Modifying transplantable organs ex VIVO with RNA lipid nanoparticle perfusion

Abstract

The present invention provides methods for modifying a donor organ, e.g., liver, or a portion thereof. The methods comprise contacting the donor liver with lipid nanoparticles comprising RNA. Compositions of lipid nanoparticles comprising RNA are also provided. The disclosed LNPs may comprise an siRNA, e.g., targeting tissue plasminogen activator (tPA) or plasminogen activator inhibitor 1 (PAI-1). The LNPs may comprise an mRNA encoding a protein, e.g., Nuclear Factor-erythroid factor 2-related factor 2 (NRF2) or thrombomodulin.

Description

[0001] MODIFYING TRANSPLANTABLE ORGANS EX VIVO WITH RNA LIPID NANOPARTICLE PERFUSION

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority to U.S. Provisional Patent Application No.

[0004] 63 / 709,856 that was filed October 21, 2024, the entire contents of which are hereby incorporated by reference.

[0005] SEQUENCE LISTING A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “160180.00196. xml” which is 27,992 bytes in size and was created on October 21, 2025. The sequence listing is electronically submitted via Patent Center and is incorporated by reference herein in its entirety.

[0006] BACKGROUND

[0007] Liver transplantation is the sole curative treatment for end-stage liver disease. However, the demand for liver transplants exceeds the available organs, which results in a substantial waiting list and high mortality rates (7). Marginal grafts (livers complicated by old age, fatty composition, or extended ischemia time) are underutilized, in part due to the risk of graft failure from ischemiareperfusion injury (IRI). IRI is a leading cause of organ damage and graft failure during procurement and reimplantation, which can be exacerbated in marginal livers. While the use of machine perfusion techniques, including normothermic machine perfusion (NMP) and hypothermic machine perfusion, show promise in mitigating IRI and graft failure in marginal livers, additional therapeutic measures are required.

[0008] SUMMARY

[0009] In an aspect of this disclosure, methods of modifying a donor organ, e.g., a liver, or a portion thereof, e.g., a liver graft, are provided. In some embodiments, the methods comprise contacting the donor liver or donor liver graft with lipid nanoparticles (LNPs) comprising an RNA.

[0010] In an aspect of this disclosure, compositions comprising lipid nanoparticles (LNPs) are provided. In some embodiments, the compositions comprise LNPs comprising an RNA, wherein the RNA is selected from: (a) an siRNA that reduces the expression of tissue plasminogen activator (tPA) or plasminogen activator inhibitor 1 (PAI-1); or an mRNA encoding a Nuclear Factor-erythroid factor 2-related factor 2 (NRF2) protein or a thrombomodulin protein.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS FIGs. 1A, IB, and 1C show lipid nanoparticles deliver functional RNA to the liver, with systemic downstream protein signal. A) Mice injected with mRNA-LNP encoding firefly luciferase express functional luciferase protein predominantly in the liver, as visualized by in vivo luminescent imaging (IVIS). Relative luminescence scale shown at right. B) Plasma levels of fibrinogen (Fgn, arrow) from Fga’’ mice injected with mRNA-LNP encoding fibrinogen alpha chain (Fga), detected by fluorescent immunoblot. C) Plasma fibrinogen levels of mice injected with LNP containing siRNA targeting fibrinogen alpha chain (siFga), control siRNA targeting luciferase (siLuc), or untreated controls (No LNP).

[0012] FIGs. 2A and 2B show mRNA-LNP transfection of rat liver in a model of perfusion and transplant. A) mRNA-LNP containing mRNA encoding reporter protein, or control RNA, was administered to rats through either intravenous route (in vivo (I. V.)), administered IV prior to liver harvest and machine perfusion (Dose pre-perfusion), or during perfusion of isolated organs (dose during perfusion). mRNA uptake was quantified using RT-qPCR, and reported as mRNA level relative to in vivo (I.V.) dosing. B) mRNA-LNP encoding reporter protein NanoLuciferase was delivered to rat liver 30 min after initializing machine perfusion. Samples were collected from peripheral tissue segments and from perfusate solution at indicated time points (x-axis). Samples were assessed for Nanoluciferase protein content by either directly (perfusate samples), or following protein extraction (liver tissue samples), by enzymatic assays of Nanoluciferase activity (y-axis). Dots represent n=l.

[0013] FIGs. 3A, 3B, 3C, and 3D show mRNA-LNP transfection of transplantable livers in a rat model of perfusion. A) Schematic of transfection models. mRNA-LNP was injected either systemically by tail vein injection (in situ) or to the liver alone by injection into the perfusate during normothermic machine perfusion (NMP). After 4 hours of incubation either in situ or during NMP, livers were harvested for protein and RNA analysis. B,C) NanoLuc protein signal (B) and mRNA uptake (C) in transfected livers was measured by enzymatic activity assays and RT-qPCR, respectively. Colors as in A for each group, bars represent mean ± SEM, n = 3; * / ? < 0.05, ns, not significant compared to untreated controls. D) Stability of mRNA-LNP in perfusate, reported as mRNA encapsulation. FTGs. 4 A, 4B, 4C, and 4D show ischemic damage and repair influences but does not abolish hepatic LNP transfection. A) Schematic of ischemia - reperfusion injury (IRI) model. Rat livers were clamped to induce warm ischemia. Clamps were released after 1 h of ischemia and liver was re-perfused for 1, 4, or 24 h before LNP injection by I.V. Livers were harvested after 4 h of LNP incubation. B,C) NanoLuc protein signal (B) and mRNA uptake (C) in transfected livers measured by enzymatic activity assays and RL-qPCR, respectively. Control livers received either no LNP (Untreated) or no ischemia (No injury). Bars represent mean ± SEM, n = 2-3. Ns, not significant; *, p < 0.05. D) Immunofluorescent staining images of transfected rat liver following IRI. Exogenous luciferase protein shown in green, DAPI shown in blue. Red staining denotes liver sinusoidal endothelial cells (top row), or liver resident Kupffer cells (bottom row).

[0014] DETAILED DESCRIPTION

[0015] The present invention provides methods for modifying a donor liver. The methods comprise contacting the donor liver with lipid nanoparticles comprising RNA. Compositions of lipid nanoparticles comprising a polynucleotide, e.g., RNA, are also provided.

[0016] Methods for modifying a donor organ

[0017] In an aspect of this disclosure, methods of modifying a donor organ, e.g., a liver, or a portion thereof, are provided. In some embodiments, the methods comprise contacting the donor organ with lipid nanoparticles (LNPs) comprising a polynucleotide, e.g., an RNA.

[0018] The donor organ may comprise, e.g., liver, kidney, pancreas, lung, or heart.

[0019] A “donor liver” is a liver or a portion thereof that is removed from one subject, i.e., the “donor”, and transplanted into another subject, i.e., the “recipient,” in an organ transplant procedure.

[0020] A “marginal liver” is a liver that has characteristics that may increase the risk of poor outcomes after transplantation. Examples of such characteristics include, but are not limited to, donor age (e.g., equal to or over 60 years old), fatty composition (steatosis approx. >30%) which may be determined by a physician, and extended ischemia time. Extended ischemia time may be determined by a physician. The disclosed methods and compositions may be used preventatively on a solid organ that is believed to have experienced ischemia.

[0021] The term “modifying” is used herein to refer to process that alters gene expression in a donor liver. Modification may result in increased expression of a native gene, reduced expression of a native gene, or expression of a heterologous gene (i.e., a gene that the subject does not natural have).

[0022] The LNPs may be “contacted” by introducing the LNPs into the circulatory vessels in the donor liver, e.g., ex vivo using a perfusion machine, or prior to or after machine perfusion; by intravenously administering the LNPs to the donor subject; or by administration to the graft recipient after or at the time of transplant.

[0023] The term “lipid nanoparticle” refers to a particle, e.g., a spheroid or spherical particle, made of lipids. The LNPs may further encapsulate a payload. Lipid nanoparticles may be used to deliver drugs and nucleic acids and may encapsulate such molecules. Examples of suitable lipids for use in lipid nanoparticles include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidyl serine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). The lipid nanoparticles of the present invention are used to deliver one or more polynucleotides, e.g., RNAs, to the cells of a donor liver.

[0024] The LNPs may comprise (a) at least one (ionizable) cationic lipid; (b) at least one helper lipid; (c) a sterol; and (d) at least one lipid-polyethylene glycol conjugate. The LNPs may comprise (i) 30-55 percent by molecular weight (mol %) of the at least one (ionizable) cationic lipid; (ii) 5-20 mol % of the at least one helper lipid; (iii) 25-50 mol % of the sterol; and (iv) 0.5 - 3 mol % of the at least one lipid-polyethylene glycol conjugate

[0025] The at least one ionizable cationic lipid may be one or more of SM-102, ALC-0315, ALC-0159, or DLin-MC3-DMA.

[0026] The at least one helper lipid may be one or more of phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), egg sphingomyelin (ESM).

[0027] The sterol in the lipid mixture may be one or more of cholesterol or a cholesterol derivative. The at least one lipid polyethylene glycol conjugate may be l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 (DSPE-PEG2000), or PEG-1, 2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG). The term “intravenous administration” refers to administration of a composition into a vein. Intravenous administration may be accomplished using catheter or a needle. It may be administered as a bolus or as an infusion.

