Rehabilitation of marginal organs

Abstract

Methods are provided for the preconditioning, conditioning, or rehabilitation of marginal organs to be used for organ or organ-derived cell transplantation. The methods of the present disclosure contact the marginal organs with a medium or perfusate comprising an effective dose of a Wnt agent. The Wnt agent reduces apoptosis of the cells in the marginal organ while also activating stem cells present in the marginal organ to promote repair, reconditioning, and / or regeneration of the marginal organ. Preconditioning or conditioning of the marginal organ with a Wnt agent reduces the risk of reperfusion injury. In some embodiments, preconditioning of the marginal organ reduces the risk of graft rejection when transplanted into an individual in need thereof.

Description

ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 REHABILITATION OF MARGINAL ORGANS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No.63 / 624,225 filed January 23, 2024, and United States Provisional Patent Application Serial No.63 / 637,825 filed April 23, 2024, the disclosures of which applications are herein incorporated by reference. SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (STAN-2128WO_SEQ-LIST.xml; Size: 3,581 bytes; and Date of Creation: January 21, 2025) is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] Currently, organ transplantation is the ultimate, life-saving treatment for patients with end- stage organ disease such as liver, kidney, lung, and heart. One of the most critical issues facing organ transplantation is the insufficient number of high-quality organs from young donors. As a result of organ shortage, organ transplantation is available for a limited number of patients. For example, even among those who qualify for liver transplant, up to ~26% die while waiting for a liver to become available.

[0003] The majority of donated organs come from older individuals but are typically deemed “marginal” due to their poor regenerative capacity and inadequate metabolic function e.g., fatty livers. Organs that experience greater preoperative damage also qualify as “marginal” organs. It is well established that using marginal organs in transplantation leads to significantly lower graft survival but nonetheless their use is widely considered one strategy to address the substantial disparity between the number of organs available for transplant and the number of individuals awaiting organ transplant.

[0004] Further contributing to morbidity and mortality in organ recipients are ischemia and reperfusion injury (IRI) and ischemic cholangiopathy (IC). Extended preservation and cold storage times, especially in marginal organs, are associated with greater incidence of IRI and IC and these conditions contribute significantly to post-operative complications in transplant patients. Approximately 80% of livers donated after cardiac death (DCD) are discarded because of irreversible ischemic damage and even in those DCD livers that are transplanted, long-term survival outcomes are inferior to those recipients of livers donated after brain death. Similarly,ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 60% of kidneys that come from deceased donors over 59 years of age are being discarded after retrieval due to concerns of their inadequate function. There are currently no established therapies for IRI.

[0005] Most approaches seek to limit the duration of cold preservation. Other strategies aim to vitrify the organ e.g., subject the organ to ice-free cryopreservation after retrieval. Another strategy involves persufflation, which involves gaseous oxygen perfusion as a means to preserve organs prior to transplant. Other strategies seek to optimize the solutions in which the organ is maintained; these solutions typically contain a combination of electrolytes, antioxidants, and other components that help maintain the integrity of cell membranes and cellular structures. In general, most strategies aimed at organ preservation are initiated upon declaration of death.

[0006] Other approaches seek to maintain organ viability via therapeutic molecules that reduce cell death, reduce vascular permeability, and reduce edema associated with organ preservation. In addition to hypothermic preservation conditions, preservation at normothermic and hyperthermic temperatures have been tested for their ability to maintain organ viability.

[0007] A preservation technology that ameliorates IRI, maintains organ viability, and allows for assessment of quality and function prior to transplantation involves the use of oxygenated hypothermic and normothermic machine perfusion (MP) devices. For example, ex vivo prolonged lung preservation has resulted in successful transplantation of high-risk donor lungs. Normothermic MP of hearts and livers has displayed safe (heart) and superior (liver) preservation in randomized controlled trials (RCT). Normothermic kidney preservation for 24 h was recently established. Early clinical outcomes beyond the market entry trials indicate bioenergetics reconditioning, improved preservation of structures subject to IRI, and significant prolongation of the preservation time. The monitoring of perfusion parameters, the biochemical investigation of preservation fluids, and the assessment of tissue viability and bioenergetics function now offer a comprehensive assessment of organ quality and function ex situ. SUMMARY OF THEINVENTION

[0008] Systems and methods utilizing a Wnt agent are provided for the rehabilitation of organs and organ-derived cell products to be used for transplantation.

[0009] In some embodiments, the Wnt agent is delivered to the donor prior to organ retrieval, referred to herein as preconditioning. For example, in DBD (donation after brain death) or in DCD (donation after cardiac death), following declaration of death the Wnt agent is delivered to the donor via portal injection, or systemic injection. In an example, for brain dead donors where the heart continues beating, the Wnt agent can be delivered via portal or systemic injection after whichATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 organs can be retrieved and maintained in storage. For donors after cardiac death (DCD), the Wnt agent may be delivered by regional perfusion to allow for circulation of the Wnt agent through the blood vessels.

[0010] In some embodiments, the Wnt agent is delivered to an organ maintained at normothermic temperatures, at hypothermic temperatures, or at hyperthermic temperatures. In some embodiments, the Wnt agent is delivered during and after thawing of a vitrified organ.

[0011] In some embodiments, the Wnt agent is provided in a preservation medium that enhances organ preservation by minimizing ischemic injury and cellular damage and maintaining tissue architecture during the period when the organ is not receiving a blood supply.

[0012] In some embodiments, the Wnt agent is provided in a preservation medium that improves organ preconditioning by enhancing organ tolerance to IRI and other insults associated with marginal organ use and transplantation. Collectively these factors reduce the risk of graft dysfunction or loss when transplanted into an individual in need thereof.

[0013] A perfusate or preservation medium of the disclosure comprises an effective amount of a Wnt agent. In some embodiments, the Wnt agent is a Wnt agonist. In some embodiments, the Wnt agent is a Wnt protein. The Wnt protein may be any Wnt protein deemed useful. For instance, the Wnt protein may be a Wnt 1, Wnt 2, Wnt 2B, Wnt 3, Wnt 3A, Wnt 4, Wnt 5A, Wnt 5B, Wnt 6, Wnt 7A, Wnt 7B, Wnt 8A, Wnt 8B, Wnt 10A, Wnt 10B, Wnt 11, Wnt 14, Wnt 15, Wnt 16A, or Wnt 16B. In some embodiments, the Wnt protein is Wnt 3A. In some embodiments, the Wnt protein is formulated in a liposome. In some embodiments, the Wnt agent is liposomal Wnt 3A.

[0014] The Wnt agent preserves viability and inhibits apoptosis of cells in the marginal organs. The Wnt agent also activates stem, progenitor, or differentiated cells present in the marginal organs to promote repair, reconditioning, and / or regeneration of the marginal organ. The Wnt agent can reduce or inhibit caspase activity. The Wnt agent promotes repair, reconditioning, and / or regenerative responses.

[0015] In some embodiments, the Wnt agent prevents, reduces, or ameliorates ischemic damage to the organ, for example where the organ is a liver or tissue derived therefrom. In some embodiments, the Wnt agent reduces ischemic cholangiopathy, IC. In some embodiments, the Wnt agent maintains viability of the marginal organ. In some embodiments, the Wnt agent reduces fatty degeneration of the liver. In some embodiments, the Wnt agent reduces cirrhosis of the liver. In some embodiments, the Wnt reduces early allograft dysfunction or primary non-function of the organ. In some embodiments, the Wnt agent promotes regeneration of the organ. In some embodiments, the Wnt agent improves metabolic, synthetic, or mechanical function. In some embodiments, the Wnt improves functional architecture of the organ such as, but not limited, toATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 reduction in scarring of the organ parenchyma, and improves diffusion surface of the organ. In some embodiments, the rehabilitation of the organ improves or ameliorates any one of the criteria listed in Tables 1-5.

[0016] A marginal organ or marginal organ-derived cell product may be any organ or organ- derived cell product intended for transplantation into an individual in need thereof. Non-limiting marginal organs include a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, a heart, a peripheral or central nerve, a uterus, and connective tissue. In some embodiments, the marginal organ is a liver. In some embodiments, the marginal organ meets one or more of the criteria in any one of table 1-5.

[0017] Systems and methods utilizing a perfusion device and a perfusate comprising a Wnt agent are also provided for the rehabilitation of organs and organ-derived cell products to be used for transplantation. The systems and methods of the present disclosure maintain cell viability in organs and cell products from marginal organs by providing the organs with a preconditioning solution comprising a Wnt agent. The systems of the current disclosure utilize a perfusate solution comprising an effective amount of a Wnt agent.

[0018] The system of the present disclosure may have a number of features that allow for the system to maintain and / or preserve the marginal organ for the above described time ranges including, without limitation, monitoring and manipulating glucose levels within the perfusate, producing a pulsatile flow within the perfusate and throughout the entire perfusion loop, mixing oxygenated and deoxygenated blood prior to delivering the mixture to the marginal organ, oscillating the marginal organ within the perfusion device, maintaining a 50-75% oxygen saturation within the perfusate, monitoring and maintaining the following parameters in a physiological range: perfusate temperature; perfusate pH; perfusate hematocrit concentration; oxygen and CO2 levels in lines such as a hepatic artery line, a vena cava line, and a portal vein line; perfusate pressure and flow in lines such as a hepatic artery line, a vena cava line, and a portal vein line, etc.

[0019] In some embodiments, the methods of the present disclosure comprise perfusing the marginal organ, or conditioning the organ in preservation medium. The marginal organ may be treated for a range of different times. For instance, the marginal organ may be treated for at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, at least 168 hours, at least 180 hours, at least 192 hours, at least 204 hours, at least 216 hours, at least 228 hours, at least 240 hours, at least 252 hours, at least 264 hours, at least 276ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 hours, at least 288 hours, at least 300 hours, at least 312 hours, at least 324 hours, at least 336 hours, or greater than 336 hours.

[0020] The system and / or preservation media of the present disclosure is capable of preserving and / or maintaining a marginal organ for a range of different times. For instance, the system and preservation media are capable of preserving and / or maintaining the marginal organ for at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, at least 168 hours, at least 180 hours, at least 192 hours, at least 204 hours, at least 216 hours, at least 228 hours, at least 240 hours, at least 252 hours, at least 264 hours, at least 276 hours, at least 288 hours, at least 300 hours, at least 312 hours, at least 324 hours, at least 336 hours, or greater than 336 hours.

[0021] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of action, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention. BRIEFDESCRIPTION OF THEDRAWINGS

[0022] FIG. 1A-FIG.1D discloses the apoptotic response of hepatocytes following cold preservation of a liver for varying lengths of time, from 0 to 120 minutes.

[0023] FIG. 2A-FIG.2D discloses the level of apoptosis of hepatocytes in a liver slice culture, maintained at 37°C for 48 hours.

[0024] FIG.3A-FIG.3B discloses the distribution of Wnt-responsive stem cells in the intact liver.

