Multiple human transgenes prolong survival of triple-carbohydrate knockout porcine kidney xenografts in nonhuman primates

Multiple human transgenes prolong survival of triple-carbohydrate knockout porcine kidney xenografts in nonhuman primates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multiple human transgenes prolong survival of triple-carbohydrate knockout porcine kidney xenografts in nonhuman primates Tatsuo Kawai, Ahmad Karadagi, Takayuki Hirose, Grace Lassiter, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6017857/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Genetically modified pigs are being developed to address the critical shortage of human organs for transplantation. We have previously demonstrated significantly prolonged survival of porcine xenografts devoid of three major carbohydrate xenoantigens (3KO) by incorporating human transgenes (HTGs). However, the optimal HTG combination and the mechanisms underlying improved xenograft survival following such genetic editing remain undefined. In the current study, we evaluated, in nonhuman primates, immune responses and transplant outcome of 3KO kidney xenografts with or without four different combinations of HTGs. We show that addition of HTGs significantly reduced transcripts associated with initial immune activation, resulting in markedly extended survival of the 3KO xenografts. Most notably, the addition of anti-inflammatory genes, TNFAIP3 and HMOX1 , was associated with improved graft survival with significantly lower expression of rejection-related gene sets in protocol xenograft biopsies, while the inclusion of coagulation-related HTGs was less effective. Although further studies are needed to define the optimal HTG combination for human recipients, we conclude that multiple combinations of HTGs can effectively prolong primate survival following 3KO kidney xenotransplantation. Biological sciences/Immunology/Transplant immunology Health sciences/Medical research/Preclinical research Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION A critical shortage of transplantable human organs has developed as kidney transplantation has become the standard of care for end stage kidney disease. Of the 95,000 patients currently awaiting kidney transplants in the USA only, approximately 30% will receive human transplants each year. 1 One promising approach to this critical healthcare challenge has been the development of xenotransplantation, using porcine organs for human transplantation. The primary barrier to successful pig-to-human xenotransplantation is the typically high level of preformed ‘natural’ anti-pig antibodies in human blood. Most of these antibodies bind to three glycan antigens expressed on porcine cells, αGal (galactose-α-1,3-galactose), Neu5GC ( N -glycolylneuraminic acid), and SD(a) (Sia-α2.3-[GalNAc-β1.4]Gal-β1.4-GlcNAc). 2 Knock-out of the pig enzyme encoding genes significantly reduces human natural antibody binding to porcine cells, making pig organs devoid of these three carbohydrate antigens (3KO) ideal for xenotransplantation 3 4 . Using a 3KO porcine donor, we recently performed the world’s first clinical kidney xenotransplantation. 5 This xenograft also contained seven human transgenes (HTGs) designed to mitigate complement dysregulation, coagulation imbalances, and inflammatory responses resulting from molecular incompatibilities between pigs and primates. However, the necessity of including HTGs remains controversial, especially in 3KO pigs. While some nonhuman primate (NHP) studies have demonstrated survival benefits of αGal knockout xenografts with CD55 6,7 and thrombomodulin (THBD), 8 no studies have specifically assessed the usefulness of incorporating HTGs in the context of 3KO pigs. The current study was undertaken in NHPs to systematically evaluate the transplant outcomes and immune responses associated with 3KO kidney xenografts, with or without various combinations of HTGs, including the gene edits used in our first clinical case. Our findings suggest that incorporating multiple HTGs, particularly those with anti-inflammatory properties, appears to be essential for prolonging 3KO kidney xenograft survival with favorable transcriptomic responses. RESULTS Addition of HTGs appears essential to achieve long-term survival of the 3KO kidney xenograft Kidneys from gene edited pigs with 3KO alone (No HTG) or 3KO with various combinations of HTGs (Table 1 ) were transplanted into cynomolgus monkeys treated with an anti-CD154 mAb – based immunosuppressive regimen (Fig. 1 ). Table 1 HTGs in 3KO xenografts Anti-inflammatory/Immune Complement Coagulation Group N KO TNF AIP3 HMOX1 CD47 HLA-E/ β 2M CD46 CD55 CD59 THBD PROCR TFPI No HTG 4 3KO HTG-A 6 3KO \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) HTG-B 6 3KO \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) HTG-C 3 3KO \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) HTG-D 16 3KO \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) \(\:\surd\:\) Transplantation of 3KO kidney xenograft without HTG (No HTG) consistently resulted in early graft loss (< 50 days), significantly shorter compared to xenografts in all four groups with HTGs (Fig. 2 A) (p = 0.0015, Log-rank test). All groups with HTGs (HTG-A - D) reduced the hazard risk (HR) of graft loss compared to No HTG with the largest reduction was seen in HTG-D in a univariate cox proportional hazards test (HR = 0.09, 95% CI: 0.02–0.30, p < 0.001, Fig. 2 B). By adding 2 self-recognition pathway regulatory molecules and 3 complement related HTGs to 3KO in HTG-A, the hazard ratio was reduced to 0.18 (CI: 0.05–0.71, p = 0.014) compared to No HTG, with maximal graft survival of 316 days (Fig. 2 A, 2 B and 2 C). Addition of 2 or even 3 coagulation pathway regulatory molecules in HTG-B and C, respectively, resulted in similar HR to HTG-A and failed to further extend graft survival (Fig. 2 A, 2 B and 2 C). Interestingly, progressive thrombocytopenia was observed in all HTG-B recipients (Fig. 2 D) despite the addition of THBD and TFPI transgenes. This progressive thrombocytopenia was not observed in Group C, where PROCR was added. Importantly, in association with adding the two anti-inflammatory HTGs, TNFAIP3 and HMOX1 in HTG-D, xenograft survival with excellent kidney function beyond 1 year was achieved, with the longest survival exceeding 2 years despite omitting HLA-E and TFPI transgenes from the donor pigs. Table 2 Graft survival days and causes of graft loss Group N Graft Survival Days (Pathology) No HTG 4 1 (ATI), 4 (ATI), 6 (TMA), 50 (ATI, TCMR) HTG-A 6 15 (TMA), 20 (thrombosis), 71 (TMA), 135 (AMR/TMA), 265 (AMR/TCMR), 316 (AMR/TCMR) HTG-B 6 37 (TMA), 82 (TMA), 90 (TMA/AMR), 202 (TMA), 242 (NER) HTG-C 3 30 (TMA), 119 (TG), 205 (TMA) HTG-D 16 6 (TMA), 9 (TMA), 9 (TMA), 16 (TMA), 25 (TMA/AMR), 25 (TMA), 103 (TMA/TCMR) 176 (TMA/AMR), 240 (AMR/TCMR), 283 (NER), 365 (TMA/AMR), 379 (NER), 457 (TMA), 511 (TMA), 691 (TMA/AMR), 758 (TMA/AMR) ATI: acute tubular injury, TMA: thrombotic microangiopathy, AMR: antibody mediated rejection, TCMR: T cell mediated rejection, CAMR: chronic antibody mediated rejection, NER: no diagnostic abnormality, TG: transplant glomerulopathy The causes of kidney xenograft loss The kidney xenograft survival and histopathologic diagnoses of terminal xenografts in each group are shown in Table 2 . The primary cause of graft loss in No HTG recipients was severe acute tubular injury (ATI) present in three NHPs (Table 2 and Fig. 3 A-C). One recipient with no HTG survived until day 50 but lost graft function due to ATI with TCMR (Table 2 ). The major cause of early graft loss throughout the HTG-A to D groups was thrombotic microangiopathy (TMA) (Fig. 3 and Fig. 4 D-F), which accounted for 77% of graft loss within 100 days (Fig. 4 ). In the later post-transplant period (> 100 days), the major cause of graft failure was a combination of TMA and antibody mediated rejection (AMR) (Fig. 4 G-I) (29.4%) or TMA and T cell mediated rejection (TCMR) (23.5%) but the graft loss with TMA alone was observed in 23.5% of recipients (Fig. 3 ). Significantly higher transcriptomic signatures associated with immune activation without HTGs in 1-hour biopsies To investigate possible differences in recipient immune responses with or without HTGs, we assessed bulk mRNA analysis in kidney biopsies. Since most No HTG recipients lost their kidney transplant function within one week, we evaluated 1-hour biopsies to define possible differences in immune responses between No-HTG and HTG-D, where sufficient number of biopsies were available for statistical analysis. For this transcriptomic analysis, we used NanoString nCounter platform and a modified Banff Human Organ Transplant (B-HOT) panel (Table S1 ). 9 The principal component analysis (PCA) demonstrated that No HTG and HTG-D recipients were two distinct populations in terms of transcriptomic responses (Fig. 5 A). In the HTG-D group, volcano plots showed markedly high expression of three HTGs, CD46 , CD47 and THBD , which were evaluated with our NanoString panel (Fig. 5 B). It also revealed significantly high mRNA expression of genes related to immune activation, including HLA-DRA (human leukocyte antigen, class II, DR alpha), IGHG3 (immunoglobulin heavy constant gamma 3) and KDR (kinase insert domain receptor) in the No HTG group compared to HTG-D. Conversely, KIT and BMP4 (Bone Morphogenetic Protein 4) were expressed at higher levels in HTG-D biopsies (Fig. 5 C). HTG-B vs. HTG-D (3-month biopsy) Kidney xenografts with additional TNFAIP3 and HMOX1 in HTG-D showed significantly lower transcriptomic signatures associated with rejection at 3 months Kidney xenograft survival beyond one year was achieved by adding TNFAIP3 and HMOX1 in HTG-D. This was achieved in the absence of HLA-E /β 2M and TFPI transgenes. To elucidate the mechanisms of this survival difference, we compared bulk mRNA expression in the HTG-B and D protocol biopsies taken around 3 months after kidney xenotransplantation, when the transplant kidney function was stable. The biopsy analyses, using the NanoString nCounter platform, revealed upregulation of seven distinct pathways in the HTG-B group, including kidney injury (AKI), antibody mediated rejection (AMR), angiogenesis, donor specific antibody transcripts (DSAST), endothelial-associated transcripts (ENDAT), and Rho GTPase signal. In contrast, the HTG-D group showed high levels of IL-7 signaling and quantitative cytotoxic T lymphocytes-associated transcripts (QCAT), along with promotion of T cell checkpoint signaling and Th2 differentiation pathways (Fig. 6 ). Transcriptomic responses in the HTG-D group Although duration of overall graft survival of the HTG-D group xenograft was the longest, six out of 16 recipients in this group lost their xenograft early, within 25 days. To identify any contributing factors causing early xenograft loss, we evaluated pre-transplant anti-porcine donor antibody (DSA) titers among the HTG-D group recipients. There was no significant difference in pre-transplant DSA titers between short ( 100 days) survivors (Fig. 7 A). We next compared the transcriptomic responses in 1-hour biopsies between short vs. long-term survivors. Although HLA-DRA and SLPI Sa, both of which may indicate immune/inflammation activation, were high in short-term vs. long-term survivors, there was no significant differences in overall transcripts between short- vs. long-term survivors (Fig. 7 B). In the current study, expression of three HTGs ( CD46 , CD47 and THBD ) were evaluable using specific probes available on the NanoString platform. As previously reported using scRNAseq 4 , we confirmed that pre-transplant levels of these three HTGs were maintained without significant changes until study termination (Fig. 8 A). To evaluate long-term transcriptomic responses, bulk mRNA analysis of