[0028] “Machine perfusion” is a technique that uses artificial blood perfusion to preserve organs and facilitate organ transplantation. It involves continuously pumping a specialized solution through an organ to mimic the body's natural blood flow, while also controlling the organ's temperature, oxygen levels, chemical composition, and mechanical stress. Suitable machine perfusion techniques include, but are not limited to, hypothermic machine perfusion (HMP), including hypothermic oxygenated machine perfusion (HOPE) (2-12 °C), sub-normothermic machine perfusion (SNMP) (20-25 °C), and normothermic machine perfusion (NMP) (35-37.5 °C).

[0029] “Silencing RNA” or “small-interfering RNA” (siRNA) is a class of double-stranded RNA molecules that can silence gene expression by interfering with mRNA translation. siRNAs are typically about 19-27 nucleotides long. They function by binding to a target transcript and triggering an RNA interference (RNAi) response, resulting in cleavage of the target transcript. The siRNA may comprise one of SEQ ID NOs: 10-17.

[0030] “Messenger RNA” (mRNA) is a single-stranded RNA molecule that corresponds to the genetic sequence of a gene and is read by a ribosome in the process of synthesizing a protein.

[0031] The mRNA may encode SEQ ID NO 1 or 3, or a sequence with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NO: 1 or 3. The mRNAs may comprise one of SEQ ID NOs: 2 or 4, or a sequence with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NO: 2 or 4.

[0032] The disclosed polynucleotides, e.g., mRNAs or siRNAs, may comprise at least one modified nucleotide. The modified nucleotides may comprise 1-methyl-pseudouridine (Mir ), pseudouridine

[0033]

[0034] 5-methoxyuridine, or other modifications. As used herein, “modified nucleotides” refer to nucleotide bases which are not found in nature, or which undergo chemical modification from the standard nucleotide structure (A, C, U, G). Exemplary modified nucleotides may include, but are not limited to, modified nucleotides such as 2'-O-methyl (2'0Me) nucleotides, 2 '-deoxy-2 '-fluoro (2'F) nucleotides, 2'-deoxy nucleotides, 2'-O-(2-methoxyethyl) (MOE) nucleotides, and the like. The preparation of modified siRNA is known by one skilled in the art. In some embodiments, the disclosed dsRNA molecules include one or more modified nucleotides at the 5 '-terminus of the passenger strand of the dsRNA that prevent incorporation of the passenger strand into RISC (See, e.g., Walton et al., Minireview: “Designing highly active siRNAs for therapeutic applications,” the FEBS Journal, 277 (2010) 4806-4813). Further, the disclosed compositions may comprise nucleotides with phosphorothioate modifications to the ribose sugar moiety on a nucleotide or “locked nucleic acids” (LNA), a type of modified nucleotide, which, as used herein, refers to nucleotides comprising a 2'-O, 4'-C methylene bridge. An advantage of LNA incorporation into siRNAs (or dsRNAs in general) comprises increased stability.

[0035] The design and generation of siRNAs is within the skill of an ordinary artisan. For example, siRNAs targeting tPA or PAI-1 may be determined using the consensus sequences for tPA (amino acid sequence SEQ ID NO: 5, mRNA, SEQ ID NO: 6) or PALI (amino acid sequence: SEQ ID NO: 7, mRNA: SEQ ID NO: 8).

[0036] A “constitutively active point mutation” is a genetic mutation that causes a gene to be always "on" or active, regardless of environmental conditions. Examples of constitutively active point mutants include, but are not limited to, Nrf2-T80K, and THDB-C537Stop. See, e g., Huppke et al., “Activating de novo mutations in NFE2L2 encoding NRF2 cause a multisystem disorder” Nat. Commun. 2017 Oct 10;8(l):818 and Dargaud et al. “Characterization of an autosomal dominant bleeding disorder caused by a thrombomodulin mutation” Blood. 2015 Feb 26; 125(9): 1497-501 , which are incorporated by reference herein in their entireties.

[0037] Mitigating Ischemia-Reperfusion Injury

[0038] Expressing Nrf2 (Nrf2 controls expression of antioxidant response element (ARE)-dependent genes to regulate the physiological and pathophysiological outcomes of oxidant exposure).

[0039] Thrombosis Reducing PAI-1 (high levels of PAI-1 are associated with thrombosis), expressing thrombomodulin (Thrombosis is a significant concern following transplant, and may be mitigated by expression of thrombomodulin or silencing PAI-1 expression)

[0040] Defatting

[0041] Reducing tPA (Hepatic tPA has a role in reducing apoB lipidation and VLDL assembly in hepatocytes; reducing hepatic tPA may promote lipid secretion and mobilization from livers) Bleeding

[0042] siRNA targeting plasminogen (downregulating plasminogen favours clot stability) The disclosed methods may comprise the use of LNPs comprising mRNA encoding gene editors, e.g., nucleic acid-guided nucleases and associated guide RNAs (gRNAs). These gene editors may be used to permanently induce the effects of upregulated protein activity (from introducing activating mutations), or downregulation (by editing for knockdown) which is otherwise achieved by transient mRNA or siRNA strategies. These targets for permanent gene editing may include, but are not limited to, the above targets of Nrf2, thrombomodulin, or tPA. The design of guide RNAs targeting, e.g., Nrf2, thrombomodulin, or tPA is within the skill of the ordinary artisan and may be performed according to known methods. See, e.g., Mohr et al., “CRISPR guide RNA design for research applications” FEBS J . 2016 Sep;283(17):3232-8, which is incorporated by reference herein.

[0043] Compositions

[0044] In an aspect of this disclosure, compositions are provided. In some embodiments, the compositions comprise lipid nanoparticles comprising an RNA, wherein the RNA is selected from: (a) an siRNA that reduces the expression of tissue plasminogen activator (tPA) or plasminogen activator inhibitor 1 (PAI-1); or an mRNA that encodes a Nrf2 protein or a thrombomodulin protein.

[0045] The lipid nanoparticles may comprise (a) at least one (ionizable) cationic lipid; (b) at least one helper lipid; (c) a sterol; and (d) at least one lipid-polyethylene glycol conjugate, as discussed above.

[0046] The siRNA for use in the methods and compositions may comprise one of SEQ ID NOs: 10-17. SEQ ID NOs 10 and 11, 12 and 13, 14 and 15, and 16 and 17 may be used as duplex pairs in siRNA compositions to reduce expression of PAI-1. See also WO2024243694A1. The mRNA may encode SEQ ID NO 1 or 3, or a sequence with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NO: 1 or 3. The mRNAs may comprise one of SEQ ID NOs: 2 or 4, or a sequence with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NO: 2 or 4.

[0047] Nrf2 (SEQ ID NO: 1) : (protein)

[0048] MMDLELPPPGLPSQQDMDLIDILWRQDIDLGVSREVFDFSQRRKEYELEKQKKLEKERQ EQLQKEQEKAFFAQLQLDEETGEFLPIQPAQHIQSETSGSANYSQVAHIPKSDALYFDDC MQLLAQTFPFVDDNEVSSATFQSLVPDIPGHIESPVFIATNQAQSPETSVAQVAPVDLDGM QQDIEQVWEELLSIPELQCLNIENDKLVETTMVPSPEAKLTEVDNYHFYSSIPSMEKEVG NC SPHFLNAFED SF S SIL STEDPNQLT VNSLNSD AT VNTDFGDEF YS AFIAEP SISNSMP SPA TLSHSLSELLNGPIDVSDLSLCKAFNQNHPESTAEFNDSDSGISLNTSPSVASPEHSVESSS YGDTLLGLSDSEVEELDSAPGSVKQNGPKTPVHSSGDMVQPLSPSQGQSTHVHDAQCE NTPEKELPVSPGHRKTPFTKDKHSSRLEAHLTRDELRAKALHIPFPVEKIINLPVVDFNEM MSKEQFNEAQLALIRDIRRRGKNKVAAQNCRKRKLENIVELEQDLDHLKDEKEKLLKEK GENDKSLHLLKKQLSTLYLEVFSMLRDEDGKPYSPSEYSLQQTRDGNVFLVPKSKKPDV KKN*

[0049] Nr£2 (mRNA, SEQ ID NO: 2):

