Methods and compositions for preventing herpes virus reactivation in organ xenograft

Abstract

Disclosed herein include methods and compositions for pig-to-human organ xenotransplantation using genetically modified pigs with triple-gene knockouts and human gene insertions. In some embodiments, the organ xenotransplantation is a pig-to-human kidney xenotransplantation demonstrating the restoration of renal function. Also disclosed are methods and compositions for preventing acute rejection and / or reactivation of porcine herpesvirus, which contributes to advancing xenotransplantation as a viable solution to the global organ shortage.

Description

Attorney Docket No. 71951-708.602METHODS AND COMPOSITIONS FOR PREVENTING HERPES VIRUS REACTIVATION IN ORGAN XENOGRAFTCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to International Patent Application No. PCT / CN2024 / 120036 filed on September 20, 2024, the disclosures of which application are incorporated herein in the entirety for all purposes.BACKGROUND

[0002] The persistent global organ shortage has spurred intense interest and innovation in transplantation medicine, leading to the exploration of xenotransplantation as a potential solution. Recent breakthroughs in biotechnology have paved the way for genetically engineered porcine kidney xenotransplantation, marked by initial progress in eight transplantations into brain-dead humans (six cases of kidney and two cases of heart xenotransplantation)1'7. These studies demonstrated that targeted gene modifications can effectively overcome the natural hyperacute rejection response encountered in pig-to-human xenotransplantation. However, the brain-dead decedent model, while offering clinical relevance beyond non-human primate (NHP) models, has inherent limitations in terms of observation duration, with the reported times ranging from 54-74 hours to a maximum of 7 days5. This limited timeframe or premature termination may not allow sufficient progression of the pathological changes of acute xenograft rejection.

[0003] Improved xenograft and methods of using the xenograft for transplantation and / or clinical observation is needed. This disclosure addresses these and other needs.SUMMARY

[0004] In some embodiments, provided herein is a genetically modified porcine cell, tissue, organ, or animal comprising in its genome: (i) inactivation of porcine genes GGTA1, P4GalNT2, and CMAH; (ii) a transgene expressing hCD55, wherein the genome comprises no more than five transgenes. In some embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal comprises no more than four transgenes. In some embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal comprises no more than three transgenes. In some embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal comprises no more than two transgenes.

[0005] In any of the preceding embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal can comprise a transgene selected from the group consisting of a human anticoagulant gene, a human complement inhibitor gene, and a humanAtorney Docket No. 71951-708.602 immunomodulatory gene, or any combination thereof. In any of the preceding embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal can comprise a transgene selected from the group consisting of hTBM, hCD39, human tissue factor pathway inhibitor, human endothelial protein C receptor, hCD46, hCD59, A20, HO-1, CD47, HLA-E, B2M, PD-L1, and FasL, or any combination thereof.

[0006] In any of the preceding embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal can comprise a transgene expressing hTBM. In any of the preceding embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal can comprise no inactivation of any other porcine gene, or alternatively, comprise inactivation of the porcine growth hormone receptor (GHR) gene.

[0007] In any of the preceding embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal can comprise (i) inactivation of porcine genes GGTA1, p4GalNT2, and CMAH, and no inactivation of any other porcine gene; and (ii) a transgene expressing hCD55, a transgene expressing hTBM, and no other human transgene.

[0008] In any of the preceding embodiments, the genetically modified porcine cell, tissue, organ, or animal can be free of porcine endogenous retroviruses. In any of the preceding embodiments, the genetically modified porcine cell, tissue, organ, or animal can be free of porcine roseoloviruses. In any of the preceding embodiments, the genetically modified porcine cell, tissue, organ, or animal can be free of porcine cytomegaloviruses.

[0009] In any of the preceding embodiments, the genetically modified porcine cell, tissue, organ, or animal can be free of red blood cell antigens. In any of the preceding embodiments, the genetically modified porcine cell, tissue, organ, or animal can be a donor and express a red blood cell antigen type that matches the red blood cell antigen type of a human recipient.

[0010] In any of the preceding embodiments, the genetically modified porcine cell, tissue, or organ can be or be from a kidney, heart, lung, liver, spleen, pancreas, or cornea.

[0011] In any of the preceding embodiments, the porcine gene inactivation can be selected from the group consisting of gene knock-out, gene replacement, point mutation, deletion, or disruption of the porcine gene.

[0012] In some embodiments, disclosed herein is a method for treating a subject in need thereof, comprising transplanting into the subject the genetically modified porcine cell, tissue, or organ of any one of the preceding embodiments. In some embodiments, the subject is a human and the genetically modified porcine organ is a kidney, heart, lung, liver, spleen, pancreas, or cornea.

[0013] In any of the preceding embodiments, the method can further comprise detecting the presence or absence of porcine endogenous retroviruses in the genetically modified porcine cell,Atorney Docket No. 71951-708.602 tissue, or organ prior to the transplanting. In any of the preceding embodiments, the method can further comprise removing or inactivating porcine endogenous retroviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting. In any of the preceding embodiments, the method can further comprise detecting the presence or absence of porcine roseoloviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting. In any of the preceding embodiments, the method can further comprise removing or inactivating porcine roseoloviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting. In any of the preceding embodiments, the method can further comprise comprising detecting the presence or absence of porcine cytomegaloviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting. In any of the preceding embodiments, the method can further comprise removing or inactivating porcine cytomegaloviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting.

[0014] In any of the preceding embodiments, the method can further comprise administering to the subject a therapeutic agent. In some embodiments, the therapeutic agent comprises an antirejection agent, an anti-inflammatory agent, an immunosuppressive agent, an immunomodulatory agent, an anti -microbial agent, or an anti-viral agent, or any combination thereof. In any of the preceding embodiments, the therapeutic agent can comprise a CD40- CD40L pathway blocking antibody, belatacept, anti-thymocyte globulin (rATG, thymoglobulin), rituximab, tacrolimus, my cophenolate mofetil, steroid, anti-C5 antibody, Cl inhibitor, or methylprednisolone, or any combination thereof.

[0015] In any of the preceding embodiments, acute rejection of the genetically modified porcine cell, tissue, or organ may not occur until at least 12 days post -transplantation.

[0016] In some embodiments, provided herein is a method for treating a human individual in need thereof, comprising: transplanting into the human individual a genetically modified porcine organ comprises in its genome: (i) inactivation of porcine genes GGTA1, p4GalNT2, and CMAH; and (ii) a transgene expressing hCD55 and optionally a transgene expressing hTBM; and administering to the human individual a therapeutic agent comprising an anti-rejection agent, an anti-inflammatory agent, an immunosuppressive agent, an immunomodulatory agent, an anti -microbial agent, or an anti-viral agent, or any combination thereof, wherein acute rejection of the genetically modified porcine organ does not occur until at least 12 days posttransplantation.

[0017] In some embodiments, the genetically modified porcine organ does not express red blood cell antigens. In some embodiments, the genetically modified porcine organ expresses a red blood cell antigen type that matches the red blood cell antigen type of the human individual.Atorney Docket No. 71951-708.602

[0018] In any of the preceding embodiments, the genetically modified porcine organ can be free of porcine endogenous retroviruses, porcine roseoloviruses, and porcine cytomegaloviruses.

[0019] In any of the preceding embodiments, the method can further comprise administering to the human individual one or more anti-viral agents that prevent infection by and / or reactivation of porcine endogenous retroviruses, porcine roseoloviruses, and porcine cytomegaloviruses.

[0020] In any of the preceding embodiments, the genome of the genetically modified porcine organ can comprise no more than nine genetic modifications including inactivation of porcine genes and human transgenes. In any of the preceding embodiments, the genome of the genetically modified porcine organ can comprise no more than six genetic modifications including inactivation of porcine genes and human transgenes. In any of the preceding embodiments, the genome of the genetically modified porcine organ can comprise no inactivation of any porcine gene other than GGTA1, p4GalNT2, and CMAH and no human transgene other than hCD55 and optionally hTBM.

[0021] In some embodiments, provided herein is a method for treating a human individual in need thereof, comprising: transplanting into the human individual a genetically modified porcine organ comprises in its genome: (i) inactivation of porcine genes GGTA1, p4GalNT2, and CMAH, and no inactivation of any other porcine gene; and (ii) a transgene expressing hCD55, optionally a transgene expressing hTBM, and no other human transgene, wherein the genetically modified porcine organ expresses a red blood cell antigen type that matches the red blood cell antigen type of the human individual; administering to the human individual an immunosuppressive agent; and administering to the human individual a complement inhibitor, wherein administration of the immunosuppressive agent is initiated earlier than administration of the complement inhibitor.

[0022] In some embodiments, the method further comprises administering to the human individual one or more anti-viral agents that prevent infection by and / or reactivation of porcine endogenous retroviruses, porcine roseoloviruses, and porcine cytomegaloviruses.

[0023] In any of the preceding embodiments, the immunosuppressive agent can comprise a CD40-CD40L pathway blocking antibody, belatacept, anti-thymocyte globulin (rATG, thymoglobulin), rituximab, tacrolimus, mycophenolate mofetil, steroid, or methylprednisolone, or any combination thereof. In any of the preceding embodiments, the complement inhibitor can comprise an anti-C5 antibody.

[0024] In any of the preceding embodiments, the genetically modified porcine organ can be a kidney.

[0025] In any of the preceding embodiments, acute rejection of the genetically modified porcine organ may not occur until at least 12 days post -transplantation.Atorney Docket No. 71951-708.602BRIEF DESCRIPTIONS OF FIGURES

[0026] FIGS. 1A-1C show exemplary processes and immunosuppression regimen. FIG. 1A shows the timeline and event summary for two cases of porcine-to-human decedent kidney transplantation. FIG. IB shows the pharmacologic immunosuppression regimen of Case 1. FIG. 1C shows the pharmacologic immunosuppression regimen of Case 2.

[0027] FIGS. 2A-2C show clinical outcomes and immunological monitoring of Case 1 throughout the postoperative course. FIG. 2A shows gross findings from the xenograft: xenograft perfusion (Al), xenograft reperfusion (A2), xenograft at termination (A3), and crosssection of the xenograft (A4). FIG. 2B shows changes in urine output, serum creatinine, platelets, hemoglobin, tacrolimus concentration, and lymphocyte subsets. FIG. 2C shows changes in antibody binding (IgM and IgG) to donor pig PBMCs and CDC against the same PBMCs. (Mix: the pooled sera of 20 healthy human volunteers were used as control sera).

