Growth factor mimetic peptide compositions and methods for the preservation and therapeutic enhancement of organs for transplantation

Abstract

The present disclosure provides methods of reducing ischemic damage in an organ removed from a subject, comprising contacting the organ with a perfusion solution comprising one or more GDFMPs, and perfusing the organ with a machine perfusion device. The disclosure also provides an organ perfusion solution comprising one or more GDFMPs of Table 1, and an organ preservation device comprising an organ perfusion solution comprising one or more GDFMPs of Table 1; and an organ perfusion machine.

Description

[0001] Atorney Docket No. 132033-00620

[0002] GROWTH FACTOR MIMETIC PEPTIDE COMPOSITIONS AND METHODS FOR THE PRESERVATION AND THERAPEUTIC ENHANCEMENT OF ORGANS FOR TRANSPLANTATION

[0003] RELATED APPLICATIONS

[0004] This instant application claims the benefit of priority to U.S. Provisional Application No. 63 / 707,974, filed on October 16, 2024, the entire contents of which are incorporated herein by reference.

[0005] REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0006] The application contains a Sequence Listing which has been submited electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on October 15, 2025, is named “132033-00620.xml” and is 17,654 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

[0007] BACKGROUND

[0008] In the past 50 years, the most effective means devised for extending the life of a patient suffering from organ failure has been the allotransplantation of an organ from a live and / or deceased donor. However, one of the biggest problems in donor organ transplantation is the storage and preservation of organs from the time of harvest from a donor to the time of transplantation into a recipient. Once harvested, cells and tissues are deprived of the oxygen that is required to maintain internal metabolism and cell volume integrity. This low oxygen state is called ischemia, and leads to hypoxia, which prevents oxygen from being delivered to the organ. Hepatic ischemia-reperfusion injury (IRI) is a pathological process that involves ischemia-mediated cellular damage, and is one of the primary causes of early organ dysfunction and failure after liver transplantation (Hirao et al. , 2022, Nature Reviews Gastroenterology & Hepatology volume 19, pages 239-256).

[0009] The classical method for limiting ischemia damage during the period from organ harvest to implantation is to store the non-perfused organ at low temperature, usually on ice, to slow down cellular metabolism. But, even during this cold ischemic period, there are changes in tissues of the harvested, ex vivo organ that result in inflammation. These cellular responses trigger the proinflammatory response that leads to reperfusion injury where, after organ implantation and contact with the recipient’s immune system, the recipient’s immune response to injured cells in the reperfused tissue results in early graft dysfunction (EGD). Ex vivo normothermic machine perfusion (NMP) is a more recent procedure where, to support normal cellular metabolism, the organ is perfused 132033-00620 with a supportive perfusate solution at body temperature. It offers a better outcome, but not enough to significantly reduce the percentage of donor organs judged unsuitable for implantation.

[0010] Heart transplantation remains the gold standard for the treatment of patients with refractory heart failure (Burchill and Ross, 2012, Future Cardiol. 8(2) 329-342). Unfortunately, despite the ever- increasing population of heart failure patients, the development of heart transplantation has been limited by the shortage of suitable donor hearts (Christie et al., 2012; J. Heart Lung. Transplant 31 1073-1086). Recently, the adoption of hearts from donation after circulatory death (DCD) has been considered as a promising approach to expanding the donor pool (Smith et al., 2019; Intensive Care Med. 45 310-321). DCD heart transplantation has the potential to significantly increase transplant activity by 30% in the United States (Jawitz et al., 2020; J. Thorac. Cardiovasc. Surg. 159 e307-e309) and 48% in the United Kingdom (Messer et al., 2020; J. Heart Lung. Transplant 39 1463-1475) and has resulted in sum decrease in waiting list mortality.

[0011] During transplantation of hearts donated, due to brain death (DBD), static cold storage (SCS) has been considered as a reliable and traditional heart preservation strategy. It is simple, inexpensive, and able to preserve DBD hearts for 4-6 hours with acceptable transplantation outcomes (Dhital et al., 2017; Curr. Opin. Organ. Transplant 22 189-197). However, there is a limited supply of DBD hearts, thus the interest in DCD hearts. The DCD procedure involves a significant period during which the donor heart is under ischemic conditions while the death of the donor is determined. The result is that, while DCD hearts are more plentiful, they are more fragile and SCS time is significantly shorter than for DBD hearts (Niederberger et al., 2019; Circ. Heart Fail 12:e005517). In recent years, ex vivo NMP has been recognized as a promising and novel strategy for DCD heart preservation. Ex vivo NMP can supply donated hearts with oxygenated, warm, and nutrient-enriched blood-based perfusate in a semi-physiological state during organ storage (Lund et al., 2017; Circ. Heart Fail 12:e005517). Therefore, compared with static cold storage, ex vivo NMP of DCD hearts can attenuate myocardial ischemia / reperfusion injury (IRI) injury, prolong storage time, and provide a unique platform for assessing the contractile function of DCD hearts in real-time, and provide recovery time DCD hearts. Moreover, compared with conventional DBD heart transplantation, DCD heart transportation and transplantation under ex vivo normothermic machine perfusion (NMP) provides a similar 30-day or 1- year postoperative survival rate (Messer et al., 2020; Messer et al., 2017; J. Heart Lung. Transplant 36 1311-1318). Nevertheless, the inevitable period of warm ischemia time due to systolic blood pressure lower than 50 mmHg that starts after the withdrawal of life -sustaining therapy during the necessary period needed to determine donor death, harvest of the organ, transportation to the recipient site, out to the point of reperfusion or cardioplegia, results in more serious myocardial IRI in the DCD hearts. The result is higher incidence of primary graft dysfunction (PGD) for the patients undergoing DCD heart transplantation (Bracey NA et al. Antioxid Redox Signaling. (2015) 22(13): 1176-87, Burchill LJ et al. Future Cardiol. (2012) 8(2): 329— 42) . To reduce myocardial IRI of the DCD hearts, it is necessary to improve the current preservation strategies. 132033-00620

[0012] In stressed, minimally injured cells, ischemia / reperfusion injury (IRI) can be prevented by inhibiting the apoptotic and pro-inflammatory response processes. Certain members of the BMP growth and differentiation factor family have been shown to inhibit the apoptotic and pro- inflammatory response processes and thereby block the IRI process during reperfusion. Several issues, however, render the native BMPs unacceptable as soft tissue therapeutics: they are subject to inhibition by natural inhibitor proteins, they are prone to stimulate ectopic bone growth, they are large enough to initiate an immunological response, and they are expensive to produce. The therapeutic alternative to them is a family of BMP-7 mimetics, small GDFMPs derived from bone morphogenetic protein 7 (BMP-7), that have been shown to be effective at blocking apoptosis, the pro-inflammatory responses and reversing fibrosis in other settings (Salido-Medina A 2022). BMP -7 mimetics are small peptide growth factors that are known to have anti-inflammatory, anti-apoptotic, anti-fibrotic, and pro-regenerative properties but lack bone-forming activity, distinguishing them from BMP-7 monomers (Carlson et al., Front Pharmacol. 2022;13:864509). These properties make BMP-7 mimetics an excellent therapeutic agent for application in graft recovery. BMP and transforming growth factor P (TGF- ) signaling pathways often act against each other; therefore, BMP-7 mimetics oppose TGF- induced fibrosis by blocking epithelial-to-mesenchymal transition (Carlson et al., 2022). Furthermore, BMP-7 mimetic (TBD 184, also called THR-184) is reported to have success in reversing fibrosis in mouse models of chronic renal injury with the potential to induce regeneration and repair (Sugimoto et al., Nat Med. 2012; 18(3) :396-404). It has also been used in phases 1 & 2 of a clinical trial of AKI for patients undergoing cardiac surgery requiring a bypass pump during the perioperative phase. Although the endpoint did not show a significant difference, there was a noticeable fall in the incidence of AKI in patients with pre-existing CKD who were treated with the highest dose of THR-184 (Himmelfarb et al., J Am Soc Nephrol. 2018;29(2):670-9). BMP-7 mimetics have also been tested in in-vivo models of kidney injury using rats with a unilateral clamp to induce ischemia-reperfusion injury. BMP-7 mimetics are found useful both as prophylactic medication before inducing injury and helping in recovering kidney post-ischemia treatment (Carlson et al., 2022).

[0013] There remains an inadequate supply of organs suitable for transplantation primarily due to limitations in procedures used for preservation of donor organs during the period between harvest and implantation. There is a need to develop improved methods, preservation solutions and devices in order to improve the success rate of organ transplantation.

[0014] SUMMARY

[0015] As the gap between the number of organs available and the number of organs required continues to grow, it is of paramount importance to utilize all available donor organs, and this requires optimizing marginal and inferior grafts through various interventions. One of the primary problems encountered with organ transplantation is ischemia / reperfusion injury (IRI) resulting in early graft 132033-00620 dysfunction and likely long-term fibrosis in the grafted organ. The present disclosure makes use of growth and differentiation factor mimetic peptides (GDFMPs) to improve the likely outcome of therapeutic organ transplantation. In particular, the present disclosure makes use of GDFMPs to improve the therapeutic suitability of donated organs for allograft or xenograft by providing GDFMPs in organ perfusates prior to static cold ischemia, during static warm ischemia, and / or during normothermic machine perfusion (NMP) or hypothermic machine perfusion (HMP) to improve the suitability of harvested organs for transplant and the likelihood of transplant success.

[0016] According to a first aspect, the disclosure provides a method of reducing ischemic damage in an organ removed from a subject, the method comprising contacting the organ with a perfusion solution comprising one or more GDFMPs, and perfusing the organ with a machine perfusion device. According to some embodiments, the one or more GDFMPs is selected from Table 1. According to some embodiments, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. According to some embodiments, the organ is in a container comprising the perfusion solution, and the machine perfusion device comprises a first and a second end, wherein the first end of the machine perfusion device is connected to the perfusion solution in the container and the second end is connected to the organ, thereby perfusing the organ. According to some embodiments of the aspects and embodiments herein, the organ is at least partially immersed in the perfusion solution. According to some embodiments of the aspects and embodiments herein, the perfusion solution is at a temperature of between about 37°C to about 38°C, e.g., a temperature of 37°C, 37.5°C or 38°C. According to some embodiments, the perfusion solution is at a temperature of about 37°C. According to some embodiments of the aspects and embodiments herein, the perfusion solution is at a temperature of between about 10°C to about 30°C, e.g., a temperature of 10°C to about 15 °C, 10°C to about 25 °C, 15 °C to about 25 °C, 20°C to about 25 °C, 20°C to about 30°C . According to some embodiments, the perfusion solution is at a temperature of between about 15 °C to about 25 °C. According to some embodiments of the aspects and embodiments herein, the organ undergoes at least 15 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments of the aspects and embodiments herein, the organ undergoes at least 30 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments of the aspects and embodiments herein, the organ undergoes 0 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments, the machine perfusion device is a normothermic machine perfusion (NMP) device. According to some embodiments, the machine perfusion device is a hypothermic machine perfusion (HMP) device. According to some embodiments of the aspects and embodiments herein, the organ is selected from the group consisting of: a liver, a heart, a pancreas, a kidney, a spleen, and a lung.

