BACKGROUND:Kidney xenotransplantation offers a potential solution to the organ shortage, but questions remain regarding durability, zoonotic infection risk, and whether the immunological response to the xenograft elicits sensitisation that could complicate subsequent allotransplantation. We report outcomes from a porcine kidney xenograft in a living recipient followed by human allotransplantation. METHODS:A patient with end-stage kidney disease, a prolonged anticipated waiting time for deceased donor transplantation, and with no suitable living donor underwent transplantation at Massachusetts General Hospital (Boston, MA, USA) with a gene-edited porcine kidney (EGEN-2784; eGenesis [Cambridge, MA, USA]) incorporating the deletion of major glycan xenoantigens, inactivation of porcine endogenous retroviruses, and insertion of seven human transgenes. The recipient received costimulation blockade-based immunosuppression with complement inhibition. Monitoring included renal function, flow cytometric crossmatch, anti-HLA antibodies, and porcine microbial surveillance, including metagenomic sequencing. This report describes the first recipient in a planned three-patient study conducted under a US Food and Drug Administration Expanded Access Investigational New Drug application. FINDINGS:The xenograft functioned immediately after transplantation on Jan 25, 2025, and sustained dialysis independence for 271 days. A biopsy on day 14 showed T-cell-mediated rejection, which resolved with treatment. Graft function remained stable for approximately 6 months until immunosuppression was reduced in the setting of non-zoonotic bacterial infection. Microvascular inflammation with endothelial injury subsequently emerged, progressing to thrombotic microangiopathy despite persistently negative donor-specific crossmatch, leading to graft failure and nephrectomy. Tissue analysis showed a macrophage and natural-killer-cell-predominant infiltrate with minimal T-cell involvement. No porcine pathogen transmission was detected. Anti-HLA antibodies remained unchanged. 82 days after explantation, the patient underwent human kidney allotransplantation with immediate graft function and no evidence of sensitisation during 231 days of follow-up. INTERPRETATION:This case shows that porcine kidney xenotransplantation can provide prolonged renal support and be discontinued without clinically significant allosensitisation or zoonotic infection. Early cellular rejection resolved with treatment, whereas later graft failure was associated with microvascular injury progressing to thrombotic microangiopathy despite a negative donor-specific crossmatch, supporting the possibility that mechanisms beyond conventional antibody-mediated rejection contributed to late graft injury. Kidney xenotransplantation has the potential to provide prolonged dialysis-free support while also serving as a bridge to subsequent human allotransplantation. FUNDING:Massachusetts General Hospital and eGenesis.

Genetically modified pigs are being developed to address the critical shortage of human organs for transplantation. Although porcine xenografts lacking the three major carbohydrate xenoantigens (3KO) have been considered ideal for human transplantation, the optimal combination of human transgenes (HTGs) to mitigate protein incompatibility remains undefined. In the current study, we evaluate immune responses and transplant outcomes of 3KO kidney xenografts with four different combinations of HTGs in a nonhuman primate model. Here, we show that the addition of HTGs significantly reduces transcripts associated with early immune activation, resulting in markedly prolonged survival of 3KO xenografts. Notably, the inclusion of the anti-inflammatory genes TNFAIP3 and HMOX1 is associated with improved graft survival, significantly reduced T-cell and CD11b⁺ myeloid cell infiltration, and lower expression of rejection-related gene sets in protocol xenograft biopsies.

The success and safety of clinical xenotransplantation are determined by technical aspects of surgery, the nature and intensity of immunosuppression, xenograft function, and the ability to avoid immunologic, hematologic, infectious, and malignant complications. In clinical xenotransplantation from swine, infectious challenges relate to the potential spread of pig pathogens to immunosuppressed humans as well as manifestations of infections of the recipient from prior exposures to common human organisms. Robust strategies are required for donor screening, recipient monitoring, and infection prevention. This report outlines the comprehensive infectious disease management strategy deployed in the first living human recipient of a genetically modified pig kidney, including the following: (1) donor screening and pathogen mitigation, (2) pretransplant and posttransplant infection monitoring of the recipient, (3) infectious complications and management, and (4) implications for future xenotransplantation protocols.