sequential biopsies of NHPs from the long-term (> 1 year) survivors in HTG-D recipients (n = 6) were analyzed. At 1-hour, although upregulation of innate and adaptive immunity, such as innate-IS, adaptive-IS, ABMR,TCMR-RATS and NK gene sets, was already observed, expression of these pathways was reduced by 3 months and remained low and stable until 1 year, suggesting innate and adaptive immune responses towards xenografts were effectively suppressed by the ongoing immunosuppressive medications and HTGs. Only in the terminal necropsy samples were significantly higher innate and adaptive immune responses detected (Fig. 8 B). Discussion In this sequential analysis of genetically engineered porcine donors for human kidney transplantation, human CD55, CD46 , and CD59 were initially added to 3 KO pigs in HTG-A to reduce activation of the complement cascade. h CD47 was also included to suppress macrophage-mediated autologous phagocytosis through the ligation to signal regulatory protein-alpha (SIRPα) as well as h HLA-E and β 2M to prevent activation of NK cells 10 , 11 . In association with these HTGs, graft survival was significantly prolonged (No-HTG vs. HTG-A), but survival beyond 6 months was unusual. Addition of human coagulation-related genes, h THBD 12 , 13 and h TFPI 14 , in the HTG-B group, failed to further extend xenograft survival compared to HTG-A and, interestingly, was consistently associated with earlier, progressive, and more severe thrombocytopenia. Notably, thrombocytopenia was not observed in recipients of gene edits lacking h TFPI (HTG-A and D) recipients, suggesting that inclusion of h TFPI may be harmful, rather than beneficial in xenotransplantation. However, a contradictory observation was that no thrombocytopenia was observed in the HTG-C xenografts despite presence of h TFPI. In HTG-C, addition of h PROCR 15 , 16 may have mitigated platelet consumption by inhibiting the coagulation cascade through the activation of protein C. Notably, kidney xenograft survival exceeding one year was achieved only in association with the HTG-D gene construct, which included the anti-inflammatory genes TNFAIP3 , encoding the protein A20, and HMOX1 , encoding the enzyme heme oxygenase-1 (HO-1) along with complement and coagulation pathway regulatory gene cassettes. To more precisely evaluate possible differences in immune responses associated with adding the HTG-D gene cassette, we first compared transcriptomic responses in 1-hour xenograft biopsies taken from no HTG vs. HTG-D recipients, in which sufficient number of biopsies was available for statistical analysis. No HTG kidneys exhibited significantly higher expression of HLA-DR and IGHG3 , which may reflect enhanced recipient immune cell activation immediately after transplantation. In addition, the higher KDR pathway transcripts indicates vascular endothelial growth factor-induced endothelial activation. Conversely, KIT and BMP4 (bone morphogenetic protein 4) were relatively elevated in HTG-D xenografts. This may suggest that BMP4, part of the TGFβ superfamily, may be associated in HTG-D recipients with promoting anti-inflammatory regulatory function 17 , though exact interpretation of high KIT remains to be defined. We further evaluated the effect of adding anti-inflammatory genes by comparing transcriptomic responses in group HTG-B and D in protocol biopsies taken at 3 months from recipients with clinically healthy xenografts that were histologically normal. Despite stable renal function in both groups, the transcriptomic analyses revealed significantly higher expression of seven distinct pathways for rejection in the HTG-B group, including AKI, AMR, angiogenesis, chronic AMR (CAMR), DSAST, ENDAT and Rho GTPase signal. These results suggest that, despite absence of histologic evidence of rejection, when compared to HTG-D grafts, 3-month HTG-B grafts already had relative enrichment of pathways associated with rejection, inflammation, and tissue damage. In contrast, HTG-D showed higher levels of IL-7 signaling and Th2 differentiation, which are associated with regulatory immune responses in several experimental transplant models 18 , 19 . We speculate that elevated cytotoxic T cell transcript (QCAT) may represent exhausted CD8 populations, but further studies will be necessary to explore this hypothesis. Overexpression of TNFAIP3 (A20) has been shown to inhibit TNF-induced apoptosis 20 , 21 . Similarly, HMOX-1 expression has been demonstrated to significantly protect porcine endothelial cells during ex vivo perfusion with human blood 22 . We presume that expression of these human anti-inflammatory proteins prolonged graft survival by reducing inflammation mediated by innate and adaptive immune cells. Although Group HTG-D recipients demonstrated the most encouraging long-term kidney xenograft survival, approximately 38% experienced graft loss due to thrombotic microangiopathy (TMA) within 25 days. Neither pre-transplant recipient serum donor-specific antibody (DSA) levels nor bulk RNA expression in one-hour post-transplant biopsies correlated with or predicted graft fate. It is possible that the human proteins encoded by the transgenes designed for clinical use do not interact physiologically with cynomolgus monkey complement, coagulation, and self-recognition analogues, 23 in which case these candidate mechanisms driving TMA may be more prominent in the monkey model that they will prove to be clinically. While underlying causes of xenograft-associated TMA in preclinical models remain a mechanistically important question, its clinical significance has not yet been established: TMA was not observed at two months in our clinical case, or in a decedent 3KO recipient, both of whom were treated with anti-C5 mAb 24 , 25 . Our current and previous 4 studies have shown that the expression of HTGs remained stable at the mRNA and protein level even in the terminal autopsy samples, suggesting that xenograft injury is not primarily caused by down-modulation of ‘protective’ human transgenes. Our longitudinal observations of transcriptomic responses in the long-term HTG-D survivors revealed that both innate and adaptive immune responses were effectively suppressed for up to one year after transplantation. However, all HTG-D recipients eventually rejected their xenografts, sometimes as late as 758 days post-transplant, in associated with elevated innate and adaptive transcriptomic responses. We conclude that maintenance immunosuppression with anti-CD154 and MMF as dosed in these studies is not sufficient to suppress rejection long-term in NHP. The limitations of the current studies include the inability to isolate and assess the contribution of each human transgene (HTG) individually at clinical or mechanistic levels. Separately evaluating the numerous possible combinations of human transgenes incorporated into the pig constructs used in this life-supporting kidney xenograft model would be both cost-prohibitive and impractical. In conclusion, the current studies demonstrated that multiple combinations of HTGs, particularly those with anti-inflammatory properties, significantly extended the survival of 3KO kidney xenografts, accompanied by more favorable transcriptomic responses. While the optimal HTG combinations and immunosuppressive protocols remain to be determined, the currently available pig constructs are promising and now justify initial application of this approach to the treatment of human patients with end-stage renal disease. MATERIALS AND METHODS Animals Cynomolgus monkeys (purchased from Charles River Primates, Wilmington, MA) weighing 6–11 kg were used. Gene edited porcine donors were provided by eGenesis (Cambridge, MA). Yucatan miniature pigs weighing 10–25 kg were used as the kidney donor. All surgeries and postoperative care of animals were performed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) certified facility, per National Institutes of Health (NIH) guidelines for the care and use of nonhuman primates. These studies were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee. Husbandry, veterinary supervision, and daily care of animals were provided by Massachusetts General Hospital, Center for Comparative Medicine (CCM). Genetic modifications of the pig kidney xenografts with four different HTG combinations. All pigs of O blood type were engineered using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-mediated nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Intentional genomic alterations in EGEN-2259 (no HTG), EGEN-2536 (HTG-A), EGEN-2383 (HTG-B), EGEN-2597 (HTG-C) and EGEN-2784 (HTG-D) with or without PERV inactivation. In HTG-A, HTGs to suppress macrophage ( CD47 ) and natural killer (NK) cells ( HLA-E/ β 2M ), as well as to inhibit complement activation ( CD46, CD55 and CD59 ) were inserted. In HTG-B, coagulation related HTGs ( THBD and TFPI ) were added to those included in HTG-A. In HTG-C, another coagulation related gene, PROCR , was added to gene edits of HTG-B. Finally, in HTG-D which was used in the clinical case, anti-inflammatory HTGs, TNFAIP3 and HMOX1 , were added but without HLA-E/ β 2M and TFPI are summarized in Table 1 as reported 4 and described below: 1. Functional inactivation of all (59) porcine endogenous retrovirus (PERV) pol genes (PERV A, B, C), resulting in inactivation (RI, retroviral inactivation) of the PERV reverse transcriptase (RT) enzyme. Functional inactivation of PERV in the porcine genome is designed to reduce the potential for cross-species transfer of the retrovirus and to make donor organs safer for human use. 2. Compound heterozygous knockout (KO) of 4 genes, including glycoprotein alpha-galactosyltransferase 1 ( GGTA1 ), cytidine monophosphate-N-acetylneuraminic acid hydroxylase ( CMAH ), and beta-1,4-N-acetyl-galactosaminyltransferase 2 ( B4GALNT2 ) and B4GALNT2-like ( B4GALNT2L ) genes, resulting in undetectable levels of three antigens, galactose-α-1,3-galactose (α-Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) glycan antigens, respectively. 3. Hemizygous insertion of a transgenic construct, containing the various combinations of human transgenes Cluster of Differentiation CD46 , CD55 , CD59, CD47 , HLA-E/ β 2M , thrombomodulin ( THBD ), tissue factor pathway inhibitor ( TFPI ), endothelial protein C receptor ( PROCR ), TNF alpha-induced protein 3 ( TNFAIP3 ), and heme oxygenase 1 ( HMOX1 ) as listed in Table 1 , into the adeno-associated virus integration site 1 ( AAVS1 ) genomic locus, causing expression of various combinations of human proteins, CD46, CD55, CD59, CD47, HLA-E/β2M, thrombomodulin(TM), TFPI, endothelial protein C receptor (EPCR), TNFAIP3 protein (A20), and heme oxygenase 1 (HO-1), respectively, from the AAVS1 genomic locus. Next-generation sequencing (NGS) was performed on the edited porcine cells and/or cells from the cloned porcine donor produced, to confirm knockouts (3KO), and human transgene insertion and expression, which were confirmed by RNAseq and the flow cytometric analysis of ear-punched derived cells (EPDCs) from the porcine donor used in this study. Pathogen testing for porcine donor release included negative assays for potential swine viruses including Hepatitis E Virus, Porcine Circovirus 1, Porcine Circovirus 2, Porcine Cytomegalovirus (serologic and nucleic acid testing), Porcine Lymphotropic Herpesvirus 1, and Porcine Lymphotropic Herpesvirus 2. Kidney Transplant procedure. Kidney transplantation was performed according to earlier established methods for NHP kidney allotransplantations 26 . Briefly, the kidney xenograft was transplanted intraperitoneally, anastomosing renal vein to recipient vena cava and renal artery to recipient abdominal aorta. Ureterovesical anastomosis was performed according to the Lich-Gregoir technique. Immunosuppressive regimen (Fig. 1) NHP recipients received 20 mg/kg of anti-CD20 