Thymoliver and methods of constructing thereof

Abstract

The present disclosure provides porcine thymolivers, methods of making thereof, and methods of using thereof. The present disclosure further provides methods of treating a disease (e.g., a liver disease) in a human subject in need thereof comprising transplanting of a porcine thymoliver.

Description

Attorney Docket No.14648-044-228 THYMOLIVER AND METHODS OF CONSTRUCTING THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application No. 63 / 648,893 filed May 17, 2024, the content of which is incorporated by reference in its entirety herein, and to which priority is claimed. 1. GOVERNMENT LICENSE RIGHTS

[0002] This invention was made with government support under AI045897 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. 2. FIELD

[0003] The present disclosure provides porcine thymolivers, methods of making thereof, and methods of using thereof. The present disclosure further provides methods of treating a disease (e.g., a liver disease) in a human subject in need thereof comprising transplanting of a porcine thymoliver. 3. BACKGROUND

[0004] Xenotransplantation is a rapidly developing area of interest, with pre-clinical studies in deceased human donors already underway (see, e.g., Moazami et al., Nat Med 29, 1989-1997 (2023); Montgomery et al., N Engl J Med 386, 1889-1898 (2022)). There is an ongoing and unmet need for liver xenografts that are appropriate for patients with liver disease. The success of liver xenotransplantation has historically lagged behind other solid-organ xenotransplantations (see, e.g., Cross-Najafi AA et al., Frontiers in Immunology (2022) Feb 23;13:827535). One of the many obstacles preventing the successful clinical use of liver xenotransplants is, in part, rejection due to immune reactions to the graft. As such, overcoming immunologic barriers to liver xenotransplantation is needed in the field. 4. SUMMARY

[0005] In an aspect, the present disclosure provides an isolated porcine thymoliver comprising: (a) a porcine liver; and (b) porcine thymic tissue, wherein the thymic tissue adheres to the liver. NAI-5000951688v1 1Attorney Docket No.14648-044-228

[0006] In certain embodiments, the thymic tissue adheres to the liver parenchyma and / or the surface of the liver. In certain embodiments, the thymic tissue adheres to at least 25%, 30%, 35%, 40%, 45%, 50% of the surface of the liver.

[0007] In certain embodiments, the thymic tissue adheres to at least one lobe of the liver. In certain embodiments, the at least one lobe of the liver is selected from the group consisting of right lobe, left lobe, caudate lobe, and quadrate lobe.

[0008] In certain embodiments, the thymic tissue adheres to at least one segment of the liver. In certain embodiments, the at least one segment of the liver is selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, the at least one segment of the liver is segment IV.

[0009] In certain embodiments, the liver and / or thymic tissue are from highly inbred porcine donors. In certain embodiments, the liver and / or thymic tissue are from a minipig. In certain embodiments, the liver and / or thymic tissue are from the same individual minipig. In certain embodiments, the minipig is a genetically modified minipig.

[0010] In certain embodiments, the genetically modified minipig has at least one genetic modification. In certain embodiments, the least one genetic modification comprises: (a) a mutation in the GGTA1 gene; (b) a mutation in the CMAH gene; (c) a mutation in the gene. In certain embodiments, genetically modified minipig comprises at least one genetic modification that reduces or eliminates expression of a protein encoded by the GGTA1 gene, the CMAH gene, and the gene. Non-limiting additional examples of the genetic modification that potential benefit for xenotransplantation include transgenic expression of human decay-accelerating factor (DAF), human CD46, human CD59, human CD39, human thrombomodulin, human endothelial protein C receptor (EPCR), human HO-1, and human A20, anti-CD2, CTLA4Ig, human CD47, PD-L1, FasL, and Class I MHC (see e.g. Sykes et al., Nat Rev Nephrol.2022 Dec;18(12):745-761). In certain embodiments, the genetically modified minipig donor comprises at least one genetic modification that results in expression of a protein for a porcine CTLA4 fused to a hinge and CH2 / CH3 regions of human IgG1 (pCTLA4-Ig). In certain embodiments, the genetically modified minipig donor comprises at least one genetic modification that results in the inactivation of porcine endogenous retrovirus (PERV). In certain embodiments, the genetically modified minipig comprises a GGTA1 genetic modification. In NAI-5000951688v1 2Attorney Docket No.14648-044-228 certain embodiments, the genetically modified minipig does not express alpha-1,3- galactosyltransferase (GalT-KO).

[0011] In certain embodiments, the thymic tissue is fully neovascularized. In certain embodiments, the thymic tissue has a cell density equal to that of a porcine thymic tissue that is not included in the isolated porcine thymoliver.

[0012] In certain embodiments, the thymic tissue is capable of supporting thymopoiesis. In certain embodiments, the thymic tissue comprises CD3-CD4+CD8+double-positive (DP) thymocytes. In certain embodiments, the thymic tissue comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD3-CD4+CD8+double- positive (DP) thymocytes.

[0013] In certain embodiments, the thymoliver has at least one genetic modification. In certain embodiments, the thymoliver does not express alpha-1,3-galactosyltransferase (GalT- KO).

[0014] In another aspect, the present disclosure provides a swine comprising a thymoliver. In certain embodiments, the swine can comprise any thymoliver as described in the present disclosure.

[0015] In another aspect, the present disclosure provides a method of treating a disease of the liver in a human subject in need thereof. In certain embodiments, the present disclosure provides a method of treating a disease of the liver in a human subject in need thereof wherein the method comprises transplanting a thymoliver as disclosed herein into the human subject.

[0016] In certain embodiments, the human subject has or is suspected of having end-stage liver disease. In certain embodiments, a disease of the liver in the human subject is selected from the group consisting of acute hepatic necrosis, alcohol-related liver disease, autoimmune hepatitis (AIH), biliary atresia, bile duct cancer, liver cancer, metabolic disease, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), viral hepatitis, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC).

[0017] In another aspect, the present disclosure provides a method of creating the thymoliver disclosed herein. In certain embodiments, the present disclosure provides a method of creating the thymoliver disclosed herein wherein the method comprises: (a) obtaining thymic tissue from a porcine donor animal; and (b) transplanting the thymic tissue obtained in step (a) into the liver of a recipient animal. NAI-5000951688v1 3Attorney Docket No.14648-044-228

[0018] In certain embodiments, the thymic tissue is obtained using cervical thymectomy.

[0019] In certain embodiments, the transplanting step (b) is conducted by inserting the thymic tissue into the liver of the recipient animal intraparenchymally. In certain embodiments, at least 1 mm3of the thymic tissue is transplanted into the liver of the recipient animal intraparenchymally. In certain embodiments, the thymic tissue is transplanted into the liver of the recipient animal intraparenchymally about 1 mm to 5 mm deep into the liver parenchyma. In certain embodiments, thymic tissue is transplanted into the liver of the recipient animal intraparenchymally in at least one location within the liver parenchyma. In certain embodiments, thymic tissue is transplanted into the liver of the recipient animal intraparenchymally in one, two, three, four, five six, seven, eight, nine, ten, or more locations within the liver parenchyma.

[0020] In certain embodiments, the transplanting step (b) is conducted by inserting the thymic tissue into a wedge resection within the liver of the recipient animal. In certain embodiments, about 1 cm3to 2 cm3liver parenchyma is resected. In certain embodiments, the thymic tissue inserted into the wedge resection comprises at least half of a thymic lobe. In certain embodiments, the transplanting step (b) can further comprise suturing the thymic tissue in the wedge resection.

[0021] In certain embodiments, the transplanting step (b) is conducted by inserting the thymic tissue into an incision within the liver of the recipient animal. In certain embodiments, the thymic tissue inserted into the incision comprises at least half of a thymic lobe. In certain embodiments, the transplanting step (b) can further comprise suturing the thymic tissue in the incision.

[0022] In certain embodiments, the transplanting step (b) comprises inserting the thymic tissue into the liver of the recipient animal intraparenchymally, into a wedge resection, and / or into an incision within the liver of the recipient animal.

[0023] In certain embodiments, the thymoliver is harvested from the recipient animal at least 8 weeks after the transplanting step (b). In certain embodiments, the thymoliver is harvested from the recipient animal at least 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the transplanting step (b).

[0024] In certain embodiments, the thymus tissue of the thymoliver harvested from the recipient animal after the transplanting step (b) is a vascularized thymus tissue. NAI-5000951688v1 4Attorney Docket No.14648-044-228

[0025] In certain embodiments, the thymus tissue of the thymoliver harvested from the recipient animal after the transplanting step (b) is adhered to the liver parenchyma. In certain embodiments, the thymoliver comprises at least about 25%, 30%, 35%, 40%, 45%, 50% thymus tissue adhered to the liver parenchyma.

[0026] In certain embodiments, the thymus tissue of the thymoliver harvested from the recipient animal after the transplanting step (b) is adhered to the liver surface. In certain embodiments, the thymoliver comprises at least about 25%, 30%, 35%, 40%, 45%, 50% thymus tissue adhered to the liver surface. 5. BRIEF DESCRIPTION OF THE FIGURES

[0027] FIG.1 depicts representative images of thymoliver construction in a porcine donor. Solid arrows point to the locations where autologous thymic tissue was seeded / inserted intraparenchymally (IP) into the porcine donor liver. Dashed arrows point to the locations where autologous thymic tissue was sutured within a wedge resection of porcine donor liver.

[0028] FIGS.2A-2C depict representative images of a macroscopic overview of the thymoliver harvested 8-weeks after construction. FIG.2A shows a macroscopic view of the isolated thymoliver. Solid arrows point to the locations where autologous thymic tissue was seeded / inserted intraparenchymally (IP) into the porcine donor liver. Dashed arrows point to the locations where autologous thymic tissue was sutured within a wedge resection of porcine donor liver. FIG.2B shows a macroscopic view of thymic tissue adhered to the liver tissue of the thymoliver where the thymic tissue was sutured within a wedge resection. FIG.2C shows a macroscopic view of thymic tissue adhered to the liver tissue of the thymoliver where the thymic tissue was seeded / inserted intraparenchymally (IP).

[0029] FIG.3 depicts a flow cytometric analysis performed on the thymic tissue of the thymoliver, showing the presence of CD3-CD4+CD8+Double Positive (DP) cells.

[0030] FIGS.4A-4B depict representative images of histopathology of the thymoliver recovered 8-weeks after thymoliver construction. FIG.4A shows a histopathological view of thymic tissue seeded / inserted intraparenchymally (IP) and liver tissue in the harvested thymoliver. FIG.4B shows a histopathological view of thymic tissue sutured within a wedge resection and liver tissue in the harvested thymoliver.

[0031] FIG.5 depicts percentage of pig CD45+cells in recipient after pig-to-baboon liver xenotransplantations (LXTx) or intra-bone marrow transplantation (IBBMTx). NAI-5000951688v1 5Attorney Docket No.14648-044-228 6. DETAILED DESCRIPTION

[0032] Provided herein is a porcine thymoliver, methods of making thereof, and methods of using thereof.

[0033] In certain embodiments, provided herein is a porcine thymoliver (see Section 6.1). In certain embodiments, a porcine thymoliver provided herein comprises a porcine liver and porcine thymic tissue. A porcine donor that provides at least one of the tissues (e.g., liver tissue and / or thymic tissue) that a porcine thymoliver disclosed herein is comprised of is described in Section 6.2 below. In certain embodiments, provided herein is a swine that comprises a thymoliver (i.e., a thymoliver swine) as described in Section 6.3 below.

[0034] In certain embodiments, provided herein are methods of making a porcine thymoliver (see Section 6.4). A porcine thymoliver of the present disclosure is generated, in part, from thymic tissue obtained from a porcine donor according to the methods described in Section 6.4.1 below. A porcine thymoliver remains in the porcine donor for further development until donor hepatectomy (see Section 6.4.3).

[0035] In certain embodiments, provided herein are methods of using a porcine thymoliver (see Section 6.5). A porcine thymoliver of the present disclosure can be transplanted into a subject in need thereof according to the methods described in Section 6.5.1 below. A subject suitable for receiving a porcine thymoliver disclosed herein is described in Section 6.5.3 below. Such a subject in need of a porcine thymoliver of the present disclosure can have, be suspected of having, or demonstrate at least one symptom of a disease described in Section 6.5.4 below. 6.1 Porcine Thymoliver

[0036] In certain embodiments, the present disclosure provides a porcine thymoliver. In certain embodiments, a thymoliver of the present disclosure comprises a modified liver organ prepared by insertion of thymic tissue harvested from a donor into a donor’s liver parenchyma. In certain embodiments, a porcine thymoliver provided herein comprises porcine liver tissue and porcine thymic tissue. In certain embodiments, a porcine thymoliver provided herein comprises a whole porcine liver and porcine thymic tissue. In certain embodiments, a porcine thymoliver provided herein comprises a partial porcine liver and porcine thymic tissue. In certain embodiments, a porcine thymoliver provided herein comprises at least one lobe of a porcine liver and porcine thymic tissue. In certain embodiments, a porcine thymoliver provided herein comprises at least one segment of a porcine liver and porcine thymic tissue. In certain NAI-5000951688v1 6Attorney Docket No.14648-044-228 embodiments, a porcine thymoliver disclosed herein is an isolated porcine thymoliver. In certain embodiments, an isolated porcine thymoliver provided herein comprises a porcine thymoliver removed from the porcine donor. Porcine donors and tissues derived thereof are further described in Section 6.2 and methods of making a porcine thymoliver disclosed herein are described in Section 6.4.

[0037] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the liver. In certain embodiments, the thymic tissue adheres to the liver by the development of a fibrous tissue between the two tissues. In certain embodiments, the thymic tissue adheres to the liver by the development of a vascular tissue between the two tissues. In certain embodiments, the thymic tissue adheres to the liver by the development of at least one blood vessel connecting the two tissues. In certain embodiments, the thymic tissue adheres to the liver by engraftment of the thymic tissue into the liver tissue. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the liver parenchyma. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface of the liver and to the liver parenchyma.