Xenotransplantation offers a potential solution to the organ shortage crisis. A 62-year-old hemodialysis-dependent man with long-standing diabetes, advanced vasculopathy, and marked dialysis-access challenges received a gene-edited porcine kidney with 69 genomic edits, including deletion of three glycan antigens, inactivation of porcine endogenous retroviruses, and insertion of seven human transgenes. The xenograft functioned immediately. The patient's creatinine levels decreased promptly and progressively, and dialysis was no longer needed. After a T-cell-mediated rejection episode on day 8, intensified immunosuppression reversed rejection. Despite sustained kidney function, the patient died from unexpected, sudden cardiac causes on day 52; autopsy revealed severe coronary artery disease and ventricular scarring without evident xenograft rejection. (Funded by Massachusetts General Hospital and eGenesis.).

OBJECTIVES: To systematically review the safety and efficacy of nonbiological (NBAL) or biological artificial liver support systems (BAL) and whole-organ extracorporeal liver perfusion (W-ECLP) systems, in adults with acute liver failure (ALF) and acute-on-chronic liver failure (ACLF). DATA SOURCES: Eligible NBAL/BAL studies from PubMed/Embase searches were randomized controlled trials (RCTs) in adult patients with ALF/ACLF, greater than or equal to ten patients per group, reporting outcomes related to survival, adverse events, transplantation rate, and hepatic encephalopathy, and published in English from January 2000 to July 2023. Separately, we searched for studies evaluating W-ECLP in adult patients with ALF or ACLF published between January1990 and July 2023. STUDY SELECTION AND DATA EXTRACTION: Two researchers independently screened citations for eligibility and, of eligible studies, retrieved data related to study characteristics, patients and interventions, outcomes definition, and intervention effects. The Cochrane Risk of Bias 2 tool and Joanna Briggs Institute checklists were used to assess individual study risk of bias. Meta-analysis of mortality at 28–30 days post-support system initiation and frequency of at least one serious adverse event (SAE) generated pooled risk ratios (RRs), based on random (mortality) or fixed (SAE) effects models. DATA SYNTHESIS: Of 17 trials evaluating NBAL/BAL systems, 11 reported 28–30 days mortality and five reported frequency of at least one SAE. Overall, NBAL/BAL was not statistically associated with mortality at 28–30 days (RR, 0.85; 95% CI, 0.67–1.07; p = 0.169) or frequency of at least one SAE (RR, 1.15; 95% CI, 0.99–1.33; p = 0.059), compared with standard medical treatment. Subgroup results on ALF patients suggest possible benefit for mortality (RR, 0.67; 95% CI, 0.44–1.03; p = 0.069). From six reports of W-ECLP (12 patients), more than half (58%) of severe patients were bridged to transplantation and survived without transmission of porcine retroviruses. CONCLUSIONS: Despite no significant pooled effects of NBAL/BAL devices, the available evidence calls for further research and development of extracorporeal liver support systems, with larger RCTs and optimization of patient selection, perfusion durability, and treatment protocols.

