WO2025250192A1 - Non-ischemic beating heart xenotransplantation - Google Patents

Non-ischemic beating heart xenotransplantation

Info

Publication number
WO2025250192A1
WO2025250192A1 PCT/US2025/013314 US2025013314W WO2025250192A1 WO 2025250192 A1 WO2025250192 A1 WO 2025250192A1 US 2025013314 W US2025013314 W US 2025013314W WO 2025250192 A1 WO2025250192 A1 WO 2025250192A1
Authority
WO
WIPO (PCT)
Prior art keywords
hours
donor heart
heart
minipig
donor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/013314
Other languages
French (fr)
Inventor
Andrew B. GOLDSTONE
Daniel H. WOLBROM
Michael Brewer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Columbia University in the City of New York
Original Assignee
Columbia University in the City of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Columbia University in the City of New York filed Critical Columbia University in the City of New York
Publication of WO2025250192A1 publication Critical patent/WO2025250192A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3621—Extra-corporeal blood circuits
    • A61M1/3666—Cardiac or cardiopulmonary bypass, e.g. heart-lung machines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/14—Mechanical aspects of preservation; Apparatus or containers therefor
    • A01N1/142—Apparatus
    • A01N1/143—Apparatus for organ perfusion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M60/00—Blood pumps; Devices for mechanical circulatory actuation; Balloon pumps for circulatory assistance
    • A61M60/10—Location thereof with respect to the patient's body
    • A61M60/122—Implantable pumps or pumping devices, i.e. the blood being pumped inside the patient's body
    • A61M60/196—Implantable pumps or pumping devices, i.e. the blood being pumped inside the patient's body replacing the entire heart, e.g. total artificial hearts [TAH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B17/00234—Surgical instruments, devices or methods for minimally invasive surgery
    • A61B2017/00238—Type of minimally invasive operation
    • A61B2017/00243—Type of minimally invasive operation cardiac
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods
    • A61B2017/00969—Surgical instruments, devices or methods used for transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2210/00—Anatomical parts of the body
    • A61M2210/12—Blood circulatory system
    • A61M2210/125—Heart

Definitions

  • Cardiac xenotransplantation is a rapidly developing area of interest as there is an ongoing and unmet need for donor hearts for patients with heart disease.
  • Use of donor hearts from swine as viable xenografts is hampered by the ischemia that develops in the donor tissue using traditional methods of cold organ storage and cardioplegia during heart transplant surgery. Ischemia in donor heart tissue correlates with perioperative cardiac xenograft dysfunction (PCXD).
  • PCXD perioperative cardiac xenograft dysfunction
  • the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises: (a) placing the primate subject on cardiopulmonary bypass (CPB) and surgically removing the heart of the primate subject; (b) placing the donor heart on a normothermic coronary perfusion circuit and then explanting the donor heart while beating from the swine; (c) implanting the beating explanted donor heart into the primate subject on CPB; and (d) removing the primate subject from CPB.
  • CPB cardiopulmonary bypass
  • the donor heart remains perfused and beating throughout the entire procedure.
  • the beating explanted donor heart comprises substantially no ischemic tissue at the time of implantation into the primate subject on CPB.
  • the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step (b) for a maximum of about 10 hours, optionally wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step (b) for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours.
  • the transplanted donor heart is substantially free of ischemic tissue at the time of the implanting step.
  • the transplanted donor heart has a left ventricle ejection fraction (LVEF), a right ventricular (RV) systolic function, a pulmonary artery (PA) pressure, a right atrial (RA) pressure, a sinus rhythm, and/or a cardiac output comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • LVEF left ventricle ejection fraction
  • RV right ventricular
  • PA pulmonary artery
  • RA right atrial
  • sinus rhythm a cardiac output comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • the normothermic coronary perfusion circuit is comprised of at least two drainage cannulas and at least one outflow cannula.
  • step (b) further comprises ligating the superior vena cava (SVC) and the inferior vena cava (IVC) and clamping the aorta distal to the outflow cannula before explanting the beating donor heart.
  • SVC superior vena cava
  • IVC inferior vena cava
  • the present disclosure provides a method of explanting a donor heart while perfused and beating from a swine, wherein the method comprises: (a) placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by: (i) placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart via the apex or across the left atrium; (ii) placing an outflow cannula in the proximal ascending aorta of the donor heart; (iii) ligating the superior vena cava (SVC) NAI-1540201368v1 Attorney Docket No.14648-049-228 and the inferior vena cava (IVC) of the donor heart; (iv) clamping the aorta of the donor heart; (b) explanting the donor heart while beating from the swine; (c) maintaining the
  • the method further comprises priming the normothermic coronary perfusion circuit with plasmalyte A before placing the donor heart on the normothermic coronary perfusion circuit.
  • the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for a maximum of about 10 hours, optionally wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step (c) for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours.
  • the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ in an organ reservoir.
  • the organ reservoir is maintained at a temperature ranging from about 32°C to about 37°C.
  • the beating donor heart maintained on the normothermic coronary perfusion circuit ex situ in an organ reservoir is placed in a sterile container suitable for transportation.
  • the beating donor heart is perfused with an oxygenated physiological solution.
  • the oxygenated physiological solution comprises blood from the swine that is sourced from the donor heart.
  • the swine is a genetically-modified minipig.
  • the genetically-modified minipig comprises a GGTA1 genetic modification. In certain embodiments, the genetically-modified minipig does not express alpha-1,3- galactosyltransferase (GalT-KO). [0019] In certain embodiments, the genetically-modified minipig is a GalT-KO minipig and: (a) is wild type for a protein encoded by the porcine genes CMAH, ⁇ 4GalNT, ASGR1, and CD46; and/or (b) does not express any recombinant proteins. In certain embodiments, the genetically- modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens.
  • the genetically-modified minipig is GalT-KO and the GalT-KO is the NAI-1540201368v1 Attorney Docket No.14648-049-228 only genetic modification in the minipig.
  • the primate subject is a human subject.
  • the human subject has advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof.
  • the human subject is a pediatric human subject.
  • the method is performed in the absence of cardioplegia.
  • the present disclosure provides a medical instrument comprising (a) at least two drainage cannulas and at least one outflow cannula, wherein one drainage cannula is suitable to be placed in the right atrium of a donor heart and another drainage cannula is suitable to be placed in the left ventricle of the donor heart; (b) an outflow cannula, wherein the outflow cannula is suitable to be placed in the proximal ascending aorta of the donor heart; and (c) a pump to maintain homeostasis with a perfusion solution; wherein the medical instrument is specifically adapted to perform normothermic machine perfusion on a heart from a minipig.
  • the medical instrument further comprises an organ reservoir.
  • the medical instrument further comprises a membrane oxygenator, a leukocyte filter, an arterial filter, a heater-cooler unit, an autonomous power unit, or any combination thereof.
  • the medical instrument further comprises a sterile container, wherein the sterile container is suitable for transportation of a donor heart that is perfused and beating during transport.
  • the medical instrument further comprises a collection chamber, wherein the collection chamber is suitable for collecting lost blood during transportation of a donor heart.
  • the present disclosure provides an explanted donor heart from a minipig, wherein the donor heart is connected to an ex situ normothermic coronary perfusion circuit prior to transplantation into a subject in need thereof.
  • the minipig is a genetically-modified minipig.
  • the genetically-modified minipig comprises a GGTA1 genetic modification.
  • the genetically-modified minipig does not express alpha-1,3- galactosyltransferase (GalT-KO).
  • the genetically-modified minipig is a GalT-KO minipig and: NAI-1540201368v1 Attorney Docket No.14648-049-228 (a) is wild type for a protein encoded by the porcine genes CMAH, ⁇ 4GalNT, ASGR1, and CD46; and/or (b) does not express any recombinant proteins.
  • the genetically-modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens.
  • the genetically-modified minipig is GalT-KO and the GalT- KO is the only genetic modification in the minipig.
  • the normothermic coronary perfusion circuit is first established in situ by: (a) placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart; and (b) placing an outflow cannula in the proximal ascending aorta of the donor heart.
  • the normothermic coronary perfusion circuit is established in situ, (a) the superior vena cava (SVC) and the inferior vena cava (IVC) of the donor heart are ligated; (b) the aorta of the donor heart is clamped; (c) the donor heart is explanted while beating from the swine; and (d) the beating explanted donor heart is maintained on the normothermic coronary perfusion circuit ex situ.
  • SVC superior vena cava
  • IVC inferior vena cava
  • the explanted donor heart comprises substantially no ischemic tissue within at least 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or at least 12 hours.
  • the present disclosure provides a method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises transplanting a donor heart into the subject according to the methods of the present disclosure.
  • the present disclosure provides a method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises use of the medical instrument of the present disclosure.
  • the present disclosure provides a method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises transplanting the explanted donor heart from a minipig of the present disclosure into the subject.
  • the present disclosure provides a method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises transplanting a donor heart into the subject according NAI-1540201368v1 Attorney Docket No.14648-049-228 to the methods of the present disclosure.
  • PCXD perioperative cardiac xenograft dysfunction
  • the present disclosure provides a method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises use of the medical instrument of the present disclosure.
  • the present disclosure provides a method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises transplanting the explanted donor heart from a minipig of the present disclosure into the subject.
  • the human subject has at least one condition that can be treated by receiving a heart transplantation.
  • the at least one condition that can be treated by receiving a heart transplantation comprises advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof.
  • the human subject is a pediatric human subject. 4. BRIEF DESCRIPTION OF THE FIGURES [0042]
  • FIG.1 depicts a schematic of the coronary perfusion system. The grey arrows indicate drainage cannulas inserted into RA and the LV of the heart. The black arrow indicates an outflow cannula placed in the proximal ascending aorta. The striped circle indicates a pump.
  • FIG.2 depicts a representative image of a porcine donor heart maintained on a normothermic coronary perfusion circuit ex situ.
  • FIG.3 depicts representative images of echocardiography performed in a baboon donor heart recipient after weaning from cardiopulmonary bypass (CPB).
  • the top left image shows echocardiographic assessment of left ventricular (LV) systolic function.
  • the bottom left image shows echocardiographic assessment of LV diastole function.
  • the top right image shows echocardiographic assessment of mitral valve (MV) function.
  • MV mitral valve
  • TV echocardiographic assessment of tricuspid valve
  • FIG.4 depicts a representative image of histopathology of the myocardium of the donor heart harvested from a baboon after the donor heart was transplanted. The image shows a hematoxylin and eosin (H&E) stained section of the tissue at 400x magnification.
  • FIGS.5A-5B depict representative images of echocardiogram data collected on post- NAI-1540201368v1 Attorney Docket No.14648-049-228 transplant Day 8 showing cardiac function.
  • FIG.5A shows left ventricle short axis systole and
  • FIG.5B shows left ventricle short axis diastole. 5.
  • an explanted beating donor heart from swine methods of preparing thereof, and methods using thereof.
  • an explanted beating donor heart from swine see Section 5.2.
  • a swine that provides the beating donor heart disclosed herein is described in Section 5.3 below.
  • a medical instrument specifically adapted for coronary perfusion during cardiac xenotransplantation see Section 5.1).
  • the medical device disclosed herein is specifically adapted for coronary perfusion of the explanted beating donor heart from swine as described in Section 5.2 below, and for use in methods of explanting a beating donor heart (see Section 5.4.1) and transplanting a beating donor heart (see Section 5.4.2).
  • a method of using an explanted beating donor heart from swine see Section 5.4). Methods of explanting a beating donor heart are disclosed in Section 5.4.1 below.
  • An explanted beating donor heart from swine can be transplanted into a subject in need thereof according to the methods described in Section 5.4.2 below.
  • a subject suitable for receiving an explanted beating donor heart disclosed herein is described in Section 5.4.4 below.
  • Such a subject in need of an explanted beating donor heart of the present disclosure can have, be suspected of having, or demonstrate at least one symptom of a disease described in Section 5.4.5 below.
  • 5.1 Coronary Perfusion Systems [0051]
  • the present disclosure provides a medical instrument specifically adapted for coronary perfusion during cardiac xenotransplantation.
  • the present disclosure provides a medical instrument specifically adapted to perform normothermic machine perfusion during cardiac xenotransplantation.
  • the present disclosure provides a medical instrument specifically adapted to perform normothermic machine perfusion of a donor heart from a porcine donor.
  • medical instruments of the present disclosure are specifically adapted for use with any porcine donor described in Section 5.3 and/or any donor heart described in Section 5.2.
  • medical instruments of the present disclosure can be used for normothermic machine perfusion of a donor heart according to the methods described herein (see Section 5.4).
  • Normothermic machine perfusion is generally used to recreate the physiological environment of a donor organ by maintaining normal temperature and providing the essential substrates needed to support organ viability prior to transplantation.
  • a medical instrument disclosed herein is a normothermic ex-vivo perfusion system specifically adapted for use in cardiac xenotransplantation.
  • a medical instrument disclosed herein is a normothermic ex-vivo perfusion system specifically adapted for use with a porcine donor described in Section 5.3 herein.
  • a medical instrument disclosed herein is a normothermic ex-vivo perfusion system specifically adapted for use with a minipig donor.
  • a medical instrument disclosed herein is a normothermic ex-vivo perfusion system suitable for circulating an oxygenated physiological solution through a donor heart explanted from a minipig donor.
  • a medical instrument disclosed herein comprises at least one cannula.
  • a “cannula” refers to a small tube that can be inserted into a body cavity, duct, or vessel.
  • a cannula that is suitable for use with a medical instrument disclosed herein can be made of any biomaterial acceptable for medical use.
  • a cannula suitable for a medical instrument disclosed herein can be made of polyvinylchloride (PVC).
  • a cannula suitable for a medical instrument disclosed herein can be wire reinforced to prevent obstruction due to kinking.
  • a medical instrument disclosed herein comprises at least two drainage cannulas.
  • a “drainage cannula” refers to a cannula for drainage of venous blood.
  • a medical instrument disclosed herein comprises at least two drainage cannulas, wherein one drainage cannula is suitable to be placed in the right atrium of a donor heart as described herein (e.g., Section 5.3).
  • a medical NAI-1540201368v1 Attorney Docket No.14648-049-228 instrument disclosed herein comprises at least two drainage cannulas, wherein one drainage cannula is suitable to be placed in the left ventricle of a donor heart as described herein (e.g., Section 5.3).
  • a medical instrument disclosed herein comprises at least two drainage cannulas, wherein one drainage cannula is suitable to be placed in the right atrium of a donor heart and another drainage cannula is suitable to be placed in the left ventricle of the donor heart.
  • a medical instrument disclosed herein comprises at least one outflow cannula.
  • an “outflow cannula” refers to a cannula for perfusing a donor heart as described herein (e.g., Section 5.3) with a perfusion solution.
  • a medical instrument disclosed herein comprises an outflow cannula, wherein the outflow cannula is suitable to be placed in the proximal ascending aorta of the donor heart.
  • a medical instrument disclosed herein comprises at least one pump to maintain homeostasis with a perfusion solution.
  • a medical instrument disclosed herein comprises at least one perfusion pump.
  • a perfusion pump as used herein is suitable for the fluid transfer of the perfusate through the various components of the perfusion system as described herein and through the donor heart.
  • types of perfusion pumps include axial flow pumps, peristaltic pumps, diaphragm pumps, pumping cassettes, roller pumps, centrifugal pumps, and pulsatile pumps.
  • a medical instrument disclosed herein further comprises an organ reservoir.
  • An organ reservoir is a container suitable for maintaining a donor heart on a normothermic coronary perfusion circuit ex situ in a sterile state.
  • an organ reservoir suitable for use in the medical instrument disclosed herein comprises an outlet so that the blood, perfused through, and out of, the organ, which collects in the organ reservoir can be collected for later reoxygenation, recirculation, and/or reperfusion.
  • the inside of an organ reservoir suitable for use herein comprises an organ- supporting means which are well-known in the art, such as a soft mesh floor, a sling or “hammock,” a tilted or non-tilted “V-shaped or “U”-shaped platform, or may contain gauze or other sterile protective material inserted by the surgeon, to provide support to the donor heart and to prevent movement of the donor heart during transport.
  • a medical instrument disclosed herein further comprises a sterile container.
  • a sterile container suitable for use in the medical instrument disclosed herein is suitable for transportation of a donor heart that is perfused and beating during transport.
  • a sterile container suitable for use in the medical instrument disclosed herein is made of a rigid material suitable for protecting the donor heart from physical contact, direct or indirect.
  • a sterile container suitable for use in the medical instrument disclosed herein is a box-shape having a hinged lid.
  • a sterile container suitable for use in the medical instrument disclosed herein comprises at least one aperture for monitoring the donor heart during transport.
  • a sterile container suitable for use in the medical instrument disclosed herein comprises an outlet suitable for later reoxygenation, recirculation, and/or reperfusion of blood and/or perfusion solution collected during transportation of a donor heart.
  • a sterile container suitable for use in the medical instrument disclosed herein comprises a means for supporting the organ reservoir as described herein.
  • a medical instrument disclosed herein further comprises a collection chamber container.
  • a collection chamber container suitable for use in the medical instrument disclosed herein is suitable for collecting lost blood and/or perfusion solution during transportation of a donor heart.
  • a collection chamber container suitable for use in the medical instrument disclosed herein comprises an outlet suitable for later reoxygenation, recirculation, and/or reperfusion of blood and/or perfusion solution collected during transportation of a donor heart.
  • a medical instrument disclosed herein further comprises a membrane oxygenator.
  • a membrane oxygenator as used herein is a device for adding oxygen to and removing carbon dioxide from the blood. Membrane oxygenators are known in the art, and a variety are commercially available.
  • a membrane oxygenator suitable for use in the medical instrument disclosed herein comprises a semipermeable polymer membrane.
  • suitable polymers include polypropylene, polyethylene, poly-4-methylpentene (PMP), polyvinylidene fluoride (PVDF), and polytetrafluoroethylene (PTFE).
  • PMP poly-4-methylpentene
  • PVDF polyvinylidene fluoride
  • PTFE polytetrafluoroethylene
  • a medical instrument disclosed herein further comprises a leukocyte filter. Leukodepletion occurs through a specific filter (i.e., a “leukocyte filter”) to NAI-1540201368v1 Attorney Docket No.14648-049-228 remove leukocytes from blood and/or hemocomponents.
  • Leukocyte filters suitable for use in the medical instrument disclosed herein can include those known in the art (see., e.g., Urias EVR et al., Rev Assoc Med Bras (2021) Jul;67(7):1056-1060).
  • a medical instrument disclosed herein further comprises an arterial filter.
  • An arterial filter for use herein filters out various material with embolic potential such as, for example, micro air, particulates, and/or aggregates composed of platelets, red blood cells and other debris.
  • arterial filter suitable for use in the medical instrument disclosed herein comprises a pore size ranging from 25 ⁇ m to about 45 ⁇ m (e.g., about 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 45 ⁇ m).
  • An arterial filter suitable for use in the medical instrument disclosed herein can include those known in the art (see., e.g., Hawkins JL et al., J Extra Corpor Technol (2010) Mar;42(1):71-4).
  • a medical instrument disclosed herein further comprises a heater-cooler unit.
  • a heater-cooler unit suitable for use in the medical instrument disclosed herein comprises a heat exchanger.
  • a heater-cooler unit is suitable for maintaining the perfusion solution, the donor heart, or both at physiological temperature. In certain embodiments, a heater-cooler unit is suitable for maintaining the perfusion solution, the donor heart, or both at a temperature ranging from about 32°C to about 37°C (e.g., about 32°C, 33°C, 34°C, 35°C, 36°C, 37°C).
  • a medical instrument disclosed herein further comprises an autonomous power unit. In certain embodiments, a medical instrument disclosed herein further comprises a portable autonomous power unit adapted to power the medical instrument.
  • the present disclosure provides an explanted donor heart for coronary perfusion during cardiac xenotransplantation.
  • the explanted donor heart as described in this section can be used with any one of the medical instruments disclosed herein (e.g., Section 5.1).
  • the explanted donor heart can be used in any one of the methods of transplantation as described in Section 5.4.2.
  • the explanted donor heart is from a porcine donor described in Section 5.3.
  • the explanted donor heart is from a minipig.
  • the explanted donor heart is from a genetically-modified minipig.
  • the explanted donor heart is from a genetically-modified minipig wherein the NAI-1540201368v1 Attorney Docket No.14648-049-228 genetically-modified minipig comprises a GGTA1 genetic modification.
  • the explanted donor heart is from a genetically-modified minipig wherein the genetically- modified minipig does not express alpha-1,3-galactosyltransferase (GalT-KO).