[0050] ATGATGGACTTGGAGCTGCCGCCGCCGGGACTCCCGTCCCAGCAGGACATGGATTTG ATTGACATACTTTGGAGGCAAGATATAGATCTTGGAGTAAGTCGAGAAGTATTTGACT TCAGTCAGCGACGGAAAGAGTATGAGCTGGAAAAACAGAAAAAACTTGAAAAGGA AAGACAAGAACAACTCCAAAAGGAGCAAGAGAAAGCCTTTTTCGCTCAGTTACAAC TAGATGAAGAGACAGGTGAATTTCTCCCAATTCAGCCAGCCCAGCACATCCAGTCAG AAACCAGTGGATCTGCCAACTACTCCCAGGTTGCCCACATTCCCAAATCAGATGCTTT GTACTTTGATGACTGCATGCAGCTTTTGGCGCAGACATTCCCGTTTGTAGATGACAAT GAGGTTTCTTCGGCTACGTTTCAGTCACTTGTTCCTGATATTCCCGGTCACATCGAGA GCCCAGTCTTCATTGCTACTAATCAGGCTCAGTCACCTGAAACTTCTGTTGCTCAGGT AGCCCCTGTTGATTTAGACGGTATGCAACAGGACATTGAGCAAGTTTGGGAGGAGCT ATTATCCATTCCTGAGTTACAGTGTCTTAATATTGAAAATGACAAGCTGGTTGAGACTA CCATGGTTCCAAGTCCAGAAGCCAAACTGACAGAAGTTGACAATTATCATTTTTACTC ATCTATACCCTCAATGGAAAAAGAAGTAGGTAACTGTAGTCCACATTTTCTTAATGCTT TTGAGGATTCCTTCAGCAGCATCCTCTCCACAGAAGACCCCAACCAGTTGACAGTGA ACTCATTAAATTCAGATGCCACAGTCAACACAGATTTTGGTGATGAATTTTATTCTGCT TTCATAGCTGAGCCCAGTATCAGCAACAGCATGCCCTCACCTGCTACTTTAAGCCATT CACTCTCTGAACTTCTAAATGGGCCCATTGATGTTTCTGATCTATCACTTTGCAAAGCT TTCAACCAAAACCACCCTGAAAGCACAGCAGAATTCAATGATTCTGACTCCGGCATT TCACTAAACACAAGTCCCAGTGTGGCATCACCAGAACACTCAGTGGAATCTTCCAGC TATGGAGACACACTACTTGGCCTCAGTGATTCTGAAGTGGAAGAGCTAGATAGTGCC CCTGGAAGTGTCAAACAGAATGGTCCTAAAACACCAGTACATTCTTCTGGGGATATGG TACAACCCTTGTCACCATCTCAGGGGCAGAGCACTCACGTGCATGATGCCCAATGTG AGAACACACCAGAGAAAGAATTGCCTGTAAGTCCTGGTCATCGGAAAACCCCATTCA CAAAAGACAAACATTCAAGCCGCTTGGAGGCTCATCTCACAAGAGATGAACTTAGGG CAAAAGCTCTCCATATCCCATTCCCTGTAGAAAAAATCATTAACCTCCCTGTTGTTGAC TTCAACGAAATGATGTCCAAAGAGCAGTTCAATGAAGCTCAACTTGCATTAATTCGGG ATATACGTAGGAGGGGTAAGAATAAAGTGGCTGCTCAGAATTGCAGAAAAAGAAAAC TGGAAAATATAGTAGAACTAGAGCAAGATTTAGATCATTTGAAAGATGAAAAAGAAA AATTGCTCAAAGAAAAAGGAGAAAATGACAAAAGCCTTCACCTACTGAAAAAACAA CTCAGCACCTTATATCTCGAAGTTTTCAGCATGCTACGTGATGAAGATGGAAAACCTT ATTCTCCTAGTGAATACTCCCTGCAGCAAACAAGAGATGGCAATGTTTTCCTTGTTCC CAAAAGTAAGAAGCCAGATGTTAAGAAAAACTAG THBD (protein, SEQ ID NO: 3):

[0051] MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALYPGPATFLNASQICDGL RGHLMTVRSSVAADVISLLLNGDGGVGRRRLWIGLQLPPGCGDPKRLGPLRGFQWVTG DNNTSYSRWARLDLNGAPLCGPLCVAVSAAEATVPSEPIWEEQQCEVKADGFLCEFHFPA TCRPLAVEPGAAAAAVSITYGTPFAARGADFQALPVGSSAAVAPLGLQLMCTAPPGAVQG HWAREAPGAWDCSVENGGCEHACNAIPGAPRCQCPAGAALQADGRSCTASATQSCNDL CEHFCVPNPDQPGSYSCMCETGYRLAADQHRCEDVDDCILEPSPCPQRCVNTQGGFECH CYPNYDLVDGECVEPVDPCFRANCEYQCQPLNQTSYLCVCAEGFAPIPHEPHRCQMFCN QTACPADCDPNTQASCECPEGYILDDGFICTDIDECENGGFCSGVCHNLPGTFECICGPDS ALARHIGTDCDSGKVDGGDSGSGEPPPSPTPGSTLTPPAVGLVHSGLLIGISIASLCLVVAL LALLCHLRKKQGAARAKMEYKCAAPSKEVVLQHVRTERTPQRL*

[0052] THBD (mRNA, SEQ ID NO:4):

[0053] ATGCTTGGGGTCCTGGTCCTTGGCGCGCTGGCCCTGGCCGGCCTGGGGTTCCC CGCACCCGCAGAGCCGCAGCCGGGTGGCAGCCAGTGCGTCGAGCACGACTGCTTCG CGCTCTACCCGGGCCCCGCGACCTTCCTCAATGCCAGTCAGATCTGCGACGGACTGC GGGGCCACCTAATGACAGTGCGCTCCTCGGTGGCTGCCGATGTCATTTCCTTGCTACT GAACGGCGACGGCGGCGTTGGCCGCCGGCGCCTCTGGATCGGCCTGCAGCTGCCAC CCGGCTGCGGCGACCCCAAGCGCCTCGGGCCCCTGCGCGGCTTCCAGTGGGTTACGG GAGACAACAACACCAGCTATAGCAGGTGGGCACGGCTCGACCTCAATGGGGCTCCCC TCTGCGGCCCGTTGTGCGTCGCTGTCTCCGCTGCTGAGGCCACTGTGCCCAGCGAGC CGATCTGGGAGGAGCAGCAGTGCGAAGTGAAGGCCGATGGCTTCCTCTGCGAGTTCC ACTTCCCAGCCACCTGCAGGCCACTGGCTGTGGAGCCCGGCGCCGCGGCTGCCGCC GTCTCGATCACCTACGGCACCCCGTTCGCGGCCCGCGGAGCGGACTTCCAGGCGCTG CCGGTGGGCAGCTCCGCCGCGGTGGCTCCCCTCGGCTTACAGCTAATGTGCACCGCG CCGCCCGGAGCGGTCCAGGGGCACTGGGCCAGGGAGGCGCCGGGCGCTTGGGACTG CAGCGTGGAGAACGGCGGCTGCGAGCACGCGTGCAATGCGATCCCTGGGGCTCCCC GCTGCCAGTGCCCAGCCGGCGCCGCCCTGCAGGCAGACGGGCGCTCCTGCACCGCA TCCGCGACGCAGTCCTGCAACGACCTCTGCGAGCACTTCTGCGTTCCCAACCCCGAC CAGCCGGGCTCCTACTCGTGCATGTGCGAGACCGGCTACCGGCTGGCGGCCGACCAA CACCGGTGCGAGGACGTGGATGACTGCATACTGGAGCCCAGTCCGTGTCCGCAGCGC TGTGTCAACACACAGGGTGGCTTCGAGTGCCACTGCTACCCTAACTACGACCTGGTG GACGGCGAGTGTGTGGAGCCCGTGGACCCGTGCTTCAGAGCCAACTGCGAGTACCA GTGCCAGCCCCTGAACCAAACTAGCTACCTCTGCGTCTGCGCCGAGGGCTTCGCGCC CATTCCCCACGAGCCGCACAGGTGCCAGATGTTTTGCAACCAGACTGCCTGTCCAGC CGACTGCGACCCCAACACCCAGGCTAGCTGTGAGTGCCCTGAAGGCTACATCCTGGA CGACGGTTTCATCTGCACGGACATCGACGAGTGCGAAAACGGCGGCTTCTGCTCCGG GGTGTGCCACAACCTCCCCGGTACCTTCGAGTGCATCTGCGGGCCCGACTCGGCCCT TGCCCGCCACATTGGCACCGACTGTGACTCCGGCAAGGTGGACGGTGGCGACAGCG GCTCTGGCGAGCCCCCGCCCAGCCCGACGCCCGGCTCCACCTTGACTCCTCCGGCCG TGGGGCTCGTGCATTCGGGCTTGCTCATAGGCATCTCCATCGCGAGCCTGTGCCTGGT GGTGGCGCTTTTGGCGCTCCTCTGCCACCTGCGCAAGAAGCAGGGCGCCGCCAGGG CCAAGATGGAGTACAAGTGCGCGGCCCCTTCCAAGGAGGTAGTGCTGCAGCACGTG CGGACCGAGCGGACGCCGCAGAGACTCTGA

[0054] The disclosed compositions may further comprise at least one pharmaceutically acceptable excipient.

[0055] The disclosed compositions may be administered to a subject or used in methods of perfusing a donor liver.

[0056] Administration, may be performed by any suitable route, e.g., intravenous, intramuscular, intrathecal, subcutaneous, topical, osseus, etc.

[0057] Additional Definitions

[0058] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof, as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements.

[0059] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0060] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or descriptions found in the cited references.

