inflammation.
  • caspase- 1 Upon activation (e.g., danger associated molecular patterns (DAMPs) induced by IRI and factors associated with reperfusion), caspase- 1 processes IL-ip from an inactive precursor to the active form. Caspase-1 also cleaves Gasdermin D (GsdmD), a key factor of pyroptosis pathway.
  • GsdmD Gasdermin D
  • GsdmD N-terminus or GsdmD- N forms transmembrane pores that lead to cell membrane injury and leakage of intracellular contents, including active IL-ip, which triggers cell death and tissue inflammation.
  • endosomal sorting complexes required for transport (ESCRT) machinery has been proposed to repair pyroptosis-induced membrane damage, ESCRT blockage does not cause 100% pyroptotic cell death’ showing that there is an alternative pathway(s) for membrane repair after GsdmD activation.
  • MG53 protects against IRI induced lung cell membrane damage: Repair of injury to the cell membrane is an important aspect of physiology, and inadequate membrane repair results in pathophysiology in many human diseases, including cardiovascular dysfunctions. MG53 is an essential component of the cell membrane repair machinery. MG53 functions in vesicle trafficking and allows for nucleation of intracellular vesicles at sites of membrane disruption. Genetic ablation of MG53 results in defective membrane repair. The benefit of MG53 in lung injury and explored the therapeutic benefit in transplantation and acute lung injury has been demonstrated. Application of rhMG53 protein reduced pyroptotic cells in IRI injured lung tissue (Figure 33). Furthermore, live cell imaging revealed RFP-MG53 translocates to cell membrane upon activation of pyroptosis ( Figure 36). In vitro biochemical study also revealed that MG53 could bind to GsdmD ( Figure 37 A) and inhibit its cleavage by caspase- 1 ( Figure 37B).
  • IRI happens universally in transplantation triggering robust inflammatory responses and cellular death which leads to PGD, organ rejection, and increased mortality;
  • IRI contributes to pyroptosis activation and release of inflammatory cytokines;
  • maintaining endothelial cellular and cell junction integrity prevents IRI;
  • MG53 is a factor that regulates pyroptosis, preserving cell integrity (Figure 28). It has been shown that an absence of MG53 in mice leaves the lung at risk of injury . It was demonstrated that rhMG53 protein can mitigate lung dysfunction associated with ventilator induced lung injury (VILI) and IRI.
  • VILI ventilator induced lung injury
  • Rodent and porcine large animal models of EVLP and the corresponding in vivo models of lung transplantation have been established ( Figures. 35, 39, 41, 43 & 44).
  • the rodent lung transplant models have allowed evaluation of the in vivo function of MG53 on the progression of transplant associated lung injury. It has been recapitulated the transplant effect of IRI using precision cut lung slices (PCLS) to simulate the transplant experience inhibition in tissue preserving the complex 3-dimensional micro- environment of the lung.
  • PCLS precision cut lung slices
  • the mechanism of MG53 in preservation of lung endothelial cell integrity in transplant related injury The potential benefit of MG53 is shown at the endothelial cell level, cellular, molecular, and live cell imaging tools are used to derive a mechanistic base for how circulating MG53 preserves endothelial cell integrity, improves cell survival, and reduces inflammation during IRI. These studies determine mechanisms by which pulmonary vascular endothelial cells contribute to leukocyte signaling and inflammation. This study establishes a set of biomarkers modulated by rhMG53 that quantifiably predicts lung injury prior to transplantation.
  • rhMG53 suppresses IRI-induced ET-1 elevation in rat lung and cultured endothelial cells: Utilizing this established rat model of EVLP, the impact of rhMG53 on lung injury was evaluated. It is demonstrated (Figure 29A) that 1 hour of warm ischemia induced structural damage to the rat lung that could be rescued by EVLP with exogenous rhMG53 (5 pg/ml perfusate concentration). Prior studies by other investigators have shown that ET-1 correlates with PGD and lung injury. ET-1 is elevated in transbronchial biopsies under suspicion of infection or rejection post lung transplantation.