mAb on day − 5 (Anti-CD20 [2B8R1F8]-Afucosylated, NIH Nonhuman Primate Reagent Resource). Anti-rhesus thymocyte [rhATG] - antibody was engineered and produced by the NIH Nonhuman Primate Reagent Resource and was administered at 5 mg/kg as induction therapy on days − 1 and 0. Immunosuppression was maintained with daily 200 mg mycophenolate mofetil (CellCept, Genentech, San Francisco, CA) given orally and weekly doses of anti-CD154 at 20 mg/kg. The Anti-CD154 [5C8H1] antibody was engineered and produced by the Nonhuman Primate Reagent Resource (NIH Nonhuman Primate Reagent Resource Cat#PR-1547, RRID:AB_2716324). Tacrolimus (Prograf, Astellas) was administered as intramuscular injection with trough levels of 8–10 ng/ml dictating the doses for the initial period and was discontinued by post operative day 60. Additionally, tapered methylprednisone (Solu-Medrol, Pfizer) was administered as intramuscular injection over the first 60 days. The recipients were followed by serial blood tests (CBC and chemistries) and ultrasound of the kidney xenograft weekly. Histological analyses Protocol renal biopsies were obtained every 2–4 months in recipients with stable function as well as “for cause” biopsy whenever a rise in serum creatinine occurred. Tissue was processed for light microscopy and a portion frozen for immunofluorescence staining. Other organs obtained surgically (lymph nodes, native kidney and spleen) were similarly processed. Following euthanasia of a xenograft recipient, a complete necropsy was performed for histopathologic examination of the renal xenograft, lymph nodes, heart, lung, liver, pancreas, thymus, and skin. Xenograft H&E and PAS-stained samples were scored by current Banff criteria 9 including C4d deposition by immunohistochemistry 27 . Nanostring mRNA expression Bulk mRNA analysis was performed from formalin-fixed paraffin embedded xenograft biopsies and a sample from the non-transplanted contralateral donor kidney, using the nCounter instrument (Bruker Spatial Biology, Seattle WA) following the methods and data analysis previously published for human allograft biopsies 28 . The B-HOT panel contains 770 probes for human transcripts 28 . Pathways were manually curated from KEGG, Gene Ontogeny and Reactome datasets (genome.jp/kegg/pathway.html, GeneOntology.org and reactome.org) and from prior publications 28 – 31 . The probe sequences were screened for homology with pig and NHP transcripts. The probe sequences that had > 85% homology with pig sequences for parenchymal cells or endothelium and probes that had > 85% homology for NHP leukocytes were used, supplemented with 35 pig specific probes. The final panel, termed “B-HOTx” had 549 probes, as listed in Table S1 . Results were compared with well-phenotyped human allograft samples analyzed with the B-HOT panel and previously published 28 , and normalized with seven housekeeping probes (NRDE2, POLR2A, ABCF1, OAZI, PPIA, G6PD, TBP) with > 90% homology with the corresponding porcine transcripts. Pathway analysis was performed in nSolver software (Bruker Spatial Biology, Seattle WA) with subsequent statistical analysis using R programming version 4.4.1 (R Foundation for Statistical Computing, Vienna, Austria). Probes used in pathway analysis is listed in Table S2 . Raw counts were used for differential expression analysis in R using the DESeq2 package version 1.44.0. Statistical analysis All statistical analyses were performed using R programming version 4.4.1 (R Foundation for Statistical Computing, Vienna, Austria). Values of P < 0.05 were considered to be significant. Post-transplant survival times were plotted and compared using Kaplan-Meier survival curves and log-rank tests. Cox proportional hazards regression analysis was conducted using the coxph function from survival package in R to test the association between genetic edits and graft survival. T-test was used when comparing two groups. Declarations Acknowledgment: We acknowledge Drs. Joanne Morris and Michael Duggan for veterinary supervision and Drs. Joren C Madsen and David Cooper for critical reading and comments. Funding: eGenesis Inc. (T.K.) Matsuko Levin Research Fund (T.K.) AK received support from Karolinska Institutet (Hirsch, and Fernströms travel grants) and the Swedish Society of Medicine. Author contributions: A.K., T.H., G.L., T.T. and T.K. participated in surgery postoperative animal care. A.K., I.R., R.B.C. and T.K. conducted in vitro experiments, interpreted data, and participated in writing the manuscript. R.P.A., J.V. L., M.C.,S.L., and W.Q. provided resources (3KO genetically modified pigs) and A.K., I.R., R.N.P., A.B.C., R.B.C. and T.K. wrote the manuscript. T.K. supervised and designed the study. Competing interests: eGenesis has filed patent applications on the transgenic pig technology described in this paper. R.P.A., J.V. L., M.C.,S.L., and W.Q. are employees of eGenesis Bio. Other authors have no conflicts of interest to disclose. Data and materials availability: All data are available in the main text or the supplementary materials. References OPTN. National Data https://optn.transplant.hrsa.gov/data/view-data-reports/national-data/#. (2024). Buhler, L., Friedman, T., Iacomini, J. & Cooper, D. K. Xenotransplantation--state of the art--update 1999. Front Biosci 4 , D416-432 (1999). Ma, D. et al. Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques. Am J Transplant 22 , 46-57 (2022). https://doi.org/10.1111/ajt.16780 Anand, R. P. et al. Design and testing of a humanized porcine donor for xenotransplantation. Nature 622 , 393-401 (2023). https://doi.org/10.1038/s41586-023-06594-4 al., K. T. e. Gene-Edited Porcine Kidney Xenotransplantation for End-Stage Kidney Disease. N Engl J Med (2025). Schuurman, H. J. et al. Incidence of hyperacute rejection in pig-to-primate transplantation using organs from hDAF-transgenic donors. Transplantation 73 , 1146-1151 (2002). https://doi.org/10.1097/00007890-200204150-00024 McGregor, C. G. et al. Human CD55 expression blocks hyperacute rejection and restricts complement activation in Gal knockout cardiac xenografts. Transplantation 93 , 686-692 (2012). https://doi.org/10.1097/TP.0b013e3182472850 Singh, A. K. et al. Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs. Xenotransplantation 26 , e12465 (2019). https://doi.org/10.1111/xen.12465 Mengel, M. et al. Banff 2019 Meeting Report: Molecular diagnostics in solid organ transplantation-Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation. Am J Transplant 20 , 2305-2317 (2020). https://doi.org/10.1111/ajt.16059 Puga Yung, G., Schneider, M. K. J. & Seebach, J. D. The Role of NK Cells in Pig-to-Human Xenotransplantation. J Immunol Res 2017 , 4627384 (2017). https://doi.org/10.1155/2017/4627384 Weiss, E. H. et al. HLA-E/human beta2-microglobulin transgenic pigs: protection against xenogeneic human anti-pig natural killer cell cytotoxicity. Transplantation 87 , 35-43 (2009). https://doi.org/10.1097/TP.0b013e318191c784 Kim, H. et al. Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno-immune response. Xenotransplantation 22 , 260-272 (2015). https://doi.org/10.1111/xen.12173 Hara, H. et al. Stable expression of the human thrombomodulin transgene in pig endothelial cells is associated with a reduction in the inflammatory response. Cytokine 148 , 155580 (2021). https://doi.org/10.1016/j.cyto.2021.155580 Lee, K. F. et al. Recombinant pig TFPI efficiently regulates human tissue factor pathways. Xenotransplantation 15 , 191-197 (2008). https://doi.org/10.1111/j.1399-3089.2008.00476.x Lee, K. F. et al. Protective effects of transgenic human endothelial protein C receptor expression in murine models of transplantation. Am J Transplant 12 , 2363-2372 (2012). https://doi.org/10.1111/j.1600-6143.2012.04122.x Salvaris, E. J. et al. Pig endothelial protein C receptor is functionally compatible with the human protein C pathway. Xenotransplantation 27 , e12557 (2020). https://doi.org/10.1111/xen.12557 Regateiro, F. S., Howie, D., Cobbold, S. P. & Waldmann, H. TGF-beta in transplantation tolerance. Curr Opin Immunol 23 , 660-669 (2011). https://doi.org/10.1016/j.coi.2011.07.003 Mazzucchelli, R. et al. Development of regulatory T cells requires IL-7Ralpha stimulation by IL-7 or TSLP. Blood 112 , 3283-3292 (2008). https://doi.org/10.1182/blood-2008-02-137414 Waaga, A. M. et al. Regulatory functions of self-restricted MHC class II allopeptide-specific Th2 clones in vivo. J Clin Invest 107 , 909-916 (2001). Ferran, C., Stroka, D. M., Badrichani, A. Z., Cooper, J. T. & Bach, F. H. Adenovirus-mediated gene transfer of A20 renders endothelial cells resistant to activation: a means of evaluating the role of endothelial cell activation in xenograft rejection. Transplant Proc 29 , 879-880 (1997). https://doi.org/10.1016/s0041-1345(96)00184-4 Oropeza, M. et al. Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation 16 , 522-534 (2009). https://doi.org/10.1111/j.1399-3089.2009.00556.x Petersen, B. et al. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. Xenotransplantation 18 , 355-368 (2011). https://doi.org/10.1111/j.1399-3089.2011.00674.x Jacobsen, F. W. et al. Molecular and functional characterization of cynomolgus monkey IgG subclasses. J Immunol 186 , 341-349 (2011). https://doi.org/10.4049/jimmunol.1001685 Jones-Carr, M. E. et al. C5 inhibition with eculizumab prevents thrombotic microangiopathy in a case series of pig-to-human kidney xenotransplantation. The Journal of Clinical Investigation (2024). https://doi.org/10.1172/JCI175996 Young, J. A., Pallas, C. R. & Knovich, M. A. Transplant-associated thrombotic microangiopathy: theoretical considerations and a practical approach to an unrefined diagnosis. Bone marrow transplantation 56 , 1805-1817 (2021). https://doi.org/10.1038/s41409-021-01283-0 Cosimi, A. B. et al. Prolonged survival of nonhuman primate renal allograft recipients treated only with anti-CD4 monoclonal antibody. Surgery 108 , 406-413; discussion 413-404 (1990). Adam, B. A. et al. Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes. Am J Transplant (2017). https://doi.org/10.1111/ajt.14327 Rosales, I. A. et al. Banff Human Organ Transplant Transcripts Correlate with Renal Allograft Pathology and Outcome: Importance of Capillaritis and Subpathologic Rejection. J Am Soc Nephrol 33 , 2306-2319 (2022). https://doi.org/10.1681/ASN.2022040444 Smith, R. N. et al. Utility of Banff Human Organ Transplant Gene Panel in Human Kidney Transplant Biopsies. Transplantation 107 , 1188-1199 (2023). https://doi.org/10.1097/TP.0000000000004389 Reeve, J. et al. Molecular diagnosis of T cell-mediated rejection in human kidney transplant biopsies. Am J Transplant 13 , 645-655 (2013). https://doi.org/10.1111/ajt.12079 Venner, J. M., Hidalgo, L. G., Famulski, K. S., Chang, J. & Halloran, P. F. The molecular landscape of antibody-mediated kidney transplant rejection: evidence for NK involvement through CD16a Fc receptors. Am J Transplant 15 , 1336-1348 (2015). https://doi.org/10.1111/ajt.13115 Additional Declarations There is NO Competing Interest. Supplementary Files AKXenoSupplemental21225finalclean.