[0017] According to other aspects, the disclosure provides a method of recovering an organ with ischemic damage, the method comprising contacting the organ with a perfusion solution comprising one or more GDFMPs, and perfusing the organ with a machine perfusion device. According to some 132033-00620 embodiments, the one or more GDFMPs is selected from Table 1. According to some embodiments of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98% OR 99% identical to SEQ ID NO: 1. According to some embodiments, the organ is in a container comprising the perfusion solution, and the machine perfusion device comprises a first and a second end, wherein the first end of the machine perfusion device is connected to the perfusion solution in the container and the second end is connected to the organ, thereby perfusing the organ. According to some embodiments of the aspects and embodiments herein, the organ is at least partially immersed in the perfusion solution. According to some embodiments of the aspects and embodiments herein, the perfusion solution is at a temperature of between about 37°C to about 38°C, e.g., a temperature of 37°C, 37.5°C or 38°C. According to some embodiments, the perfusion solution is at a temperature of about 37°C. According to some embodiments of the aspects and embodiments herein, the perfusion solution is at a temperature of between about 10°C to about 30°C, e.g., a temperature of 10°C to about 15°C, 10°C to about 25°C, 15°C to about 25°C, 20°C to about 25 °C, 20°C to about 30°C . According to some embodiments, the perfusion solution is at a temperature of between about 15 °C to about 25 °C. According to some embodiments of the aspects and embodiments herein, the organ undergoes at least 15 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments of the aspects and embodiments herein, the organ undergoes at least 30 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments of the aspects and embodiments herein, the organ undergoes 0 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments, the machine perfusion device is a normothermic machine perfusion (NMP) device. According to some embodiments, the machine perfusion device is a hypothermic machine perfusion (HMP) device. According to some embodiments of the aspects and embodiments herein, the organ is selected from the group consisting of: a liver, a heart, a pancreas, a kidney, a spleen, and a lung. According to some embodiments of the aspects and embodiments herein, the subject is administered the one or more GDFMPs postmortem in vivo, postmortem for the perfusion of said organ prior to harvesting.

[0018] According to other aspects, the disclosure provides a method of improving the acceptability of an ex vivo donor organ for therapeutic allograft or xenograft transplant into a human recipient, the method comprising contacting the ex vivo donor organ with one or more GDFMPs of Table 1. According to some embodiments, the one or more GDFMPs are administered postmortem in vivo for the perfusion of said organ prior to harvesting. According to other embodiments, the one or more GDFMPs are used as an additive to an organ appropriate perfusate prior to and during static ex vivo storage of said organ during the period post-harvest and prior to implantation into a human recipient. According to further embodiments, the one or more GDFMPs are used an additive to an organ appropriate perfusate during therapeutic, active ex vivo machine perfusion prior to implantation of said organ into a human recipient. 132033-00620

[0019] According to some aspects, the disclosure provides an organ preservation device comprising an organ perfusion solution comprising one or more GDFMPs of Table 1; and an organ perfusion machine. According to some embodiments, the one or more GDFMPs is selected from Table 1. According to some aspects of the above aspects and embodiments, the organ perfusion machine is a normothermic machine perfusion (NMP) device. According to other aspects of the aspects and embodiments herein, the device further monitors metabolic markers in the organ perfusion solution.

[0020] According to other aspects, the disclosure provides an organ perfusion solution comprising one or more GDFMPs of Table 1.

[0021] According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 1. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 2. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs 132033-00620 from Table 1 consists of SEQ ID NO: 3. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 4. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 5. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 6. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 8. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 9. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 10. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 11. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 12. According to some aspects of any of the aspects and embodiments herein, the one or more GDFMPs from Table 1 consists of SEQ ID NO: 13.

[0022] DESCRIPTION OF THE DRAWINGS

[0023] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0024] Embodiments of the present disclosure, briefly summarized above and discussed in greater detail below, can be understood by reference to the illustrative embodiments of the disclosure depicted in the appended drawings. However, the appended drawings illustrate only typical embodiments of the disclosure and are therefore not to be considered limiting of scope, for the disclosure may admit to other equally effective embodiments.

[0025] FIG. 1 is a graph that shows oxygen consumption by hearts in the three study arms over the 4 hr of NMP.

[0026] FIG. 2 is a graph that shows lactate concentration in the aortic perfusate of hearts in the three study arms over the 4 hr of NMP.

[0027] FIG. 3 is a graph that shows lactate concentration in the left ventricular perfusate of hearts in the three study arms over the 4 hr of NMP.

[0028] FIG. 4 is a graph that shows percent weight gain (relative to time 0) of hearts for the three study arms at the end of the 4 hr of NMP period.

[0029] FIG. 5 is a graph that shows mean heart rates for the three study arms over the 4 hr of NMP period.

[0030] FIG. 6 is a graph that shows mean perfusate flow rates through hearts in the three study arms over the 4 hr of NMP period. 132033-00620

[0031] FIG. 7 is a graph that shows mean vascular resistance to perfusate flow through hearts in the three study arms over the 4 hr of NMP period.

[0032] FIG. 8 is a graph that shows mean aortic coronary pressure in hearts in the three study arms over the 4 hr of NMP period.

[0033] FIG. 9 shows a schematic diagram of the study arms and time course of the study described in Example 2. Livers in arml, “Fresh”, experienced no warm, static ischemia and was not contacted with THR-184 during the 6 hr NMP period; livers in arm 2, “WI Control”, did experience warm, static ischemia for 30 min. prior to the 6 hr NMP period, and were no contacted with THR-184 at any time; livers in arm 3, “WI THR-184”, were contacted with THR-184 during both the 30 min of static, warm ischemia and the subsequent 6 hr of NMP. Perfusate was sampled at 30 min intervals during NMP for pH, 02 usage, lactate concentration.

[0034] FIG. 10 is a graph that shows the mean oxygen uptake rate (normalized to liver mass) for livers in each of the study arms during the 6 hr NMP.

[0035] FIG. 11 is a graph that shows the mean pH for liver perfusates entering the liver during the 6 hr of NMP. Dotted lines are the bounds of the normal plasma pH range for the rat.

[0036] FIG. 12 is a graph that shows the mean lactate concentration in perfusate entering the livers during the 6 hr NMP. Dashed lines are the mean slope of lactate decrease during the last 3 hr of NMP.

[0037] FIG. 13 is a graph that shows the mean concentration of Alanine Transaminase (ALT) in each study arm at 3, 4 and 6 hr. NMP. The dotted line is the normal level for in vivo rat plasma.

[0038] FIG. 14 is a graph that shows the mean concentration of Aspartate Transaminase (AST) in each study arm at 3, 4 and 6 hr. NMP. The dotted line is the normal level for in vivo rat plasma.

[0039] FIG. 15 shows H&E-stained slices of representative livers from both the “WI Control” and “WI THR-184” arms at the end of 6 hr of NMP. Pores in the liver are indicated by dashed circles.

[0040] FIG. 16 shows TUNEL stained (for apoptosis) slices of representative livers from both the “WI Control” and “WI THR-184” arms at the end of 6 hr of NMP. Nuclei that are stained brown and marked with red circles are undergoing apoptosis. There were fewer cells undergoing apoptosis when THR-184 is present in the perfusate.

[0041] FIG. 17 is a schematic that shows the study design for application of TBD-184 (BMP-7 mimetic) as a prophylactic measure for liver graft in donation after brain death (DBD) model simulated on a pump (NMP: normothermic machine perfusion) along with exploration of dual upstream (prophylactic) and downstream management for reduction in ischemia reperfusion injury (IRI). CIT: cold ischemia time, SCS: static cold storage.

[0042] FIG. 18A is an illustration of abdominal NRP. FIG. 18B is a schematic of the study design for application of TBD 184 (BMP-7 mimetics) as a treatment measure during NRP for liver graft procurement simulated on a pump (NMP: normothermic machine perfusion) along with exploration of potential dual upstream (prophylactic) and downstream management for reduction in ischemia 132033-00620 reperfusion injury (IRI). CIT: cold ischemic time, WIT: warm ischemic time, IVC: inferior venacava.

[0043] Illustration created with BioRender.com.

[0044] DETAILED DESCRIPTION

[0045] I. Definitions

[0046] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0047] The term “organ” as used herein is meant to refer to a structure of bodily tissue in a subject, e.g., a mammalian subject such as a human, wherein the tissue structure as a whole is specialized to perform a particular bodily function. Organs and tissues include, but are not limited to, heart, kidney, liver, lung, pancreas, intestine, thymus, bone, tendon, cornea, skin, heart valve, nerve, and blood vessel (e.g., vein). In some embodiments, the term “organ” also encompasses decellularized and recellularized organs, as well as engineered and artificial organs and tissues, including engineered organs (e.g., tissue engineered constructs), engineered organs comprising a bioscaffold, tissues, organ slices and partial organs.

[0048] The term “cold ischemia time” (CIT) is meant to refer to the duration of time an organ spends in cold storage prior to transplantation.

[0049] The term “warm ischemia time” (WIT) is meant to refer to the time an organ remains at body temperature after its blood supply has been reduced or cut off but before it is cooled or reconnected to a blood supply. WIT may be defined as removal of an organ from cold storage to completion of reperfusion of warm blood following anastomosis.

[0050] The term “viability” as used herein is meant to refer to the state of an organ's survival capability, e.g., capable of survival after transplantation into a recipient. Viability can be used as a measure of the entire organ's survival or a part of the organ, or the viability of cells within the organ.

[0051] The term “reliability” as used herein is meant to refer to the extent to which a measure, procedure or instrument yields the same result on repeated trials. Stated another way, reliability as used herein refers how well a particular assessment method provides consistent results, regardless of who uses the method or when it is used.