[0028] FIGS. 3A-3D show clinical outcomes and immunological monitoring of Case 2 throughout the postoperative course. FIG. 3A shows gross findings from the xenograft: xenograft perfusion (Al), xenograft reperfusion (A2), xenograft at termination (A3), and crosssection of the xenograft (A4). FIG. 3B shows changes in urine output, serum creatinine, platelets, hemoglobin, tacrolimus concentration and lymphocyte subsets. FIG. 3C shows changes in antibody binding (IgM and IgG) to donor pig PBMCs and CDC against the same PBMCs. (Mix: the pooled sera of 20 healthy human volunteers were used as control sera). FIG. 3D shows expression of blood group A antigen on xenografts and the changes in serum anti-A antibodies against human type A RBCs.

[0029] FIGS. 4A-4C show histopathology of xenograft of Case 1. FIG. 4A shows HE staining of the Case 1 xenograft on postoperative day 7, day 10, day 12, and EM on postoperative day 12. Thick arrows show mild intimal arteritis (Al) and acute tubular injury(A3). Arrowheads show mild glomerulitis (A5, 7). Arrows show mild peritubular capillaritis (A5,6,7). EM of graft on day 12 shows endothelial cell swelling and a widened subendothelial space with loss of endothelial fenestrations (black asterisk) and a small amount of fibrin tactoid in subendothelial area (white arrow), but no fibrin thrombi (A4). (A8) tubule epithelial cell swelling and disintegration (white asterisk); PTCs are dilated (black asterisk) and monocyte-infiltrated (black arrow). Bars represent 100 microns (Al-3), 50 microns (A5-7), 2 microns (A4), and 10 microns (A8), respectively. FIG. 4B shows immunohistochemical staining of C4d and immunofluorescent staining of immunoglobulin and complement of the Case 1 xenograft on postoperative day 7 (Bl-4), day 10 (B5-8), and day 12 (B9-13). C4d staining is focally positive in PTC on day 7 and diffusely positive on day 10 (Bl, B5), C4d-positive tubular epithelium on day 10 (B5), and diffusely C4d-positive PTC on day 12 (B9). Immunofluorescent staining forAttomey Docket No. 71951-708.602IgM, IgG, and C3c was weakly and sporadically positive on day 7 and day 10 (B2-4,6-8), but prominently positive on day 12 (BIO-12). A prominently positive deposition of C5b-9 was found on day 12 (B13). Arrowheads show positive staining of glomeruli. White arrows show positive peritubular capillary (BIO) or positive staining of tubular epithelial cells (Bl 1,12). Bars represent 100 microns (B2-4,6-8,10-13) and 50 microns (B 1,5,9), respectively. FIG. 4C shows immunohistochemical staining of infiltrated inflammatory cell phenotypes of the Case 1 xenograft on postoperative day 7 (Cl-5), day 10 (C6-10), and day 12 (Cl 1-15). Staining is shown for CD3 (Cl, 6, 11), CD4 (C2,7,12), CD8 (C3,8,13), CD16 (C4,9,14), CD68 (C5,10,15). Thick arrows show multifocal lymphocyte infiltrated into the renal interstitium (C 1 -3,6-8, 11- 13), and arrowheads show infiltrated CD8+lymphocytes (C8), CD16+NK cells (C4,14), and CD68+macrophages (C5,15) in the glomerulus. Arrows show peritubular capillaritis and infiltrated CD16+NK cells (C4,9,14) and CD68+macrophages (C5,10,15). Bars represent 50 microns.

[0030] FIGS. 5A-5C show histopathology of xenograft of Case 2. FIG. 5A shows HE staining of the Case 2 xenograft on postoperative day 3 (Al, 5), day 6 (A2, 6), day 12 (A3, 7) and EM on postoperative day 12 (A4, 8). There was no significant mononuclear cell infiltration in the interstitium during the postoperative observation period. Arrows show mild peritubular capillaritis (A7). Dayl2 EM observation of the xenograft revealed monocyte infiltration into glomerular capillary loops and adhesion to endothelial cells, and neutrophil infiltration into peritubular capillaries (arrows). Bars represent 100 microns (Al -3), 50 microns (A5-7), 2 microns (A4), and 10 microns (A8), respectively. FIG. 5B shows immunohistochemical staining for C4d and immunofluorescent staining for immunoglobulin and complement in the Case 2 xenograft on postoperative day 3 (Bl-4), day 6 (B5-8), and day 12 (B9-13). C4d staining was negative on day 3 (Bl) and diffusely positive on day 6 and day 12 (B5,9). The immunofluorescent staining for IgM, IgG, and C3c were weakly sporadically positive on day 3 and day 6 (B2-4,6-8), while a prominent positive deposition was found on day 12 (BIO-12). A weakly positive deposition of C5b-9 was observed on day 12 (B13). Arrowheads show positive staining of glomeruli. Bars represent 100 microns (B2-4, 6-8, 10-13) and 50 microns (Bl, 5, 9), respectively. FIG. 5C shows immunohistochemical staining of infiltrated inflammatory cell phenotypes of the Case 2 xenograft on postoperative day 3 (Cl-5), day 6 (C6-10), and day 12 (Cl 1-15). Staining is shown for CD3 (Cl, 6, 11), CD4 (C2,7,12), CD8 (C3,8,13), CD16 (C4,9,14), and CD68 (C5,10,15). Arrowheads show glomerulitis and infiltrated CD68+macrophages (CIO, 15) and CD16+NK cells (C14). Arrows show peritubular capillaritis and infiltrated CD68+macrophages (C5,10,15) and CD16+NK cells (C14). Bars represent 50 microns.Attomey Docket No. 71951-708.602

[0031] FIGS. 6A-6D show analysis of PERVs and PCMV / PRV in xenografts and decedent tissues. In FIG. 6A, “W” represents water, as a negative control. Lane 1-3: recipient PBMCs of Pre-Tx, Post-Tx and termination, Lane 4-7: recipient heart, liver, spleen, lung after termination; Lane 8: kidney from a PERVA / B / C-positive pig, Lane 9: Pig-1 kidney after termination. In FIG. 6B, Lanel-7: recipient PBMCs of Pre-Tx, Post-Txl, 3, 5, 7, 10 and termination, Lane 8- 12: recipient heart, liver, spleen, lung, and lymph nodes after termination; Lane 13: the kidney from a PERVA / B / C-positive pig; Lane 14: Pig-2 kidney after termination. FIG. 6C shows PCMV / PRV detected by nested PCR using xenografts and Case 1 samples. FIG. 6D shows PCMV / PRV detected by nested PCR using xenografts and Case 2 samples. The transplanted transgenic kidney from the donor pig, and the heart, liver, spleen, and lung of the recipient were analyzed. Pig (+) is a PCMV / PRV-positive pig; water is shown as a negative control.

[0032] FIGS. 7A-7C show manufacturing overview of gene-engineered pigs used in the examples. FIG. 7A shows primary ear fibroblasts from GTKO pigs were used for targeted insertion of the human CD55 or TBM genes. GTKO / hCD55 and GTKO / hTBM pigs were generated via somatic cell cloning and embryo transfer. Fl piglets were screened, and selected GTKO / hCD55 pigs were mated with GTKO / hTBM pigs. The B4GalNT2 and CMAH genes were then knocked out in fibroblasts from these pigs. FIG. 7B shows CRISPR-Cas9 was used to knock out three xenoantigens in pigs for potential pig-to-human xenotransplantation targets. Guide RNAs targeted GGTA1, CMAH, and B4GALNT2 / B4GalNT2-like genes, which are family genes with products having the same function. FIG. 7C shows the human CD55 gene, driven by the EFl -a promoter, was inserted into the Rosa26 site of the porcine genome. The porcine TBM promoter drove the expression of the human TBM gene, which was integrated between the PCDH17 and DIAPH3 genes on chromosome 11.

[0033] ABBREVIATIONS: AMR, antibody-mediated rejection; p4GalNT2, 1,4-N-acetyl- galactosaminyl transferase 2; CDC, complement-dependent cytotoxicity; CMAH, CMP-N- acetylneuraminic acid hydroxylase; EM, electron microscopy; MVI, microvascular inflammation; NHP, non-human primate; PBMCs, peripheral blood mononuclear cells; PCMV / PRV, porcine cytomegalovirus / porcine roseolovirus; POD, post-operative day; PTC, peritubular capillaritis; SCr, serum creatinine; TKO, triple-knockout; TCMR, T cell-mediated rejection.DETAILED DESCRIPTION

[0034] All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. IfAtorney Docket No. 71951-708.602 a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0035] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0036] In some embodiments, provided herein are pig-to-human kidney transplants in braindead human recipients, with renal function restoration in kidney xenografts from genetically modified minipigs. In some cases, acute rejection can occur by day 12 without CD40-CD154 blockade, and reactivation of porcine cytomegalovirus (PCMV) may lead to significant immunopathological changes in recipients.

[0037] In some aspects, disclosed herein is an observational model to assess kidney function recovery and xenogeneic immune responses in kidney xenotransplants, focusing on gene editing and immunosuppression. Two brain-dead patients undergo single kidney xenotransplantation, with kidneys donated by minipigs genetically modified to include triple gene knockouts (GGTA1, p4GalNT2, CMAH) and human gene transfers (hCD55 or hCD55 / hTBM). Renal xenograft functions are fully restored, and immunosuppression such as by using CD40-CD154 pathway blockade may prevent acute rejection by day 12. When rejection does occur, this rejection manifests as both T cell-mediated rejection and antibody-mediated rejection (AMR), confirmed by NK cell and macrophage infiltration in sequential xenograft biopsies. Despite donor pigs being pathogen-free before transplantation, xenografts and recipient organs test positive for PCMV / PRV by the end of the observation period, indicating reactivation and contributing to significant immunopathological changes. The extended clinical observation (until day 12 post-transplantation) provided by the genetically modified pig organ is unexpected (e.g., Porrett et al.2used 10 gene-edited pigs where the xenograft function appeared unsatisfactory during the 74-hour observation period), and provides comprehensive evaluation using deceased human models to advance xenograft success.