[0038] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least one lobe of the liver. The liver has four lobes – right, left, caudate, and quadrate. The quadrate lobe is located on the inferior surface of the right lobe whereas the caudate lobe is located between the left and right lobes in an anterior and superior location. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least two lobes of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least three lobes of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of four lobes of the liver. NAI-5000951688v1 7Attorney Docket No.14648-044-228

[0039] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the right, left, caudate, and / or quadrate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the right, left, and / or caudate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the right, left, and / or quadrate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the right, caudate, and / or quadrate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the left, caudate, and / or quadrate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the right and / or left lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the right and / or quadrate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the right and / or caudate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the caudate and / or quadrate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the left and / or quadrate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the left and / or caudate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the right lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and NAI-5000951688v1 8Attorney Docket No.14648-044-228 porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the left lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the caudate lobe of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of the quadrate lobe of the liver.

[0040] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least one segment of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of more than one segment of the liver. The Couinaud classification system divides the liver into eight functionally independent segments, each with a center consisting of a portal vein, hepatic artery branches, and bile duct drainage (see, e.g., Fasel and Schenk, J Clin Imaging Sci (2013) Oct 29;3:48). The numbering system consists of the following segments: (I) caudate lobe; (II) superior subsegment of the lateral segment; (III) inferior subsegment of the lateral segment; (IV) superior (IVa) and inferior (IVb) subsegment of the medial segment; (V) inferior subsegment of the anterior segment; (VI) inferior subsegment of the posterior segment; (VII) superior subsegment of the posterior segment; and (VIII) superior subsegment of the anterior segment.

[0041] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segments I, II, III, IV, V, VI, VII, and / or VIII of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least one segment of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least two segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least three segments of the liver selected from the group consisting of segments NAI-5000951688v1 9Attorney Docket No.14648-044-228 I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least four segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least five segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least six segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of at least seven segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segments I, II, III, IV, V, VI, VII, and VIII of the liver.

[0042] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment I of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment II of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment III of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment V of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment VI of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment VII of the liver. In certain embodiments, a porcine NAI-5000951688v1 10Attorney Docket No.14648-044-228 thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment VIII of the liver.

[0043] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment IV of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment IVa of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment IVb of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface and / or parenchyma of segment Iva and / or IVb of the liver.

[0044] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue adheres to the surface of the liver. In certain embodiments, the thymic tissue adheres to the surface of the liver by the development of a fibrous tissue between the two tissues. In certain embodiments, the thymic tissue adheres to the surface of the liver by engraftment into the liver tissue. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue partially covers the surface of the liver.

[0045] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue covers at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the surface of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue covers more than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the surface of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue covers about 1% to about 5%, about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50% of the surface of the liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a NAI-5000951688v1 11Attorney Docket No.14648-044-228 porcine liver and porcine thymic tissue wherein the thymic tissue covers about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the surface of the liver.

[0046] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue is neovascularized. As used herein, “neovascularized” or “neovascularization” refers to the formation of new blood vessels in a tissue (e.g., a thymic tissue). In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue is partially neovascularized. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue is fully neovascularized. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% neovascularized tissue. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises about 50% to about 99%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 90% to about 95%, or about 95% to about 99% neovascularized tissue. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% neovascularized tissue.

[0047] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue has a cell density comparable to that of a porcine thymic tissue that is not included in the porcine thymoliver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue has a cell density equal to that of a porcine thymic tissue that is not included in the porcine thymoliver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue has a cell density that is about equal to a porcine thymic tissue that is not included in the porcine thymoliver.

[0048] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue has a cell density within at least about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 5% NAI-5000951688v1 12Attorney Docket No.14648-044-228 to about 6%, about 6% to about 7%, about 8% to about 9%, or about 9% to about 10% to that of the cell density of a porcine thymic tissue that is not included in the porcine thymoliver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue has a cell density within at least about or about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% or more than about 10% to that of the cell density of a porcine thymic tissue that is not included in the porcine thymoliver.

[0049] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue is obtained from the porcine donor of the porcine liver. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue is fetal thymus tissue. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue is provided from neonatal porcine donor. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine fetal thymus tissue, wherein the fetal thymus tissue is provided from at least one fetal pig aged about 30 days to about 115 days from date of fertilization. Obtaining fetal and / or neonatal thymic tissue from a porcine donor can be performed using methods as described in U.S. Provisional Application No.63 / 768,408 filed on 03 / 07 / 2025. In certain embodiments, the fetal porcine thymic tissue used for transplanting according to the method disclosed herein is obtained from an inbred herd of fetal porcine donor (see Section 6.2) and is maintained in a medium at physiological conditions that comprising immunosuppressive agents to remove the residual T cells from the fetal thymus. Non-limiting types of immunosuppressive agents suitable for use in the method are described in Section 6.5.2(a). Without being bound by theory, immunosuppression may not be needed if the thymus is obtained from a highly inbred herd of fetal porcine donor (e.g., a herd with a COI of at least 0.9, or at least 0.95, or at least 0.98 (see Section 6.2)).

[0050] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue is capable of supporting thymopoiesis. Thymopoiesis is the process within the thymus that transforms thymocytes into mature T cells. In order for a thymocyte to differentiate into a mature T cell, the thymocyte goes through several maturational stages including the progression and maturation to double-positive (DP) thymocytes NAI-5000951688v1 13Attorney Docket No.14648-044-228 expressing both CD4 and CD8 co-receptors. Accordingly, without wishing to be bound by any particular theory, the presence of CD3-CD4+CD8+double-positive (DP) thymocytes in the porcine thymic tissue disclosed herein is indicative of a tissue that is capable of supporting thymopoiesis. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises CD3-CD4+CD8+double-positive (DP) thymocytes. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises at least about or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD3-CD4+CD8+DP thymocytes. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 40% to about 60%, about 50% to about 70%, or about 40% to about 50% CD3-CD4+CD8+DP thymocytes.

[0051] In certain embodiments, a porcine thymoliver disclosed herein comprises at least one gene modification. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the liver comprises at least one gene modification. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the liver comprises more than one gene modification. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the liver comprises at least two gene modifications. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the liver comprises more than two gene modifications.

[0052] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises at least one gene modification. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises more than one gene modification. In certain embodiments, a porcine thymoliver disclosed herein comprises a NAI-5000951688v1 14Attorney Docket No.14648-044-228 porcine liver and porcine thymic tissue wherein the thymic tissue comprises at least two gene modifications. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises more than two gene modifications.

[0053] In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the liver comprises the gene modification(s) of the porcine donor. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises the gene modification(s) of the porcine donor. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the liver comprises the gene modification(s) of any of the porcine donors described in Section 6.2. In certain embodiments, a porcine thymoliver disclosed herein comprises a porcine liver and porcine thymic tissue wherein the thymic tissue comprises the gene modification(s) of any of the porcine donors described in Section 6.2. In certain embodiments, a porcine thymoliver disclosed herein comprises at least one gene modification in the gene encoding alpha-1,3-galactosyltransferase. In certain embodiments, a porcine thymoliver disclosed herein does not express alpha-1,3- galactosyltransferase (i.e., GalT-KO). 6.2 Porcine Donors

[0054] In certain embodiments, a porcine thymoliver provided herein comprises porcine liver tissue and porcine thymic tissue provided from a porcine donor. 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 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 NAI-5000951688v1 15Attorney Docket No.14648-044-228 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.

[0055] In certain embodiments, the breed of a porcine donor for use in the present disclosureis 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.

[0056] In certain embodiments, a porcine donor of the present disclosure can be a wholly orpartially inbred swine. In certain embodiments, a porcine donor of the present disclosure can be a highly inbred swine. The determination of how inbred a swine is can be determined by calculating the coefficient of inbreeding (COI). The coefficient of inbreeding (f) of an individual swine can be calculated by formula (I): (I), wherein n is the number of of inbreeding of the common ancestor of X’sruns from an individual up to the common ancestor through one parent and back down to the other parent, without going NAI-5000951688v1 16Attorney Docket No.14648-044-228 through any individual twice. In certain embodiments, a porcine donor of the present disclosure can be an inbred swine having a COI of less than about 25, less than about 20, less than about 15, or less than about 10. In certain embodiments, a porcine donor of the present disclosure can be an inbred swine having a COI of less than about 1 to about 2, about 2 to about 3, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, or about 9 to about 10. In certain embodiments, a porcine donor of the present disclosure can be an inbred swine having a COI of less than about 0.1 to about 0.2, about 0.2 to about 0.3, about 0.3 to about 0.4, about 0.4 to about 0.5, about 0.5 to about 0.6, about 0.6 to about 0.7, about 0.7 to about 0.8, about 0.8 to about 0.9, or about 0.9 to about 1. In certain embodiments, a porcine donor of the present disclosure can be highly inbred swine having a COI of less than about 1.

[0057] 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 embodiments, a porcine donor of the present disclosure is from a herd of wholly or partially inbred swine (e.g., highly 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).

[0058] In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a porcine donor disclosed herein. In certain embodiments, a thymoliver NAI-5000951688v1 17Attorney Docket No.14648-044-228 disclosed herein comprises liver tissue and / or thymic tissue from the same porcine donor disclosed herein. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different porcine donors from the same breed. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different porcine donors from the same herd (e.g., a highly inbred herd). In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different porcine donors that are major histocompatibility complex (MHC) (i.e., swine leukocyte antigen (SLA))-matched.

[0059] In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a minipig. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from the same minipig disclosed herein. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different minipigs from the same breed. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different minipigs from the same herd (e.g., a highly inbred herd). In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different minipigs that are major histocompatibility complex (MHC) (i.e., swine leukocyte antigen (SLA))-matched

[0060] 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 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 liver and / or thymus). In certain embodiments, a genetically modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein is higher compared to that of a non-genetically modified porcine donor. In certain embodiments, a genetically modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein is higher in at least one organ, tissue, or cell type (e.g., in the liver and / or thymus) compared to the at least one organ, tissue or cell type of a non-genetically modified porcine donor. In certain embodiments, a genetically modified porcine donor comprises at least NAI-5000951688v1 18Attorney Docket No.14648-044-228 one genetic modification in which the expression of a porcine protein is lower compared to that of a non-genetically modified porcine donor. In certain embodiments, a genetically modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein is lower in at least one organ, tissue, or cell type (e.g., in the liver and / or thymus) compared to the at least one organ, tissue or cell type of a non-genetically modified porcine donor. In certain embodiments, a genetically modified porcine donor comprises at least one genetic modification in which a human protein is expressed in the genetically modified porcine donor. In certain embodiments, a genetically modified porcine donor comprises at least one genetic modification in which a human protein is expressed in at least one organ, tissue, or cell type (e.g., in the liver and / or thymus) of the genetically modified porcine donor. In certain embodiments, a genetically modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein is prevented and at least one genetic modification in which a human protein is expressed in the genetically modified porcine donor. 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 liver and / or thymus) and at least one genetic modification in which a human protein is expressed in at least one organ, tissue or cell type (e.g., in the liver and / or thymus) of the genetically modified porcine donor.

[0061] In certain embodiments, a genetically modified porcine donor (e.g., a transgenic, chimeric, or mosaic swine) comprises at least one genetic modification wherein the genetic modification comprises a mutation of a gene. A genetically modified porcine donor known in the art can be suitable for use in the present disclosure as such, but not limited to, those described in Denner, J Transplant Technol Res (2014) 4(2):133; Wei et al., Front Cell Dev Biol (2022) Oct 17;10:1031812; and Xi et al., Front Cell Dev Biol (2023) Jan 12;10:1093534, the disclosures of which are incorporated herein by reference. In certain embodiments, a genetically modified porcine donor (e.g., a minipig) comprises at least one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene, the CMAH gene, and / or the gene. In certain embodiments, a genetically modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein encoded by the GGTA1 gene, the CMAH gene, the gene, the ASGR1 gene, and / or the gene is prevented (i.e., knocked out, or “KO”) in the genetically modified porcine donor. In certain embodiments, a NAI-5000951688v1 19Attorney Docket No.14648-044-228 genetically modified porcine donor comprises at least one genetic modification in which the expression of a porcine protein encoded by the GGTA1 gene, the CMAH gene, and / or the gene is prevented in at least one organ, tissue, or cell type (e.g., in the liver and / or thymus) of the genetically modified porcine donor. Non-limiting additional examples of the genetic modification that potential benefit for xenotransplantation include transgenic expression of human decay-accelerating factor (DAF), human CD46, human CD59, human CD39, human thrombomodulin, human endothelial protein C receptor (EPCR), human HO-1, and human A20, anti-CD2, CTLA4Ig, human CD47, PD-L1, FasL, and Class I MHC (see e.g. Sykes et al., Nat Rev Nephrol.2022 Dec;18(12):745-761).. In certain embodiments, a genetically modified porcine donor comprises at least one genetic modification that results in expression of a protein for a porcine CTLA4 fused to a hinge and CH2 / CH3 regions of human IgG1 (pCTLA4-Ig) in the genetically modified porcine donor. In certain embodiments, a genetically modified porcine donor comprises at least one genetic modification that results in the inactivation of porcine endogenous retrovirus (PERV) (i.e., a PERV knockout). In certain embodiments, a genetically modified porcine donor (e.g., a transgenic, chimeric, or mosaic swine) comprises at least one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene..

[0062] In certain embodiments, a genetically modified porcine donor is a genetically modified minipig. In certain embodiments, a genetically modified minipig comprises at least one genetic modification wherein the genetic modification comprises a mutation in the GGTA1 gene. 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.

[0063] 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; and 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 NAI-5000951688v1 20Attorney Docket No.14648-044-228 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.