[0025] FIG. 4 discloses the stability of liposomal WNT3A at a normothermic temperature as a function of time.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270

[0026] FIG. 5 discloses liposomal WNT3A endocytosis by cells as a function of time and temperature.

[0027] FIG.6 discloses the distribution of liposomal WNT3A following intrasplenic injection.

[0028] FIG.7A-FIG.7K discloses the level of apoptosis of hepatocytes in a liver graft slice treated with liposomal Wnt3A, maintained at 37°C for 48 hours.

[0029] FIG.8A-FIG.8D discloses the apoptotic response of hepatocytes following preconditioning with saline versus liposomal Wnt3A and cold preservation for 120 min. DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0030] Systems and methods are provided for the rehabilitation of marginal organs to be used for organ transplantation. The systems and methods of the present disclosure rehabilitate marginal organs by perfusing the marginal organs with a perfusate comprising a Wnt agent. The systems of the present disclosure comprise a perfusion device comprising a dialysis system and a perfusate comprising an effective dose of a Wnt agent. The Wnt agent reduces apoptosis of the cells in the marginal organ while also activating stem cells present in the marginal organ to promote repair, reconditioning, and / or regeneration of the marginal organ. In some embodiments, preconditioning of the marginal organ reduces the risk of graft rejection when transplanted into an individual in need thereof. In some embodiments, the perfusion device is a normothermic perfusion device.

[0031] Before the present methods are described, it is to be understood that this invention is not limited to particular methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0032] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges encompassed within the invention, subject to any specifically excluded limit in the stated range.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods andATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0034] It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a microsphere” includes a plurality of such microspheres and reference to “the stent” includes reference to one or more stents and equivalents thereof known to those skilled in the art, and so forth.

[0035] All publications, patents, and patent applications mentioned herein are expressly incorporated herein by reference to disclose and describe the systems, methods and / or materials in connection with which the publications are cited.

[0036] “Ischemic episode” as used herein refers to any circumstance that results in a deficient supply of blood to a tissue, usually due to a restriction in blood supply and / or decreased availability of oxygen to and / or in an organ or tissue of an individual, wherein the restriction may be a constriction and / or an obstruction, for example. The restriction may be due to factors in the blood vessels, in certain cases, and in particular aspects the ischemic episode results in damage or dysfunction of tissue of the organ or tissue and, in some cases, of the function of the organ or tissue itself.

[0037] “Reperfusion injury” as used herein refers to tissue damage caused when blood supply returns to the tissue after a period of ischemia. The absence of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function It is known that restoration of blood flow following an ischemic episode can be equally if not more damaging than the ischemic episode, because reintroduction of oxygen results in an increased production of damaging free radicals that results in reperfusion injury. Necrosis can be greatly accelerated upon reperfusion.

[0038] “Ischemic cholangiopathy” as used herein refers to focal or extensive damage to the bile ducts due to impaired blood supply. Ischemic cholangiopathy is characterized by bile duct necrosis, bile leakage, biloma, bile duct fibrosis or stenosis. Bile duct necrosis and bilomas develop predominantly where there is an abrupt and complete interruption of arterial blood supply, for example when HA thrombose in a liver transplant recipient.

[0039] “Normothermic perfusion” as used here in refers to a method where the perfusion or exposure to preservation medium is performed at or near physiological temperatures, e.g. fromATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 about 25oC to about 38oC, from about 30oto about 37o, from about 35-37o, and may be around 37oC. The principle of normothermic perfusion is to recreate the physiological environment by maintaining normal temperature and providing the essential substrates for cellular metabolism, oxygen and nutrition. Normothermic perfusion circuits may comprise components developed for cardiopulmonary bypass. Oxygen carriage is achieved by using either whole blood, pyridoxylated bovine haemoglobin or a combination of preservation solution and purified red blood cells. Other critical components of the perfusate include nutrition (glucose, insulin, amino acids), drugs to prevent thrombosis or micro-circulatory failure (heparin, prostacycline) and other agents to reduce cellular oedema, cholestasis and free radical injury. In addition to a reduction in ischaemia- reperfusion injury, a further potential advantage of normothermic perfusion is the assessment of viability: because the organ is metabolically active, it is possible to measure function, in order to predict post-transplant outcome before subjecting the patient to surgery.

[0040] “Hypothermic perfusion” as used herein refers to methods where the perfusion or exposure to preservation medium is performed below physiologic temperatures. Hypothermic oxygenated perfusion allows a redirect from anaerobic metabolism to aerobic metabolism under hypothermic conditions and protecting grafts from oxidative species–related damage. The temperature for hypothermic perfusion may be from about 1oC to about 25oC, usually from about 4oC to about 10oC, and may be from about 4oC to about 8oC.

[0041] “Hyperthermic perfusion” as used herein refers to methods where the perfusion or exposure to preservation medium is performed above physiologic temperatures. The temperature for hyperthermic perfusion may be from about 38oC to about 43oC, usually from about 39oC to about 43oC, and may be from about 40oC to about 42oC.

[0042] “Vitrification” as used herein refers to a cryopreservation process that rapidly freezes cells, tissues, and / or solutions in the absence of ice crystallization. Vitrification may employ the usage of cryoprotectants and additives that assist in the process to rapidly dehydrate cells. A “vitrified organ” refers to an organ that has undergone vitrification resulting in an organ that is cryopreserved and is capable of being thawed and is able to function physiologically.

[0043] “Marginal organ” as used herein refers to organs that are: from suboptimal cadaveric grafts, from non-heart beating donors, from living donors with some acceptable medical risks, from older donors (e.g., from individuals aged 50 or older), and, in the case of livers, steatotic livers, livers from hepatitis C positive donors, or split livers. Marginal organs may be defined using specific criteria such as the criteria disclosed below. In some cases, marginal organs are organs who satisfy one or more of the criteria below. Criteria for classifying an organ as a marginal organATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 are known in the art and have been disclosed by, for example, Resch et al. (Front Immunol.2020 May 12:11:631) which is specifically incorporated by reference herein.

[0044] “Donation after brain death (DBD)”, “brain-dead donor”, or “donor after brain death” as used herein refers to a donor who has had primary brain death but whose circulatory system remains functional, either naturally or by medical measures (e.g., mechanical ventilation, drugs, intra-aortic balloon pump, or extracorporeal machine oxygenation device).

[0045] “Donation after cardiac death” or “donor after cardiac death” as used herein refers to a donor whose circulatory function had been lost (e.g., heart death) prior to organ harvest. In some examples, artificial circulation such as by heart-lung bypass instruments may be needed to deliver the compound administered to the DCD donor to the organ(s) contemplated for harvest and transplant. DCD donors can be further classified into further subgroups. In some examples, the DCD donor is a controlled DCD (cDCD) donor. A cDCD donor is a donor whose life support will be withdrawn and whose family has given written consent for organ donation in the controlled environment of the operating room. In some examples, the DCD donor is an uncontrolled DCD (uDCD) donor. A uDCD donor is a donor, for example, who expires in the emergency room or elsewhere in the hospital before consent for organ donation is obtained and catheters are placed in the femoral vessels and peritoneum to cool organs until consent can be obtained. A uDCD donor is also a donor who has consented for organ donation but suffers a cardiac arrest requiring CPR during procurement of the organs. Table 1. Commonly used criteria for marginal kidneys. CVA= cerebrovascular accident was the cause of death; HTN = History of hypertension Donor Condition Donor Age Category (years)Table 2. Commonly used criteria for marginal livers. Donation after brain deathATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 Cardiac arrest (min) >15 Prolonged hypotensive periods <60 mmHg for >1 hr s virus;HCV=hepatitis C virus; BMI=body mass index; ICU=intensive care unit; AST=aspartate aminotransferase; ALT=Alanin-aminotransferase. Table 3. Commonly used criteria for marginal pancreas. Age (yrs) <10 or >45ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 Table 4. Commonly used criteria for marginal hearts. Age (yrs) >55 BMI mismatch donor / recipient (%) >20. y g g . Age (yrs) >55

[0046] Wnt protein. Wnt proteins form a family of highly conserved secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis. The terms "Wnts" or "Wnt gene product" or "Wnt protein" or "Wnt polypeptide" are used interchangeable and encompass native sequence Wnt polypeptides, Wnt polypeptide variants, Wnt polypeptide fragments and chimeric Wnt polypeptides. In some embodiments of the invention, the Wnt protein comprises palmitateATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 covalently bound to a cysteine residue. A "native sequence" polypeptide is one that has the same amino acid sequence as a Wnt polypeptide derived from nature, regardless of the method used for its production. Such native sequence polypeptides can be isolated from cells producing endogenous Wnt protein or can be produced by recombinant or synthetic means. Thus, a native sequence polypeptide can have the amino acid sequence of, e.g., naturally occurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species, or from non- mammalian species, e.g. Drosophila, C. elegans, and the like.

[0047] The term "native sequence Wnt polypeptide" includes, without limitation, human and murine Wnt polypeptides. Human Wnt proteins include the following: Wnt1, Genbank reference NP005421.1; Wnt2, Genbank reference NP003382.1, which is expressed in brain in the thalamus, in fetal and adult lung and in placenta; two isoforms of Wnt2B, Genbank references NP004176.2 and NP078613.1. Isoform 1 is expressed in adult heart, brain, placenta, lung, prostate, testis, ovary, small intestine, and colon. In the adult brain, it is mainly found in the caudate nucleus, subthalamic nucleus and thalamus. Also detected in fetal brain, lung, and kidney. Isoform 2 is expressed in fetal brain, fetal lung, fetal kidney, caudate nucleus, testis, and cancer cell lines. Wnt 3 and Wnt3A play distinct roles in cell-cell signaling during morphogenesis of the developing neural tube and have the Genbank references NP110380.1 and X56842 (Swiss-Prot P56704), respectively. . Wnt3 has the Genbank reference AB060284.1 (see also GenBank Nos. BAB61052.1 and AAI03924.1). Wnt3A has the Genbank accession BC103922 and the accession number BC103921. In some instances, the term "native sequence Wnt protein" or "native sequence Wnt polypeptide" includes the Wnt3A native polypeptides (e.g., polypeptides of accession numbers BC103921 and BC103922) with or without the initiating N-terminal methionine (Met), and with or without the native signal sequence. In some cases, the terms include the 352 amino acids native human Wnt3A polypeptide of SEQ ID NO:2, without or without its N-terminal methionine (Met), and with or without the native signal sequence.