[0061] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0062] EXAMPLES

[0063] Example 1 - RNA-Lipid nanoparticles modify “donor” livers and are expected to provide therapeutic benefits upon transplantation Lipid nanoparticles (LNP) containing therapeutic RNA (i.e. siRNA, mRNA) are effective agents to deliver cargo RNA to target cells. Recent improvements to LNPs enabled the FDA approved siRNA therapeutic, Onpattro, and the Pfizer and Moderna COVID-19 vaccines (2, 3). The inventors have evaluated LNP formulations that are efficiently internalized by hepatocytes in vivo, comprised of ALC-0315, DSPC, PEG-2000 and cholesterol, with subsequent intracellular RNA release and either synthesis of the encoded protein (in cases of mRNA delivery) or silencing of the target gene (in cases of siRNA delivery). LNP containing RNA agents are predominantly taken up by the liver when administered via I.V., supporting the liver’s significant capacity for LNP specificity. Thus, the inventors expect that administration of therapeutic RNA - LNP prior to transplant, either before collection from the donor or during ex vivo liver perfusion, will be effective as a mechanism to genetically modify transplantable livers by delivering therapeutic RNA agents. These agents in turn are expected to enhance or decrease target protein levels, leveraging native or non-native biochemical pathways to promote organ viability, health, or function. To modify transplant livers for improved transplant outcomes, the inventors propose to deliver therapeutic RNA - LNP to the organ before transplant, during or prior to ex vivo perfusion.

[0064] Here we describe a system comprising a donor liver, a machine perfusion system, and an LNP containing exogenous nucleic acids, and propose that this strategy can be developed to directly and effectively modify donor livers for enhanced recovery after transplantation, improving outcomes and expanding the use of marginalized livers.

[0065] Composition, Methods and Results: Modification of transplantable livers ex vivo by RNA - LNP perfusion

[0066] Outlined here, is the first-ever method describing successful transfection of liver ex vivo through machine perfusion with administration of RNA - LNP.

[0067] Upon intravenous (I V.) administration in vivo, RNA - LNP are preferentially taken up by the liver (4). LNP are particularly adept delivery vehicles of RNA, including silencing RNA (siRNA) and messenger RNA (mRNA). Both mRNA and siRNA cargo encapsulated in LNP are effective and functional following hepatic delivery (Fig. 1). Delivery of these therapeutic RNA agents result in decreased expression of target endogenous genes (siRNA) or enhanced expression of encoded genes (mRNA) in the liver (Fig. 1). Both strategies may be beneficial in improving liver transplant outcomes, depending on the desired outcome. Thus, transfecting liver ex vivo with RNA - LNP is a promising strategy to effectively modify the target organ, improving outcomes after transplant by specifically modulating the levels of therapeutic protein targets using encapsulated mRNA or siRNA.

[0068] Machine perfusion ex vivo can be a strategy to extend the preservation time of livers prior to transplant (5). During this time, the organ is kept external to both the donor and the recipient patient, and thus any therapeutics introduced have limited systemic exposure to the recipient. Administering therapeutics at this point can also be beneficial in that they can be taken up and begin to induce their physiological effects before they reach the patient.

[0069] A significant strength of this approach is the efficacy timeline. While genetic liver modification may enhance recovery after IRI, treatment with mRNA or siRNA would not result in a permanent genetic change which could be carried over permanently to the recipient. When used in vivo, standard mRNA expression persists for approximately 1-2 days (6), while the effects of siRNA can persist for weeks (7). These timelines would cover the time frame prior to and immediately following transplant into a recipient, while not extending far beyond the scope of recovery, when the modification would no longer be desirable. RNA can be effectively delivered by LNP during or prior to machine perfusion, as RNA delivered prior to organ harvest persists through perfusion to reach the organ recipient (Fig. 2A). Furthermore, the immune reactions to RNA - LNP are minimal, which will be crucial to not introduce additional immune responses immediately following transplant (3).

[0070] To effectively demonstrate the capabilities of our RNA delivery system to liver during NMP, we delivered mRNA - LNP encoding luciferase, a substance not naturally found in rat livers, to provide a clear, measurable indication of the delivery system's performance and impact. A donor rat liver was surgically collected and maintained on normothermic perfusion (NMP) for four hours. After an initial 30 minutes of perfusion, mRNA-LNP encoding reporter protein Nanoluciferase was added to the reservoir solution and delivered via perfusion at a dose of 0.5 mg RNA per kg rat total weight. At baseline, 30 minutes, and every subsequent hour time points, samples were collected from peripheral sections of liver tissue, as well as from the perfusate solution, and assessed for the presence of Nanoluciferase reporter protein using a luminescence-based enzymatic activity assay (Fig. 2B). Luciferase expression was detectable in liver tissue after 1 h of NMP, and continued to increase throughout the 4 h experiment time. Given the longest reported time of clinical NMP at 3 days (5), our results highlight the potential of RNA - LNP modification during NMP to effectively modify donor livers. This technology has a number of potential uses, as outlined below.

[0071] Potential Uses

[0072] (1) Improved ischemia reperfusion injury (IRI) Recovery by Increased levels of Nrf2 Mitogen-activated protein kinases (MAPKs) play a vital role in cellular responses to hypoxia and reactive oxygen species (ROS), by activating downstream cascades mediated by the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) (9-11). MAPK is highly activated during IRI and NMP, and pharmacological inhibition of the MAPK pathway significantly worsens hepatic IRI, suggesting that MAPK play key roles in preventing liver damage during IRI and NMP. Nrf2 knockout (KO) rats have worse hepatic IRI than WT rats, further supporting that Nrf2 is important in recovery from IRI. Nrf2 activation promotes antioxidant gene transcription after oxidative stress, in turn ameliorating liver IRI. Delivery of mRNA - LNP encoding Nrf2, either the native coding sequence or a constitutively active point mutant (T80K) (72), may promote enhanced recovery from IRI in the period following transplantation, and better patient outcomes.

[0073] (2) Rapid Liver Defatting by Silencing tPA

[0074] The hepatic protein tissue plasminogen activator (tPA) has recently demonstrated a novel role in reducing apolipoprotein B (apoB) lipidation and very low-density lipoprotein (VLDL) assembly in hepatocytes (73). Modulating the amount of tPA in transplantable livers through siRNA knockdown will promote apoB lipidation, and pack lipids onto VLDL for secretion. This metabolic modulation will increase lipid mobilization from the liver, as an approach to defat livers on the timeline of 1-3 days, a feasible timeline for liver transplantation. Further, the temporary silencing of the tPA gene may permit the use of steatotic livers which would otherwise be discarded as ineligible for transplant.

[0075] (3) Managing Thrombosis by Expression of Thrombomodulin, Silencing of PAL I Liver transplantation is commonly accompanied by hemostatic imbalance and coagulopathies (14). Delivery of mRNA encoding hemostatic enzymes or proteins within the coagulation cascade may improve hemostatic imbalances associated with the acute phase following liver transplant. The time period following liver transplant is highly prothrombotic, commonly address by treatment with anticoagulants (75). Delivering mRNA encoding hemostatic agents such as thrombomodulin (THBD which acts as an antithrombotic by altering the substrate specificity of thrombin, may be an alternate approach to control thrombosis following liver transplant. Similarly, genetic approaches targeting fibrinolysis may also mitigate post-transplant thrombosis. Plasminogen activator inhibitor 1 (PAI-1) is a circulating protein synthesized in the liver, which inhibits fibrinolysis (16). Silencing PAI-1 inhibitory activity of fibrinolysis may ameliorate thrombosis post-transplant, and can be achieved in transplant tissues through delivery of silencing RNA (siRNA) in LNPs. Notably, graft thrombosis is a well-recognized complication and mechanism of failure for solid organ transplant beyond livers, including kidneys (77). Thus, the antithrombotic strategies listed here may be able to mitigate the hemostatic complications associated in transplants of other organ systems and tissues, as well.

[0076] References:

[0077] 1. OPTN National Data. (http: / / optn.transplant.hrsa.gov / latestData / rptData.asp).

[0078] 2. J. A. Kulkami, D. Witzigmann, S. Chen, P. R. Cullis, R. van der Meel, Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics. Acc Chem Res 52, 2435-2444 (2019).

[0079] 3. Y. Lee, M. Jeong, J. Park, H. Jung, H. Lee, Immunogenicity of lipid nanoparticles and its impact on the efficacy of mRNA vaccines and therapeutics. Exp Mol Med 55, 2085-2096 (2023).

[0080] 4. A. Akinc, W. Querbes, S. De, J. Qin, M. Frank-Kamenetsky, K. N. Jayaprakash, M.

[0081] Jayaraman, K. G. Rajeev, W. L. Cantley, J. R. Dorkin, J. S. Butler, L. Qin, T. Racie, A. Sprague, E. Fava, A. Zeigerer, M. J. Hope, M. Zerial, D. W. Sah, K. Fitzgerald, M. A. Tracy, M. Manoharan, V. Koteliansky, A. Fougerolles, M. A. Maier, Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther 18, 1357-1364 (2010).

[0082] 5. C. D. L. Ceresa, D. Nasralla, J. M. Pollok, P. J. Friend, Machine perfusion of the liver: applications in transplantation and beyond. Nat Rev Gastroenterol Hepatol 19, 199-209 (2022).

[0083] 6. N. Pardi, S. Tuyishime, H. Muramatsu, K. Kariko, B. L. Mui, Y. K. Tam, T. D. Madden, M. J. Hope, D. Weissman, Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J Control Release 217, 345-351 (2015). 7. A. Akinc, M. A. Maier, M. Manoharan, K. Fitzgerald, M. Jayaraman, S. Barros, S.

[0084] Ansell, X. Du, M. J. Hope, T. D. Madden, B. L. Mui, S. C. Semple, Y. K. Tam, M.