[0064] In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified porcine donor (e.g., a transgenic, chimeric, or mosaic swine) disclosed herein. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from the same genetically modified porcine donor. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different genetically modified porcine donors from the same breed. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different genetically modified porcine donors from the same herd. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified porcine donor wherein the genetic modification comprises a mutation of a gene. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified porcine donor wherein the genetic modification comprises a mutation in the GGTA1 gene. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified porcine donor that does not express alpha-1,3- galactosyltransferase.

[0065] In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified minipig (e.g., a transgenic, chimeric, or mosaic minipig) disclosed herein. In certain embodiments, a thymoliver disclosed herein comprises NAI-5000951688v1 21Attorney Docket No.14648-044-228 liver tissue and / or thymic tissue from the same genetically modified minipig. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different genetically modified minipigs from the same breed. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from two different genetically modified minipigs from the same herd. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified minipig wherein the genetic modification comprises a mutation of a gene. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified minipig wherein the genetic modification comprises a mutation in the GGTA1 gene. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified minipig that does not express alpha-1,3-galactosyltransferase.

[0066] In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a male or female porcine donor (e.g., a minipig). In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified male porcine donor (e.g., a minipig). In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a male GalT-KO minipig. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a genetically modified female porcine donor (e.g., a minipig). In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a female GalT-KO minipig.

[0067] In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a porcine donor (e.g., a minipig) that is at least about 6 weeks of age. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a porcine donor (e.g., a minipig) that is about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, or more than about 10 weeks of age. NAI-5000951688v1 22Attorney Docket No.14648-044-228

[0068] In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue from genetically modified minipig that is 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a GalT-KO minipig that is about 8 weeks of age.

[0069] In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a porcine donor (e.g., a minipig) wherein the porcine donor weight is 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.

[0070] In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 thymoliver disclosed herein comprises liver tissue and / or thymic tissue 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 NAI-5000951688v1 23Attorney Docket No.14648-044-228 more than about 12 kg. In certain embodiments, a thymoliver disclosed herein comprises liver tissue and / or thymic tissue from a GalT-KO minipig wherein the weight of the GalT-KO minipig ranges from about 8 kg to 10 kg. 6.3 Thymoliver Swine

[0071] In certain embodiments, the present disclosure provides a swine that comprises a thymoliver (i.e., a thymoliver swine). In certain embodiments, a thymoliver swine is comprised of a thymoliver described in Section 6.1. In certain embodiments, a thymoliver swine is comprised of a thymoliver generated from tissues from a porcine donor described in Section 6.2. In certain embodiments, a thymoliver swine is a porcine donor described in Section 6.2. In certain embodiments, a thymoliver swine is comprised of a thymoliver created according to the methods described in Section 6.4. In certain embodiments, a method of xenotransplantation compromises harvesting of the thymoliver from a thymoliver swine and transplanting the thymoliver into a subject in need thereof (see Section 6.5). 6.4 Methods of Making

[0072] In certain embodiments, the present disclosure provides a method of creating a thymoliver described herein (see Section 6.1). In certain embodiments, a method of creating a thymoliver of the present disclosure comprises (a) obtaining thymic tissue from a porcine donor and (b) transplanting the thymic tissue obtained in step (a) into the liver of a recipient animal. Methods of obtaining thymic tissue from a porcine donor are described in Section 6.4.1 and methods of transplanting the obtained thymic tissue into the liver of a recipient animal are described in Section 6.4.2 below. In certain embodiments, the thymoliver resulting from steps (a) and (b) is incubated in the recipient animal for a period of time prior to harvesting for use. Methods of incubating a thymoliver created by methods of the present disclosure prior to harvesting are described in Section 6.4.3. 6.4.1 Obtaining Thymic Tissue from a Porcine Donor

[0073] In certain embodiments, a method of creating a thymoliver of the present disclosure comprises obtaining thymic tissue from a porcine donor (e.g., a minipig) described herein (see Section 6.2). In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a thymectomy. A “thymectomy” as referred to herein is a surgical method to remove some or all of the thymus. The thymus is a soft organ with two lobes that is located anterior to the ascending aorta and posterior to the sternum. Any suitable method toward performing a NAI-5000951688v1 24Attorney Docket No.14648-044-228 thymectomy known in the art can be used to obtain thymic tissue from a porcine donor disclosed herein (see, e.g., Yamada et al., Methods Mol Biol (2020) 2110:151-171).

[0074] In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a thymectomy to remove one or both lobes of the thymus. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a partial or a total (i.e., 100%) thymectomy. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a partial thymectomy wherein at least about or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of the thymus is removed from the porcine donor.

[0075] In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a cervical or thoracic thymectomy. Cervical and thoracic thymic tissue have similar stromal organization. Cervical thymic tissue is typically lateral to the groove between the trachea and the underlying esophagus whereas thoracic thymic tissue is adjacent to parathyroid tissue and surrounded by thyroid tissue (see, e.g., Dooley et al., J Immunol (2006) Jun 1;176(11):6484-90). In certainmethod of obtaining thymic tissue from a porcine donor comprises a thoracic thymectomy. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a partial or a total (i.e., 100%) thoracic thymectomy. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a partial thoracic thymectomy wherein at least about or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of thoracic thymic tissue is removed from the porcine donor. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a partial thoracic thymectomy and a total cervical thymectomy. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a cervical thymectomy. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a partial or a total (i.e., 100%) cervical thymectomy. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a partial cervical thymectomy wherein at least about or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of cervical thymic tissue is removed from the porcine donor. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a cervical thymectomy wherein one or both cervical thymus lobes are removed from the porcine donor. In certain embodiments, a method of obtaining thymic tissue from a porcine donor comprises a total cervical thymectomy. NAI-5000951688v1 25Attorney Docket No.14648-044-228

[0076] In certain embodiments, thymic tissue obtained from a porcine donor according to methods herein is placed in a suitable physiological solution until transplanted into a recipient animal (e.g., into the liver of the recipient animal). Suitable physiological solutions include, but are not limited to, normal saline (about 0.9% w / v of NaCl), phosphate-buffered saline, Ringer's solution, Tris-buffered saline, and HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid)-buffered saline. In certain embodiments, thymic tissue obtained from a porcine donor according to a method disclosed herein is placed in normal saline until transplanted into a recipient animal. In certain embodiments, thymic tissue obtained from a porcine donor according to a method disclosed herein is placed in a suitable physiological solution (e.g., normal saline) until transplanted into a recipient animal wherein the physiological solution is at a temperature that is less than ambient temperature. In certain embodiments, thymic tissue obtained from a porcine donor according to a method disclosed herein is placed in a suitable physiological solution (e.g., normal saline) until transplanted into a recipient animal wherein the physiological solution is at a temperature that is at least about or about 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, or more than 8°C. In certain embodiments, thymic tissue obtained from a porcine donor according to a method disclosed herein is placed in a suitable physiological solution (e.g., normal saline) until transplanted into a recipient animal wherein the physiological solution is at a temperature that is about 4°C.

[0077] In certain embodiments, thymic tissue obtained from a porcine donor according to a method disclosed herein is prepared in advance of transplanting into the liver of a recipient animal. In certain embodiments, thymic tissue obtained from a porcine donor is prepared in advance of transplanting by stripping of the thymic capsule. In certain embodiments, thymic tissue obtained from a porcine donor is prepared in advance of transplanting by cutting the thymic tissue into pieces. In certain embodiments, thymic tissue obtained from a porcine donor is prepared in advance of transplanting by cutting the thymic tissue into pieces that range in size from about 0.5 mm3to about 5 mm3, about 1 mm3to about 4.5 mm3, about 1.5 mm3to about 4 mm3, about 2 mm3to about 3.5 mm3, about 0.5 mm3to about 1 mm3, about 1 mm3to about 1.5 mm3, about 1.5 mm3to about 2 mm3, about 2 mm3to about 2.5 mm3, or about 1 mm3to about 2 mm3. In certain embodiments, thymic tissue obtained from a porcine donor is prepared in advance of transplanting by cutting the thymic tissue into pieces that are at least about or about 0.5 mm3, 1 mm3, 1.5 mm3, 2 mm3, 2.5 mm3, 3 mm3, 3.5 mm3, 4 mm3, 4.5 mm3, or 5 mm3. NAI-5000951688v1 26Attorney Docket No.14648-044-228

[0078] In certain embodiments, a method of creating a thymoliver of the present disclosure comprises obtaining fetal thymic tissue from a porcine donor. Obtaining fetal thymic tissue from a porcine donor can be performed using methods as described in U.S. Provisional Application No.63 / 768,408 filed on 03 / 07 / 2025. 6.4.2 Thymoliver Surgery

[0079] In certain embodiments, a method of creating a thymoliver of the present disclosure comprises (a) obtaining thymic tissue from a porcine donor according to a method described in Section 6.4.1 and (b) transplanting the thymic tissue obtained in step (a) into a liver of a recipient animal. In certain embodiments, a recipient animal comprises a porcine donor as described herein (see Section 6.2). In certain embodiments, the recipient animal becomes the thymoliver swine (see Section 6.3) after completing step (b). In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal (e.g., a porcine donor) are the same animal. In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from two different porcine donors (e.g., minipigs). In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from two different porcine donors (e.g., minipigs) from the same breed. In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from two different porcine donors (e.g., minipigs) from the same herd. In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from two different porcine donors (e.g., minipigs) from the same highly inbred herd. In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from two different genetically modified porcine donors (e.g., genetically modified minipigs). In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from two different genetically modified porcine donors (e.g., genetically modified minipigs), wherein the genetically modified porcine donors have the same genetic modification. In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from two different genetically modified porcine donors (e.g., genetically modified minipigs), wherein the genetically modified porcine donors have a different genetic modification. In certain embodiments, the porcine donor from which thymic tissue is obtained is a non-genetically modified porcine donor and the recipient animal is a genetically NAI-5000951688v1 27Attorney Docket No.14648-044-228 modified porcine donor. In certain embodiments, the porcine donor from which thymic tissue is obtained is a non-genetically modified porcine donor and the recipient animal is a genetically modified porcine donor, wherein the non-genetically modified porcine donor and the genetically modified porcine donor are the same breed. In certain embodiments, the porcine donor from which thymic tissue is obtained is a non-genetically modified porcine donor and the recipient animal is a genetically modified porcine donor, wherein the non-genetically modified porcine donor and the genetically modified porcine donor are the same herd (e.g., highly inbred herd). In certain embodiments, the porcine donor from which thymic tissue is obtained is a non- genetically modified porcine donor and the recipient animal is a genetically modified porcine donor, wherein the non-genetically modified porcine donor and the genetically modified porcine donor are major histocompatibility complex (MHC) (i.e., swine leukocyte antigen (SLA))-matched. In certain embodiments, the porcine donor from which thymic tissue is obtained is a genetically modified porcine donor and the recipient animal is a non-genetically modified porcine donor. In certain embodiments, the porcine donor from which thymic tissue is obtained is a genetically modified porcine donor and the recipient animal is a non-genetically modified porcine donor, wherein the non-genetically modified porcine donor and the genetically modified porcine donor are the same breed. In certain embodiments, the porcine donor from which thymic tissue is obtained is a genetically modified porcine donor and the recipient animal is a non-genetically modified porcine donor, wherein the non-genetically modified porcine donor and the genetically modified porcine donor are the same herd (e.g., highly inbred herd). In certain embodiments, the porcine donor from which thymic tissue is obtained is a genetically modified porcine donor and the recipient animal is a non-genetically modified porcine donor, wherein the non-genetically modified porcine donor and the genetically modified porcine donor are MHC (i.e., SLA)-matched. In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from two different genetically modified porcine donors (e.g., genetically modified minipigs), wherein the genetically modified porcine donors both comprise a GGTA1 genetic modification. In certain embodiments, the porcine donor from which thymic tissue is obtained is a non-genetically modified porcine donor and the recipient animal is a genetically modified porcine donor comprising a GGTA1 genetic modification. In certain embodiments, the porcine donor from which thymic tissue is obtained is a genetically modified porcine donor comprising a GGTA1 genetic modification and the recipient animal is a NAI-5000951688v1 28Attorney Docket No.14648-044-228 non-genetically modified porcine donor. In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from the same genetically modified porcine donor (e.g., a genetically modified minipig), wherein the genetically modified porcine donor comprises a GGTA1 genetic modification. In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from the same genetically modified minipig, wherein the genetically modified minipig comprises a GGTA1 genetic modification. In certain embodiments, the porcine donor from which thymic tissue is obtained and the recipient animal are from the same genetically modified minipig, wherein the genetically modified minipig does not express alpha-1,3-galactosyltransferase (i.e., a GalT-KO minipig).

[0080] In certain embodiments, the transplanting of the thymic tissue obtained according to a method described herein (see Section 6.4.1) into the liver of a recipient animal (e.g., porcine donor) is conducted using abdominal surgery. In certain embodiments, abdominal surgery comprises accessing the liver by dissection into the abdominal space of the porcine donor.

[0081] In certain embodiments, the transplanting of the thymic tissue obtained according to a method described herein into a liver of a recipient animal (e.g., porcine donor) comprises inserting the thymic tissue into the liver parenchyma of the recipient animal. As used herein, the term “parenchyma” is understood to refer to the liver parenchyma. In certain embodiments, thymic tissue is inserted into the liver intraparenchymally. The term “intraparenchymally” as used herein refers to the “seeding” or insertion of pieces of thymic tissue into the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein (see Section 6.4.1) is inserted intraparenchymally into the liver parenchyma wherein at least about or about 0.5 mm3, 1 mm3, 1.5 mm3, 2 mm3, 2.5 mm3or 3 mm3of thymic tissue is inserted intraparenchymally per location in the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted intraparenchymally into the liver parenchyma wherein about 0.5 mm3to about 1 mm3, about 1 mm3to about 1.5 mm3, about 1.5 mm3to about 2 mm3, about 2 mm3to about 2.5 mm3, about 2.5 mm3to about 3 mm3, or about 1 mm3to about 2 mm3of thymic tissue is inserted intraparenchymally per location in the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted intraparenchymally into the liver parenchyma wherein 1 mm3to 2 mm3of thymic tissue is inserted intraparenchymally per location in the liver. In certain embodiments, the transplanting of the thymic tissue obtained according to a method described herein into a liver of a recipient NAI-5000951688v1 29Attorney Docket No.14648-044-228 animal (e.g., porcine donor) comprises a local resection of liver parenchyma. Without being bound by theory, a proper local resection of liver parenchyma could enhance thymic tissue engraftment efficiency.