[0052] The term “damaged organ” as used herein is meant to refer to indicate an organ that is in less than ideal condition for transplantation, such that the expected probability of transplant success is reduced. Examples of damaged organs include, but are not limited to, organs that suffer warm 132033-00620 ischemia for more than 30 minutes, organs that suffer cold ischemia for more than 12 hours, moderate or highly steatotic livers (e.g. , livers with greater than 30% fat), fibrotic livers, cirrhotic livers, livers from patients afflicted with hepatitis C or HIV, and the like.

[0053] The term “transplant” as used herein is meant to refer to an organ, a part of an organ, an engineered tissue, or other body tissue that has been transferred from its site of origin in one subject to a recipient site in the same or a different subject. Specifically in an allograft transplant procedure, the site of origin of the transplant is in a donor individual and the recipient site is in another, recipient individual.

[0054] The term “transplantation” as used herein is meant to refer to the method of transferring of an organ, or other bodily tissue from its site of origin in one subject to a recipient site in the same or a different subject, whether or not autologous, homologous or heterologous and whether or not it is performed directly or subsequently to further processing or preservation of the tissue or organ.

[0055] The term “hypothermic” as used herein is meant to refer to temperatures below room temperature. For example, “hypothermic” temperatures include, but are not limited to, temperatures between about 0° C to about 37° C, temperatures between about 15° C to about 37° C, temperatures between about 10° C to about 20° C, temperatures between about 5° C to about 25° C, temperatures between about 15° C to about 20° C, temperatures between about 1° C to about 10° C, temperatures between about 3° C to about 5° C or temperatures between about 1° C to about 5° C.

[0056] The term “room temperature” as used herein is meant to refer to a temperature between about 15° C and about 25° C For example, “room temperature” includes, but is not limited to, temperatures between about 18° C and about 23° C, temperatures between about 19° C and about 21° C, temperatures between about 24° C and about 25° C, or temperatures between about 20° C and about 21° C.

[0057] The term “normothermic” as used herein is meant to refer to temperatures approximately at room temperature. For example, “normothermic” temperatures are temperatures about 37° C, and can include temperatures between about 36° C and about 38° C, between about 37° C and about 38° C, between about 37.5° C and about 38° C temperatures between about 37° C and about 37.5° C.

[0058] The term “subzero” as used herein is meant to refer to any temperature below 0° C. For example, a sub-zero temperature can be in a range, for example, but is not limited to, about -5° C and about -10° C, a temperature of about -10° C and about -15° C, a temperature of about -15° C and about -25° C, a temperature of about -25° C and about -30° C, a temperature of about -30° C and about -50° C, a temperature of about -50° C and about -80° C, or below about -80° C.

[0059] The term “perfusion” as used herein is meant to refer to the flowing of fluid through the tissue or organ. Techniques for perfusing organs and tissue are well known in the art, and are disclosed in International Patent Application WO2011 / 002926, and U.S. Pat. Nos. 5,723,282 and 5,699,793 which are both incorporated herein in their entirety by reference. 132033-00620

[0060] The term a “perfusate” as used herein is meant to refer to any fluid capable of improving or maintaining the vitality of a cell, tissue, organ (including decellularized and recellularized organs), bioscaffold, and the like. Improving or maintaining vitality can include one or more of the following: maintenance of appropriate osmotic pressure, maintenance of appropriate oncotic pressure, maintenance of appropriate temperature, inhibition of decay, inhibition of microbial growth, and the like.

[0061] The term “storage medium” as used herein as used herein is meant to refer to be any substance for preserving vitality of a cell, tissue, organ (including decellularized and recellularized organs), bioscaffold, and the like. Preservation of vitality can include one or more of the following: maintenance of appropriate osmotic pressure, maintenance of appropriate oncotic pressure, maintenance of appropriate temperature, inhibition of decay, inhibition of microbial growth, and the like.

[0062] The term “supercoolant agent” as used herein is meant to refer to an agent which, when added to a solution or organ, prevents freezing, or the solution becoming a solid at the normal freezing temperatures, and allows the solution or organ to stay in a liquid state at the desired sub-zero temperature. A supercooling agent aids supercooling, which is the cooling of a liquid below its freezing point without it becoming solid. Supercoolant agents are often confused with cryoprotectant agents, which may also aid in freezing point depression but are actually aimed at preserving the tissue where the water has reached a solid state (either ice of vitrification). A supercooling agent prevents freezing of a tissue or organ when the tissue is cooled to subzero temperatures and results in an preserving the viability after warming, in comparison to the effect of cooling without a supercoolant.

[0063] The term “cryoprotectant” as used herein is meant to refer to an agent which minimizes ice crystal formation in a tissue or organ when the tissue is cooled to subzero temperatures or freezes.

[0064] The term “recovery” or “organ recovery” as used herein is meant to refer to the restoration of clinical signs to a degree that the organ is acceptable for transplantation. According to some embodiments, organ recovery can take 60 minutes or less. According to some embodiments, organ recovery can take 30 minutes or less. According to some embodiments, organ recovery can take 15 minutes or less. According to some embodiments, organ recovery can take 10 minutes or less. According to some embodiments, organ recovery can take 5 minutes or less. According to some embodiments, organ recovery can take 120 minutes or more. According to some embodiments, organ recovery can take 90 minutes or more. According to some embodiments, organ recovery can take 80 minutes or more. According to some embodiments, organ recovery can take 70 minutes or more. According to some embodiments, organ recovery can take 60 minutes or more. According to some embodiments, organ recovery can take 30 minutes or more. According to some embodiments, organ recovery can take 15 minutes or more. According to some embodiments, organ recovery can take 10 minutes or more. According to some embodiments, organ recovery can take 5 minutes or more. 132033-00620

[0065] The term “rejection” as used herein is meant to refer to the process or processes by which the immune response of an organ transplant recipient mounts a reaction against the transplanted organ, cell or tissue, whether native or bioartificial, such as a recellularized tissue, sufficient to impair or destroy normal function of the organ. The immune system response can involve specific (antibody and T cell-dependent) or non-specific (phagocytic, complement-dependent, etc.) mechanisms, or both.

[0066] The term “effective” as used herein is meant to refer to a characteristic of an amount of preservation perfusion solution, and / or metabolic suppression, and / or subzero storage, or any combination thereof, to achieve the goal of preventing, avoiding or retarding tissue damage in tissue, such as a harvested organ, whether the tissue damage results from ischemia, reperfusion, degradation of high-energy phosphates, inflammatory responses, edema, or any other tissue response to a stimulus such as the disruption of function and the manipulation that attends harvesting and storage of the organ.

[0067] The term “cells,” “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to a particular cell type, but to the progeny or potential progeny of such a cell.

[0068] The term “subject” and “patient” as used herein are meant to be used interchangeably to refer to any vertebrate, including, but not limited to, a mammal and a human. In some embodiments, the subject is a human or a non-human. “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, llamas, camels, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, rabbits, guinea pigs, and the like. The subject may be any age or sex.

[0069] The term “agent” as used herein is meant to refer to any entity which is normally not present or not present at the levels being administered in the cell. Agent can be selected from a group comprising: chemicals; small molecules; nucleic acid sequences; nucleic acid analogues; proteins; peptides; aptamers; antibodies; or fragments thereof. Agents can be known to have a desired activity and / or property, or can be selected from a library of diverse compounds.

[0070] The terms “activate” or “increased” or “increase” as used in the context of biological activity of a cell herein generally means an increase in the biological activity of a cell by a statically significant amount relative to in a control condition. For the avoidance of doubt, an “increase”, or “activation” of a cell means a statistically significant increase of at least about 10% of the level and / or activity of ATP as compared to non-treatment of an organ according to the methods including an increase of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more, including, for example at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold increase or greater of level of ATP as compared to if the organ was not treated according to the methods and compositions as disclosed herein. 132033-00620

[0071] The terms “increased”, “increase” or “enhance” or “higher” are all used herein to generally mean an increase compared to a control. In some embodiments, the terms “increased”, “increase” or “enhance” or “higher” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0072] The terms “lower”, “reduced”, “reduction” or “decrease” or “inhibit” are all used herein generally to mean a decrease compared to a control. In some embodiments, the terms “lower”, “reduced”, “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (z.e., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0073] The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.

[0074] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0075] The articles “a” and “an” are used herein to refer to one or to more than one (z. e. , at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a pharmaceutical composition comprising “an agent” includes reference to two or more agents.

[0076] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation. The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment. As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. 132033-00620

[0077] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. II. Growth and Differentiation Factor Mimetic Peptides (GDFMPs)

[0078] As described herein, the present disclosure provides growth and differentiation factor mimetic peptides (GDFMPs) that can be used to improve the acceptability of an ex vivo donor organ for therapeutic allograft or xenograft transplant into a subject, for example a human recipient.

[0079] According to some embodiments, the GDFMP comprises a sequence selected from Table 1. Table 1. Growth and differentiation factor mimetic peptides (GDFMPs) 132033-00620

[0080] According to some embodiments, the GDFMP comprises SEQ ID NO: 1. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 1. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 1. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO: 1.

[0081] According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 1.

[0082] According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 1.

[0083] According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 1.

[0084] According to some embodiments, SEQ ID NO: 1 comprises an N-terminal (H) and a C-terminal (OH).

[0085] According to some embodiments, the GDFMP comprises SEQ ID NO: 2. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 2. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 2. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO: 2.

[0086] According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 2.

[0087] According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 2.

[0088] According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 2.

[0089] According to some embodiments, SEQ ID NO: 2 comprises an N-terminal (H) and a C-terminal (OH).

[0090] According to some embodiments, the GDFMP comprises SEQ ID NO: 3. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 3. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 3. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO: 3.

[0091] According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 3.

[0092] According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 3.

[0093] According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 3.

[0094] According to some embodiments, SEQ ID NO: 3 comprises an N-terminal (H) and a C-terminal (OH).

[0095] According to some embodiments, the GDFMP comprises SEQ ID NO: 4. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 4. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 4. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO: 4.

[0096] According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 4. According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 4. 132033-00620

[0097] According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 4. According to some embodiments, SEQ ID NO: 4 comprises an N-terminal (H) and a C-terminal (OH).

[0098] According to some embodiments, the GDFMP comprises SEQ ID NO: 5. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 5. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 5. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO: 5.

[0099] According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 5.

[0100] According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 5.

[0101] According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 5.

[0102] According to some embodiments, SEQ ID NO: 5 comprises an N-terminal (H) and a C-terminal (OH).

[0103] According to some embodiments, the GDFMP comprises SEQ ID NO: 6. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 6. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 6. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO: 6.