  • rhMG53 protects against injury to endothelial cells: Live cell imaging was performed with GFP-MG53 transiently expressed in aortic endothelial cells (AoEC). Following microelectrode induced injury to the plasma membrane, rapid translocation of MG53 was observed at the injury site, similar to what has been previously observed with other cell types. Using the established glass-beads induced membrane damage assays, it was showed that rhMG53 displayed dose-dependent reduction of LDH release, supporting the membrane repair function of rhMG53 in AoEC and pulmonary artery endothelial cells (PAEC). Moreover, it is shown that rhMG53 is effective in protection of H2O2 and IRI induced endothelial cell injury.
  • AoEC aortic endothelial cells
  • Alexa647-rhMG53 studies were conducted with fluorescent conjugated Alexa647-rhMG53, and it was found that the hypoxia and reoxygenation (H/R) induced injury to AoECs led to rapid uptake of Alexa647-rhMG53 into the cytosol ( Figure 30).
  • PS phosphatidylserine
  • Alexa647-BSA did not show uptake into the cells.
  • the protective function of rhMG53 in endothelial cells may involve both plasma membrane and intracellular signaling.
  • rhMG53 is an effective reagent to preserve endothelial cell integrity.
  • This study aimed to define the mechanistic function of circulating MG53 or exogenous rhMG53 in preservation of endothelial cell integrity.
  • These studies test the hypothesis that, in addition to plasma membrane preservation, endocytic uptake of rhMG53 can contribute to the survival of endothelial cells under ischemic conditions that occur during lung transplantation.
  • the data shows a candidate pathway is the VEGF receptor ( Figure 31). Live-cell imaging and CRISPR- gene editing are utilized to show the mechanism of VEGFR2-mediated MG53 uptake in endothelial cell protection.
  • MG53 endocytic uptake to preserve endothelial cell integrity It was previously shown that extracellular rhMG53 can recognize exposed PS to preserve plasma membrane integrity of lung epithelial cells under A/R conditions. To test if MG53 binding to PS mediates membrane repair function of endothelial cells, experiments are conducted by co-treating the hypoxic cells with different lipids (PS and phosphatidylcholine) and rhMG53 to test whether competitive binding with the cell membrane disruption would facilitate or inhibit rhMG53- mediated endothelial membrane repair. In a co-culture model of endothelial cells and THP-1 macrophages, the response to H/R results in an exacerbated inflammatory and VEGF response.
  • PAECs function in a relative hypoxic physiologic environment as compared to AoECs and would potentially respond to transplant related hypoxia and IRI differently.
  • Published studies from Ulyatt et al. and Nadeau demonstrate that hypoxia decreases VEGFR2 expression showing a role of VEGFR2 in the lung under hypoxic, transplant related conditions. It was proposed to use CRISPR to knockout VEGFR2 from PAEC and AoEC to test the contribution of VEGFR2 mediated rhMG53 uptake into endothelial cells, and its impact on rhMG53-mediated preservation of endothelial cell integrity and survival under hypoxic conditions.
  • pharmacological agents that influence endocytosis or pinocytosis pathways for rhMG53 entry into AoEC and PAEC i.e., receptor mediated via clathrin coated pits or fluid phase mediated via non-clathrin coated endosomal uptake across the cell membrane are used.
  • ET-1 expression is controlled by numerous stressors on endothelial cells (e.g., cytokine, oxidative stress, hypoxia shear) and increased ET-1 levels are modulated by hypoxia or cytokine induction of endothelin converting enzyme (ECE) activity to cleave the precursor big ET-1.
  • ECE endothelin converting enzyme
  • the role of ET-1 as a marker of lung injury or rejection post-transplant has been well established.
  • Endothelial cell IRI induces generation of reactive oxygen species (ROS) which may contribute to elevation of ET-1. It has previously been demonstrated that rhMG53 can enter cells, protect mitochondria from injury, and reduce ROS generation.