[0038] In the present disclosure, with full informed consent and graciously written authorization from the next of kin of the brain-dead donors, the observation period was extended as long as feasible by maintaining hemodynamic stability until xenograft dysfunction or specific conditions necessitated termination. This extended timeframe enabled comprehensive investigation of the process of xenograft rejection and dysfunction, especially the posthyperacute rejection phase, shedding light on critical aspects of this groundbreaking procedure, especially transplantation immunology, with an ultimate goal of achieving successful long term kidney function restoration.Attomey Docket No. 71951-708.602

[0039] In some embodiments, the present disclosure includes transgenic animals that are particularly useful as a source of organs, organ fragments, tissues or cells for xenotransplantation. In particular, the present disclosure includes transgenic porcine animals useful as a source of organs, organ fragments, tissues or cells for xenotransplantation. In particular, the present disclosure includes the organs, organ fragments, tissues or cells derived from such donor animals, methods of producing such donor animals, as well as the use of organs, organ fragments, tissues or cells derived from such animal in the treatment of diseases and disorders.

[0040] The organ or organ fragment may be any suitable organ, for example, a lung, heart, liver or pancreas. The tissue may be any suitable tissue, for example, epithelial or connective tissue. The cell may be any suitable cell. The cell may be any suitable cell, for example, an islet cell.

[0041] In some embodiments, the present disclosure includes a transgenic animal (e.g., porcine animal) particularly useful as a source of organs (e.g., kidney), organ fragments, tissues or cells for lung xenotransplantation, and extends to organs (e.g., kidney), organ fragments, tissues and cells derived therefrom, as well as methods of producing the transgenic animal and methods of using the organs, tissues and cells derived therefrom for organ xenotransplantation.

[0042] In some embodiments, a transplant or a graft (e.g., xenograft) as used herein includes the act of inserting tissue or an organ into a subject under conditions that allow the tissue or organ to become vascularized; and shall also refer to the so-inserted (e.g., “implanted” or “transplanted” or “grafted”) tissue or organ. Conditions favoring vascularization of a graft in a mammal comprise a localized tissue bed at the site of the graft having an extensive blood supply network.

[0043] In some embodiments, an immunomodulator includes a transgene with the ability to modulate the immune responses. In exemplary embodiments, an immunomodulator can be a complement inhibitor or an immunosuppressant. In specific embodiments, the immunomodulator is a complement inhibitor. The complement inhibitor can be CD46 (or MCP), CD55, CD59 and / or CD35 / CR1. In a specific embodiment, at least two complement inhibitors can be expressed. In one embodiment, the complement inhibitors can be CD55 and CD59. In another embodiment, the immunomodulator can be a class II transactivator or mutants thereof. In certain embodiments, the immunomodulator can be a class II transactivator dominant negative mutant (CIITA-DN). In another specific embodiment, the immunomodulator is an immunosuppressant. The immunosuppressor can be CTLA4-Ig. Other immunomodulators can be selected from the group but not limited to CIITA-DN, PD-L1, PD-L2, or tumor necrosisAtorney Docket No. 71951-708.602 factor-a related-inducing ligand (TRAIL), Fas ligand (FasL, CD95L), CD47, HLA-E, HLA-DP, HLA-DQ, and / or HLA-DR.

[0044] In some embodiments, an organ includes a collection of tissues joined in a structural unit to serve a common function. The organ may be a solid organ. Solid organs are internal organs that has a firm tissue consistency and is neither hollow (such as the organs of the gastrointestinal tract) nor liquid (such as blood). Examples of solid organs include the heart, kidney, liver, lungs, pancreas, spleen and adrenal glands.

[0045] In some embodiments, a tissue includes cellular organizational level intermediate between cells and a complete organ. A tissue is an ensemble of similar cells from the same origin that together carry out a specific function. Organs are then formed by the functional grouping together of multiple tissues. Examples of tissues contemplated by the present invention include, without limitation, connective tissue, muscle tissue, nervous tissue, epithelial tissue and mineralized tissue. Blood, bone, tendon, ligament, adipose and areolar tissues are examples of connective tissues — which may also be classified as fibrous connective tissue, skeletal connective tissue, and fluid connective tissue. Muscle tissue is separated into three distinct categories: visceral or smooth muscle, found in the inner linings of organs; skeletal muscle, typically attached to bones and which generates gross movement; and cardiac muscle, found in the heart where it contracts to pump blood throughout an organism. Cells comprising the central nervous system and peripheral nervous system are classified as nervous (or neural) tissue. In the central nervous system, neural tissues form the brain and spinal cord. In the peripheral nervous system, neural tissues forms the cranial nerves and spinal nerves, inclusive of the motor neurons.

[0046] In some embodiments, a porcine, porcine animal, pig and swine includes the same type of animal without regard to gender, size, or breed.

[0047] In some embodiments, a subject includes any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like (e.g., that is to be the recipient of a particular treatment (e.g., transplant graft) or that is a donor of a graft. The terms “subject” and “patient” are used interchangeably in reference to a human subject, unless indicated otherwise herein (e.g., wherein a subject is a graft donor).

[0048] In some embodiments, a transgene herein includes a gene or genetic material that has been transferred from one organism to another. When a transgene is transferred into an organism, the organism can then be referred to as a transgenic organism Typically, the term describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporatedAtorney Docket No. 71951-708.602 into the organisms germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum or via somatic cell nuclear transfer where a somatic cell, with the desired transgene(s) is incorporated into the host genome, is transferred to an enucleated oocyte and results in live offspring after transplantation into a surrogate mother. When inserted into a cell, a transgene can be either a cDNA (complementary DNA) segment, which is a copy of mRNA (messenger RNA), or the gene itself residing in its original region of genomic DNA. The transgene can be a genome sequence, in particular when introduced as large clones in BACs (bacterial artificial chromosomes) or cosmid, or could be a form of “minigene” often characterized by a combination of both genomic DNA (including intron regions, e.g. intron 1), 5' or 3' regulatory regions, along with cDNA regions. Transgene “expression” in the context of the present specification, unless otherwise specified, means that a peptide sequence from a non-native nucleic acid is expressed in at least one cell in a host. The peptide can be expressed from a transgene that is incorporated in the host genome. A transgene can comprise a polynucleotide encoding a protein or a fragment (e.g., a functional fragment) thereof. A fragment (e.g., a functional fragment) of a protein can comprise at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amino acid sequence of the protein. A fragment of a protein can be a functional fragment of the protein. A functional fragment of a protein can retain part or all of the function of the protein.

[0049] In some embodiments, using a genetically modified cell, tissue, or organ as a xenograft in a human recipient achieves transplant tolerance. In some embodiments, transplant tolerance includes a state of donor-specific unresponsiveness without a need for ongoing pharmacologic immunosuppression. Transplantation tolerance could eliminate many of the adverse events associated with immunosuppressive agents. As such, induction of tolerance may result in improved receipt of a xenograft. In an embodiment, induction of tolerance may be identified by a decrease in clinical symptoms of xenograft rejection. In another embodiment, induction of tolerance may ameliorate or prevent the metabolic, inflammatory and proliferative pathological conditions or diseases associated with xenograft transplantation. In still another embodiment, induction of tolerance may ameliorate or decrease or prevent the adverse clinical conditions or diseases associated with the administration of immunosuppressive therapy used to prevent xenograft rejection. In still yet another embodiment, induction of tolerance may promote xenograft survival. In a different embodiment, induction of tolerance may prevent relapses in patients exhibiting these diseases or conditions.

[0050] In some embodiments, provided herein is a genetically modified porcine cell, tissue, organ, or animal comprising in its genome: (i) inactivation of porcine genes GGTA1, P4GalNT2, and CMAH; (ii) a transgene expressing hCD55, wherein the genome comprises noAtorney Docket No. 71951-708.602 more than five transgenes. In some embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal comprises no more than four transgenes. In some embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal comprises no more than three transgenes. In some embodiments, the genome of the genetically modified porcine cell, tissue, organ, or animal comprises no more than two transgenes.

[0051] In some embodiments, disclosed herein is a method for treating a subject in need thereof, comprising implanting into the subject the genetically modified porcine cell, tissue, organ, or animal of any one of the preceding embodiments. In some embodiments, the subject is a human and the genetically modified porcine organ is a kidney, heart, lung, liver, spleen, pancreas, or cornea.

[0052] In some embodiments, provided herein is a method for treating a human individual in need thereof, comprising: transplanting into the human individual a genetically modified porcine organ comprises in its genome: (i) inactivation of porcine genes GGTA1, p4GalNT2, and CMAH; and (ii) a transgene expressing hCD55 and optionally a transgene expressing hTBM; and administering to the human individual a therapeutic agent comprising an anti-rejection agent, an anti-inflammatory agent, an immunosuppressive agent, an immunomodulatory agent, an anti -microbial agent, or an anti-viral agent, or any combination thereof, wherein acute rejection of the genetically modified porcine organ does not occur until at least 12 days posttransplantation.

[0053] In some embodiments, provided herein is a method for treating a human individual in need thereof, comprising: transplanting into the human individual a genetically modified porcine organ comprises in its genome: (i) inactivation of porcine genes GGTA1, p4GalNT2, and CMAH, and no inactivation of any other porcine gene; and (ii) a transgene expressing hCD55, optionally a transgene expressing hTBM, and no other human transgene, wherein the genetically modified porcine organ expresses a red blood cell antigen type that matches the red blood cell antigen type of the human individual; administering to the human individual an immunosuppressive agent; and administering to the human individual a complement inhibitor, wherein administration of the immunosuppressive agent is initiated earlier than administration of the complement inhibitor.

[0054] In some embodiments, the method comprises administering to the human individual one or more anti-viral agents that prevent infection by and / or reactivation of porcine endogenous retroviruses, porcine roseoloviruses, and porcine cytomegaloviruses.

[0055] In some embodiments, the immunosuppressive agent comprises a CD40-CD40L pathway blocking antibody, belatacept, anti-thymocyte globulin (rATG, thymoglobulin), rituximab, tacrolimus, mycophenolate mofetil, steroid, or methylprednisolone, or anyAtorney Docket No. 71951-708.602 combination thereof. In some embodiments, the complement inhibitor comprises an anti-C5 antibody.