[0082] In certain embodiments, thymic tissue obtained according to a method described herein (see Section 6.4.1) is inserted intraparenchymally at least about or about 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm deep into the liver parenchyma as measured from the surface of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted intraparenchymally at about 1 mm to about 1.5 mm, about 1.5 mm to about 2 mm, about 2 mm to about 2.5 mm, about 2.5 mm to about 3 mm, about 3 mm to about 3.5 mm, about 3.5 mm to about 4 mm, about 4 mm to about 4.5 mm, or about 4.5 mm to about 5 mm deep into the liver parenchyma as measured from the surface of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted intraparenchymally no deeper than about 5 mm deep into the liver parenchyma as measured from the surface of the liver.

[0083] In certain embodiments, thymic tissue obtained according to a method described herein is inserted into the liver of the recipient animal intraparenchymally in at least one location within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into the liver of the recipient animal intraparenchymally in more than one location within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into the liver of the recipient animal intraparenchymally in at least about or about one, two, three, four, five, six, seven, eight, nine, ten, or more than about ten locations within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into the liver of the recipient animal intraparenchymally in about one to about two, about two to about three, about three to about four, about four to about five, about five to about six, about six to about seven, about seven to about eight, about eight to about nine, about nine to about ten, about one to about four, about one to about six, about one to about eight, or about one to about ten locations within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into the liver of the recipient animal intraparenchymally in at least about or about four locations within the liver parenchyma. NAI-5000951688v1 30Attorney Docket No.14648-044-228

[0084] In certain embodiments, thymic tissue obtained according to a method described herein (see Section 6.4.1), is inserted intraparenchymally into the liver parenchyma wherein the total about of thymic tissue transplanted into the liver is at least about or about 1 mm3, 1.5 mm3, 2 mm3, 2.5 mm3, 3 mm3, 3.5 mm3, 4 mm3, 4.5 mm3, 5 mm3, 5.5 mm3, 6 mm3, 6.5 mm3, 7 mm3, 7.5 mm3, 8 mm3, 8.5 mm3, 9 mm3, 9.5 mm3, 10 mm3, 10.5 mm3, 11 mm3, 11.5 mm3, 12 mm3, 12.5 mm3, 13 mm3, 13.5 mm3, 14 mm3, 14.5 mm3, 15 mm3, 15.5 mm3, 16 mm3, 16.5 mm3, 17 mm3, 17.5 mm3, 18 mm3, 18.5 mm3, 19 mm3, 19.5 mm3, or 20 mm3. In certain embodiments, thymic tissue obtained according to a method described herein, is inserted intraparenchymally into the liver parenchyma wherein the total about of thymic tissue transplanted into the liver is between about 1 mm3to about 1.5 mm3, about 1.5 mm3to about 2 mm3, about 2 mm3to about 2.5 mm3, about 2.5 mm3to about 3 mm3, about 3 mm3to about 3.5 mm3, about 3.5 mm3to about 4 mm3, about 4 mm3to about 4.5 mm3, about 4.5 mm3to about 5 mm3, about 5 mm3to about 5.5 mm3, about 5.5 mm3to about 6 mm3, about 6 mm3to about 6.5 mm3, about 6.5 mm3to about 7 mm3, about 7 mm3to about 7.5 mm3, about 7.5 mm3to about 8 mm3, about 8 mm3to about 8.5 mm3, about 8.5 mm3to about 9 mm3, about 9 mm3to about 9.5 mm3, about 9.5 mm3to about 10 mm3, about 10 mm3to about 10.5 mm3, about 10.5 mm3to about 11 mm3, about 11 mm3to about 11.5 mm3, about 11.5 mm3to about 12 mm3, about 12 mm3to about 12.5 mm3, about 12.5 mm3to about 13 mm3, about 13 mm3to about 13.5 mm3, about 13.5 mm3to about 14 mm3, about 14 mm3to about 14.5 mm3, about 14.5 mm3to about 15 mm3, about 15 mm3to about 15.5 mm3, about 15.5 mm3to about 16 mm3, about 16 mm3to about 16.5 mm3, about 16.5 mm3to about 17 mm3, about 17 mm3to about 17.5 mm3, about 17.5 mm3to about 18 mm3, about 18 mm3to about 18.5 mm3, about 18.5 mm3to about 19 mm3, about 19 mm3to about 19.5 mm3, or about 19.5 mm3to about 20 mm3.

[0085] In certain embodiments, thymic tissue obtained according to a method described herein is inserted into the liver of the recipient animal intraparenchymally in at least one location within the liver parenchyma, wherein optionally the site of insertion is sutured shut at the liver surface.

[0086] In certain embodiments, the transplanting of the thymic tissue obtained according to a method described herein into a liver of a recipient animal (e.g., porcine donor) comprises inserting the thymic tissue into a wedge resection within the liver of the recipient animal. A wedge resection removes a portion of the liver parenchyma. In certain embodiments, thymic NAI-5000951688v1 31Attorney Docket No.14648-044-228 tissue obtained according to a method described herein (see Section 6.4.1) is inserted into a wedge resection wherein the wedge resection is at least about or about 1 cm, 1.5 cm, 2 cm, 2.5 cm, 3 cm, 3.5 cm, 4 cm, 4.5 cm, or 5 cm deep into the liver parenchyma as measured from the surface of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection wherein the wedge resection is about 1 cm to about 1.5 cm, about 1.5 cm to about 2 cm, about 2 cm to about 2.5 cm, about 2.5 cm to about 3 cm, about 3 cm to about 3.5 cm, about 3.5 cm to about 4 cm, about 4 cm to about 4.5 cm, or about 4.5 cm to about 5 cm deep into the liver parenchyma as measured from the surface of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection wherein the wedge resection is no deeper than about 3 cm deep into the liver parenchyma as measured from the surface of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection wherein at least about or about 1 cm3, 1.5 cm3, 2 cm3, 2.5 cm3, 3 cm3, 3.5 cm3, or 4 cm3of liver parenchyma is resected. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection wherein about 1 cm3to about 1.5 cm3, about 1.5 cm3to about 2 cm3, about 2 cm3to about 2.5 cm3, about 2.5 cm3to about 3 cm3, about 3 cm3to about 3.5 cm3, or about 3.5 cm3to about 4 cm3of liver parenchyma is resected. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection wherein at least about 2 cm3of liver parenchyma is resected. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection wherein the thymic tissue is sutured into the wedge resection.

[0087] In certain embodiments, the transplanting of the thymic tissue obtained according to a method described herein into a liver of a recipient animal (e.g., porcine donor) comprises inserting the thymic tissue into an incision within the liver of the recipient animal. In certain embodiments, an incision is made at the surface of the liver into the liver parenchyma. In certain embodiments, an incision into the liver parenchyma does not remove a portion of the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein (see Section 6.4.1) is inserted into an incision wherein the incision is at least about or about 1 cm, 1.5 cm, 2 cm, 2.5 cm, 3 cm, 3.5 cm, 4 cm, 4.5 cm, or 5 cm deep into the liver parenchyma as measured from the surface of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into an incision wherein the incision NAI-5000951688v1 32Attorney Docket No.14648-044-228 is about 1 cm to about 1.5 cm, about 1.5 cm to about 2 cm, about 2 cm to about 2.5 cm, about 2.5 cm to about 3 cm, about 3 cm to about 3.5 cm, about 3.5 cm to about 4 cm, about 4 cm to about 4.5 cm, or about 4.5 cm to about 5 cm deep into the liver parenchyma as measured from the surface of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into an incision wherein the incision is no deeper than about 3 cm deep into the liver parenchyma as measured from the surface of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into an incision wherein the thymic tissue is sutured into the incision.

[0088] In certain embodiments, thymic tissue obtained according to a method described herein (see Section 6.4.1), is inserted into a wedge resection and / or an incision into the liver parenchyma wherein the total about of thymic tissue transplanted into the liver is at least about or about 1 / 3, about 1 / 2, or about 2 / 3 of one thymus lobe. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection and / or an incision into the liver parenchyma wherein the total about of thymic tissue transplanted into the liver is from about 1 / 3 to about 2 / 3 or from about 1 / 3 to about 1 / 2 of one thymus lobe. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection and / or an incision into the liver parenchyma wherein the total about of thymic tissue transplanted into the liver is at least 1 / 2 of one thymus lobe.

[0089] In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection and / or an incision into the liver parenchyma of the recipient animal in at least one location within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection and / or an incision into the liver parenchyma of the recipient animal in more than one location within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection and / or an incision into the liver parenchyma of the recipient animal in at least about or about one, two, three, four, five, six, seven, eight, nine, ten, or more than about ten locations within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection and / or an incision into the liver parenchyma of the recipient animal in about or about one to about two, about two to about three, about three to about four, about four to about five, about five to about six, about six to about seven, about seven to about eight, about eight to NAI-5000951688v1 33Attorney Docket No.14648-044-228 about nine, about nine to about ten, about one to about four, about one to about six, about one to about eight, or about one to about ten locations within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection and / or an incision into the liver parenchyma of the recipient animal in at least about or about two locations within the liver parenchyma.

[0090] In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection and intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into an incision and intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection and into an incision of the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma.

[0091] In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the right, left, and / or caudate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the right, left, and / or quadrate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the right, caudate, and / or quadrate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or NAI-5000951688v1 34Attorney Docket No.14648-044-228 intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the left, caudate, and / or quadrate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the right and / or left lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the right and / or quadrate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the right and / or caudate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the caudate and / or quadrate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the left and / or quadrate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the left and / or caudate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the right lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the left lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver NAI-5000951688v1 35Attorney Docket No.14648-044-228 parenchyma of the caudate lobe of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of the quadrate lobe of the liver.

[0092] In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal in one or more locations within the liver parenchyma of at least one segment of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal in one or more locations within the liver parenchyma of more than one segments of the liver.

[0093] In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of segments I, II, III, IV, V, VI, VII, and / or VIII of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of at least one segment of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of at least two segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of at least three segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of at least four segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In NAI-5000951688v1 36Attorney Docket No.14648-044-228 certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of at least five segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of at least six segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of at least seven segments of the liver selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at one or more locations within the liver parenchyma of segments I, II, III, IV, V, VI, VII, and VIII of the liver.

[0094] In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment I of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment II of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment III of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment V of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment VI of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver NAI-5000951688v1 37Attorney Docket No.14648-044-228 parenchyma of the recipient animal at segment VII of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment VIII of the liver.

[0095] In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment IV of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment IVa of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment IVb of the liver. In certain embodiments, thymic tissue obtained according to a method described herein is inserted into a wedge resection, into an incision, and / or intraparenchymally into the liver parenchyma of the recipient animal at segment IVa and / or IVb of the liver.

[0096] In certain embodiments, the thymic tissue obtained according to a method described herein (see Section 6.4.1) is inserted into the liver of a recipient animal (e.g., porcine donor), wherein the recipient animal is subjected to a thymectomy prior to transplanting the thymic tissue. In certain embodiments, a recipient animal of the present disclosure is subjected to a thymectomy to remove one or both lobes of the cervical thymus prior to transplanting the thymic tissue into the liver. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial or a total (i.e., 100%) thymectomy prior to transplanting the thymic tissue into the liver. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial thymectomy prior to transplanting the thymic tissue into the liver, wherein at least about or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of the thymus is removed. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial thymectomy prior to transplanting the thymic tissue into the liver, wherein about 20% to about 40%, about 40% to about 60%, about 60% to about 80%, or about 80% to more than about 95% of the thymus is removed. In certain embodiments, the thymectomy can be omitted prior to transplanting the thymic tissue into the liver. NAI-5000951688v1 38Attorney Docket No.14648-044-228

[0097] In certain embodiments, a recipient animal of the present disclosure is subjected to a cervical and / or thoracic thymectomy prior to transplanting the thymic tissue into the liver. Cervical and thoracic thymic tissue have similar stromal organization. Cervical thymic tissue is lying on the trachea and thyroid gland with parathyroid whereas thoracic thymic tissue is in mediastinum and anterior to pericardium. In certain embodiments, a recipient animal of the present disclosure is subjected to a thoracic thymectomy prior to transplanting the thymic tissue into the liver. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial or a total (i.e., 100%) thoracic thymectomy prior to transplanting the thymic tissue into the liver. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial thoracic thymectomy prior to transplanting the thymic tissue into the liver, wherein at least about or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of thoracic thymic tissue is removed. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial thoracic thymectomy prior to transplanting the thymic tissue into the liver, wherein about 20% to about 40%, about 40% to about 60%, about 60% to about 80%, or about 80% to more than about 95% of the thoracic thymus tissue is removed. In certain embodiments, a recipient animal of the present disclosure is subjected to a cervical thymectomy prior to transplanting the thymic tissue into the liver. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial or a total (i.e., 100%) cervical thymectomy prior to transplanting the thymic tissue into the liver. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial cervical thymectomy prior to transplanting the thymic tissue into the liver, wherein at least about or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of cervical thymic tissue is removed. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial cervical thymectomy prior to transplanting the thymic tissue into the liver, wherein about 20% to about 40%, about 40% to about 60%, about 60% to about 80%, or about 80% to more than about 95% of the cervical thymus tissue is removed. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial thoracic thymectomy and a total cervical thymectomy prior to transplanting the thymic tissue into the liver. In certain embodiments, a recipient animal of the present disclosure is subjected to a total thoracic thymectomy and a partial cervical thymectomy prior to transplanting the thymic tissue into the liver. In certain embodiments, a recipient animal of the present disclosure is subjected to a total and / or partial thoracic NAI-5000951688v1 39Attorney Docket No.14648-044-228 thymectomy prior to transplanting the thymic tissue into the liver, wherein the cervical thymic tissue remains intact. In certain embodiments, a recipient animal of the present disclosure is subjected to a total and / or partial cervical thymectomy prior to transplanting the thymic tissue into the liver, wherein the thoracic thymic tissue remains intact. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial cervical thymectomy prior to transplanting the thymic tissue into the liver, wherein the thoracic thymic tissue remains intact and at least about or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of cervical thymic tissue is removed. In certain embodiments, a recipient animal of the present disclosure is subjected to a partial cervical thymectomy prior to transplanting the thymic tissue into the liver, wherein the thoracic thymic tissue remains intact and about 20% to about 40%, about 40% to about 60%, about 60% to about 80%, or about 80% to more than about 95% of the cervical thymus tissue is removed. 6.4.3 Post Thymoliver Surgery

[0098] In certain embodiments, the thymoliver resulting from a method described in Sections 6.4.1 and 6.4.2 is incubated in the recipient animal for a period of time prior to harvesting for use.