  • ROS reactive oxygen species
  • the pulmonary vascular endothelium is the gateway to leukocyte infiltration in the lung parenchyma: THP-1 macrophages were isolated with mg53-/- genetic ablation and through co-culture with endothelial cells and when the co-culture model of where mg53-/- THP-1 cells are utilized in a H/R model, increased inflammasome response was demonstrated (Figure 32, IL-ip, TNF-a, LDH) which is mitigated by administration of rhMG53.
  • ROS and rhMG53 uptake into PAEC and AoEC cells are simultaneously monitored, and tests are performed to find whether rhMG53- mediated suppression in ROS correlates with reduction of ET-1 release from these cells.
  • PCLS Precision Cut Lung Slices
  • Figures. 30-34 While cell culture ( Figures. 30-34) models have the ability to provide insight into mechanistic approaches to the MG53 protection of endothelium and the ability to regulate inflammation and indirectly regulate cell survival, there are limitations.
  • non- transplantable human tissue and diseased tissue are obtained from a local organ procurement organization (Lifeline of Ohio OPO) and a transplant repository.
  • PCLS undergo intervals of H/R with the addition of dose escalating rhMG53, disulfiram (as pyroptotic inhibitor control) and the combination.
  • This disease modeling allows the assessment of the secretome, cytokines and environmental stressors as well as the interaction of the micro-environment with the endothelial cells, leukocytes, and pneumocytes. Supernatant is collected for analysis and to be used as conditioned medium for cell culture and analysis. Advanced imaging (Leica Thunder) is utilized to perform high resolution imaging and time lapse microscopy. Additionally, Spatial Transcriptomics via Visium (lOx Genomics) is employed as a functional discovery system to analyze whole transcriptomes in healthy and IRI of non-transplantable human donor lung sections.
  • LPS lipopolysaccharide
  • Ni Nigericin
  • pyroptosis might be a key pathway of neutrophil death in lung tissue after IRI contributing to development of PGD and rhMG53 treatment could inhibit neutrophil pyroptosis and might preserve lung function.
  • mg53-/ ⁇ lung recipient mice show higher levels of pyroptosis and inflammation: Previous studies have established that MG53 repairs cell membrane injury and plays a critical role in protecting against lung injury. In addition, rhMG53 treatment reduced pyroptotic cells in IRI injury model ( Figure 34), Next, the role of MG53 and pyroptosis was tested in a murine lung transplantation model.
  • MG53 translocates to the plasma membrane but cannot inhibit activation of caspase- 1:
  • THP-1 cells neurotrophils are known to have a short lifetime of less than 10 hours in culture condition was used. Thus, THP-1 cells were used).
  • First live cell imaging was performed with THP-1 cells overexpressing RFP-MG53.
  • THP-1 cells were treated using various concentrations of MG53 (0, 0.1, 1, 10 pg/ml) together with LPS+Ni (induction of pyroptosis). It was found that the activation of caspase- 1, evaluated by the band intensity of p20 and plO, was not affected by MG53 ( Figure 36B). Thus, MG53 might affect caspase- 1 mediated GsdmD cleavage.
  • MG53 directly binds to GsdmD to inhibit its cleavage by caspase-1: To test the second possibility, a co-immunoprecipitation experiment was first performed. As shown in Figure 37A, MG53 and GsdmD could interact with each other. To test the potential function of MG53 on GsdmD cleavage by caspase-1, rhMG53 was incubated with caspase-1 before adding to GsdmD. As shown in Figure 37B (left), the inhibition of cleavage was prominent after 10 min, where a significant fraction of GsdmD was cleaved.
  • the inhibition of the cleavage can be caused by direct inhibition of the enzyme caspase- 1, or by an interaction of GsdmD and MG53 that protects the exposure of the cleavage site. Subsequently GsdmD fused with an N-terminal Sumo (small ubiquitin-related modifier) domain was used, which often increases the solubility of the fusion proteins using its surface hydrophilic residues. If MG53 interacts with caspase- 1, the interaction should not be altered by the presence of Sumo domain on GsdmD.