[0048] In some embodiments, the native human Wnt3A amino acid sequence is specifically disclosed as the following: MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMP SVAEGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSC AEGTAAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNN EAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESR GWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCC GRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCKNPGSRAGNSAHQPPHPQPPV RFHPPLRRAGKVP (SEQ ID NO: 01).ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270

[0049] Wnt3A is expressed in bone marrow. Wnt 4 has the Genbank reference NP110388.2. Wnt 5A and Wnt 5B have the Genbank references NP003383.1 and AK013218. Wnt 6 has the Genbank reference NP006513.1; Wnt 7A is expressed in placenta, kidney, testis, uterus, fetal lung, and fetal and adult brain, Genbank reference NP004616.2. Wnt 7B is moderately expressed in fetal brain, weakly expressed in fetal lung and kidney, and faintly expressed in adult brain, lung, and prostate, Genbank reference NP478679.1. Wnt 8A has two alternative transcripts, Genbank references NP114139.1 and NP490645.1. Wnt 8B is expressed in the forebrain and has the Genbank reference NP003384.1. Wnt 10A has the Genbank reference NP079492.2. Wnt 10B is detected in most adult tissues, with highest levels in heart and skeletal muscle. It has the Genbank reference NP003385.2. Wnt 11 is expressed in fetal lung, kidney, adult heart, liver, skeletal muscle, and pancreas, and has the Genbank reference NP004617.2. Wnt 14 has the Genbank reference NP003386.1. Wnt 15 is moderately expressed in fetal kidney and adult kidney and is also found in brain. It has the Genbank reference NP003387.1. Wnt 16 has two isoforms, Wnt- 16a and Wnt-16b, produced by alternative splicing. Isoform Wnt-16B is expressed in peripheral lymphoid organs such as spleen, appendix, and lymph nodes, in kidney but not in bone marrow. Isoform Wnt-16a is expressed at significant levels only in the pancreas. The Genbank references are NP057171.2 and NP476509.1. All GenBank, SwissProt and other database sequences listed are expressly incorporated by reference herein.

[0050] The term "native sequence Wnt protein" or "native sequence Wnt polypeptide" includes the native proteins with or without the initiating N-terminal methionine (Met), and with or without the native signal sequence. The terms specifically include the 352 amino acids long native human Wnt3a polypeptide, without or without its N terminal methionine (Met), and with or without the native signal sequence.

[0051] A "variant" polypeptide means a biologically active polypeptide as defined below having less than 100% sequence identity with a native sequence polypeptide. Such variants include polypeptides wherein one or more amino acid residues are added at the N- or C- terminus of, or within, the native sequence; from about one to forty amino acid residues are deleted, and optionally substituted by one or more amino acid residues; and derivatives of the above polypeptides, wherein an amino acid residue has been covalently modified so that the resulting product has a non-naturally occurring amino acid. Ordinarily, a biologically active Wnt variant will have an amino acid sequence having at least about 90% amino acid sequence identity with a native sequence Wnt polypeptide, preferably at least about 95%, more preferably at least about 99%.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270

[0052] In some instances, a biologically active Wnt variant has an amino acid sequence having at least about 80% amino acid sequence identity with a native sequence Wnt polypeptide. In some instances, the biologically active Wnt variant has an amino acid sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, or 99% amino acid sequence identity with a native sequence Wnt polypeptide. In some cases, the biologically active Wnt variant has an amino acid sequence having at least about 95% amino acid sequence identity with a native sequence Wnt polypeptide. In some cases, the biologically active Wnt variant has an amino acid sequence having at least about 99% amino acid sequence identity with a native sequence Wnt polypeptide. In some embodiments, the biologically active Wnt variant is a Wnt3A variant. In some embodiments, the biologically active Wnt variant is a human Wnt3A variant.

[0053] In some instances, a biologically active Wnt variant comprises a truncation of a native sequence of a Wnt polypeptide. In some instances, a biologically active Wnt variant comprises an amino acid sequence according to: MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGLVPKQLRFCRNYVEIMP SVAEGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRESAFVHAIASAGVAFAVTRSC AEGTAAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDARSAMNRHNN EAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQPDFRAIGDFLKDKYDSASEMVVEKHRESR GWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVSSHGIDGCDLLCC GRGHNARAERRREKCRCVFHWCCYVSCQECTRVYDVHTCK (SEQ ID NO: 02). In some instances, a biologically active Wnt variant comprises a lipid modification at one or more amino acid positions. In some cases, the lipid modification is at a position on a Wnt variant that is equivalent to position 77 set forth in SEQ ID NO: 1. In some cases, the lipid modification is at a position on a Wnt variant that is equivalent to position 209 set forth in SEQ ID NO: 1. In some cases, the lipid modification comprises both positions that are equivalent to positions 77 and 209 set forth in SEQ ID NO: 1. In some instances, the Wnt variant is Wnt3A, Wnt5A or Wnt 10B. In some cases, the Wnt variant is Wnt3A. In some cases, the Wnt3A variant comprises a lipid modification at a position equivalent to residue 77 set forth in SEQ ID NO: 1. In some cases, the Wnt3A variant comprises a lipid modification at a position equivalent to residue 209 set forth in SEQ ID NO: 1. In some cases, the Wnt3A variant comprises lipid modifications at positions equivalent to residues 77 and 209 set forth in SEQ ID NO: 1. In some cases, the modification is palmitoylation.

[0054] In some instances, a biologically active Wnt variant further comprises a residue modified by glycosylation. In some cases, the modification occurs at a position equivalent to position 82 and / or 298 set forth in SEQ ID NO: 1. In some cases, the Wnt variant is Wnt3A. In some cases,ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 a Wnt3A variant further comprises a residue modified by glycosylation. In some cases, a Wnt3A variant further comprises a glycosylated residue at one or more positions equivalent to residue 82 and / or residue 298 set forth in SEQ ID NO: 1.

[0055] A "chimeric" Wnt polypeptide is a polypeptide comprising a Wnt polypeptide or portion (e.g., one or more domains) thereof fused or bonded to heterologous polypeptide. The chimeric Wnt polypeptide will generally share at least one biological property in common with a native sequence Wnt polypeptide. Examples of chimeric polypeptides include immunoadhesins, combine a portion of the Wnt polypeptide with an immunoglobulin sequence, and epitope tagged polypeptides, which comprise a Wnt polypeptide or portion thereof fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made yet is short enough such that it does not interfere with biological activity of the Wnt polypeptide. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 6-60 amino acid residues.

[0056] A "functional derivative" of a native sequence Wnt polypeptide is a compound having a qualitative biological property in common with a native sequence Wnt polypeptide. "Functional derivatives" include, but are not limited to, fragments of a native sequence and derivatives of a native sequence Wnt polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence Wnt polypeptide. The term "derivative" encompasses both amino acid sequence variants of Wnt polypeptide and covalent modifications thereof.

[0057] Biologically Active Wnt. The methods of the disclosure utilize Wnt compositions that are active when administered to an animal, e.g., a mammal, such as a human, in vivo. One may determine the specific activity of a Wnt protein in a composition by determining the level of activity in a functional cell-based assay, for example in an in vitro assay, or after in vivo administration in a test model, e.g. accelerating bone regeneration, upregulation of stem cell proliferation, etc.

[0058] Alternative assays for quantitating the amount of Wnt protein present include non- functional assays, e.g. immunostaining, ELISA, quantitation on Coomasie or silver stained gel, etc., and determining the ratio of in vivo biologically active Wnt to total Wnt.

[0059] Lipid Structure. As used in the systems and methods of the disclosure, lipid structures are found to benefit the activity of Wnt proteins following in vivo administration. These Wnt proteins are not encapsulated in the aqueous phase of these structures, but are rather integrated into the lipid membrane, and may be inserted in the outer layer of a membrane. Such a structure is not predicted from conventional methods of formulating proteins in, for example, liposomes. A Wnt polypeptide with such lipid structure is referred herein as L-Wnt, such as L-Wnt3a. The methodsATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 used for tethering Wnt proteins to the external surface of a liposome or micelle may utilize a sequence so as to emphasize the exo-liposomal display of the protein, where crude liposomes are first pre-formed; Wnt protein is then added to the crude mixture, which will favor addition of exo-liposomal Wnt, followed by various formulation steps, which may include size filtering; dialysis, and the like. Suitable lipids include fatty acids, neutral fats such as triacylglycerols, fatty acid esters and soaps, long chain (fatty) alcohols and waxes, sphingoids, and other long chain bases, glycolipids, sphingolipids, carotenes, polyprenols, sterols, and the like, as well as terpenes and isoprenoids. For example, molecules such as diacetylene phospholipids may find use. Included are cationic molecules, including lipids, synthetic lipids, and lipid analogs, having hydrophobic and hydrophilic moieties, a net positive charge, and which by itself can form spontaneously into bilayer vesicles or micelles in water. Liposomes manufactured with a neutral charge, e.g., DMPC, are preferred. The term also includes any amphipathic molecules that can be stably incorporated into lipid micelle or bilayers in combination with phospholipids, with its hydrophobic moiety in contact with the interior, hydrophobic region of the micelle or bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane.

[0060] The term "cationic amphipathic molecules" is intended to encompass molecules that are positively charged at physiological pH, and more particularly, constitutively positively charged molecules, comprising, for example, a quaternary ammonium salt moiety. Cationic amphipathic molecules typically consist of a hydrophilic polar head group and lipophilic aliphatic chains. Similarly, cholesterol derivatives having a cationic polar head group may also be useful. See, for example, Farhood et al. (1992) Biochim. Biophys. Acta 1111:239- 246; Vigneron et al. (1996) Proc. Natl. Acad. Sci. (USA) 93:9682-9686. Cationic amphipathic molecules of interest include, for example, imidazolinium derivatives (WO 95 / 14380), guanidine derivatives (WO 95 / 14381), phosphatidyl choline derivatives (WO 95 / 35301), and piperazine derivatives (WO 95 / 14651). Examples of cationic lipids that may be used in the present invention include DOTIM (also called BODAI) (Saladin et al., (1995) Biochem. 34: 13537-13544), DDAB (Rose et al., (1991) BioTechniques 10(4):520-525), DOTMA (U.S. Pat. No. 5,550,289), DOTAP (Eibl and Wooley (1979) Biophys. Chern.10:261-271), DMRIE (Feigner et al., (1994) J. Bioi. Chern.269(4): 2550- 2561), EDMPC (commercially available from Avanti Polar Lipids, Alabaster, Ala.), DCC hoi (Gau and Huang (1991) Biochem. Biophys. Res. Comm.179:280-285), DOGS (Behr et al., (1989) Proc. Nat!. Acad. Sci. USA, 86:6982-6986), MBOP (also called MeBOP) (WO 95 / 14651 ), and those described in WO 97 / 00241.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270

[0061] While not required for activity, in some embodiments a lipid structure may include a targeting group, e.g., a targeting moiety covalently or non-covalently bound to the hydrophilic head group. Head groups useful to bind to targeting moieties include, for example, biotin, amines, cyano, carboxylic acids, isothiocyanates, thiols, disulfides, ahalocarbonyl compounds, a,p- unsaturated carbonyl compounds, alkyl hydrazines, etc. Chemical groups that find use in linking a targeting moiety to an amphipathic molecule also include carbamate; amide (amine plus carboxylic acid); ester (alcohol plus carboxylic acid), thioether (haloalkane plus sulfhydryl; maleimide plus sulfhydryl), Schiffs base (amine plus aldehyde), urea (amine plus isocyanate), thiourea (amine plus isothiocyanate), sulfonamide (amine plus sulfonyl chloride), disulfide; hyrodrazone, lipids, and the like, as known in the art. For example, targeting molecules may be formed by converting a commercially available lipid, such as DAGPE, a PEG-PDA amine, DOTAP, etc. into an isocyanate, followed by treatment with triethylene glycol diamine spacer to produce the amine terminated thiocarbamate lipid which by treatment with the para- isothiocyanophenyl glycoside of the targeting moiety produces the desired targeting glycolipids. This synthesis provides a water soluble flexible linker molecule spaced between the amphipathic molecule that is integrated into the nanoparticle, and the ligand that binds to cell surface receptors, allowing the ligand to be readily accessible to the protein receptors on the cell surfaces. Further information about liposomal Wnt compositions and their use is found in U.S. Patent No.10183057 which is specifically incorporated by reference herein.