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6017857","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":416025626,"identity":"ade0f451-4565-48a8-83c0-220385d6ef50","order_by":0,"name":"Tatsuo Kawai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBAC+xuJbRIJf2wYGCQY2KBiCQwMPHi0sEkcPibxwSCNFC2SbWmSMwwOk6JFmsdMmsfgfLR8dAPbg597DsubtycwPnjbhkeLHI/ZbR6D27kb7xxgN+x5dthwzpkHzIZzidIyI4FNgudAGuMMiQQ2aV58WqT5v93+Y3AOrEXyz4E0e6AW9t94tUj2mP/mMTiQOx9kOM8Bm0SQLcx4tUicMbfmMUjO3SCR2CYtc8AmeQbPw2bJOefwackBhtgfu9z5M5KPSb45IGE7gz354Ic3Zbi1wIHBAcYGKBPOIADkiVQ3CkbBKBgFIxAAAJnlUY+LdBuFAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-6900-4603","institution":"Massachusetts General Hospital","correspondingAuthor":true,"prefix":"","firstName":"Tatsuo","middleName":"","lastName":"Kawai","suffix":""},{"id":416025627,"identity":"5d1e45e7-1bb4-4fe6-baf3-0b8f1e9385ec","order_by":1,"name":"Ahmad Karadagi","email":"","orcid":"","institution":"Karolinska Institute","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Karadagi","suffix":""},{"id":416025628,"identity":"f105648e-4e3a-427c-a691-92bf75076c7a","order_by":2,"name":"Takayuki Hirose","email":"","orcid":"","institution":"Massachusetts General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Takayuki","middleName":"","lastName":"Hirose","suffix":""},{"id":416025629,"identity":"85abd598-30f2-4cff-a5fd-c0910f6aa2e0","order_by":3,"name":"Grace Lassiter","email":"","orcid":"","institution":"Massachusetts General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Grace","middleName":"","lastName":"Lassiter","suffix":""},{"id":416025630,"identity":"736a63aa-3b27-4571-a28f-9aedf990fd49","order_by":4,"name":"Ivy Rosales","email":"","orcid":"https://orcid.org/0000-0003-0621-3202","institution":"Massachusetts General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ivy","middleName":"","lastName":"Rosales","suffix":""},{"id":416025631,"identity":"c757473c-a9ed-4d49-a85e-5dfd74933b5b","order_by":5,"name":"Toshihide Tomosugi","email":"","orcid":"","institution":"Massachusetts General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Toshihide","middleName":"","lastName":"Tomosugi","suffix":""},{"id":416025632,"identity":"ea2d4c4b-d359-48f7-99fe-67a2cf5d15be","order_by":6,"name":"Ryo Otsuka","email":"","orcid":"https://orcid.org/0000-0002-3730-9867","institution":"Massachusetts General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ryo","middleName":"","lastName":"Otsuka","suffix":""},{"id":416025633,"identity":"929eeaf8-0b2a-4178-a6aa-e486ba68f073","order_by":7,"name":"Ranjith Anand","email":"","orcid":"","institution":"eGenesis","correspondingAuthor":false,"prefix":"","firstName":"Ranjith","middleName":"","lastName":"Anand","suffix":""},{"id":416025634,"identity":"004317b0-3f95-4d27-ab9a-9864ed8c876e","order_by":8,"name":"Richard Pierson III","email":"","orcid":"https://orcid.org/0000-0003-3764-4590","institution":"Massachusetts General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"Pierson","lastName":"III","suffix":""},{"id":416025635,"identity":"14667fa0-a07c-493a-8848-54f4a3c03eb3","order_by":9,"name":"A Cosimi","email":"","orcid":"","institution":"Massachusetts General Hospital","correspondingAuthor":false,"prefix":"","firstName":"A","middleName":"","lastName":"Cosimi","suffix":""},{"id":416025636,"identity":"61359df0-74d8-42da-b62e-b1c23aa63f4b","order_by":10,"name":"Michael Curtis","email":"","orcid":"","institution":"eGenesis","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Curtis","suffix":""},{"id":416025637,"identity":"091706e0-ca14-4d58-bfd6-38e03ef294e5","order_by":11,"name":"Susan Low","email":"","orcid":"","institution":"eGenesis","correspondingAuthor":false,"prefix":"","firstName":"Susan","middleName":"","lastName":"Low","suffix":""},{"id":416025638,"identity":"beb55615-1727-42d6-9690-f24f3acc6c8a","order_by":12,"name":"Wenning Qin","email":"","orcid":"","institution":"eGenesis","correspondingAuthor":false,"prefix":"","firstName":"Wenning","middleName":"","lastName":"Qin","suffix":""},{"id":416025639,"identity":"3b1acc25-a96a-43c5-8ab3-e4b1dc7cfa40","order_by":13,"name":"Robert Colvin","email":"","orcid":"","institution":"Massachusetts General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Colvin","suffix":""},{"id":416025640,"identity":"6abaaffc-e51a-4ed4-8666-34d5a9386277","order_by":14,"name":"Jacob Layer","email":"","orcid":"","institution":"eGenesis Inc","correspondingAuthor":false,"prefix":"","firstName":"Jacob","middleName":"","lastName":"Layer","suffix":""},{"id":416025641,"identity":"0e7a6f86-0dbd-4038-9873-6e69547c807c","order_by":15,"name":"David Ma","email":"","orcid":"","institution":"Andover Surgical Associates","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Ma","suffix":""}],"badges":[],"createdAt":"2025-02-12 20:15:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6017857/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6017857/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78735992,"identity":"b87c39c3-b538-4401-b6d6-956190c7d1a5","added_by":"auto","created_at":"2025-03-18 08:11:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eImmunosuppressive protocol includes anti-CD20 mAb and rabbit ATG induction, followed by anti-CD154 mAb, mycophenolate mofetil. Tacrolimus and steroids were also administered for the first 60 days.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/964fc6b66940e26a9faf639c.png"},{"id":78735993,"identity":"ff0b5d65-13b0-4f3d-a3d3-ce56c5fd9a56","added_by":"auto","created_at":"2025-03-18 08:11:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":324690,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTransplant outcome of 3KO kidney xenografts with or without HTGs.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003e(A)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eKidney xenograft survival by Kaplan-Meier analysis. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(B) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ekidney xenograft survival by a univariate cox proportional hazards test. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(C)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Serum creatinine (mg/dl) and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(D)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Platelet counts (X1000/µl) in each group.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/807e6651720bdcd5647b88d4.png"},{"id":78736209,"identity":"15996650-c8e6-416f-977c-aa5202e6b144","added_by":"auto","created_at":"2025-03-18 08:19:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":88459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCauses of graft loss in HTG A-D group recipients.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e TMA is the major cause of graft loss prior to 100 days after transplantation. After 100 days, AMR or TCMR combined with TMA were the cause of the graft loss but the graft loss by TMA alone was still observed.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/d9a9538f990595c01828c057.png"},{"id":78737383,"identity":"3342de98-53a8-4bca-b30f-f424b7c5f458","added_by":"auto","created_at":"2025-03-18 08:35:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2758632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePatho histology of kidney xenografts.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(A-C):\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Representative histologic images of xenografts at autopsy from No-HTG showing extensive acute tubular injury with tubular necrosis and unremarkable glomeruli by light and electron microscopy. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(D-F)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e: TMA is seen along an artery and in a glomerulus in an animal from Group B. Marked endothelial loss in glomerular capillaries is present by light and electron microscopy.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e (G-I)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e: Images from a long-term surviving xenograft showing intact arteries and prominent glomerulitis with peritubular capillaritis and C4d deposition which are features of antibody-mediated rejection.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/0ca519cf143d715f3ec19b29.png"},{"id":78737058,"identity":"e7557625-91f6-4b7c-ad29-9e9591dff1c9","added_by":"auto","created_at":"2025-03-18 08:27:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":189829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003emRNA expression at 1-hour biopsy.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. PCA of two groups (No-HTG vs. HTG-D). and \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003evolcano plots of bulk mRNA analysis showed markedly high HTG expression (CD46, CD47 and THBD) in HTG-D. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Significantly higher expression of HLA-DRA, IGHG3 and KDR was detected in No HTG group, compared to HTG-D biopsies.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/8bed77815177e472e36ce739.png"},{"id":78736000,"identity":"940f5675-95b8-4fd5-8594-2fbeccad044a","added_by":"auto","created_at":"2025-03-18 08:11:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":330798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eBulk mRNA expression in Group B and D protocol biopsies taken around 3 months\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. Despite stable biopsy results in both groups, significantly lower transcriptomic signatures representing rejection by adding anti-inflammatory HTGs in HTG-D.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/be35a73478dad3e146a587ea.png"},{"id":78736005,"identity":"86c48469-dd70-45eb-a4b2-d1bf565e62b3","added_by":"auto","created_at":"2025-03-18 08:11:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":196659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePre transplant DSA and\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBulk mRNA analysis between short-term (\u0026lt;25 days) and long-term (\u0026gt;100 days) survivors among the Group HTG-D recipients.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e There was no significant difference in pre-transplant serum IgM and IgG DSA levels in either short or long-term survivors. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Although volcano plots showed\u003c/em\u003e \u003cem\u003ehigher HLA-DRA and SLAPI Sa in short-term vs. long-term survivors, there was no significant difference in overall transcripts.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/b0a02e68a6d6713ae038d428.png"},{"id":78736018,"identity":"70bce07e-883a-4cc4-a713-715896a72040","added_by":"auto","created_at":"2025-03-18 08:11:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":355334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA. Sequential analyses of expression of hCD46, hCD47 and hTHBD\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ein the HTG-D kidney xenografts. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eExpression of HTGs in the contralateral kidney (not transplanted) was measured for comparison. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eBulk mRNA analysis of sequential biopsies of NHPs from Group D long-term survivors. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e\u0026nbsp;Analysis of sequential biopsies using the NanoString nCounter platform revealed elevated innate and adaptive responses immediately after transplant subsided by 3 months and continued to be suppressed until one year. \u0026nbsp;In the terminal biopsies, marked elevation of both innate and adaptive immune responses was observed.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/70d22866a36c72eae984b667.png"},{"id":78738175,"identity":"c91f0758-3208-4e18-92f2-38dfbe77e38d","added_by":"auto","created_at":"2025-03-18 08:44:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7107837,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/03a3c286-b45c-4dfb-9c0e-a1eccade7e6b.pdf"},{"id":78737057,"identity":"edaff4d1-5a10-470b-bcec-a94966aa1445","added_by":"auto","created_at":"2025-03-18 08:27:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":95626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"AKXenoSupplemental21225finalclean.docx","url":"https://assets-eu.researchsquare.com/files/rs-6017857/v1/e956325b7fd6156b59dc4053.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Multiple human transgenes prolong survival of triple-carbohydrate knockout porcine kidney xenografts in nonhuman primates","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eA critical shortage of transplantable human organs has developed as kidney transplantation has become the standard of care for end stage kidney disease. Of the 95,000 patients currently awaiting kidney transplants in the USA only, approximately 30% will receive human transplants each year.