  • MG53 suppresses the leakage of the liposome induced by GsdmD-N: While experiments in Figure 37 demonstrated that MG53 could inhibit caspase-1 induced GsdmD cleavage, it was still unclear whether MG53 could directly inhibit pyroptosis activity.
  • a liposome reconstitution assay was performed as shown in previous publication, where GsdmD-N transmembrane pore formation can be quantified to serve as a functional indicator of pyroptosis activity. Briefly, liposomes containing fluorophore are treated with GsdmD and caspase- 1. Caspase- 1 could cleave and activate GsdmD forming transmembrane pores inducing the release of fluorescent dye inside the liposome.
  • the released fluorophore is quenched by polyclonal antibodies present in solution outside of liposome.
  • polyclonal antibodies present in solution outside of liposome.
  • MG53 is a key component of cell membrane damage repair machinery in the lung, and it quickly accumulates on cell membrane upon pyroptosis (Figure 36), it was hypothesized that GsdmD-N formed plasma membrane pores can be repaired by MG53 and the data in Figure 37 show a direct binding between MG53 and cleaved GsdmD-N. Thus, it is determined how MG53 interacts with GsdmD to prevent its cleavage.
  • MG53 may interact with GsdmD-N (after pore formation). This is supported by the facts that 1. MG53 translocates to the plasma membrane after pyroptosis activation ( Figure 36), 2. MG53 interacts with GsdmD (intact) but does not interact with sumoGsdmD (GsdmD-N might be blocked by sumo motif) ( Figure 37), 3. Both GsdmD-N and MG53 have a high binding affinity to PS during cell membrane damage and repair. Time course of interaction between MG53 and GsdmD-N is determined by live cell imaging.
  • Annexin V (a known PS binding protein) is used to show the involvement of PS in the interaction between GsdmD-N and MG53. Colocalization between MG53 and GsdmD-N is observed therefore, native gels are performed to investigate the possible physical interaction between MG53 & GsdmD-N after pyroptosis activation. MG53 repairs membrane to inhibit pyroptosis.
  • the ESCRT machinery and MG53 are two parallel systems that sense and repair the damaged membrane using a similar mechanism. ESCRT repairs GsdmD-N induced membrane injury, but deletion of ESCRT does not lead to 100% cell pyroptosis, showing there can be an alternative pathway for membrane repair after GsdmD activation.
  • Neutrophils from mg53-/-, tPA-mg53, GsdmD-/-, and WT littermate mice are isolated freshly.
  • Dose and time effects of rhMG53 are investigated upon induction of pyroptosis in neutrophils (Figure 39).
  • the protocol for induction of neutrophil pyroptosis is: 1-hour LPS priming, followed by administration of Ni.
  • rhMG53 (0, 0.1, 1, 10, 100 pg/ml) are used to determine an effective dose of rhMG53 in protecting neutrophils against pyroptosis.
  • Live cell imaging of FM1-43 dye entry into cells is used as an index for cell membrane injury.
  • pyroptotic markers, GsdmD-N, and caspase- 1 activation are assessed using WB as endpoint measurement.
  • the release of LDH into the culture medium in response to various experimental manipulations are used as an alternative method to assess cell membrane integrity.
  • MG53 The impact of MG53 is investigated on time course of inflammation and pyroptosis evolution.
  • GsdmD-/- cells are a negative control for neutrophil pyroptosis and membrane damage; tPA-MG53 neutrophils and overexpressing endogenous MG53 are protected from pyroptosis without rhMG53.
  • dose effects of rhMG53 Dose-effects of rhMG53 on WT neutrophils elucidate whether MG53 could fully block pyroptosis at high concentrations.
  • the data of MG53’s effects on liposome leakage (Figure 38) showed an incomplete blockage of leakage. Either complete or partial blockage highlights a new mechanism of MG53 to protect lung IRI.