[0099] In certain embodiments, a method of incubating the thymoliver in the recipient animal comprises incubating the liver for at least about or about 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the transplanting step. In certain embodiments, a method of incubating the thymoliver in the recipient animal comprises incubating the liver for at least about or about 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the transplanting step. In certain embodiments, a method of incubating the thymoliver in the recipient animal comprises incubating the liver for about 6 weeks to about 10 weeks, about 6 weeks to about 9 weeks, or about 6 weeks to about 8 weeks after the transplanting step.

[0100] In certain embodiments, a method of incubating the thymoliver in the recipient animal comprises incubating the liver after the transplanting step until the thymus tissue is fully neovascularized. In certain embodiments, a method of incubating the thymoliver in the recipient animal comprises incubating the liver after the transplanting step until the thymus tissue is partially neovascularized. In certain embodiments, a method of incubating the thymoliver in the recipient animal comprises incubating the liver after the transplanting step until the thymus tissue comprises at least about or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or NAI-5000951688v1 40Attorney Docket No.14648-044-228 99% neovascularized thymus tissue. In certain embodiments, a method of creating a thymoliver of the present disclosure results in a thymoliver comprised of fully neovascularized thymic tissue at least about or at about 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the transplanting step. In certain embodiments, a method of creating a thymoliver of the present disclosure results in a thymoliver comprised of at least about or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% neovascularized thymus tissue at least about or at about 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the transplanting step. 6.5 Methods of Use

[0101] In certain embodiments, the present disclosure provides a method of using a thymoliver described herein (see Section 6.1). In certain embodiments, a method of using a thymoliver of the present disclosure comprises (a) obtaining the thymoliver from a porcine donor (see Section 6.5.1(a)) and (b) transplanting the thymoliver obtained in step (a) into a subject (i.e., a recipient) in need thereof (see Section 6.5.1(b)). Subjects suitable for receiving a thymoliver according to the methods disclosed herein are described in Section 6.5.3. In certain embodiments, one or more additional treatments can be administered to the thymoliver recipient as described in Section 6.5.2.

[0102] In certain embodiments, the present disclosure provides methods of treating a disease in a subject in need thereof, wherein the method comprises transplanting a thymoliver described herein (see Section 6.1). Methods of evaluating the effectiveness of the treatment of the disease in the thymoliver recipient is described in Section 6.5.4. 6.5.1 Method of Transplantation of Thymoliver (a) Thymoliver Harvest from Donor

[0103] In certain embodiments, the present disclosure provides methods of harvesting a thymoliver prepared according to the methods described in Section 6.4. In certain embodiments, a porcine thymoliver is harvested from a porcine donor by donor hepatectomy procedure. A “donor hepatectomy” is a surgical procedure to remove a donor liver (e.g., a thymoliver) from the donor for transplant into a patient in need thereof (e.g., the “recipient”). In certain embodiments, a thymoliver prepared according to the methods described herein is harvested NAI-5000951688v1 41Attorney Docket No.14648-044-228 from the donor using either an open donor hepatectomy procedure, a laparoscopic donor hepatectomy procedure, or a robotic-assisted donor hepatectomy procedure.

[0104] In certain embodiments, a thymoliver is harvested from the donor at least about or about 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after creating the thymoliver according to the methods described in Section 6.4. In certain embodiments, a thymoliver is harvested about 6 weeks to about 7 weeks, about 7 weeks to about 8 weeks, about 8 weeks to about 9 weeks, about 9 weeks to about 10 weeks, or about 6 weeks to about 8 weeks after creating the thymoliver according to the methods described in Section 6.4.

[0105] In certain embodiments, during the transition period after the thymoliver is harvested from the donor animal but before being transplanted in the recipient, the thymoliver is stored in certain container to maintain the viability of the thymoliver. In certain embodiments, the container is capable of perfusion of the thymoliver at warm and / or cold temperatures to extend the useful life of the thymoliver. In certain embodiments, the thymoliver is stored in the container with proper fluid samples. In certain embodiments, the thymoliver is stored in the container with ample supply of oxygen. An example of such a container is provided in U.S. Pat. No.6,673,594, which is hereby incorporated by reference in its entirety. (b) Transplantation of Thymoliver

[0106] In certain embodiments, the present disclosure provides a method of transplanting a thymoliver prepared according to the methods described in Section 6.4 into a subject in need thereof. A subject suitable for receiving a thymoliver disclosed herein is described in Section 6.5.3 below. Methods of transplanting a porcine donor liver into a recipient animal are generally known in the art. See, e.g., Cooper DK et al., Transplantation (2016) Oct;100(10):2039-47; and Zhang X et al., Xenotransplantation (2019) May;26(3):e12497.

[0107] In certain embodiments, a total hepatectomy of the recipient’s liver is performed prior to transplanting the thymoliver into the recipient. In certain embodiments, a partial hepatectomy of the recipient’s liver is performed prior to transplanting the thymoliver into the recipient. In certain embodiments, a thymoliver disclosed herein can be used in an external liver perfusion procedure (see, e.g., Mallapaty S. Nature (2024) Mar;627(8005):710-711). 6.5.2 Additional Treatments

[0108] In certain embodiments, a recipient treated in accordance with the methods described herein (e.g., transplantation of a thymoliver) undergoes additional treatment. A recipient may NAI-5000951688v1 42Attorney Docket No.14648-044-228 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 thymoliver).

[0109] Additional treatments are generally intended to improve the tolerance of the thymoliver in recipients, but other treatments are contemplated. In certain embodiments, a method of transplantation of a thymoliver 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 thymoliver. In certain embodiments, a recipient is thymectomized and / or splenectomized.

[0110] 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.

[0111] The blood of a subject undergoing transplantation by a method described herein can contain preexisting, xenoreactive antibodies that target the thymoliver. Without being bound by theory, these pre-existing antibodies can be major effectors of rejection. Any method known to the skilled artisan can be used to eliminate or reduce the titer of these pre-existing antibodies. Such methods include for example immunoadsorption by donor-species organ hemoperfusion, immunoaffinity columns of synthetic oligosaccharides and treatment with anti-Ig antibodies. In certain embodiments, the thymoliver may be genetically modified such that it is not recognized by these pre-existing antibodies present in the host (e.g., the donor thymic tissue and / or the donor liver is a-1,3-galactosyltransferase deficient) per Section 6.2. (a) Immunosuppressive Therapy

[0112] In certain embodiments, a patient receiving a thymoliver in accordance with the methods described herein can further receive immunosuppressive therapy. The immunosuppressive therapy can 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, NAI-5000951688v1 43Attorney Docket No.14648-044-228 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.

[0113] 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(6):409 and Benvenuto et al., J Thorac Dis (2018)10:3141-3155.

[0114] 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 as prednisone; and (7) antibodies to any one of a number of cellular or serum targets (including anti-lymphocyte globulin and anti-thymocyte globulin).

[0115] 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, NAI-5000951688v1 44Attorney Docket No.14648-044-228 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).

[0116] 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.provided herein comprise steps to induce tolerance and mixed chimerism in the recipient without bone marrow or mobilized peripheral blood stem cell transplantation. (b) Hematopoietic Stem Cell Transplant

[0117] In certain embodiments, a patient receiving a thymoliver 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.

[0118] 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. NAI-5000951688v1 45Attorney Docket No.14648-044-228

[0119] 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). In certain embodiments, a method provided herein comprise steps to induce tolerance and mixed chimerism in the recipient without bone marrow transplantation, mobilized peripheral blood stem cell transplantation, or hematopoietic stem cell transplantation. (c) Thymus derived Regulatory T-cells (thyTreg) co-transplantation with thymoliver

[0120] In certain embodiments, a patient receiving a thymoliver in accordance with the methods described herein can further receive a co-transplantation of donor-specific thymus- derived Tregs (thyTreg) to promote dominant regulatory tolerance. See, e.g., Bernaldo-de- Quirós E et al., . In certain embodiments, a patient treated in accordance with the method described herein is administered about 2x107to about 3x107cells / kg thyTregs, wherein the thyTregs are isolated from the thoracic and cervical thymus harvested from the donor of the thymoliver. In certain embodiments, a patient treated in accordance with the method described herein is administered about 2x107to about 3x107cells / kg thyTregs, wherein the thyTregs are isolated from the thymus harvested from the donor of the thymus. In certain embodiments, the thyTregs described herein can be isolated by sorting NAI-5000951688v1 46Attorney Docket No.14648-044-228 CD3+CD4+CD8-CD25+CD127- thymocytes through a combination of FACS (Fluorescence- activated cell sorting) and MASC (Magnetic activated cell sorting). In certain embodiments, the isolated thyTregs described herein can be administered intravenously immediately following the thymoliver transplantation. In certain embodiments, the isolated thyTregs described herein can be administered intravenously a few days (e.g., about 10 to 14 days) after the thymoliver transplantation. 6.5.3 Thymoliver Recipients

[0121] In certain embodiments, the present disclosure provides a method of treating a patient in need thereof with a thymoliver described herein (see Section 6.1). In certain embodiments, a patient treated with a thymoliver described herein can be a thymoliver recipient. In certain embodiments, a patient treated in accordance with the methods described herein (e.g., the recipient of a thymoliver) is a human patient. As used herein, the terms “subject” and “patient” are used interchangeably and include any human or non-human mammal. Non-limiting examples include members of the human, equine, porcine, bovine, rattus, murine, canine and feline species. In certain embodiments, the subject is a non-human primate. 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 human and receives one or more donor grafts from a porcine donor. 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. Methods of evaluating the effectiveness of treatment of a disease in the thymoliver recipient is described in Section 6.5.4. In certain embodiments, the subject is a human subject having a disease of the liver.

[0122] In certain embodiments, a patient treated in accordance with the methods described herein is in need of a liver transplant. A patient can be in need of a liver transplant due to liver failure or the rejection of a donor liver. A patient whose liver has been damaged by means including injury, disease, or birth defect may meet the criteria to receive a liver transplant and can be treated in accordance with the methods described herein. A patient treated in accordance with the methods described herein can have required a liver transplant for any reason. Generally, a patient suffering from end-stage liver disease whose life expectancy is predicted to be extended by a liver transplant beyond the life expectancy without the liver transplant can be treated in accordance with the methods described herein. Examples of indications of a liver transplant are NAI-5000951688v1 47Attorney Docket No.14648-044-228 described, for example, in the EASL Clinical Practice Guidelines: Liver transplantation J Hepatol (2016) Feb;64(2):433-485.

[0123] In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having a hepatic cancer, such as fibrolamellar carcinoma or epitheliod hemangioendothelioma. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having a hepatocellular carcinoma. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having choloangiocarcinoma. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having hepatic metastases of a cancer.

[0124] In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having acute hepatic necrosis. Acute hepatic necrosis, also referred to as acute liver failure, can be caused by paracetamol (acetaminophen) toxicity, ischemia, drug-induced liver injury, hepatitis A, B, and E, autoimmunity, heat stroke, pregnancy-associated injury (e.g., acute fatty liver of pregnancy and HELLP [hemolysis, elevated liver enzyme, and low platelet] syndrome), Budd-Chiari syndrome, nonhepatotrophic viral infections such as herpes simplex, intestinal endotoxins, and diffusely infiltrating malignancies. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has at least one symptom of acute hepatic necrosis. Non-limiting symptoms of acute hepatic necrosis include yellowing of your skin and eyeballs (i.e., jaundice), pain in the upper right abdomen, a swollen belly (i.e., ascites), nausea, vomiting, a general sense of feeling unwell (i.e., malaise), disorientation or confusion, sleepiness, breath may have a musty or sweet odor, and tremors.

[0125] In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having viral hepatitis. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having a Hepatitis B-related liver disease. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having a Hepatitis C-related liver disease. In certain NAI-5000951688v1 48Attorney Docket No.14648-044-228 embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having autoimmune hepatitis (AIH).

[0126] In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having an alcoholic liver disease. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having non-alcoholic fatty liver disease (NAFLD). In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having non-alcoholic steatohepatitis (NASH).

[0127] In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having a metabolic disease. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having alpha 1-antitrypsin deficiency. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having hemochromatosis. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having tyrosinemia. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having Wilson’s Disease.

[0128] In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having biliary atresia. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having primary biliary cirrhosis (PBC). In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having primary sclerosing cholangitis (PSC). In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having a bile duct cancer.