[0062] Chemical groups that find use in linking a targeting moiety to an amphipathic molecule also include carbamate; amide (amine plus carboxylic acid); ester (alcohol plus carboxylic acid), thioether (haloalkane plus sulfhydryl; maleimide plus sulfhydryl), Schiff’s base (amine plus aldehyde), urea (amine plus isocyanate), thiourea (amine plus isothiocyanate), sulfonamide (amine plus sulfonyl chloride), disulfide; hyrodrazone, lipids, and the like, as known in the art.

[0063] For example, targeting molecules may be formed by converting a commercially available lipid, such as DAGPE, a PEG-PDA amine, DOTAP, etc. into an isocyanate, followed by treatment with triethylene glycol diamine spacer to produce the amine terminated thiocarbamate lipid which by treatment with the para-isothiocyanophenyl glycoside of the targeting moiety produces the desired targeting glycolipids. This synthesis provides a water soluble flexible linker molecule spaced between the amphipathic molecule that is integrated into the nanoparticle, and the ligand that binds to cell surface receptors, allowing the ligand to be readily accessible to the protein receptors on the cell surfaces.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270

[0064] A targeting moiety, as used herein, refers to all molecules capable of specifically binding to a particular target molecule and forming a bound complex as described above. Thus, the ligand and its corresponding target molecule form a specific binding pair.

[0065] The term "specific binding" refers to that binding which occurs between such paired species as enzyme / substrate, receptor / agonist, antibody / antigen, and lectin / carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non- covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, "specific binding" occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody / antigen or enzyme / substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. Thus, for example, an antibody preferably binds to a single epitope and to no other epitope within the family of proteins.

[0066] Examples of targeting moieties include, but are not limited to antibodies, lymphokines, cytokines, receptor proteins such as CD4 and CD8, solubilized receptor proteins such as soluble CD4, hormones, growth factors, peptidomimetics, synthetic ligands, and the like which specifically bind desired target cells, and nucleic acids which bind corresponding nucleic acids through base pair complementarity. Targeting moieties of particular interest include peptidomimetics, peptides, antibodies and antibody fragments (e.g. the Fab' fragment). For example, b-D-lactose has been attached on the surface to target the aloglysoprotein (ASG) found in liver cells which are in contact with the circulating blood pool.

[0067] Cellular targets include tissue specific cell surface molecules, for targeting to specific sites of interest, e.g. neural cells, liver cells, bone marrow cells, kidney cells, pancreatic cells, muscle cells, and the like. For example, nanoparticles targeted to hematopoietic stem cells may comprise targeting moieties specific for CD34, ligands for c-kit, etc. Nanoparticles targeted to lymphocytic cells may comprise targeting moieties specific for a variety of well known and characterized markers, e.g. B220, Thy-1, and the like.

[0068] The use of liposomes or micelles as a delivery vehicle is one method of interest. A liposome is a spherical vesicle with a membrane composed of a phospholipid bilayer. Liposomes can be composed of naturally-derived phospholipids with mixed lipid chains (like egg phosphatidylethanolamine), or of pure surfactant components like DOPE (dioleolylphosphatidylethanolamine). Liposomes often contain a core of encapsulated aqueousATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 solution; while lipid spheres that contain no aqueous material are referred to as micelles. As the Wnt proteins are present in the lipid phase and not the encapsulated aqueous phase, micelles may be used interchangeably with liposome for the compositions of the present invention. The lipids may be any useful combination of known liposome or micelle forming lipids, including cationic lipids, such as phosphatidylcholine, or neutral lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.

[0069] In another embodiment, the vesicle-forming lipid is selected to achieve a specified degree of fluidity or rigidity, to control the stability of the structure in vivo, etc. Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer, are achieved by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g., up to 60oC. Rigid, i.e., saturated, lipids contribute to greater membrane rigidity in the lipid bilayer. Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity in lipid bilayer structures. Lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g., at or below room temperature.

[0070] The liposomes may be prepared by a variety of techniques, such as those detailed in Szoka, F., Jr., et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980). Typically, the liposomes are multilamellar vesicles (MLVs), which can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome-forming lipids of the type detailed above dissolved in a suitable organic solvent is evaporated in a vessel to form a thin film, which is then covered by an aqueous medium. The lipid film hydrates to form MLVs, typically with sizes between about 0.05 to 1 micron.

[0071] The liposomal micelles, etc. of the invention may have substantially homogeneous sizes in a selected size range, typically between about 0.01 to 0.5 microns, more preferably between 0.03-0.10 microns. One effective sizing method for REVs and MLVs involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2 microns. The pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane. Homogenization methods are also useful for down- sizing liposomes to sizes of 100 nm or less.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 METHODS FOR REHABILITATING, CONDITIONING, OR PRECONDITIONING MARGINAL ORGANS

[0072] The present disclosure provides methods for the rehabilitation, conditioning, or preconditioning of marginal organs comprising contacting a marginal organ with a perfusate or preservation medium comprising an effective amount of a Wnt agent. The medium or perfusate may be any deemed useful, for example, Stanford University solution; Collins solution; modified Collins solution; University of Wisconsin solution; modified University of Wisconsin solution; Columbia University solution; Vasosol; histidine-tryptophan-ketoglutarate (HTK) solution; Celsior; isotonic saline solutions, that may contain, in various proportions, salts, sugars, osmotic agents, local anesthetic, buffers, and other such agents; ViaSpan®; solutions comprising pyruvate, inorganic salts supporting cell membrane potential and albumin or fetal calf serum; solutions comprising one or more phosphatidic acids or sugars, and lysophosphotidic acids or sugars, together with enhancers such as albumen, optionally delivered in liposomal compositions; whole blood; parts of whole blood (e.g., plasma with synthetic hemoglobin); other organ preservation solutions (see, e.g., U.S. Pat. No. 7,220,538); or any combination of the foregoing. Vasosol is known in the art and has been described by, for example, Polyak et al. (J Surg Res. 2008 Dec;150(2):255-60) which is specifically incorporated by reference herein. In some embodiments, the perfusate comprises whole blood. In some embodiments, the perfusate comprises parts of whole blood. In some embodiments, the perfusate comprises a clinical grade organ preservation solution. Clinical grade organ preservation solutions include, without limitation, University of Wisconsin solution, histidine-tryptophan-ketoglutarate, Celsior, Institut Georges Lopez (IGL-1) solution, etc.

[0073] In some embodiments, the method of rehabilitating, conditioning, or preconditioning a marginal organ comprises obtaining a marginal organ, contacting the marginal organ with a preservation medium comprising an effective dose of a Wnt agent, thereby rehabilitating the marginal organ. The marginal organ may be in vivo or ex vivo. When the marginal organ is in vivo, the marginal organ is in a donor after brain death (DBD) or a donor after cardiac death (DCD). When the donor is a DBD, the donor has a functional circulatory system and the contacting may comprise local or systemic intravenous or intraarterial delivery into veins and / or arteries that flow to the marginal organ. For instance, when the marginal organ is a liver in a DBD, the contacting includes delivery through the portal vein. When the donor is a DCD, the donor does not have a functional circulatory system and the contacting may comprise regional prefusion into the marginal organ. Additionally, the contacting includes, without limitation, intra-arterial delivery, intravenous delivery, intrabronchial delivery, intracardiac delivery, intracerebral delivery, intracisternal delivery, intracorneal delivery, intraesophageal delivery, intragastric delivery,ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 intradural delivery, intradermal delivery, intraepidermal delivery, intraileal delivery, intrapulmonary delivery, intraspinal, intrasynovial delivery, intrathecal delivery, intrauterine delivery, etc. When the marginal organ is ex vivo, the contacting includes perfusing the marginal organ with a perfusate comprising an effective amount of the Wnt agent.

[0074] When the marginal organ is ex vivo, the marginal organ may be maintained in normothermic, hyperthermic, or hypothermic conditions. When the marginal organ is maintained at normothermic conditions, the marginal organ may be maintained from about 25oC to about 38oC, from about 30oto about 37o, or from about 35-37oC. When the marginal organ is maintained at hyperthermic conditions, the marginal organ may be maintained from about 38oC to about 43oC, from about 39oC to about 43oC, or from about 40oC to about 42oC. When the marginal organ is maintained at hypothermic conditions, the marginal organ may be maintained from 1oC to about 25oC, from about 4oC to about 10oC, or from about 4oC to about 8oC. In some embodiments, the marginal organ is a vitrified organ. When the marginal organ is a vitrified organ, the contacting occurs during and / or after the thawing of the vitrified organ. In some embodiments, the contacting occurs during the thawing of the vitrified organ. In some embodiments, the contacting occurs after the thawing of the vitrified organ. In some embodiments, the contacting occurs during and after the thawing of the vitrified organ.

[0075] The marginal organ may be a range of different marginal organs. Non-limiting marginal organs include a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, a heart, a uterus, a peripheral or central nerve, and connective tissue. In some embodiments, the marginal organ is a liver. In some embodiments, the marginal organ is a brain. In some embodiments, the marginal organ is a spinal cord. In some embodiments, the marginal organ is a kidney. In some embodiments, the marginal organ is a uterus. In some embodiments, the marginal organ is a lung. In some embodiments, the marginal organ is an eye. In some embodiments, the marginal organ is a pancreas. In some embodiments, the marginal organ is a spleen. In some embodiments, the marginal organ is an intestine. In some embodiments, the marginal organ is a cornea. In some embodiments, the marginal organ is skin. In some embodiments, the marginal organ is a heart. In some embodiments, the marginal organ is a peripheral or central nerve. In some embodiments, the marginal organ is a connective tissue. In some embodiments, the marginal organ meets one or more of the criteria in any one of table 1-5.