  • the explanted donor heart is from a genetically-modified minipig wherein the genetically-modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens.
  • the explanted donor heart is from a genetically-modified minipig wherein the genetically-modified minipig is GalT-KO and the GalT-KO is the only genetic modification in the minipig.
  • the explanted donor heart is from a GalT-KO minipig.
  • the explanted donor heart is from a GalT-KO minipig that is otherwise wildtype for other proteins, protein expression levels, or both.
  • the explanted donor heart is from a GalT-KO minipig that is wild type for a protein encoded by the porcine genes CMAH, ⁇ 4GalNT, ASGR1, and/or CD46.
  • the explanted donor heart is from a GalT-KO minipig that does not overexpress any recombinant proteins. In certain embodiments, the explanted donor heart is from a GalT-KO minipig that does not overexpress any recombinant human proteins. In certain embodiments, the explanted donor heart is from a GalT-KO minipig that does not overexpress any recombinant proteins encoded by a human gene.
  • the explanted donor heart is from a GalT-KO minipig that does not overexpress any recombinant proteins encoded by the human genes A20/TNFAIP3, CD39, CD46, CD47, CD55, CD59, ENTPD1, EPCR, HO-1, SERPING1, TBM, TFPI, VWF, HLA-E, and/or HLA-G.
  • the explanted donor heart is connected to a normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart is connected to a normothermic coronary perfusion circuit in situ.
  • the normothermic coronary perfusion circuit is first established in situ by (a) placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart; and (b) placing an outflow cannula in the proximal ascending aorta of the donor heart.
  • the explanted donor heart is connected to an ex situ normothermic coronary perfusion circuit.
  • the explanted donor heart is connected to an ex situ normothermic coronary perfusion circuit after the normothermic coronary NAI-1540201368v1 Attorney Docket No.14648-049-228 perfusion circuit is established in situ.
  • the explanted donor heart of the present disclosure comprises a beating heart connected to an ex situ normothermic coronary perfusion circuit.
  • the explanted donor heart of the present disclosure comprises a beating heart connected to an ex situ normothermic coronary perfusion circuit with a sinus rhythm rate ranging from about 50–180 beats per minute.
  • the explanted donor heart of the present disclosure comprises a beating heart connected to an ex situ normothermic coronary perfusion circuit with a sinus rhythm rate ranging from about 50-180, about 50-170, about 50- 160, about 50-150, about 50-140, about 50-130, about 50-120, about 50-110, about 50-100, about 60-180, about 60-170, about 60-160, about 60-150, about 60-140, about 60-130, about 60- 120, about 60-110, about 60-100, about 70-180, about 70-170, about 70-160, about 70-150, about 70-140, about 70-130, about 70-120, about 70-110, or about 70-100 beats per minute.
  • the explanted donor heart of the present disclosure comprises a beating heart connected to an ex situ normothermic coronary perfusion circuit with an average sinus rhythm rate of at least about or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 beats per minute.
  • the explanted donor heart of the present disclosure comprises substantially no ischemic tissue.
  • the explanted donor heart of the present disclosure comprises substantially no ischemic tissue within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart of the present disclosure comprises less than about 1% to 20% ischemic tissue within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit.
  • the explanted donor heart of the present disclosure comprises less than about 1% to 20%, 1% to 15%, 1% to NAI-1540201368v1 Attorney Docket No.14648-049-228 10% or 1% to 5% ischemic tissue within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit.
  • the explanted donor heart of the present disclosure comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% ischemic tissue within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit.
  • the explanted donor heart of the present disclosure comprises substantially no cardiac edema.
  • the explanted donor heart of the present disclosure comprises substantially no cardiac edema within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart of the present disclosure comprises less than about 1% to 20% cardiac edema within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit.
  • the explanted donor heart of the present disclosure comprises less than about 1% to 20%, 1% to 15%, 1% to 10% or 1% to 5% cardiac edema within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit.
  • the explanted donor heart of the present disclosure comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% cardiac edema within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit.
  • the explanted donor heart of the present disclosure comprises substantially no evidence of acute T cell rejection.
  • the explanted donor heart of the present disclosure comprises substantially no evidence of acute T cell rejection within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit.
  • the explanted donor heart of the present disclosure comprises substantially no evidence of antibody-mediated rejection.
  • an explanted donor heart of the present disclosure comprises substantially no evidence of antibody-mediated rejection within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit.
  • an explanted donor heart disclosed herein comprises at least one gene modification.
  • an explanted donor heart disclosed herein comprises the gene modification of the porcine donor.
  • an explanted donor heart disclosed herein comprises one gene modification in the gene encoding alpha-1,3- galactosyltransferase. In certain embodiments, an explanted donor heart disclosed herein does not express alpha-1,3-galactosyltransferase (i.e., GalT-KO).
  • a donor heart provided herein comprises a heart harvested from a swine.
  • the term “porcine” is used interchangeably herein with the terms “pig” and “swine” and refers to mammals in the family Suidae.
  • Non-limiting examples of the breeds of swine suitable for use herein includes any of the following: American Landrace, American Yorkshire, Aksai Black Pied, Angeln saddleback, Appalachian English, Arapawa Island, Auckland Island, Australian Yorkshire, Babi Kampung, Ba Xuyen, Bantu, Basque, Bazna, Beijing Black, else Black Pied, Belgian Landrace, Bengali Brown Shannaj, Bentheim Black Pied, Berkshire, Bisaro, Bangur, Black Slavonian, Black Canarian, Breitovo, British Landrace, British Lop, British Saddleback, Bulgarian White, Cambrough, Cantonese, Celtic, Chato NAI-1540201368v1 Attorney Docket No.14648-049-228 Murciano, Chester White, Chiangmai Blackpig, Choctaw Hog, Creole, Czech Improved White, Danish Landrace, Danish Protest, Dermantsi Pied, Li Yan, Duroc, Dutch Landrace, East Landrace, East Balkan, Essex, Estonia
  • the breed of a porcine donor for use in the present disclosure is a breed of minipig.
  • minipig breeds include Bama, Berlin, Chinese, Clawn, Czech-Republic, Göttingen, Hanford, Lee Sung, Mini-Lewe, Mini-Sib, Kunststoff, National Institutes of Health (NIH), Ohmini, Panepinto, Sinclair, Vietnamese potbellied, Wuzhishan, WZS, and Yucatan.
  • a porcine donor for use herein is a NIH minipig.
  • a porcine donor for use herein is a Yucatan minipig.
  • a porcine donor for use herein is a Göttingen minipig.
  • a porcine donor of the present disclosure includes wholly or partially inbred swine.
  • a porcine donor of the present disclosure is from the same herd.
  • a “herd,” as used herein, refers to a group of at least one male and one female which can breed to produce fertile male and female offspring.
  • the herd of swine includes at least one male swine and at least one female swine capable of reproduction, e.g., at least one male and one female which can produce functional gametes.
  • a porcine donor of the present disclosure is from a herd of wholly or partially inbred swine. In certain embodiments, a porcine donor of the present disclosure is from a herd of inbred swine homozygous for a major histocompatibility complex haplotype. In certain embodiments, a porcine donor of the present disclosure is from a herd of swine homozygous for a major histocompatibility complex haplotype and at least about 50% homozygous at all other genetic loci.
  • a porcine donor of the present disclosure is from a herd of swine homozygous for a major histocompatibility complex haplotype and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homozygous at all other genetic loci.
  • a porcine donor of the present disclosure is from a herd of swine homozygous at swine leukocyte antigens A, B, C, DR, and/or DQ.
  • the swine leukocyte antigens A, B, C, DR, and DQ can comprise haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc) haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), and/or haplotype j (Aj, Bj, Cj, DRj, DQj).
  • haplotype a Aa, Ba, Ca, DRa, DQa
  • haplotype c Ac, Bc, Cc, DRc, DQc
  • haplotype d Ad, Bd, Cd, DRd, DQd
  • haplotype g Ag, Bg, Cg, DRg, DQg
  • a donor heart disclosed herein comprises a heart from a porcine donor disclosed herein. In certain embodiments, a donor heart disclosed herein comprises a heart from a minipig.
  • a porcine donor of the present disclosure is genetically modified. In certain embodiments, a genetically-modified porcine donor (e.g., a transgenic, chimeric, or mosaic swine) comprises at least one genetic modification. In certain embodiments, a genetically-modified porcine donor comprises only one genetic modification. In certain embodiments, a genetically-modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein is prevented (i.e., knocked out).
  • a genetically-modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein is prevented in at least one organ, tissue, or cell type (e.g., in the heart).
  • a genetically-modified porcine donor e.g., a transgenic, chimeric, or mosaic swine
  • a genetically-modified porcine donor e.g., a minipig
  • a genetically-modified porcine donor NAI-1540201368v1 Attorney Docket No.14648-049-228 comprises one genetic modification in which the expression of a porcine protein encoded by the GGTA1 gene is prevented (i.e., knocked out, or “KO”) in the genetically-modified porcine donor.
  • a genetically-modified porcine donor comprises one genetic modification in which the expression of a porcine protein encoded by the GGTA1 gene is prevented in at least one organ, tissue, or cell type (e.g., in the heart) of the genetically-modified porcine donor.
  • a genetically-modified porcine donor (e.g., a minipig) comprises one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene and no other genes are genetically modified.
  • a genetically-modified minipig comprises one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene and no other genes are genetically modified.
  • a genetically-modified minipig does not express alpha-1,3- galactosyltransferase (i.e., the minipig is a GalT-KO minipig).
  • a genetically-modified porcine donor is a genetically- modified minipig that is a GalT-KO and the GalT-KO is the only genetic modification in the minipig.
  • a genetically-modified minipig comprises one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene and there are no genetic modifications of the CMAH gene, the ⁇ 4GalNT gene, the ASGR1 gene, or the porcine CD46 (pCD46) gene.
  • a genetically-modified porcine donor is a genetically-modified minipig that is a GalT-KO and has normal expression of proteins encoded by the CMAH gene, the ⁇ 4GalNT gene, the ASGR1 gene, and the porcine CD46 (pCD46) gene.
  • a genetically-modified porcine donor is a genetically- modified minipig that is a GalT-KO that does not express any recombinant proteins.
  • a genetically-modified porcine donor is a genetically-modified minipig that is a GalT-KO and that does not express any recombinant human proteins.
  • a genetically-modified porcine donor is a genetically-modified minipig that is a GalT-KO and that NAI-1540201368v1 Attorney Docket No.14648-049-228 does not express any recombinant human proteins encoded by the human CD46 (hCD46) gene, the human CD55 (hCD55) gene, the human CD59 (hCD59) gene, the human thrombomodulin (hTBM) gene, the human CD47 (hCD47) gene; the human heme oxygenase-1 (hHO-1) gene, the human A20 (hA20) gene, the human ectonucleoside triphosphate diphosphohydrolase-1 (hENTPD1) gene, the human tissue factor pathway inhibitor (hTFPI); or the human leukocyte antigen G (HLA-G) gene.
  • HLA-G human leukocyte antigen G
  • a genetically-modified porcine donor e.g., a genetically- modified minipig
  • a genetically-modified porcine donor can be generated using any number of suitable methods known in the art for generating transgenic swine (see, e.g., Lai et al., Science (2002) Feb 8;295(5557):1089-92; Hryhorowicz et al., Genes (Basel) (2020) Jun 19;11(6):670).
  • a method of generating a genetically-modified porcine donor comprises micro-injection of DNA material into the male pronucleus, RNA material into the cytoplasm, or proteins into the cytoplasm or pronucleus at early embryonic stage (usually the zygote/one-cell stage).
  • a method of generating a genetically-modified porcine donor comprises sperm-mediated gene transfer (SMGT) (see, e.g., Lavitrano et al., Reprod Fertil Dev (2006)18(1-2):19-23).
  • SMGT sperm-mediated gene transfer
  • a method of generating a genetically-modified porcine donor comprises somatic cell nuclear transfer (SCNT).
  • a method of generating a genetically-modified porcine donor comprises a gene- targeting technique.
  • a gene-targeting technique suitable for use herein include homologous recombination (HR), non-homologous DNA end joining (NHEJ), zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR- based systems.
  • a donor heart disclosed herein comprises a heart from a male or female porcine donor (e.g., a minipig). In certain embodiments, a donor heart disclosed herein comprises a heart from a genetically-modified male porcine donor (e.g., a minipig). In certain embodiments, a donor heart disclosed herein comprises a heart from a male GalT-KO minipig. In certain embodiments, a donor heart disclosed herein comprises a heart from a genetically- modified female porcine donor (e.g., a minipig). In certain embodiments, a donor heart disclosed herein comprises a heart from a female GalT-KO minipig.
  • a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) that is at least about 6 weeks of age. In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) that is at least about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age.
  • a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) that is at least about 6 weeks to 10 weeks, about 6 weeks to 9 weeks, about 6 weeks to 8 weeks, or about 7 weeks to 8 weeks.
  • a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) that is 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age.
  • a donor heart disclosed herein comprises a heart from a genetically-modified minipig that is at least about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age.
  • a donor heart disclosed herein comprises a heart from genetically-modified minipig that is at least about 6 weeks to 10 weeks, about 6 weeks to 9 weeks, about 6 weeks to 8 weeks, or about 7 weeks to 8 weeks. In certain embodiments, a donor heart disclosed herein comprises a heart from genetically-modified minipig that is 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age. In certain embodiments, a donor heart disclosed herein comprises a heart from a GalT-KO minipig that is at least about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age.
  • a donor heart disclosed herein comprises a heart from a GalT-KO minipig that is at least about 6 weeks to 10 weeks, about 6 weeks to 9 weeks, about 6 weeks to 8 weeks, or about 7 weeks to 8 weeks. In certain embodiments, a donor heart disclosed herein comprises a heart from a GalT-KO minipig that is about 8 weeks of age. [0090] In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) wherein the porcine donor weighs at least about 6 kg, about 7 kg, about 8 kg, about 9 kg, about 10 kg, about 11 kg, about 12 kg, or more than about 12 kg.
  • porcine donor e.g., a minipig
  • a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) wherein the weight of the porcine donor ranges from about 6 kg to 12 kg, about 7 kg to 11 kg, or about 8 kg to 10 kg.
  • a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) wherein the porcine donor weight is 6 NAI-1540201368v1 Attorney Docket No.14648-049-228 kg, about 7 kg, about 8 kg, about 9 kg, about 10 kg, about 11 kg, about 12 kg, or more than about 12 kg.
  • a donor heart disclosed herein comprises a heart from a genetically-modified minipig wherein the genetically-modified minipig weighs at least about 6 kg, about 7 kg, about 8 kg, about 9 kg, about 10 kg, about 11 kg, about 12 kg, or more than about 12 kg.
  • a donor heart disclosed herein comprises a heart from a GalT- KO minipig wherein the GalT-KO minipig weighs at least about 6 kg, about 7 kg, about 8 kg, about 9 kg, about 10 kg, about 11 kg, about 12 kg, or more than about 12 kg.
  • a donor heart disclosed herein comprises a heart from a GalT-KO minipig wherein the weight of the GalT-KO minipig ranges from about 8 kg to 10 kg.
  • the present disclosure provides a method of using a medical instrument described herein (see Section 5.1).
  • the present disclosure provides a method of using an explanted donor heart described herein (see Section 5.2).
  • a method of using an explanted donor heart of the present disclosure comprises a method of explanting a donor heart while perfused and beating from a swine that is described in Section 5.3.
  • the present disclosure provides a method of transplanting a donor heart from a swine into a subject (see Section 5.4.2). Subjects suitable for receiving a donor heart from a swine according to a method disclosed herein are described in Section 5.4.4. In certain embodiments, one or more additional treatments can be administered to the donor heart recipient as described in Section 5.4.3. In certain embodiments, the present disclosure provides a method of treating a disease in a subject in need thereof, wherein the method comprises transplanting an explanted donor heart described herein (see Section 5.2). Methods of evaluating the effectiveness of the treatment of the disease in the donor heart recipient is described in Section 5.4.5.
  • the present disclosure provides a method of explanting a beating donor heart from a swine.
  • a donor heart suitable for use in a method described in this section can be from a swine as described in Section 5.3.
  • the present disclosure provides a method of explanting a donor heart from a swine wherein the donor heart remains perfused and beating throughout the entire explant procedure.
  • the present disclosure provides a method of explanting a donor heart while perfused and beating from a swine wherein the method comprises placing the donor heart on a normothermic coronary perfusion circuit.
  • a method of explanting a donor heart while perfused and beating from a swine comprises placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ.
  • a method of establishing a normothermic coronary perfusion circuit in situ comprises placement of cannulas into the donor heart.
  • a method of establishing a normothermic coronary perfusion circuit in situ comprises placement of at least two drainage cannulas into the donor heart. Drainage cannulas as described in Section 5.1 are suitable for use in the methods disclosed herein toward establishing a normothermic coronary perfusion circuit in situ.
  • a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas.
  • a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas, wherein one drainage cannula is placed in the right atrium.
  • a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas, wherein one drainage cannula is placed in the left ventricle via the apex. In certain embodiments, a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas, wherein one drainage cannula is placed in the left ventricle via across from the left atrium. In certain embodiments, a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas, wherein one drainage cannula is placed in the left ventricle via the apex or across the left atrium.
  • a method of explanting a donor heart while perfused and beating from a swine comprises placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart via the apex or across the left atrium.
  • a method of establishing a normothermic coronary perfusion circuit in situ comprises placement of at least one outflow cannula into the donor heart. Outflow cannulas as described in Section 5.1 are suitable for use in the methods disclosed herein toward establishing a normothermic coronary perfusion circuit in situ.
  • a NAI-1540201368v1 Attorney Docket No.14648-049-228 normothermic coronary perfusion circuit for use in the method disclosed herein is comprised of one outflow cannula.
  • a normothermic coronary perfusion circuit for use in the method disclosed herein is comprised of one outflow cannula that is placed in the aorta.
  • a normothermic coronary perfusion circuit for use in the method disclosed herein is comprised of one outflow cannula that is placed in the proximal ascending aorta.
  • a method of explanting a donor heart while perfused and beating from a swine comprises placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by placing an outflow cannula in the proximal ascending aorta of the donor heart.
  • a method of establishing a normothermic coronary perfusion circuit in situ comprises ligation of at least one vein that drains into the donor heart.
  • methods of establishing a normothermic coronary perfusion circuit in situ comprise ligation of at least the superior vena cava (SVC).
  • a method of establishing a normothermic coronary perfusion circuit in situ comprises ligation of at least the inferior vena cava (IVC). In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises ligation of the SVC and the IVC of the donor heart. In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises clamping the aorta of the donor heart. [0098] In certain embodiments, provided herein is a method of placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ.
  • a method of placing the donor heart on a normothermic coronary perfusion circuit wherein the normothermic coronary perfusion circuit is established in situ by (i) placing at least one cannula into the donor heart; (ii) placing an outflow cannula in the aorta of the donor heart; (iii) ligating least one vein that drains into the donor heart; and (iv) clamping the aorta of the donor heart.
  • a method of placing the donor heart on a normothermic coronary perfusion circuit wherein the normothermic coronary perfusion circuit is established in situ by (i) placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart via the apex or across the left atrium; (ii) placing an outflow cannula in the proximal ascending aorta of the donor heart; (iii) ligating the SVC and the IVC of the donor heart; and (iv) clamping the aorta of the donor heart.
  • a method of establishing a normothermic coronary perfusion circuit in situ comprises priming the normothermic coronary perfusion circuit with a priming solution before placing the donor heart on the normothermic coronary perfusion circuit.
  • a priming solution for use in a method disclosed herein comprises, for example but not limited to, buffered electrolytes, mannitol, vitamins, and/or steroids.
  • a priming solution for use in a method disclosed herein has a physiological pH.
  • a priming solution for use in a method disclosed herein has a pH ranging from about 7.0 to 8.0 (e.g., about 7.0; 7.1; 7.2; 7.3; 7.4; 7.5; 7.6; 7.7; 7.8; 7.9; 8.0).
  • a priming solution for use in a method disclosed herein comprises sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and/or magnesium chloride.
  • a priming solution for use in a method disclosed herein comprises plasmalyte A.
  • a method of establishing a normothermic coronary perfusion circuit in situ comprises priming the normothermic coronary perfusion circuit with plasmalyte A before placing the donor heart on the normothermic coronary perfusion circuit.
  • a method of explanting a donor heart while perfused and beating from a swine wherein the method comprises explanting the donor heart while beating from the swine. Methods of explanting (e.g., surgically removing) the donor heart from swine are generally known in the art.