[0056] In some embodiments, the genetically modified porcine organ is a kidney. In some embodiments, acute rejection of the genetically modified porcine organ does occur until at least 12 days post-transplantation.EXAMPLE 1 : PIG-TO-BRAIN-DEAD HUMAN KIDNEY XENOGRAFT

[0057] This example describes the xenotransplantation procedure and the immunosuppression regimen for two cases of pig-to-brain-dead human kidney xenotransplantation, as illustrated in FIGS. 1A-1C

[0058] Normal renal function was restored rapidly after transplantation in Case 1 (FIGS. 2A- 2C). The mean daily urine output was 4000 mL, with serum creatinine (SCr) fluctuating around 100 pmol / L during the first 4-day period. On post-operative day (POD) 5, urine output decreased significantly to around 1000 mL and then returned to normal until POD 11 after dosing with 500 mg of methylprednisolone. However, SCr levels rose to 238 pmol / L on POD 6 and then fluctuated between 175 and 300 pmol / L, ending at 221 pmol / L on POD 12 (FIG. 2B). During the observation period, the trough level of tacrolimus fluctuated between 5 to 20 ng / ml. Peripheral blood B lymphocyte counts remained low after rituximab administration. However, satisfactory T-lymphocyte counts were not achieved following the administration of rabbit antithymocyte globulin (rATG), with a noticeable temporary rebound on POD 5 (FIG. 2B). At the time of termination on POD 12, the xenograft appeared firm, dark red, and significantly enlarged, with scattered hemorrhagic spots; focal hemorrhages at the corticomedullary junction were seen on the kidney surface (FIG. 2A).

[0059] Case 2 (FIGS. 3A-3D) had a urinary output of >2000 mL / day for the first 9 days after transplantation, and the SCr decreased from 216 pmol / L (POD 0) to 100 pmol / L (POD 5). However, because of a rise in SCr to 135 pmol / L and an elevated renal arterial resistive index (RI) of 0.9 on POD 6, a high dose of methylprednisolone (500 mg / d) was administered. Despite methylprednisolone therapy, the SCr did not decrease significantly and began to increase progressively on POD 9; urinary output decreased significantly, and the SCr was 585 pmol / L at the end of the study on POD 12 (FIG. 3B). The trough level of tacrolimus fluctuated between 5 and 15 ng / mL during the observation period. Peripheral blood T- and B-cell counts remained at very low levels after administering rituximab and rATG (FIG. 3B). A significant increase in SCr level and a marked reduction in urinary output led to terminate the trial on day 12. At the time of termination, the transplanted kidney was grayish-brown in color, markedly enlarged in size, and hard in texture, and focally hemorrhagic foci were visible on the kidney surface (FIG. 3A)Atorney Docket No. 71951-708.602

[0060] Color Doppler ultrasound was used to monitor the changes in blood flow and size of the kidney xenografts. Initially, both xenografts exhibited abundant blood flow signals with no significant volume increase in the first 5-6 days post-xenotransplantation. Subsequently, the blood flow signal of both renal xenografts gradually decreased, and xenograft volume progressively increased, basically consistent with the deterioration of renal function (data not shown).

[0061] Throughout the postoperative period, serum levels of K+, CT, and Ca2+remained within normal range. However, as renal function declined, both recipients experienced a significant increase in serum Na+levels. Notably, elevated serum HCCU levels were observed even during periods of normal renal function. Prothrombin time (PT) and activated partial thromboplastin time (APTT) generally increased with worsening renal function. Concurrently, fibrinogen and antithrombin III levels exhibited an overall downward trend post-transplantation. D-dimer remained at a high level throughout the postoperative process. Urinary microalbumin levels were almost negative when renal function was normal but then were significantly elevated with increased SCr. Erythropoietin (EPO) levels remained consistently low after transplantation in Case 1. In contrast, Case 2 did not experience a decrease in EPO levels as a result of exogenous EPO supplementation (data not shown).

[0062] The presence of anti-donor xenoantibodies

[0063] To determine the levels of anti-donor pig antibodies and complement-dependent cytotoxicity (CDC), serum samples from the two cases before and after transplantation were tested by flow cytometry; peripheral blood mononuclear cells (PBMCs) were isolated from the donor blood samples and used for analysis. The pooled sera of 20 healthy human volunteers were used as control sera.

[0064] Before transplantation, the levels of anti-pig IgM and IgG in the serum of Case 1 were approximately two-fold higher than control pooled human sera, implying significantly higher levels of natural xenoantibodies against triple knockout (TKO) pig PBMCs in this case than in the general population. Consistent with the antibody binding results, the cytotoxicity of pretransplant serum against pig PBMCs was relatively higher than that of the control sera (9.6% vs. 4.4%) (FIG. 2C). At various time points after transplantation, the serum levels of anti -pig IgM and IgG were not significantly increased; this result indicates that the recipient did not produce a significant level of induced xenoantibodies (FIG. 2C), although the possibility of anti -pig antibodies being removed from the serum through absorption by antibody bound to the xenograft could not be completely ruled out.

[0065] Unlike Case 1, the levels of anti -donor pig IgM and IgG antibodies in the pre-transplant serum of Case 2 were initially comparable to those in the pooled human sera. However, aAtorney Docket No. 71951-708.602 significant increase was observed at 6 to 9 days after transplantation, leading to a significant elevation in CDC (FIG. 3C). In addition, since Case 2 was an ABO-incompatible (A-to-B) xenotransplantation, flow cytometry was used to detect changes in the anti -A antibody levels. Serum levels of both anti-A-IgM and IgG were low before transplantation; although the levels of anti-A-IgG did not change significantly afterward, the levels of anti-A-IgM markedly increased (FIG. 3D)

[0066] Histopathological findings

[0067] In Case 1, the multifocal infiltration of mononuclear cells and mild intimal arteritis of the biopsy specimen were observed on POD 7. Furthermore, microvascular inflammation (MVI) characterized by glomerulitis (g) and peritubular capillaritis (PTC) also appeared (FIG. 4A). These changes suggested the presence of acute T cell -mediated rejection (TCMR) and antibody- mediated rejection (AMR). C4d staining was focally positive in the PTC on POD 7 and diffusely positive in POD 10 and POD 12 (FIG. 4B). Immunofluorescent staining on POD 7 and 10 showed only weakly positive, scatered deposition of IgM, IgG, and C3. In contrast, strongly positive IgM, IgG, C3c, and C5b-9 deposits were found in dysfunctional anatomical specimens on POD 12 (FIG. 4B). The infiltrated mononuclear cells included CD3-, CD4-, and CD8- positive lymphocytes, along with an increased number of CD16+NK cells and CD68+macrophages. In the autopsy specimens on POD 12, the degree of glomerulitis was aggravated, and infiltrated CD68+macrophages were significantly increased. CD68+macrophages and CD16+NK cells were mainly infiltrated into the MVI; according to the Banff score, the total score for the MVI was equal to 5 (g3 + ptc2) (FIG. 4C). Electron microscopy (EM) revealed infiltration of monocytes and neutrophils into capillary loops of the glomerulus and peritubular capillaries, significant endothelial cell swelling, and widening of the subendothelial space with near occlusion of the capillary loops, and a small amount of fibrin tactoid deposition in the subendothelium; however, no fibrin thrombi were observed on POD 12 (FIG. 4A).

[0068] In Case 2, no significant acute TCMR changes were observed during the postoperative observation period, but MVI changes were seen in biopsy samples on POD 6 and in autopsy specimens on POD 12 (FIG. 5A and FIG. 5C). Furthermore, C4d was diffusely positive on both POD 6 and 12. The immunofluorescent staining of Case 2 was similar to that of Case 1 in that there were weakly positive, scattered deposits of IgM, IgG, and C3; prominently positive IgM, IgG, C3c, and C5b-9 were also evident in dysfunctional specimens on POD 12 (FIG. 5B). The infiltrating inflammatory cells were mainly CD16+NK cells and CD68+macrophages on POD 6 and POD 12 (FIG. 5C). EM revealed frequent monocytic infiltration into glomerular capillary loops and adhesion to the endothelial cells, as well as infiltration of neutrophils into the peritubular capillaries (FIG. 5A).Atorney Docket No. 71951-708.602

[0069] Although microvascular endothelial injury was evident in the two xenografts, no microthrombosis, renal parenchymal hemorrhage, or necrosis was observed. Acute tubular injury and partial tubular epithelial necrosis, however, were seen in both cases in biopsy and anatomical specimens (FIG. 4A and FIG. 5A). In both kidney grafts, no reduction in human transgene expression was observed on day 12 when compared to that on day 0 (data not shown).

[0070] Donor screening and viral findings

[0071] Donor pigs underwent comprehensive pathogen screening before transplantation, yielding negative results for all pathogens except PERV-A and PERV-B (data not shown). Although the donor pig nasal swab was PCMV / PRV-negative before transplantation, subsequent post-transplant autopsy outcomes revealed the presence of PCMV / PRV DNA in both xenografted kidneys and selected recipient tissues (heart, liver, spleen, and lung) in both cases (FIGS. 6A-6D). It is essential to emphasize that typical PCMV / PRV inclusion bodies or viral particles were conspicuously absent from the histopathological sections and EM specimens, even after meticulous EM scrutiny. Furthermore, PCMV / PRV particles were conspicuously absent from renal tubule epithelial cells, glomerular capillary loop endothelial cells, and peritubular capillary (PTC) endothelial cells (FIG. 4A and FIG. 5A).

[0072] Previous studies have had limited kidney xenotransplantation observation periods ranging from 2 to 7 days1'5, suitable for demonstrating hyperacute rejection scenarios in pig-to- human decedent models. In the present example, with a timeframe extended to 12 days, additional immunological events and definitively pathological manifestations became evident.

[0073] In the previously reported pre-clinical trials of pig-to-human kidney xenotransplantation, donor pigs were either single-gene (GGTA1) knockout or 10 gene-edited (4 gene knockouts and 6 human transgene insertions) animals. Montgomery et al.1transplanted “thymokidneys” from GGTA1 -knockout pigs into two brain-dead human decedents, employing daily high-dose methylprednisolone and twice-daily intravenous mycophenolate mofetil. Both xenografts maintained good function for 54 hours, showing no signs of hyperacute or antibody- mediated rejection. Porrett et al.2transplanted two kidneys from a 10 gene-edited pig into a human deceased recipient and used an immunosuppressive regimen. Although no hyperacute rejection was observed, the xenograft function appeared unsatisfactory during the 74-hour observation period2. They later reported another case by Locke et al.5using the same gene-edited pig, with the addition of anti-C5 monoclonal antibody in the immunosuppressive regimen and found that xenograft function remained normal for 7 days. However, due to the limited observation time, the final outcome remained unknown. There is also a recent case report with an extended observation period of 61 days, which is another step forward in xenotransplantation; however, the detailed results of this case have not yet been published6.Atorney Docket No. 71951-708.602

[0074] In this example, the donor pigs employed were triple-gene knockouts. In addition, in corresponding to the protective effect of hCD55 on renal xenografts in pig-to-NHP models, the donor pigs were either transferred with the hCD55 gene alone or in combination with the human anti coagulation gene thrombomodulin (TBM) (FIGS. 7A-7C). The detection results for the donor pigs confirmed the gene knockout effect and the expression level of the transgenes met the requirements (data not shown). Results of in vitro experiments also showed that human serum IgG and IgM binding to the PBMCs of the gene-edited pigs was almost negligible8.