Introduction: Xenotransplantation holds immense potential as replacement therapy for end-stage renal disease and a solution to the global organ shortage. Development of porcine xenografts with triple knockouts (3KO) of known xenoantigens (GGTA1, CMAH, B4GALNT2) have partly mitigated the hyperacute rejection by natural antibodies to unmodified porcine organs1. Transgenic expression of human transgenes has further considerably improved xenograft survival by overcoming the molecular incompatibilities between pigs and humans2. Methods: 3KO donors (n=3), and 3KO donors also expressing selected human transgenes that control complement (CD46 and CD55), coagulation (THBD and PROCR), innate immunity (CD47), and inflammation (TNFAIP3, and HMOX1) were produced by eGenesis (EGEN-2734, n=10; EGEN-2784, n=7) on a Yucatan miniature swine background. EGEN-2784 expresses the same human transgenes with additional retroviral inactivation to minimize the possible zoonosis risk of porcine endogenous retroviral elements (Figure 1)3,4. Life-sustaining kidney transplantation was performed in cynomolgus macaques using these genetically modified porcine grafts to evaluate the addition of human transgenes. T and B cell depletion was followed by a corticosteroid taper and short course of tacrolimus. Immunosuppression was maintained with anti-CD154 monoclonal antibody +/- mycophenolate mofetil. Pre-transplant IgG/IgM antibodies binding to porcine endothelial cells were surveyed by flow cytometry. Results: Recipient survival was significantly increased using donor organs expressing human transgenes compared to 3KO only grafts (median, 103 vs. 6 days, p=0.001, Log-rank) (Figure 1). Pre-transplant anti-porcine titers showed no correlation to survival, long-term animals, including >2 year surviving animal showed high comparative pre-transplant IgM binding. The major cause of the early (<60 days) graft loss was thrombotic microangiopathy (TMA). Biopsies from three recipients currently in progress on >244, >420 and >670 days showed no rejection or TMA. Four other long-term (>240 days) survivors also showed no rejection/TMA until close to the graft loss which was due to rapidly developed antibody-mediated rejection (AMR) and TMA. In contrast to allotransplantation, in which T cell-mediated rejection (TCMR) is the major pathology, TCMR was observed in only two EGEN-2734 recipients after reduction of anti-CD154 mAb dose (Table 1).Conclusions: Life-sustaining renal xenograft survival exceeding two years was achieved in cynomolgus macaques using triple knock-out (3KO) porcine kidneys expressing seven human transgenes. Although the outcome is heterogenous, pre-transplant anti-porcine antibody titer binding do not correlate with clinical outcome. Less xenograft damage in early rejection might indicate complement activation and protein incompatibilities as causes for graft dysfunction, which may suggest further modifications of transgenes may be necessary to improve the consistency of the results.References: 1. D. K. Cooper, A. H. Good, E. Koren, R. Oriol, A. J. Malcolm, R. M. Ippolito, F. A. Neethling, Y. Ye, E. Romano, N. Zuhdi, Identification of alpha-galactosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies: relevance to discordant xenografting in man. Transpl Immunol. 1, 198–205 (1993). 2. Ma, D., Hirose, T., Lassiter, G., Sasaki, H., Rosales, I., Coe, T. M., Rickert, C. G., Matheson, R., Colvin, R. B., Qin, W., Kan, Y., Layer, J. v., Paragas, V. B., Stiede, K., Hall, K. C., Youd, M. E., Queiroz, L. M., Westlin, W. F., Curtis, M., … Kawai, T. (2022). Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques. American Journal of Transplantation, 22(1), 46–57. 3. C. Patience, Y. Takeuchi, R. A. Weiss, Infection of human cells by an endogenous retrovirus of pigs. Nature medicine. 3, 282–6 (1997). 4. D. Niu, H.-J. Wei, L. Lin, H. George, T. Wang, I.-H. Lee, H.-Y. Zhao, Y. Wang, Y. Kan, E. Shrock, E. Lesha, G. Wang, Y. Luo, Y. Qing, D. Jiao, H. Zhao, X. Zhou, S. Wang, H. Wei, M. Güell, G. M. Church, L. Yang, Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. 357, 1303–1307 (2017).