[0085] Ciufolini, D. Witzigmann, J. A. Kulkami, R. van der Meel, P. R. Cullis, The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat Nanotechnol 14, 1084-1087 (2019).

[0086] 8. P. A. Clavien, P. Dutkowski, M. Mueller, D. Eshmuminov, L. Bautista Borrego, A.

[0087] Weber, B. Muellhaupt, R. X. Sousa Da Silva, B. R. Burg, P. Rudolf von Rohr, M. J. Schuler, D. Becker, M. Hefti, M. W. Tibbitt, Transplantation of a human liver following 3 days of ex situ normothermic preservation. Nat Biotechnol 40, 1610-1616 (2022). 9. A. N. Kong, E. Owuor, R. Yu, V. Hebbar, C. Chen, R. Hu, S. Mandlekar, Induction of xenobiotic enzymes by the MAP kinase pathway and the antioxidant or electrophile response element (ARE / EpRE). Drug Metab Rev 33, 255-271 (2001).

[0088] 10. Z. Sun, Z. Huang, D. D. Zhang, Phosphorylation of Nrf2 at multiple sites by MAP kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response. PLoS One 4, e6588 (2009).

[0089] 11. M. Zhao, P. Zhu, M. Fujino, Y. Nishio, J. Chen, H. Ito, K. Takahashi, M. Nakajima, T.

[0090] Tanaka, L. Zhao, J. Zhuang, X. K. Li, 5-Aminolevulinic acid with sodium ferrous citrate induces autophagy and protects cardiomyocytes from hypoxia-induced cellular injury through MAPK-Nrf-2-HO- 1 signaling cascade. Biochem Biophys Res Cornmun 479, 663- 669 (2016).

[0091] 12. P. Huppke, S. Weissbach, J. A. Church, R. Schnur, M. Krusen, S. Dreha-Kulaczewski, W. N. Kuhn-Velten, A. Wolf, B. Huppke, F. Millan, A. Begtrup, F. Almusafri, H. Thiele, J. Altmuller, P. Numberg, M. Muller, J. Gartner, Activating de novo mutations in NFE2L2 encoding NRF2 cause a multisystem disorder. Nat Commun 8, 818 (2017). 13. W. Dai, H. Zhang, H. Lund, Z. Zhang, M. Castleberry, M. Rodriguez, G. Kuriakose, S.

[0092] Gupta, M. Lewandowska, H. R. Powers, S. Valmiki, J. Zhu, A. D. Shapiro, M. M.

[0093] Hussain, J. A. Lopez, M. G. Sorci-Thomas, R. L. Silverstein, H. N. Ginsberg, D. Sahoo, I. Tabas, Z. Zheng, Intracellular tPA-P Al- 1 interaction determines VLDL assembly in hepatocytes. Science 381, eadh5207 (2023).

[0094] 14. A. Saracoglu, K. T. Saracoglu, Coagulopathy during liver transplantation. J Anaesthesiol Clin Pharmacol 34, 289-295 (2018). 15. A. A. Pillai, M. Kriss, D. P. ALAdra, R. M. Chadha, M. M. Cushing, K. Farsad, B. E. Fortune, A. S. Hess, R. Lewandowski, M. K. Nadim, T. Nydam, P. Sharma, C. J.

[0095] Karvellas, N. Intagliata, Coagulopathy and hemostasis management in patients undergoing liver transplantation: Defining a dynamic spectrum across phases of care. Liver Transpl 28, 1651-1663 (2022).

[0096] 16. G. Cesarman-Maus, K. A. Hajj ar, Molecular mechanisms of fibrinolysis. Br J Haematol 129, 307-321 (2005).

[0097] 17. V. Surianarayanan, T. J. Hoather, S. J. Tingle, E. R. Thompson, J. Hanley, C. H. Wilson, Interventions for preventing thrombosis in solid organ transplant recipients. Cochrane Database SystRev 3, CD011557 (2021).

[0098] Example 2 - Methods of delivering lipid nanoparticles to a donor liver

[0099] FIG. 3A shows models of dosing LNP for transplant, including dosing the liver while on machine perfusion, and dosing the liver prior to machine perfusion (‘dosing the donor’). Bottom dosing model used as control. FIGs. 3B and C show that all models enable RNA-LNP uptake, and subsequent translation of the delivered mRNA. FIG. 3D shows that RNA-LNP are stable in standard perfusates over hours.

[0100] FIG. 4 shows that livers are modifiable in Ischemia-reperfusion (a significant mechanism of graft damage in transplant). FIG. 4A demonstrates that the disclosed methods are effective in a model of warm ischemia. FIGs. 4B and 4C show that RNA-LNP transfection is maintained in ischemia-reperfusion injury grafts, as shown by RNA-LNP uptake (B), and subsequent translation of the delivered mRNA (C). FIG 4D shows that RNA-LNP transfection and exogenous protein expression is maintained in IRI at various stages of recovery

[0101] Sequences

[0102] SEQ ID NO Description Sequence MMDLELPPPGLPSQQDMDLIDILWRQDIDLGVSREVFDFSQR RKEYELEKQKKLEKERQEQLQKEQEKAFFAQLQLDEETGEFL PIQPAQHIQSETSGSANYSQVAHIPKSDALYFDDCMQLLAQTF PF VDDNE VS S ATFQ SLVPDIPGHIESP VFIATNQ AQ SPET S VAQ VAPVDLDGMQQDIEQVWEELLSIPELQCLNIENDKLVETTMV P SPE AKLTEVDNYHF YS SIP SMEKEVGNC SPHFLNAFED SF S SI

[0103]

[0104] 1 Nrf2 AA LSTEDPNQLTVNSLNSDATVNTDFGDEFYSAFIAEPSISNSMPS P ATL SHSLSELLNGPID VSDL SLCK AFNQNHPEST AEFND SD SG ISLNT SP S VASPEHS VES S S YGDTLLGL SD SEVEELD S APGS VK QNGPKTPVHSSGDMVQPLSPSQGQSTHVHDAQCENTPEKELP VSPGHRKTPFTKDKHSSRLEAHLTRDELRAKALHIPFPVEKIIN LPVVDFNEMMSKEQFNEAQLALIRDIRRRGKNKVAAQNCRK RKLENIVELEQDLDHLKDEKEKLLKEKGENDKSLHLLKKQLS TLYLEVFSMLRDEDGKPYSPSEYSLQQTRDGNVFLVPKSKKP DVKKN

[0105] atgatggacttggagctgccgccgccgggactcccgtcccagcaggacatggatttgattgacat actttggaggcaagatatagatcttggagtaagtcgagaagtatttgacttcagtcagcgacggaa agagtatgagctggaaaaacagaaaaaacttgaaaaggaaagacaagaacaactccaaaagga gcaagagaaagcctttttcgctcagttacaactagatgaagagacaggtgaatttctcccaattcag ccagcccagcacatccagtcagaaaccagtggatctgccaactactcccaggttgcccacattcc caaatcagatgcttgtactttgatgactgcatgcagcttttggcgcagacattcccgtttgtagatga caatgaggtttcttcggctacgtttcagtcacttgttcctgatattcccggtcacatcgagagcccagt cttcatgctactaatcaggctcagtcacctgaaacttctgttgctcaggtagcccctgttgatttaga cggtatgcaacaggacattgagcaagtttgggaggagctattatccattcctgagttacagtgtctta atattgaaaatgacaagctggttgagactaccatggttccaagtccagaagccaaactgacagaag ttgacaattatcatttttactcatctataccctcaatggaaaaagaagtaggtaactgtagtccacatttt cttaatgcttttgaggattccttcagcagcatcctctccacagaagaccccaaccagttgacagtga actcattaaattcagatgccacagtcaacacagattttggtgatgaatttattctgctttcatagctga gcccagtatcagcaacagcatgccctcacctgctactttaagccattcactctctgaacttctaaatg ggcccattgatgtttctgatctatcactttgcaaagctttcaaccaaaaccaccctgaaagcacagca gaattcaatgattctgactccggcatttcactaaacacaagtcccagtgtggcatcaccagaacact cagtggaatcttccagctatggagacacactacttggcctcagtgattctgaagtggaagagctag atagtgcccctggaagtgtcaaacagaatggtcctaaaacaccagtacatcttctggggatatggt acaacccttgtcaccatctcaggggcagagcactcacgtgcatgatgcccaatgtgagaacacac cagagaaagaattgcctgtaagtcctggtcatcggaaaaccccattcacaaaagacaaacattcaa gccgcttggaggctcatctcacaagagatgaacttagggcaaaagctctccatatcccattccctgt agaaaaaatcattaacctccctgttgttgacttcaacgaaatgatgtccaaagagcagttcaatgaa gctcaacttgcattaattcgggatatacgtaggaggggtaagaataaagtggctgctcagaattgc agaaaaagaaaactggaaaatatagtagaactagagcaagatttagatcatttgaaagatgaaaaa gaaaaattgctcaaagaaaaaggagaaaatgacaaaagccttcacctactgaaaaaacaactcag caccttatatctcgaagttttcagcatgctacgtgatgaagatggaaaaccttattctcctagtgaata Nrf2 ctccctgcagcaaacaagagatggcaatgttttccttgttcccaaaagtaagaagccagatgttaag mRNA aaaaactag MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALYPGP ATFLNASQICDGLRGHLMTVRSSVAADVISLLLNGDGGVGRR RLWIGLQLPPGCGDPKRLGPLRGFQWVTGDNNTSYSRWARL DLNGAPLCGPLCVAVSAAEATVPSEPIWEEQQCEVKADGFLC EFHFPATCRPLAVEPGAAAAAVSITYGTPFAARGADFQALPV GSSAAVAPLGLQLMCTAPPGAVQGHWAREAPGAWDCSVEN GGCEHACNAIPGAPRCQCPAGAALQADGRSCTASATQSCNDL CEHFCVPNPDQPGSYSCMCETGYRLAADQHRCEDVDDCILEP SPCPQRCVNTQGGFECHCYPNYDLVDGECVEPVDPCFRANCE