[0075] Since clinical practice only allows the use of existing FDA-approved immunosuppressants, the study followed almost the strongest regimen consisting of clinically available immunosuppressants. The two renal xenografts in the example underwent acute xenograft rejections within 12 days, suggesting that acute rejection may pose a significant immunological barrier to the long-term survival of future clinical kidney xenotransplantations under current clinically feasible immunosuppressive protocols. Nevertheless, this example did not employ a CD154-CD40 pathway -blocking antibody or thymic tissue to reduce the xenogeneic immune response9. These additional treatments may control acute xenograft rejection and consequently significantly prolong graft survival.

[0076] Cytokine or inflammatory factor storms typically occur in the early stage of fatal brain trauma. However, the cases in this example received prolonged treatment in the emergency department and ICU before brain death. By the time of organ donation and xenotransplantation, the storm phase had likely passed, as indicated by the absence of central diabetes insipidus and near-normal cytokine levels (data not shown). Throughout the observation period, cytokines and inflammatory factors levels, including IL-2, IL-4, IL-6, IL- 10, TNF-a and IFN-y levels were monitored. In Case 1, the levels of all cytokines and inflammatory factors were normal or near normal during the first 7 days after transplantation, and only IL- 10 continued to increase from day 7 onwards. In Case 2, IL-6 levels were high in the early stage and gradually decreased, while other cytokines and inflammatory factors were almost at normal levels (data not shown). These findings suggest that initiating xenograft studies after brain death or cytokine storm should not significantly impact outcomes.

[0077] The results indicated that graft loss was primarily due to specific immune responses (such as AMR and TCMR or their combination), rather than nonspecific inflammatory factors. Histopathological observations definitively revealed the presence of TCMR and AMR in Case 1, and AMR in Case 2. Cellular rejection was evident through multifocal inflammatory cell infiltration in the renal interstitium, significant MVI, diffuse C4d deposition, and positive immunofluorescent staining for immunoglobulin and complement. These findings highlight the crucial role of complement inhibitors (e.g., anti-C5 therapy) even when the xenograft expressesAtorney Docket No. 71951-708.602 hCD55. At present, there are only two kinds of complement inhibitors in clinical practice: anti- C5 mAh and Cl inhibitor, among which anti-C5 mAb has a stronger inhibitory effect on the complement activation. AMR occurred despite the use of anti-C5 mAb in case 2, probably due to the use of a lower dose of anti-C5 mAb (300mg / dose) than the standard dose (900mg / dose). Increasing the dose of anti-C5 mAb may be beneficial to improve the outcome. In addition, a low-dose rATG therapy (approximately 2.5 mg / kg) was used in both cases, which is consistent with common practice of clinical renal allotransplantation in China, but below the US FDA- recommended standard dose (4.5 mg / kg). According to the recommended dose of 0.04-1.5 mg / (kg d) in the Chinese guidelines, the total dose of rATG given to both cases was 150mg. With this dose of rATG, desired T cell depletion was achieved in Case 2. However, Case 1 exhibited relatively weak T cell depletion, which may have contributed to the TCMR in this Case. Therefore, in order to avoid TCMR, future pig-to-human xenotransplantation may require the use of higher doses of rATG. It is noteworthy that initiating ATG treatment earlier than anti- C5 therapy is optimal, since C5 inhibition may reduce the effectiveness of ATG.

[0078] Monocytic infiltration and endothelial cell damage at the site of MVI were both detected. The involvement of AMR was also clearly suggested by electron microscopy, especially in the form of monocytic infiltration and endothelial damage in the capillaries of the microcirculation. These findings indicate that both CDC and antibody -dependent cell-mediated cytotoxicity (ADCC) may play an important role in the initialization and development of AMR.

[0079] Furthermore, it is important to mention a comprehensive study led by Loupy and coworkers4, utilizing specimens provided by Montgomery et al.1, which further demonstrated that potential rejection was evident within 2-3 days after xenotransplantation. Using the latest multidimensional spatial molecular assessment analysis, they found early signs of AMR in renal xenografts from single-gene knockout (GGTA1) pigs transplanted into brain-dead human decedents. Their findings were characterized by microvascular inflammation, primarily representing activation of monocytes and macrophages in the glomeruli, as well as increased natural killer cells and endothelial cell activation. However, the limited observation time prevented these investigators from observing the consequential immunopathological damage and outcomes.

[0080] It is necessary to select recipients with low titers of anti -pig antibodies to ensure successful long-term xenograft survival. In case 1, the recipient exhibited higher anti -pig antibody levels than did the control pooled human sera. However, the lack of a definitive threshold for antibody levels complicates the categorization of low, moderate, and high titers that affect xenograft survival. The results of previous studies8,10,11, which involved pig-to- monkey kidney xenotransplantation, revealed no consistent correlation between anti-pigAtorney Docket No. 71951-708.602 antibody titers and xenograft survival (data not shown). Various factors, including the immunosuppressive regimens, followed and the genetic characteristics of the engineered pigs, also contributed to the outcome.

[0081] No definitive reports exist regarding the development of anti -blood type antibodies (e.g., anti-A) in NHPs and humans following xenotransplantation of genetically modified pig organs, primarily because blood type O pigs are commonly employed. Due to the limited availability of gene-edited pigs, gene-edited pigs and human brain-dead organ donors were randomly paired. In addition, it is often difficult to accurately determine blood type in pigs using commonly used clinical methods, which resulted in the ABO-incompatible (A-to-B) xenotransplantation in case 2. Although pre-transplant serum anti-A IgM and IgG levels were low before transplantation, serum anti-A IgM levels were markedly increased after transplantation, suggesting that blood group antibodies may be involved in AMR in this case. Several significant observations were made concerning case 2, as follows: (i) the study identified antibody-mediated rejection in ABO-incompatible xenotransplants within 2 weeks, an uncommon phenomenon when compared to the same regimen in clinical ABO-incompatible allotransplantation, (ii) the immunosuppressive regimen employed in this example seemed to suppress the development of anti-A IgG antibodies, but not anti-A IgM production. This study highlights the importance of matching blood group types for xenotransplantation. An effective pig blood type testing and screening protocol can be used to ensure the use of O-type pigs as donors for future subclinical and clinical studies.

[0082] Before transplantation, the health of the donor pigs was confirmed, and serum samples obtained on the day of transplantation detected no signs of PCMV / PRV (data not shown). A critical observation was that PCMV / PRV remained absent from the sera of the transplant recipients until 12 (case 1) or 6 days (case 2) post-transplantation, respectively (data not shown). These findings suggest that latent PCMV / PRV present in the donor pig kidneys was reactivated as a result of the transplantation and subsequent immunosuppression. It is essential to note that there was no evidence of active PCMV / PRV infection in the donor pigs before transplantation.

[0083] The pigs used in this example were initially screened for pathogens and were negative for PCMV / PRV through throat swabs. They were kept as "clean grade animals" under non-DFP conditions. Although PCMV / PRV testing was negative for pre-transplant serum, positive PCMV / PRV in both xenografts was detected after transplantation. A latent PCMV / PRV infection may have evaded the previous detection methods (nasal swab samples and nested PCR assay). Prior to transplantation, donor pigs older than 6 months may be in the incubation period of carrying the virus, and detection may be ineffective due to low viral load. Newborn piglets may acquire PCMV / PRV-reactive antibodies via colostrum from infected mothers, furtherAtorney Docket No. 71951-708.602 complicating detection tests. Thus, a sensitive detection method, along with the collection of diverse samples from animals of varying ages, are essential12'13. The main strategy for obtaining PCMV / PRV-free donor pigs is to prevent a PCMV / PRV infection in the piglets by Cesarean section and early weaning12, and then raising them in PCMV / PRV-free facilities. In addition, ensuring a pathogen-free environment for the pig post-acquisition is paramount. This highlights the need for future studies to utilize DPF pigs directly, rather than clean grade animals that rely on throat swab-screening.

[0084] As early as 2014, Yamada et al. clearly warned that PCMV / PRV infection was associated with early rejection of kidney grafts in a pig-to-baboon xenotransplantation model14. Recently published reports on the dysfunction of the first human heart xenotransplant13,15also indicated that PCMV / PRV infection and reactivation in vivo is one of the potential causes of endothelial cell damage in xenografts.

[0085] In addition, this example has demonstrated the presence of PCMV / PRV in all recipient organs tested (heart, liver, spleen, lung) in both cases. This finding is consistent with the observations reported by Muhiuddin et al.13. However, it is crucial to note that their comprehensive study did not detect transcription of the virus in any recipient tissues.Furthermore, the identification of PCMV / PRV DNA was only made in conjunction with porcine cell DNA. This result suggests that PCMV / PRV DNAemia may have originated from lysed or circulating porcine cells or PCMV / PRV virions generated within the xenograft, rather than from active replication of PCMV / PRV within the recipient organs.

[0086] While PCMV / PRV DNA was identified through a nested PCR assay, no pathological features indicative of PCMV / PRV infection were found in the recipients’ tissues (data not shown). Even though viral DNA was detected, there was no evidence of the presence of a replicating virus capable of causing infection. Nevertheless, PCMV / PRV was reactivated in porcine organs and cells thereof, likely contributing to rejection of the xenograft.

[0087] While hyperacute rejection due to ABO incompatibility was not observed in the second case, the ABO incompatibility was present (with latent PCMV / PRV reactivation in both cases). These factors require careful consideration when attempting to understand xenograft rejection dynamics in brain-dead individuals. It is essential to use PCMV / PRV-negative (i.e., no latent PCMV / PRV infection) and O blood-type donor pigs for both preclinical and clinical xenotransplantation. Upgraded, designated pathogen-free (DPF) facilities, especially with regard to PCMV / PRV, have been established.