Purpose Previous studies showed ischemia minimization (IM), accomplished by perfusing the heart xenograft during storage, prevented 'initial cardiac xenograft dysfunction' (ICXD) and prolonged survival following in vivo orthotopic cardiac xenotransplantation. We report initial observations in a model designed to evaluate effects associated with IM and genetic modifications on heart performance in a working ex vivo model. Methods Hearts from genetically modified (gm) and wildtype (WT) pigs were procured and stored for 3 hours either in cold saline (cold storage (CS)) or were perfused with oxygenated Steen's solution with RBCs (IM). The gm hearts either had combined knockouts of 3 xenogenic carbohydrate genes (GTKO, CMAHKO, β4GALNT2KO: TKO) with variable expression of human transgenes (n=13); or GTKO with expression of hCD55 (GTKO.hCD55; n=2). Heart function and troponin I as a marker for myocardial injury were assessed on a working heart rig while perfused with whole human blood. Results 19 hearts were perfused ex vivo, ten with CS (TKO n=6, GTKO.hCD55 n=1, WT n=3), 9 with IM (TKO n=7, GTKO.hCD55 n=1, WT n=1). Mean troponin I levels were significantly reduced after 1 hour of ex vivo perfusion in the IM group (70.2 ng/mL vs. 279.5 ng/mL; p = .038), but not at final time points (see fig. 1a-c). Cardiac function (cardiac output in response to increased filling pressures) decreased over time after CS, whereas the IM-stored heart function tended to improve over time (Fig. 2). Conclusion IM significantly decreased myocardial injury during the first hour of ex vivo working heart perfusion relative to CS hearts: heart injury (troponin release) was attenuated, and graft failure was delayed in some TKO hearts treated with IM. Evaluation of whether variation in expression of human transgenes may have influenced TKO graft protection from ICXD is in progress. We provisionally conclude that IM attenuates ICXD of genetically modified pig hearts during initial exposure to human blood. Previous studies showed ischemia minimization (IM), accomplished by perfusing the heart xenograft during storage, prevented 'initial cardiac xenograft dysfunction' (ICXD) and prolonged survival following in vivo orthotopic cardiac xenotransplantation. We report initial observations in a model designed to evaluate effects associated with IM and genetic modifications on heart performance in a working ex vivo model. Hearts from genetically modified (gm) and wildtype (WT) pigs were procured and stored for 3 hours either in cold saline (cold storage (CS)) or were perfused with oxygenated Steen's solution with RBCs (IM). The gm hearts either had combined knockouts of 3 xenogenic carbohydrate genes (GTKO, CMAHKO, β4GALNT2KO: TKO) with variable expression of human transgenes (n=13); or GTKO with expression of hCD55 (GTKO.hCD55; n=2). Heart function and troponin I as a marker for myocardial injury were assessed on a working heart rig while perfused with whole human blood. 19 hearts were perfused ex vivo, ten with CS (TKO n=6, GTKO.hCD55 n=1, WT n=3), 9 with IM (TKO n=7, GTKO.hCD55 n=1, WT n=1). Mean troponin I levels were significantly reduced after 1 hour of ex vivo perfusion in the IM group (70.2 ng/mL vs. 279.5 ng/mL; p = .038), but not at final time points (see fig. 1a-c). Cardiac function (cardiac output in response to increased filling pressures) decreased over time after CS, whereas the IM-stored heart function tended to improve over time (Fig. 2). IM significantly decreased myocardial injury during the first hour of ex vivo working heart perfusion relative to CS hearts: heart injury (troponin release) was attenuated, and graft failure was delayed in some TKO hearts treated with IM. Evaluation of whether variation in expression of human transgenes may have influenced TKO graft protection from ICXD is in progress. We provisionally conclude that IM attenuates ICXD of genetically modified pig hearts during initial exposure to human blood.

Pigs with deletion of 3 carbohydrate xenoantigens (triple knock-out, TKO) are expected to be optimal donors for human xenotransplantation. We hypothesized that concomitantly inserted human transgenes (hTGs) are important to attenuate anti-xenograft immune responses. In the current study, kidney xenotransplants from four TKO pig lines with different hTGs as well as TKO without hTGs were evaluated. Nineteen cynomolgus monkeys received kidneys from four different TKO pig lines (TKO-A to D) with various expression of human immune regulatory proteins (ImmuRPs), complement regulatory proteins (CompRPs) and coagulation regulatory proteins (CoagRPs) or no hTGs. Recipients were treated with anti-thymocyte globulin (ATG) and rituximab induction followed by anti-CD154 antibody (every 1-2 weeks) and daily mycophenolate mofetil (MMF). Prednisone and either rapamycin or tacrolimus were also administered for the first two months. Two recipients of TKO-A, which expressed higher ImmuRP with lower CompRPs, survived for 2 and 61 days, while recipients of TKO-B with high CompRPs and lower ImmuRPs survived for 15, 20, 71, 135, 265 and 316 days (Table 2). 15 NHPs received xenografts from TKO-C with CompRP, ImmuRP, CoagRPs TM/EPCR, and with or without endogenous retrovirus inactivation (RI). Ongoing recipients (7) show no signs of rejection or thrombotic microangiopathy (TMA), currently at days >489, >482, >292, >160, >104, and >48, treated with only anti-CD154 mAb and MMF after 2 months. Rejection and TMA appeared to contribute to graft loss in the remaining 8 recipients between 8 and 240 days following transplant. Both recipients of TKO-D, in which CoagRPs TM and TFPI were present, survived for 243 and 267 days without rejection or TMA but were euthanized due to infectious complications. Finally, both recipients of TKO without hTG lost their xenografts early on day 4 and 50, due to severe tubular injury and significant proteinuria (final pathologic diagnosis pending but AMR is suspected) respectively. Prolonged (>1 year) rejection and TMA-free survival of kidney xenografts with TKO and multiple hTGs have been achieved. Whether the hTGs are essential for long-term xenograft survival remains to be determined with more control animals without hTGs. Our preliminary results that two recipients of TKO without hTG lost their xenografts early, suggest an essential role of hTGs for long-term xenograft survival. Funding provided by NIH Grant 5T32AI007529-22 & eGenesis Inc.