Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

Novel factors potentially initiating acute antibody-mediated rejection in pig kidney xenografts despite an efficient immunosuppressive regimen.

Antibody-mediated rejection (AMR) is a common cause of graft failure after pig-to-nonhuman primate organ transplantation, even when the graft is from a pig with multiple genetic modifications. The specific factors that initiate AMR are often uncertain. We report two cases of pig kidney transplantation into immunosuppressed baboons in which we identify novel factors associated with the initiation of AMR. In the first, membranous nephropathy was the initiating factor that was then associated with the apparent loss of the therapeutic anti-CD154 monoclonal antibody in the urine when severe proteinuria was present. This observation suggests that proteinuria may be associated with the loss of any therapeutic monoclonal antibody, for example, anti-CD154 or eculizumab, in the urine, resulting in xenograft rejection. In the second case, the sequence of events and histopathology tentatively suggested that pyelonephritis may have initiated acute-onset AMR. The association of a urinary infection with graft rejection has been well-documented in ABO-incompatible kidney allotransplantation based on the expression of an antigen on the invading microorganism shared with the kidney graft, generating an immune response to the graft. To our knowledge, these potential initiating factors of AMR in pig xenografts have not been highlighted previously.

Two-day Static Cold Preservation of α1,3-Galactosyltransferase Knockout Kidney Grafts Before Simulated Xenotransplantation.

Transplantation remains the preferred treatment for end-stage kidney disease but is critically limited by the number of available organs. Xenografts from genetically modified pigs have become a promising solution to the loss of life while waiting for transplantation. However, the current clinical model for xenotransplantation will require off-site procurement, leading to a period of ischemia during transportation. As of today, there is limited understanding regarding the preservation of these organs, including the duration of viability, and the associated molecular changes. Thus, our aim was to evaluate the effects of static cold storage (SCS) on α1,3-galactosyltransferase knockout (GGTA1 KO) kidney. After SCS, viability was further assessed using acellular sub-normothermic ex vivo perfusion and simulated transplantation with human blood. Compared to baseline, tubular and glomerular interstitium was preserved after 2 days of SCS in both WT and GGTA1 KO kidneys. Bulk RNA-sequencing demonstrated that only eight genes were differentially expressed after SCS in GGTA1 KO kidneys. During sub-normothermic perfusion, kidney function, reflected by oxygen consumption, urine output, and lactate production was adequate in GGTA1 KO grafts. During a simulated transplant with human blood, macroscopic and histological assessment revealed minimal kidney injury. However, GGTA1 KO kidneys exhibited higher arterial resistance, increased lactate production, and reduced oxygen consumption during the simulated transplant. In summary, our study suggests that SCS is feasible for the preservation of porcine GGTA1 KO kidneys. However, alternative preservation methods should be evaluated for extended preservation of porcine grafts.

TNX-1500, a crystallizable fragment-modified anti-CD154 antibody, prolongs nonhuman primate renal allograft survival.

The blockade of the CD154-CD40 pathway with anti-CD154 monoclonal antibody has been a promising immunomodulatory approach to prevent allograft rejection. However, clinical trials of immunoglobulin G1 antibodies targeting this pathway revealed thrombogenic properties, which were subsequently shown to be mediated by crystallizable fragment (Fc)-gamma receptor IIa-dependent platelet activation. To prevent thromboembolic complications, an immunoglobulin G4 anti-CD154 monoclonal antibody, TNX-1500, which retains the fragment antigen binding region of ruplizumab (humanized 5c8, BG9588), was modified by protein engineering to decrease Fc binding to Fc-gamma receptor IIa while retaining certain other effector functions and pharmacokinetics comparable with natural antibodies. Here, we report that TNX-1500 treatment is not associated with platelet activation in vitro and consistently inhibits kidney allograft rejection in vivo without clinical or histologic evidence of prothrombotic phenomena. We conclude that TNX-1500 retains efficacy similar to that of 5c8 to prevent kidney allograft rejection while avoiding previously identified pathway-associated thromboembolic complications.

A liver-humanized mouse model of carbamoyl phosphate synthetase 1-deficiency.