[0088] The findings from the two cases are closely aligned with expectations derived from prior NHP studies: in particular, with the following two points: (i) PCMV / PRV reactivation may accelerate xenograft rejection14, and (ii) long-term xenograft survival may be achieved byAtorney Docket No. 71951-708.602 immunosuppressive therapy (e.g., CD40-CD154 pathway blockade)16. These findings highlight the fact that comprehensive and long-term observations in pig-to-NHP preclinical xenotransplantation models are still essential. Eisenson et al. recently demonstrated that belatacept, as an alternative to CD40-CD154 antibodies, can also achieve long-term survival in pig-to-NHP xenotransplantation17.

[0089] It is important to note that the immune response to pig cells between humans and Old World NHPs differ, stemming from the inherent differences between the immune systems of humans and Old World NHPs18. Moreover, using NHP models poses challenges, as recipient animals are typically housed in standard cages under suboptimal conditions without constant supervision. The brain death decedent serves as a new research model and a bridge between NHP studies and initial clinical trials. In fact, the brain-dead decedent model used in the example can more closely resemble the human clinic and allow continuous care 24 hours a day by the entire clinical staff, including surgeons, intensive care unit nurses, doctors, and anesthesiologists. Although the number of cases in this study was limited and the duration was relatively short, it demonstrated that the 12-day observation period was sufficient to effectively test anti -hyperacute rejection regimens and observe delayed or accelerated xenograft rejection. It is demonstrated that the gene editing protocol effectively controlled hyperacute rejection, and clinical immunosuppressive regimens such as CD40-CD40L blockade can be used to control subsequent acute rejection.

[0090] Hemodynamic stability of the brain-dead decedents was effectively maintained for at least 12 days with the support of ventilation and standard ICU medications, including norepinephrine and dopamine for maintaining blood pressure to prolong this pre-clinical xenotransplantation study. This study established that decedents in a state of brain death, despite their many pathophysiological features that differ from those of healthy humans, remain intact in terms of instinctive immune response capacity throughout the study period and were suitable for xenotransplant investigation.

[0091] Factors that should be considered include donor genetic modifications, the immunosuppressive regimen, elimination of PCMV reactivation, suppression of cytokine storms in a brain-dead recipient patient19.

[0092] This example shows: (i) The extended observation period revealed significant changes such as the reactivation of latent PCMV / PRV in immunosuppressed recipients. The extended 12-day observation unveiled important implications for zoonotic considerations, highlighting the necessity of a prolonged observation period, (ii) Antibody-mediated rejection in ABO- incompatible xenotransplants within 12 days using the same regimen, a phenomenon seldom observed in clinical ABO-incompatible allotransplants. Revealing the need for concern aboutAtorney Docket No. 71951-708.602 potential underestimation of the immune response during pig antigen sensitization when the observation period is 7 days or less, this finding underscores the importance of an extended observation for a comprehensive assessment, particularly in xenotransplantation scenarios, (iii) Immune responses against pig blood type A antigens, in addition to non-Gal antigens, following ABO-incompatible kidney xenotransplantation, (iv) Sequential xenograft biopsies provided evidence of innate cell dynamics, including NK cells and macrophage infiltration into the kidney xenografts, as confirmed by immunohistochemistry, the first demonstration of such dynamics in xenotranspl antati on .

[0093] In summary, the observations underscore the importance of an extended observation period and a comprehensive evaluation of preclinical deceased human models for advancing the understanding of xenograft dynamics and survival towards an ultimate goal of achieving successful long-term kidney function restoration, for instance by using refined genetic modifications of donor pigs, optimized immunosuppressive regimens, and effective strategies to prevent porcine cytomegalovirus (PCMV) reactivation.

[0094] METHOD DETAILS

[0095] Selection of gene-engineered pigs

[0096] Two genetically modified Barna miniature pigs were created by Clonorgan Biotechnology Co., Ltd., located in Chengdu, China (data not shown), and housed in the DPF facility. The donor pigs were transported separately under strictly controlled conditions. During land-air-land travel, precautions were taken to ensure that the donor pigs were isolated from other animals.

[0097] Pig 1 was male, 662 days old, weighing 55kg with blood type O; this pig was selected from animals with TKO of three genes (GGTA1, p4GalNT2 and CMAH); one gene (hCD55) was transferred (data not shown).

[0098] Pig 2 was female, 447 days old, weighing 60kg with blood type A. This was the only suitable candidate available at that time and had been modified by three genes TKO (GGTA1, P4GalNT2 and CMAH) and two genes (hCD55 / hTBM) transferred (data not shown).