  • a method of explanting a donor heart while perfused and beating from a swine comprising maintaining the beating donor heart on a normothermic coronary perfusion circuit ex situ in an organ reservoir.
  • Organ reservoirs as described in Section 5.1 are suitable for use in a method disclosed herein toward maintaining the beating donor heart on a normothermic coronary perfusion circuit ex situ.
  • an organ reservoir for use in a method disclosed herein is maintained at a temperature ranging from about 32°C to about 37°C. In certain embodiments, an organ reservoir for use in a method disclosed herein is maintained at a temperature ranging from about 32°C to about 33°C, about 33°C to about 34°C, about 34°C to about 35°C, about 35°C to about 36°C, or about 36°C to about 37°C. In certain embodiments, an organ reservoir for use in a method disclosed herein is maintained at a temperature of about 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.
  • an organ reservoir for use in a method disclosed herein comprises a perfusion solution and the beating donor heart.
  • an organ reservoir for use in a method disclosed herein comprises a perfusion solution, the beathing donor heart, or both that are maintained at a temperature ranging from about 32°C to about 37°C.
  • an organ reservoir for use in a method disclosed herein comprises a perfusion solution, the beathing donor heart, or both that are maintained at a temperature ranging from about 32°C to about 33°C, about 33°C to about 34°C, about 34°C to about 35°C, about 35°C to about 36°C, or about 36°C to about 37°C.
  • an organ reservoir for use in a method disclosed herein comprises a perfusion solution, the beathing donor heart, or both that are maintained at a temperature of about 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.
  • a method of explanting a donor heart while perfused and beating from a swine comprising perfusing the beating donor heart with a physiological solution.
  • the method comprises perfusing the beating donor heart with an oxygenated physiological solution.
  • the method comprises perfusing the beating donor heart with an oxygenated physiological solution comprising more than about 90% oxygen.
  • the method comprises perfusing the beating donor heart with an oxygenated physiological solution comprising about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% oxygen.
  • the method comprises perfusing the beating donor heart with an oxygenated physiological solution comprising about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% oxygen.
  • the method comprises perfusing the beating donor heart with an oxygenated physiological solution comprising blood from the swine that sourced the donor heart.
  • the method comprises perfusing the beating donor heart with an oxygenated physiological solution, optionally wherein the oxygenated physiological solution may comprise an agent suitable for use with the methods herein.
  • an agent provides nutrition or treatment to the cardiac tissue of the donor heart.
  • an agent is to be delivered locally to the cardiac tissue of the donor heart.
  • an agent comprises a polynucleotide or fragment thereof, a small molecule, NAI-1540201368v1 Attorney Docket No.14648-049-228 an antibody or fragment thereof, a polypeptide or fragment thereof, a gene editing system, or any combination thereof.
  • an agent comprises a vasodilator (e.g., nitrates, nicorandil). In certain embodiments, an agent comprises a vasopressor (e.g., norepinephrine, epinephrine). In certain embodiments, a method disclosed herein comprises perfusing the beating donor heart with an oxygenated physiological solution, wherein the oxygenated physiological solution is free from additional agents. In certain embodiments, a method disclosed herein comprises perfusing the beating donor heart with an oxygenated physiological solution, wherein the oxygenated physiological solution is free of cardioplegia.
  • a vasodilator e.g., nitrates, nicorandil
  • an agent comprises a vasopressor (e.g., norepinephrine, epinephrine).
  • a method disclosed herein comprises perfusing the beating donor heart with an oxygenated physiological solution, wherein the oxygenated physiological solution is free from additional agents.
  • a method of explanting a donor heart while perfused and beating from a swine comprising maintaining the beating donor heart on a normothermic coronary perfusion circuit ex situ.
  • the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for a maximum of about 10 hours.
  • the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for about 30 minutes to about 1 hour, about 1 to about 1.5 hours, about 1.5 hours to about 2 hours, about 2 hours to about 2.5 hours, about 2.5 hours to about 3 hours, about 3 hours to about 3.5 hours, about 3.5 hours to about 4 hours, about 4 hours to about 4.5 hours, about 4.5 hours to about 5 hours, about 5 hours to about 5.5 hours, about 5.5 hours to about 6 hours, about 6 hours to about 6.5 hours, about 6.5 hours to about 7 hours, about 7 hours to about 7.5 hours, about 7.5 hours to about 8 hours, about 8 hours to about 8.5 hours, about 8.5 hours to about 9 hours, about 9 hours to about 9.5 hours, about 9.5 hours to about 10 hours, or more than about 10 hours.
  • the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours.
  • the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours.
  • the present disclosure provides a method of transplanting an explanted beating donor heart described in Section 5.2 into a subject in need thereof.
  • a subject NAI-1540201368v1 Attorney Docket No.14648-049-228 suitable for receiving a beating donor heart as disclosed herein is described in Section 5.4.4 below.
  • the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises placing the primate subject on cardiopulmonary bypass (CPB) and surgically removing the heart of the primate subject. Methods of surgically removing the heart from a primate subject in preparation for transplant are generally known in the art.
  • CPB cardiopulmonary bypass
  • the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises placing the donor heart on a normothermic coronary perfusion circuit and then explanting the donor heart while beating from the swine.
  • the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises implanting the beating explanted donor heart into the primate subject on CPB.
  • Methods of transplanting a porcine donor heart into a primate subject are generally known in the art.
  • the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises removing the primate subject from CPB after implantation of the beating explanted donor heart according to standard methods generally known in the art.
  • the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises (a) placing the primate subject on cardiopulmonary bypass (CPB) and surgically removing the heart of the primate subject; (b) placing the donor heart on a normothermic coronary perfusion circuit and then explanting the donor heart while beating from the swine; (c) implanting the beating NAI-1540201368v1 Attorney Docket No.14648-049-228 explanted donor heart into the primate subject on CPB; and (d) removing the primate subject from CPB.
  • CPB cardiopulmonary bypass
  • the beating donor heart remains perfused throughout the procedure (e.g., the method of transplanting the beating donor heart). In certain embodiments, the beating donor heart remains perfused for at least 95% of the procedure. In certain embodiments, the beating donor heart remains perfused for about 95% to 99%, 96% to 99%, 97% to 99%, or 98 to 99% of the procedure. [00111] In certain embodiments, the beating donor heart remains beating throughout the procedure (e.g., the method of transplanting the beating donor heart). In certain embodiments, the beating donor heart maintains a steady sinus rhythm rate throughout the procedure. In certain embodiments, the beating donor heart maintains a sinus rhythm rate ranging from about 50–180 beats per minute.
  • the beating donor heart maintains a sinus rhythm rate ranging from about 50-180, about 50-170, about 50-160, about 50-150, about 50-140, about 50- 130, about 50-120, about 50-110, about 50-100, about 60-180, about 60-170, about 60-160, about 60-150, about 60-140, about 60-130, about 60-120, about 60-110, about 60-100, about 70- 180, about 70-170, about 70-160, about 70-150, about 70-140, about 70-130, about 70-120, about 70-110, or about 70-100 beats per minute.
  • the beating donor heart maintains an average sinus rhythm rate of about, of at least about, or of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 beats per minute.
  • the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the transplanted donor heart is substantially free of ischemic tissue at the time of implanting the beating explanted donor heart into the primate subject on CPB.
  • the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the transplanted donor heart comprises less than about 1% to 20% ischemic tissue at the time of implanting the beating explanted donor heart into the primate subject on CPB. In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the transplanted donor heart comprises less than about 1% to 20%, 1% to 15%, 1% to 10% or 1% to 5% ischemic tissue at the time of implanting the beating explanted donor heart into the primate subject on CPB.
  • the NAI-1540201368v1 Attorney Docket No.14648-049-228 present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the transplanted donor heart comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% ischemic tissue at the time of implanting the beating explanted donor heart into the primate subject on CPB.
  • a donor heart recipient treated in accordance with the methods described herein undergoes additional treatment.
  • a donor heart recipient may undergo additional treatment by one or more different methods. Additional treatment may occur prior to, concurrently with, or subsequent to the method of treatment provided herein (e.g., transplantation of a donor heart).
  • Additional treatments are generally intended to improve the tolerance of the donor heart in recipients, but other treatments are contemplated.
  • a method of transplantation of a donor heart in accordance with the methods described herein can thus include administering one or more additional treatments, e.g., a treatment that inhibits T cells, blocks complement, or otherwise down regulates the recipient’s immune response to the donor heart.
  • a recipient is thymectomized and/or splenectomized.
  • a recipient receives radiation, for example, total body irradiation. In certain embodiments, a recipient receives 5-10 Gy or 10-15 Gy irradiation. In certain embodiments, thymic irradiation can be used.
  • the recipient is administered low dose radiation (e.g., a sub lethal dose of between 100 rads and 400 rads whole body radiation).
  • local thymic radiation is administered to the recipient.
  • the blood of a subject undergoing transplantation by a method described herein may contain antibodies that target the donor heart. Such antibodies can be eliminated by organ perfusion, and/or transplantation of tolerance-inducing bone marrow. Natural antibodies can be absorbed from the recipient’s blood by hemoperfusion of a liver of the donor species. Similarly, antibody-producing cells may be present in the recipient. Such antibody producing cells may be eliminated by, for example, irradiation or drug treatments.
  • the donor heart may be genetically-modified such that it is not recognized by antibodies present in the host (e.g., the donor heart is a-1,3-galactosyltransferase deficient) per Section 5.3.
  • the donor heart is a-1,3-galactosyltransferase deficient
  • a patient receiving a donor heart in accordance with the methods described herein can further receive immunosuppressive therapy.
  • the immunosuppressive therapy may be any FDA-approved treatment indicated to reduce transplant rejection and/or ameliorate the outcome of xenotransplantation.
  • Non-limiting examples of immunosuppressive therapy include calcineurin inhibitors (e.g., tacrolimus or cyclosporine), antiproliferative agents (e.g., anti-metabolites such a mycophenolate, 6-mercaptopurine or its prodrug azathioprine), inhibitors of mammalian target of rapamycin (mTOR) (e.g., sirolimus, rapamycin), steroids (e.g., prednisone), cell cycle inhibitors (azathioprine or mycophenolate mofetil), lymphocyte-depleting agents (e.g., anti-thymocyte globulin or antibodies such as alemtuzumab, siplizumab or basiliximab) and co-stimulation blockers (e.g., belatacept).
  • calcineurin inhibitors e.g., tacrolimus or cyclosporine
  • antiproliferative agents e.g., anti-metabolites such
  • immunosuppressive therapy can be administered as induction therapy (perioperative, or immediately after surgery), a maintenance dose, or for an acute rejection.
  • Induction therapy commonly includes basiliximab, anti-thymocyte globulin or alemtuzumab.
  • Immunosuppressive therapy can also be administered as maintenance therapy, which is often required to continue for the life of the recipient.
  • Maintenance immunosuppressive therapy commonly includes a calcineurin inhibitor (tacrolimus or cyclosporine), an antiproliferative agent (mycophenolate or azathioprine), and corticosteroids.
  • Immunosuppressive therapy for acute rejections commonly includes thymoglobulin or mycophenolate. See, e.g., Chung et al., Ann Transl Med (2020) Mar; 8: 409 and Benvenuto et al., J Thorac Dis (2016)10:3141-3155.
  • Non-limiting examples of immunosuppressants include, (1) antimetabolites, such as purine synthesis inhibitors (such as inosine monophosphate dehydrogenase (IMPDH) inhibitors, e.g., azathioprine, mycophenolate, and mycophenolate mofetil), pyrimidine synthesis inhibitors (e.g., leflunomide and teriflunomide), and antifolates (e.g., methotrexate); (2) calcineurin inhibitors, such as tacrolimus, cyclosporine A, pimecrolimus, and voclosporin; (3) TNF-alpha inhibitors, such as thalidomide and lenalidomide; (4) IL-1 receptor antagonists, such as anakinra; (5) mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin (sirolimus), deforolimus, everolimus, temsirolimus, zotarolimus,
  • Non-limiting exemplary cellular targets and their respective inhibitor compounds include, but are not limited to, complement component 5 (e.g., eculizumab); tumor necrosis factors (TNFs) (e.g., infliximab, adalimumab, certolizumab pegol, afelimomab and golimumab); IL-5 (e.g., mepolizumab ); IgE (e.g., omalizumab ); BAYX (e.g., nerelimomab ); interferon (e.g., faralimomab); IL-6 (e.g., elsilimomab); IL-12 and IL-13 (e.g., lebrikizumab and ustekinumab); CD3 (e.g., muromonab-CD3, otelixizumab, teplizumab, visilizumab); CD4 (e.
  • a method provided herein can comprise steps to induce tolerance in the recipient, e.g., by inducing mixed chimerism.
  • Mixed chimerism is commonly understood to describe a state in which the lymphohematopoietic system of the recipient of allogeneic hematopoietic stem cells comprises a mixture of host and donor cells. This state is usually attained through either bone marrow or mobilized peripheral blood stem cell transplantation.
  • Mixed chimerism can be transient or stable. See, e.g., Sachs et al., Cold Spring Harb Perspect Med (2014) 4:a015529; U.S. Patent No.6,296,846; and U.S. Patent No. 6,306,651.
  • a patient receiving a donor heart in accordance with the methods described herein can further receive a hematopoietic stem cell transplant.
  • Stem cell engraftment and hematopoiesis across disparate species barriers can be enhanced by providing a hematopoietic stromal environment from the donor species.
  • the stromal matrix supplies species-specific factors that are required for interactions between hematopoietic stem cells and their stromal environment, such as hematopoietic growth factors, adhesion molecules, and their ligands.
  • NAI-1540201368v1 Attorney Docket No.14648-049-228
  • fetal liver can also serve as an alternative to bone marrow as a source of hematopoietic stem cells.
  • fetal liver cells can be administered in fluid suspension.
  • the thymus is the major site of T cell maturation.
  • Each organ includes an organ specific stromal matrix that can support differentiation of the respective undifferentiated stem cells implanted into the host. Thymic stromal tissue can be irradiated prior to transplantation.
  • Porcine hematopoietic chimeras can lead to donor-specific nonresponsiveness in the mixed lymphocyte reaction, lack of anti-donor IgG antibody production, and acceptance of donor grafts. Accordingly, mixed chimerism is capable of inducing tolerance in a highly disparate xenogeneic combination and can have the clinical potential to prevent xenograft rejection. See, e.g., Griesemer et al., Immunol. Rev (2014) 258(1): 241-258; Sachs et al., Cold Spring Harb Perspect Med (2014) 4:a015529.
  • Bone marrow cells or another source of hematopoietic stem cells, e.g., a fetal liver suspension, of the donor can be injected into the recipient in order to induce mixed chimerism.
  • the hematopoietic stem cells may be taken from any source, for example from the bone marrow or peripheral blood stem cells. See, e.g., Sachs et al., Cold Spring Harb Perspect Med (2014) 4:a015529.
  • Donor BMC home to appropriate sites of the recipient and grow contiguously with remaining host cells and proliferate, forming a chimeric lymphohematopoietic population.
  • the present disclosure provides a method of treating a patient in need thereof with a donor heart described herein (see Section 5.2).
  • a patient treated with a donor heart described herein can be a donor heart recipient.
  • a patient treated in accordance with a method described herein e.g., the recipient of a donor heart
  • a primate subject e.g., a patient treated in accordance with a method described herein (e.g., the recipient of a donor heart) is a human patient.
  • a patient treated in accordance with a method described herein e.g., the NAI-1540201368v1 Attorney Docket No.14648-049-228 recipient of a donor heart
  • the terms “subject” and “patient” are used interchangeably and include any human or non-human mammal.
  • the subject is a primate.
  • the subject is a non-human primate (e.g., a baboon, a cynomolgus monkey or a rhesus macaque) and receives one or more grafts from a porcine donor.
  • the subject is human.
  • the subject is a human adult.
  • the subject is a human child.
  • the subject is a pediatric patient (e.g., 0–18 years old).
  • the subject is a geriatric patient (e.g., ⁇ 65 years old).
  • the subject is human and receives one or more donor grafts from a porcine donor. Methods of evaluating the effectiveness of treatment of a disease in the donor heart recipient is described in Section 5.4.5.
  • the subject is a human subject having a disease of the heart.
  • a patient treated in accordance with the methods described herein is in need of a heart transplant.
  • a patient may be in need of a heart transplant due to heart failure or the rejection of a donor heart.
  • Heart failure can have a number of causes, including but not limited to high blood pressure (hypertension), physical injury, diabetes, and autoimmune disorders.
  • a patient treated in accordance with the methods described herein has advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof.
  • Heart diseases and conditions can be diagnosed by laboratory tests (cholesterol, high- sensitivity C-reactive protein (hs-CRP), lipoprotein a (Lp(a)), plasma ceramides, natriuretic peptides, troponin), electrocardiogram (ECG), echocardiogram (ultrasound)), exercise stress test, nuclear cardiac stress test, coronary angiogram, magnetic resonance imaging (MRI), coronary computed tomography angiogram (CCTA), and/or a heart biopsy.
  • hs-CRP high- sensitivity C-reactive protein
  • Lp(a) lipoprotein a
  • ECG electrocardiogram
  • ECG echocardiogram
  • exercise stress test nuclear cardiac stress test
  • coronary angiogram magnetic resonance imaging (MRI), coronary computed tomography angiogram (CCTA
  • Classes and stages of heart failure classifies patients by measuring a patient’s overall heart function and severity of symptoms (see, e.g., Dolgin M et al., New York Heart Association Criteria Committee. NOMENCLATURE AND CRITERIA FOR DIAGNOSIS OF DISEASES OF THE HEART AND GREAT VESSELS.9th ed. Boston, MA: Lippincott Williams and Wilkins; March 1, 1994).
  • a patient treated in accordance with a method described herein has Stage C heart failure.
  • a patient treated in accordance with a method described herein has Stage D heart failure.
  • a patient treated in accordance with a NAI-1540201368v1 Attorney Docket No.14648-049-228 method described herein has a class I New York Heart Association (NYHA) Functional Classification.
  • a patient treated in accordance with a method described herein has a class II NYHA Functional Classification.
  • a patient treated in accordance with a method described herein has a class III NYHA Functional Classification.
  • a patient treated in accordance with a method described herein has a class IV NYHA Functional Classification.
  • a method of transplanting a beating donor heart according to the present disclosure prevents perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft.
  • a method of transplanting a beating donor heart according to the present disclosure reduces the occurrence of perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure.
  • a method of transplanting a beating donor heart reduces the occurrence of perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure by at least about 25%.
  • PCXD perioperative cardiac xenograft dysfunction
  • a method of transplanting a beating donor heart reduces the occurrence of perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure by about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 75% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99%.
  • PCXD perioperative cardiac xenograft dysfunction
  • a method of transplanting a beating donor heart reduces the occurrence of transplant rejection in a human subject in need of a cardiac xenograft compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure.
  • a method of transplanting a beating donor heart reduces the occurrence of transplant rejection in a human subject in need of a cardiac xenograft by about 10% to about NAI-1540201368v1 Attorney Docket No.14648-049-228 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 90% compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure.
  • a method of transplanting a beating donor heart according to the present disclosure reduces the occurrence of transplant rejection in a human subject in need of a cardiac xenograft by least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure.
  • a method of transplanting a beating donor heart according to the present disclosure results in a more viable transplant compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure.
  • a method of transplanting a beating donor heart results in a transplant comprising about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 90% more viability compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure.
  • a method of transplanting a beating donor heart according to the present disclosure results in a transplant comprising about at least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% more viability compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure.
  • a method of transplanting a beating donor heart according to the present disclosure results in a more functional transplant compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure.
  • Examples of methods for assessing heart transplant function include measuring left ventricle ejection fraction (LVEF), a right ventricular (RV) systolic function, a pulmonary artery (PA) pressure, a right atrial (RA) pressure, a sinus rhythm, and/or a cardiac output.
  • LVEF left ventricle ejection fraction
  • RV right ventricular
  • PA pulmonary artery
  • RA right atrial
  • a cardiac output a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having comparable cardiac function to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a left ventricle ejection fraction (LVEF) comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • LVEF left ventricle ejection fraction
  • a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having right ventricular (RV) systolic function comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • RV right ventricular
  • a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a pulmonary artery (PA) pressure comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a right atrial (RA) pressure comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • RA right atrial
  • a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a sinus rhythm comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a cardiac output comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • transplantation of a donor heart according to the present disclosure results in a better rate of survival of the donor heart recipient as compared to the rate of survival before transplantation of the donor heart to the patient.