A liver-humanized mouse model for CPS1-deficiency was generated by the high-level repopulation of the mouse liver with CPS1-deficient human hepatocytes. When compared with mice that are highly repopulated with CPS1-proficient human hepatocytes, mice that are repopulated with CPS1-deficient human hepatocytes exhibited characteristic symptoms of human CPS1 deficiency including an 80% reduction in CPS1 metabolic activity, delayed clearance of an ammonium chloride infusion, elevated glutamine and glutamate levels, and impaired metabolism of [15 N]ammonium chloride into urea, with no other obvious phenotypic differences. Because most metabolic liver diseases result from mutations that alter critical pathways in hepatocytes, a model that incorporates actual disease-affected, mutant human hepatocytes is useful for the investigation of the molecular, biochemical, and phenotypic differences induced by that mutation. The model is also expected to be useful for investigations of modified RNA, gene, and cellular and small molecule therapies for CPS1-deficiency. Liver-humanized models for this and other monogenic liver diseases afford the ability to assess the therapy on actual disease-affected human hepatocytes, in vivo, for long periods of time and will provide data that are highly relevant for investigations of the safety and efficacy of gene-editing technologies directed to human hepatocytes and the translation of gene-editing technology to the clinic.

Characteristics and risk factors for recurrence of nonalcoholic steatohepatitis following liver transplantation.

OBJECTIVES: Nonalcoholic steatohepatitis (NASH), which is a common and increasing indication for liver transplantation (LT), is known to recur after LT. Since the recurrence of NASH can lead to graft failure, the identification of predictive factors is needed and preventive strategies should be implemented. METHODS: We retrospectively examined 95 patients who had undergone LT for NASH or alcoholic liver disease (ALD) as a primary indication. We evaluated peritransplant characteristics and histological findings 1-year post LT among liver transplant patients due to NASH or ALD. RESULTS: Pre-LT body mass index (BMI) was higher and pre-LT diabetes was more prevalent in NASH patients than in ALD patients (p < .01). The difference of BMI persisted at 3 months and 1 year after LT. There were no differences between the groups regarding histopathological findings including the degree of steatosis and fibrosis in 1-year biopsy. In multivariate analysis, recipient age and 1-year BMI were independent risk factors for post-LT fatty liver disease development. Regarding predictive factors of NASH recurrence, the prevalence of pre-LT insulin-dependent diabetes was significantly higher in patients who developed NASH recurrence than those who did not. The increase of HbA1c at 1-year post-LT checkup was higher in patients who developed recurrence than those who did not, although the difference did not reach statistical significance. CONCLUSIONS: The results of this study suggest that insulin-dependent diabetes has detrimental effects on NASH recurrence following LT. Optimal glycemic control should be recommended, but studies are needed to prove its preventive effect on NASH recurrence.

Selective Bcl-2 inhibition promotes hematopoietic chimerism and allograft tolerance without myelosuppression in nonhuman primates.

Hematopoietic stem cell transplantation (HSCT) has many potential applications beyond current standard indications, including treatment of autoimmune disease, gene therapy, and transplant tolerance induction. However, severe myelosuppression and other toxicities after myeloablative conditioning regimens have hampered wider clinical use. To achieve donor hematopoietic stem cell (HSC) engraftment, it appears essential to establish niches for the donor HSCs by depleting the host HSCs. To date, this has been achievable only by nonselective treatments such as irradiation or chemotherapeutic drugs. An approach that is capable of more selectively depleting host HSCs is needed to widen the clinical application of HSCT. Here, we show in a clinically relevant nonhuman primate model that selective inhibition of B cell lymphoma 2 (Bcl-2) promoted hematopoietic chimerism and renal allograft tolerance after partial deletion of HSCs and effective peripheral lymphocyte deletion while preserving myeloid cells and regulatory T cells. Although Bcl-2 inhibition alone was insufficient to induce hematopoietic chimerism, the addition of a Bcl-2 inhibitor resulted in promotion of hematopoietic chimerism and renal allograft tolerance despite using only half of the dose of total body irradiation previously required. Selective inhibition of Bcl-2 is therefore a promising approach to induce hematopoietic chimerism without myelosuppression and has the potential to render HSCT more feasible for a variety of clinical indications.

Procurement and Evaluation of Hepatocytes for Transplantation From Neonatal Donors After Circulatory Death.