[0099] Porcine pathogenic microbial surveillance[000100] The ear tissues, nasal swabs, and sera from donor pigs were used to detect porcine endogenous retrovirus (PERV) type A / B / C, PCMV / PRV, influenza A, Mycoplasma hyopneumoniae, porcine circovirus type 1 / 2 / 3, and porcine reproductive and respiratory syndrome virus pre-transplantation (Table SI). Samples taken from xenografts and recipient heart, liver, spleen, lung and serum were tested for pathogenic microorganisms when the study was terminated (Table S2). The nest PCR method was used to detect PCMV / PRV23.Specifically, a genomic DNA extraction kit was used to extract the total genomic DNA of the- 1 -Atorney Docket No. 71951-708.602 sample, and nested PCR to screen for PCMV / PRV DNA in the total DNA. For the first run of PCR, the total genomic DNA of the sample was used for the template, and PCMV-P1-1 (5 - CGTGGGTTACTATGCTTCTC-3') and PCMV-P1-2 (5 '- CTTTCTAACGAGTTCTACGC-3') were used for primers (Table S3). PCR was performed at 94°C for 3 min; followed by 35 cycles of 98°C for 10 sec, 55°C for 30 sec, 68°C for 30 sec; and a final extension of 68°C for 5 min; For the second PCR run, the template was the PCR product of the first run, with PCMV-P2-1 (5'- TGGCTCAGGAAGAGAAAGGAAGTG-3') and PCMV-P2-2 (5'- GACGAGAGGACATTGTTGATAAAG-31) as primers. PCR was performed at 94°C for 3 min; followed by 35 cycles of 98°C for 10 sec, 58°C for 30 sec, 68 °C for 30 sec; and a final extension of 68 °C for 5 min. PCR products were electrophoresed in a 1 .5% agarose gel, stained with ethidium bromide, and visualized under ultraviolet (UV) light on a Bio-Rad gel imaging system, then finally confirmed by DNA sequencing.[000101] Detection of the Expression of Gal, Sda, Neu5Gc, hCD55, and hTBM on Donor Pig VECs[000102] The vascular endothelial cells (VECs) of Donor 1 and Donor 2 pigs were isolated, and PBMCs were isolated from the whole blood of each pig. Pig VECs were stained for the expression of Gal (by isolectin BSI-B4, 1 :500, Sigma-Aldrich, St. Louis, MO), Sda (with Dolichos biflorus agglutinin, DBA, 1 : 1000, Vector Labs, Burlingame, CA, USA), and Neu5Gc (with chicken anti-Neu5Gc mAb, 1 :800, Biolegend, San Diego, CA). The expression of human CD55 was detected by using an allophycocyanin (APC)- conjugated anti-human CD55 (1 : 100, Biolegend, San Diego, CA) and APC-conjugated anti-human CD141 (TBM) antibody (1 : 100, BD Bioscience, San Francisco CA, USA). Samples were analyzed on a flow cytometer (Cytoflex S, Beckman Coulter, USA), and data were analyzed using FlowJo software (Treestar, San Carlos, CA); results are expressed as the geometric mean.[000103] Surgery[000104] In a deceased human, a mid-abdominal skin incision was made following standard organ donation procedures. Bilateral kidney retrieval was then performed in accordance with established protocols. Subsequently, the kidneys were allocated through the China Organ Transplant Response System to the transplantation center and transplanted into four patients with end-stage renal disease. At the time of this paper submission, all recipients exhibited normal kidney function. Two recipients who received kidneys from Case 1 were followed up for one year, and the other two recipients who received kidneys from Case 2 were followed up for 6 months. All four recipients remain stable, with serum creatinine levels ranging from 100 to 110 pmol / L. There were no significant infections or rejections during the follow-up period.Atorney Docket No. 71951-708.602[000105] The porcine kidney procurement procedure mimics a clinical living kidney retrieval protocol conducted under general anesthesia and open surgery. Porcine kidneys were perfused with 4°C hi stidine-tryptophan -ketoglutarate (HTK) solution, placed in cold HTK solution, and transported to the recipient's operating room for transplantation.[000106] Porcine kidney transplantations follow a routine clinical protocol, with the graft located in the right retroperitoneal space. The right peritoneum was separated to fully expose the right external iliac arterial and vein. The porcine renal artery was anastomosed to the recipient's iliac artery in an end-to-side fashion. Similarly, venous anastomosis was performed end-to-side, attaching the porcine renal vein to the side of the recipient's iliac vein. The right porcine ureter was anastomosed to the recipient's bladder, followed by the placement of a pair of J tubes in the renal pelvis.[000107] Kidney 1, for Case 1, measured 13 * 6 * 4.5 cm and weighed 210g. Kidney 2, for Case 2, measured 12.5 x 7 x 4 cm and weighed 225g. The warm and cold ischemia times for Kidney 1 were 2 and 136 min, and for Kidney 2 were 1 and 222 min.[000108] Immunosuppression regimens[000109] For Case 1, a conventional immunosuppressive protocol used in clinical pre-sensitized renal allotransplantation was applied, including rabbit anti -thymocyte globulin (rATG, thymoglobulin), rituximab, tacrolimus, mycophenolate mofetil, and steroids (FIGS. 1A-1C). Medications that have not been approved for clinical use, such as CD40 and CD 154 antibodies, were excluded. Although Case 2 was an ABO-incompatible xenotransplantation, the level of preformed anti-donor blood type antibodies was quite low. To avoid the effect of blood group antibodies on the xenograft outcome, an anti-C5 monoclonal antibody (Eculizumab, 300 mg, iv, per week) was added to the standard protocol for Case 2.[000110] In both cases, the doses of tacrolimus were adjusted according to the blood concentration (targeted to 7-12 ng / ml) and administered via nasogastric feeding. Pulse doses of methylprednisolone (250-500mg / d) were given when acute rejection was of concern.[000111] Xenograft function and immunological investigation[000112] The post-operative investigation included: 1. urine output, SCr level, platelet count, hemoglobin, and microscale albuminuria; 2. serum levels of K+, Na+, C1-, Ca2+, and HCO3-; 3. erythropoietin (EPO); 4. prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimer, fibrinogen, and antithrombin III.[000113] Immunological monitoring included assessment of anti -donor pig IgG and IgM antibodies, CDC, lymphocyte subset, and tacrolimus blood concentration.[000114] Various parameters such as lymphocyte sub-population, tacrolimus concentration, creatinine levels, electrolyte balance, blood routine examination, coagulation function, arterialAtorney Docket No. 71951-708.602 blood gas analysis, and EPO levels were measured at regular intervals in both xenograft transplant recipients. Baseline data were also collected before xenotransplantation. All samples were processed at the Clinical Laboratory of the Second Affiliated Hospital of Hainan Medical University and disposed of safely after testing. The testing standards and methods aligned completely with those used in clinical kidney transplantation. The findings were reviewed by kidney transplant surgeons and critical care physicians.[000115] Also, color Doppler ultrasonography was conducted twice daily to assess perfusion and blood flow distribution in the xenograft.[000116] Detection of the binding of serum IgM and IgG to pig cells by flow cytometry [000117] The binding of serum IgM and IgG to pig PBMCs was measured by flow cytometry, as previously described8,25’26. To ensure the specificity and reliability of the assays, pooled human sera from healthy individuals were utilized as a control in the experiments. This approach allowed assessment of whether the levels of anti-donor porcine antibodies in the pre-transplant sera of brain-dead recipients were within typical population ranges, providing insight into potential immune responses during xenotransplantation in humans. In brief, 100 pl of isolated donor pig PBMCs (5 xl05 cells / tube) or RBCs (5 xl06 cells / tube) were incubated with 100 pl of diluted pooled human sera or decedent sera (10% for PBMCs and 25% for RBCs, final concentration, heat-inactivated for 30 min at 56°C in advance) for 30 min at 4°C. Cells incubated with 100 pl of phosphate-buffered saline (PBS) served as negative controls. After incubation, the cells were washed twice in PBS and centrifuged at 400 g for 5 min. The supernatant was then discarded. To prevent non-specific binding, 200 pl of 10% heat-inactivated goat serum (Solarbio, Beijing, China) was added, and incubation was performed for 15 min in the dark at 4°C. After washing with FACS buffer, the cells were incubated for 30 min with donkey anti-human IgM (1 : 1000) conjugated to Alexa-647 or goat anti-human IgG (1 : 1600) conjugated to Alexa-488 (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). After incubation, the samples were washed twice and then resuspended with 200 pl of PBS. The stained cells were then analyzed by flow cytometry (Cytoflex S, Beckman Coulter, USA); data were analyzed by FlowJo software (Treestar, SanCarlos, CA), and results are expressed as the relative geometric mean (rGM), which was calculated by dividing the geometric mean value for each sample by the negative control (no serum with secondary antibody only), as previously described25.[000118] Pooled human sera were obtained from 20 healthy human volunteers (22-44 years of age; both genders) who had no history of previous exposure to pig antigens or to alloantigens (such as blood transfusions, a previous failed renal allograft, or pregnancy). When required, decomplementation was carried out by heat-inactivation for 30 min at 56°C. All proceduresAtorney Docket No. 71951-708.602 involving humans were performed in accordance with the relevant guidelines and regulations and had no adverse effects on the subjects.[000119] Detection of human serum CDC in pig PBMCs by flow cytometry[000120] PBMCs (5* 105cells in 250pl of FACS buffer) were incubated with 50 pl of heat- inactivated human serum or decedent sera at 4°C for 1 h. After washing with PBS, FACS buffer (200 pl) and rabbit complement (50 pL, Cedarlane, Hornby, CA, USA) were added (final concentration, 20%), and incubation was carried out at 37°C for 30 min. After washing with PBS, the cells were incubated in the dark at 4°C for 15 min with propidium iodide; finally, 200 pL of FACS buffer was added. Flow cytometry was then carried out using a BD FACSCelesta (Becton Dickinson, San Jose, CA, USA).[000121] Detection of anti-A antibody levels by flow cytometry using human type A RBCs [000122] The concentration of anti-A antibodies in sera taken from the potential recipients was evaluated by measuring the binding of antibodies to human type Al reagent RBCs (Shanghai Blood / Biomedical Co., Ltd, Shanghai, CHN). In brief, serially diluted serum (50 pl) from Case 2 was incubated with 50 pl of human type Al RBCs (1 x 107 / ml) for 30 min at 4°C and subsequently washed twice in FACS buffer (phosphate-buffered saline containing 1% bovine serum albumin). The binding of anti-A antibodies was measured by indirect flow cytometry using donkey anti-human IgM (1 : 1000) or goat anti-human IgG (1 : 1000) antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). A minimum of 10,000 events were acquired by the flow cytometer (BD FACSAria Flow Cytometer, BD Biosciences, CA, USA); results are expressed as the geometric mean.[000123] Histopathology[000124] Needle core biopsies and autopsy examinations were performed for both xenografts. All specimens were examined by routine staining, immunofluorescent staining, immunohistochemical staining, and EM. Histopathological sections were examined by a renal pathologist, and lesions were classified and scored according to the Banff 2019 Schema27. [000125] For light microscopy, 10% buffered formalin-fixed and paraffin-embedded sections were processed and stained with hematoxylin and eosin (HE). Immunofluorescent staining for Gal, Sda, Neu5Gc, hCD55, hTBM, IgG, IgM, C3c, and C5b-9 proteins was performed on frozen or paraffin-embedded sections (-80°C) as previously reported. Immunohistochemical staining for C4d, CD3, CD4, CD8, CD 16, and CD68 proteins was performed on paraffin-embedded sections using immunoperoxidase methods, as described previously28.[000126] The primary antibodies included: anti-IgM (polyclonal rabbit anti-human IgM, Dako- Agilent), anti-IgG (goat anti-human IgG, Jackson ImmunoResearch Laboratories Inc., West Grove, PA), anti-human C4d rabbit monoclonal antibody (clone A24-T, Abeam), anti-CD68Attorney Docket No. 71951-708.602(clone KPI, GeneTech), anti-CD4 (clone EP204, ZSGB-BIO), anti- CD8 (clone SP16, ZSGB- BIO), anti-CD3 (clone GR107, GeneTech), anti-CD16 (clone EP364, GeneTech), anti-CD55 (clone BRIC 216, Santa Cruz), anti- TM (clone SC-13164, Santa Cruz), anti-Neu5Gc (clone Poly21469, Biolegend), Isolectin BSLB4 (L2895-1, Sigma-Aldrich), and Dolichos biflorus agglutinin (FL-1031-5, Vector).[000127] Experimental endpoints[000128] The preset endpoint was irreversible graft dysfunction unless one of the following scenarios occurred: heart-lung dysfunction, uncontrollable infections, or termination requested by the next of kin or the ethics committee.[000129] QUANTIFICATION AND STATISTICAL ANALYSIS[000130] If not explicitly stated, figures are presented as mean ± SEM. P-values are determined using unpaired two-tailed t-tests assuming normal distributions, or two-way ANOVA mixed- model. Statistical calculations are performed in Prism 7 (GraphPad Software, La Jolla CA). Significance was set at p<0.05. Time-course data, such as electrolyte levels, coagulation parameters, and inflammatory cytokines, were analyzed using repeated-measures ANOVA to account for within-subject correlations over time. Where appropriate, post hoc corrections were applied to control for multiple comparisons. Data were visualized using scatter plots, line graphs, and bar charts, with error bars representing either SD or IQR, as specified in the figure legends. Where relevant, individual data points are shown to provide insight into the variability within groups.[000131] References1. Montgomery RA, Stern JM, Lonze BE, et al. Results of two cases of pig-to-human kidney xenotransplantation. N Engl J Med 386, 1889-1898 (2022).2. Porrett PM, Orandi BJ, Kumar V, et al. First clinical -grade porcine kidney xenotransplant using a human decedent model. Am J Transplant 22, 1037-1053 (2022).3. Moazami N, Stern JM, Khalil K, et al. Pig-to-human heart xenotransplantation in two recently deceased human recipients. Nat Med 29, 1989-1997 (2023).4. Loupy A, Goutaudier V, Giarraputo A, et al. Immune response after pig-to-human kidney xenotransplantation: a multimodal phenotyping study. Lancet 402, 1158-1169 (2023).5. Locke JE, Kumar V, Anderson D, Porrett PM. Normal Graft Function After Pig-to- Human Kidney Xenotransplant. JAMA Surg 158, 1106-1108 (2023).6. nvuiangone.or / news / two-month-studv-piR-kidnev-xenotransolantation-gives-aew-hqAtorney Docket No. 71951-708.6027. _ Jones-Carr ME, Porrett PM, Jayme E, Locke JE, et al.C5 inhibition with eculizumab prevents thrombotic microangiopathy in a case series of pig-to-human kidney xenotransplantation. J Clin Invest, (2024), (In-Press Preview), https: / / doi.org / 10.1172 / JCI175996.8. Feng H, Li T, Du J, et al. Both Natural and Induced Anti-Sda Antibodies Play Important Roles in GTKO Pig-to-Rhesus Monkey Xenotransplantation. Front. Immunol 13, 1-10 (2022).9. Yamada K, Yazawa K, Shimizu A, et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha 1,3 -galactosyltransferase gene-knockout donors and the co-transplantation of vascularized thymic tissue. Nat Med; 11(1), 32-34 (2005).10. Feng H, Zhang M, Du J, et al. Swine leukocyte antigen (SLA) induces significant antibody production after pig-to-rhesus monkey kidney xenotransplantation. Transplantation 107(10S2): p 100(2023). (Abstract #312.9)11. Li T, Feng H, Du J, et al. Antibodies to unknown antigens on GTKO / p4GalNT2KO pig cells are associated with AHXR after pig-to rhesus monkey kidney transplantation. Transplantation 107(10S2): p 55(2023). (Abstract #217.4)12. Halecker S, Hansen S, Krabben L, Ebner F, Kaufer B, Denner J. How, where and when to screen for porcine cytomegalovirus (PCMV) in donor pigs for xenotransplantation. Sci Rep12. 21545 (2022).13. Mohiuddin MM, Singh AK, Scobie L, et al. Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report. Lancet 402, 397-410 (2023).14. Yamada K, Tasaki M, Sekijima M, et al. Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model. Transplantation 98, 411-418 (2014).15. Griffith BP, Goerlich CE, Singh AK, et al. Genetically Modified Porcine -to-Human Cardiac Xenotransplantation. N Engl J Med 387, 35-44 (2022).16. Yamamoto T, Hara H, Foote J, et al. Life-supporting kidney xenotransplantation from genetically engineered pigs in baboons: A comparison of two immunosuppressive regimens. Transplantation 103, 2090-2104(2019).17. Eisenson D, Hisadome Y, Santillan M, et al. Consistent survival in consecutive cases of life-supporting porcine kidney xenotransplantation using 10GE source pigs. Nat Commun 15, 3361(2024).18. Yamamoto T, Iwase H, Patel D, et al. Old World Monkeys are less than ideal transplantation models for testing pig organs lacking three carbohydrate antigens (tripleknockout). Sci Rep 10,1-13(2020).Atorney Docket No. 71951-708.60219. Cooper DKC, Kobayashi T. Xenotransplantation experiments in brain-dead human subjects-A critical appraisal. Am J Transplant 24, 520-525 (2024).20. Expert Consensus on Xenotransplantation, China Organ Transplantation Development Foundation, 2022-04-06, (in Chinese), cotdf.org.cn / article / 1214)21. Opinions on strengthening ethical governance of science and technology, General Office of the State Council (2022-NolO, General No: 1765) 2022-4-19, (in Chinese),(gov . cn / gongb ao / content / 2022 / content_5683838. htm)22. Law of the People's Republic of China on the Prevention and Control of Infectious, June 29, 2013, (in Chinese) ( npc. ov.cn / npc / c2 / c238 / 202001 / t20200122304251.html)23. Liu X, Liao S, Zhu L, et al. Molecular epidemiology of porcine Cytomegalovirus (PCMV) in Sichuan Province, China: 2010-2012. PLoS One 8, e64648 (2013).24. Criteria and practical guidance for determination of brain death in adults (2nd edition), Brain Injury Evaluation Quality Control Center of National Health Commission; Neurocritical Care Committee of the Chinese Society of Neurology (NCC / CSN); Neurocritical Care Commitee of China Neurologist Association (NCC / CNA). Chinese Medical Journal 132, 329- 335 (2019).25. Chen G, Qian H, Starzl T, et al. Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys. Nat. Med. 11, 1295-1298 (2005).26. Li T, Feng H, Du J, et al. Serum Antibody Binding and Cytotoxicity to Pig Cells in Chinese Subjects: Relevance to Clinical Renal Xenotransplantation. Front. Immunol. 13, 1-13 (2022).27. Loupy A, Haas M, Roufosse C, et al. The Banff 2019 Kidney Meeting Report (I): Updates on and classification of criteria for T cell- and antibody -mediated rejection. Am J Transplant 20, 2018-2331 (2020).28. Chen S, Zhong S, Xiang Y, et al. Complement inhibition enables renal allograft accommodation and long-term engraftment in presensitized nonhuman primates. Am. J. Transplant. 11, 2057-2066 (2011).[000132] The present disclosure is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the present disclosure. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