  • Timing effects of rhMG53 Protective effects are observed when rhMG53 is added before adding Ni or LPS. When MG53 repairs pyroptosis induced membrane damage, significant protection with MG53 added 10 min post Ni is observed.
  • mice models There are many cell types in the pulmonary parenchyma, e.g., alveolar macrophages, interstitial macrophages, ATI and AT2 pneumocytes, neutrophils, fibroblasts, and endothelial cells.
  • the ischemic insult could lead to cell death of all types of cells in the lung, with the release of many cellular factors with varying biological roles.
  • GsdmD-N is primarily located in neutrophils after IRI ( Figure 34), it does not rule out the possibility that other cell types do not undergo pyroptosis or contribute to pulmonary damage.
  • mice consist of: (1) sham injury, (2) IRI + vehicle, (3) IRI + dose escalation of rhMG53, (4) IRI + disulfiram (0-40 pM), and (5) IRI + rhMG53 + disulfiram.
  • One cohort of mice is used to perform histological, biochemical, and biomolecular analysis with tissue. For histology, a portion of the lung is processed for H&E and lung injury scores are assessed. BAL is collected to look for protein, total cells, and neutrophils. Lung tissue is collected for WB (IL-1, IL-6, TNF- a, ET-1), wet/dry ratio, MPO activity, and qPCR for inflammation.
  • WB IL-1, IL-6, TNF- a, ET-1
  • Pulmonary function is assessed by plethysmography and Flexivent prior to sacrifice of the survivors.
  • raw survival from injury is assessed via the Kaplan-Meier method, and body weight is monitored with daily plethysmography.
  • BAL and serum are collected at the time of sacrifice.
  • the right upper lobe is collected for histology and immunohistochemistry.
  • Efficacy is assessed by physiologic and molecular metrics established in Pls’ labs ( Figures. 33-34).
  • B iometric signature of molecular markers is employed (ET-1, BigET- 1, LDH, HA, IL-1B, NLRP-3, caspase- 1/3/7, flow cytometry) and pulmonary function (Flexivent and plethysmography) and arterial blood oxygenation (ABG).
  • Lung injury is assessed by lung permeability index (the ratio of BAL) protein to serum protein concentrations. Wet-to-dry ratio is evaluated by the severity of pulmonary edema and lung tissue is used to create lysate to measure biomarkers.
  • ET-1 expression is controlled by numerous stressors on endothelial cells (e.g., cytokine, oxidative stress, hypoxia shear) and increased ET-1 levels are modulated by hypoxia or cytokine induction of endothelin converting enzyme (ECE) activity.
  • ECE endothelin converting enzyme
  • GsdmD-/- mice are ideal as a recipient mouse line to eliminate interference caused by pyroptosis from whole animal.
  • the following series of transplantation experiments are used to specifically assess the role of neutrophils in transplant IRI. All mice of listed genotypes are available for the protocol. Completion of the studies have led to show that 1, the extent that neutrophils contribute to pyroptosis-induced post-IRI damage, 2, if MG53 directly inhibits neutrophil pyroptosis in vivo, 3, if the cell type origin of MG53 to inhibit post-IRI pyroptosis.
  • left donor lung allografts with 60-min warm ischemia are transplanted GsdmD-/- mice.
  • a group of mice are sacrificed 24 hours after surgery, with lungs, serum, and BAL being collected.
  • recipient mice survive a week from transplantation with serum collection at 1-, 3-, 7-days.
  • Standard histology, ELISA and immunohistochemistry are employed.
  • Longitudinal pulmonary function assessed via plethysmography at 1-, 3-, 7-days post-transplant (Plethysmography enables non-invasive, longitudinal pulmonary function, and lung mechanics data over the post IRI period. This enables the study to conserve animals and minimize stress due to sedation) and Flexivent is conducted prior to sacrifice.
  • Transplanted lungs contain cell types other than neutrophils, such as macrophages that might contribute to IRI.
  • GsdmD-/- mice have been successfully generated neutrophil specific GsdmD-/- mice by breeding LysMCre mice with GsdmD fl/fl mice.