[0106]

[0107] THBD AA YQCQPLNQTSYLCVCAEGFAPIPHEPHRCQMFCNQTACPADC DPNTQASCECPEGYILDDGFICTDIDECENGGFCSGVCHNLPG TFECICGPDSALARHIGTDCDSGKVDGGDSGSGEPPPSPTPGST LTPPAVGLVHSGLLIGISIASLCLVVALLALLCHLRKKQGAAR AKMEYKCAAPSKEVVLQHVRTERTPQRL ATGCTTGGGGTCCTGGTCCTTGGCGCGCTGGCCCTGGCCGG CCTGGGGTTCCCCGCACCCGCAGAGCCGCAGCCGGGTGGC AGCCAGTGCGTCGAGCACGACTGCTTCGCGCTCTACCCGG GCCCCGCGACCTTCCTCAATGCCAGTCAGATCTGCGACGGA CTGCGGGGCCACCTAATGACAGTGCGCTCCTCGGTGGCTGC CGATGTCATTTCCTTGCTACTGAACGGCGACGGCGGCGTTG GCCGCCGGCGCCTCTGGATCGGCCTGCAGCTGCCACCCGG CTGCGGCGACCCCAAGCGCCTCGGGCCCCTGCGCGGCTTCC AGTGGGTTACGGGAGACAACAACACCAGCTATAGCAGGTG GGCACGGCTCGACCTCAATGGGGCTCCCCTCTGCGGCCCGT TGTGCGTCGCTGTCTCCGCTGCTGAGGCCACTGTGCCCAGC GAGCCGATCTGGGAGGAGCAGCAGTGCGAAGTGAAGGCC GATGGCTTCCTCTGCGAGTTCCACTTCCCAGCCACCTGCAG GCCACTGGCTGTGGAGCCCGGCGCCGCGGCTGCCGCCGTC TCGATCACCTACGGCACCCCGTTCGCGGCCCGCGGAGCGG ACTTCCAGGCGCTGCCGGTGGGCAGCTCCGCCGCGGTGGC TCCCCTCGGCTTACAGCTAATGTGCACCGCGCCGCCCGGAG CGGTCCAGGGGCACTGGGCCAGGGAGGCGCCGGGCGCTTG GGACTGCAGCGTGGAGAACGGCGGCTGCGAGCACGCGTGC AATGCGATCCCTGGGGCTCCCCGCTGCCAGTGCCCAGCCG GCGCCGCCCTGCAGGCAGACGGGCGCTCCTGCACCGCATC CGCGACGCAGTCCTGCAACGACCTCTGCGAGCACTTCTGCG TTCCCAACCCCGACCAGCCGGGCTCCTACTCGTGCATGTGC GAGACCGGCTACCGGCTGGCGGCCGACCAACACCGGTGCG AGGACGTGGATGACTGCATACTGGAGCCCAGTCCGTGTCC GCAGCGCTGTGTCAACACACAGGGTGGCTTCGAGTGCCAC TGCTACCCTAACTACGACCTGGTGGACGGCGAGTGTGTGG AGCCCGTGGACCCGTGCTTCAGAGCCAACTGCGAGTACCA GTGCCAGCCCCTGAACCAAACTAGCTACCTCTGCGTCTGCG CCGAGGGCTTCGCGCCCATTCCCCACGAGCCGCACAGGTG CCAGATGTTTTGCAACCAGACTGCCTGTCCAGCCGACTGCG ACCCCAACACCCAGGCTAGCTGTGAGTGCCCTGAAGGCTA CATCCTGGACGACGGTTTCATCTGCACGGACATCGACGAGT GCGAAAACGGCGGCTTCTGCTCCGGGGTGTGCCACAACCT CCCCGGTACCTTCGAGTGCATCTGCGGGCCCGACTCGGCCC TTGCCCGCCACATTGGCACCGACTGTGACTCCGGCAAGGTG GACGGTGGCGACAGCGGCTCTGGCGAGCCCCCGCCCAGCC CGACGCCCGGCTCCACCTTGACTCCTCCGGCCGTGGGGCTC GTGCATTCGGGCTTGCTCATAGGCATCTCCATCGCGAGCCT THBD GTGCCTGGTGGTGGCGCTTTTGGCGCTCCTCTGCCACCTGC

[0108]

[0109] mRNA GCAAGAAGCAGGGCGCCGCCAGGGCCAAGATGGAGTACA AGTGCGCGGCCCCTTCCAAGGAGGTAGTGCTGCAGCACGT GCGGACCGAGCGGACGCCGCAGAGACTCTGA MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGARSYQVICR DEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPV KSCSEPRCFNGGTCQQALYFSDFVCQCPEGFAGKCCEIDTRAT CYEDQGISYRGTWSTAESGAECTNWNSSALAQKPYSGRRPD AIRLGLGNHNYCRNPDRD SKPWCYVFKAGKYS SEFC STPACS EGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTA QNPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWEYC DVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRR SPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRV VPGEEEQKFEVEKYIVHKEFDDDT YDNDIALLQLKSD S SRC A QESSVVRTVCLPPADLQLPDWTECELSGYGKHEALSPFYSERL KEAHVRL YPS SRCT SQHLLNRT VTDNMLC AGDTRSGGPQ AN LHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGV

[0110] tPAAA YTKVTNYLDWIRDNMRP

[0111] at ggatgcaatg aagagagggc

[0112] 121 tctgctgtgt gctgctgctg tgtggagcag tcttcgtttc gcccagccag gaaatccatg

[0113] 181 cccgattcag aagaggagcc agatcttacc aagtgatctg cagagatgaa aaaacgcaga

[0114] 241 tgatatacca gcaacatcag tcatggctgc gccctgtgct cagaagcaac cgggtggaat

[0115] 301 attgctggtg caacagtggc agggcacagt gccactcagt gcctgtcaaa agttgcagcg

[0116] 361 agccaaggtg tttcaacggg ggcacctgcc agcaggccct gtacttctca gatttcgtgt

[0117] 421 gccagtgccc cgaaggattt gctgggaagt gctgtgaaat agataccagg gccacgtgct

[0118] 481 acgaggacca gggcatcagc tacaggggca cgtggagcac agcggagagt ggcgccgagt

[0119] 541 gcaccaactg gaacagcagc gcgttggccc agaagcccta cagcgggcgg aggccagacg

[0120] 601 ccatcaggct gggcctgggg aaccacaact actgcagaaa cccagatcga gactcaaagc

[0121] 661 cctggtgcta cgtctttaag gcggggaagt acagctcaga gttctgcagc acccctgcct

[0122] 721 gctctgaggg aaacagtgac tgctactttg ggaatgggtc agcctaccgt ggcacgcaca

[0123] 781 gcctcaccga gtcgggtgcc tcctgcctcc cgtggaattc catgatcctg ataggcaagg

[0124] 841 tttacacagc acagaacccc agtgcccagg cactgggcct gggcaaacat aattactgcc

[0125] 901 ggaatcctga tggggatgcc aagccctggt gccacgtgct gaagaaccgc aggctgacgt

[0126]

[0127] tPA mRN A 961 gggagtactg tgatgtgccc tcctgctcca cctgcggcct gagacagtac agccagcctc

[0128] 1021 agtttcgcat caaaggaggg ctcttcgccg acatcgcctc ccacccctgg caggctgcca

[0129] 1081 tctttgccaa gcacaggagg tcgcccggag agcggttcct gtgcgggggc atactcatca

[0130] 1141 gctcctgctg gattctctct gccgcccact gcttccagga gaggtttccg ccccaccacc

[0131] 1201 tgacggtgat cttgggcaga acataccggg tggtccctgg cgaggaggag cagaaatttg

[0132] 1261 aagtcgaaaa atacattgtc cataaggaat tcgatgatga cacttacgac aatgacattg

[0133] 1321 cgctgctgca gctgaaatcg gattcgtccc gctgtgccca ggagagcagc gtggtccgca

[0134] 1381 ctgtgtgcct tcccccggcg gacctgcagc tgccggactg gacggagtgt gagctctccg

[0135] 1441 gctacggcaa gcatgaggcc ttgtctcctt tctattcgga gcggctgaag gaggctcatg

[0136] 1501 tcagactgta cccatccagc cgctgcacat cacaacattt acttaacaga acagtcaccg

[0137] 1561 acaacatgct gtgtgctgga gacactcgga gcggcgggcc ccaggcaaac ttgcacgacg

[0138] 1621 cctgccaggg cgattcggga ggccccctgg tgtgtctgaa cgatggccgc atgactttgg

[0139] 1681 tgggcatcat cagctggggc ctgggctgtg gacagaagga tgtcccgggt gtgtacacca

[0140] 1741 aggttaccaa ctacctagac tggattcgtg acaacatgcg accgtga

[0141] MQMSPALTCLVLGLALVFGEGSAVHHPPSYVAHLASDFGVR VFQQVAQASKDRNVVFSPYGVASVLAMLQLTTGGETQQQIQ AAMGFKIDDKGMAPALRHLYKELMGPWNKDEISTTDAIFVQ RDLKL VQGFMPHFFRLFRST VKQ VDF SEVERARFIIND WVKT HTKGMISNLLGKGAVDQLTRL VLVNAL YFNGQWKTPFPD S S THRRLFHKSDGSTVSVPMMAQTNKFNYTEFTTPDGHYYDILE LPYHGDTLSMFIAAPYEKEVPLSALTNILSAQLISHWKGNMTR LPRLL VLPKF SLETEVDLRKPLENLGMTDMFRQFQ ADFT SLSD QEPLHVAQALQKVKIEVNESGTVAS S STAVIVS ARMAPEEIIM