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e One promising approach to this critical healthcare challenge has been the development of xenotransplantation, using porcine organs for human transplantation.\u003c/p\u003e \u003cp\u003eThe primary barrier to successful pig-to-human xenotransplantation is the typically high level of preformed \u0026lsquo;natural\u0026rsquo; anti-pig antibodies in human blood. Most of these antibodies bind to three glycan antigens expressed on porcine cells, αGal (galactose-α-1,3-galactose), Neu5GC (\u003cem\u003eN\u003c/em\u003e-glycolylneuraminic acid), and SD(a) (Sia-α2.3-[GalNAc-β1.4]Gal-β1.4-GlcNAc).\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Knock-out of the pig enzyme encoding genes significantly reduces human natural antibody binding to porcine cells, making pig organs devoid of these three carbohydrate antigens (3KO) ideal for xenotransplantation \u003csup\u003e3 4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUsing a 3KO porcine donor, we recently performed the world\u0026rsquo;s first clinical kidney xenotransplantation.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e This xenograft also contained seven human transgenes (HTGs) designed to mitigate complement dysregulation, coagulation imbalances, and inflammatory responses resulting from molecular incompatibilities between pigs and primates. However, the necessity of including HTGs remains controversial, especially in 3KO pigs. While some nonhuman primate (NHP) studies have demonstrated survival benefits of αGal knockout xenografts with CD55\u003csup\u003e6,7\u003c/sup\u003e and thrombomodulin (THBD),\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e no studies have specifically assessed the usefulness of incorporating HTGs in the context of 3KO pigs.\u003c/p\u003e \u003cp\u003eThe current study was undertaken in NHPs to systematically evaluate the transplant outcomes and immune responses associated with 3KO kidney xenografts, with or without various combinations of HTGs, including the gene edits used in our first clinical case. Our findings suggest that incorporating multiple HTGs, particularly those with anti-inflammatory properties, appears to be essential for prolonging 3KO kidney xenograft survival with favorable transcriptomic responses.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAddition of HTGs appears essential to achieve long-term survival of the 3KO kidney xenograft\u003c/h2\u003e \u003cp\u003eKidneys from gene edited pigs with 3KO alone (No HTG) or 3KO with various combinations of HTGs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) were transplanted into cynomolgus monkeys treated with an anti-CD154 mAb \u0026ndash; based immunosuppressive regimen (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e HTGs in 3KO xenografts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003eAnti-inflammatory/Immune\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eComplement\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e \u003cp\u003eCoagulation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTNF\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eAIP3\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eHMOX1\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eCD47\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eHLA-E/\u003c/em\u003e β\u003cem\u003e2M\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eCD46\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eCD55\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eCD59\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003eTHBD\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cem\u003ePROCR\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cem\u003eTFPI\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNo HTG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3KO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTG-A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3KO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTG-B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3KO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTG-C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3KO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTG-D\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3KO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\surd\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e Transplantation of 3KO kidney xenograft without HTG (No HTG) consistently resulted in early graft loss (\u0026lt;\u0026thinsp;50 days), significantly shorter compared to xenografts in all four groups with HTGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) (p\u0026thinsp;=\u0026thinsp;0.0015, Log-rank test). All groups with HTGs (HTG-A - D) reduced the hazard risk (HR) of graft loss compared to No HTG with the largest reduction was seen in HTG-D in a univariate cox proportional hazards test (HR\u0026thinsp;=\u0026thinsp;0.09, 95% CI: 0.02\u0026ndash;0.30, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy adding 2 self-recognition pathway regulatory molecules and 3 complement related HTGs to 3KO in HTG-A, the hazard ratio was reduced to 0.18 (CI: 0.05\u0026ndash;0.71, p\u0026thinsp;=\u0026thinsp;0.014) compared to No HTG, with maximal graft survival of 316 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Addition of 2 or even 3 coagulation pathway regulatory molecules in HTG-B and C, respectively, resulted in similar HR to HTG-A and failed to further extend graft survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Interestingly, progressive thrombocytopenia was observed in all HTG-B recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) despite the addition of \u003cem\u003eTHBD\u003c/em\u003e and \u003cem\u003eTFPI\u003c/em\u003e transgenes. This progressive thrombocytopenia was not observed in Group C, where \u003cem\u003ePROCR\u003c/em\u003e was added. Importantly, in association with adding the two anti-inflammatory HTGs, \u003cem\u003eTNFAIP3\u003c/em\u003e and \u003cem\u003eHMOX1\u003c/em\u003e in HTG-D, xenograft survival with excellent kidney function beyond 1 year was achieved, with the longest survival exceeding 2 years despite omitting \u003cem\u003eHLA-E\u003c/em\u003e and \u003cem\u003eTFPI\u003c/em\u003e transgenes from the donor pigs.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e Graft survival days and causes of graft loss\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGraft Survival Days (Pathology)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNo HTG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e (ATI), \u003cb\u003e4\u003c/b\u003e (ATI), \u003cb\u003e6\u003c/b\u003e (TMA), \u003cb\u003e50\u003c/b\u003e (ATI, TCMR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTG-A\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e (TMA), \u003cb\u003e20\u003c/b\u003e (thrombosis), \u003cb\u003e71\u003c/b\u003e(TMA), \u003cb\u003e135\u003c/b\u003e(AMR/TMA), \u003cb\u003e265\u003c/b\u003e(AMR/TCMR), \u003cb\u003e316\u003c/b\u003e(AMR/TCMR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTG-B\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e37\u003c/b\u003e(TMA), \u003cb\u003e82\u003c/b\u003e(TMA), \u003cb\u003e90\u003c/b\u003e(TMA/AMR), \u003cb\u003e202\u003c/b\u003e(TMA), \u003cb\u003e242\u003c/b\u003e(NER)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTG-C\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e30\u003c/b\u003e(TMA), \u003cb\u003e119\u003c/b\u003e(TG), \u003cb\u003e205\u003c/b\u003e(TMA)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHTG-D\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e(TMA), \u003cb\u003e9\u003c/b\u003e(TMA), \u003cb\u003e9\u003c/b\u003e(TMA), \u003cb\u003e16\u003c/b\u003e(TMA), \u003cb\u003e25\u003c/b\u003e(TMA/AMR), \u003cb\u003e25\u003c/b\u003e (TMA), \u003cb\u003e103\u003c/b\u003e(TMA/TCMR)\u003c/p\u003e \u003cp\u003e\u003cb\u003e176\u003c/b\u003e(TMA/AMR), \u003cb\u003e240\u003c/b\u003e(AMR/TCMR), \u003cb\u003e283\u003c/b\u003e(NER), \u003cb\u003e365\u003c/b\u003e(TMA/AMR), \u003cb\u003e379\u003c/b\u003e(NER), \u003cb\u003e457\u003c/b\u003e(TMA), \u003cb\u003e511\u003c/b\u003e(TMA), \u003cb\u003e691\u003c/b\u003e(TMA/AMR), \u003cb\u003e758\u003c/b\u003e(TMA/AMR)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eATI: acute tubular injury, TMA: thrombotic microangiopathy, AMR: antibody mediated rejection, TCMR: T cell mediated rejection, CAMR: chronic antibody mediated rejection, NER: no diagnostic abnormality, TG: transplant glomerulopathy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe causes of kidney xenograft loss\u003c/h3\u003e\n\u003cp\u003eThe kidney xenograft survival and histopathologic diagnoses of terminal xenografts in each group are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The primary cause of graft loss in No HTG recipients was severe acute tubular injury (ATI) present in three NHPs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). One recipient with no HTG survived until day 50 but lost graft function due to ATI with TCMR (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The major cause of early graft loss throughout the HTG-A to D groups was thrombotic microangiopathy (TMA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F), which accounted for 77% of graft loss within 100 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the later post-transplant period (\u0026gt;\u0026thinsp;100 days), the major cause of graft failure was a combination of TMA and antibody mediated rejection (AMR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-I) (29.4%) or TMA and T cell mediated rejection (TCMR) (23.5%) but the graft loss with TMA alone was observed in 23.5% of recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSignificantly higher transcriptomic signatures associated with immune activation without HTGs in 1-hour biopsies\u003c/h3\u003e\n\u003cp\u003eTo investigate possible differences in recipient immune responses with or without HTGs, we assessed bulk mRNA analysis in kidney biopsies. Since most No HTG recipients lost their kidney transplant function within one week, we evaluated 1-hour biopsies to define possible differences in immune responses between No-HTG and HTG-D, where sufficient number of biopsies were available for statistical analysis. For this transcriptomic analysis, we used NanoString nCounter platform and a modified Banff Human Organ Transplant (B-HOT) panel (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e The principal component analysis (PCA) demonstrated that No HTG and HTG-D recipients were two distinct populations in terms of transcriptomic responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the HTG-D group, volcano plots showed markedly high expression of three HTGs, \u003cem\u003eCD46\u003c/em\u003e, CD47 and \u003cem\u003eTHBD\u003c/em\u003e, which were evaluated with our NanoString panel (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). It also revealed significantly high mRNA expression of genes related to immune activation, including \u003cem\u003eHLA-DRA\u003c/em\u003e (human leukocyte antigen, class II, DR alpha), \u003cem\u003eIGHG3\u003c/em\u003e (immunoglobulin heavy constant gamma 3) and \u003cem\u003eKDR\u003c/em\u003e (kinase insert domain receptor) in the No HTG group compared to HTG-D. Conversely, \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eBMP4\u003c/em\u003e (Bone Morphogenetic Protein 4) were expressed at higher levels in HTG-D biopsies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eHTG-B vs. HTG-D (3-month biopsy)\u003c/h3\u003e\n\u003cp\u003e \u003cb\u003eKidney xenografts with additional\u003c/b\u003e \u003cb\u003eTNFAIP3\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eHMOX1\u003c/b\u003e \u003cb\u003ein HTG-D showed significantly lower transcriptomic signatures associated with rejection at 3 months\u003c/b\u003e\u003c/p\u003e \u003cp\u003eKidney xenograft survival beyond one year was achieved by adding \u003cem\u003eTNFAIP3\u003c/em\u003e and \u003cem\u003eHMOX1\u003c/em\u003e in HTG-D. This was achieved in the absence of \u003cem\u003eHLA-E\u003c/em\u003e/β\u003cem\u003e2M\u003c/em\u003e and \u003cem\u003eTFPI\u003c/em\u003e transgenes. To elucidate the mechanisms of this survival difference, we compared bulk mRNA expression in the HTG-B and D protocol biopsies taken around 3 months after kidney xenotransplantation, when the transplant kidney function was stable. The biopsy analyses, using the NanoString nCounter platform, revealed upregulation of seven distinct pathways in the HTG-B group, including kidney injury (AKI), antibody mediated rejection (AMR), angiogenesis, donor specific antibody transcripts (DSAST), endothelial-associated transcripts (ENDAT), and Rho GTPase signal. In contrast, the HTG-D group showed high levels of IL-7 signaling and quantitative cytotoxic T lymphocytes-associated transcripts (QCAT), along with promotion of T cell checkpoint signaling and Th2 differentiation pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptomic responses in the HTG-D group\u003c/b\u003e \u003c/p\u003e \u003cp\u003e Although duration of overall graft survival of the HTG-D group xenograft was the longest, six out of 16 recipients in this group lost their xenograft early, within 25 days. To identify any contributing factors causing early xenograft loss, we evaluated pre-transplant anti-porcine donor antibody (DSA) titers among the HTG-D group recipients. There was no significant difference in pre-transplant DSA titers between short (\u0026lt;\u0026thinsp;25 days) and long-term(\u0026gt;\u0026thinsp;100 days) survivors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We next compared the transcriptomic responses in 1-hour biopsies between short vs. long-term survivors. Although HLA-DRA and SLPI Sa, both of which may indicate immune/inflammation activation, were high in short-term vs. long-term survivors, there was no significant differences in overall transcripts between short- vs. long-term survivors (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the current study, expression of three HTGs (\u003cem\u003eCD46\u003c/em\u003e, \u003cem\u003eCD47\u003c/em\u003e and \u003cem\u003eTHBD\u003c/em\u003e) were evaluable using specific probes available on the NanoString platform. As previously reported using scRNAseq \u003csup\u003e4\u003c/sup\u003e, we confirmed that pre-transplant levels of these three HTGs were maintained without significant changes until study termination (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). To evaluate long-term transcriptomic responses, bulk mRNA analysis of sequential biopsies of NHPs from the long-term (\u0026gt;\u0026thinsp;1 year) survivors in HTG-D recipients (n\u0026thinsp;=\u0026thinsp;6) were analyzed. At 1-hour, although upregulation of innate and adaptive immunity, such as innate-IS, adaptive-IS, ABMR,TCMR-RATS and NK gene sets, was already observed, expression of these pathways was reduced by 3 months and remained low and stable until 1 year, suggesting innate and adaptive immune responses towards xenografts were effectively suppressed by the ongoing immunosuppressive medications and HTGs. Only in the terminal necropsy samples were significantly higher innate and adaptive immune responses detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this sequential analysis of genetically engineered porcine donors for human kidney transplantation, human \u003cem\u003eCD55, CD46\u003c/em\u003e, and \u003cem\u003eCD59\u003c/em\u003e were initially added to 3 KO pigs in HTG-A to reduce activation of the complement cascade. h\u003cem\u003eCD47\u003c/em\u003e was also included to suppress macrophage-mediated autologous phagocytosis through the ligation to signal regulatory protein-alpha (SIRPα) as well as h\u003cem\u003eHLA-E\u003c/em\u003e and β\u003cem\u003e2M\u003c/em\u003e to prevent activation of NK cells\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In association with these HTGs, graft survival was significantly prolonged (No-HTG vs. HTG-A), but survival beyond 6 months was unusual. Addition of human coagulation-related genes, h\u003cem\u003eTHBD\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e and h\u003cem\u003eTFPI\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, in the HTG-B group, failed to further extend xenograft survival compared to HTG-A and, interestingly, was consistently associated with earlier, progressive, and more severe thrombocytopenia. Notably, thrombocytopenia was not observed in recipients of gene edits lacking h\u003cem\u003eTFPI\u003c/em\u003e (HTG-A and D) recipients, suggesting that inclusion of h\u003cem\u003eTFPI\u003c/em\u003e may be harmful, rather than beneficial in xenotransplantation. However, a contradictory observation was that no thrombocytopenia was observed in the HTG-C xenografts despite presence of h\u003cem\u003eTFPI.\u003c/em\u003e In HTG-C, addition of h\u003cem\u003ePROCR\u003c/em\u003e \u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e may have mitigated platelet consumption by inhibiting the coagulation cascade through the activation of protein C.\u003c/p\u003e \u003cp\u003eNotably, kidney xenograft survival exceeding one year was achieved only in association with the HTG-D gene construct, which included the anti-inflammatory genes \u003cem\u003eTNFAIP3\u003c/em\u003e, encoding the protein A20, and \u003cem\u003eHMOX1\u003c/em\u003e, encoding the enzyme heme oxygenase-1 (HO-1) along with complement and coagulation pathway regulatory gene cassettes. To more precisely evaluate possible differences in immune responses associated with adding the HTG-D gene cassette, we first compared transcriptomic responses in 1-hour xenograft biopsies taken from no HTG vs. HTG-D recipients, in which sufficient number of biopsies was available for statistical analysis. No HTG kidneys exhibited significantly higher expression of \u003cem\u003eHLA-DR\u003c/em\u003e and \u003cem\u003eIGHG3\u003c/em\u003e, which may reflect enhanced recipient immune cell activation immediately after transplantation. In addition, the higher KDR pathway transcripts indicates vascular endothelial growth factor-induced endothelial activation. Conversely, \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eBMP4\u003c/em\u003e (bone morphogenetic protein 4) were relatively elevated in HTG-D xenografts. This may suggest that BMP4, part of the TGFβ superfamily, may be associated in HTG-D recipients with promoting anti-inflammatory regulatory function\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, though exact interpretation of high \u003cem\u003eKIT\u003c/em\u003e remains to be defined.\u003c/p\u003e \u003cp\u003eWe further evaluated the effect of adding anti-inflammatory genes by comparing transcriptomic responses in group HTG-B and D in protocol biopsies taken at 3 months from recipients with clinically healthy xenografts that were histologically normal. Despite stable renal function in both groups, the transcriptomic analyses revealed significantly higher expression of seven distinct pathways for rejection in the HTG-B group, including AKI, AMR, angiogenesis, chronic AMR (CAMR), DSAST, ENDAT and Rho GTPase signal. These results suggest that, despite absence of histologic evidence of rejection, when compared to HTG-D grafts, 3-month HTG-B grafts already had relative enrichment of pathways associated with rejection, inflammation, and tissue damage. In contrast, HTG-D showed higher levels of IL-7 signaling and Th2 differentiation, which are associated with regulatory immune responses in several experimental transplant models\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We speculate that elevated cytotoxic T cell transcript (QCAT) may represent exhausted CD8 populations, but further studies will be necessary to explore this hypothesis. Overexpression of \u003cem\u003eTNFAIP3\u003c/em\u003e (A20) has been shown to inhibit TNF-induced apoptosis \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Similarly, \u003cem\u003eHMOX-1\u003c/em\u003e expression has been demonstrated to significantly protect porcine endothelial cells during ex vivo perfusion with human blood\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We presume that expression of these human anti-inflammatory proteins prolonged graft survival by reducing inflammation mediated by innate and adaptive immune cells.\u003c/p\u003e \u003cp\u003eAlthough Group HTG-D recipients demonstrated the most encouraging long-term kidney xenograft survival, approximately 38% experienced graft loss due to thrombotic microangiopathy (TMA) within 25 days. Neither pre-transplant recipient serum donor-specific antibody (DSA) levels nor bulk RNA expression in one-hour post-transplant biopsies correlated with or predicted graft fate. It is possible that the human proteins encoded by the transgenes designed for clinical use do not interact physiologically with cynomolgus monkey complement, coagulation, and self-recognition analogues,\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e in which case these candidate mechanisms driving TMA may be more prominent in the monkey model that they will prove to be clinically. While underlying causes of xenograft-associated TMA in preclinical models remain a mechanistically important question, its clinical significance has not yet been established: TMA was not observed at two months in our clinical case, or in a decedent 3KO recipient, both of whom were treated with anti-C5 mAb \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur current and previous\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e studies have shown that the expression of HTGs remained stable at the mRNA and protein level even in the terminal autopsy samples, suggesting that xenograft injury is not primarily caused by down-modulation of \u0026lsquo;protective\u0026rsquo; human transgenes. Our longitudinal observations of transcriptomic responses in the long-term HTG-D survivors revealed that both innate and adaptive immune responses were effectively suppressed for up to one year after transplantation. However, all HTG-D recipients eventually rejected their xenografts, sometimes as late as 758 days post-transplant, in associated with elevated innate and adaptive transcriptomic responses. We conclude that maintenance immunosuppression with anti-CD154 and MMF as dosed in these studies is not sufficient to suppress rejection long-term in NHP.\u003c/p\u003e \u003cp\u003eThe limitations of the current studies include the inability to isolate and assess the contribution of each human transgene (HTG) individually at clinical or mechanistic levels. Separately evaluating the numerous possible combinations of human transgenes incorporated into the pig constructs used in this life-supporting kidney xenograft model would be both cost-prohibitive and impractical.\u003c/p\u003e \u003cp\u003eIn conclusion, the current studies demonstrated that multiple combinations of HTGs, particularly those with anti-inflammatory properties, significantly extended the survival of 3KO kidney xenografts, accompanied by more favorable transcriptomic responses. While the optimal HTG combinations and immunosuppressive protocols remain to be determined, the currently available pig constructs are promising and now justify initial application of this approach to the treatment of human patients with end-stage renal disease.