[0076] The methods of the present disclosure comprise a perfusate or medium comprising an effective amount of a Wnt agent. The Wnt agent is capable of inhibiting programmed cell death within the marginal organ and preventing or reducing ischemic damage. The Wnt agent is capableATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 of activating stem cells within the marginal organ and inducing repair, reconditioning, and / or regenerative responses. The Wnt agent can reduce or inhibit caspase activity, e.g., caspase 2 activity, a caspase 8 activity, a caspase 9 activity, a caspase 10 activity, a caspase 3 activity, a caspase 6 activity, and / or a caspase 7 activity. In some embodiments, the Wnt agent is a Wnt agonist. In some embodiments, the Wnt agent is a Wnt protein. The Wnt protein may be any Wnt protein deemed useful. For instance, the Wnt protein may be a Wnt 1, Wnt 2, Wnt 2B, Wnt 3, Wnt 3A, Wnt 4, Wnt 5A, Wnt 5B, Wnt 6, Wnt 7A, Wnt 7B, Wnt 8A, Wnt 8B, Wnt 10A, Wnt 10B, Wnt 11, Wnt 14, Wnt 15, Wnt 16A, or Wnt 16B. In some embodiments, the Wnt protein is Wnt 3A. In some embodiments, the Wnt protein is formulated in a liposome. In some embodiments, the Wnt agent is liposomal Wnt 3A. Liposomal Wnt 3A is known in the art and have been described in, for example, US Patent Nos 9,937,126 and 9,301,980 and US Patent Application No 16 / 067,944 each of which is specifically incorporated by reference herein. In some embodiments, the Wnt agent is a GSK-3 inhibitor. GSK-3 inhibitors include, without limitation, GSK 3 Inhibitor IX (6- Bromoindirubin-3'-oxime; BIO), SB-216763, SB-415286, tideglusib, CHIR99021, Bisindolylmaleimide I (GF109203X), LY2090314, COB-187, Kenpaullone, TWS119, AR- A014418, Lithium chloride hydrate, CHIR-98014, TDZD-8, etc.

[0077] An effective amount of a Wnt agent that may be administered to an individual or a cell in a tissue or organ thereof includes a dose of about 0.0001 nM to about 2000 μM, for example. More specifically, doses are from about 0.01 nM to about 2000 μM; about 0.01 μM to about 0.05 μM; about 0.05 μM to about 1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5 μM to about 2.0 μM; about 2.0 μM to about 3.0 μM; about 3.0 μM to about 4.0 μM; about 4.0 μM to about 5.0 μM; about 5.0 μM to about 10 μM; about 10 μM to about 50 μM; about 50 μM to about 100 μM; about 100 μM to about 200 μM; about 200 μM to about 300 μM; about 300 μM to about 500 μM; about 500 μM to about 1000 μM; about 1000 μM to about 1500 μM and about 1500 μM to about 2000 μM, for example. Of course, all of these amounts are exemplary, and any amount in-between these points is also expected to be of use in the invention.

[0078] In some embodiments, the Wnt agent maintains viability of the marginal organ. In some embodiments, the Wnt agent prevents ischemic damage to the organ. In some embodiments, preconditioning or conditioning of the marginal organ reduces the risk of reperfusion injury. In some embodiments, the Wnt reduces early allograft dysfunction or primary non-function of the organ. In some embodiments, the Wnt agent promotes regeneration of the organ. In some embodiments, preconditioning of the marginal organ reduces the risk of graft rejection when transplanted into an individual in need thereof. In some embodiments, the Wnt agent reduces ischemic cholangiopathy. In some embodiments, the Wnt agent improves metabolic, synthetic orATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 mechanical function. In some embodiments, the Wnt improves the functional architecture of the organ such as but not limited to reduction in scarring of the organ parenchyma, improved diffusion surface of the organ. In some embodiments, the rehabilitation of the marginal organ improves or ameliorates any one of the criteria listed in Tables 1-5.

[0079] The methods of the present disclosure are capable of preconditioning, conditioning, preserving, and rehabilitating marginal organs. The effects on marginal organs in the present disclosure may provide a number of benefits including, without limitation, improving the quality of the marginal organ, reducing the risk of reperfusion injury once the marginal organ has been transplanted into an individual in need thereof, reducing the risk of graft failure, etc. In some embodiments, the method inhibits apoptosis of cells in the marginal organ.

[0080] In some embodiments, the rehabilitation, conditioning, or preconditioning of the marginal organ reduces the risk of reperfusion injury. In some embodiments, rehabilitation, conditioning, or preconditioning of the marginal organ reduces the risk of reperfusion injury. In some embodiments, the Wnt reduces early allograft dysfunction or primary non-function of the organ. In some embodiments, the Wnt agent promotes regeneration of the organ. In some embodiments, the Wnt agent reduces ischemic cholangiopathy. In some embodiments, the preconditioning of the marginal organ reduces the risk of graft rejection when transplanted into an individual in need thereof. In some embodiments, the Wnt agent improves metabolic, synthetic, or mechanical function. In some embodiments, the Wnt improves functional architecture of the organ such as but not limited to reduction in scarring of the organ parenchyma, improved diffusion surface of the organ. In some embodiments, the Wnt agent is provided in a preservation medium that enhances organ preservation by minimizing ischemic injury and cellular damage and maintaining tissue architecture during the period when the organ is not receiving a blood supply. In some embodiments, the Wnt agent is provided in a preservation medium that improves organ preconditioning by enhancing organ tolerance to IRI and other insults associated with marginal organ use and transplantation. In some embodiments, the rehabilitation of the marginal organ improves or ameliorates any one of the criteria listed in Tables 1-5.

[0081] The present disclosure provides methods of preserving an organ comprising contacting an organ with a preservation medium comprising an effective amount of a Wnt agent. The Wnt agent may be any of the Wnt agents discussed above. The organ may be a marginal organ or a non-marginal organ. In some embodiments, the organ is a marginal organ. In some embodiments, the organ is a non-marginal organ. Non-limiting organs include a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, a heart, a uterus, a peripheral or central nerve, and connective tissue. The contacting may include a range of differentATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 contacting means. For instance, the contacting may be incubating the organ in the preservation medium, perfusing the organ with the preservation medium, etc.

[0082] The organ may be maintained at a temperature that assists in preserving the organ. In some embodiments, the organ is maintained at hypothermic temperatures. For instance, the organ may be maintained from 1oC to about 25oC, from about 4oC to about 10oC, or from about 4oC to about 8oC. The organ may be preserved for a range of different times. For instance, the organ may be preserved for at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 37 hours, at least about 38 hours, at least about 39 hours, at least about 40 hours at least about 41 hours, at least about 42 hours, at least about 43 hours, at least about 44 hours, at least about 45 hours, at least about 46 hours, at least about 47 hours, at least about 48 hours, or greater than 48 hours.

[0083] The preserving of the organ in a perseveration medium comprising an effective amount of a Wnt agent reduces the amount of programed cell death / apoptosis compared to an organ preserved in a preservation medium in the absence of a Wnt agent. The preserving of the organ may provide a range of different benefits. For instance, the preserving may reduce the risk ischemic damage to the organ, reduce the risk of early allograft dysfunction or primary non- function of the organ, reduce apoptosis of the cells in the organ, and reduce the risk of reperfusion injury, etc. SYSTEMS FOR REHABILITATING MARGINAL ORGANS

[0084] In some embodiments of the present disclosure, systems for the rehabilitation of marginal organs comprise a perfusion device and a perfusate comprising an effective amount of a Wnt agent. In some embodiments, the perfusion device comprises a dialysis system.

[0085] The marginal organ may be a range of different marginal organs. Non-limiting marginal organs include a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, a heart, a uterus, a peripheral or central nerve, and connective tissue.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 In some embodiments, the marginal organ is a liver. In some embodiments, the marginal organ is a brain. In some embodiments, the marginal organ is a spinal cord. In some embodiments, the marginal organ is a kidney. In some embodiments, the marginal organ is a uterus. In some embodiments, the marginal organ is a lung. In some embodiments, the marginal organ is an eye. In some embodiments, the marginal organ is a pancreas. In some embodiments, the marginal organ is a spleen. In some embodiments, the marginal organ is an intestine. In some embodiments, the marginal organ is a cornea. In some embodiments, the marginal organ is skin. In some embodiments, the marginal organ is a heart. In some embodiments, the marginal organ is a peripheral or central nerve. In some embodiments, the marginal organ is a connective tissue. In some embodiments, the marginal organ meets one or more of the criteria in any one of table 1-5.

[0086] The systems of the present disclosure comprise a perfusion device. The perfusion device may be any perfusion device that is capable of rehabilitating a marginal organ using the perfusate of the present disclosure. In some embodiments, the perfusion device is a normothermic perfusion device. Normothermic perfusion devices are known in the art and have been described in, for example, Eshmuminov et al. (Nat Biotechnol 2020 Feb;38(2):189-198), Croome K. (J Clin Med. 2023 Jan 23;12(3):909) and US Patent Publication Nos US 2020 / 0375178 A1 and US 2020 / 0253194 A1, each of which are specifically incorporated by reference herein. Non-limiting normothermic perfusion devices include OCSTMliver system (TransMedics), OrganOx metra®System, the normothermic perfusion device disclosed in Eshmuminov et al, and the normothermic perfusion device disclosed in US 2020 / 0375178 A1. In some embodiments, the perfusion device is a hypothermic perfusion device. Non-limiting hypothermic perfusion devices include LifePort®Liver Transporter and VitaSmartTMLiver Machine Perfusion System (Bridge to Life).

[0087] In some embodiments, the perfusion devices of the present disclosure comprise a dialysis system. Any of the perfusion devices disclosed above may be modified to comprise a dialysis system when a dialysis system is not present. The term “dialysis system” as used herein refers to a system in which the perfusate is passed through a filter (e.g., a dialyzer, high-flux or low-flux filter) that removes toxins from the perfusate while equilibrating a physiological dialysate solution through the filter with respect to glucose, electrolytes, osmolarity, pH-value and other relevant physiological factors. In some embodiments, the filter is a membrane that separates the dialysate and the perfusate. Through the filter (e.g., a membrane), mass transfer (e.g. by diffusion) and also fluid transfer (e.g. by convection) takes place between dialysate and perfusion fluid according to concentration and pressure gradients across the membrane.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270

[0088] The system of the present disclosure is capable of preserving and / or maintaining the marginal organ for a range of different times. For instance, the system is capable of preserving and / or maintaining the marginal organ for at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, at least 132 hours, at least 144 hours, at least 156 hours, at least 168 hours, at least 180 hours, at least 192 hours, at least 204 hours, at least 216 hours, at least 228 hours, at least 240 hours, at least 252 hours, at least 264 hours, at least 276 hours, at least 288 hours, at least 300 hours, at least 312 hours, at least 324 hours, at least 336 hours, or greater than 336 hours.