[0142]

[0143] PALI AA DRPFLFVVRHNPTGTVLFMGQVMEP ACAGCTGTGTTTGGCTGCAGGGCCAAGAGCGCTGTCAAGA AGACCCACACGCCCCCCTCCAGCAGCTGAATTCCTGCAGCT CAGCAGCCGCCGCCAGAGCAGGACGAACCGCCAATCGCAA GGCACCTCTGAGAACTTCAGGATGCAGATGTCTCCAGCCCT CACCTGCCTAGTCCTGGGCCTGGCCCTTGTCTTTGGTGAAG GGTCTGCTGTGCACCATCCCCCATCCTACGTGGCCCACCTG GCCTCAGACTTCGGGGTGAGGGTGTTTCAGCAGGTGGCGC AGGCCTCCAAGGACCGCAACGTGGTTTTCTCACCCTATGGG GTGGCCTCGGTGTTGGCCATGCTCCAGCTGACAACAGGAG GAGAAACCCAGCAGCAGATTCAAGCAGCTATGGGATTCAA GATTGATGACAAGGGCATGGCCCCCGCCCTCCGGCATCTGT ACAAGGAGCTCATGGGGCCATGGAACAAGGATGAGATCAG CACCACAGACGCGATCTTCGTCCAGCGGGATCTGAAGCTG GTCCAGGGCTTCATGCCCCACTTCTTCAGGCTGTTCCGGAG CACGGTCAAGCAAGTGGACTTTTCAGAGGTGGAGAGAGCC AGATTCATCATCAATGACTGGGTGAAGACACACACAAAAG GTATGATCAGCAACTTGCTTGGGAAAGGAGCCGTGGACCA GCTGACACGGCTGGTGCTGGTGAATGCCCTCTACTTCAACG GCCAGTGGAAGACTCCCTTCCCCGACTCCAGCACCCACCGC CGCCTCTTCCACAAATCAGACGGCAGCACTGTCTCTGTGCC CATGATGGCTCAGACCAACAAGTTCAACTATACTGAGTTCA CCACGCCCGATGGCCATTACTACGACATCCTGGAACTGCCC TACCACGGGGACACCCTCAGCATGTTCATTGCTGCCCCTTA TGAAAAAGAGGTGCCTCTCTCTGCCCTCACCAACATTCTGA GTGCCCAGCTCATCAGCCACTGGAAAGGCAACATGACCAG GCTGCCCCGCCTCCTGGTTCTGCCCAAGTTCTCCCTGGAGA CTGAAGTCGACCTCAGGAAGCCCCTAGAGAACCTGGGAAT GACCGACATGTTCAGACAGTTTCAGGCTGACTTCACGAGTC TTTCAGACCAAGAGCCTCTCCACGTCGCGCAGGCGCTGCA GAAAGTGAAGATCGAGGTGAACGAGAGTGGCACGGTGGC CTCCTCATCCACAGCTGTCATAGTCTCAGCCCGCATGGCCC CCGAGGAGATCATCATGGACAGACCCTTCCTCTTTGTGGTC CGGCACAACCCCACAGGAACAGTCCTTTTCATGGGCCAAG TGATGGAACCCTGACCCTGGGGAAAGACGCCTTCATCTGG GACAAAACTGGAGATGCATCGGGAAAGAAGAAACTCCGA AGAAAAGAATTTTAGTGTTAATGACTCTTTCTGAAGGAAG AGAAGACATTTGCCTTTTGTTAAAAGATGGTAAACCAGATC TGTCTCCAAGACCTTGGCCTCTCCTTGGAGGACCTTTAGGT CAAACTCCCTAGTCTCCACCTGAGACCCTGGGAGAGAAGT TTGAAGCACAACTCCCTTAAGGTCTCCAAACCAGACGGTG ACGCCTGCGGGACCATCTGGGGCACCTGCTTCCACCCGTCT CTCTGCCCACTCGGGTCTGCAGACCTGGTTCCCACTGAGGC CCTTTGCAGGATGGAACTACGGGGCTTACAGGAGCTTTTGT GTGCCTGGTAGAAACTATTTCTGTTCCAGTCACATTGCCAT PALI CACTCTTGTACTGCCTGCCACCGCGGAGGAGGCTGGTGAC

[0144]

[0145] mRNA AGGCCAAAGGCCAGTGGAAGAAACACCCTTTCATCTCAGA GTCCACTGTGGCACTGGCCACCCCTCCCCAGTACAGGGGTG CTGCAGGTGGCAGAGTGAATGTCCCCCATCATGTGGCCCA ACTCTCCTGGCCTGGCCATCTCCCTCCCCAGAAACAGTGTG CATGGGTTATTTTGGAGTGTAGGTGACTTGTTTACTCATTG AAGCAGATTTCTGCTTCCTTTTATTTTTATAGGAATAGAGG AAGAAATGTCAGATGCGTGCCCAGCTCTTCACCCCCCAATC TCTTGGTGGGGAGGGGTGTACCTAAATATTTATCATATCCT TGCCCTTGAGTGCTTGTTAGAGAGAAAGAGAACTACTAAG GAAAATAATATTATTTAAACTCGCTCCTAGTGTTTCTTTGT GGTCTGTGTCACCGTATCTCAGGAAGTCCAGCCACTTGACT GGCACACACCCCTCCGGACATCCAGCGTGACGGAGCCCAC ACTGCCACCTTGTGGCCGCCTGAGACCCTCGCGCCCCCCGC GCCCCTCTTTTTCCCCTTGATGGAAATTGACCATACAATTTC ATCCTCCTTCAGGGGATCAAAAGGACGGAGTGGGGGGACA GAGACTCAGATGAGGACAGAGTGGTTTCCAATGTGTTCAA TAGATTTAGGAGCAGAAATGCAAGGGGCTGCATGACCTAC CAGGACAGAACTTTCCCCAATTACAGGGTGACTCACAGCC GCATTGGTGACTCACTTCAATGTGTCATTTCCGGCTGCTGT GTGTGAGCAGTGGACACGTGAGGGGGGGGTGGGTGAGAG AGACAGGCAGCTCGGATTCAACTACCTTAGATAATATTTCT GAAAACCTACCAGCCAGAGGGTAGGGCACAAAGATGGAT GTAATGCACTTTGGGAGGCCAAGGCGGGAGGATTGCTTGA GCCCAGGAGTTCAAGACCAGCCTGGGCAACATACCAAGAC CCCCGTCTCTTTAAAAATATATATATTTTAAATATACTTAA ATATATATTTCTAATATCTTTAAATATATATATATATTTTAA AGACCAATTTATGGGAGAATTGCACACAGATGTGAAATGA ATGTAATCTAATAGAAGCCTAATCAGCCCACCATGTTCTCC ACTGAAAAATCCTCTTTCTTTGGGGTTTTTCTTTCTTTCTTTT TTGATTTTGCACTGGACGGTGACGTCAGCCATGTACAGGAT CCACAGGGGTGGTGTCAAATGCTATTGAAATTGTGTTGAAT TGTATGCTTTTTCACTTTTGATAAATAAACATGTAAAAATG TTTCAAAAAAATAATAAAATAAATAAATACGAA ATGATGGACTTGGAGCTGCCGCCGCCGGGACTCCCGTCCC AGCAGGACATGGATTTGATTGACATACTTTGGAGGCAAGA TATAGATCTTGGAGTAAGTCGAGAAGTATTTGACTTCAGTC AGCGACGGAAAGAGTATGAGCTGGAAAAACAGAAAAAAC TTGAAAAGGAAAGACAAGAACAACTCCAAAAGGAGCAAG AGAAAGCCTTTTTCGCTCAGTTACAACTAGATGAAGAGAA AGGTGAATTTCTCCCAATTCAGCCAGCCCAGCACATCCAGT CAGAAACCAGTGGATCTGCCAACTACTCCCAGGTTGCCCA CATTCCCAAATCAGATGCTTTGTACTTTGATGACTGCATGC AGCTTTTGGCGCAGACATTCCCGTTTGTAGATGACAATGAG GTTTCTTCGGCTACGTTTCAGTCACTTGTTCCTGATATTCCC GGTCACATCGAGAGCCCAGTCTTCATTGCTACTAATCAGGC