  • transplantation of a donor heart according to the present disclosure improves the rate of survival of the donor heart recipient by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 90% compared to the rate of survival before transplantation of the donor heart to the patient.
  • transplantation of a donor heart according to the present disclosure improves the rate of survival of the donor heart recipient by at least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% compared to the rate of survival before transplantation of the donor heart to the patient.
  • transplantation of a donor heart according to the NAI-1540201368v1 Attorney Docket No.14648-049-228 present disclosure improves the rate of survival of the donor heart recipient by about 3 months to about 6 months, about 6 months to about 1 year, about 1 year to about 3 years, about 3 years to about 5 years, about 5 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, about 20 years to about 25 years, about 25 years to about 30 years, about 30 years to about 40 years, about 40 years to about 50 years, or about more than 50 years compared to the rate of survival before transplantation of the donor heart to the patient.
  • transplantation of a donor heart according to the present disclosure improves the rate of survival of the donor heart recipient by at least about or about 3 months, 6 months, 1 year, 3 years, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, 40 years, 50 years, or more than 50 years compared to the rate of survival before transplantation of the donor heart to the patient.
  • transplantation of a donor heart according to the present disclosure results in an improvement of the corresponding disease that the transplantation aims to intervene within the donor heart recipient as compared to the disease before transplantation of the donor heart to the patient.
  • transplantation of a donor heart according to the present disclosure results in a greater improvement of the corresponding disease that the transplantation aims to intervene within the donor heart recipient compared to that of a patient transplanted with a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure.
  • an improvement of the corresponding disease following transplantation of a donor heart according to the present disclosure is assessed according to the disease itself, severity of the disease, the health of the patient before and after transplant, and a myriad of other factors than can be disease and/or patient specific.
  • an improvement of the corresponding disease can include, but is not limited to, amelioration and/or ablation of at least one symptom associated with the disease, slowing progression of the disease, preventing further progression of the disease, reversing disease progression, and/or returning the donor heart recipient to a healthy status (e.g., comparable to a healthy, disease-free patient of comparable age, gender, and background).
  • transplantation of a donor heart according to the present disclosure results in an improvement of the corresponding disease that the transplantation aims to intervene within the donor heart recipient as indicated by at least one biomarker corresponding to the disease.
  • transplantation of a donor heart results in an improvement of heart disease and/or at least one symptom associated with heart disease within the donor heart recipient as compared to before transplantation of the donor heart to the patient.
  • transplantation of a donor heart results in at least about or about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than 90% improvement of heart disease and/or at least one symptom associated with heart disease within the donor heart recipient as compared to before transplantation of the donor heart to the patient.
  • EXAMPLES [00137] The following is a description of various methods and materials used in the studies.
  • Perioperative cardiac xenograft dysfunction is a major limitation to long-term xenograft survival and is attributed to ischemia during the heart procurement and transplant. Described herein is an exemplary method of explanting and transplanting perfused, beating donor hearts without any ischemia in a pig-to-baboon model. 6.1.1 Methods [00139] An ⁇ -1,3 galactosyltransferase knock-out (GalT-KO) pig weighing 16 kilograms (kg), was selected as the donor for a 24 kg male baboon. First, the baboon was placed on cardiopulmonary bypass (CPB) and the native heart was explanted.
  • CPB cardiopulmonary bypass
  • the donor pig heart was then placed on a normothermic coronary perfusion circuit (FIG.1) with drainage cannulas in the NAI-1540201368v1 Attorney Docket No.14648-049-228 right atrium and the left ventricle and an outflow cannula in the proximal ascending aorta.
  • the superior vena cava (SVC) and the inferior vena cava (IVC) were ligated, the aorta was clamped, and then the heart was explanted while beating (FIG.2).
  • the pig heart was implanted into the baboon, and hemodynamic, EKG, arterial blood gas (ABG), and echocardiography data was collected for 60 minutes post-CPB separation.
  • RV right ventricular
  • LV left ventricular
  • MR trivial mitral regurgitation
  • FOG.3 mild tricuspid regurgitation
  • FIG.4 shows no evidence of acute T cell or antibody-mediated rejection, ischemic injury, or edema (FIG.4).
  • Echocardiogram data taken on post-transplant Day 8 demonstrated excellent cardiac function.
  • FIG.5A shows left ventricle short axis systole and
  • FIG.5B shows left ventricle short axis diastole.
  • the left ventricle ejection fraction calculated from the echocardiogram was ⁇ 55%.
  • Echocardiogram data taken on post-transplant Day 14 demonstrated similar cardiac function (data not shown). 6.1.3 Conclusions [00142] Eliminating ischemia in cardiac xenografts was not previously achieved before the exemplary methods described herein.
  • the present disclosure provides the following non-limiting embodiments: 1. A method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises: a. placing the primate subject on cardiopulmonary bypass (CPB) and surgically removing the heart of the primate subject; b.
  • CPB cardiopulmonary bypass
  • any one of embodiments 1 to 3 wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step b for a maximum of about 10 hours, optionally wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step b for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours. 5.
  • the method of any one of embodiments 1 to 4 wherein the transplanted donor heart is substantially free of ischemic tissue at the time of the implanting step. 6.
  • LVEF left ventricle ejection fraction
  • RV right ventricular
  • PA pulmonary artery
  • RA right atrial
  • sinus rhythm a cardiac output comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject.
  • the normothermic coronary perfusion circuit is comprised of at least two drainage cannulas and at least one outflow cannula.
  • step b further comprises ligating the superior vena cava (SVC) and the inferior vena cava (IVC), and clamping the aorta distal to the outflow cannula before explanting the beating donor heart.
  • a method of explanting a donor heart while perfused and beating from a swine comprises: a. placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by: i. placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart via the apex or across the left atrium; ii. placing an outflow cannula in the proximal ascending aorta of the donor heart; iii. ligating the superior vena cava (SVC) and the inferior vena cava (IVC) of the donor heart; iv.
  • SVC superior vena cava
  • IVC inferior vena cava
  • the oxygenated physiological solution comprises blood from the swine that sourced the donor heart.
  • the genetically-modified minipig comprises a GGTA1 genetic modification.
  • the genetically-modified minipig does not express alpha-1,3-galactosyltransferase (GalT-KO).
  • the genetically-modified minipig is a GalT-KO minipig and: a. is wild type for a protein encoded by the porcine genes CMAH, ⁇ 4GalNT, ASGR1, and CD46; and/or b. does not express any recombinant proteins.
  • 25. The method of any one of the preceding embodiments, wherein the primate subject is a human subject.
  • 26. The method of embodiment 25, wherein the human subject has advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof.
  • a medical instrument comprising a. at least two drainage cannulas and at least one outflow cannula, wherein one drainage cannula is suitable to be placed in the right atrium of a donor heart and another drainage cannula is suitable to be placed in the left ventricle of the donor heart; b. an outflow cannula, wherein the outflow cannula is suitable to be placed in the proximal ascending aorta of the donor heart; and c.
  • a pump to maintain homeostasis with a perfusion solution; wherein the medical instrument is specifically adapted to perform normothermic machine perfusion on a heart from a minipig.
  • 44. A method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises transplanting a donor heart into the subject according to the methods of any one of embodiments 1 to 28.
  • 45. A method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises use of the medical instrument of embodiments 29 to 33. 46.
  • a method of treating a disease of the heart in a human subject in need thereof, wherein the NAI-1540201368v1 Attorney Docket No.14648-049-228 method comprises transplanting the explanted donor heart from a minipig of embodiments 34 to 43 into the subject.
  • a method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft wherein the method comprises transplanting a donor heart into the subject according to the methods of any one of embodiments 1 to 28.
  • a method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft wherein the method comprises use of the medical instrument of embodiments 29 to 33.
  • a method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft wherein the method comprises transplanting the explanted donor heart from a minipig of embodiments 34 to 43 into the subject.

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Abstract

The present disclosure provides beating explanted donor hearts from swine, methods of making thereof, and methods of using thereof. The present disclosure further provides methods of treating a disease (e.g., a heart disease) in a human subject in need thereof comprising transplanting of a beating explanted donor heart from swine. The present disclosure also provides medical instruments suitable for transplanting a beating explanted donor heart from swine.

Description

Attorney Docket No.14648-049-228 NON-ISCHEMIC BEATING HEART XENOTRANSPLANTATION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to United States Provisional Application No. 63/654,727 filed May 31, 2024, the content of which is incorporated by reference in its entirety herein, and to which priority is claimed. 1. FIELD [0002] The present disclosure provides beating explanted donor hearts from swine, methods of making thereof, and methods of using thereof. The present disclosure further provides methods of treating a disease (e.g., a heart disease) in a human subject in need thereof comprising transplanting a beating explanted donor heart from swine. 2. BACKGROUND [0003] Cardiac xenotransplantation is a rapidly developing area of interest as there is an ongoing and unmet need for donor hearts for patients with heart disease. Use of donor hearts from swine as viable xenografts is hampered by the ischemia that develops in the donor tissue using traditional methods of cold organ storage and cardioplegia during heart transplant surgery. Ischemia in donor heart tissue correlates with perioperative cardiac xenograft dysfunction (PCXD). Using traditional methods of cardiac xenotransplantation not only limits the time frame for conducting the transplantation procedure but also reduces the distance that donor hearts can be transported. As such, there is a need in the art to develop improved methods for obtaining explanted donor hearts comprising substantially no ischemic tissue for use in cardiac xenotransplantation. 3. SUMMARY [0004] In an aspect, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises: (a) placing the primate subject on cardiopulmonary bypass (CPB) and surgically removing the heart of the primate subject; (b) placing the donor heart on a normothermic coronary perfusion circuit and then explanting the donor heart while beating from the swine; (c) implanting the beating explanted donor heart into the primate subject on CPB; and (d) removing the primate subject from CPB. NAI-1540201368v1 Attorney Docket No.14648-049-228 [0005] In certain embodiments, the donor heart remains perfused and beating throughout the entire procedure. [0006] In certain embodiments, the beating explanted donor heart comprises substantially no ischemic tissue at the time of implantation into the primate subject on CPB. [0007] In certain embodiments, the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step (b) for a maximum of about 10 hours, optionally wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step (b) for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours. [0008] In certain embodiments, the transplanted donor heart is substantially free of ischemic tissue at the time of the implanting step. [0009] In certain embodiments, the transplanted donor heart has a left ventricle ejection fraction (LVEF), a right ventricular (RV) systolic function, a pulmonary artery (PA) pressure, a right atrial (RA) pressure, a sinus rhythm, and/or a cardiac output comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject. [0010] In certain embodiments, the normothermic coronary perfusion circuit is comprised of at least two drainage cannulas and at least one outflow cannula. In certain embodiments, one drainage cannula is placed in the right atrium and another drainage cannula is placed in the left ventricle via the apex or across the left atrium. In certain embodiments, the outflow cannula is placed in the proximal ascending aorta. [0011] In certain embodiments, step (b) further comprises ligating the superior vena cava (SVC) and the inferior vena cava (IVC) and clamping the aorta distal to the outflow cannula before explanting the beating donor heart. [0012] In another aspect, the present disclosure provides a method of explanting a donor heart while perfused and beating from a swine, wherein the method comprises: (a) placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by: (i) placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart via the apex or across the left atrium; (ii) placing an outflow cannula in the proximal ascending aorta of the donor heart; (iii) ligating the superior vena cava (SVC) NAI-1540201368v1 Attorney Docket No.14648-049-228 and the inferior vena cava (IVC) of the donor heart; (iv) clamping the aorta of the donor heart; (b) explanting the donor heart while beating from the swine; (c) maintaining the beating donor heart on the normothermic coronary perfusion circuit ex situ. [0013] In certain embodiments, the method further comprises priming the normothermic coronary perfusion circuit with plasmalyte A before placing the donor heart on the normothermic coronary perfusion circuit. [0014] In certain embodiments, the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for a maximum of about 10 hours, optionally wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step (c) for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours. [0015] In certain embodiments, the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ in an organ reservoir. In certain embodiments, the organ reservoir is maintained at a temperature ranging from about 32°C to about 37°C. [0016] In certain embodiments, the beating donor heart maintained on the normothermic coronary perfusion circuit ex situ in an organ reservoir is placed in a sterile container suitable for transportation. [0017] In certain embodiments, the beating donor heart is perfused with an oxygenated physiological solution. In certain embodiments, the oxygenated physiological solution comprises blood from the swine that is sourced from the donor heart. [0018] In certain embodiments, the swine is a genetically-modified minipig. In certain embodiments, the genetically-modified minipig comprises a GGTA1 genetic modification. In certain embodiments, the genetically-modified minipig does not express alpha-1,3- galactosyltransferase (GalT-KO). [0019] In certain embodiments, the genetically-modified minipig is a GalT-KO minipig and: (a) is wild type for a protein encoded by the porcine genes CMAH, β4GalNT, ASGR1, and CD46; and/or (b) does not express any recombinant proteins. In certain embodiments, the genetically- modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens. In certain embodiments, the genetically-modified minipig is GalT-KO and the GalT-KO is the NAI-1540201368v1 Attorney Docket No.14648-049-228 only genetic modification in the minipig. [0020] In certain embodiments, the primate subject is a human subject. In certain embodiments, the human subject has advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof. In certain embodiments, the human subject is a pediatric human subject. [0021] In certain embodiments, the method is performed in the absence of cardioplegia. [0022] In another aspect, the present disclosure provides a medical instrument comprising (a) at least two drainage cannulas and at least one outflow cannula, wherein one drainage cannula is suitable to be placed in the right atrium of a donor heart and another drainage cannula is suitable to be placed in the left ventricle of the donor heart; (b) an outflow cannula, wherein the outflow cannula is suitable to be placed in the proximal ascending aorta of the donor heart; and (c) a pump to maintain homeostasis with a perfusion solution; wherein the medical instrument is specifically adapted to perform normothermic machine perfusion on a heart from a minipig. [0023] In certain embodiments, the medical instrument further comprises an organ reservoir. [0024] In certain embodiments, the medical instrument further comprises a membrane oxygenator, a leukocyte filter, an arterial filter, a heater-cooler unit, an autonomous power unit, or any combination thereof. [0025] In certain embodiments, the medical instrument further comprises a sterile container, wherein the sterile container is suitable for transportation of a donor heart that is perfused and beating during transport. [0026] In certain embodiments, the medical instrument further comprises a collection chamber, wherein the collection chamber is suitable for collecting lost blood during transportation of a donor heart. [0027] In another aspect, the present disclosure provides an explanted donor heart from a minipig, wherein the donor heart is connected to an ex situ normothermic coronary perfusion circuit prior to transplantation into a subject in need thereof. [0028] In certain embodiments, the minipig is a genetically-modified minipig. In certain embodiments, the genetically-modified minipig comprises a GGTA1 genetic modification. In certain embodiments, the genetically-modified minipig does not express alpha-1,3- galactosyltransferase (GalT-KO). [0029] In certain embodiments, the genetically-modified minipig is a GalT-KO minipig and: NAI-1540201368v1 Attorney Docket No.14648-049-228 (a) is wild type for a protein encoded by the porcine genes CMAH, β4GalNT, ASGR1, and CD46; and/or (b) does not express any recombinant proteins. [0030] In certain embodiments, the genetically-modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens. [0031] In certain embodiments, the genetically-modified minipig is GalT-KO and the GalT- KO is the only genetic modification in the minipig. [0032] In certain embodiments, the normothermic coronary perfusion circuit is first established in situ by: (a) placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart; and (b) placing an outflow cannula in the proximal ascending aorta of the donor heart. [0033] In certain embodiments, after the normothermic coronary perfusion circuit is established in situ, (a) the superior vena cava (SVC) and the inferior vena cava (IVC) of the donor heart are ligated; (b) the aorta of the donor heart is clamped; (c) the donor heart is explanted while beating from the swine; and (d) the beating explanted donor heart is maintained on the normothermic coronary perfusion circuit ex situ. [0034] In certain embodiments, the explanted donor heart comprises substantially no ischemic tissue within at least 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or at least 12 hours. [0035] In certain embodiments, the present disclosure provides a method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises transplanting a donor heart into the subject according to the methods of the present disclosure. [0036] In certain embodiments, the present disclosure provides a method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises use of the medical instrument of the present disclosure. [0037] In certain embodiments, the present disclosure provides a method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises transplanting the explanted donor heart from a minipig of the present disclosure into the subject. [0038] In certain embodiments, the present disclosure provides a method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises transplanting a donor heart into the subject according NAI-1540201368v1 Attorney Docket No.14648-049-228 to the methods of the present disclosure. [0039] In certain embodiments, the present disclosure provides a method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises use of the medical instrument of the present disclosure. [0040] In certain embodiments, the present disclosure provides a method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises transplanting the explanted donor heart from a minipig of the present disclosure into the subject. [0041] In certain embodiments, the human subject has at least one condition that can be treated by receiving a heart transplantation. In certain embodiments, the at least one condition that can be treated by receiving a heart transplantation comprises advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof. In certain embodiments, the human subject is a pediatric human subject. 4. BRIEF DESCRIPTION OF THE FIGURES [0042] FIG.1 depicts a schematic of the coronary perfusion system. The grey arrows indicate drainage cannulas inserted into RA and the LV of the heart. The black arrow indicates an outflow cannula placed in the proximal ascending aorta. The striped circle indicates a pump. RA = right atrium; LA = left atrium; RV = right ventricle; LV = left ventricle. [0043] FIG.2 depicts a representative image of a porcine donor heart maintained on a normothermic coronary perfusion circuit ex situ. [0044] FIG.3 depicts representative images of echocardiography performed in a baboon donor heart recipient after weaning from cardiopulmonary bypass (CPB). The top left image shows echocardiographic assessment of left ventricular (LV) systolic function. The bottom left image shows echocardiographic assessment of LV diastole function. The top right image shows echocardiographic assessment of mitral valve (MV) function. The bottom right image shows echocardiographic assessment of tricuspid valve (TV) function. [0045] FIG.4 depicts a representative image of histopathology of the myocardium of the donor heart harvested from a baboon after the donor heart was transplanted. The image shows a hematoxylin and eosin (H&E) stained section of the tissue at 400x magnification. [0046] FIGS.5A-5B depict representative images of echocardiogram data collected on post- NAI-1540201368v1 Attorney Docket No.14648-049-228 transplant Day 8 showing cardiac function. FIG.5A shows left ventricle short axis systole and FIG.5B shows left ventricle short axis diastole. 5. DETAILED DESCRIPTION [0047] Provided herein is an explanted beating donor heart from swine, methods of preparing thereof, and methods using thereof. Also provided herein are medical instruments specifically adapted for coronary perfusion of the explanted beating donor heart from swine as disclosed herein, and methods using thereof. [0048] In certain embodiments, provided herein is an explanted beating donor heart from swine (see Section 5.2). A swine that provides the beating donor heart disclosed herein is described in Section 5.3 below. [0049] In certain embodiments, provided herein is a medical instrument specifically adapted for coronary perfusion during cardiac xenotransplantation (see Section 5.1). The medical device disclosed herein is specifically adapted for coronary perfusion of the explanted beating donor heart from swine as described in Section 5.2 below, and for use in methods of explanting a beating donor heart (see Section 5.4.1) and transplanting a beating donor heart (see Section 5.4.2). [0050] In certain embodiments, provided herein is a method of using an explanted beating donor heart from swine (see Section 5.4). Methods of explanting a beating donor heart are disclosed in Section 5.4.1 below. An explanted beating donor heart from swine can be transplanted into a subject in need thereof according to the methods described in Section 5.4.2 below. A subject suitable for receiving an explanted beating donor heart disclosed herein is described in Section 5.4.4 below. Such a subject in need of an explanted beating donor heart of the present disclosure can have, be suspected of having, or demonstrate at least one symptom of a disease described in Section 5.4.5 below. 