[0089] The system of the present disclosure may have a number of features that allow for the system to maintain and / or preserve the marginal organ for the above described time ranges including, without limitation, monitoring and manipulating glucose levels within the perfusate, producing a pulsatile flow within the perfusate and throughout the entire perfusion loop, mixing oxygenated and deoxygenated blood prior to delivering the mixture to the marginal organ, oscillating the marginal organ within the perfusion device, maintaining a 50-75% oxygen saturation within the perfusate, monitoring and maintaining the following parameters in a physiological range: perfusate temperature; perfusate pH; perfusate hematocrit concentration; oxygen and CO2 levels in lines such as a hepatic artery line, a vena cava line, and a portal vein line; perfusate pressure and flow in lines such as a hepatic artery line, a vena cava line, and a portal vein line, etc. In some embodiments, the perfusion device monitors glucose levels in the perfusate and administers glucagon or insulin to the perfusate when the glucose levels in the perfusate are outside of a preset range. In some embodiments, the present range has a lower limit of about 6 mmol / l of glucose. In some embodiments, the perfusion device comprises a pump that is capable of producing pulsatile flow of the perfusate within the perfusion device. In some embodiments, the perfusion device mixes oxygenate blood and deoxygenated blood to produce a blood mixture prior to delivering the blood mixture to the marginal organ. In some embodiments, the perfusion device comprises a diaphragm that oscillates the marginal organ in the perfusion device. In some embodiments, the oscillation is lifting and lowering. In some embodiments, the diaphragm is a flexible material that is lifted and lowered by the inflating and deflating of a balloon device. In some embodiments, the perfusion device of the present disclosure maintains a 50-75% oxygen saturation level with the perfusate.

[0090] The systems of the present disclosure comprise a perfusate. The perfusate may be any perfusate deemed useful. A perfusate includes, for example, Stanford University solution (see, e.g., Swanson et al., 1988, Journal of Heart Transplantation, 7(6): 456-467); Collins solution; modified Collins solution (see, e.g., Maurer et al., 1990, Transplantation Proceedings, 22(2): 548-ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 550; Swanson et al., supra); University of Wisconsin solution (see, e.g., U.S. Pat. No.4,798,824, issued to Belzer et al.); modified University of Wisconsin solution (Yeh et al., Ann Thorac Surg. 1990 June; 49(6):932-9); Columbia University solution (see, e.g., U.S. Pat. Nos.5,552,267 and 5,370,989, and Kayano et al., 1999, J. Thoracic Cardiovascular Surg.118: 135-144); histidine- tryptophan-ketoglutarate (HTK) solution (see, e.g., Ku et al., Transplantation. 1997 Oct 15;64(7):971-5); Celsior (see, e.g., Janssen et al., Transplant International (2003), 16(7): pp.515- 522); isotonic saline solutions, that may contain, in various proportions, salts, sugars, osmotic agents, local anesthetic, buffers, and other such agents (see, e.g., Berdyaev et al., U.S. Pat. No. 5,432,053; Belzer et al.); ViaSpan® (see, e.g., U.S. Pat. Nos. 4,798,824, 4,879,283; and 4,873,230; Taylor, U.S. Pat. No.5,405,742; Dohi et al., U.S. Pat. No.5,565,317; Stern et al., U.S. Pat. Nos. 5,370,989 and 5,552,267); solutions comprising pyruvate, inorganic salts supporting cell membrane potential and albumin or fetal calf serum (see, e.g., U.S. Pat. No. 5,066,578); solutions comprising one or more phosphatidic acids or sugars, and lysophosphotidic acids or sugars, together with enhancers such as albumen, optionally delivered in liposomal compositions (see, e.g., U.S. Pat. Nos. 6,495,532 and 6,004,579); whole blood; parts of whole blood (e.g., plasma with synthetic hemoglobin); other organ preservation solutions (see, e.g., U.S. Pat. No. 7,220,538); or any combination of the foregoing. In some embodiments, the perfusate comprises whole blood. In some embodiments, the perfusate comprises parts of whole blood. In some embodiments, the perfusate comprises a clinical grade organ preservation solution. Clinical grade organ preservation solutions include, without limitation, University of Wisconsin solution, histidine- tryptophan-ketoglutarate, Celsior, Institut Georges Lopez (IGL-1) solution, etc.

[0091] The systems of the present disclosure comprise a perfusate comprising an effective amount of a Wnt agent. The Wnt agent is capable of activating stem cells within the marginal organ and inducing repair, reconditioning, and / or regenerative responses. In some embodiments, the Wnt agent is a Wnt agonist. In some embodiments, the Wnt agent is a Wnt protein. The Wnt protein may be any Wnt protein deemed useful. For instance, the Wnt protein may be a Wnt 1, Wnt 2, Wnt 2B, Wnt 3, Wnt 3A, Wnt 4, Wnt 5A, Wnt 5B, Wnt 6, Wnt 7A, Wnt 7B, Wnt 8A, Wnt 8B, Wnt 10A, Wnt 10B, Wnt 11, Wnt 14, Wnt 15, Wnt 16A, or Wnt 16B. In some embodiments, the Wnt protein is Wnt 3A. In some embodiments, the Wnt protein is formulated in a liposome. In some embodiments, the Wnt agent is liposomal Wnt 3A. Liposomal Wnt 3A is known in the art and has been described in, for example, US Patent Nos 9,937,126 and 9,301,980 and US Patent Application No 16 / 067,944 each of which is specifically incorporated by reference herein. In some embodiments, the Wnt agent is a GSK-3 inhibitor. GSK-3 inhibitors include, without limitation, GSK 3 Inhibitor IX (6-Bromoindirubin-3'-oxime; BIO), SB-216763, SB-415286, tideglusib,ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 CHIR99021, Bisindolylmaleimide I (GF109203X), LY2090314, COB-187, Kenpaullone, TWS119, AR-A014418, Lithium chloride hydrate, CHIR-98014, TDZD-8, etc. In some embodiments, the Wnt agent is a GSK-3 inhibitor. GSK-3 inhibitors include, without limitation, GSK 3 Inhibitor IX (6- Bromoindirubin-3'-oxime; BIO), SB-216763, SB-415286, tideglusib, CHIR99021, Bisindolylmaleimide I (GF109203X), LY2090314, COB-187, Kenpaullone, TWS119, AR- A014418, Lithium chloride hydrate, CHIR-98014, TDZD-8, etc.

[0092] An effective amount of a Wnt agent that may be administered to an individual or a cell in a tissue or organ thereof includes a dose of about 0.0001 nM to about 2000 μM, for example. More specifically, doses are from about 0.01 nM to about 2000 μM; about 0.01 μM to about 0.05 μM; about 0.05 μM to about 1.0 μM; about 1.0 μM to about 1.5 μM; about 1.5 μM to about 2.0 μM; about 2.0 μM to about 3.0 μM; about 3.0 μM to about 4.0 μM; about 4.0 μM to about 5.0 μM; about 5.0 μM to about 10 μM; about 10 μM to about 50 μM; about 50 μM to about 100 μM; about 100 μM to about 200 μM; about 200 μM to about 300 μM; about 300 μM to about 500 μM; about 500 μM to about 1000 μM; about 1000 μM to about 1500 μM and about 1500 μM to about 2000 μM, for example. Of course, all of these amounts are exemplary, and any amount in-between these points is also expected to be of use in the invention.

[0093] The systems of the present disclosure are capable of rehabilitating marginal organs. The rehabilitation of the marginal organs of the present disclosure may provide a number of benefits including, without limitation, improving the quality of the marginal organ, reducing the risk of reperfusion injury once the marginal organ has been transplanted into an individual in need thereof, reducing the risk of graft failure, reducing the risk of ischemic cholangiopathy, improves functional architecture of the organ such as but not limited to reduction in scarring of the organ parenchyma, improved diffusion surface of the organ etc. In some embodiments, the system inhibits apoptosis of cells in the marginal organ.

[0094] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270

[0095] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0096] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. Due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims. EXPERIMENTAL Example 1

[0097] FIG.1 discloses the apoptotic response of hepatocytes following cold preservation of a liver for varying lengths of time, from 0 to 120 minutes. The objective of this experiment was to determine the extent of cellular apoptosis in the liver following its retrieval and preservation in cold storage. The rationale for this experiment was to provide a baseline of cellular apoptosis associated with retrieval and cold preservation. To carry out this analysis, adult wild-type mice were sacrificed; immediately thereafter, livers were retrieved and placed in media and maintained at 4ºC for a maximum of 2 hours (experimental design indicated in panel A). At intervals indicated e.g., 0 min, 30 min, 60 min, 90 min and 120 min, livers were rinsed in ice-cold phosphate-buffered saline and prepared for cryo-embedding. Tissue sections were generated at 8µm, then stained using a TUNEL assay (Roche) to identify free 3′-OH termini in single-stranded breaks in high- molecular-weight nuclear DNA fragments (example is indicated in panel B), which are abundant in cells undergoing apoptosis.

[0098] FIG.1 also discloses the apoptotic response of hepatocytes following cold preservation of a liver in a solution containing L-WNT for 120 minutes. The objective of this experiment was to determine the extent to which L-WNT affected the level of cellular apoptosis in the liver following its retrieval and preservation in cold storage. The distribution of green, TUNEL-positive cells was visualized in control livers (panel B) and in livers treated with L-WNT (panel C). Quantification of the number of TUNEL-positive cells is presented in panel D.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 Example 2

[0099] FIG.2 discloses the level of apoptosis of hepatocytes in a liver slice culture, maintained at 37°C for 48 hours. The objective of this experiment was to develop an ex vivo system in which programmed cell death in the liver could be examined as a function of time. The rationale for this experiment was to develop and validate a physiologically relevant, functional ex vivo model of liver function. To carry out this analysis, livers were retrieved from adult mice, immediately embedded in 3% low-melting point agarose, then sectioned at ~500µm. Each liver section was placed on a Millipore polycarbonate filter. Williams media was added and liver slices were maintained in a tissue culture dish at the air-media interface for 48 hours. After 48 hours, liver slices were fixed, embedded and cryo-sectioned (experimental design show in panel A). TUNEL staining verified apoptotic hepatocytes in the liver slices (panel B). Caspase3 immunohistochemical staining identified cells undergoing apoptosis (shown in panel C). Example 3

[0100] FIG. 3 discloses the distribution of Wnt-responsive stem cells in the intact liver. The objective of this experiment was to identify Wnt-responsive cells in the intact liver. The rationale for this experiment was to identify those cell populations that would be activated in response to a liposomal WNT3A stimulus. To carry out this analysis, Wnt reporter e.g. Axin2LacZ / + mice were employed. In this strain, the LacZ gene replaces one copy of Axin2 and is under control of the Axin2 promoter. Xgal staining is then used to identify the LacZ gene product, beta galactosidase 18. Wnt-responsive cells were identified, surrounding the central vein (shown in panel FIG.3A). The fates of these Wnt-responsive cells were followed using the lineage tracing strain, Axin2CreERT2 / +; R26RmTmG / + 19. The broader distribution of cells immunopositive for Green Fluorescent Protein (GFP) indicates that hepatic stem cells that give rise to hepatocytes are Wnt- responsive (FIG.3B). Example 4