[0129] In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having a genetic disease, such as genetic cholestatic disorders, hereditary haemochromatosis, tyrosinemia, alpha-1-antitrypsin deficiency, urea cycle disorders, Crigler-Najjar syndrome, familial amyloid polyneuropathy, NAI-5000951688v1 49Attorney Docket No.14648-044-228 primary hyperoxaluria type 1, or atypical hemolytic uremic syndrome-1. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having a genetic cholestatic disorder. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having hereditary haemochromatosis. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having tyrosinemia, alpha-1-antitrypsin deficiency. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having urea cycle disorder. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having Crigler-Najjar syndrome. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having familial amyloid polyneuropathy. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having primary hyperoxaluria type 1. In certain embodiments, a patient treated in accordance with the methods described herein who is in need of a liver transplant has or is suspected of having atypical hemolytic uremic syndrome-1. 6.5.4 Methods of Evaluating Transplantation

[0130] In certain embodiments, use of a thymoliver produced according to the present disclosure results in a decreased occurrence of transplant rejection compared to that of current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced according to the present disclosure results in a decreased occurrence of acute transplant rejection compared to that of current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced according to the present disclosure results in a decreased occurrence of short-term transplant rejection compared to that of current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced according to the present disclosure results in an 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% decreased occurrence of transplant rejection (e.g., acute, short-term, long-term) compared to that of current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced according to the present NAI-5000951688v1 50Attorney Docket No.14648-044-228 disclosure results in an at least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% decreased occurrence of transplant rejection (e.g., acute, short-term, long-term) compared to that of current standard of care (i.e., a liver transplant).

[0131] In certain embodiments, use of a thymoliver produced according to the present disclosure results in a more viable transplant compared to that of current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced 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 current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced 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 current standard of care (i.e., a liver transplant). In certain embodiments, the viability of the thymoliver produced according to the present disclosure is prolonged by about 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, or more than about 30 more years compared to that of current standard of care (i.e., a liver transplant).

[0132] In certain embodiments, a biopsy of the transplanted thymoliver can be performed to examine the viability of the transplanted thymoliver and determine induction of tolerance or evidence of graft rejection. Tissue biopsies can be examined using routine light microscopy, immunofluorescence, and electron microscopy. In certain embodiments, the viability of the transplanted thymoliver can be determined by detecting biopsy-proven acute rejection (BPAR) in a recipient. Herein, BPAR is defined as acute rejection with a locally confirmed rejection 3 according to the Banff 1997 criteria. The rejection activity index (RAI) is based on scoring features in 3 categories: portal inflammation (PI), bile duct inflammation damage (BDD), and venous endothelial inflammation (VEI) (see., e.g., Banff Schema for Grading Liver Allograft Rejection: An International Consensus Document. Hepatology (1997);25(3):658-63). In certain embodiments, use of a thymoliver produced according to the present disclosure results in no BPAR is detected in a recipient at 1 week, 2 weeks, 3 week, 4 weeks, 2 months , 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 12 months, 15 months, 18 months, 20 months, 25 months, 30 months, 35 months, 40 NAI-5000951688v1 51Attorney Docket No.14648-044-228 months, 45 months, 50 months, 55 months, or 60 months post-transplant. In certain embodiments, use of a thymoliver produced according to the present disclosure results in a RAI less 3 detected in a recipient at 1 week, 2 weeks, 3 week, 4 weeks, 2 months , 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 12 months, 15 months, 18 months, 20 months, 25 months, 30 months, 35 months, 40 months, 45 months, 50 months, 55 months, or 60 months post-transplant.

[0133] In certain embodiments, use of a thymoliver produced according to the present disclosure results in a more functional transplant compared to that of current standard of care (i.e., a liver transplant). Examples of methods for assessing liver transplant function include laboratory tests such as measuring alanine transaminase (ALT) and aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), serum bilirubin, prothrombin time (PT), the international normalized ratio (INR), total protein and albumin, imaging tests (e.g., ultrasound, computer tomography), and / or liver biopsy. In certain embodiments, use of a thymoliver produced according to the present disclosure results in a transplant comprising more than 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% function compared to that of current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced according to the present disclosure results in a transplant comprising more than about at least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% function compared to that of current standard of care (i.e., a liver transplant).

[0134] In certain embodiments, transplantation of a thymoliver produced according to the present disclosure results in normal and / or improved function of at least one untargeted organ system within the thymoliver recipient as compared to the function of the other untargeted organ system before transplantation of the thymoliver to the patient. In certain embodiments, use of a thymoliver produced according to the present disclosure results in more than 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% improved function of at least one untargeted organ system compared to that of current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced according to the present disclosure results in at least about or about 10%, NAI-5000951688v1 52Attorney Docket No.14648-044-228 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% improved function of at least one untargeted organ system compared to that of current standard of care (i.e., a liver transplant).

[0135] In certain embodiments, transplantation of a thymoliver produced according to the present disclosure results in better rate of survival of the thymoliver recipient as compared to the rate of survival before transplantation of the thymoliver to the patient. In certain embodiments, transplantation of a thymoliver produced according to the present disclosure improves the rate of survival of the thymoliver 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 thymoliver to the patient. In certain embodiments, transplantation of a thymoliver produced according to the present disclosure improves the rate of survival of the thymoliver 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 thymoliver to the patient. In certain embodiments, transplantation of a thymoliver produced according to the present disclosure improves the rate of survival of the thymoliver 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 thymoliver to the patient. In certain embodiments, transplantation of a thymoliver produced according to the present disclosure improves the rate of survival of the thymoliver 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 thymoliver to the patient.

[0136] In certain embodiments, a method of treating a patient (i.e., a recipient) in need thereof with a thymoliver produced according to the present disclosure can result in reduced administration of immunosuppressive therapy to the recipient when compared to current standard of care after the transplantation of the donor organs (i.e., a liver transplant). In certain embodiments, transplantation of a thymoliver produced according to the present disclosure results in a reduced amount of immunosuppressive agents administered to the recipient when NAI-5000951688v1 53Attorney Docket No.14648-044-228 compared to the amount of immunosuppressive agents generally administered to a comparable transplant recipient (e.g., a person of the same sex and of comparable age, height, and / or weight). In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by about 10% administered to the recipient. In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by about 10% to about 20% administered to the recipient. In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by about 20% to about 30% administered to the recipient. In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by about 30% to about 40% administered to the recipient. In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by about 40% to about 50% administered to the recipient. In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by about 50% to about 60% administered to the recipient. In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by about 60% to about 70% administered to the recipient. In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by about 70% to about 80% administered to the recipient. In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by about 80% to about 90% administered to the recipient. In certain embodiments, the method provided herein results in a reduced amount of immunosuppressive agents by more than about 90% administered to the recipient.

[0137] In certain embodiments, transplantation of a thymoliver produced according to the present disclosure results in a reduced administration frequency of immunosuppressive therapy to the recipient when compared to the one which is typically administered to a comparable recipient (e.g., a person of the same sex and of comparable age, height, and / or weight). In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by about 10% administered to the recipient. In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by about 10% to about 20% administered to the recipient. In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by about 20% to about 30% administered to the recipient. In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by about 30% to about 40% NAI-5000951688v1 54Attorney Docket No.14648-044-228 administered to the recipient. In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by about 40% to about 50% administered to the recipient. In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by about 50% to about 60% administered to the recipient. In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by about 60% to about 70% administered to the recipient. In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by about 70% to about 80% administered to the recipient. In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by about 80% to about 90% administered to the recipient. In certain embodiments, the method provided herein results in a reduced frequency of immunosuppressive agents by more than about 90% administered to the recipient.

[0138] In certain embodiments, transplantation of a thymoliver produced according to the present disclosure results in a shortened treatment duration of immunosuppressive therapy administered to the recipient when compared to the one which is typically administered to a comparable recipient (e.g., a person of the same sex and of comparable age, height, and / or weight). In certain embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by about 10%. In certain embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by about 10% to about 20%. In certain embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by about 20% to about 30%. In certain embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by about 30% to about 40%. In certain embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by about 40% to about 50%. In certain embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by about 50% to about 60%. In certain embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by about 60% to about 70%. In certain embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by about 70% to about 80%. In certain embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by about 80% to about 90%. In certain NAI-5000951688v1 55Attorney Docket No.14648-044-228 embodiments, the method provided herein results in a shortened treatment duration of immunosuppressive therapy by more than about 90%.

[0139] In certain embodiments, transplantation of a thymoliver produced according to the present disclosure results in an improvement of the corresponding disease that the transplantation aims to intervene within the thymoliver recipient as compared to the disease before transplantation of the thymoliver to the patient. In certain embodiments, transplantation of a thymoliver produced according to the present disclosure results in a greater improvement of the corresponding disease that the transplantation aims to intervene within the thymoliver recipient compared to that of a patient transplanted according to the current standard of care (i.e., a liver transplant). One of skill in the art can appreciate that an improvement of the corresponding disease following transplantation of a thymoliver produced 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 thymoliver 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 thymoliver produced according to the present disclosure results in an improvement of the corresponding disease that the transplantation aims to intervene within the thymoliver recipient as indicated by at least one biomarker corresponding to the disease. In certain embodiments, use of a thymoliver produced according to the present disclosure results in 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% improvement of the corresponding disease compared to that of current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced according to the present disclosure results in at least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% improvement of the corresponding disease compared to that of current standard of care (i.e., a liver transplant). NAI-5000951688v1 56Attorney Docket No.14648-044-228

[0140] In certain embodiments, transplantation of a thymoliver produced according to the present disclosure results in an improvement of liver disease and / or at least one symptom associated with liver disease within the thymoliver recipient as compared to before transplantation of the thymoliver to the patient. In certain embodiments, transplantation of a thymoliver produced according to the present disclosure results in a greater improvement of liver disease and / or at least one symptom associated with liver disease within the thymoliver recipient compared to that of a patient transplanted according to the current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced according to the present disclosure results in 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% improvement of liver disease and / or at least one symptom associated with liver disease compared to that of current standard of care (i.e., a liver transplant). In certain embodiments, use of a thymoliver produced according to the present disclosure results in at least about or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% improvement of liver disease and / or at least one symptom associated with liver disease compared to that of current standard of care (i.e., a liver transplant).

[0141] In certain embodiments, assessments of the outcome transplantation of a thymoliver produced according to the present disclosure can include the monitoring of the function of the transplanted thymoliver in the recipient. For example, the efficacy of a method of treatment described herein (e.g., transplantation of a thymoliver) may be assessed by measuring liver function at 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7.5 months, 9 months, 10.5 months, 12 months, 13.5 months, 15 months, 16.5 months, 18 months, 19.5 months, 21 months, 22.5 months, 24 months, 27 months, 30 months, 36 months, 42 months, 48 months, or , 54 months post-transplant, or at 1-5 months, 5-10 months, 10-15 months, 15-20 months, 20-25 months, 25-30 months, 30-35 months, 35-40 months, 40-45 months, 45-50 months, 50-55 months, or 55-60 months post-transplant.

[0142] Liver function may be determined, for example, by conducting liver function tests, such as measurements of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), serum bilirubin, prothrombin time (PT), the international normalized ratio (INR) and / or albumin. In certain embodiments, liver NAI-5000951688v1 57Attorney Docket No.14648-044-228 function is determined by measurement of alanine transaminase (ALT). In certain embodiments, liver function is determined by measurement of aspartate transaminase (AST). In certain embodiments, liver function is determined by measurement of alkaline phosphatase (ALP). In certain embodiments, liver function is determined by measurement of gamma-glutamyl transferase (GGT). In certain embodiments, liver function is determined by measurement of serum bilirubin. In certain embodiments, liver function is determined by measurement of prothrombin time (PT). In certain embodiments, liver function is determined by measurement of the international normalized ratio (INR) and albumin. In certain embodiments, liver function is determined by measurement of albumin. In certain embodiments, liver function is determined by measurement of alanine transaminase (ALT) and aspartate transaminase (AST). In certain embodiments, liver function is determined by measurement of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), serum bilirubin, and the international normalized ratio (INR). In certain embodiments, liver function is determined by measurement of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma- glutamyl transferase (GGT), serum bilirubin, prothrombin time (PT), the international normalized ratio (INR), and albumin.

[0143] In certain embodiments, a method of treatment described herein results in a thymoliver recipient having normal liver function (as determined by liver function tests). In certain embodiments, a method of treatment described herein results in a recipient having normal liver function (as determined by liver function tests) for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 25 months, at least 30 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver.

[0144] In certain embodiments, a method of treatment described herein (e.g., transplantation of a thymoliver) results in a thymoliver recipient having better liver function as determined by at least one liver function test. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having better liver function as determined by at least one liver function test for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 NAI-5000951688v1 58Attorney Docket No.14648-044-228 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 25 months, at least 30 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver as compared to a patient post liver transplant.

[0145] In certain embodiments, a method of treatment described herein (e.g., transplantation of a thymoliver) results in a thymoliver recipient having improved ALT values compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having an at least about or an about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% improvement in ALT values as compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having ALT values of 5-60 U / L, 7-55 U / L, 10-55 U / L, 15-50 U / L, 20-40 U / L or 25-35 U / L. In certain embodiments, a method of treatment described herein results in a recipient having ALT values of 5-60 U / L, 7-55 U / L, 10-55 U / L, 15-50 U / L, 20-40 U / L or 25-35 U / L for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 25 months, at least 30 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver.