5.1 Coronary Perfusion Systems [0051] In certain embodiments, the present disclosure provides a medical instrument specifically adapted for coronary perfusion during cardiac xenotransplantation. In certain embodiments, the present disclosure provides a medical instrument specifically adapted to perform normothermic machine perfusion during cardiac xenotransplantation. In certain embodiments, the present disclosure provides a medical instrument specifically adapted to perform normothermic machine perfusion of a donor heart from a porcine donor. In certain NAI-1540201368v1 Attorney Docket No.14648-049-228 embodiments, medical instruments of the present disclosure are specifically adapted for use with any porcine donor described in Section 5.3 and/or any donor heart described in Section 5.2. [0052] In certain embodiments, medical instruments of the present disclosure can be used for normothermic machine perfusion of a donor heart according to the methods described herein (see Section 5.4). Normothermic machine perfusion is generally used to recreate the physiological environment of a donor organ by maintaining normal temperature and providing the essential substrates needed to support organ viability prior to transplantation. In certain embodiments, a medical instrument disclosed herein is a normothermic ex-vivo perfusion system specifically adapted for use in cardiac xenotransplantation. For example, differences in pig anatomy compared with human anatomy are known in the art (see, e.g., Shah A. et al., JACC Case Rep. (2022) Jul 7;4(16):1049-1052). In certain embodiments, a medical instrument disclosed herein is a normothermic ex-vivo perfusion system specifically adapted for use with a porcine donor described in Section 5.3 herein. In certain embodiments, a medical instrument disclosed herein is a normothermic ex-vivo perfusion system specifically adapted for use with a minipig donor. In certain embodiments, a medical instrument disclosed herein is a normothermic ex-vivo perfusion system suitable for circulating an oxygenated physiological solution through a donor heart explanted from a minipig donor. [0053] In certain embodiments, a medical instrument disclosed herein comprises at least one cannula. As used herein, a “cannula” refers to a small tube that can be inserted into a body cavity, duct, or vessel. One of skill in the art would understand that the opposite end of a cannula that is not inserted into a cavity, duct, or vessel can be connected to a tube, device, or path of flow (e.g., a perfusion system). A cannula that is suitable for use with a medical instrument disclosed herein can be made of any biomaterial acceptable for medical use. In certain embodiments, a cannula suitable for a medical instrument disclosed herein can be made of polyvinylchloride (PVC). In certain embodiments, a cannula suitable for a medical instrument disclosed herein can be wire reinforced to prevent obstruction due to kinking. [0054] In certain embodiments, a medical instrument disclosed herein comprises at least two drainage cannulas. As used herein, a “drainage cannula” refers to a cannula for drainage of venous blood. In certain embodiments, a medical instrument disclosed herein comprises at least two drainage cannulas, wherein one drainage cannula is suitable to be placed in the right atrium of a donor heart as described herein (e.g., Section 5.3). In certain embodiments, a medical NAI-1540201368v1 Attorney Docket No.14648-049-228 instrument disclosed herein comprises at least two drainage cannulas, wherein one drainage cannula is suitable to be placed in the left ventricle of a donor heart as described herein (e.g., Section 5.3). In certain embodiments, a medical instrument disclosed herein comprises at least two drainage cannulas, wherein one drainage cannula is suitable to be placed in the right atrium of a donor heart and another drainage cannula is suitable to be placed in the left ventricle of the donor heart. [0055] In certain embodiments, a medical instrument disclosed herein comprises at least one outflow cannula. One of skill in the art would understand that, as applied to heart surgery, the terms “inflow” and “outflow” refer to the direction of the flow relative to the device/pump rather than the heart. As used herein, an “outflow cannula” refers to a cannula for perfusing a donor heart as described herein (e.g., Section 5.3) with a perfusion solution. In certain embodiments, a medical instrument disclosed herein comprises an outflow cannula, wherein the outflow cannula is suitable to be placed in the proximal ascending aorta of the donor heart. [0056] In certain embodiments, a medical instrument disclosed herein comprises at least one pump to maintain homeostasis with a perfusion solution. In certain embodiments, a medical instrument disclosed herein comprises at least one perfusion pump. A perfusion pump as used herein is suitable for the fluid transfer of the perfusate through the various components of the perfusion system as described herein and through the donor heart. Non-limiting examples of types of perfusion pumps that may be used include axial flow pumps, peristaltic pumps, diaphragm pumps, pumping cassettes, roller pumps, centrifugal pumps, and pulsatile pumps. [0057] In certain embodiments, a medical instrument disclosed herein further comprises an organ reservoir. An organ reservoir is a container suitable for maintaining a donor heart on a normothermic coronary perfusion circuit ex situ in a sterile state. In certain embodiments, an organ reservoir suitable for use in the medical instrument disclosed herein comprises an outlet so that the blood, perfused through, and out of, the organ, which collects in the organ reservoir can be collected for later reoxygenation, recirculation, and/or reperfusion. In certain embodiments, the inside of an organ reservoir suitable for use herein comprises an organ- supporting means which are well-known in the art, such as a soft mesh floor, a sling or “hammock,” a tilted or non-tilted “V-shaped or “U”-shaped platform, or may contain gauze or other sterile protective material inserted by the surgeon, to provide support to the donor heart and to prevent movement of the donor heart during transport. NAI-1540201368v1 Attorney Docket No.14648-049-228 [0058] In certain embodiments, a medical instrument disclosed herein further comprises a sterile container. In certain embodiments, a sterile container suitable for use in the medical instrument disclosed herein is suitable for transportation of a donor heart that is perfused and beating during transport. In certain embodiments, a sterile container suitable for use in the medical instrument disclosed herein is made of a rigid material suitable for protecting the donor heart from physical contact, direct or indirect. In certain embodiments, a sterile container suitable for use in the medical instrument disclosed herein is a box-shape having a hinged lid. In certain embodiments, a sterile container suitable for use in the medical instrument disclosed herein comprises at least one aperture for monitoring the donor heart during transport. In certain embodiments, a sterile container suitable for use in the medical instrument disclosed herein comprises an outlet suitable for later reoxygenation, recirculation, and/or reperfusion of blood and/or perfusion solution collected during transportation of a donor heart. In certain embodiments, a sterile container suitable for use in the medical instrument disclosed herein comprises a means for supporting the organ reservoir as described herein. [0059] In certain embodiments, a medical instrument disclosed herein further comprises a collection chamber container. In certain embodiments, a collection chamber container suitable for use in the medical instrument disclosed herein is suitable for collecting lost blood and/or perfusion solution during transportation of a donor heart. In certain embodiments, a collection chamber container suitable for use in the medical instrument disclosed herein comprises an outlet suitable for later reoxygenation, recirculation, and/or reperfusion of blood and/or perfusion solution collected during transportation of a donor heart. [0060] In certain embodiments, a medical instrument disclosed herein further comprises a membrane oxygenator. A membrane oxygenator as used herein is a device for adding oxygen to and removing carbon dioxide from the blood. Membrane oxygenators are known in the art, and a variety are commercially available. In certain embodiments, a membrane oxygenator suitable for use in the medical instrument disclosed herein comprises a semipermeable polymer membrane. Non-limiting examples of suitable polymers include polypropylene, polyethylene, poly-4-methylpentene (PMP), polyvinylidene fluoride (PVDF), and polytetrafluoroethylene (PTFE). [0061] In certain embodiments, a medical instrument disclosed herein further comprises a leukocyte filter. Leukodepletion occurs through a specific filter (i.e., a “leukocyte filter”) to NAI-1540201368v1 Attorney Docket No.14648-049-228 remove leukocytes from blood and/or hemocomponents. Leukocyte filters suitable for use in the medical instrument disclosed herein can include those known in the art (see., e.g., Urias EVR et al., Rev Assoc Med Bras (2021) Jul;67(7):1056-1060). [0062] In certain embodiments, a medical instrument disclosed herein further comprises an arterial filter. An arterial filter for use herein filters out various material with embolic potential such as, for example, micro air, particulates, and/or aggregates composed of platelets, red blood cells and other debris. In certain embodiments, arterial filter suitable for use in the medical instrument disclosed herein comprises a pore size ranging from 25 µm to about 45 µm (e.g., about 25 µm, 30 µm, 35 µm, 40 µm, 45 µm). An arterial filter suitable for use in the medical instrument disclosed herein can include those known in the art (see., e.g., Hawkins JL et al., J Extra Corpor Technol (2010) Mar;42(1):71-4). [0063] In certain embodiments, a medical instrument disclosed herein further comprises a heater-cooler unit. In certain embodiments, a heater-cooler unit suitable for use in the medical instrument disclosed herein comprises a heat exchanger. In certain embodiments, a heater-cooler unit is suitable for maintaining the perfusion solution, the donor heart, or both at physiological temperature. In certain embodiments, a heater-cooler unit is suitable for maintaining the perfusion solution, the donor heart, or both at a temperature ranging from about 32°C to about 37°C (e.g., about 32°C, 33°C, 34°C, 35°C, 36°C, 37°C). [0064] In certain embodiments, a medical instrument disclosed herein further comprises an autonomous power unit. In certain embodiments, a medical instrument disclosed herein further comprises a portable autonomous power unit adapted to power the medical instrument. 5.2 Explanted Donor Hearts [0065] In certain embodiments, the present disclosure provides an explanted donor heart for coronary perfusion during cardiac xenotransplantation. In certain embodiments, the explanted donor heart as described in this section can be used with any one of the medical instruments disclosed herein (e.g., Section 5.1). In certain embodiments, the explanted donor heart can be used in any one of the methods of transplantation as described in Section 5.4.2. [0066] In certain embodiments, the explanted donor heart is from a porcine donor described in Section 5.3. In certain embodiments, the explanted donor heart is from a minipig. In certain embodiments, the explanted donor heart is from a genetically-modified minipig. In certain embodiments, the explanted donor heart is from a genetically-modified minipig wherein the NAI-1540201368v1 Attorney Docket No.14648-049-228 genetically-modified minipig comprises a GGTA1 genetic modification. In certain embodiments, the explanted donor heart is from a genetically-modified minipig wherein the genetically- modified minipig does not express alpha-1,3-galactosyltransferase (GalT-KO). [0067] In certain embodiments, the explanted donor heart is from a genetically-modified minipig wherein the genetically-modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens. In certain embodiments, the explanted donor heart is from a genetically-modified minipig wherein the genetically-modified minipig is GalT-KO and the GalT-KO is the only genetic modification in the minipig. [0068] In certain embodiments, the explanted donor heart is from a GalT-KO minipig. In certain embodiments, the explanted donor heart is from a GalT-KO minipig that is otherwise wildtype for other proteins, protein expression levels, or both. In certain embodiments, the explanted donor heart is from a GalT-KO minipig that is wild type for a protein encoded by the porcine genes CMAH, β4GalNT, ASGR1, and/or CD46. In certain embodiments, the explanted donor heart is from a GalT-KO minipig that does not overexpress any recombinant proteins. In certain embodiments, the explanted donor heart is from a GalT-KO minipig that does not overexpress any recombinant human proteins. In certain embodiments, the explanted donor heart is from a GalT-KO minipig that does not overexpress any recombinant proteins encoded by a human gene. In certain embodiments, the explanted donor heart is from a GalT-KO minipig that does not overexpress any recombinant proteins encoded by the human genes A20/TNFAIP3, CD39, CD46, CD47, CD55, CD59, ENTPD1, EPCR, HO-1, SERPING1, TBM, TFPI, VWF, HLA-E, and/or HLA-G. [0069] In certain embodiments, the explanted donor heart is connected to a normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart is connected to a normothermic coronary perfusion circuit in situ. In certain embodiments, the normothermic coronary perfusion circuit is first established in situ by (a) placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart; and (b) placing an outflow cannula in the proximal ascending aorta of the donor heart. [0070] In certain embodiments, the explanted donor heart is connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart is connected to an ex situ normothermic coronary perfusion circuit after the normothermic coronary NAI-1540201368v1 Attorney Docket No.14648-049-228 perfusion circuit is established in situ. In certain embodiments, after the normothermic coronary perfusion circuit is established in situ, (a) the superior vena cava (SVC) and the inferior vena cava (IVC) of the donor heart are ligated; (b) the aorta of the donor heart is clamped; (c) the donor heart is explanted while beating from the swine; and (d) the beating explanted donor heart is maintained on a normothermic coronary perfusion circuit ex situ. [0071] In certain embodiments, the explanted donor heart of the present disclosure comprises a beating heart connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart of the present disclosure comprises a beating heart connected to an ex situ normothermic coronary perfusion circuit with a sinus rhythm rate ranging from about 50–180 beats per minute. In certain embodiments, the explanted donor heart of the present disclosure comprises a beating heart connected to an ex situ normothermic coronary perfusion circuit with a sinus rhythm rate ranging from about 50-180, about 50-170, about 50- 160, about 50-150, about 50-140, about 50-130, about 50-120, about 50-110, about 50-100, about 60-180, about 60-170, about 60-160, about 60-150, about 60-140, about 60-130, about 60- 120, about 60-110, about 60-100, about 70-180, about 70-170, about 70-160, about 70-150, about 70-140, about 70-130, about 70-120, about 70-110, or about 70-100 beats per minute. In certain embodiments, the explanted donor heart of the present disclosure comprises a beating heart connected to an ex situ normothermic coronary perfusion circuit with an average sinus rhythm rate of at least about or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 beats per minute. [0072] In certain embodiments, the explanted donor heart of the present disclosure comprises substantially no ischemic tissue. In certain embodiments, the explanted donor heart of the present disclosure comprises substantially no ischemic tissue within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart of the present disclosure comprises less than about 1% to 20% ischemic tissue within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart of the present disclosure comprises less than about 1% to 20%, 1% to 15%, 1% to NAI-1540201368v1 Attorney Docket No.14648-049-228 10% or 1% to 5% ischemic tissue within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart of the present disclosure comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% ischemic tissue within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. [0073] In certain embodiments, the explanted donor heart of the present disclosure comprises substantially no cardiac edema. In certain embodiments, the explanted donor heart of the present disclosure comprises substantially no cardiac edema within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart of the present disclosure comprises less than about 1% to 20% cardiac edema within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart of the present disclosure comprises less than about 1% to 20%, 1% to 15%, 1% to 10% or 1% to 5% cardiac edema within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. In certain embodiments, the explanted donor heart of the present disclosure comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% cardiac edema within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. [0074] In certain embodiments, the explanted donor heart of the present disclosure comprises substantially no evidence of acute T cell rejection. One of skill in the art will appreciate that NAI-1540201368v1 Attorney Docket No.14648-049-228 evidence of acute T cell rejection is demonstrated by a host T-lymphocyte-mediated response mounted against the allograft tissue. In certain embodiments, the explanted donor heart of the present disclosure comprises substantially no evidence of acute T cell rejection within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. [0075] In certain embodiments, the explanted donor heart of the present disclosure comprises substantially no evidence of antibody-mediated rejection. One of skill in the art will appreciate that evidence of antibody-mediated rejection is demonstrated by allograft injury resulting from activation of the complement system, typically by recipient-generated antibodies directed against the allograft tissue. In certain embodiments, the explanted donor heart of the present disclosure comprises substantially no evidence of antibody-mediated rejection within at least about 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or 12 hours of being connected to an ex situ normothermic coronary perfusion circuit. [0076] In certain embodiments, an explanted donor heart disclosed herein comprises at least one gene modification. In certain embodiments, an explanted donor heart disclosed herein comprises the gene modification of the porcine donor. In certain embodiments, an explanted donor heart disclosed herein comprises one gene modification in the gene encoding alpha-1,3- galactosyltransferase. In certain embodiments, an explanted donor heart disclosed herein does not express alpha-1,3-galactosyltransferase (i.e., GalT-KO). 5.3 Porcine Donors [0077] In certain embodiments, a donor heart provided herein comprises a heart harvested from a swine. The term “porcine” is used interchangeably herein with the terms “pig” and “swine” and refers to mammals in the family Suidae. Non-limiting examples of the breeds of swine suitable for use herein includes any of the following: American Landrace, American Yorkshire, Aksai Black Pied, Angeln saddleback, Appalachian English, Arapawa Island, Auckland Island, Australian Yorkshire, Babi Kampung, Ba Xuyen, Bantu, Basque, Bazna, Beijing Black, Belarus Black Pied, Belgian Landrace, Bengali Brown Shannaj, Bentheim Black Pied, Berkshire, Bisaro, Bangur, Black Slavonian, Black Canarian, Breitovo, British Landrace, British Lop, British Saddleback, Bulgarian White, Cambrough, Cantonese, Celtic, Chato NAI-1540201368v1 Attorney Docket No.14648-049-228 Murciano, Chester White, Chiangmai Blackpig, Choctaw Hog, Creole, Czech Improved White, Danish Landrace, Danish Protest, Dermantsi Pied, Li Yan, Duroc, Dutch Landrace, East Landrace, East Balkan, Essex, Estonian Bacon, Fengjing, Finnish Landrace, Forest Mountain, French Landrace, Gascon, German Landrace, Gloucestershire Old Spots, Gottingen minipig, Grice, Guinea Hog, Hampshire, Hante, Hereford, Hezuo, Hogan Hog, Huntington Black Hog, Iberian, Italian Landrace, Japanese Landrace, Jeju Black, Jinhua, Kakhetian, Kele, Kemerovo, Korean Native, Krskopolje, Kunekune, Lamcombe, Large Black, Large Black-White, Large White, Latvian White, Leicoma, Lithuanian Native, Lithuanian White, Lincolnshire Curly- Coated, Livny, Malhado de Alcobaca, Mangalitsa, Meishan, Middle White, Minzhu, Minokawa Buta, Mong Cai, Mora Romagnola, Moura, Mukota, Mulefoot, Murom, Myrhorod, Nero dei Nebrodi, Neijiang, New Zealand, Ningxiang, North Caucasian, North Siberian, Norwegian Landrace, Norwegian Yorkshire, Ossabaw Island, Oxford Sandy and Black, Pakchong 5, Philippine Native, Pietrain, Poland China, Red Wattle, Saddleback, Semirechensk, Siberian Black Pied, Small Black, Small White, Spots, Surabaya Babi, Swabian-Hall, Swedish Landrace, Swallow Belied Mangalitza, Taihu pig, Tamworth, Thuoc Nhieu, Tibetan, Tokyo-X, Tsivilsk, Turopolje, Ukrainian Spotted Steppe, Ukrainian White Steppe, Urzhum, Vietnamese Potbelly, Welsh, Wessex Saddleback, West French White, Windsnyer, Wuzhishanm, Yanan, Yorkshire and Yorkshire Blue and White. [0078] In certain embodiments, the breed of a porcine donor for use in the present disclosure is a breed of minipig. Non-limiting examples of minipig breeds include Bama, Berlin, Chinese, Clawn, Czech-Republic, Göttingen, Hanford, Lee Sung, Mini-Lewe, Mini-Sib, Munich, National Institutes of Health (NIH), Ohmini, Panepinto, Sinclair, Vietnamese potbellied, Wuzhishan, WZS, and Yucatan. In certain embodiments, a porcine donor for use herein is a NIH minipig. In certain embodiments, a porcine donor for use herein is a Yucatan minipig. In certain embodiments, a porcine donor for use herein is a Göttingen minipig. [0079] In certain embodiments, a porcine donor of the present disclosure includes wholly or partially inbred swine. In certain embodiments, a porcine donor of the present disclosure is from the same herd. A “herd,” as used herein, refers to a group of at least one male and one female which can breed to produce fertile male and female offspring. In certain embodiments, the herd of swine includes at least one male swine and at least one female swine capable of reproduction, e.g., at least one male and one female which can produce functional gametes. In certain NAI-1540201368v1 Attorney Docket No.14648-049-228 embodiments, a porcine donor of the present disclosure is from a herd of wholly or partially inbred swine. In certain embodiments, a porcine donor of the present disclosure is from a herd of inbred swine homozygous for a major histocompatibility complex haplotype. In certain embodiments, a porcine donor of the present disclosure is from a herd of swine homozygous for a major histocompatibility complex haplotype and at least about 50% homozygous at all other genetic loci. In certain embodiments, a porcine donor of the present disclosure is from a herd of swine homozygous for a major histocompatibility complex haplotype and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% homozygous at all other genetic loci. In certain embodiments, a porcine donor of the present disclosure is from a herd of swine homozygous at swine leukocyte antigens A, B, C, DR, and/or DQ. In certain embodiments, the swine leukocyte antigens A, B, C, DR, and DQ can comprise haplotype a (Aa, Ba, Ca, DRa, DQa), haplotype c (Ac, Bc, Cc, DRc, DQc) haplotype d (Ad, Bd, Cd, DRd, DQd), haplotype g (Ag, Bg, Cg, DRg, DQg), haplotype h (Ah, Bh, Ch, DRh, DQh), and/or haplotype j (Aj, Bj, Cj, DRj, DQj). [0080] In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor disclosed herein. In certain embodiments, a donor heart disclosed herein comprises a heart from a minipig. [0081] In certain embodiments, a porcine donor of the present disclosure is genetically modified. In certain embodiments, a genetically-modified porcine donor (e.g., a transgenic, chimeric, or mosaic swine) comprises at least one genetic modification. In certain embodiments, a genetically-modified porcine donor comprises only one genetic modification. In certain embodiments, a