[0101] FIG. 4 discloses the stability of liposomal WNT3A at a normothermic temperature over time. Purified WNT proteins are unstable at 37ºC 20. The objective of this experiment was to determine whether liposomal packaging of WNT3A could stabilize the protein in an aqueous environment. The rationale for this experiment was to gain insights into the stability of liposomal WNT3A at 37ºC e.g., the temperature of an organ at the time of retrieval. To carry out this analysis, a cell-based LSL assay was employed, where The LSL assay is widely used to assess the activity of Wnt proteins 21. The LSL potency assay is an appropriate surrogate for Wnt activity in human cells. The method consists of mouse L cells stably transfected with a Wnt-responsive luciferaseATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 reporter plasmid, pSuperTOPFlash (Addgene) containing 3 TCF / LEF binding sites regulating expression of luciferase. TCF / LEF transcription factors are the major endpoint mediators of Wnt signaling 22. The procedure is referred to as an “LSL” assay. When LSL cells are exposed to liposomal WNT3A, the Wnt protein binds to Frizzled (Fz) receptors on the surface of the LSL cells, which initiates a cascade of Wnt-dependent intracellular events 23 that leads to the expression of luciferase. LSL cells are incubated for 18hours at 37°C, 5% CO2 then prior to quantification, LSL cells are washed to remove liposomal WNT3A, then lysed with Lysis Buffer (Applied Biosystems) and a portion of the lysate is combined with luciferin. Using the LSL assay, the stability of liposomal WNT3A at 37ºC was compared to freshly thawed liposomal WNT3A (green bar). Naked, non-liposome stabilized WNT3A loses activity within minutes at 37ºC (red bar), indicating its labile nature. Analyses conducted over 7 days demonstrate that liposomal WNT3A largely maintains its activity for extended periods of time at body temperature. Example 5

[0102] FIG. 5 discloses liposomal WNT3A endocytosis by cells as a function of time and temperature. The objective of this experiment was to quantify the rate of liposomal WNT3A uptake by endocytosis. The rationale for this experiment was that the rate of endocytosis is influenced by time and temperature. To carry out this analysis, primary cells from the bone marrow were plated and grown in vitro until attachment was achieved. DiI-labeled liposomal WNT3A was added to the cells. Cells were maintained either at 37ºC or 23ºC. DiI fluoresces when it is bound to a cell membrane; consequently, endocytosed liposomal WNT3A can be distinguished from free DiI- labeled liposomal WNT3A in the media. At timepoints indicated e.g., from 0 min to 120 min, the level of fluorescent was measured using a plate reader. The resulting data demonstrate that the amount of liposomal WNT3A that is endocytosed increases as a function of time, and that endocytosis is significantly increased at 37ºC compared to 23ºC. Example 6

[0103] FIG.6 discloses the distribution of liposomal WNT3A following intrasplenic injection. The objective of this experiment was to test the distribution of liposomal WNT3A delivered via the hepatic vasculature. The rationale for this experiment was that the patency of hepatic vessels will determine the extent to which liposomal WNT3A can activate hepatic stem cells and curtail hepatocyte apoptosis. To carry out this analysis, a mouse model of regional perfusion was employed, where rhodamine-labeled liposomes (dia. = 100 nm) were delivered via intrasplenic injection to anesthetized mice over a 15 min period. Immediately thereafter, animals wereATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 sacrificed and livers were retrieved; fixed in paraformaldehyde, and cryo-sectioned. Fluorescence was visualized via a compound microscope. These analyses revealed extensive filling of the hepatic vasculature in a model of regional perfusion. Example 7

[0104] FIG. 7 discloses the level of apoptosis of hepatocytes in a liver graft slice treated with liposomal Wnt3A, maintained at 37°C for 48 hours. The objective of this experiment was to develop an ex vivo system in which programmed cell death in the liver could be examined as a function of time. The rationale for this experiment was to use a physiologically relevant, functional ex vivo model of liver function, and specifically to screen and test the effects of liposomal WNT3A on hepatocyte viability. To carry out this analysis, Wnt reporter e.g. Axin2LacZ / + mice were employed. In this strain, the LacZ gene replaces one copy of Axin2 and is under control of the Axin2 promoter. Xgal staining is then used to identify the LacZ gene product, beta galactosidase 18. Livers were retrieved from adult Axin2LacZ / + mice, immediately embedded in 3% low-melting point agarose, then sectioned at ~500µm. Each liver section was placed on a Millipore polycarbonate filter. Williams media was added and liver slices were maintained in a tissue culture dish at the air-media interface for 48 hours (shown in panel A). After 48 hours, liver slices were fixed, embedded and cryo-sectioned. Xgal staining identified Wnt-responsive cells surrounding the central vein (shown in panel B). Caspase3 immunohistochemical staining identified cells undergoing apoptosis (panel C). TUNEL staining verified apoptotic hepatocytes in the liver slices (panel D). Livers were also retrieved from adult Axin2LacZ / + mice, immediately embedded in 3% low-melting point agarose, then sectioned at ~500µm. Each liver section was placed on a Millipore polycarbonate filter. Williams media and liposomal WNT3A at a concentration of 0.5ng / µL was added once. Liver slices were maintained in a tissue culture dish at the air-media interface for 48 hours (panel E). After 48 hours, WNT-treated liver slices were fixed, embedded and cryo- sectioned. Xgal staining identified significantly more Wnt-responsive cells surrounding the central vein in liver slices treated with liposomal WNT3A (panels F and G). Caspase3 immunohistochemical staining identified significantly fewer cells undergoing apoptosis in liver slices treated with liposomal WNT3A (panels H and I). TUNEL staining verified that apoptotic hepatocytes in the liver slices was reduced in liver slices treated with liposomal WNT3A (panels J and K). Example 8

[0105] FIG. 8 discloses the apoptotic response of hepatocytes following preconditioning with saline versus liposomal Wnt3A followed by cold preservation for 120 min. Some strategies forATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 mitigating IRI include initiating organ preservation while the heart continues beating, for example in brain dead donors. In these cases, the Wnt agent can be delivered via portal injection after which organs can be retrieved and maintained in preservation solution. The objective of this experiment was to determine whether portal administration of liposomal WNT3A followed by cold storage had a measurable impact on cell viability in the liver over that seen following portal administration of saline. The rationale for this experiment was to provide evidence that liposomal WNT3A enhances cell viability by actively repressing apoptosis associated with organ retrieval and cold storage. To carry out this analysis, adult wild-type mice were deeply anesthetized, following which 500µL liposomal WNT3A at a concentration of 2.5ng / µL was delivered to the liver via portal injection over 15 minutes. In controls, the same volume, 500µL of saline was delivered via portal injection (shown in panel A). Immediately thereafter, livers were retrieved and placed in Williams media and maintained at 4ºC for 2 hours (panel A). Afterwards, livers were rinsed in ice- cold phosphate-buffered saline and prepared for cryo-embedding. Tissue sections were generated at 8µm, then stained using a TUNEL assay (Roche) to identify cells undergoing apoptosis 17. The distribution of green, TUNEL-positive cells was significantly reduced in livers treated with liposomal WNT3A compared to livers treated with saline (panels B,C; quantified in D). References 1. Jochmans, I., van Rosmalen, M., Pirenne, J. & Samuel, U. Adult Liver Allocation in Eurotransplant. Transplantation 101, 1542-1550 (2017). 2. Croome, K.P., Lee, D.D., Keaveny, A.P. & Taner, C.B. Noneligible Donors as a Strategy to Decrease the Organ Shortage. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 17, 1649-1655 (2017). 3. Su, F., et al. Aging of Liver Transplant Registrants and Recipients: Trends and Impact on Waitlist Outcomes, Post-Transplantation Outcomes, and Transplant-Related Survival Benefit. Gastroenterology 150, 441-453 e446; quiz e416 (2016). 4. Schmucker, D.L. & Sanchez, H. Liver regeneration and aging: a current perspective. Curr Gerontol Geriatr Res 2011, 526379 (2011). 5. Neri, A.A., Dontas, I.A., Iliopoulos, D.C. & Karatzas, T. Pathophysiological Changes During Ischemia-reperfusion Injury in Rodent Hepatic Steatosis. In Vivo 34, 953-964 (2020). 6. Eslam, M., et al. Metabolic (dysfunction)-associated fatty liver disease in individuals of normal weight. Nature reviews. Gastroenterology & hepatology 19, 638-651 (2022). 7. Resch, T., et al. Transplanting Marginal Organs in the Era of Modern Machine Perfusion and Advanced Organ Monitoring. Frontiers in immunology 11, 631 (2020). 8. Querard, A.H., et al. Comparison of survival outcomes between Expanded Criteria Donor and Standard Criteria Donor kidney transplant recipients: a systematic review and meta- analysis. Transpl Int 29, 403-415 (2016).ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 9. Aubert, O., et al. Disparities in Acceptance of Deceased Donor Kidneys Between the United States and France and Estimated Effects of Increased US Acceptance. JAMA Intern Med 179, 1365-1374 (2019). 10. Dayoub, J.C., Cortese, F., Anzic, A., Grum, T. & de Magalhaes, J.P. The effects of donor age on organ transplants: A review and implications for aging research. Exp Gerontol 110, 230-240 (2018). 11. Haque, O., Yuan, Q., Uygun, K. & Markmann, J.F. Evolving utilization of donation after circulatory death livers in liver transplantation: The day of DCD has come. Clin Transplant 35, e14211 (2021). 12. Eggenhofer, E., et al. Steatotic Livers Are More Susceptible to Ischemia Reperfusion Damage after Transplantation and Show Increased gammadelta T Cell Infiltration. International journal of molecular sciences 22(2021). 13. Rampes, S. & Ma, D. Hepatic ischemia-reperfusion injury in liver transplant setting: mechanisms and protective strategies. J Biomed Res 33, 221-234 (2019). 14. van Beekum, C.J., et al. Normothermic Machine Perfusion (NMP) of the Liver - Current Status and Future Perspectives. Ann Transplant 26, e931664 (2021). 15. Eden, J., et al. Utilization of livers donated after circulatory death for transplantation - An international comparison. J Hepatol 78, 1007-1016 (2023). 16. Mohan, S., Yu, M., King, K.L. & Husain, S.A. Increasing Discards as an Unintended Consequence of Recent Changes in United States Kidney Allocation Policy. Kidney Int Rep 8, 1109-1111 (2023).