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eCynomolgus monkeys (purchased from Charles River Primates, Wilmington, MA) weighing 6\u0026ndash;11 kg were used. Gene edited porcine donors were provided by eGenesis (Cambridge, MA). Yucatan miniature pigs weighing 10\u0026ndash;25 kg were used as the kidney donor. All surgeries and postoperative care of animals were performed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) certified facility, per National Institutes of Health (NIH) guidelines for the care and use of nonhuman primates. These studies were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee. Husbandry, veterinary supervision, and daily care of animals were provided by Massachusetts General Hospital, Center for Comparative Medicine (CCM).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic modifications of the pig kidney xenografts with four different HTG combinations.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll pigs of O blood type were engineered using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-mediated nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Intentional genomic alterations in EGEN-2259 (no HTG), EGEN-2536 (HTG-A), EGEN-2383 (HTG-B), EGEN-2597 (HTG-C) and EGEN-2784 (HTG-D) with or without PERV inactivation. In HTG-A, HTGs to suppress macrophage (\u003cem\u003eCD47\u003c/em\u003e) and natural killer (NK) cells (\u003cem\u003eHLA-E/\u003c/em\u003eβ\u003cem\u003e2M\u003c/em\u003e), as well as to inhibit complement activation (\u003cem\u003eCD46, CD55\u003c/em\u003e and \u003cem\u003eCD59\u003c/em\u003e) were inserted. In HTG-B, coagulation related HTGs (\u003cem\u003eTHBD and TFPI\u003c/em\u003e) were added to those included in HTG-A. In HTG-C, another coagulation related gene, \u003cem\u003ePROCR\u003c/em\u003e, was added to gene edits of HTG-B. Finally, in HTG-D which was used in the clinical case, anti-inflammatory HTGs, \u003cem\u003eTNFAIP3\u003c/em\u003e and \u003cem\u003eHMOX1\u003c/em\u003e, were added but without \u003cem\u003eHLA-E/\u003c/em\u003eβ\u003cem\u003e2M\u003c/em\u003e and \u003cem\u003eTFPI\u003c/em\u003e are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e as reported\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and described below:\u003c/p\u003e\u003cp\u003e\u003cspan\u003e1. Functional inactivation of all (59) porcine endogenous retrovirus (PERV)\u0026nbsp;\u003cem\u003epol\u003c/em\u003e genes (PERV A, B, C), resulting in inactivation (RI, retroviral inactivation) of the PERV reverse transcriptase (RT) enzyme. Functional inactivation of PERV in the porcine genome is designed to reduce the potential for cross-species transfer of the retrovirus and to make donor organs safer for human use.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e2. Compound heterozygous knockout (KO) of 4 genes, including glycoprotein alpha-galactosyltransferase 1 (\u003cem\u003eGGTA1\u003c/em\u003e), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (\u003cem\u003eCMAH\u003c/em\u003e), and beta-1,4-N-acetyl-galactosaminyltransferase 2 (\u003cem\u003eB4GALNT2\u003c/em\u003e) and B4GALNT2-like (\u003cem\u003eB4GALNT2L\u003c/em\u003e) genes, resulting in undetectable levels of three antigens, galactose-\u0026alpha;-1,3-galactose (\u0026alpha;-Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) glycan antigens, respectively.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e3. Hemizygous insertion of a transgenic construct, containing the various combinations of human transgenes Cluster of Differentiation \u003cem\u003eCD46\u003c/em\u003e, \u003cem\u003eCD55\u003c/em\u003e, \u003cem\u003eCD59, CD47\u003c/em\u003e, \u003cem\u003eHLA-E/\u003c/em\u003e\u0026beta;\u003cem\u003e2M\u003c/em\u003e, thrombomodulin (\u003cem\u003eTHBD\u003c/em\u003e), tissue factor pathway inhibitor (\u003cem\u003eTFPI\u003c/em\u003e), endothelial protein C receptor (\u003cem\u003ePROCR\u003c/em\u003e), TNF alpha-induced protein 3 (\u003cem\u003eTNFAIP3\u003c/em\u003e), and heme oxygenase 1 (\u003cem\u003eHMOX1\u003c/em\u003e) as listed in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, into the adeno-associated virus integration site 1 (\u003cem\u003eAAVS1\u003c/em\u003e) genomic locus, causing expression of various combinations of human proteins, CD46, CD55, CD59, CD47, HLA-E/\u0026beta;2M, thrombomodulin(TM), TFPI, endothelial protein C receptor (EPCR), TNFAIP3 protein (A20), and heme oxygenase 1 (HO-1), respectively, from the AAVS1 genomic locus.\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eNext-generation sequencing (NGS) was performed on the edited porcine cells and/or cells from the cloned porcine donor produced, to confirm knockouts (3KO), and human transgene insertion and expression, which were confirmed by RNAseq and the flow cytometric analysis of ear-punched derived cells (EPDCs) from the porcine donor used in this study. Pathogen testing for porcine donor release included negative assays for potential swine viruses including Hepatitis E Virus, Porcine Circovirus 1, Porcine Circovirus 2, Porcine Cytomegalovirus (serologic and nucleic acid testing), Porcine Lymphotropic Herpesvirus 1, and Porcine Lymphotropic Herpesvirus 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKidney Transplant procedure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKidney transplantation was performed according to earlier established methods for NHP kidney allotransplantations\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Briefly, the kidney xenograft was transplanted intraperitoneally, anastomosing renal vein to recipient vena cava and renal artery to recipient abdominal aorta. Ureterovesical anastomosis was performed according to the Lich-Gregoir technique.\u003c/p\u003e\n\u003ch3\u003eImmunosuppressive regimen (Fig. 1)\u003c/h3\u003e\n\u003cp\u003eNHP recipients received 20 mg/kg of anti-CD20 mAb on day \u0026minus;\u0026thinsp;5 (Anti-CD20 [2B8R1F8]-Afucosylated, NIH Nonhuman Primate Reagent Resource). Anti-rhesus thymocyte [rhATG] - antibody was engineered and produced by the NIH Nonhuman Primate Reagent Resource and was administered at 5 mg/kg as induction therapy on days \u0026minus;\u0026thinsp;1 and 0. Immunosuppression was maintained with daily 200 mg mycophenolate mofetil (CellCept, Genentech, San Francisco, CA) given orally and weekly doses of anti-CD154 at 20 mg/kg. The Anti-CD154 [5C8H1] antibody was engineered and produced by the Nonhuman Primate Reagent Resource (NIH Nonhuman Primate Reagent Resource Cat#PR-1547, RRID:AB_2716324). Tacrolimus (Prograf, Astellas) was administered as intramuscular injection with trough levels of 8\u0026ndash;10 ng/ml dictating the doses for the initial period and was discontinued by post operative day 60. Additionally, tapered methylprednisone (Solu-Medrol, Pfizer) was administered as intramuscular injection over the first 60 days. The recipients were followed by serial blood tests (CBC and chemistries) and ultrasound of the kidney xenograft weekly.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eHistological analyses\u003c/h2\u003e\n \u003cp\u003eProtocol renal biopsies were obtained every 2\u0026ndash;4 months in recipients with stable function as well as \u0026ldquo;for cause\u0026rdquo; biopsy whenever a rise in serum creatinine occurred. Tissue was processed for light microscopy and a portion frozen for immunofluorescence staining. Other organs obtained surgically (lymph nodes, native kidney and spleen) were similarly processed. Following euthanasia of a xenograft recipient, a complete necropsy was performed for histopathologic examination of the renal xenograft, lymph nodes, heart, lung, liver, pancreas, thymus, and skin. Xenograft H\u0026amp;E and PAS-stained samples were scored by current Banff criteria \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e including C4d deposition by immunohistochemistry \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eNanostring mRNA expression\u003c/h2\u003e\n \u003cp\u003eBulk mRNA analysis was performed from formalin-fixed paraffin embedded xenograft biopsies and a sample from the non-transplanted contralateral donor kidney, using the nCounter instrument (Bruker Spatial Biology, Seattle WA) following the methods and data analysis previously published for human allograft biopsies \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The B-HOT panel contains 770 probes for human transcripts \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Pathways were manually curated from KEGG, Gene Ontogeny and Reactome datasets (genome.jp/kegg/pathway.html, GeneOntology.org and reactome.org) and from prior publications \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The probe sequences were screened for homology with pig and NHP transcripts. The probe sequences that had\u0026thinsp;\u0026gt;\u0026thinsp;85% homology with pig sequences for parenchymal cells or endothelium and probes that had\u0026thinsp;\u0026gt;\u0026thinsp;85% homology for NHP leukocytes were used, supplemented with 35 pig specific probes. The final panel, termed \u0026ldquo;B-HOTx\u0026rdquo; had 549 probes, as listed in \u003cstrong\u003eTable \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e. Results were compared with well-phenotyped human allograft samples analyzed with the B-HOT panel and previously published \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and normalized with seven housekeeping probes (NRDE2, POLR2A, ABCF1, OAZI, PPIA, G6PD, TBP) with \u0026gt;\u0026thinsp;90% homology with the corresponding porcine transcripts. Pathway analysis was performed in nSolver software (Bruker Spatial Biology, Seattle WA) with subsequent statistical analysis using R programming version 4.4.1 (R Foundation for Statistical Computing, Vienna, Austria). Probes used in pathway analysis is listed in \u003cstrong\u003eTable S2\u003c/strong\u003e. Raw counts were used for differential expression analysis in R using the DESeq2 package version 1.44.0.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll statistical analyses were performed using R programming version 4.4.1 (R Foundation for Statistical Computing, Vienna, Austria). Values of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered to be significant. Post-transplant survival times were plotted and compared using Kaplan-Meier survival curves and log-rank tests. Cox proportional hazards regression analysis was conducted using the coxph function from survival package in R to test the association between genetic edits and graft survival. T-test was used when comparing two groups.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Drs. Joanne Morris and Michael Duggan for veterinary supervision and Drs. Joren C Madsen and David Cooper for critical reading and comments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eeGenesis Inc. (T.K.)\u003c/p\u003e\n\u003cp\u003eMatsuko Levin Research Fund (T.K.)\u003c/p\u003e\n\u003cp\u003eAK received support from Karolinska Institutet (Hirsch, and Fernstr\u0026ouml;ms travel grants) and the Swedish Society of Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u0026nbsp; A.K., T.H., G.L., T.T. and T.K. participated in surgery postoperative animal care. A.K., I.R., R.B.C. and T.K. conducted in vitro experiments, interpreted data, and participated in writing the manuscript. R.P.A., J.V. L., M.C.,S.L., and W.Q. provided resources (3KO genetically modified pigs) and A.K., I.R., R.N.P., A.B.C., R.B.C. and T.K. wrote the manuscript. T.K. supervised and designed the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e eGenesis has filed patent applications on the transgenic pig technology described in this paper.\u0026nbsp;R.P.A., J.V. L., M.C.,S.L., and W.Q.\u0026nbsp;are employees of eGenesis Bio.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOther authors have no conflicts of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data are available in the main text or the supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOPTN. National Data https://optn.transplant.hrsa.gov/data/view-data-reports/national-data/#. (2024).