[0104] According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 6.

[0105] According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 6.

[0106] According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 6.

[0107] According to some embodiments, SEQ ID NO: 6 comprises an N-terminal (H) and a C-terminal (OH).

[0108] According to some embodiments, the GDFMP comprises SEQ ID NO: 8. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 8. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 8. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO: 8.

[0109] According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 8.

[0110] According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 8.

[0111] According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 8.

[0112] According to some embodiments, SEQ ID NO: 8 comprises an N-terminal (H) and a C-terminal (OH).

[0113] According to some embodiments, the GDFMP comprises SEQ ID NO: 9. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 9. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 9. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO: 9.

[0114] According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 9.

[0115] According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 9.

[0116] According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 9.

[0117] According to some embodiments, SEQ ID NO: 9 comprises an N-terminal (H) and a C-terminal (OH).

[0118] According to some embodiments, the GDFMP comprises SEQ ID NO: 10. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 10. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 10. 132033-00620

[0119] According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO:

[0120] 10. According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 10. According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 10. According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 10. According to some embodiments, SEQ ID NO: 10 comprises an N-terminal (H) and a C- terminal (OH).

[0121] According to some embodiments, the GDFMP comprises SEQ ID NO: 11. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 11. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 11. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO:

[0122] 11. According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 11. According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 11. According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 11. According to some embodiments, SEQ ID NO: 11 comprises an N-terminal (H) and a C- terminal (OH).

[0123] According to some embodiments, the GDFMP comprises SEQ ID NO: 12. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 12. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 12. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO:

[0124] 12. According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 12. According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 12. According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 12. According to some embodiments, SEQ ID NO: 12 comprises an N-terminal (H) and a C- terminal (OH).

[0125] According to some embodiments, the GDFMP comprises SEQ ID NO: 13. According to some embodiments, the GDFMP comprises a sequence 95% identical to SEQ ID NO: 13. According to some embodiments, the GDFMP comprises a sequence 96% identical to SEQ ID NO: 13. According to some embodiments, the GDFMP comprises a sequence 97% identical to SEQ ID NO:

[0126] 13. According to some embodiments, the GDFMP comprises a sequence 98% identical to SEQ ID NO: 13. According to some embodiments, the GDFMP comprises a sequence 99% identical to SEQ ID NO: 13. According to some embodiments, the GDFMP comprises a sequence consisting of SEQ ID NO: 13. According to some embodiments, SEQ ID NO: 13 comprises an N-terminal (H) and a C- terminal (OH).

[0127] According to some embodiments, the GDFMP comprises one or more members of the BMP-7 mimetic family.

[0128] According to some embodiments, the GDFMP is THR-184. 132033-00620

[0129] III. Organ Perfusates Comprising GDFMPs

[0130] The original perfusates, which were used for SCS, were the University of Wisconsin solution (VIASPAN™ or the University of Wisconsin (UW) solution) and the Euro-Collins solution. There have been approximately 150 perfusate solutions tested at this time. (Demmy TE., 1997; Minasian SM., 2014) Nevertheless, the single significant issue that none have addressed thus far is the incidence of inflammation, apoptosis and the proinflammatory response that result in posttransplantation early graft dysfunction and likely the development of fibrosis in the transplanted organ. The present inventors have surprisingly found that including one or more GDFMPs in the perfusate promoted levels and / or changes in levels of markers (e.g., metabolic markers (e.g., 02 consumption)) that are indicative of ex vivo organ injury and, therefore, predictive of a successful organ implantation.

[0131] The perfusates described herein comprising one or more GDFMPs may also include therapeutic components to help maintain the organs and protect them against ischemia, reperfusion injury and other ill effects during perfusion. Therapeutics may also help mitigate edema, provide general endothelial tissue support for the organs, and otherwise provide preventative or prophylactic treatment.

[0132] According to some aspects, the disclosure provides a perfusate (also called a perfusion solution) for maintaining the ex vivo organ during normothermic machine perfusion comprising at least one GDFMP. According to some embodiments, the perfusate further comprises one more of the following: an isotonic set of salts (e.g., Ringer Solution, Isotonic sodium chloride), antioxidants (e.g., glutathione), buffers (e.g., sodium bicarbonate), oxygen carriers, or energy sources. In some embodiments, the oxygen carriers may be packed red blood cells or synthetic substances such as HemoPure®. In some embodiments, the energy sources may be glucose or glycogen. In some embodiments, the perfusate may contain buffers to maintain the solution at an optimal pH. These may include disodium phosphate anhydrate, a physiologic balancing buffer or monopotassium phosphate to maintain the average pH of the solution during perfusion. In some embodiments, the perfusion fluid can comprise one or more amino acids such as L-argenine, L-glutamine, and the like. In some embodiments, the perfusion fluid can comprise one or more buffers such as phosphate buffered saline (“PBS”), Krebs-Ringer buffer (“KRB”) (available from Sigma Aldrich, Inc. of St. Louis, Mo.), and the like. In some embodiments, the perfusion fluid can comprise one or more inorganic salts such as sodium chloride, calcium chloride, potassium chloride, and the like. In some embodiments, the perfusion fluid can comprise one or more substrates for metabolism such as glucose and other carbohydrates, lactate, fatty acids, other energy sources, vitamins, and the like. In some embodiments, the perfusion fluid can comprise one or more hormones such as insulin. In some embodiments, the perfusion fluid can comprise one or more antibiotics such as penicillin and / or streptomycin. In some embodiments, the perfusion fluid can comprise plasma. The one or more anticoagulants can include heparin. 132033-00620

[0133] In some embodiments, the perfusate is for the heart, and comprises a modified Krebs- Henseleit buffer enhanced with the addition of packed and washed red blood cells as oxygen carriers.

[0134] In some embodiments, the perfusion fluid can also comprise decellularization agents, for example, one or more agents selected from the group consisting of detergents, vasodilators, buffers, inorganic salts, and enzymes. Non-limiting examples of inorganic salts include calcium chloride, cupric sulfate, ferric nitrate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium chloride, and zinc sulfate.

[0135] The solution may contain an antimicrobial or antifungal agent to prevent infection. These may include bacterial and fungal antimicrobial agents that provide protection against both gram negative and gram positive bacteria. Suitable antimicrobial or antifungal agents include cefazolin, ciprofloxacin, and voriconazole. Alternatively the solution may contain any effective antimicrobial or antifungal agent.

[0136] In some embodiments, the solution is used for machine perfusion. In some embodiments, the solution is used for normothermic machine perfusion (NMP). In some embodiments, the solution is used for hypothermic machine perfusion (HMP).

[0137] According to some embodiments, the amount of GDFMP in the perfusate is from about O.lpg / ml to about 1 pg / ml or more, O.lpg / ml to about 10 pg / ml or more, O.lpg / ml to about 100 pg / ml or more, or O.lpg / ml to about 1000 pg / ml or more. According to some embodiments, the amount of GDFMP in the perfusate is from about 0.01 pg / ml to about 1 pg / ml or more, about 0.01 pg / ml to about 10 pg / ml or more, about O.Olpg / ml to about 100 pg / ml or more, about O.Olpg / ml to about 1000 pg / ml or more. According to some embodiments, the amount of GDFMP in the perfusate is from about O.OOlpg / ml to about 1 pg / ml or more, about O.OOlpg / ml to about 10 pg / ml or more, about O.OOlpg / ml to about 100 pg / ml or more, about O.OOlpg / ml to about 1000 pg / ml or more. According to some embodiments, the amount of GDFMP in the perfusate is about O.OOlpg / ml or more, 0.05 pg / ml or more, O.lpg / ml or more, 0.5pg / ml or more, Ipg / ml or more, 10 pg / ml or more, 50 pg / ml or more, 100 pg / ml or more, 200 pg / ml or more, 500 pg / ml or more, 750 pg / ml or more, 1000 pg / ml or more, 1500 pg / ml or more, or 2000 pg / ml or more.

[0138] IV. Methods of improving therapeutic suitability of donated organs with GDFMPs

[0139] The present inventors have found that growth and differentiation factor mimetic peptides (GDFMPs) (e.g., SEQ ID NOs 1-6 and 8-13, as shown in Table 1 above) in organ perfusates prior to static cold ischemia, during static warm ischemia, and / or during normothermic machine perfusion (NMP) or hypothermic machine perfusion (HMP) can be used to improve the suitability of harvested organs for transplant and the likelihood of transplant success.

[0140] The present disclosure provides methods for reducing ischemic damage in an organ, the method comprising contacting an organ removed from a subject with a perfusion solution comprising one or more GDFMPs, and perfusing the organ with a machine perfusion device. The present 132033-00620 disclosure also provides methods for recovering an organ with ischemic damage, the method comprising contacting the organ with a perfusion solution comprising one or more GDFMPs, and perfusing the organ with a machine perfusion device.

[0141] The present disclosure provides in some aspects a method of reducing ischemic damage in an organ removed from a subject, the method comprising contacting the organ with a perfusion solution comprising one or more GDFMPs, and perfusing the organ with a machine perfusion device. According to some embodiments, the one or more GDFMPs is selected from Table 1. According to some embodiments, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. According to some embodiments, the organ is in a container comprising the perfusion solution, and the machine perfusion device comprises a first and a second end, wherein the first end of the machine perfusion device is connected to the perfusion solution in the container and the second end is connected to the organ, thereby perfusing the organ. According to some embodiments of the aspects and embodiments herein, the organ is at least partially immersed in the perfusion solution. According to some embodiments of the aspects and embodiments herein, the perfusion solution is at a temperature of between about 37°C to about 38°C, e.g., a temperature of 37°C, 37.5°C or 38°C. According to some embodiments, the perfusion solution is at a temperature of about 37°C. According to some embodiments of the aspects and embodiments herein, the perfusion solution is at a temperature of between about 10°C to about 30°C, e.g., a temperature of 10°C to about 15 °C, 10°C to about 25 °C, 15 °C to about 25 °C, 20°C to about 25 °C, 20°C to about 30°C . According to some embodiments, the perfusion solution is at a temperature of between about 15 °C to about 25 °C. According to some embodiments of the aspects and embodiments herein, the organ undergoes at least 15 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments of the aspects and embodiments herein, the organ undergoes at least 30 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments of the aspects and embodiments herein, the organ undergoes 0 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments, the machine perfusion device is a normothermic machine perfusion (NMP) device. According to some embodiments, the machine perfusion device is a hypothermic machine perfusion (HMP) device.