genetically-modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein is prevented (i.e., knocked out). In certain embodiments, a genetically-modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein is prevented in at least one organ, tissue, or cell type (e.g., in the heart). [0082] In certain embodiments, a genetically-modified porcine donor (e.g., a transgenic, chimeric, or mosaic swine) comprises one genetic modification wherein the genetic modification comprises a mutation of a gene. In certain embodiments, a genetically-modified porcine donor (e.g., a minipig) comprises one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene. In certain embodiments, a genetically-modified porcine donor NAI-1540201368v1 Attorney Docket No.14648-049-228 comprises one genetic modification in which the expression of a porcine protein encoded by the GGTA1 gene is prevented (i.e., knocked out, or “KO”) in the genetically-modified porcine donor. In certain embodiments, a genetically-modified porcine donor comprises one genetic modification in which the expression of a porcine protein encoded by the GGTA1 gene is prevented in at least one organ, tissue, or cell type (e.g., in the heart) of the genetically-modified porcine donor. [0083] In certain embodiments, a genetically-modified porcine donor (e.g., a minipig) comprises one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene and no other genes are genetically modified. In certain embodiments, a genetically-modified minipig comprises one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene and no other genes are genetically modified. In certain embodiments, a genetically-modified minipig does not express alpha-1,3- galactosyltransferase (i.e., the minipig is a GalT-KO minipig). Examples of genetically- modified donor minipigs that lack functional GGTA1 expression suitable for use in the present disclosure include those described in Kolber-Simonds et al., PNAS (2004) 101:7335-7340 and Lai et al., Science (2002) Feb 8;295(5557):1089-92, the disclosures of which are incorporated herein by reference. [0084] In certain embodiments, a genetically-modified porcine donor is a genetically- modified minipig that is a GalT-KO and the GalT-KO is the only genetic modification in the minipig. In certain embodiments, a genetically-modified minipig comprises one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene and there are no genetic modifications of the CMAH gene, the β4GalNT gene, the ASGR1 gene, or the porcine CD46 (pCD46) gene. In certain embodiments, a genetically-modified porcine donor is a genetically-modified minipig that is a GalT-KO and has normal expression of proteins encoded by the CMAH gene, the β4GalNT gene, the ASGR1 gene, and the porcine CD46 (pCD46) gene. [0085] In certain embodiments, a genetically-modified porcine donor is a genetically- modified minipig that is a GalT-KO that does not express any recombinant proteins. In certain embodiments, a genetically-modified porcine donor is a genetically-modified minipig that is a GalT-KO and that does not express any recombinant human proteins. In certain embodiments, a genetically-modified porcine donor is a genetically-modified minipig that is a GalT-KO and that NAI-1540201368v1 Attorney Docket No.14648-049-228 does not express any recombinant human proteins encoded by the human CD46 (hCD46) gene, the human CD55 (hCD55) gene, the human CD59 (hCD59) gene, the human thrombomodulin (hTBM) gene, the human CD47 (hCD47) gene; the human heme oxygenase-1 (hHO-1) gene, the human A20 (hA20) gene, the human ectonucleoside triphosphate diphosphohydrolase-1 (hENTPD1) gene, the human tissue factor pathway inhibitor (hTFPI); or the human leukocyte antigen G (HLA-G) gene. [0086] In certain embodiments, a genetically-modified porcine donor (e.g., a genetically- modified minipig) can be generated using any number of suitable methods known in the art for generating transgenic swine (see, e.g., Lai et al., Science (2002) Feb 8;295(5557):1089-92; Hryhorowicz et al., Genes (Basel) (2020) Jun 19;11(6):670). In certain embodiments, a method of generating a genetically-modified porcine donor comprises micro-injection of DNA material into the male pronucleus, RNA material into the cytoplasm, or proteins into the cytoplasm or pronucleus at early embryonic stage (usually the zygote/one-cell stage). In certain embodiments, a method of generating a genetically-modified porcine donor comprises sperm-mediated gene transfer (SMGT) (see, e.g., Lavitrano et al., Reprod Fertil Dev (2006)18(1-2):19-23). In certain embodiments, a method of generating a genetically-modified porcine donor comprises somatic cell nuclear transfer (SCNT). SCNT involves the transfer of the nucleus of a donor cell into an oocyte or early embryo from which the chromosomes have been removed (see, e.g., Wilmut et al., Philos Trans R Soc Lond B Biol Sci (2015) Oct 19;370(1680):20140366). In certain embodiments, a method of generating a genetically-modified porcine donor comprises a gene- targeting technique. Non-limiting examples of a gene-targeting technique suitable for use herein include homologous recombination (HR), non-homologous DNA end joining (NHEJ), zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR- based systems. [0087] In certain embodiments, a donor heart disclosed herein comprises a heart from a male or female porcine donor (e.g., a minipig). In certain embodiments, a donor heart disclosed herein comprises a heart from a genetically-modified male porcine donor (e.g., a minipig). In certain embodiments, a donor heart disclosed herein comprises a heart from a male GalT-KO minipig. In certain embodiments, a donor heart disclosed herein comprises a heart from a genetically- modified female porcine donor (e.g., a minipig). In certain embodiments, a donor heart disclosed herein comprises a heart from a female GalT-KO minipig. NAI-1540201368v1 Attorney Docket No.14648-049-228 [0088] In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) that is at least about 6 weeks of age. In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) that is at least about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age. In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) that is at least about 6 weeks to 10 weeks, about 6 weeks to 9 weeks, about 6 weeks to 8 weeks, or about 7 weeks to 8 weeks. In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) that is 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age. [0089] In certain embodiments, a donor heart disclosed herein comprises a heart from a genetically-modified minipig that is at least about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age. In certain embodiments, a donor heart disclosed herein comprises a heart from genetically-modified minipig that is at least about 6 weeks to 10 weeks, about 6 weeks to 9 weeks, about 6 weeks to 8 weeks, or about 7 weeks to 8 weeks. In certain embodiments, a donor heart disclosed herein comprises a heart from genetically-modified minipig that is 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age. In certain embodiments, a donor heart disclosed herein comprises a heart from a GalT-KO minipig that is at least about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age. In certain embodiments, a donor heart disclosed herein comprises a heart from a GalT-KO minipig that is at least about 6 weeks to 10 weeks, about 6 weeks to 9 weeks, about 6 weeks to 8 weeks, or about 7 weeks to 8 weeks. In certain embodiments, a donor heart disclosed herein comprises a heart from a GalT-KO minipig that is about 8 weeks of age. [0090] In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) wherein the porcine donor weighs at least about 6 kg, about 7 kg, about 8 kg, about 9 kg, about 10 kg, about 11 kg, about 12 kg, or more than about 12 kg. In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) wherein the weight of the porcine donor ranges from about 6 kg to 12 kg, about 7 kg to 11 kg, or about 8 kg to 10 kg. In certain embodiments, a donor heart disclosed herein comprises a heart from a porcine donor (e.g., a minipig) wherein the porcine donor weight is 6 NAI-1540201368v1 Attorney Docket No.14648-049-228 kg, about 7 kg, about 8 kg, about 9 kg, about 10 kg, about 11 kg, about 12 kg, or more than about 12 kg. [0091] In certain embodiments, a donor heart disclosed herein comprises a heart from a genetically-modified minipig wherein the genetically-modified minipig weighs at least about 6 kg, about 7 kg, about 8 kg, about 9 kg, about 10 kg, about 11 kg, about 12 kg, or more than about 12 kg. In certain embodiments, a donor heart disclosed herein comprises a heart from a GalT- KO minipig wherein the GalT-KO minipig weighs at least about 6 kg, about 7 kg, about 8 kg, about 9 kg, about 10 kg, about 11 kg, about 12 kg, or more than about 12 kg. In certain embodiments, a donor heart disclosed herein comprises a heart from a GalT-KO minipig wherein the weight of the GalT-KO minipig ranges from about 8 kg to 10 kg. 5.4 Methods of Use [0092] In certain embodiments, the present disclosure provides a method of using a medical instrument described herein (see Section 5.1). In certain embodiments, the present disclosure provides a method of using an explanted donor heart described herein (see Section 5.2). In certain embodiments, a method of using an explanted donor heart of the present disclosure comprises a method of explanting a donor heart while perfused and beating from a swine that is described in Section 5.3. In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a subject (see Section 5.4.2). Subjects suitable for receiving a donor heart from a swine according to a method disclosed herein are described in Section 5.4.4. In certain embodiments, one or more additional treatments can be administered to the donor heart recipient as described in Section 5.4.3. In certain embodiments, the present disclosure provides a method of treating a disease in a subject in need thereof, wherein the method comprises transplanting an explanted donor heart described herein (see Section 5.2). Methods of evaluating the effectiveness of the treatment of the disease in the donor heart recipient is described in Section 5.4.5. 5.4.1 Method of Explanting a Beating Donor Heart [0093] In certain embodiments, the present disclosure provides a method of explanting a beating donor heart from a swine. A donor heart suitable for use in a method described in this section can be from a swine as described in Section 5.3. In certain embodiments, the present disclosure provides a method of explanting a donor heart from a swine wherein the donor heart remains perfused and beating throughout the entire explant procedure. NAI-1540201368v1 Attorney Docket No.14648-049-228 [0094] In certain embodiments, the present disclosure provides a method of explanting a donor heart while perfused and beating from a swine wherein the method comprises placing the donor heart on a normothermic coronary perfusion circuit. In certain embodiments, a method of explanting a donor heart while perfused and beating from a swine comprises placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ. [0095] In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises placement of cannulas into the donor heart. In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises placement of at least two drainage cannulas into the donor heart. Drainage cannulas as described in Section 5.1 are suitable for use in the methods disclosed herein toward establishing a normothermic coronary perfusion circuit in situ. In certain embodiments, a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas. In certain embodiments, a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas, wherein one drainage cannula is placed in the right atrium. In certain embodiments, a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas, wherein one drainage cannula is placed in the left ventricle via the apex. In certain embodiments, a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas, wherein one drainage cannula is placed in the left ventricle via across from the left atrium. In certain embodiments, a normothermic coronary perfusion circuit for use in the method disclosed herein comprises two drainage cannulas, wherein one drainage cannula is placed in the left ventricle via the apex or across the left atrium. In certain embodiments, a method of explanting a donor heart while perfused and beating from a swine comprises placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart via the apex or across the left atrium. [0096] In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises placement of at least one outflow cannula into the donor heart. Outflow cannulas as described in Section 5.1 are suitable for use in the methods disclosed herein toward establishing a normothermic coronary perfusion circuit in situ. In certain embodiments, a NAI-1540201368v1 Attorney Docket No.14648-049-228 normothermic coronary perfusion circuit for use in the method disclosed herein is comprised of one outflow cannula. In certain embodiments, a normothermic coronary perfusion circuit for use in the method disclosed herein is comprised of one outflow cannula that is placed in the aorta. In certain embodiments, a normothermic coronary perfusion circuit for use in the method disclosed herein is comprised of one outflow cannula that is placed in the proximal ascending aorta. In certain embodiments, a method of explanting a donor heart while perfused and beating from a swine comprises placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by placing an outflow cannula in the proximal ascending aorta of the donor heart. [0097] In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises ligation of at least one vein that drains into the donor heart. In certain embodiments, methods of establishing a normothermic coronary perfusion circuit in situ comprise ligation of at least the superior vena cava (SVC). In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises ligation of at least the inferior vena cava (IVC). In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises ligation of the SVC and the IVC of the donor heart. In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises clamping the aorta of the donor heart. [0098] In certain embodiments, provided herein is a method of placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ. In certain embodiments, provided herein is a method of placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by (i) placing at least one cannula into the donor heart; (ii) placing an outflow cannula in the aorta of the donor heart; (iii) ligating least one vein that drains into the donor heart; and (iv) clamping the aorta of the donor heart. In certain embodiments, provided herein is a method of placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by (i) placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart via the apex or across the left atrium; (ii) placing an outflow cannula in the proximal ascending aorta of the donor heart; (iii) ligating the SVC and the IVC of the donor heart; and (iv) clamping the aorta of the donor heart. NAI-1540201368v1 Attorney Docket No.14648-049-228 [0099] In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises priming the normothermic coronary perfusion circuit with a priming solution before placing the donor heart on the normothermic coronary perfusion circuit. In certain embodiments, a priming solution for use in a method disclosed herein comprises, for example but not limited to, buffered electrolytes, mannitol, vitamins, and/or steroids. In certain embodiments, a priming solution for use in a method disclosed herein has a physiological pH. In certain embodiments, a priming solution for use in a method disclosed herein has a pH ranging from about 7.0 to 8.0 (e.g., about 7.0; 7.1; 7.2; 7.3; 7.4; 7.5; 7.6; 7.7; 7.8; 7.9; 8.0). In certain embodiments, a priming solution for use in a method disclosed herein comprises sodium chloride, sodium gluconate, sodium acetate, potassium chloride, and/or magnesium chloride. In certain embodiments, a priming solution for use in a method disclosed herein comprises plasmalyte A. In certain embodiments, a method of establishing a normothermic coronary perfusion circuit in situ comprises priming the normothermic coronary perfusion circuit with plasmalyte A before placing the donor heart on the normothermic coronary perfusion circuit. [00100] In certain embodiments, provided herein is a method of explanting a donor heart while perfused and beating from a swine, wherein the method comprises explanting the donor heart while beating from the swine. Methods of explanting (e.g., surgically removing) the donor heart from swine are generally known in the art. See, e.g., Mohiuddin MM et al., Int J Surg (2015) Nov;23(Pt B):234-239; McGregor CG et al., J Thorac Cardiovasc Surg. (2005) Sep;130(3):844-51. [00101] In certain embodiments, provided herein is a method of explanting a donor heart while perfused and beating from a swine, wherein the method comprises maintaining the beating donor heart on a normothermic coronary perfusion circuit ex situ in an organ reservoir. Organ reservoirs as described in Section 5.1 are suitable for use in a method disclosed herein toward maintaining the beating donor heart on a normothermic coronary perfusion circuit ex situ. In certain embodiments, an organ reservoir for use in a method disclosed herein is maintained at a temperature ranging from about 32°C to about 37°C. In certain embodiments, an organ reservoir for use in a method disclosed herein is maintained at a temperature ranging from about 32°C to about 33°C, about 33°C to about 34°C, about 34°C to about 35°C, about 35°C to about 36°C, or about 36°C to about 37°C. In certain embodiments, an organ reservoir for use in a method disclosed herein is maintained at a temperature of about 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. NAI-1540201368v1 Attorney Docket No.14648-049-228 In certain embodiments, an organ reservoir for use in a method disclosed herein comprises a perfusion solution and the beating donor heart. In certain embodiments, an organ reservoir for use in a method disclosed herein comprises a perfusion solution, the beathing donor heart, or both that are maintained at a temperature ranging from about 32°C to about 37°C. In certain embodiments, an organ reservoir for use in a method disclosed herein comprises a perfusion solution, the beathing donor heart, or both that are maintained at a temperature ranging from about 32°C to about 33°C, about 33°C to about 34°C, about 34°C to about 35°C, about 35°C to about 36°C, or about 36°C to about 37°C. In certain embodiments, an organ reservoir for use in a method disclosed herein comprises a perfusion solution, the beathing donor heart, or both that are maintained at a temperature of about 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. [00102] In certain embodiments, provided herein is a method of explanting a donor heart while perfused and beating from a swine, wherein the method comprises perfusing the beating donor heart with a physiological solution. In certain embodiments, the method comprises perfusing the beating donor heart with an oxygenated physiological solution. In certain embodiments, the method comprises perfusing the beating donor heart with an oxygenated physiological solution comprising more than about 90% oxygen. In certain embodiments, the method comprises perfusing the beating donor heart with an oxygenated physiological solution comprising about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% oxygen. In certain embodiments, the method comprises perfusing the beating donor heart with an oxygenated physiological solution comprising about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% oxygen. In certain embodiments, the method comprises perfusing the beating donor heart with an oxygenated physiological solution comprising blood from the swine that sourced the donor heart. [00103] In certain embodiments, the method comprises perfusing the beating donor heart with an oxygenated physiological solution, optionally wherein the oxygenated physiological solution may comprise an agent suitable for use with the methods herein. In certain embodiments, an agent provides nutrition or treatment to the cardiac tissue of the donor heart. In certain embodiments, an agent is to be delivered locally to the cardiac tissue of the donor heart. In certain embodiments, an agent comprises a polynucleotide or fragment thereof, a small molecule, NAI-1540201368v1 Attorney Docket No.14648-049-228 an antibody or fragment thereof, a polypeptide or fragment thereof, a gene editing system, or any combination thereof. In certain embodiments, an agent comprises a vasodilator (e.g., nitrates, nicorandil). In certain embodiments, an agent comprises a vasopressor (e.g., norepinephrine, epinephrine). In certain embodiments, a method disclosed herein comprises perfusing the beating donor heart with an oxygenated physiological solution, wherein the oxygenated physiological solution is free from additional agents. In certain embodiments, a method disclosed herein comprises perfusing the beating donor heart with an oxygenated physiological solution, wherein the oxygenated physiological solution is free of cardioplegia. [00104] In certain embodiments, provided herein is a method of explanting a donor heart while perfused and beating from a swine, wherein the method comprises maintaining the beating donor heart on a normothermic coronary perfusion circuit ex situ. In certain embodiments, the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for a maximum of about 10 hours. In certain embodiments, the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for about 30 minutes to about 1 hour, about 1 to about 1.5 hours, about 1.5 hours to about 2 hours, about 2 hours to about 2.5 hours, about 2.5 hours to about 3 hours, about 3 hours to about 3.5 hours, about 3.5 hours to about 4 hours, about 4 hours to about 4.5 hours, about 4.5 hours to about 5 hours, about 5 hours to about 5.5 hours, about 5.5 hours to about 6 hours, about 6 hours to about 6.5 hours, about 6.5 hours to about 7 hours, about 7 hours to about 7.5 hours, about 7.5 hours to about 8 hours, about 8 hours to about 8.5 hours, about 8.5 hours to about 9 hours, about 9 hours to about 9.5 hours, about 9.5 hours to about 10 hours, or more than about 10 hours. In certain embodiments, the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours. In certain embodiments, the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours. 5.4.2 Transplantation of Beating Donor Heart [00105] In certain embodiments, the present disclosure provides a method of transplanting an explanted beating donor heart described in Section 5.2 into a subject in need thereof. A subject NAI-1540201368v1 Attorney Docket No.14648-049-228 suitable for receiving a beating donor heart as disclosed herein is described in Section 5.4.4 below. [00106] In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises placing the primate subject on cardiopulmonary bypass (CPB) and surgically removing the heart of the primate subject. Methods of surgically removing the heart from a primate subject in preparation for transplant are generally known in the art. See, e.g., Mohiuddin MM et al., Int J Surg (2015) Nov;23(Pt B):234-239; McGregor CG et al., J Thorac Cardiovasc Surg. (2005) Sep;130(3):844- 51. [00107] In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises placing the donor heart on a normothermic coronary perfusion circuit and then explanting the donor heart while beating from the swine. Methods for placing the donor heart on a normothermic coronary perfusion circuit and then explanting the donor heart while beating from the swine are described in Section 5.4.1 and are suitable for use in the methods of transplanting an explanted beating donor heart as described in this section. [00108] In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises implanting the beating explanted donor heart into the primate subject on CPB. Methods of transplanting a porcine donor heart into a primate subject are generally known in the art. See, e.g., Mohiuddin MM et al., Int J Surg (2015) Nov;23(Pt B):234-239; McGregor CG et al., J Thorac Cardiovasc Surg. (2005) Sep;130(3):844-51. In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises removing the primate subject from CPB after implantation of the beating explanted donor heart according to standard methods generally known in the art. [00109] In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises (a) placing the primate subject on cardiopulmonary bypass (CPB) and surgically removing the heart of the primate subject; (b) placing the donor heart on a normothermic coronary perfusion circuit and then explanting the donor heart while beating from the swine; (c) implanting the beating NAI-1540201368v1 Attorney Docket No.14648-049-228 explanted donor heart into the primate subject on CPB; and (d) removing the primate subject from CPB. [00110] In certain embodiments, the beating donor heart remains perfused throughout the procedure (e.g., the method of transplanting the beating donor heart). In certain embodiments, the beating donor heart