\u003c/li\u003e\n\u003cli\u003eBuhler, L., Friedman, T., Iacomini, J. \u0026amp; Cooper, D. K. Xenotransplantation--state of the art--update 1999. \u003cem\u003eFront Biosci\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, D416-432 (1999).\u003c/li\u003e\n\u003cli\u003eMa, D.\u003cem\u003e et al.\u003c/em\u003e Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques. \u003cem\u003eAm J Transplant\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 46-57 (2022). https://doi.org/10.1111/ajt.16780\u003c/li\u003e\n\u003cli\u003eAnand, R. P.\u003cem\u003e et al.\u003c/em\u003e Design and testing of a humanized porcine donor for xenotransplantation. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e622\u003c/strong\u003e, 393-401 (2023). https://doi.org/10.1038/s41586-023-06594-4\u003c/li\u003e\n\u003cli\u003eal., K. T. e. Gene-Edited Porcine Kidney Xenotransplantation for End-Stage Kidney Disease. \u003cem\u003eN Engl J Med\u003c/em\u003e (2025).\u003c/li\u003e\n\u003cli\u003eSchuurman, H. J.\u003cem\u003e et al.\u003c/em\u003e Incidence of hyperacute rejection in pig-to-primate transplantation using organs from hDAF-transgenic donors. \u003cem\u003eTransplantation\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 1146-1151 (2002). https://doi.org/10.1097/00007890-200204150-00024\u003c/li\u003e\n\u003cli\u003eMcGregor, C. G.\u003cem\u003e et al.\u003c/em\u003e Human CD55 expression blocks hyperacute rejection and restricts complement activation in Gal knockout cardiac xenografts. \u003cem\u003eTransplantation\u003c/em\u003e \u003cstrong\u003e93\u003c/strong\u003e, 686-692 (2012). https://doi.org/10.1097/TP.0b013e3182472850\u003c/li\u003e\n\u003cli\u003eSingh, A. K.\u003cem\u003e et al.\u003c/em\u003e Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs. \u003cem\u003eXenotransplantation\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, e12465 (2019). https://doi.org/10.1111/xen.12465\u003c/li\u003e\n\u003cli\u003eMengel, M.\u003cem\u003e et al.\u003c/em\u003e Banff 2019 Meeting Report: Molecular diagnostics in solid organ transplantation-Consensus for the Banff Human Organ Transplant (B-HOT) gene panel and open source multicenter validation. \u003cem\u003eAm J Transplant\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 2305-2317 (2020). https://doi.org/10.1111/ajt.16059\u003c/li\u003e\n\u003cli\u003ePuga Yung, G., Schneider, M. K. J. \u0026amp; Seebach, J. D. The Role of NK Cells in Pig-to-Human Xenotransplantation. \u003cem\u003eJ Immunol Res\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, 4627384 (2017). https://doi.org/10.1155/2017/4627384\u003c/li\u003e\n\u003cli\u003eWeiss, E. H.\u003cem\u003e et al.\u003c/em\u003e HLA-E/human beta2-microglobulin transgenic pigs: protection against xenogeneic human anti-pig natural killer cell cytotoxicity. \u003cem\u003eTransplantation\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 35-43 (2009). https://doi.org/10.1097/TP.0b013e318191c784\u003c/li\u003e\n\u003cli\u003eKim, H.\u003cem\u003e et al.\u003c/em\u003e Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno-immune response. \u003cem\u003eXenotransplantation\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 260-272 (2015). https://doi.org/10.1111/xen.12173\u003c/li\u003e\n\u003cli\u003eHara, H.\u003cem\u003e et al.\u003c/em\u003e Stable expression of the human thrombomodulin transgene in pig endothelial cells is associated with a reduction in the inflammatory response. \u003cem\u003eCytokine\u003c/em\u003e \u003cstrong\u003e148\u003c/strong\u003e, 155580 (2021). https://doi.org/10.1016/j.cyto.2021.155580\u003c/li\u003e\n\u003cli\u003eLee, K. F.\u003cem\u003e et al.\u003c/em\u003e Recombinant pig TFPI efficiently regulates human tissue factor pathways. \u003cem\u003eXenotransplantation\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 191-197 (2008). https://doi.org/10.1111/j.1399-3089.2008.00476.x\u003c/li\u003e\n\u003cli\u003eLee, K. F.\u003cem\u003e et al.\u003c/em\u003e Protective effects of transgenic human endothelial protein C receptor expression in murine models of transplantation. \u003cem\u003eAm J Transplant\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2363-2372 (2012). https://doi.org/10.1111/j.1600-6143.2012.04122.x\u003c/li\u003e\n\u003cli\u003eSalvaris, E. J.\u003cem\u003e et al.\u003c/em\u003e Pig endothelial protein C receptor is functionally compatible with the human protein C pathway. \u003cem\u003eXenotransplantation\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, e12557 (2020). https://doi.org/10.1111/xen.12557\u003c/li\u003e\n\u003cli\u003eRegateiro, F. S., Howie, D., Cobbold, S. P. \u0026amp; Waldmann, H. TGF-beta in transplantation tolerance. \u003cem\u003eCurr Opin Immunol\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 660-669 (2011). https://doi.org/10.1016/j.coi.2011.07.003\u003c/li\u003e\n\u003cli\u003eMazzucchelli, R.\u003cem\u003e et al.\u003c/em\u003e Development of regulatory T cells requires IL-7Ralpha stimulation by IL-7 or TSLP. \u003cem\u003eBlood\u003c/em\u003e \u003cstrong\u003e112\u003c/strong\u003e, 3283-3292 (2008). https://doi.org/10.1182/blood-2008-02-137414\u003c/li\u003e\n\u003cli\u003eWaaga, A. M.\u003cem\u003e et al.\u003c/em\u003e Regulatory functions of self-restricted MHC class II allopeptide-specific Th2 clones in vivo. \u003cem\u003eJ Clin Invest\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 909-916 (2001).\u003c/li\u003e\n\u003cli\u003eFerran, C., Stroka, D. M., Badrichani, A. Z., Cooper, J. T. \u0026amp; Bach, F. H. Adenovirus-mediated gene transfer of A20 renders endothelial cells resistant to activation: a means of evaluating the role of endothelial cell activation in xenograft rejection. \u003cem\u003eTransplant Proc\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 879-880 (1997). https://doi.org/10.1016/s0041-1345(96)00184-4\u003c/li\u003e\n\u003cli\u003eOropeza, M.\u003cem\u003e et al.\u003c/em\u003e Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. \u003cem\u003eXenotransplantation\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 522-534 (2009). https://doi.org/10.1111/j.1399-3089.2009.00556.x\u003c/li\u003e\n\u003cli\u003ePetersen, B.\u003cem\u003e et al.\u003c/em\u003e Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys. \u003cem\u003eXenotransplantation\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 355-368 (2011). https://doi.org/10.1111/j.1399-3089.2011.00674.x\u003c/li\u003e\n\u003cli\u003eJacobsen, F. W.\u003cem\u003e et al.\u003c/em\u003e Molecular and functional characterization of cynomolgus monkey IgG subclasses. \u003cem\u003eJ Immunol\u003c/em\u003e \u003cstrong\u003e186\u003c/strong\u003e, 341-349 (2011). https://doi.org/10.4049/jimmunol.1001685\u003c/li\u003e\n\u003cli\u003eJones-Carr, M. E.\u003cem\u003e et al.\u003c/em\u003e C5 inhibition with eculizumab prevents thrombotic microangiopathy in a case series of pig-to-human kidney xenotransplantation. \u003cem\u003eThe Journal of Clinical Investigation\u003c/em\u003e (2024). https://doi.org/10.1172/JCI175996\u003c/li\u003e\n\u003cli\u003eYoung, J. A., Pallas, C. R. \u0026amp; Knovich, M. A. Transplant-associated thrombotic microangiopathy: theoretical considerations and a practical approach to an unrefined diagnosis. \u003cem\u003eBone marrow transplantation\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 1805-1817 (2021). https://doi.org/10.1038/s41409-021-01283-0\u003c/li\u003e\n\u003cli\u003eCosimi, A. B.\u003cem\u003e et al.\u003c/em\u003e Prolonged survival of nonhuman primate renal allograft recipients treated only with anti-CD4 monoclonal antibody. \u003cem\u003eSurgery\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 406-413; discussion 413-404 (1990).\u003c/li\u003e\n\u003cli\u003eAdam, B. A.\u003cem\u003e et al.\u003c/em\u003e Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes. \u003cem\u003eAm J Transplant\u003c/em\u003e (2017). https://doi.org/10.1111/ajt.14327\u003c/li\u003e\n\u003cli\u003eRosales, I. A.\u003cem\u003e et al.\u003c/em\u003e Banff Human Organ Transplant Transcripts Correlate with Renal Allograft Pathology and Outcome: Importance of Capillaritis and Subpathologic Rejection. \u003cem\u003eJ Am Soc Nephrol\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 2306-2319 (2022). https://doi.org/10.1681/ASN.2022040444\u003c/li\u003e\n\u003cli\u003eSmith, R. N.\u003cem\u003e et al.\u003c/em\u003e Utility of Banff Human Organ Transplant Gene Panel in Human Kidney Transplant Biopsies. \u003cem\u003eTransplantation\u003c/em\u003e \u003cstrong\u003e107\u003c/strong\u003e, 1188-1199 (2023). https://doi.org/10.1097/TP.0000000000004389\u003c/li\u003e\n\u003cli\u003eReeve, J.\u003cem\u003e et al.\u003c/em\u003e Molecular diagnosis of T cell-mediated rejection in human kidney transplant biopsies. \u003cem\u003eAm J Transplant\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 645-655 (2013). https://doi.org/10.1111/ajt.12079\u003c/li\u003e\n\u003cli\u003eVenner, J. M., Hidalgo, L. G., Famulski, K. S., Chang, J. \u0026amp; Halloran, P. F. The molecular landscape of antibody-mediated kidney transplant rejection: evidence for NK involvement through CD16a Fc receptors. \u003cem\u003eAm J Transplant\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 1336-1348 (2015). https://doi.org/10.1111/ajt.13115\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6017857/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6017857/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenetically modified pigs are being developed to address the critical shortage of human organs for transplantation. We have previously demonstrated significantly prolonged survival of porcine xenografts devoid of three major carbohydrate xenoantigens (3KO) by incorporating human transgenes (HTGs). However, the optimal HTG combination and the mechanisms underlying improved xenograft survival following such genetic editing remain undefined. In the current study, we evaluated, in nonhuman primates, immune responses and transplant outcome of 3KO kidney xenografts with or without four different combinations of HTGs. We show that addition of HTGs significantly reduced transcripts associated with initial immune activation, resulting in markedly extended survival of the 3KO xenografts. Most notably, the addition of anti-inflammatory genes, \u003cem\u003eTNFAIP3\u003c/em\u003e and \u003cem\u003eHMOX1\u003c/em\u003e, was associated with improved graft survival with significantly lower expression of rejection-related gene sets in protocol xenograft biopsies, while the inclusion of coagulation-related HTGs was less effective. Although further studies are needed to define the optimal HTG combination for human recipients, we conclude that multiple combinations of HTGs can effectively prolong primate survival following 3KO kidney xenotransplantation.\u003c/p\u003e","manuscriptTitle":"Multiple human transgenes prolong survival of triple-carbohydrate knockout porcine kidney xenografts in nonhuman primates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-18 08:11:50","doi":"10.21203/rs.3.rs-6017857/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ce5da723-2d9b-4cdb-b7ab-da171db51d5c","owner":[],"postedDate":"March 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44366993,"name":"Biological sciences/Immunology/Transplant immunology"},{"id":44366994,"name":"Health sciences/Medical research/Preclinical research"}],"tags":[],"updatedAt":"2025-03-18T08:11:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-18 08:11:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6017857","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6017857","identity":"rs-6017857","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}