[0142] According to some embodiments of the aspects and embodiments herein, the organ is selected from the group consisting of: a liver, a heart, a pancreas, a kidney, a spleen, and a lung.

[0143] Measuring ischemic damage may be assessed using the measures of organ “acceptability” for transplant, as described below.

[0144] According to other aspects, the disclosure provides a method of recovering an organ with ischemic damage, the method comprising contacting the organ with a perfusion solution comprising one or more GDFMPs, and perfusing the organ with a machine perfusion device. According to some embodiments, the one or more GDFMPs is selected from Table 1. According to some embodiments 132033-00620 of the aspects and embodiments herein, the one or more GDFMPs from Table 1 comprises a sequence at least 95%, 96%, 97%, 98% OR 99% identical to SEQ ID NO: 1. According to some embodiments, the organ is in a container comprising the perfusion solution, and the machine perfusion device comprises a first and a second end, wherein the first end of the machine perfusion device is connected to the perfusion solution in the container and the second end is connected to the organ, thereby perfusing the organ. According to some embodiments of the aspects and embodiments herein, the organ is at least partially immersed in the perfusion solution. According to some embodiments of the aspects and embodiments herein, the perfusion solution is at a temperature of between about 37°C to about 38°C, e.g., a temperature of 37°C, 37.5°C or 38°C. According to some embodiments, the perfusion solution is at a temperature of about 37°C. According to some embodiments of the aspects and embodiments herein, the perfusion solution is at a temperature of between about 10°C to about 30°C, e.g., a temperature of 10°C to about 15°C, 10°C to about 25°C, 15°C to about 25°C, 20°C to about 25 °C, 20°C to about 30°C . According to some embodiments, the perfusion solution is at a temperature of between about 15 °C to about 25 °C. According to some embodiments of the aspects and embodiments herein, the organ undergoes at least 15 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments of the aspects and embodiments herein, the organ undergoes at least 30 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments of the aspects and embodiments herein, the organ undergoes 0 min of static warm ischemia (WI) prior to contact with the perfusion solution. According to some embodiments, the machine perfusion device is a normothermic machine perfusion (NMP) device. According to some embodiments, the machine perfusion device is a hypothermic machine perfusion (HMP) device. According to some embodiments of the aspects and embodiments herein, the organ is selected from the group consisting of: a liver, a heart, a pancreas, a kidney, a spleen, and a lung. According to some embodiments of the aspects and embodiments herein, the subject is administered the one or more GDFMPs postmortem in vivo, postmortem for the perfusion of said organ prior to harvesting.

[0145] Measuring ischemic recovery may be assessed using the measures of organ “acceptability” for transplant, as described below.

[0146] In one aspect, the disclosure provides the use of GDFMPs to improve the therapeutic suitability of donated organs for allograft or xenograft by the postmortem in vivo perfusion of said organs with one or more GDFMPs prior to harvesting.

[0147] In one aspect, the disclosure provides the ex vivo perfusion of donated organs with one or more GDFMPs prior to and during static warm storage (SWS) or static cold storage (SCS) of an organ after harvesting and during transportation to the recipient transplant site.

[0148] In one aspect, the disclosure provides the therapeutic use of one or more GDFMPs by addition to the perfusate during ex vivo NMP or HMP.

[0149] Measures of organ “acceptability” for transplant during NMP for the heart include: 132033-00620

[0150] • Robust 02 consumption as a measure of the level of oxidative metabolism. The concentration of 02 in the perfusate is measured as it enters the heart chamber and as it exits the chamber, or as it enters and exits the coronary vasculature, the difference, normalized to the mass of the heart and to the perfusate flow rate through the heart, being the measure of oxidative metabolism.

[0151] • Lactate concentration in the perfusate: little or no rise; no reduction due to absence of CORI cycle in heart tissue.

[0152] • Beat Rate (bps) near normal.

[0153] • Ejection fraction greater than 50%

[0154] • Perfusate pH = 7.4

[0155] • Percent change in heart weight: during the period of ex vivo NMP or HMP (a measure of edema due to inflammation)

[0156] Measures of organ “acceptability” for transplant during NMP for the liver include:

[0157] • Robust rate of 02 consumption as a measure of the level of oxidative metabolism. The concentration of 02 in the perfusate is measured as it enters the liver chamber and as it exits the chamber, or as it enters and exits the hepatic vasculature, the difference, normalized to the mass of the liver and to the perfusate flow rate through the liver, being the measure of oxidative metabolism

[0158] • The rate decrease of Lactate concentration in the perfusate (in the absence of perfusate filtration or addition): is a measure of a robust CORI cycle, which converts lactate in the perfusate back to glucose and requires ATP generated by oxidative metabolism to function.

[0159] • Perfusate pH: 7.4: Should increase toward pH 7.4 as lactate is converted back to glucose.

[0160] • ALT and AST concentrations in the perfusate and their ratio.

[0161] • Percent change of liver weight during the period of NMP or HMP (measure of edema due to inflammation)

[0162] In some embodiments, the methods are used for ex vivo preservation of organ or composite tissue allografts, for example in which an organ or tissue is removed from a donor and preserved during storage and / or transport prior to implantation in a recipient. In other embodiments, the methods can also be used for autotransplantation, for example, in which an organ or tissue is temporarily removed for ex vivo therapy (such as resection of a tumor or gene therapy), followed by reimplantation. In further embodiments, the methods can be used to preserve or enhance ex vivo organ function, for example, prior to or during isolation of cells from an organ for further use (such as transplantation of cells into a donor). The disclosed methods can be used with any organ or tissue which can be machine perfused, including, but not limited to liver, heart, kidney, lung, pancreas, 132033-00620 small intestine, or any portion thereof. In some examples, the disclosed methods can be used for more than one organ in combination, for example heart and lung(s).

[0163] In some embodiments, the organ is perfused for about 1 hour to about 14 days, such as about 1-72 hours, 2-48 hours, 4-24 hours, 1-14 days, 1-10 days, 1-7 days, 2-14 days, 2-10 days, or 5-10 days. In particular examples, the organ is perfused for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more hours or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.

[0164] The perfusion solution is delivered to the organ via one or more cannulas which are inserted in a vessel of the organ (such as an artery or vein), for example a vessel that supplies blood (such as oxygenated blood) to an organ. One of ordinary skill in the art can select appropriate vessels for perfusion of an organ. For example, a kidney may be perfused through a cannula inserted in the renal artery, while a liver may be perfused through a cannula inserted in the hepatic artery and / or a cannula inserted in the portal vein, a heart may be perfused through one or more cannulas inserted in the coronary arteries, and lungs may be perfused through one or more cannulas inserted in the pulmonary arteries. In other examples, a CTA may be perfused through a cannula inserted in an artery of the CTA. In some embodiments, the flow of the perfusion solution to the organ is a continuous flow, such as a flow without substantial variations of flow rate, for example to mimic venous blood flow under most physiologic conditions. In other embodiments, the flow of the perfusion solution to the organ is a pulsatile flow (such as having flow rate variations that mimic arterial pulsatile blood flow), for example, pulsatile flow of the perfusion solution through a cannula inserted in an artery of the organ or CTA.

[0165] In some embodiments, the method of organ perfusion with a perfusion fluid as disclosed herein permits one to maintain or extend the viability and storage time of cells, tissues, and organs beyond current limits. In some embodiments, the method of perfusing an organ or tissue with a perfusion fluid as disclosed herein is at least about 20% reliable, or about 30% reliable, or about 40% reliable, or about 50% reliable, or about 60% reliable, or about 70% reliable, or about 80% reliable, or about 90% reliable, or about 100% reliable, in that, after perfusing a perfusion fluid (with or without subsequent steps of metabolic suppression via chemical or environmental means) the organ reaches the required threshold of viability for transplantability, or a reaches the required threshold of viability for cell viability for cell harvesting.

[0166] In some embodiments, the method of perfusing an organ or tissue with a perfusion fluid as disclosed herein prevents ischemic damage in the organ by about 10%, or by about 20%, or by about 30%, or by about 40%, or by about 50%, or by about 60%, or by about 70%, or by about 80%, or by about 90%, or by about 100% as compared to an organ, or tissue which has not been perfused with the perfusion fluid as disclosed herein.

[0167] In another embodiment, the amount of ischemic damage to an organ or tissue is decreased upon perfusion with the perfusion fluid according to the methods as disclosed herein by about at least 132033-00620

[0168] 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, greater than 90% as compared to an organ which has not undergone perfusion with the perfusion fluid as disclosed herein. Stated another way, in some embodiments, an organ or tissue which has undergone perfusion with the perfusion fluid according to the methods as disclosed herein has at least about 10% more viable cells than an organ or tissue which has not undergone the perfusion with the perfusion fluid, or about 20% more viable cells, or about 30% more viable cells, or about 40% more viable cells, or about 50% more viable cells, or about 60% more viable cells, or about 70% more viable cells, or about 80% more viable cells, or about 90% more viable cells, or about 100% more viable cells, or about 200% more viable cells, or about 1000% more viable cells, or more than 1000% more viable cells as compared to an organ or tissue which has not undergone the perfusion with the perfusion fluid as disclosed herein.

[0169] V. Organ preservation devices

[0170] Machine perfusion (MP) has the clinical advantage of reducing cold ischemic time significantly in long-distance procurements or complex recipient operations, such as explanation of mechanical circulatory support. According to some aspects, the disclosure provides organ preservation devices comprising an organ perfusion machine and an organ perfusate, wherein the organ perfusate comprises one or more GDFMPs (e.g., one or more GDFMPs selected from Table 1). According to some embodiments, the organ preservation devices described herein can be used in a variety of applications including, but not limited to, organ preservation, prevention of an organ from ischemic damage, recovery of an ischemically damaged organ, organ rehabilitation, and recovery of cells from damaged organs.

[0171] In some embodiments, the organ can be donated by a donor subject and transplanted into a recipient subject. In some embodiments, the organ can be isolated from a subject for rehabilitation using the organ perfusion devices described herein. In some embodiments, the perfusion is normothermic. In some embodiments, the perfusion is hypothermic. In some embodiments, the organ perfusion machine comprises a normothermic machine perfusion (NMP) device. In some embodiments, the organ perfusion machine comprises a hypothermic machine perfusion (HMP) device. The organ is then transplanted back to the same subject once it is determined to be functional. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0172] For example, hypothermic ex vivo heart perfusion involves pumping a cold crystalloid solution into the coronary arteries of the arrested heart to deliver oxygen and nutrients while removing toxic metabolites. Several hypothermic ex vivo heart perfusion platforms have been studied or are currently under investigation. Normothermic machine perfusion maintains the donor heart in a warm, contractile, near-physiologic state during transport from the donor to the recipient hospital (Kounatidis et al., Int J Mol Sci. 2023 Dec; 24(23): 16693). 132033-00620

[0173] In some embodiments, the organ is selected from the group consisting of a liver, a pancreas, a kidney, a spleen, a heart, and a lung. In some embodiments, the organ comprises a portal vein and an artery.