remains perfused for at least 95% of the procedure. In certain embodiments, the beating donor heart remains perfused for about 95% to 99%, 96% to 99%, 97% to 99%, or 98 to 99% of the procedure. [00111] In certain embodiments, the beating donor heart remains beating throughout the procedure (e.g., the method of transplanting the beating donor heart). In certain embodiments, the beating donor heart maintains a steady sinus rhythm rate throughout the procedure. In certain embodiments, the beating donor heart maintains a sinus rhythm rate ranging from about 50–180 beats per minute. In certain embodiments, the beating donor heart maintains a sinus rhythm rate ranging from about 50-180, about 50-170, about 50-160, about 50-150, about 50-140, about 50- 130, about 50-120, about 50-110, about 50-100, about 60-180, about 60-170, about 60-160, about 60-150, about 60-140, about 60-130, about 60-120, about 60-110, about 60-100, about 70- 180, about 70-170, about 70-160, about 70-150, about 70-140, about 70-130, about 70-120, about 70-110, or about 70-100 beats per minute. In certain embodiments, the beating donor heart maintains an average sinus rhythm rate of about, of at least about, or of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, or 180 beats per minute. [00112] In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the transplanted donor heart is substantially free of ischemic tissue at the time of implanting the beating explanted donor heart into the primate subject on CPB. In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the transplanted donor heart comprises less than about 1% to 20% ischemic tissue at the time of implanting the beating explanted donor heart into the primate subject on CPB. In certain embodiments, the present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the transplanted donor heart comprises less than about 1% to 20%, 1% to 15%, 1% to 10% or 1% to 5% ischemic tissue at the time of implanting the beating explanted donor heart into the primate subject on CPB. In certain embodiments, the NAI-1540201368v1 Attorney Docket No.14648-049-228 present disclosure provides a method of transplanting a donor heart from a swine into a primate subject, wherein the transplanted donor heart comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% ischemic tissue at the time of implanting the beating explanted donor heart into the primate subject on CPB. 5.4.3 Additional Treatments [00113] In certain embodiments, a donor heart recipient treated in accordance with the methods described herein (e.g., transplantation of a donor heart) undergoes additional treatment. A donor heart recipient may undergo additional treatment by one or more different methods. Additional treatment may occur prior to, concurrently with, or subsequent to the method of treatment provided herein (e.g., transplantation of a donor heart). [00114] Additional treatments are generally intended to improve the tolerance of the donor heart in recipients, but other treatments are contemplated. In certain embodiments, a method of transplantation of a donor heart in accordance with the methods described herein can thus include administering one or more additional treatments, e.g., a treatment that inhibits T cells, blocks complement, or otherwise down regulates the recipient’s immune response to the donor heart. In certain embodiments, a recipient is thymectomized and/or splenectomized. [00115] In certain embodiments, a recipient receives radiation, for example, total body irradiation. In certain embodiments, a recipient receives 5-10 Gy or 10-15 Gy irradiation. In certain embodiments, thymic irradiation can be used. In certain embodiments, the recipient is administered low dose radiation (e.g., a sub lethal dose of between 100 rads and 400 rads whole body radiation). In certain embodiments, local thymic radiation is administered to the recipient. [00116] The blood of a subject undergoing transplantation by a method described herein may contain antibodies that target the donor heart. Such antibodies can be eliminated by organ perfusion, and/or transplantation of tolerance-inducing bone marrow. Natural antibodies can be absorbed from the recipient’s blood by hemoperfusion of a liver of the donor species. Similarly, antibody-producing cells may be present in the recipient. Such antibody producing cells may be eliminated by, for example, irradiation or drug treatments. In certain embodiments, the donor heart may be genetically-modified such that it is not recognized by antibodies present in the host (e.g., the donor heart is a-1,3-galactosyltransferase deficient) per Section 5.3. (a) Immunosuppressive Therapy NAI-1540201368v1 Attorney Docket No.14648-049-228 [00117] In certain embodiments, a patient receiving a donor heart in accordance with the methods described herein can further receive immunosuppressive therapy. The immunosuppressive therapy may be any FDA-approved treatment indicated to reduce transplant rejection and/or ameliorate the outcome of xenotransplantation. Non-limiting examples of immunosuppressive therapy include calcineurin inhibitors (e.g., tacrolimus or cyclosporine), antiproliferative agents (e.g., anti-metabolites such a mycophenolate, 6-mercaptopurine or its prodrug azathioprine), inhibitors of mammalian target of rapamycin (mTOR) (e.g., sirolimus, rapamycin), steroids (e.g., prednisone), cell cycle inhibitors (azathioprine or mycophenolate mofetil), lymphocyte-depleting agents (e.g., anti-thymocyte globulin or antibodies such as alemtuzumab, siplizumab or basiliximab) and co-stimulation blockers (e.g., belatacept). See, e.g., Chung et al., Ann Transl Med (2020) Mar; 8(6): 409; van der Mark et al., Eur Respir Rev (2020) 29: 190132; and Benvenuto et al., J Thorac Dis (2018) 10:3141-3155. [00118] In certain embodiments, immunosuppressive therapy can be administered as induction therapy (perioperative, or immediately after surgery), a maintenance dose, or for an acute rejection. Induction therapy commonly includes basiliximab, anti-thymocyte globulin or alemtuzumab. Immunosuppressive therapy can also be administered as maintenance therapy, which is often required to continue for the life of the recipient. Maintenance immunosuppressive therapy commonly includes a calcineurin inhibitor (tacrolimus or cyclosporine), an antiproliferative agent (mycophenolate or azathioprine), and corticosteroids. Immunosuppressive therapy for acute rejections commonly includes thymoglobulin or mycophenolate. See, e.g., Chung et al., Ann Transl Med (2020) Mar; 8: 409 and Benvenuto et al., J Thorac Dis (2018)10:3141-3155. [00119] Non-limiting examples of immunosuppressants include, (1) antimetabolites, such as purine synthesis inhibitors (such as inosine monophosphate dehydrogenase (IMPDH) inhibitors, e.g., azathioprine, mycophenolate, and mycophenolate mofetil), pyrimidine synthesis inhibitors (e.g., leflunomide and teriflunomide), and antifolates (e.g., methotrexate); (2) calcineurin inhibitors, such as tacrolimus, cyclosporine A, pimecrolimus, and voclosporin; (3) TNF-alpha inhibitors, such as thalidomide and lenalidomide; (4) IL-1 receptor antagonists, such as anakinra; (5) mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin (sirolimus), deforolimus, everolimus, temsirolimus, zotarolimus, and biolimus A9; (6) corticosteroids, such NAI-1540201368v1 Attorney Docket No.14648-049-228 as prednisone; and (7) antibodies to any one of a number of cellular or serum targets (including anti-lymphocyte globulin and anti-thymocyte globulin). [00120] Non-limiting exemplary cellular targets and their respective inhibitor compounds include, but are not limited to, complement component 5 (e.g., eculizumab); tumor necrosis factors (TNFs) (e.g., infliximab, adalimumab, certolizumab pegol, afelimomab and golimumab); IL-5 (e.g., mepolizumab ); IgE (e.g., omalizumab ); BAYX (e.g., nerelimomab ); interferon (e.g., faralimomab); IL-6 (e.g., elsilimomab); IL-12 and IL-13 (e.g., lebrikizumab and ustekinumab); CD3 (e.g., muromonab-CD3, otelixizumab, teplizumab, visilizumab); CD4 (e.g., clenoliximab, keliximab and zanolimumab); CDI la (e.g., efalizumab); CD18 (e.g., erlizumab); CD20 (e.g., afutuzumab, ocrelizumab, pascolizumab ); CD23 (e.g., lumiliximab ); CD40 (e.g., teneliximab, toralizumab); CD62L/L-selectin (e.g., aselizumab); CD80 (e.g., galiximab); CD147/basigin (e.g., gavilimomab); CD154 (e.g., ruplizumab); BlyS (e.g., belimumab); CTLA-4 (e.g., ipilimumab, tremelimumab); CAT (e.g., bertilimumab, lerdelimumab, metelimumab); integrin (e.g., natalizumab); IL-6 receptor (e.g., tocilizumab); LFA-1 (e.g., odulimomab); and IL-2 receptor/CD25 (e.g., basiliximab, daclizumab, inolimomab). [00121] In certain embodiments, a method provided herein can comprise steps to induce tolerance in the recipient, e.g., by inducing mixed chimerism. “Mixed chimerism” is commonly understood to describe a state in which the lymphohematopoietic system of the recipient of allogeneic hematopoietic stem cells comprises a mixture of host and donor cells. This state is usually attained through either bone marrow or mobilized peripheral blood stem cell transplantation. Mixed chimerism can be transient or stable. See, e.g., Sachs et al., Cold Spring Harb Perspect Med (2014) 4:a015529; U.S. Patent No.6,296,846; and U.S. Patent No. 6,306,651. (b) Hematopoietic Stem Cell Transplant [00122] In certain embodiments, a patient receiving a donor heart in accordance with the methods described herein can further receive a hematopoietic stem cell transplant. Stem cell engraftment and hematopoiesis across disparate species barriers can be enhanced by providing a hematopoietic stromal environment from the donor species. The stromal matrix supplies species-specific factors that are required for interactions between hematopoietic stem cells and their stromal environment, such as hematopoietic growth factors, adhesion molecules, and their ligands. NAI-1540201368v1 Attorney Docket No.14648-049-228 [00123] As liver is the major site of hematopoiesis in the fetus, fetal liver can also serve as an alternative to bone marrow as a source of hematopoietic stem cells. As an alternative or an adjunct to implantation, fetal liver cells can be administered in fluid suspension. The thymus is the major site of T cell maturation. Each organ includes an organ specific stromal matrix that can support differentiation of the respective undifferentiated stem cells implanted into the host. Thymic stromal tissue can be irradiated prior to transplantation. [00124] Porcine hematopoietic chimeras can lead to donor-specific nonresponsiveness in the mixed lymphocyte reaction, lack of anti-donor IgG antibody production, and acceptance of donor grafts. Accordingly, mixed chimerism is capable of inducing tolerance in a highly disparate xenogeneic combination and can have the clinical potential to prevent xenograft rejection. See, e.g., Griesemer et al., Immunol. Rev (2014) 258(1): 241-258; Sachs et al., Cold Spring Harb Perspect Med (2014) 4:a015529. Bone marrow cells (BMC), or another source of hematopoietic stem cells, e.g., a fetal liver suspension, of the donor can be injected into the recipient in order to induce mixed chimerism. The hematopoietic stem cells may be taken from any source, for example from the bone marrow or peripheral blood stem cells. See, e.g., Sachs et al., Cold Spring Harb Perspect Med (2014) 4:a015529. Donor BMC home to appropriate sites of the recipient and grow contiguously with remaining host cells and proliferate, forming a chimeric lymphohematopoietic population. By this process, newly forming B cells (and the antibodies they produce) are exposed to donor antigens, so that the transplant will be recognized as self. Tolerance to the donor is also observed at the T cell level in animals in which hematopoietic stem cell, e.g., bone marrow cell, engraftment has been achieved. For bone marrow transplant, the recipient can be administered low dose radiation. In certain embodiments, the recipient can be treated with an agent that depletes complement, such as cobra venom factor (e.g., at day -1). 5.4.4 Donor Heart Recipients [00125] In certain embodiments, the present disclosure provides a method of treating a patient in need thereof with a donor heart described herein (see Section 5.2). In certain embodiments, a patient treated with a donor heart described herein can be a donor heart recipient. In certain embodiments, a patient treated in accordance with a method described herein (e.g., the recipient of a donor heart) is a primate subject. In certain embodiments, a patient treated in accordance with a method described herein (e.g., the recipient of a donor heart) is a human patient. In certain embodiments, a patient treated in accordance with a method described herein (e.g., the NAI-1540201368v1 Attorney Docket No.14648-049-228 recipient of a donor heart) is a pediatric human patient. [00126] As used herein, the terms “subject” and “patient” are used interchangeably and include any human or non-human mammal. In certain embodiments, the subject is a primate. In certain embodiments, the subject is a non-human primate (e.g., a baboon, a cynomolgus monkey or a rhesus macaque) and receives one or more grafts from a porcine donor. In certain embodiments, the subject is human. In certain embodiments, the subject is a human adult. In certain embodiments, the subject is a human child. In certain embodiments, the subject is a pediatric patient (e.g., 0–18 years old). In certain embodiments, the subject is a geriatric patient (e.g., ≥65 years old). In certain embodiments, the subject is human and receives one or more donor grafts from a porcine donor. Methods of evaluating the effectiveness of treatment of a disease in the donor heart recipient is described in Section 5.4.5. In certain embodiments, the subject is a human subject having a disease of the heart. [00127] In certain embodiments, a patient treated in accordance with the methods described herein is in need of a heart transplant. A patient may be in need of a heart transplant due to heart failure or the rejection of a donor heart. Heart failure can have a number of causes, including but not limited to high blood pressure (hypertension), physical injury, diabetes, and autoimmune disorders. In certain embodiments, a patient treated in accordance with the methods described herein has advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof. [00128] Heart diseases and conditions can be diagnosed by laboratory tests (cholesterol, high- sensitivity C-reactive protein (hs-CRP), lipoprotein a (Lp(a)), plasma ceramides, natriuretic peptides, troponin), electrocardiogram (ECG), echocardiogram (ultrasound)), exercise stress test, nuclear cardiac stress test, coronary angiogram, magnetic resonance imaging (MRI), coronary computed tomography angiogram (CCTA), and/or a heart biopsy. [00129] Classes and stages of heart failure classifies patients by measuring a patient’s overall heart function and severity of symptoms (see, e.g., Dolgin M et al., New York Heart Association Criteria Committee. NOMENCLATURE AND CRITERIA FOR DIAGNOSIS OF DISEASES OF THE HEART AND GREAT VESSELS.9th ed. Boston, MA: Lippincott Williams and Wilkins; March 1, 1994). In certain embodiments, a patient treated in accordance with a method described herein has Stage C heart failure. In certain embodiments, a patient treated in accordance with a method described herein has Stage D heart failure. In certain embodiments, a patient treated in accordance with a NAI-1540201368v1 Attorney Docket No.14648-049-228 method described herein has a class I New York Heart Association (NYHA) Functional Classification. In certain embodiments, a patient treated in accordance with a method described herein has a class II NYHA Functional Classification. In certain embodiments, a patient treated in accordance with a method described herein has a class III NYHA Functional Classification. In certain embodiments, a patient treated in accordance with a method described herein has a class IV NYHA Functional Classification. 5.4.5 Methods of Evaluating Transplantation [00130] In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure prevents perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure reduces the occurrence of perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure reduces the occurrence of perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure by at least about 25%. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure reduces the occurrence of perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure by about 25% to 99%, about 30% to 99%, about 35% to 99%, about 40% to 99%, about 45% to 99%, about 50% to 99%, about 55% to 99%, about 60% to 99%, about 65% to 99%, about 70% to 99%, about 75% to 99%, about 80% to 99%, about 85% to 99%, about 90% to 99%, or about 95% to 99%. [00131] In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure reduces the occurrence of transplant rejection in a human subject in need of a cardiac xenograft compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure reduces the occurrence of transplant rejection in a human subject in need of a cardiac xenograft by about 10% to about NAI-1540201368v1 Attorney Docket No.14648-049-228 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 90% compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure reduces the occurrence of transplant rejection in a human subject in need of a cardiac xenograft by least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure. [00132] In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a more viable transplant compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a transplant comprising about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 90% more viability compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a transplant comprising about at least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% more viability compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure. [00133] In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a more functional transplant compared to that of a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure. Examples of methods for assessing heart transplant function include measuring left ventricle ejection fraction (LVEF), a right ventricular (RV) systolic function, a pulmonary artery (PA) pressure, a right atrial (RA) pressure, a sinus rhythm, and/or a cardiac output. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having comparable cardiac function to that of a heart in a healthy primate subject of the same age and species as the primate subject. In certain NAI-1540201368v1 Attorney Docket No.14648-049-228 embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a left ventricle ejection fraction (LVEF) comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having right ventricular (RV) systolic function comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a pulmonary artery (PA) pressure comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a right atrial (RA) pressure comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a sinus rhythm comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject. In certain embodiments, a method of transplanting a beating donor heart according to the present disclosure results in a transplanted donor heart having a cardiac output comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject. [00134] In certain embodiments, transplantation of a donor heart according to the present disclosure results in a better rate of survival of the donor heart recipient as compared to the rate of survival before transplantation of the donor heart to the patient. In certain embodiments, transplantation of a donor heart according to the present disclosure improves the rate of survival of the donor heart recipient by about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than about 90% compared to the rate of survival before transplantation of the donor heart to the patient. In certain embodiments, transplantation of a donor heart according to the present disclosure improves the rate of survival of the donor heart recipient by at least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% compared to the rate of survival before transplantation of the donor heart to the patient. In certain embodiments, transplantation of a donor heart according to the NAI-1540201368v1 Attorney Docket No.14648-049-228 present disclosure improves the rate of survival of the donor heart recipient by about 3 months to about 6 months, about 6 months to about 1 year, about 1 year to about 3 years, about 3 years to about 5 years, about 5 years to about 10 years, about 10 years to about 15 years, about 15 years to about 20 years, about 20 years to about 25 years, about 25 years to about 30 years, about 30 years to about 40 years, about 40 years to about 50 years, or about more than 50 years compared to the rate of survival before transplantation of the donor heart to the patient. In certain embodiments, transplantation of a donor heart according to the present disclosure improves the rate of survival of the donor heart recipient by at least about or about 3 months, 6 months, 1 year, 3 years, 5 years, 10 years, 15 years, 20 years, 25 years, 30 years, 40 years, 50 years, or more than 50 years compared to the rate of survival before transplantation of the donor heart to the patient. [00135] In certain embodiments, transplantation of a donor heart according to the present disclosure results in an improvement of the corresponding disease that the transplantation aims to intervene within the donor heart recipient as compared to the disease before transplantation of the donor heart to the patient. In certain embodiments, transplantation of a donor heart according to the present disclosure results in a greater improvement of the corresponding disease that the transplantation aims to intervene within the donor heart recipient compared to that of a patient transplanted with a cardiac xenograft prepared and transplanted by methods other than those of the present disclosure. One of skill in the art can appreciate that an improvement of the corresponding disease following transplantation of a donor heart according to the present disclosure is assessed according to the disease itself, severity of the disease, the health of the patient before and after transplant, and a myriad of other factors than can be disease and/or patient specific. In certain embodiments, an improvement of the corresponding disease can include, but is not limited to, amelioration and/or ablation of at least one symptom associated with the disease, slowing progression of the disease, preventing further progression of the disease, reversing disease progression, and/or returning the donor heart recipient to a healthy status (e.g., comparable to a healthy, disease-free patient of comparable age, gender, and background). In certain embodiments, transplantation of a donor heart according to the present disclosure results in an improvement of the corresponding disease that the transplantation aims to intervene within the donor heart recipient as indicated by at least one biomarker corresponding to the disease. NAI-1540201368v1 Attorney Docket No.14648-049-228 [00136] In certain embodiments, transplantation of a donor heart according to the present disclosure results in an improvement of heart disease and/or at least one symptom associated with heart disease within the donor heart recipient as compared to before transplantation of the donor heart to the patient. In certain embodiments, transplantation of a donor heart according to the present disclosure results in at least about or about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or more than 90% improvement of heart disease and/or at least one symptom associated with heart disease within the donor heart recipient as compared to before transplantation of the donor heart to the patient. 6. EXAMPLES [00137] The following is a description of various methods and materials used in the studies. They are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for. 6.1 Example 1 [00138] Xenotransplantation offers a clinically viable solution to the organ shortage problem. Perioperative cardiac xenograft dysfunction is a major limitation to long-term xenograft survival and is attributed to ischemia during the heart procurement and transplant. Described herein is an exemplary method of explanting and transplanting perfused, beating donor hearts without any ischemia in a pig-to-baboon model. 6.1.1 Methods [00139] An α-1,3 galactosyltransferase knock-out (GalT-KO) pig weighing 16 kilograms (kg), was selected as the donor for a 24 kg male baboon. First, the baboon was placed on cardiopulmonary bypass (CPB) and the native heart was explanted. The donor pig heart was then placed on a normothermic coronary perfusion circuit (FIG.1) with drainage cannulas in the NAI-1540201368v1 Attorney Docket No.14648-049-228 right atrium and the left ventricle and an outflow cannula in the proximal ascending aorta. The superior vena cava (SVC) and the inferior vena cava (IVC) were ligated, the aorta was clamped, and then the heart was explanted while beating (FIG.2). The pig heart was implanted into the baboon, and hemodynamic, EKG, arterial blood gas (ABG), and echocardiography data was collected for 60 minutes post-CPB separation. 