[0146] Nrf2, TCAGTCACCTGAAACTTCTGTTGCTCAGGTAGCCCCTGTTG

[0147]

[0148] T80K ATTTAGACGGTATGCAACAGGACATTGAGCAAGTTTGGGA GGAGCTATTATCCATTCCTGAGTTACAGTGTCTTAATATTG AAAATGACAAGCTGGTTGAGACTACCATGGTTCCAAGTCC AGAAGCCAAACTGACAGAAGTTGACAATTATCATTTTTACT CATCTATACCCTCAATGGAAAAAGAAGTAGGTAACTGTAG TCCACATTTTCTTAATGCTTTTGAGGATTCCTTCAGCAGCAT CCTCTCCACAGAAGACCCCAACCAGTTGACAGTGAACTCA TTAAATTCAGATGCCACAGTCAACACAGATTTTGGTGATGA ATTTTATTCTGCTTTCATAGCTGAGCCCAGTATCAGCAACA GCATGCCCTCACCTGCTACTTTAAGCCATTCACTCTCTGAA CTTCTAAATGGGCCCATTGATGTTTCTGATCTATCACTTTGC AAAGCTTTCAACCAAAACCACCCTGAAAGCACAGCAGAAT TCAATGATTCTGACTCCGGCATTTCACTAAACACAAGTCCC AGTGTGGCATCACCAGAACACTCAGTGGAATCTTCCAGCT ATGGAGACACACTACTTGGCCTCAGTGATTCTGAAGTGGA AGAGCTAGATAGTGCCCCTGGAAGTGTCAAACAGAATGGT CCTAAAACACCAGTACATTCTTCTGGGGATATGGTACAACC CTTGTCACCATCTCAGGGGCAGAGCACTCACGTGCATGATG CCCAATGTGAGAACACACCAGAGAAAGAATTGCCTGTAAG TCCTGGTCATCGGAAAACCCCATTCACAAAAGACAAACAT TCAAGCCGCTTGGAGGCTCATCTCACAAGAGATGAACTTA GGGCAAAAGCTCTCCATATCCCATTCCCTGTAGAAAAAATC ATTAACCTCCCTGTTGTTGACTTCAACGAAATGATGTCCAA AGAGCAGTTCAATGAAGCTCAACTTGCATTAATTCGGGAT ATACGTAGGAGGGGTAAGAATAAAGTGGCTGCTCAGAATT GCAGAAAAAGAAAACTGGAAAATATAGTAGAACTAGAGC AAGATTTAGATCATTTGAAAGATGAAAAAGAAAAATTGCT CAAAGAAAAAGGAGAAAATGACAAAAGCCTTCACCTACTG AAAAAACAACTCAGCACCTTATATCTCGAAGTTTTCAGCAT GCTACGTGATGAAGATGGAAAACCTTATTCTCCTAGTGAAT ACTCCCTGCAGCAAACAAGAGATGGCAATGTTTTCCTTGTT CCCAAAAGTAAGAAGCCAGATGTTAAGAAAAACTAG PALI

[0149] siRNA 1.1 gtgacttgtttactcattgaagcag

[0150] PALI

[0151] siRNA 1.2 ctgcttcaatgagtaaacaagtcacct

[0152] PALI

[0153] siRNA 2.1 caactaccttagataatatttctga

[0154] PALI

[0155] siRNA 2.2 tcagaaatattatctaaggtagttgaa

[0156] PALI

[0157] siRNA 3.1 gattcaactaccttagataatatt

[0158] PALI

[0159] siRNA 3.2 aatattatctaaggtagttgaatccga

[0160] PALI

[0161]

[0162] siRNA 4.1 aggggtggtgtcaaatgctattgaa PALI

[0163]

[0164] 17 siRNA 4.2 ttcaatagcatttgacaccacccctgt

Claims

CLAIMSWhat is claimed:

1. A method of modifying a donor liver, or a portion thereof, the method comprising contacting the donor liver with lipid nanoparticles (LNPs) comprising an RNA.

2. The method of claim 1, wherein the method is performed before the donor liver is removed from a donor.

3. The method of claim 2, wherein the lipid nanoparticles are delivered to the donor liver via intravenous administration to the donor.

4. The method of claim 1, wherein the method is performed after the donor liver is removed from a donor and before the donor liver is transplanted into a recipient.

5. The method of claim 4, wherein the lipid nanoparticles are delivered to the donor liver using machine perfusion.

6. The method of claim 5, wherein the machine perfusion is normothermic perfusion.

7. The method of claim 6, wherein the normothermic perfusion is performed at about 37 degrees Celsius.

8. The method of claim 5, wherein the machine perfusion is hypothermic machine perfusion.

9. The method of claim 8, wherein the hypothermic perfusion is performed at about 2 to about 12 degrees Celsius.

10. The method of claim 1, wherein the donor liver is a marginal liver.

11. The method of claim 10, wherein the marginal liver is from a donor with an age equal to or greater than 60 years old, is a fatty liver, or the liver or the donor has experienced extended ischemia.

12. The method of claim 1, wherein the RNA is a silencing RNA (siRNA).

13. The method of claim 12, wherein the siRNA reduces the expression of tissue plasminogen activator (tPA) or plasminogen activator inhibitor 1 (PAI-1).

14. The method of claim 12, wherein the siRNA further comprises one of SEQ ID NOs: 10-17.

15. The method of claim 1, wherein the RNA is a messenger RNA (mRNA).

16. The method of claim 15, wherein the mRNA encodes a nuclear factor-erythroid factor 2-related factor 2 (NRF2) protein or a thrombomodulin protein.

17. The method of claim 16, wherein the mRNA encodes SEQ ID NO: 1 or 3.

18. The method of claim 16, wherein the Nrf2 protein comprises a constitutively active point mutation, optionally, wherein theNrf2 comprises a lysine at position 80, with reference to SEQ ID NO: 1.

19. The method of any one of the preceding claims, wherein the RNA comprises multiple different RNA molecules.

20. The method of claim 15, wherein the mRNA comprises at least one modified nucleotide.

21. The method of claim 20, wherein the at least one modified nucleotide comprises 1-methyl-pseudouridine (Ml\|r).

22. The method of any one of claims 1-6, wherein the lipid nanoparticles comprise(a) at least one (ionizable) cationic lipid;(b) at least one helper lipid;(c) a sterol; and(d) at least one lipid-polyethylene glycol conjugate.

23. The method of claim 22, wherein the lipid nanoparticles comprise(i) 30-55 mol % of the at least one (ionizable) cationic lipid;(ii) 5-20 mol % of the at least one helper lipid;(iii) 25-50 mol % of sterol; and(iv) 0.5 - 3 mol % of the at least one lipid-polyethylene glycol conjugate.

24. The method of claim 22, wherein the at least one ionizable cationic lipid is one or more of SM-102, ALC-0315, ALC-0159, or DLin-MC3-DMA.

25. The method of claim 22, wherein the at least one helper lipid is one or more of phosphatidylcholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), egg sphingomyelin (ESM).

26. The method of claim 22, wherein the sterol is cholesterol or a cholesterol derivative.

27. The method of claim 22, wherein the at least one lipid polyethylene glycol conjugate is one or more of l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 (DSPE- PEG2000), or PEG-1, 2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG).

28. A composition comprising lipid nanoparticles comprising an RNA, wherein the RNA is selected from:(a) an siRNA that reduces the expression of tissue plasminogen activator (tPA) or plasminogen activator inhibitor 1 (PAI-1); or(b) an mRNA encodes a Nrf2 protein or a thrombomodulin protein.

29. The composition of claim 28, wherein the siRNA of (a) comprises at least one modified nucleotide.

30. The composition of claim 28, wherein the mRNA of (b) encodes one of SEQ ID NOs: 1 or 3.

31. The composition claim 28, wherein the lipid nanoparticles comprise(a) at least one (ionizable) cationic lipid;(b) at least one helper lipid;(c) a sterol; and(d) at least one lipid-polyethylene glycol conjugate.

32. The composition of claim 31, wherein the lipid nanoparticles comprise(i) 30-55 mol % of the at least one (ionizable) cationic lipid;(ii) 5-20 mol % of the at least one helper lipid;(iii) 25-50 mol % of sterol; and(iv) 0.5 - 3 mol % of the at least one lipid-polyethylene glycol conjugate.

33. The composition of claim 31, wherein the at least one ionizable cationic lipid is one or more ofCL4H6, SM-102, ALC-0315, CL1 H6, CL15H6, CL1 D6, orALC-0159.

34. The composition of claim 31, wherein the at least one helper lipid is one or more of phosphatidylcholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), dioleoyl phosphatidylglycerol (DOPG), egg sphingomyelin (ESM).

35. The composition of claim 31, wherein the sterol is cholesterol or a cholesterol derivative.

36. The composition of claim 31, wherein the at least one lipid polyethylene glycol conjugate is one or more of l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 (DSPE- PEG2000), or PEG-1, 2-distearoyl-rac-glycero-3-methylpolyoxyethylene 2000 (DSG-PEG).

37. The composition of claim 30, wherein the mRNA comprises at least one modified nucleotide.

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