[0174] In some embodiments, the organ perfusion device is portable.

[0175] In one aspect, the disclosure provides an organ preservation device comprising an organ appropriate perfusion machine containing an organ appropriate perfusate comprising one or more GDFMPs. According to one embodiment, the one or more GDFMPs is selected from Table 1, as described above. According to some embodiments, the perfusion machine is equipped to monitor metabolic markers in the perfusate.

[0176] An organ appropriate perfusion machine comprises the necessary facilities beyond the basics, for example, for the heart plumbing such that oxygenated perfusate passes through the coronary arteries. Langendorff prefusion employs retrograde perfusion of the coronary vessels, which means perfusate entering coronary vessels from the venous side and exiting the arterial side; provisions for measuring heart rate, coronary arterial flow rate, the pressure across the coronary arterial network; mechanism for dampening perfusion pump pulsations. An organ appropriate perfusion machine for the liver may also comprise a bile collection port.

[0177] Kounatidis D, et al. (2023), incorporated by reference in its entirety herein, provides a review of machine perfusion The now classical approach to preservative storage of harvested ex vivo organs is static cold storage (SCS) whereby hypothermic conditions are maintained using freezer packs to slow metabolism in order to minimize ischemic injury, (c.f., the SHERPA PAK)

[0178] Current practice utilizes normothermic machine perfusion with OCS and temperature controlled cold preservation. The OCS® device creates near normal conditions of temperature, oxygenation with a supportive perfusate that is pumped through the ex vivo organ to support natural metabolism and avoid conditions that lead to ischemia / reperfiision injury after transplantation (c.f., the Organ Care System®(OCS®)) . The ideal constitution of the perfusion system and perfusate necessarily differ to meet the needs of each specific type of organ. For instance, during normothermic machine perfusion (NMP), the ex vivo hearts pumps on its own, but requires plumbing to insure that the entire organ is adequately perfused; the ex vivo lung requires a synthetic diaphragm to maintain oxygenation of the perfusate; the ex vivo liver requires an additional port for the collection of bile; and the ex vivo kidney requires a collecting port for urine as well as the provision of new perfusate to makeup that lost to the system through urine filtration. The NMP system must consider other issues that result from the isolation of the ex vivo organ from the normal services of other organs. Without lungs, the perfusate must be artificially oxygenated and provide some manner of oxygen transport, be it red blood cells or some synthetic carrier (i.e., HEMOPURE); without kidneys and a liver, a means for removing metabolic products may be required, although the use of a sufficiently large volume of perfusate may suffice for short periods of NMP. 132033-00620

[0179] Devices or systems that can be used in the disclosed methods are also known to one of ordinary skill in the art. Such devices include one or more chambers for holding an organ and a perfusion solution (such as the solutions disclosed herein) and one or more pumps (for example, one or more rotary pumps or peristaltic pumps) for delivery of the perfusion solution to the organ. Such devices also include one or more means to regulate temperature of the perfusion solution, such as one or more heat exchangers and one or more means to oxygenate the perfusion solution (such as an oxygenator in the perfusion circuit).

[0180] Exemplary devices are available from Organ Assist, Groningen, Netherlands (such as Kidney Assist or Liver Assist), Organ Recovery Systems, Itasca, Ill. (such as Life Port kidney transporter or liver transporter), Transmedics, Andover, Mass, (such as the heart or lung Organ Care System), OrganOx, Oxford, UK (such as OrganOx Metra), and XVIVO Perfusion Engelwood, Colo. Exemplary devices and systems are also described in U.S. Pat. Nos. 6,994,954; 6,953,655; 6,977,1420; 7,678,563; 7,811,808; 7,897,357; 8,268,547; 8,268,612; and 8,287,580; U.S. Pat. Publ. No. 2010 / 0028850; and International Pat. Publ. No. WO 2009 / 041806; all of which are incorporated herein by reference in their entirety.

[0181] One of ordinary skill in the art can identify additional organ perfusion devices or systems that can be used with the methods and solutions disclosed herein.

[0182] According to some embodiments, the normothermic perfusion machine comprises: a. A water-jacketed organ cell containing: i. inlet and outlet ports from which oxygenated perfusate is supplied to the hepatic artery and from which outgoing perfusate is collected from the hepatic vein, ii. a port to collect excretion of e.g., bile(liver), urine(kidney), oxygenated perfusate (Lung). iii. Inlet and outlet ports for the cell’s water jacket b. A temperature bath to regulate the liver sample cell, the membrane oxygenator cell and the perfusate bubble trap cell at normothermic temperature. c. A membrane oxygenator cell connected to a source of 95% 02, 5% CO2 to maintain the physiologically proper 02 saturation in the perfusate for the liver. d. A bubble trap for removing bubbles from the perfusate, providing a site for injecting into the perfusate nutrients and THR-184 and for damping pressure fluctuations in the perfusate. e. A peristaltic pump for maintaining a proper rate of flow of perfusate through the liver. f. Inflow and outflow sampling ports for sampling, respectively, perfusion solution entering the sample cell and perfusion solution leaving the sample cell. g. Exit ports for bile (Liver), urine (kidney) 132033-00620

[0183] This invention is further illustrated by the examples which should not be construed as limiting. The contents of all references cited throughout this application, as well as the figures and tables are incorporated herein by reference.

[0184] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0185] EXAMPLES

[0186] Example 1. The Effect of THR-184 on Rat hearts in normothermic machine perfusion (NMP) after experiencing 30 min warm ischemia.

[0187] A description of the experimental setup and the perfusate solution used, and to which the test article (THR-184, SEQ ID NO: 1) in Arm 3 is added, is provided in Pendexter etal., 2024, incorporated by reference in its entirety herein.

[0188] Arm 1 - WI Control: Untreated rat hearts that undergo 30 min. static warm ischemia (WI) prior to 4 hr. NMP without THR-184.

[0189] Arm 2 - WI THR-184: THR-184 treated rat hearts that undergo 30 min. of static WI prior to 4 hr. NMP with THR-184

[0190] Arm 3 - Fresh: Untreated rat hearts that go directly in NMP (no static WI) for 4 hr without THR- 184.

[0191] All data were sampled 20 min into NMP, then on the hour throughout NMP.

[0192] The rate of oxygen consumption is a direct, positive marker of oxidative metabolism.

[0193] Ischemia (lack of O2) shuts down oxidative metabolism. When the supply of oxygen is returned during the NMP phase, the level of O2 consumption is indicative of the degree to which ischemia damages the mitochondria in which oxidative metabolism occurs. FIG. 1 shows oxygen consumption by hearts in the three study arms over the 4 hr of NMP. In FIG. 1, hearts in the “WI THR-184” and “Fresh” arms displayed a high and consistent rate of oxygen consumption during NMP phase while those in the “WI Control” arm displayed lower and more erratic O2 consumption. The hearts in the “WI THR-184” arm emerged from warm ischemia behaving like those in the “Fresh” arm that had not experienced ischemia.

[0194] Another commonly used marker for evaluating the acceptability of an organ for transplantation is the level of lactate in the perfusate. During ischemia, where the lack of oxygen blocks oxidative metabolism, there is a buildup of pyruvate, the product of glycolysis. An alternate metabolic process reduces the buildup of pyruvate by converting it to lactate, which also produces a minimal amount of metabolic energy to maintain the homeostasis of the tissue. In addition to the generation of only a minimum of metabolic energy (2 ATPs per molecule of glucose) to maintain cellular homeostasis, the lactate formed during ischemia converts to lactic acid resulting in acidosis 132033-00620 and cellular injury. A heart that has experienced a period of ischemia will enter NMP with a significant level of lactate that will diffuse into and remain in the perfusate. FIG. 2 shows lactate concentration in the Aortic perfusate of hearts in the three study arms over the 4 hr of NMP. FIG. 3 shows lactate concentration in the Left Ventricular perfusate of hearts in the three study arms over the 4 hr of NMP. This is because the process that converts lactate back to glucose, the CORI cycle, exists only in the liver and the kidney cortex. In FIG. 2 and FIG. 3, the hearts in the “WI Control” and “WI with THR-184” arms entered the NMP process with raised lactate levels whereas the hearts in the “Fresh” control arm, which have not experienced a pre-NMP period of warm ischemia, entered NMP with nearly no lactate in the perfusate. In this study a steady increase in perfusate lactate was observed in all three arms during NMP. While this may be a marker of persistent ischemic injury from the WI period, the rise of lactate in the “Fresh” arm indicates ongoing ischemia, likely due to inadequate perfusion of the coronary arteries. During NMP, the heart continues to pump on its own. Coronary perfusion starts at the output in the aorta, which requires a pressure differential between the aorta and outside perfusate. In the in vivo setting the resistance to flow arises from the capillary network of the body, whereas in the ex vivo setting an aortic-venal pressure difference must be created by partial clamping of the aorta. The practical problem is that over-clamping can overload injured heart muscles. In this study, the rate of rise in lactate in the “WI Control” arm, relative to the rate of rise in “Fresh” arm, was 2.9 times for the aorta (FIG. 2) and 2.4 times for the left ventricle (FIG. 3). The corresponding ratios for the “WI THR-184” arm were 1.7 times and 1.2 times. In the “WI THR- 184” arm, the effect of the presence of THR-184 in the perfusate during both the WI and NMP periods was to reduce the rate of lactate rise during NMP by half.

[0195] Tissue injury from warm ischemia results in inflammation, which leads to edema (swelling) and an increase in the resistance to vascular flow resulting in a rise in aortic-coronary pressure. Edema is reflected in an increase in the weight of the hearts at the end of NMP compared to the start of the NMP phase. FIG. 4 shows that by the end of the 4 hour NMP run, the hearts in the “WI with THR-184” arm had gained the least weight, slightly less than the hearts in the “Fresh” arm, and less than half the weight increase of hearts in the “WI Control” arm. There was also noticeably little variation in weights in the “Fresh” and “WI THR-184” arms compared to the “WI Control” arm.