Hepatocyte transplantation is a promising treatment for liver failure and inborn metabolic liver diseases, but progress has been hampered by a scarcity of available organs. Here, hepatocytes isolated from livers procured for a neonatal hepatocyte donation program within a research setting were assessed for metabolic function and suitability for transplantation. Organ donation was considered for infants who died in neonatal intensive care in the Stockholm region during 2015-2021. Inclusion was assessed when a decision to discontinue life-sustaining treatment had been made and hepatectomy performed after declaration of death. Hepatocyte isolation was performed by three-step collagenase perfusion. Hepatocyte viability, yield, and function were assessed using fresh and cryopreserved cells. Engraftment and maturation of cryopreserved neonatal hepatocytes were assessed by transplantation into an immunodeficient mouse model and analysis of the gene expression of phase I, phase II, and liver-specific enzymes and proteins. Twelve livers were procured. Median warm ischemia time (WIT) was 190 [interquartile range (IQR): 80-210] minutes. Median viability was 86% (IQR: 71%-91%). Median yield was 6.9 (IQR: 3.4-12.8) x106 viable hepatocytes/g. Transplantation into immunodeficient mice resulted in good engraftment and maturation of hepatocyte-specific proteins and enzymes. A neonatal organ donation program including preterm born infants was found to be feasible. Hepatocytes isolated from neonatal donors had good viability, function, and engraftment despite prolonged WIT. Therefore, neonatal livers should be considered as a donor source for clinical hepatocyte transplantation, even in cases with extended WIT.

Effects of a Domino Liver Transplantation Program on Patient Survival and Waiting List Time: A Single-Center Retrospective Study.

Explanted livers from patients with familial amyloid polyneuropathy have often been used for domino liver transplantation (DLT). This has expanded the organ pool for liver transplantation. We evaluated the effects of a single-center DLT program on waiting list duration and patient survival. Liver transplants conducted from 2007 to 2017 were analyzed. Selected patients, all liver transplant candidates above the age of 60 years and patients with hepatocellular carcinoma, were offered DLT. Survival, time on waiting list, and operative factors were evaluated. The study group included 485 patients transplanted with grafts from deceased donors (conventional liver transplantation) and 149 patients who were offered and accepted a potential DLT, of whom 34 underwent DLT and 115 did not; these patients received a deceased donor graft (non-DLT). Five-year and overall estimated survival rates respectively were 79% and 54.4% for DLT and 67.6% and 46.7% for non-DLT (P = .67, log rank test). No differences were noted in survival (P = .816) or waiting times (P = 1.0) between DLT and non-DLT groups. As expected, survival time in the conventional liver transplantation group was longer (84.7% and 60.6%, P < .001). Donor age and ischemia time were significantly different between DLT and non-DLT (P < .001). DLT has enabled 6% additional transplantations without affecting waiting time or survival (34/600).

Transfer of Hemolysis, Elevated Liver Enzymes, and Low Platelets Syndrome by a Liver Graft From a Pregnant Female Donor to a Male Recipient: A Case Report.

Eclampsia with hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome is a rare complication of pregnancy. HELLP syndrome may occur up to a week postpartum in women with eclampsia. CASE REPORT: We report a case of liver transplantation with the organ procured from a pregnant (gestation week 28) female donor who suffered brain death after cerebellar hemorrhage owing to eclampsia. Liver function tests were normal at the time of liver procurement. The liver graft was matched to a 62-year-old man with primary sclerosing cholangitis. On day 7 after an uneventful transplantation, the recipient presented with increased aminotransferases and severe thrombocytopenia. The recipient also developed hypertension and hyperthermia and a clinical picture of HELLP syndrome. The patient underwent emergency liver retransplantation on day 12 after the first transplantation. Intraoperatively, massive necrosis of the liver graft with diffuse subcapsular hematomas was seen. CONCLUSIONS: It appears that in our case, HELLP syndrome was transferred to and occurred in a male recipient. Eclampsia in the donor without overt HELLP syndrome may persist and be transferred by liver graft, developing into HELLP syndrome even in a male recipient. Therefore, liver grafts from female donors with eclampsia should be used with caution. Emergency retransplantation may be necessary.

Blood Group Antigen Expression in Isolated Human Liver Cells in Preparation for Implementing Clinical ABO-Incompatible Hepatocyte Transplantation.