[0146] In certain embodiments, a method of treatment described herein (e.g., transplantation of a thymoliver) results in a thymoliver recipient having improved AST values compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having an at least about or an about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% improvement in AST values as compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having AST values of 5-50 U / L, 8-48 U / L, 10-45 U / L, 15-40 U / L, or 20-30 U / L. In certain embodiments, a method of treatment described herein results in a recipient having AST values of 5-50 U / L, 8-48 U / L, 10-45 U / L, 15-40 U / L, or 20-30 U / L for at leat 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 NAI-5000951688v1 59Attorney Docket No.14648-044-228 months, at least 10 months, at least 15 months, at least 20 months, at least 25 months, at least 30 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver.

[0147] In certain embodiments, a method of treatment described herein (e.g., transplantation of a thymoliver) results in a thymoliver recipient having improved ALP values compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having an at least about or an about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% improvement in ALP values as compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having ALP values of 30-150 U / L, 40-129 U / L, 50-120 U / L, 60-110 U / L, 70-100 U / L, or 80-90 U / L. In certain embodiments, a method of treatment described herein results in a recipient having ALP values of 30-150 U / L, 40-129 U / L, 50-120 U / L, 60-110 U / L, 70-100 U / L, or 80-90 U / L for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 25 months, at least 30 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver.

[0148] In certain embodiments, a method of treatment described herein (e.g., transplantation of a thymoliver) results in a thymoliver recipient having improved GGT values compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having an at least about or an about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% improvement in GGT values as compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having GGT values of 5-70 U / L, 8-61 U / L, 10-50 U / L, 15-45 U / L, 20-40 U / L, or 25-35 U / L. In certain embodiments, a method of treatment described herein results in a recipient having GGT values of 5-70 U / L, 8-61 U / L, 10-50 U / L, 15-45 U / L, 20-40 U / L, or 25-35 U / L for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 25 NAI-5000951688v1 60Attorney Docket No.14648-044-228 months, at least 30 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver.

[0149] In certain embodiments, a method of treatment described herein (e.g., transplantation of a thymoliver) results in a thymoliver recipient having improved serum bilirubin values compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having an at least about or an about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% improvement in serum bilirubin values as compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymokideny recipient having serum bilirubin values of 0.05-2 mg / dL, 0.1-1.5 mg / dL, 0.1-1.2 mg / dL, 0.5-1 mg / dL or 0.7-1 mg / dL. In certain embodiments, a method of treatment described herein results in a recipient having serum bilirubin values of 0.05-2 mg / dL, 0.1-1.5 mg / dL, 0.1-1.2 mg / dL, 0.5-1 mg / dL or 0.7-1 mg / dL for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 25 months, at least 30 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver.

[0150] In certain embodiments, a method of treatment described herein (e.g., transplantation of a thymoliver) results in a thymoliver recipient having improved PT compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having an at least about or an about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% improvement in PT as compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having a PT of 7-15 seconds, 8-14 seconds, 9-13 seconds, 9.4-12.5 seconds, or 10-12 seconds. In certain embodiments, a method of treatment described herein results in a recipient having a PT of 7-15 seconds, 8-14 seconds, 9-13 seconds, 9.4-12.5 seconds, or 10-12 seconds for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 25 months, at least 30 NAI-5000951688v1 61Attorney Docket No.14648-044-228 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver.

[0151] In certain embodiments, a method of treatment described herein (e.g., transplantation of a thymoliver) results in a thymoliver recipient having improved albumin values compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having an at least about or an about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% improvement in albumin values as compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having albumin values of 2-6 g / dL, 2.5-5.5 g / dL, 3-5 g / Dl, 3.5-5 g / dL, or 4- 4.5 g / dL. In certain embodiments, a method of treatment described herein results in a recipient having albumin values of 2-6 g / dL, 2.5-5.5 g / dL, 3-5 g / Dl, 3.5-5 g / dL, or 4-4.5 g / dL for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 25 months, at least 30 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver.

[0152] In certain embodiments, a method of treatment described herein (e.g., transplantation of a thymoliver) results in a thymoliver recipient having an improved INR compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having an at least about or an about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% improvement in INR as compared to before transplant. In certain embodiments, a method of treatment described herein results in a thymoliver recipient having an INR 1-3, 1.5-2.5, 1.5-2 or below 1.1. In certain embodiments, a method of treatment described herein results in a recipient having an INR 1-3, 1.5-2.5, 1.5-2 or below 1.1 for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 15 months, at least 20 months, at least 25 months, at least 30 months, at least 35 months, at least 40 months, at least 45 months, at least 50 months, at least 55 months, or at least 60 months post-transplant of a thymoliver. NAI-5000951688v1 62Attorney Docket No.14648-044-228 7. EXAMPLES

[0153] 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. 7.1 Example 1

[0154] The ability of the liver to create transplant tolerance after allotransplantation compared to other solid organ transplants is due, in part, to the liver’s reservoir of donor hematopoietic stem cells (HSCs) and leukocytes that create mixed chimerism. In xenotransplantation models, due to circulating anti-pig antibodies, liver grafts lose their advantage in promoting tolerance by hyperacute loss of circulating donor cells. In the exemplary study herein, a thymoliver organ was constructed. Without being bound to any particular theory, the thymoliver organ constructed according to the exemplary method described herein can ameliorate the immunologic barriers that currently occur after liver xenotransplantation. 7.1.1 Methods and Materials (a) Animals

[0155] An eight (8)-week-old male GalT-KO Miniature Swine (8-10 kg) was used for constructing the autologous thymoliver. The properties of these animals have been previously described (see, e.g., Sachs et al., Transplantation 22, 559-567 (1976); Pennington et al., Transplantation 31, 66-71 (1981); Lai et al., Science 295, 1089-1092 (2002); Kolber-Simonds et al., PNAS 101, 7335-7340 (2004)). (b) Thymoliver Construction

[0156] The animals were sedated, intubated, and draped. To recover the cervical thymus for thymoliver construction, a midline incision on the neck was made between the sternum and mandibular angles. A total cervical thymectomy was performed according to methods similar to NAI-5000951688v1 63Attorney Docket No.14648-044-228 those described in LaMattina JC, et al. Transplantation (2002) Mar 15;73(5):826-31, the disclosure of which is incorporated herein in its entirety.

[0157] On the same animal, a midline abdominal incision was made to expose the liver. One thymic lobe was stripped of its capsule, minced into 1-2 mm3pieces, and seeded / inserted intraparenchymally (IP) (FIG.1, solid arrows). Half of the contralateral thymic lobe was sutured within a wedge resection made in the liver (FIG.1, dashed arrows). The abdomen was closed, and the animal was observed for 8 weeks to allow for thymus revascularization. (c) Necropsy and Tissue Collection

[0158] The animals were euthanized 8 weeks after thymoliver construction. The thoracic and abdominal cavities were opened and the thymoliver recovered. Macroscopically, the thymus portion of the thymoliver was identified. Thymoliver samples were recovered as 1 cm3pieces for histopathology. The thymus portion of the thymoliver was further stripped from the liver for subsequent processing and flow cytometric analysis. A piece of the liver and the native thoracic thymus were collected for histopathology and flow cytometric analysis, to be used as negative and positive controls, respectively. (d) Flow Cytometry

[0159] The single cell suspensions from the tissues were Fc-blocked with normal porcine serum and stained with mouse-anti-pig CD3 (Clone: BB23-8E6-8C8), CD4 (Clone: 74-12-4), -2-11) (BD, Franklin Lakes, NJ) antibodies for 30 minutes at 4 C. Thetubes were washed twice with Fluorescent Activated Cell Sorting (FACS) Buffer (BD, Franklin Lakes, NJ). DAPI (4’,6-diamidino-2-phenylindole; Abcam, Waltham, MA) was added before acquisition to distinguish dead cells. The data was acquired on a 5 Laser Cytek Aurora Flow Cytometer (Cytek, Fremont, CA) and analyzed using FCS Express (De Novo Software, Pasadena, CA). Live and singlet cells were selected and plotted using CD4 and CD8 to select CD4+CD8+double-positive (DP) thymocytes. CD4+CD8+DP thymocytes were further gated using CD3 to indicate the presence of CD3-CD4+CD8+DP thymocytes, which is a specific cell phenotype found only in the thymus. (e) Histopathology

[0160] Tissues were collected and fixed in 10% buffered formalin (McKesson, Irving, TX) for 24-48 hours for histopathology studies. Samples were processed and stained with H&E (hematoxylin and eosin) according to standard methods known in the art. Slides were analyzed NAI-5000951688v1 64Attorney Docket No.14648-044-228 using Aperio ImageScope (Leica Camera, Teaneck, NJ) and HALO, a computational pathology software for image analysis (Indica Labs, Albuquerque, New Mexico). 7.1.2 Results

[0161] Macroscopically, there was successful engraftment of thymic tissues in the liver (FIG.2A). Both the sewn-in (FIG.2B) and the IP (FIG.2C) thymus were visible and adhered to the liver. While the further increase in size of the IP thymic tissues appeared restricted, the sewn-in thymus adhered, covered the liver, and increased in size (FIGS.2A-2C). Flow cytometric analysis of both the sewn-in and the IP thymic tissue proved the presence of thymocytes and showed no difference in cellular composition compared to the native thoracic thymus (FIG.3). Histopathological analysis showed the presence of hypercellular thymic cortex, medulla, and Hassall’s corpuscles in both the IP (FIG.4A) and sewn-in (FIG.4B) thymus. 7.1.3 Conclusion

[0162] The exemplary study described above demonstrated thymic engraftment in the liver tissue, which allowed for the construction of a thymoliver organ that can be utilized in liver xenotransplantation. 7.2 Example 2

[0163] Despite the liver's inherent tolerogenic properties in allotransplantation, xenotransplantation faces significant immunological and metabolic challenges. Mixed chimerism, involving the coexistence of donor and recipient cells, is crucial for transplant tolerance. In the exemplary study herein, xenogeneic mixed chimerism early after pig-to-baboon liver xenotransplantations (LXTx) was investigated, assessing the liver graft’s role in establishing chimerism. 7.2.1 Methods

[0164] Four pig-to-baboon liver xenotransplantations (LXTx) were performed using GalT- KO miniature swine as donors. A non-survival experimental model was established to enable monitoring of peripheral blood and collection of liver samples at multiple time points before and after portal vein reperfusion. Peripheral blood and liver mononuclear cells were isolated and analyzed using flow cytometry. Pretransplant samples from both donors and recipients served as baseline controls. Additionally, peripheral chimerism levels were compared with those obtained NAI-5000951688v1 65Attorney Docket No.14648-044-228 from intra-bone marrow transplantation (IBBMTx) models utilizing human CD47 transgenic and GalT-KO swine. Non-Gal donor-specific antibodies (DSA) were quantified to assess the immune response. 7.2.2 Results

[0165] As shown in FIG.5, recipients of LXTx exhibited an average peripheral blood chimerism of 12% pig CD45+cells within 30min. Peripheral mixed chimerism subsequently stabilized between 4.4% and 7.1%, maintaining this range for up to 8h post-reperfusion. Baboon CD45+cells infiltrated the pig liver graft, peaking at 43.5% at 30 minutes post-reperfusion (data not shown). In contrast, the intra-bone marrow transplantation (IBBMTx) model initially showed higher chimerism, but this rapidly declined within 30min and remained low (0.1%) thereafter. Peripheral chimerism in LXTx was predominantly characterized by pig CD4+cells, which peaked at 66.7% at 2.5h post-reperfusion (data not shown). DSA assays indicated decreased levels of IgM and IgG post-reperfusion, with IgA also trending downward, except for one LXTx recipient (data not shown). 7.2.3 Conclusion

[0166] The exemplary study described above demonstrated that xenogeneic mixed chimerism was achievable in a pig-to-baboon LXTx model. Chimerism persisted for at least 8h following portal vein reperfusion and was notably higher compared to that observed in IBBMTx. 8. ILLUSTRATIVE EMBODIMENTS