[0225] Notwithstanding the appended claims, the disclosure set forth herein is also described by the following clauses: 1. A system for rehabilitation of a marginal organ, the system comprising: a perfusion device; and a perfusate comprising an effective amount of a Wnt agent. 2. The system of clause 1, wherein the perfusate comprises whole blood, parts of whole blood, or clinical grade organ preservation solution. 3. The system of clauses 1 or 2, wherein the perfusion device comprises a dialysis system. 4. The system of clauses any of the preceding clauses, wherein the perfusion device is a normothermic perfusion loop. 5. The system of any of the preceding clauses, wherein the perfusion device monitors glucose levels in the perfusate and administers glucagon or insulin to the perfusate when the glucose levels in the perfusate are outside of a preset range. 6. The system of any of the preceding clauses, wherein the perfusion device comprises a pump that is capable of pulsatile flow within the perfusion device. 7. The system of any of clauses 2-6, wherein the perfusion device mixes oxygenated blood and deoxygenated blood in the perfusate to produce a blood mixture prior to delivering the blood mixture in the perfusate to the marginal organ.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 8. The system of any of the preceding clauses, wherein the perfusion device comprises a diaphragm that oscillates the marginal organ in the perfusion device. 9. The system of any of the preceding clauses, wherein the perfusion device maintains a 50-75% oxygen saturation within the perfusate. 10. The system of any of the preceding clauses, wherein the Wnt agent is a Wnt protein. 11. The system of clause 10, wherein the Wnt protein is a human Wnt protein. 12. The system of clause 11, wherein the Wnt protein is formulated in association with a liposome. 13. The system of clauses 11 or 12, wherein the Wnt protein is a Wnt protein selected from the group consisting of Wnt 1, Wnt 2, Wnt 2B, Wnt 3, Wnt 3A, Wnt 4, Wnt 5A, Wnt 5B, Wnt 6, Wnt 7A, Wnt 7B, Wnt 8A, Wnt 8B, Wnt 10A, Wnt 10B, Wnt 11, Wnt 14, Wnt 15, Wnt 16A, or Wnt 16B. 14. The system of any of clauses 10-13, wherein the Wnt protein is Wnt 3A. 15. The system of any of the preceding clauses, wherein the marginal organ is an organ selected from the group consisting of a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, uterus, a heart, a peripheral or central nerve, and connective tissue. 16. The system of clause 15, wherein the marginal organ is a liver. 17. The system of any of the preceding clauses, wherein the system is capable of preserving the marginal organ for at least 168 hours. 18. The system of any of the preceding clauses, wherein the system reduces apoptosis in cells in the marginal organ. 19. The system of any of the preceding clauses, wherein the Wnt agent performs one or more of: reduces or ameliorates ischemic damage to the marginal organ, reduces ischemic cholangiopathy, reduces early allograft dysfunction or primary non-function of the marginal organ, promotes the regeneration of the marginal organ, reduces scarring of the organ parenchyma, improves diffusion surface of the marginal organ, reduces apoptosis of the cells in the marginal organ, and reduces the risk of reperfusion injury. 20. A method of rehabilitating a marginal organ, the method comprising: obtaining a marginal organ, connecting the marginal organ to the perfusion device of any of clauses 1-17, and perfusing the marginal organ with a perfusate comprising an effective amount of a Wnt agent;ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 thereby rehabilitating the marginal organ. 21. The method of clauses 20, wherein the marginal organ is an organ selected from the group consisting of a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, uterus, a heart, a peripheral or central nerve, and connective tissue. 22. The method of clauses 20 or 21, wherein the marginal organ has reduced risk of a reperfusion injury. 23. The method of any of clauses 20-22, wherein the marginal organ has reduced risk of graft rejection. 24. The method of any of clauses 20-23wherein the Wnt agent is a Wnt protein. 25. The method of clause 24, wherein the Wnt protein is a human Wnt protein. 26. The method of clause 25, wherein the Wnt protein is formulated in association with a liposome. 27. The method of any of clauses 24-26, wherein the Wnt protein is a Wnt protein selected from the group consisting of Wnt 1, Wnt 2, Wnt 2B, Wnt 3, Wnt 3A, Wnt 4, Wnt 5A, Wnt 5B, Wnt 6, Wnt 7A, Wnt 7B, Wnt 8A, Wnt 8B, Wnt 10A, Wnt 10B, Wnt 11, Wnt 14, Wnt 15, Wnt 16A, or Wnt 16B. 28. The method of any of clauses 24-27, wherein the Wnt protein is Wnt3A. 29. The method of any of clauses 20-28, wherein the marginal organ is perfused for at least about 168 hours. 30. A method of rehabilitating a marginal organ, the method comprising: obtaining a marginal organ, contacting the marginal organ with preservation medium comprising an effective dose of a Wnt agent; thereby rehabilitating the marginal organ. 31. The method of clause 30, wherein the marginal organ is an organ selected from the group consisting of a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, uterus, a heart, a peripheral or central nerve, and connective tissue. 32. The method of clauses 30 or 31, wherein the marginal organ has reduced risk of a reperfusion injury following the contacting step. 33. The method of any of clauses 30-32, wherein the marginal organ has reduced risk of graft rejection following the contacting step. 34. The method of any of clauses 30-33, wherein the Wnt agent is a Wnt protein.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 35. The method of clause 34, wherein the Wnt protein is a human Wnt protein. 36. The method of clause 34, wherein the Wnt protein is formulated in association with a liposome. 37. The method of any of clauses 34-36, wherein the Wnt protein is a Wnt protein selected from the group consisting of Wnt 1, Wnt 2, Wnt 2B, Wnt 3, Wnt 3A, Wnt 4, Wnt 5A, Wnt 5B, Wnt 6, Wnt 7A, Wnt 7B, Wnt 8A, Wnt 8B, Wnt 10A, Wnt 10B, Wnt 11, Wnt 14, Wnt 15, Wnt 16A, or Wnt 16B. 38. The method of any of clauses 34-37, wherein the Wnt protein is Wnt3A 39. The method of any of clauses 30-38, wherein the marginal organ is contacted with preservation medium for at least about 168 hours. 40. A method of preserving an organ, the method comprising: obtaining an organ, contacting the marginal organ with a preservation medium comprising an effective dose of a Wnt agent; thereby preserving the organ. 41. The method of clause 40, wherein the organ is selected from the group consisting of: a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, uterus, a heart, a peripheral or central nerve, and connective tissue. 42. The method of clause 40 or 41, wherein the organ is a liver. 42. The method of clauses 40-42, wherein the organ is a marginal organ. 43. The method of clauses 40-42, wherein the organ is a non-marginal organ. 44. The method of any of clauses 40-43, wherein the organ is maintained at a hypothermic temperature. 45. The method of any of clauses 40-44, wherein the Wnt agent is a Wnt protein. 46. The method of clause 45, wherein the Wnt protein is a human Wnt protein. 47. The method of clause 46, wherein the Wnt protein is formulated in association with a liposome. 48. The method of any of clauses 45-47, wherein the Wnt protein is a Wnt protein selected from the group consisting of Wnt 1, Wnt 2, Wnt 2B, Wnt 3, Wnt 3A, Wnt 4, Wnt 5A, Wnt 5B, Wnt 6, Wnt 7A, Wnt 7B, Wnt 8A, Wnt 8B, Wnt 10A, Wnt 10B, Wnt 11, Wnt 14, Wnt 15, Wnt 16A, or Wnt 16B. 49. The method of any of clauses 45-48, wherein the Wnt protein is Wnt3A. 51. The method of any of clauses 40-49, wherein the Wnt agent reduces apoptosis.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 52. The method of any of clauses 40-51, wherein the organ is capable of being preserved for at least two hours.

Claims

ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 WHAT IS CLAIMED IS:

1. A method of rehabilitating, conditioning, or pre-conditioning a marginal organ, the method comprising: contacting a marginal organ with preservation medium or perfusate comprising an effective dose of a Wnt agent; thereby rehabilitating, conditioning, or pre-conditioning the marginal organ.

2. The method of claim 1, wherein the marginal organ is an organ selected from the group consisting of a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, uterus, a heart, a peripheral or central nerve, and connective tissue.

3. The method of claims 1 or 2, wherein the marginal organ is present in a donor after brain death.

4. The method of claim 3, wherein the contacting comprises delivering the preservation medium or perfusate comprising an effective dose of a Wnt agent intravenously or intraarterially.

5. The method of claims 1 or 2, wherein the marginal organ is present in a donor after cardiac death.

6. The method of claim 5, wherein the contacting comprises delivering the preservation medium or perfusate comprising an effective dose of a Wnt agent through regional perfusion.

7. The method of claims 1 or 2, wherein the marginal organ is ex vivo.

8. The method of claim 7, wherein the marginal organ is maintained at hyperthermic, normothermic, or hypothermic temperatures.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 9. The method of claims 7 or 8, wherein the contacting comprises perfusing or incubating the marginal organ with the preservation medium or perfusate comprising an effective dose of a Wnt agent.

10. The method of any of claims 1-9, wherein the Wnt agent is a Wnt protein.

11. The method of claim 10, wherein the Wnt protein is a Wnt protein selected from the group consisting of Wnt 1, Wnt 2, Wnt 2B, Wnt 3, Wnt 3A, Wnt 4, Wnt 5A, Wnt 5B, Wnt 6, Wnt 7A, Wnt 7B, Wnt 8A, Wnt 8B, Wnt 10A, Wnt 10B, Wnt 11, Wnt 14, Wnt 15, Wnt 16A, or Wnt 16B.

12. The method of claims 10 or 11, wherein the Wnt protein is Wnt3A.

13. The method of any of claims 10-12, wherein the Wnt protein is formulated in association with a liposome.

14. The method of any of claims 1-13, wherein the Wnt agent performs one or more of: reduces or ameliorates ischemic damage to the marginal organ, reduces ischemic cholangiopathy, reduces early allograft dysfunction or primary non-function of the marginal organ, promotes the regeneration of the marginal organ, reduces scarring of the organ parenchyma, improves diffusion surface of the marginal organ, reduces apoptosis of the cells in the marginal organ, and reduces the risk of reperfusion injury.

15. A system for rehabilitation of a marginal organ, the system comprising: a perfusion device; and a perfusate comprising an effective amount of a Wnt agent.

16. The system of claim 15, wherein the perfusate comprises whole blood, parts of whole blood, or clinical grade organ preservation solution.

17. The system of claims 15 or 16, wherein the perfusion device comprises one or more of: a dialysis system, a pump that is capable of pulsatile flow with the perfusion device, and a diaphragm that oscillates the marginal organ in the perfusion device.ATTORNEY DOCKET NAME: STAN-2128WO CLIENT REFERENCE: S23-270 18. The system of any of claims 16-17, wherein the perfusion device performs one or more of: monitors glucose levels in the perfusate and administers glucagon or insulin to the perfusate when the glucose levels in the perfusate are outside of a preset range, mixes oxygenated blood and deoxygenated blood in the perfusate to produce a blood mixture prior to delivering the blood mixture in the perfusate to the marginal organ, and maintains a 50-75% oxygen saturation within the perfusate.

19. The system of any of claims 15-18, wherein the Wnt agent is a Wnt protein selected from the group consisting of: Wnt 1, Wnt 2, Wnt 2B, Wnt 3, Wnt 3A, Wnt 4, Wnt 5A, Wnt 5B, Wnt 6, Wnt 7A, Wnt 7B, Wnt 8A, Wnt 8B, Wnt 10A, Wnt 10B, Wnt 11, Wnt 14, Wnt 15, Wnt 16A, or Wnt 16B.

20. The system of any of claims 15-19, wherein the marginal organ is an organ selected from the group consisting of a brain, a spinal cord, a kidney, a lung, a liver, an eye, a pancreas, a spleen, an intestine, a cornea, skin, uterus, a heart, a peripheral or central nerve, and connective tissue.

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