Claims

Attorney Docket No. 71951-708.602CLAIMS1. A genetically modified porcine cell, tissue, organ, or animal comprising in its genome: (i) inactivation of porcine genes GGTA1, p4GalNT2, and CMAH; and (ii) a transgene expressing hCD55, wherein the genome comprises no more than five transgenes.

2. The genetically modified porcine cell, tissue, organ, or animal of claim 1, wherein the genome comprises no more than four transgenes.

3. The genetically modified porcine cell, tissue, organ, or animal of claim 1, wherein the genome comprises no more than three transgenes.

4. The genetically modified porcine cell, tissue, organ, or animal of claim 1, wherein the genome comprises no more than two transgenes.

5. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 4, wherein the genome comprises a transgene selected from the group consisting of a human anticoagulant gene, a human complement inhibitor gene, and a human immunomodulatory gene.

6. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 5, wherein the genome comprises a transgene selected from the group consisting of hTBM, hCD39, human tissue factor pathway inhibitor, human endothelial protein C receptor, hCD46, hCD59, A20, HO-1, CD47, HLA-E, B2M, PD-L1, and FasL.

7. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 6, wherein the genome comprises a transgene expressing hTBM.

8. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 7, wherein the genome comprises no inactivation of any other porcine gene.

9. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 7, wherein the genome comprises inactivation of the porcine growth hormone receptor (GHR) gene.

10. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 7, wherein the genome comprises (i) inactivation of porcine genes GGTA1, P4GalNT2, and CMAH, and no inactivation of any other porcine gene; and (ii) a transgene expressing hCD55, a transgene expressing hTBM, and no other human transgene.Atorney Docket No. 71951-708.60211. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 10, which is free of porcine endogenous retroviruses.

12. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 11, which is free of porcine roseoloviruses.

13. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 12, which is free of porcine cytomegaloviruses.

14. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 13, which does not express red blood cell antigens.

15. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 13, which is a donor and expresses a red blood cell antigen type that matches the red blood cell antigen type of a human recipient.

16. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 15, wherein the porcine cell, tissue, or organ is or is from a kidney, heart, lung, liver, spleen, pancreas, or cornea.

17. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 16, wherein the porcine organ is a kidney.

18. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 16, wherein the porcine organ is a lung.

19. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 16, wherein the porcine organ is a heart.

20. The genetically modified porcine cell, tissue, organ, or animal of any one of claims 1 to 19, wherein the porcine gene inactivation is selected from the group consisting of gene knock-out, gene replacement, point mutation, deletion, or disruption of the porcine gene.

21. A method for treating a subject in need thereof, comprising transplanting into the subject the genetically modified porcine cell, tissue, or organ of any one of claims 1 to 20.

22. The method of claim 21, wherein the subject is a human and the genetically modified porcine organ is a kidney, heart, lung, liver, spleen, pancreas, or cornea.Attomey Docket No. 71951-708.60223. The method of claim 21 or claim 22, further comprising detecting the presence or absence of porcine endogenous retroviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting.

24. The method of any one of claims 21 to 23, further comprising removing or inactivating porcine endogenous retroviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting.

25. The method of any one of claims 21 to 24, further comprising detecting the presence or absence of porcine roseoloviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting.

26. The method of any one of claims 21 to 25, further comprising removing or inactivating porcine roseoloviruses in the modified porcine cell, tissue, or organ prior to the transplanting.

27. The method of any one of claims 21 to 26, further comprising detecting the presence or absence of porcine cytomegaloviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting.

28. The method of any one of claims 21 to 27, further comprising removing or inactivating porcine cytomegaloviruses in the genetically modified porcine cell, tissue, or organ prior to the transplanting.

29. The method of any one of claims 21 to 28, further comprising administering to the subject a therapeutic agent.

30. The method of claim 29, wherein the therapeutic agent comprises an antirejection agent, an anti-inflammatory agent, an immunosuppressive agent, an immunomodulatory agent, an anti -microbial agent, or an anti-viral agent, or any combination thereof.

31. The method of claim 29 or claim 30, wherein the therapeutic agent comprises a CD40-CD40L pathway blocking antibody, belatacept, anti-thymocyte globulin (rATG, thymoglobulin), rituximab, tacrolimus, my cophenolate mofetil, steroid, anti-C5 antibody, Cl inhibitor, or methylprednisolone, or any combination thereof.Atorney Docket No. 71951-708.60232. The method of any one of claims 21 to 31, wherein acute rejection of the genetically modified porcine cell, tissue, or organ does not occur until at least 12 days posttransplantation.

33. A method for treating a human individual in need thereof, comprising: transplanting into the human individual a genetically modified porcine organ comprises in its genome: (i) inactivation of porcine genes GGTA1, p4GalNT2, and CMAH; and (ii) a transgene expressing hCD55 and optionally a transgene expressing hTBM; and administering to the human individual a therapeutic agent comprising an anti -rejection agent, an anti-inflammatory agent, an immunosuppressive agent, an immunomodulatory agent, an anti -microbial agent, or an anti-viral agent, or any combination thereof, wherein acute rejection of the genetically modified porcine organ does not occur until at least 12 days post-transplantation.

34. The method of claim 33, wherein the genetically modified porcine organ does not express red blood cell antigens.

35. The method of claim 33, wherein the genetically modified porcine organ expresses a red blood cell antigen type that matches the red blood cell antigen type of the human individual.

36. The method of any one of claims 33 to 35, wherein the genetically modified porcine organ is free of porcine endogenous retroviruses, porcine roseoloviruses, and porcine cytomegaloviruses.

37. The method of any one of claims 33 to 36, comprising administering to the human individual one or more anti-viral agents that prevent infection by and / or reactivation of porcine endogenous retroviruses, porcine roseoloviruses, and porcine cytomegaloviruses.

38. The method of any one of claims 33 to 37, wherein the genome of the genetically modified porcine organ comprises no more than nine genetic modifications including inactivation of porcine genes and human transgenes.

39. The method of any one of claims 33 to 38, wherein the genome of the genetically modified porcine organ comprises no more than six genetic modifications including inactivation of porcine genes and human transgenes.Attomey Docket No. 71951-708.60240. The method of any one of claims 33 to 39, wherein the genome of the genetically modified porcine organ comprises no inactivation of any porcine gene other than GGTA1, P4GalNT2, and CMAH and no human transgene other than hCD55 and optionally hTBM.

41. A method for treating a human individual in need thereof, comprising: transplanting into the human individual a genetically modified porcine organ comprises in its genome: (i) inactivation of porcine genes GGTA1, p4GalNT2, and CMAH, and no inactivation of any other porcine gene; and (ii) a transgene expressing hCD55, optionally a transgene expressing hTBM, and no other human transgene, wherein the genetically modified porcine organ expresses a red blood cell antigen type that matches the red blood cell antigen type of the human individual; administering to the human individual an immunosuppressive agent; and administering to the human individual a complement inhibitor, wherein administration of the immunosuppressive agent is initiated earlier than administration of the complement inhibitor.

42. The method of claim 41, further comprising administering to the human individual one or more anti-viral agents that prevent infection by and / or reactivation of porcine endogenous retroviruses, porcine roseoloviruses, and porcine cytomegaloviruses.

43. The method of claim 41 or claim 42, wherein the immunosuppressive agent comprises a CD40-CD40L pathway blocking antibody, belatacept, anti-thymocyte globulin (rATG, thymoglobulin), rituximab, tacrolimus, mycophenolate mofetil, steroid, or methylprednisolone, or any combination thereof.

44. The method of any one of claims 41 to 43, wherein the complement inhibitor comprises an anti-C5 antibody.

45. The method of any one of claims 33 to 44, wherein the genetically modified porcine organ is a kidney.

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