[0167] The present disclosure provides the following non-limiting embodiments: 1. An isolated porcine thymoliver comprising: a. a porcine liver; and b. porcine thymic tissue, wherein the thymic tissue adheres to the liver. 2. The thymoliver of embodiment 1, wherein the thymic tissue adheres to the liver parenchyma and / or the surface of the liver. 3. The thymoliver of embodiment 1 or embodiment 2, wherein the thymic tissue adheres to at least 25%, 30%, 35%, 40%, 45%, 50% of the surface of the liver. 4. The thymoliver of any one of embodiments 1-3, wherein the thymic tissue adheres to at least one lobe of the liver. NAI-5000951688v1 66Attorney Docket No.14648-044-228 5. The thymoliver of embodiment 4, wherein the at least one lobe of the liver is selected from the group consisting of right lobe, left lobe, caudate lobe, and quadrate lobe. 6. The thymoliver of any one of embodiments 1-5, wherein the thymic tissue adheres to at least one segment of the liver. 7. The thymoliver of embodiment 6, wherein the at least one segment of the liver is selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII. 8. The thymoliver of embodiment 6 or embodiment 7, wherein the at least one segment of the liver is segment IV. 9. The thymoliver of any one of embodiments 1-8, wherein the liver and / or thymic tissue are from highly inbred porcine donors. 10. The thymoliver of any one of embodiments 1-9, wherein the liver and / or thymic tissue are from a minipig. 11. The thymoliver of embodiment 10, wherein the liver and / or thymic tissue are from the same individual minipig. 12. The thymoliver of embodiment 10 or 11, wherein the minipig is a genetically modified minipig. 13. The thymoliver of embodiment 12, wherein the genetically modified minipig has at least one genetic modification. 14. The thymoliver of embodiment 13, wherein the least one genetic modification comprises: a. a mutation in the GGTA1 gene; b. a mutation in the CMAH gene; c. a mutation in the gene. 15. The thymoliver of embodiment 14, wherein the least one genetic modification comprises no expression of a protein encoded by the GGTA1 gene, the CMAH gene, and / or the gene. 16. The thymoliver of embodiment 13, wherein the least one genetic modification comprises expression of a protein for a porcine CTLA4 fused to a hinge and CH2 / CH3 regions of human IgG1 (pCTLA4-Ig). 17. The thymoliver of embodiment 13, wherein the least one genetic modification results in the inactivation of porcine endogenous retrovirus (PERV) . NAI-5000951688v1 67Attorney Docket No.14648-044-228 18. The thymoliver any one of embodiments 12-17, wherein the genetically modified minipig comprises a GGTA1 genetic modification. 19. The thymoliver any one of embodiments 12-18, wherein the genetically modified minipig does not express alpha-1,3-galactosyltransferase (GalT-KO). 20. The thymoliver of any one of embodiments 1-19, wherein the thymic tissue is fully neovascularized. 21. The thymoliver of any one of embodiments 1-20, wherein the thymic tissue has a cell density equal to that of a porcine thymic tissue that is not included in the isolated porcine thymoliver. 22. The thymoliver of any one of embodiments 1-21, wherein the thymic tissue is capable of supporting thymopoiesis. 23. The thymoliver of embodiment 22, wherein the thymic tissue comprises CD3-CD4+CD8+double-positive (DP) thymocytes. 24. The thymoliver of embodiment 23, wherein the thymic tissue comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD3- CD4+CD8+double-positive (DP) thymocytes. 25. The thymoliver of any one of embodiments 1-24, wherein the thymoliver has at least one genetic modification. 26. The thymoliver of any one of embodiments 1-25, wherein the thymoliver does not express alpha-1,3-galactosyltransferase (GalT-KO). 27. A swine comprising the thymoliver of any one of embodiments 1-26. 28. A method of treating a disease of the liver in a human subject in need thereof, wherein the method comprises transplanting the thymoliver of any one of embodiments 1-26 into the subject. 29. The method of embodiment 28, wherein the human subject has or is suspected of having end-stage liver disease. 30. The method of embodiment 28 or embodiment 29, wherein a disease of the liver in the human subject is selected from the group consisting of acute hepatic necrosis, alcohol-related liver disease, autoimmune hepatitis (AIH), biliary atresia, bile duct cancer, liver cancer, metabolic disease, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis NAI-5000951688v1 68Attorney Docket No.14648-044-228 (NASH), viral hepatitis, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC). 31. A method of creating the thymoliver of any one of embodiments 1 to 26 or the swine of embodiment 27 wherein the method comprises: a. obtaining thymic tissue from a porcine donor animal; b. transplanting the thymic tissue obtained in step a into the liver of a recipient animal. 32. The method of embodiment 31, wherein the thymic tissue is obtained using cervical thymectomy. 33. The method of embodiment 31, wherein the transplanting step 31.b is conducted by inserting the thymic tissue into the liver of the recipient animal intraparenchymally. 34. The method of embodiment 33, wherein at least 1 mm3of the thymic tissue is transplanted into the liver of the recipient animal intraparenchymally. 35. The method of embodiment 33 or embodiment 34, wherein the thymic tissue is transplanted into the liver of the recipient animal intraparenchymally about 1 mm to 5 mm deep into the liver parenchyma. 36. The method of any one of embodiments 33-35, wherein thymic tissue is transplanted into the liver of the recipient animal intraparenchymally in at least one location within the liver parenchyma. 37. The method of embodiment 36, wherein thymic tissue is transplanted into the liver of the recipient animal intraparenchymally in one, two, three, four, five six, seven, eight, nine, ten, or more locations within the liver parenchyma. 38. The method of embodiment 31, wherein the transplanting step 31.b is conducted by inserting the thymic tissue into a wedge resection within the liver of the recipient animal. 39. The method of embodiment 38, wherein about 1 cm3to 2 cm3liver parenchyma is resected. 40. The method of embodiment 38 or embodiment 39, wherein the thymic tissue inserted into the wedge resection comprises at least half of a thymic lobe. 41. The method of any one of embodiments 38-40, further comprising suturing the thymic tissue in the wedge resection. NAI-5000951688v1 69Attorney Docket No.14648-044-228 42. The method of embodiment 31, wherein the transplanting step 31.b is conducted by inserting the thymic tissue into an incision within the liver of the recipient animal. 43. The method of embodiment 42, wherein the thymic tissue inserted into the incision comprises at least half of a thymic lobe. 44. The method of any one embodiment 42 or embodiment 43, further comprising suturing the thymic tissue in the incision. 45. The method of any one of embodiments 31-44, wherein the transplanting step 31.b comprises inserting the thymic tissue into the liver of the recipient animal intraparenchymally, into a wedge resection, and / or into an incision within the liver of the recipient animal. 46. The method of any one of embodiments 31-45, wherein the thymoliver is harvested from the recipient animal at least 8 weeks after the transplanting step 31.b. 47. The method of embodiment 46, wherein the thymoliver is harvested from the recipient animal at least 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the transplanting step 31.b. 48. The method of any one of embodiments 31-47, wherein the thymus tissue of the thymoliver harvested from the recipient animal after the transplanting step 31.b is a vascularized thymus tissue. 49. The method of any one of embodiments 31-48, wherein the thymus tissue of the thymoliver harvested from the recipient animal after the transplanting step 31.b is adhered to the liver parenchyma. 50. The method of embodiment 49, wherein the thymoliver comprises at least about 25%, 30%, 35%, 40%, 45%, 50% thymus tissue adhered to the liver parenchyma. 51. The method of any one of embodiments 31-50, wherein the thymus tissue of the thymoliver harvested from the recipient animal after the transplanting step 31.b is adhered to the liver surface. 52. The method of embodiment 51, wherein the thymoliver comprises at least about 25%, 30%, 35%, 40%, 45%, 50% thymus tissue adhered to the liver surface. 9. EQUIVALENTS

[0168] 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 NAI-5000951688v1 70Attorney Docket No.14648-044-228 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.

[0169] 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 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-5000951688v1 71

Claims

Attorney Docket No.14648-044-228 WHAT IS CLAIMED:

1. An isolated porcine thymoliver comprising: a. a porcine liver; and b. porcine thymic tissue, wherein the thymic tissue adheres to the liver.

2. The thymoliver of claim 1, wherein the thymic tissue adheres to the liver parenchyma and / or the surface of the liver.

3. The thymoliver of claim 1 or claim 2, wherein the thymic tissue adheres to at least 25%, 30%, 35%, 40%, 45%, 50% of the surface of the liver.

4. The thymoliver of any one of claims 1-3, wherein the thymic tissue adheres to at least one lobe of the liver.

5. The thymoliver of claim 4, wherein the at least one lobe of the liver is selected from the group consisting of right lobe, left lobe, caudate lobe, and quadrate lobe.

6. The thymoliver of any one of claims 1-5, wherein the thymic tissue adheres to at least one segment of the liver.

7. The thymoliver of claim 6, wherein the at least one segment of the liver is selected from the group consisting of segments I, II, III, IV, V, VI, VII, and VIII.

8. The thymoliver of claim 6 or claim 7, wherein the at least one segment of the liver is segment IV.

9. The thymoliver of any one of claims 1-8, wherein the liver and / or thymic tissue are from highly inbred porcine donors. NAI-5000951688v1 72Attorney Docket No.14648-044-228 10. The thymoliver of any one of claims 1-9, wherein the liver and / or thymic tissue are from a minipig.

11. The thymoliver of claim 10, wherein the liver and / or thymic tissue are from the same individual minipig.

12. The thymoliver of claim 10 or 11, wherein the minipig is a genetically modified minipig.

13. The thymoliver of claim 12, wherein the genetically modified minipig has at least one genetic modification.

14. The thymoliver of claim 13, wherein the least one genetic modification comprises: a. a mutation in the GGTA1 gene; b. a mutation in the CMAH gene; c. a mutation in the gene.

15. The thymoliver of claim 14, wherein the least one genetic modification comprises no expression of a protein encoded by the GGTA1 gene, the CMAH gene, and / or the gene.

16. The thymoliver of claim 13, wherein the least one genetic modification comprises expression of a protein for a porcine CTLA4 fused to a hinge and CH2 / CH3 regions of human IgG1 (pCTLA4-Ig).

17. The thymoliver of claim 13, wherein the least one genetic modification results in the inactivation of porcine endogenous retrovirus (PERV).

18. The thymoliver any one of claims 12-17, wherein the genetically modified minipig comprises a GGTA1 genetic modification. NAI-5000951688v1 73Attorney Docket No.14648-044-228 19. The thymoliver any one of claims 12-18, wherein the genetically modified minipig does not express alpha-1,3-galactosyltransferase (GalT-KO).

20. The thymoliver of any one of claims 1-19, wherein the thymic tissue is fully neovascularized.

21. The thymoliver of any one of claims 1-20, wherein the thymic tissue has a cell density equal to that of a porcine thymic tissue that is not included in the isolated porcine thymoliver.

22. The thymoliver of any one of claims 1-21, wherein the thymic tissue is capable of supporting thymopoiesis.

23. The thymoliver of claim 22, wherein the thymic tissue comprises CD3-CD4+CD8+double-positive (DP) thymocytes.

24. The thymoliver of claim 23, wherein the thymic tissue comprises at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CD3- CD4+CD8+double-positive (DP) thymocytes.

25. The thymoliver of any one of claims 1-24, wherein the thymoliver has at least one genetic modification.

26. The thymoliver of any one of claims 1-25, wherein the thymoliver does not express alpha-1,3-galactosyltransferase (GalT-KO).

27. A swine comprising the thymoliver of any one of claims 1-26.

28. A method of treating a disease of the liver in a human subject in need thereof, wherein the method comprises transplanting the thymoliver of any one of claims 1-26 into the subject. NAI-5000951688v1 74Attorney Docket No.14648-044-228 29. The method of claim 28, wherein the method does not comprise bone marrow transplantation, mobilized peripheral blood stem cell transplantation, and / or hematopoietic stem cell transplantation.

30. The method of claim 28, wherein the method does not comprise bone marrow transplantation.

31. The method of claim 28, wherein the method does not comprise mobilized peripheral blood stem cell transplantation.

32. The method of claim 28, wherein the method does not comprise hematopoietic stem cell transplantation.

33. The method of claim 28, wherein the human subject has or is suspected of having end-stage liver disease.

34. The method of claim 28 or claim 33, wherein a disease of the liver in the human subject is selected from the group consisting of acute hepatic necrosis, alcohol-related liver disease, autoimmune hepatitis (AIH), biliary atresia, bile duct cancer, liver cancer, metabolic disease, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), viral hepatitis, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC).

35. A method of creating the thymoliver of any one of claims 1 to 26 or the swine of claim 27 wherein the method comprises: a. obtaining thymic tissue from a porcine donor animal; b. transplanting the thymic tissue obtained in step a into the liver of a recipient animal.

36. The method of claim 35, wherein the thymic tissue is obtained using cervical thymectomy. NAI-5000951688v1 75Attorney Docket No.14648-044-228 37. The method of claim 35, wherein the transplanting step 35.b is conducted by inserting the thymic tissue into the liver of the recipient animal intraparenchymally.

38. The method of claim 37, wherein at least 1 mm3of the thymic tissue is transplanted into the liver of the recipient animal intraparenchymally.

39. The method of claim 37 or claim 38, wherein the thymic tissue is transplanted into the liver of the recipient animal intraparenchymally about 1 mm to 5 mm deep into the liver parenchyma.

40. The method of any one of claims 37-39, wherein thymic tissue is transplanted into the liver of the recipient animal intraparenchymally in at least one location within the liver parenchyma.

41. The method of claim 40, wherein thymic tissue is transplanted into the liver of the recipient animal intraparenchymally in one, two, three, four, five six, seven, eight, nine, ten, or more locations within the liver parenchyma.

42. The method of claim 35, wherein the transplanting step 35.b is conducted by inserting the thymic tissue into a wedge resection within the liver of the recipient animal.

43. The method of claim 42, wherein about 1 cm3to 2 cm3liver parenchyma is resected.

44. The method of claim 42 or claim 43, wherein the thymic tissue inserted into the wedge resection comprises at least half of a thymic lobe.

45. The method of any one of claims 42-44, further comprising suturing the thymic tissue in the wedge resection.

46. The method of claim 35, wherein the transplanting step 35.b is conducted by inserting the thymic tissue into an incision within the liver of the recipient animal. NAI-5000951688v1 76Attorney Docket No.14648-044-228 47. The method of claim 46, wherein the thymic tissue inserted into the incision comprises at least half of a thymic lobe.

48. The method of any one claim 46 or claim 47, further comprising suturing the thymic tissue in the incision.

49. The method of any one of claims 35-48, wherein the transplanting step 35.b comprises inserting the thymic tissue into the liver of the recipient animal intraparenchymally, into a wedge resection, and / or into an incision within the liver of the recipient animal.

50. The method of any one of claims 35-49, wherein the thymoliver is harvested from the recipient animal at least 8 weeks after the transplanting step 35.b.

51. The method of claim 50, wherein the thymoliver is harvested from the recipient animal at least 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks after the transplanting step 35.b.

52. The method of any one of claims 35-51, wherein the thymus tissue of the thymoliver harvested from the recipient animal after the transplanting step 35.b is a vascularized thymus tissue.

53. The method of any one of claims 35-52, wherein the thymus tissue of the thymoliver harvested from the recipient animal after the transplanting step 35.b is adhered to the liver parenchyma.

54. The method of claim 53, wherein the thymoliver comprises at least about 25%, 30%, 35%, 40%, 45%, 50% thymus tissue adhered to the liver parenchyma.

55. The method of any one of claims 35-54, wherein the thymus tissue of the thymoliver harvested from the recipient animal after the transplanting step 35.b is adhered to the liver surface. NAI-5000951688v1 77Attorney Docket No.14648-044-228 56. The method of claim 55, wherein the thymoliver comprises at least about 25%, 30%, 35%, 40%, 45%, 50% thymus tissue adhered to the liver surface. NAI-5000951688v1 78

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