6.1.2 Results [00140] The time on the coronary perfusion circuit was 34 minutes. The donor heart remained in sinus rhythm while on the circuit and the ABG with minimal sweep was pH = 7.64, pCO2 = 23 mmHg, pO2 = 699 mmHg, and bicarbonate (HCO3) = 24 mEq/L. The baboon was easily weaned off CPB in sinus rhythm on no inotropes or pressors. Echocardiography performed 60 minutes after weaning from CPB demonstrated normal qualitative right ventricular (RV) systolic function, mildly decreased left ventricular (LV) systolic function (LV shortening fraction 24-25%), trivial mitral regurgitation (MR), and mild tricuspid regurgitation (FIG.3). The right atrial (RA) pressure was 6 mmHg, and the pulmonary artery (PA) pressure was 32/12 mmHg in the donor heart at 60 minutes off CPB. The xenograft provided life-sustaining cardiac output with a venous oxygen saturation (SvO2) of 77% and ABG reading was pH = 7.35, pCO2 = 32 mmHg, and pO2 = 400 mmHg. Histology demonstrated no evidence of acute T cell or antibody-mediated rejection, ischemic injury, or edema (FIG.4). [00141] Echocardiogram data taken on post-transplant Day 8 demonstrated excellent cardiac function. FIG.5A shows left ventricle short axis systole and FIG.5B shows left ventricle short axis diastole. The left ventricle ejection fraction calculated from the echocardiogram was ~55%. Echocardiogram data taken on post-transplant Day 14 demonstrated similar cardiac function (data not shown). 6.1.3 Conclusions [00142] Eliminating ischemia in cardiac xenografts was not previously achieved before the exemplary methods described herein. Data provided herein demonstrate that ex-vivo coronary perfusion without using cardioplegia to arrest the heart is technically feasible. This “never ischemic” beating heart transplant and the techniques involved in the preparing thereof have important implications for cardiac xenotransplantation in the clinic and also have broader translatability in allotransplantation. 7. ILLUSTRATIVE EMBODIMENTS NAI-1540201368v1 Attorney Docket No.14648-049-228 [00143] The present disclosure provides the following non-limiting embodiments: 1. A method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises: a. placing the primate subject on cardiopulmonary bypass (CPB) and surgically removing the heart of the primate subject; b. placing the donor heart on a normothermic coronary perfusion circuit and then explanting the donor heart while beating from the swine; c. implanting the beating explanted donor heart into the primate subject on CPB; d. removing the primate subject from CPB. 2. The method of embodiment 1, wherein the donor heart remains perfused and beating throughout the entire procedure. 3. The method of embodiment 1 or embodiment 2, wherein the beating explanted donor heart comprises substantially no ischemic tissue at the time of implantation into the primate subject on CPB. 4. The method of any one of embodiments 1 to 3, wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step b for a maximum of about 10 hours, optionally wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step b for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours. 5. The method of any one of embodiments 1 to 4, wherein the transplanted donor heart is substantially free of ischemic tissue at the time of the implanting step. 6. The method any one of embodiments 1 to 5, wherein the transplanted donor heart has a left ventricle ejection fraction (LVEF), a right ventricular (RV) systolic function, a pulmonary artery (PA) pressure, a right atrial (RA) pressure, a sinus rhythm, and/or a cardiac output comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject. 7. The method of any one of embodiments 1 to 6, wherein the normothermic coronary perfusion circuit is comprised of at least two drainage cannulas and at least one outflow cannula. 8. The method of embodiment 7, wherein one drainage cannula is placed in the right atrium and another drainage cannula is placed in the left ventricle via the apex or across the left atrium. 9. The method of embodiment 7, wherein the outflow cannula is placed in the proximal NAI-1540201368v1 Attorney Docket No.14648-049-228 ascending aorta. 10. The method of any one of embodiments 1 to 9, wherein step b further comprises ligating the superior vena cava (SVC) and the inferior vena cava (IVC), and clamping the aorta distal to the outflow cannula before explanting the beating donor heart. 11. A method of explanting a donor heart while perfused and beating from a swine, wherein the method comprises: a. placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by: i. placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart via the apex or across the left atrium; ii. placing an outflow cannula in the proximal ascending aorta of the donor heart; iii. ligating the superior vena cava (SVC) and the inferior vena cava (IVC) of the donor heart; iv. clamping the aorta of the donor heart; b. explanting the donor heart while beating from the swine; c. maintaining the beating donor heart on the normothermic coronary perfusion circuit ex situ. 12. The method of embodiment 11, further comprising priming the normothermic coronary perfusion circuit with plasmalyte A before placing the donor heart on the normothermic coronary perfusion circuit. 13. The method of embodiment 11 or 12, wherein the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for a maximum of about 10 hours, optionally wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step c for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours. 14. The method of any one of embodiments 11 to 13, wherein the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ in an organ reservoir. 15. The method of embodiment 14, wherein the organ reservoir is maintained at a temperature NAI-1540201368v1 Attorney Docket No.14648-049-228 ranging from about 32°C to about 37°C. 16. The method of embodiment 14 or 15, wherein the beating donor heart maintained on the normothermic coronary perfusion circuit ex situ in an organ reservoir is placed in a sterile container suitable for transportation. 17. The method of any one of embodiments 11 to 16, wherein the beating donor heart is perfused with an oxygenated physiological solution. 18. The method of embodiment 17, wherein the oxygenated physiological solution comprises blood from the swine that sourced the donor heart. 19. The method of any one of embodiments 1 to 18, wherein the swine is a genetically- modified minipig. 20. The method of embodiment 19, wherein the genetically-modified minipig comprises a GGTA1 genetic modification. 21. The method of embodiment 19, wherein the genetically-modified minipig does not express alpha-1,3-galactosyltransferase (GalT-KO). 22. The method of any one of embodiments 1 to 21, wherein the genetically-modified minipig is a GalT-KO minipig and: a. is wild type for a protein encoded by the porcine genes CMAH, β4GalNT, ASGR1, and CD46; and/or b. does not express any recombinant proteins. 23. The method of any one of embodiments 1 to 21, wherein the genetically-modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens. 24. The method of any one of embodiments 1 to 21, wherein the genetically-modified minipig is GalT-KO and the GalT-KO is the only genetic modification in the minipig. 25. The method of any one of the preceding embodiments, wherein the primate subject is a human subject. 26. The method of embodiment 25, wherein the human subject has advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof. 27. The method of any one of embodiments 25 to 26, wherein the human subject is a pediatric human subject. 28. The method of any one of embodiments 1 to 27, wherein the method is performed in the NAI-1540201368v1 Attorney Docket No.14648-049-228 absence of cardioplegia. 29. A medical instrument comprising a. at least two drainage cannulas and at least one outflow cannula, wherein one drainage cannula is suitable to be placed in the right atrium of a donor heart and another drainage cannula is suitable to be placed in the left ventricle of the donor heart; b. an outflow cannula, wherein the outflow cannula is suitable to be placed in the proximal ascending aorta of the donor heart; and c. a pump to maintain homeostasis with a perfusion solution; wherein the medical instrument is specifically adapted to perform normothermic machine perfusion on a heart from a minipig. 30. The medical instrument of embodiment 29, further comprising an organ reservoir. 31. The medical instrument of embodiment 29 or embodiment 30, further comprising a membrane oxygenator, a leukocyte filter, an arterial filter, a heater-cooler unit, an autonomous power unit, or any combination thereof. 32. The medical instrument of any one of embodiments 29 to 31, further comprising a sterile container, wherein the sterile container is suitable for transportation of a donor heart that is perfused and beating during transport. 33. The medical instrument of any one of embodiments 29 to 32, further comprising a collection chamber, wherein the collection chamber is suitable for collecting lost blood during transportation of a donor heart. 34. An explanted donor heart from a minipig, wherein the donor heart is connected to an ex situ normothermic coronary perfusion circuit prior to transplantation into a subject in need thereof. 35. The explanted donor heart from a minipig according to embodiment 34, wherein the minipig is a genetically-modified minipig. 36. The explanted donor heart from a minipig according to embodiment 35, wherein the genetically-modified minipig comprises a GGTA1 genetic modification. 37. The explanted donor heart from a minipig according to embodiment 36, wherein the genetically-modified minipig does not express alpha-1,3-galactosyltransferase (GalT-KO). 38. The explanted donor heart from a minipig according to embodiments 34 to 37, wherein the genetically-modified minipig is a GalT-KO minipig and: a. is wild type for a protein encoded by the porcine genes CMAH, β4GalNT, ASGR1, NAI-1540201368v1 Attorney Docket No.14648-049-228 and CD46; and/or b. does not express any recombinant proteins. 39. The explanted donor heart from a minipig according to embodiments 34 to 38, wherein the genetically-modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens. 40. The explanted donor heart from a minipig according to embodiments 34 to 39, wherein the genetically-modified minipig is GalT-KO and the GalT-KO is the only genetic modification in the minipig. 41. The explanted donor heart from a minipig according to any one of embodiments 34 to 40, wherein the normothermic coronary perfusion circuit is first established in situ by: a. placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart; and b. placing an outflow cannula in the proximal ascending aorta of the donor heart. 42. The explanted donor heart from a minipig according to any one of embodiments 34 to 41, wherein after the normothermic coronary perfusion circuit is established in situ, a. the superior vena cava (SVC) and the inferior vena cava (IVC) of the donor heart are ligated; b. the aorta of the donor heart is clamped; c. the donor heart is explanted while beating from the swine; and d. the beating explanted donor heart is maintained on the normothermic coronary perfusion circuit ex situ. 43. The explanted donor heart from a minipig according to any one of embodiments 34 to 42, wherein the explanted donor heart comprises substantially no ischemic tissue within at least 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or at least 12 hours. 44. A method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises transplanting a donor heart into the subject according to the methods of any one of embodiments 1 to 28. 45. A method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises use of the medical instrument of embodiments 29 to 33. 46. A method of treating a disease of the heart in a human subject in need thereof, wherein the NAI-1540201368v1 Attorney Docket No.14648-049-228 method comprises transplanting the explanted donor heart from a minipig of embodiments 34 to 43 into the subject. 47. A method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises transplanting a donor heart into the subject according to the methods of any one of embodiments 1 to 28. 48. A method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises use of the medical instrument of embodiments 29 to 33. 49. A method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises transplanting the explanted donor heart from a minipig of embodiments 34 to 43 into the subject. 50. The method of any one of embodiments 44 to 49, wherein the human subject has at least one condition that can be treated by receiving a heart transplantation. 51. The method of embodiment 50, wherein the at least one condition that can be treated by receiving a heart transplantation comprises advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof. 52. The method of any one of embodiments 44 to 51, wherein the human subject is a pediatric human subject. 8. EQUIVALENTS [00144] Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. [00145] All patents and publications mentioned in this specification are incorporated herein by reference in their entireties. From the foregoing description, it will be apparent that variations NAI-1540201368v1 Attorney Docket No.14648-049-228 and modifications can be made to the invention described herein to adopt it to various uses and conditions. Such embodiments are also within the scope of the following claims. NAI-1540201368v1

Claims

Attorney Docket No.14648-049-228 WHAT IS CLAIMED: 1. A method of transplanting a donor heart from a swine into a primate subject, wherein the method comprises: a. placing the primate subject on cardiopulmonary bypass (CPB) and surgically removing the heart of the primate subject; b. placing the donor heart on a normothermic coronary perfusion circuit and then explanting the donor heart while beating from the swine; c. implanting the beating explanted donor heart into the primate subject on CPB; d. removing the primate subject from CPB. 2. The method of claim 1, wherein the donor heart remains perfused and beating throughout the entire procedure. 3. The method of claim 1 or claim 2, wherein the beating explanted donor heart comprises substantially no ischemic tissue at the time of implantation into the primate subject on CPB. 4. The method of any one of claims 1 to 3, wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step b for a maximum of about 10 hours, optionally wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step b for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours. 5. The method of any one of claims 1 to 4, wherein the transplanted donor heart is substantially free of ischemic tissue at the time of the implanting step. 6. The method any one of claims 1 to 5, wherein the transplanted donor heart has a left ventricle ejection fraction (LVEF), a right ventricular (RV) systolic function, a pulmonary artery (PA) pressure, a right atrial (RA) pressure, a sinus rhythm, and/or a cardiac output NAI-1540201368v1 Attorney Docket No.14648-049-228 comparable to that of a heart in a healthy primate subject of the same age and species as the primate subject. 7. The method of any one of claims 1 to 6, wherein the normothermic coronary perfusion circuit is comprised of at least two drainage cannulas and at least one outflow cannula. 8. The method of claim 7, wherein one drainage cannula is placed in the right atrium and another drainage cannula is placed in the left ventricle via the apex or across the left atrium. 9. The method of claim 7, wherein the outflow cannula is placed in the proximal ascending aorta. 10. The method of any one of claims 1 to 9, wherein step b further comprises ligating the superior vena cava (SVC) and the inferior vena cava (IVC), and clamping the aorta distal to the outflow cannula before explanting the beating donor heart. 11. A method of explanting a donor heart while perfused and beating from a swine, wherein the method comprises: a. placing the donor heart on a normothermic coronary perfusion circuit, wherein the normothermic coronary perfusion circuit is established in situ by: i. placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart via the apex or across the left atrium; ii. placing an outflow cannula in the proximal ascending aorta of the donor heart; iii. ligating the superior vena cava (SVC) and the inferior vena cava (IVC) of the donor heart; iv. clamping the aorta of the donor heart; b. explanting the donor heart while beating from the swine; c. maintaining the beating donor heart on the normothermic coronary perfusion circuit ex situ. NAI-1540201368v1 Attorney Docket No.14648-049-228 12. The method of claim 11, further comprising priming the normothermic coronary perfusion circuit with plasmalyte A before placing the donor heart on the normothermic coronary perfusion circuit. 13. The method of claim 11 or 12, wherein the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ for a maximum of about 10 hours, optionally wherein the beating explanted donor heart is placed on a normothermic coronary perfusion circuit in step c for about 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours. 14. The method of any one of claims 11 to 13, wherein the beating donor heart is maintained on the normothermic coronary perfusion circuit ex situ in an organ reservoir. 15. The method of claim 14, wherein the organ reservoir is maintained at a temperature ranging from about 32°C to about 37°C. 16. The method of claim 14 or 15, wherein the beating donor heart maintained on the normothermic coronary perfusion circuit ex situ in an organ reservoir is placed in a sterile container suitable for transportation. 17. The method of any one of claims 11 to 16, wherein the beating donor heart is perfused with an oxygenated physiological solution. 18. The method of claim 17, wherein the oxygenated physiological solution comprises blood from the swine that sourced the donor heart. 19. The method of any one of claims 1 to 18, wherein the swine is a genetically- modified minipig. 20. The method of claim 19, wherein the genetically-modified minipig comprises a GGTA1 genetic modification. NAI-1540201368v1 Attorney Docket No.14648-049-228 21. The method of claim 19, wherein the genetically-modified minipig does not express alpha-1,3-galactosyltransferase (GalT-KO). 22. The method of any one of claims 1 to 21, wherein the genetically-modified minipig is a GalT-KO minipig and: a. is wild type for a protein encoded by the porcine genes CMAH, β4GalNT, ASGR1, and CD46; and/or b. does not express any recombinant proteins. 23. The method of any one of claims 1 to 21, wherein the genetically-modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens. 24. The method of any one of claims 1 to 21, wherein the genetically-modified minipig is GalT-KO and the GalT-KO is the only genetic modification in the minipig. 25. The method of any one of the preceding claims, wherein the primate subject is a human subject. 26. The method of claim 25, wherein the human subject has advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof. 27. The method of any one of claims 25 to 26, wherein the human subject is a pediatric human subject. 28. The method of any one of claims 1 to 27, wherein the method is performed in the absence of cardioplegia. 29. A medical instrument comprising a. at least two drainage cannulas and at least one outflow cannula, wherein one drainage cannula is suitable to be placed in the right atrium of a donor heart and another drainage cannula is suitable to be placed in the left ventricle of the donor heart; NAI-1540201368v1 Attorney Docket No.14648-049-228 b. an outflow cannula, wherein the outflow cannula is suitable to be placed in the proximal ascending aorta of the donor heart; and c. a pump to maintain homeostasis with a perfusion solution; wherein the medical instrument is specifically adapted to perform normothermic machine perfusion on a heart from a minipig. 30. The medical instrument of claim 29, further comprising an organ reservoir. 31. The medical instrument of claim 29 or claim 30, further comprising a membrane oxygenator, a leukocyte filter, an arterial filter, a heater-cooler unit, an autonomous power unit, or any combination thereof. 32. The medical instrument of any one of claims 29 to 31, further comprising a sterile container, wherein the sterile container is suitable for transportation of a donor heart that is perfused and beating during transport. 33. The medical instrument of any one of claims 29 to 32, further comprising a collection chamber, wherein the collection chamber is suitable for collecting lost blood during transportation of a donor heart. 34. An explanted donor heart from a minipig, wherein the donor heart is connected to an ex situ normothermic coronary perfusion circuit prior to transplantation into a subject in need thereof. 35. The explanted donor heart from a minipig according to claim 34, wherein the minipig is a genetically-modified minipig. 36. The explanted donor heart from a minipig according to claim 35, wherein the genetically-modified minipig comprises a GGTA1 genetic modification. 37. The explanted donor heart from a minipig according to claim 36, wherein the genetically-modified minipig does not express alpha-1,3-galactosyltransferase (GalT-KO). NAI-1540201368v1 Attorney Docket No.14648-049-228 38. The explanted donor heart from a minipig according to claims 34 to 37, wherein the genetically-modified minipig is a GalT-KO minipig and: a. is wild type for a protein encoded by the porcine genes CMAH, β4GalNT, ASGR1, and CD46; and/or b. does not express any recombinant proteins. 39. The explanted donor heart from a minipig according to claims 34 to 38, wherein the genetically-modified minipig is GalT-KO but the minipig is otherwise wildtype for other surface antigens. 40. The explanted donor heart from a minipig according to claims 34 to 39, wherein the genetically-modified minipig is GalT-KO and the GalT-KO is the only genetic modification in the minipig. 41. The explanted donor heart from a minipig according to any one of claims 34 to 40, wherein the normothermic coronary perfusion circuit is first established in situ by: a. placing one drainage cannula in the right atrium of the donor heart and placing another drainage cannula in the left ventricle of the donor heart; and b. placing an outflow cannula in the proximal ascending aorta of the donor heart. 42. The explanted donor heart from a minipig according to any one of claims 34 to 41, wherein after the normothermic coronary perfusion circuit is established in situ, a. the superior vena cava (SVC) and the inferior vena cava (IVC) of the donor heart are ligated; b. the aorta of the donor heart is clamped; c. the donor heart is explanted while beating from the swine; and d. the beating explanted donor heart is maintained on the normothermic coronary perfusion circuit ex situ. 43. The explanted donor heart from a minipig according to any one of claims 34 to 42, wherein the explanted donor heart comprises substantially no ischemic tissue within at least NAI-1540201368v1 Attorney Docket No.14648-049-228 15 minutes, 30 minutes, 45 minutes, one hour, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, nine hours, 10 hours, 11 hours, or at least 12 hours. 44. A method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises transplanting a donor heart into the subject according to the methods of any one of claims 1 to 28. 45. A method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises use of the medical instrument of claims 29 to 33. 46. A method of treating a disease of the heart in a human subject in need thereof, wherein the method comprises transplanting the explanted donor heart from a minipig of claims 34 to 43 into the subject. 47. A method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises transplanting a donor heart into the subject according to the methods of any one of claims 1 to 28. 48. A method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises use of the medical instrument of claims 29 to 33. 49. A method of preventing perioperative cardiac xenograft dysfunction (PCXD) in a human subject in need of a cardiac xenograft, wherein the method comprises transplanting the explanted donor heart from a minipig of claims 34 to 43 into the subject. 50. The method of any one of claims 44 to 49, wherein the human subject has at least one condition that can be treated by receiving a heart transplantation. 51. The method of claim 50, wherein the at least one condition that can be treated by receiving a heart transplantation comprises advanced heart failure, cardiomyopathy, arrhythmia, congenital heart disease, coronary artery disease, valvular heart disease, or any condition thereof. NAI-1540201368v1 Attorney Docket No.14648-049-228 52. The method of any one of claims 44 to 51, wherein the human subject is a pediatric human subject. NAI-1540201368v1
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