[0196] The effect of edema in the “WI Control” arm on heart rate (FIG. 5), perfusate flow rate (FIG. 6), resistance to perfusate flow (FIG. 7) and aortic / coronary pressure (FIG. 8) were consistent with a large increase in edema. FIG. 5 shows the effect of THR-184 on the ex vivo heart rate during NMP. While the mean rate of the hearts in the “WI THR-184” arm tracked with those in the “Fresh” arm, the average “WI Control” arm rate was 30% lower indicating persistent myocardial injury from the WI.

[0197] FIG. 6 shows the perfusate flow rate through the hearts during the NMP. Flow rate is the combined effect of heart rate and flow resistance. Hearts in the “WI THR-184” arm had the highest and most consistent flow rates. Hearts in the “WI Control” group started NMP with a flow rate that is 132033-00620

[0198] 89% of the mean rate for the “WI THR-184” arm, but that ratio dropped to 78% over the course of 4 hr NMP. The mean rate in the” Fresh” arm started with the “WI THR-184” arm, then dropped to 89% of the “WI THR-184” flow rate. FIG. 7 shows the mean vascular resistance to perfusate flow through hearts in the three study arms over the 4 hr of NMP period. During NMP, the resistance to perfusate flow (FIG. 7) through the hearts in the “WI THR-184” arm, like those in the “Fresh” arm, are constant while the resistance in hearts in the “WI Control” arm increases with time. The same pattern was seen in FIG. 8 for the aortic / coronary pressure. FIG. 8 shows the mean aortic coronary pressure in hearts in the three study arms over the 4 hr of NMP period. These results are all consistent with an increase in edema brought on by inflammation resulting from the period of warm ischemia that is observed for the “WI Control” arm but is inhibited by the presence of THR-184 in the “WI THR-184” arm.

[0199] Example 2. Effects of supplementing normothermic machine perfusion perfusate with THR-184 on the recovery of a liver graft from warm ischemic damage in a rat liver model.

[0200] Donor organ scarcity has limited the degree to which liver transplantation can impact patient's lives. While multiple attempts have been made to expand the donor pool, such as the development of new preservative solutions and machine perfusion as well as the use of ECMO for in-situ organ procurement, popularly known as NRP (Meinders et al., Current Surgery Reports. 2024; 12(3): 15-25), yet many patients die waiting for a liver each year (Kwong et al., OPTN / SRTR 2021 Annual Data Report: Liver. Am J Transplant. 2023;23(2 Suppl l):S178-s263). The majority of grafts are usually obtained from deceased donors who have experienced either cardiac death in a controlled setting (Kwong et al., 2023) or are declared brain-dead (Martin-Loeches et al., Intensive Care Medicine. 2019;45(3) : 322-30), with a minority of grafts coming from living donors (Kwong et al., 2023). Every graft source presents distinct problems for transplantation. Grafts obtained from controlled DCD experience warm ischemia injury, which causes PNF and non-anastomotic biliary strictures and often can lead to retransplantation (Taner et al., Transpl Int. 2012;25(8) : 838-46). While DBD grafts often do not experience significant warm ischemia injury, as seen in DCD grafts, both graft types are susceptible to cold ischemic damage during transport and warm ischemic damage during the anhepatic phase of graft implantation in the recipient (Ijtsma et al. Liver Transpl. 2009; 15(9): 1050-5). Therefore, enhancing the usage of liver grafts necessitates the investigation of methods to reduce ischemia injury at different phases of transplantation, and the application of a drug offers a more straightforward and practical approach.

[0201] Donation after cardiac death (DCD) is the major source of livers; however, long warm ischemic time (WIT) in DCD livers can result in primary graft dysfunction, delayed graft function, and ischemic cholangiopathy, which require biliary interventions and retransplantation due to the development of non-anastomotic biliary strictures, which is a long-term complication. These issues make DCD grafts inferior to brain death donation (DBD). Normothermic Machine Perfusion (NMP) technology has been used to recover DCD liver grafts from warm ischemia, allowing for liver 132033-00620 assessment and the simultaneous deployment of interventions to recover the graft. Ex vivo models of perfusion offer an opportunity to investigate treatments that limit ischemic injury and promote recovery, thus expanding the number of livers available for transplant. In addition, these treatments reduce the PGD and provide a path for the development of new treatments.

[0202] This example investigated the effects of supplementing NMP perfusate with TBD-184 (a BMP-7 mimetic) on the recovery of a liver graft from warm ischemic (WI) damage in a donation after cardiac death (DCD) rat liver model. These experiments focused on biliary parameters.

[0203] The study design is shown in FIG. 9 , and included three arms:

[0204] Arm 1 - Fresh: Untreated rat livers that go directly in NMP (no static WI) for 6 hr without THR- 184. (n = 3).

[0205] Arm 2 - WI Control: Untreated rat livers that undergo 30 min. of static WI prior to 6 hr. NMP without THR- 184 (n = 7);

[0206] Arm 3 -WI THR-184: THR-184 treated rat livers that undergo 30 min. WI prior to 6 hr. NMP with THR- 184 (n = 5)

[0207] All data were sampled 20 min into NMP, then on the half hour throughout NMP.

[0208] FIG. 10 shows the mean oxygen uptake rate (normalized to liver mass) for livers in each of the study arms during the 6 hr NMP. As shown in FIG. 10 the rate of oxygen uptake by livers in the presence of THR-184 (“WI THR-184” arm 3) was higher throughout the six hours of NMP than those in the “WI Control” arm (arm 2) and even those in the “Fresh” arm (arm 1), which had not experienced WI. Robust oxygen consumption was evidence of robust oxidative metabolism due to healthy mitochondria, which for the livers in the “WI THR-184” arm, was the case despite having endured 30 minutes of static WI. In fact, the rate of O2 consumption by livers in the “WI THR-184” arm, like those in the “Fresh” arm remained relatively constant throughout the 6 hr. of NMP whereas the O2 consumption rate of those in the “WI Control” arm declined.

[0209] FIG. 11 shows the mean pH for liver perfusates entering the liver during the 6 hr of NMP and FIG. 12 shows the mean lactate concentration in perfusate entering the livers during the 6 hr NMP. The pH of the perfusate (FIG. 11) was lowered by the production of lactate, which is converted to lactic acid, during the 30 min period of static WI to which livers in the “WI Control” and “WI THR- 184” arms were subjected. The pH of the perfusate in the “WI THR-184” arm and the “Fresh” arm remained closer to the normal pH range for the rat (pH 3.5 to 4.5) than that in the “WI Control” arm throughout the 6 hr. of NMP. The effect of THR-184 on the concentration of lactate in the perfusate (FIG. 12) was less clear.

[0210] During the 30 min static WI period, glucose is metabolized only through glycolysis to form two molecules of pyruvate producing two ATP, but in the absence of O2, pyruvate cannot enter oxidative metabolism and is instead reduced to lactate. The ATP produced during glycolysis helps to maintain the cell and protect its mitochondria (the site of oxidative metabolism). Thus a high rate of lactate formation is indicative of robust glycolytic metabolism, and the livers in both the “WI 132033-00620

[0211] Control” and “WI THR-184” arms enter NMP with significant interstitial lactate concentrations that then diffuse into the respective perfusates. The liver, however, is the primary site of the CORI cycle, through which lactate is metabolized back to glucose at the expense of 6 ATP. To produce the molecules of ATP needed for the CORI cycle, oxidative metabolism must restart during the NMP period. A measure of the health of the mitochondria in the livers in both the “WI Control” and “WI THR-184” arms is the rate of decrease of lactate in the perfusate, which can be seen in the “WI THR- 184” treated arm to start at around 3 hr. into the NMP period (FIG. 12). During the last 3 hr. of NMP, lactate in the “WI THR-184” arm decreased at a mean rate of about 8% per hr. whereas lactate in the “WI Control” arm perfusate decreased at about 1% per hr.

[0212] When liver tissue is injured the levels of enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are released into the bloodstream, and hence are observed to rise in the ex vivo perfusate (FIG. 13 and FIG. 14, respectively; the dotted line is the normal level for in vivo rat plasma). FIG. 13 shows the mean concentration of Alanine Transaminase (ALT) in each study arm at 3, 4 and 6 hr. NMP. FIG. 14 shows the mean concentration of Aspartate Transaminase (AST) in each study arm at 3, 4 and 6 hr. NMP. The effect of the presence of THR-184 in the perfusate was, in both cases, to reduce the degree of ALT and AST rise, which implied a lower degree of liver tissue injury.

[0213] Histology

[0214] Results from histological staining and imaging are shown in FIG. 15 and FIG. 16. FIG. 15 shows hematoxylin and eosin (H&E) -stained slices of representative livers from both the “WI Control” and “WI THR-184” arms at the end of 6 hr of NMP. Pores in the liver are indicated by dashed circles. As can be seen in the representative images shown in FIG. 15, cells in the “WI THR- 184” arm slice were narrower (less bloated), have better defined outer membranes, and were better organized around pores (indicated by circles) than those in the “WI Control” arm slice. This was consistent with there being less edema when THR-184 was present. FIG. 16 shows TUNEL stained (for apoptosis) slices of representative livers from both the “WI Control” and “WI THR-184” arms at the end of 6 hr of NMP. Nuclei that are stained brown and marked with red circles are undergoing apoptosis. As can be seen in FIG. 16, there was a marked decrease in apoptosis observed in the “WI THR-184” arm as compared to the “WI Control” arm as judged by the delineation of the cell walls (rectangular areas) and the cellular structure around pores (circular areas).

[0215] The results from this Example demonstrated that when the BMP -7 mimetic (THR-184) was added to the perfusate, it alleviated the damage incurred during the warm ischemic time, as seen by the improved hepatocellular function and lowering of injury markers. 132033-00620

[0216] Example 3. In vivo model for the treatment of organ donor and recipient with TBD-184 to minimize ischemic reperfusion injury.

[0217] Next, in vivo experiments will be performed where organ donor and recipient will be treated with TBD-184 for upstream and downstream management, respectively, for minimizing ischemic reperfusion injury.

[0218] Example 4. Determining the efficacy of adding BMP-7 mimetics in UW solution for the reduction of ischemia reperfusion injury (IRI).