  • LysMCre mice with GsdmD fl/fl mice.
  • six LysMCre/+ :GsdmD fl/fl mice were present in the mouse colonies.
  • the mice show similar phenotypes as their littermate controls, showing conditional GsdmD-/- does not interference with mouse physiology in basal condition. This interrogates the neutrophil contribution.
  • This study allows to study a) the use of porcine model of EVLP and lung transplantation to test whether the addition of rhMG53 the preservation process or after transplantation can have beneficial effects to prevent allograft injury; and b) test human lung tissue to support the premise of rhMG53 supplementation in transplant relevant IRI.
  • Therapeutic window of rhMG53 administration for organ preservation and EVLP assessment followed by transplantation Based on these data, the following set of porcine studies as outlined in Figure 59 are performed.
  • the proposed experimental groups are designed to test the injury associated with warm ischemia, the cold ischemia during storage and transport, and the injury associated with normo-thermic EVLP.
  • rhMG53 is added prior to cardiac arrest in the donor animal to determine the preventive effect of rhMG53 against ischemic injuries (Point A).
  • Second, rhMG53 can then be added to the normothermic EVLP Steen® solution (Point C), to assay the resuscitative effect of MG53 in preservation of to the lung during the EVLP reconditioning procedure or prior to reperfusion (Point D).
  • isolated left single lung transplant are performed in the heparinized recipient.
  • the explanted donor lung is used for assessment of endothelial cell health and lung integrity.
  • the native right and transplanted left lung is recovered and analyzed.
  • Standard clinical endpoints assess allograft integrity prior to lung transplantation and tissue and perfusate are banked for post-hoc analysis.
  • the native and transplanted lung is recovered and evaluated.
  • the right pulmonary artery is occluded and a 100% ventilated FiO2 is used with assessment of pulmonary mechanics to calculate terminal PaO2/FiO2 (P/F) ratio and function.
  • P/F PaO2/FiO2
  • IRI ischemia reperfusion injury
  • PGD primary graft dysfunction
  • exogenous MG53 administration had the ability to rescue injured allografts after prolonged CSS, prevent injury and return them close to a physiologic baseline.
  • This study builds on the premise that exogenous MG53 administration functions as a therapeutic during lung transplantation mitigating IRI.
  • LDH Lactate dehydrogenase
  • Ringoir SM Serum lactate dehydrogenase isozymes in human lung homotransplantation. Clin Chim Acta. 1975;58:291-4.

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Abstract

La présente divulgation concerne des compositions et des procédés comprenant de la mitsugumine-53 humaine recombinante (rhMG53) et leurs utilisations.
PCT/US2024/040744 2023-08-03 2024-08-02 Compositions de mitsugumine-53 humaine recombinante et leurs procédés d'utilisation Pending WO2025030108A2 (fr)

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WO2025030108A2 true WO2025030108A2 (fr) 2025-02-06
WO2025030108A3 WO2025030108A3 (fr) 2025-04-10
WO2025030108A9 WO2025030108A9 (fr) 2025-05-30

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WO2011002926A2 (fr) * 2009-07-01 2011-01-06 The General Hospital Corporation Cellules adultes isolées, organes artificiels, organes réhabilités, outils de recherche, revêtements d'organe, systèmes de perfusion d'organe, et méthodes de préparation et d'utilisation de ceux-ci
WO2016109638A1 (fr) * 2014-12-30 2016-07-07 Rutgers, The State University Of New Jersey Compositions et méthodes pour la prévention et la réparation d'une lésion rénale aiguë
WO2018024110A1 (fr) * 2016-08-01 2018-02-08 北京大学 Mutant de mg53, son procédé de préparation et ses utilisations
EP3982999B1 (fr) * 2019-06-17 2026-01-28 Trim-Edicine, Inc. Composition et méthode de traitement d'une lésion tissulaire hépatique

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WO2025030108A3 (fr) 2025-04-10

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