BACKGROUND: ABO blood group antigens in the liver are expressed mainly on endothelial cells or biliary epithelial cells but not on hepatocytes. This suggests that ABO-incompatible hepatocyte transplantation (ABOi-HTx) is theoretically feasible. However, the effects of stress on ABO blood group antigen expression caused by isolation and intraportal infusion require thorough investigation before ABOi-HTx can be implemented in clinical settings. METHODS: Human hepatocytes were isolated from liver tissue obtained from liver resection or deceased donor livers. The expression of blood group antigens on cryopreserved human liver tissues and isolated hepatocyte smear specimens were examined by immunofluorescent staining. The effect of proinflammatory cytokines on blood group antigen expression of hepatocytes was evaluated by flow cytometry. Instant blood-mediated inflammatory reaction after hepatocyte incubation with ABO-incompatible whole blood was examined using the tubing loop model. RESULTS: Blood group antigens were mainly expressed on vessels in the portal area. In hepatocyte smear specimens, isolated hepatocytes did not express blood group antigens. In contrast, a subset of cells in the smear specimens of nonparenchymal liver cells stained positive. In the flow cytometry analysis, isolated hepatocytes were negative for blood group antigens, even after 4-h incubation with cytokines. Platelet counts and complement activation were not significantly different in ABO-identical versus ABO-incompatible settings in the tubing loop model. CONCLUSION: Our study showed that blood group antigens were not expressed on hepatocytes, even after isolation procedures or subsequent incubation with cytokines. This finding is an important step toward removing the restriction of ABO matching in hepatocyte transplantation. Our results suggest that ABOi-HTx is a feasible therapeutic option, especially in patients who require urgent treatment with freshly isolated hepatocytes, such as those with acute liver failure.

Systemic modified messenger RNA for replacement therapy in alpha 1-antitrypsin deficiency.

Alpha 1-antitrypsin (AAT) deficiency arises from an inherited mutation in the SERPINA1 gene. The disease causes damage in the liver where the majority of the AAT protein is produced. Lack of functioning circulating AAT protein also causes uninhibited elastolytic activity in the lungs leading to AAT deficiency-related emphysema. The only therapy apart from liver transplantation is augmentation with human AAT protein pooled from sera, which is only reserved for patients with advanced lung disease caused by severe AAT deficiency. We tested modified mRNA encoding human AAT in primary human hepatocytes in culture, including hepatocytes from AAT deficient patients. Both expression and functional activity were investigated. Secreted AAT protein increased from 1,14 to 3,43 µg/ml in media from primary human hepatocytes following mRNA treatment as investigated by ELISA and western blot. The translated protein showed activity and protease inhibitory function as measured by elastase activity assay. Also, mRNA formulation in lipid nanoparticles was assessed for systemic delivery in both wild type mice and the NSG-PiZ transgenic mouse model of AAT deficiency. Systemic intravenous delivery of modified mRNA led to hepatic uptake and translation into a functioning protein in mice. These data support the use of systemic mRNA therapy as a potential treatment for AAT deficiency.

Exogenous alpha 1-antitrypsin down-regulates SERPINA1 expression.

The main goal of the therapy with purified human plasma alpha1-antitrypsin (A1AT) is to increase A1AT levels and to prevent lungs from elastolytic activity in patients with PiZZ (Glu342Lys) A1AT deficiency-related emphysema. Potential hepatic gains of this therapy are unknown. Herein, we investigated the effect of A1AT therapy on SERPINA1 (gene encoding A1AT) expression. The expression of SERPINA1 was determined in A1AT or A1AT plus Oncostatin M (OSM) treated primary human hepatocytes isolated from liver tissues from A1AT deficient patients and control liver tissues. In addition, SERPINA1 mRNA was assessed in lung tissues from PiZZ emphysema patients with and without A1AT therapy, and in adherent human peripheral blood mononuclear cells (PBMC) isolated from healthy PiMM donors. In a dose-dependent manner purified A1AT lowered SERPINA1 expression in hepatocytes. This latter effect was more prominent in hepatocytes stimulated with OSM. Although it did not reach statistical significance (P = 0.0539)-analysis of lung tissues showed lower SERPINA1 expression in PiZZ emphysema patients receiving augmentation therapy relative to those without therapy. Finally, exogenously added purified A1AT (1mg/ml) reduced SERPINA1 expression in naïve as well as in lipopolysaccharide (LPS)-stimulated human adherent PBMCs. Exogenous A1AT protein reduces its own endogenous expression. Hence, augmentation with native M-A1AT protein and a parallel reduction in expression of dysfunctional mutant Z-A1AT may be beneficial for PiZZ liver, and this motivates further studies.