[0219] In cases with donation after brain death (DBD), graft damage predominantly results from sterile inflammation due to the cytokine storm, dysregulation of the autonomic nerve system, and disruption of the hypothalamic -pituitary-adrenal axis, leading to electrolyte and hemodynamic imbalances (Patel et al., Front Immunol. 2021 ; 12:681504). Therefore, it is crucial to manage braindead donors with the infusion of fluids and maintain blood pressure and cardiac function with medications like norepinephrine, dopamine, vasopressin, and hydrocortisone (Martin-Loeches et al. Intensive Care Medicine. 2019;45(3): 322-30). Brain stem damage prevents spontaneous breathing, which is a criterion for brain death; thus, mechanical ventilatory support is needed to sustain oxygenation. Since DBD meets death criteria, organ donation intervention does not pose the same ethical issues as DCD. DBD cases give a chance to undertake medication intervention to maintain organ health for optimal donation; hence, BMP-7 mimetics may aid liver grafts to withstand cold ischemia better. BMP-7 mimetics like THR-184 have been successfully used in the in vivo model of unilateral AKI in rats with pre-ischemia, i.e., prophylactic and post-ischemic treatment for AKI (Carlson et al., Front Pharmacol. 2022;13:864509). Also, it has been used in phase II clinical trials to explore the prevention of AKI in the setting of patients going for cardiac surgery requiring cardiopulmonary bypass (Himmelfab et al., J Am Soc Nephrol. 2018;29(2):670-9), indicating its safety profile for use in a DBD for enhancing graft quality as a prophylactic measure with its added advantage of being anti-apoptotic, anti-inflammatory, anti-fibrotic, and pro-regenerative (Carlson et al., 2022), which could work as a supplement in maintaining hemodynamic stability of DBD donors.

[0220] Initial 6 hr of NMP 1 will be used as a platform to simulate DBD, followed by 48 hr of cold ischemia time (CIT) for static cold storage (SCS), then another 6 hr NMP 2 will be used as a simulation of transplantation in the recipient along with the assessment of graft function. Group 2 will test the prophylactic role, and Group 3 will test the combined prophylactic and post-cold ischemia treatment efficacy of BMP-7 mimetic (TBD-184), as illustrated in FIG. 17. Perfusion parameters for NMP 1 & 2 will be recorded similarly to NMP in specific aim # 1. Stats for NMP 1 : both groups 2 & 3 NMP 1 data will be combined and compared with group 1 : NMP 1 using unpaired two-sided student's t-test with Welch's correction. Stats for NMP 2: ANOVA with post-hoc Tukey's test, one-way ANOVA followed by two-way ANOVA for finding differences at individual time points. 132033-00620

[0221] Example 5. Determining efficacy of BMP-7 mimetics in donation after cardiac death (DCD) model during in-situ NRP and NRP + NMP.

[0222] Donation after cardiac death (DCD) is a major source for a donor pool; however, long warm ischemic time (WIT) in DCD livers can result in primary and delayed graft dysfunction and especially ischemic cholangiopathy, which can require multiple biliary interventions and even retransplantation due to the development of non-anastomotic biliary strictures as a long-term complication (Taner et al. , Transpl Int. 2012;25(8): 838-46). Historically, these issues made DCD grafts inferior to brain death donation (DBD). In-situ NRP offers recovering grafts from warm ischemia during DCD. In addition, ex vivo NMP offers an additional opportunity to recover livers for transplant.

[0223] The hypothesis tested is that NRP restores bio-energetics (ATP) and helps graft recover from warm ischemic damage. Restored graft ATP will help in handling cold ischemia during SCS. Combined NRP and NMP will have a synergistic effect in recovering and restoring graft function. 30 min of WIT will be simulated ex-situ as described in the preliminary study. An initial 6 hr of NMP 1 will be used as a platform to simulate the in situ NRP process for the DCD model, followed by 12 hr of CIT for SCS. Then another 6 hr NMP 2 will be used as a simulation of transplantation in the recipient along with the assessment of graft function. Group 2 will test for BMP-7 mimetics during the NRP phase, and Group 3 will test combined upstream and downstream management with this drug. NMP perfusion parameters will be recorded similarly as described herein. A schematic is shown in FIG. 18A and FIG. 18B.

[0224] Improved viability parameters are expected with BMP -7 mimetics supplementation of UW and perfusate supplementation during NMP for the DBD model and NRP. RNAseq will be run on the flash-frozen samples if there is a positive outcome to explore the hepato-protective factors' upregulation and regeneration pathways activation.

[0225] It is possible that growth factors related to the activation of pathways may be slower at 4°C compared to the normothermic condition of 37°C, which may obscure the beneficiary effect. Also, liver grafts are obtained from healthy rats without prior damage like in fatty (steatotic) and aged grafts, due to which the difference seen may not be large enough to reach statistical significance. Alternatives include fatty liver and DCD models as well as a longer duration of SCS than 48 hours, and different levels of injury which will magnify the effect size and find the functional range.

[0226] REFERENCES

[0227] All publications and references, including but not limited to patents and patent applications, cited in this specification and Examples herein are incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this 132033-00620 application claims priority is also incorporated by reference herein in the manner described above for publications and references.

[0228] References

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Claims

132033-00620CLAIMSWhat is claimed is:

1. A method of reducing ischemic damage in an organ removed from a subject, the method comprising contacting the organ with a perfusion solution comprising one or more GDFMPs, and perfusing the organ with a machine perfusion device.

2. The method of claim 1, wherein the one or more GDFMPs is selected from Table 1.

3. The method of claim 2, wherein the one or more GDFMPs from Table 1 comprises a sequence at least 95% identical to SEQ ID NO: 1.

4. The method of claim 1, wherein the organ is in a container comprising the perfusion solution, and the machine perfusion device comprises a first and a second end, wherein the first end of the machine perfusion device is connected to the perfusion solution in the container and the second end is connected to the organ, thereby perfusing the organ.

5. The method of any one of claims 1-4, wherein the organ is at least partially immersed in the perfusion solution.

6. The method of any of claims 1-5, wherein the perfusion solution is at a temperature of between about 37°C to about 38°C.

7. The method of claim 6, wherein the perfusion solution is at a temperature of about 37°C.

8. The method of any one of claims 1-5, wherein the perfusion solution is at a temperature of between about 10°C to about 30°C.

9. The method of claim 8, wherein the perfusion solution is at a temperature of between about 15°C to about 25°C.

10. The method of any one of claims 1-9, wherein the organ undergoes at least 15 min of static warm ischemia (WI) prior to contact with the perfusion solution.

11. The method of any one of claims 1-10, wherein the organ undergoes at least 30 min of static warm ischemia (WI) prior to contact with the perfusion solution.

12. The method of any one of claims 1-9, wherein the organ undergoes 0 min of static warm ischemia (WI) prior to contact with the perfusion solution.

13. The method of any one of claims 1-7 or 11-12, wherein the machine perfusion device is a normothermic machine perfusion (NMP) device.132033-0062014. The method of any one of claims 1-5 or 11-12, wherein the machine perfusion device is a hypothermic machine perfusion (HMP) device.

15. The method of any one of claims 1-14, wherein the organ is selected from the group consisting of: a liver, a heart, a pancreas, a kidney, a spleen, and a lung.

16. A method of recovering an organ with ischemic damage, the method comprising contacting the organ with a perfusion solution comprising one or more GDFMPs, and perfusing the organ with a machine perfusion device.

17. The method of claim 16, wherein the one or more GDFMPs is selected from Table 1.

18. The method of claim 17, wherein the one or more GDFMPs from Table 1 comprises a sequence at least 95% identical to SEQ ID NO: 1.

19. The method of claim 16, wherein the organ is in a container comprising the perfusion solution, and the machine perfusion device comprises a first and a second end, wherein the first end of the machine perfusion device is connected to the perfusion solution in the container and the second end is connected to the organ, thereby perfusing the organ.

20. The method of any one of claims 16-19, wherein the organ is at least partially immersed in the perfusion solution.

21. The method of any of claims 16-19, wherein the perfusion solution is at a temperature of between about 37°C to about 38°C.

22. The method of claim 21, wherein the perfusion solution is at a temperature of about 37°C.

23. The method of any one of claims 16-19, wherein the perfusion solution is at a temperature of between about 10°C to about 30°C.

24. The method of claim 23, wherein the perfusion solution is at a temperature of between about 15 °C to about 25 °C.

25. The method of any one of claims 16-24, wherein the organ undergoes at least 15 min of static warm ischemia (WI) prior to contact with the perfusion solution.

26. The method of any one of claims 16-25, wherein the organ undergoes at least 30 min of static warm ischemia (WI) prior to contact with the perfusion solution.132033-0062027. The method of any one of claims 16-24, wherein the organ undergoes 0 min of static warm ischemia (WI) prior to contact with the perfusion solution.

28. The method of any one of claims 16-22 or 23-24, wherein the machine perfusion device is a normothermic machine perfusion (NMP) device.

29. The method of any one of claims 16-19 or 23-24, wherein the machine perfusion device is a hypothermic machine perfusion (HMP) device.

30. The method of any one of claims 16-29, wherein the organ is selected from the group consisting of: a liver, a heart, a pancreas, a kidney, a spleen, and a lung.

31. The method of any one of claims 1-15, wherein the subject is administered the one or more GDFMPs postmortem in vivo, postmortem for the perfusion of said organ prior to harvesting.

32. A method of improving the acceptability of an ex vivo donor organ for therapeutic allograft or xenograft transplant into a human recipient, the method comprising contacting the ex vivo donor organ with one or more GDFMPs of Table 1.

33. The method of claim 31, wherein the one or more GDFMPs are administered postmortem in vivo for the perfusion of said organ prior to harvesting.

34. The method of claim 31, wherein the one or more GDFMPs are used as an additive to an organ appropriate perfusate prior to and during static ex vivo storage of said organ during the period post-harvest and prior to implantation into a human recipient.

35. The method of claim 31, wherein the one or more GDFMPs are used an additive to an organ appropriate perfusate during therapeutic, active ex vivo machine perfusion prior to implantation of said organ into a human recipient.

36. An organ preservation device comprising: an organ perfusion solution comprising one or more GDFMPs of Table 1; and an organ perfusion machine.

37. The organ preservation device of claim 36, wherein the one or more GDFMPs is selected from Table 1.

38. The organ preservation device of claim 37, wherein the one or more GDFMPs from Table 1 comprises a sequence at least 95% identical to SEQ ID NO: 1.132033-0062039. The organ preservation device of any one of clams 36-38, wherein the organ perfusion machine is a normothermic machine perfusion (NMP) device.

40. The organ preservation device of any one of claims 36-39, wherein the device further monitors metabolic markers in the organ perfusion solution.

41. An organ perfusion solution comprising one or more GDFMPs of Table 1.

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