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.

Kidney transplantation from triple-knockout pigs expressing multiple human proteins in cynomolgus macaques.

Porcine cells devoid of three major carbohydrate xenoantigens, αGal, Neu5GC, and SDa (TKO) exhibit markedly reduced binding of human natural antibodies. Therefore, it is anticipated that TKO pigs will be better donors for human xenotransplantation. However, previous studies on TKO pigs using old world monkeys (OWMs) have been disappointing because of higher anti-TKO pig antibodies in OWMs than humans. Here, we show that long-term survival of renal xenografts from TKO pigs that express additional human transgenes (hTGs) can be achieved in cynomolgus monkeys. Kidney xenografts from TKO-hTG pigs were transplanted into eight cynomolgus recipients without pre-screening for low anti-pig antibody titers. Two recipients of TKO-hTG xenografts with low expression of human complement regulatory proteins (CRPs) (TKO-A) survived for 2 and 61 days, whereas six recipients of TKO-hTG xenografts with high CRP expression (TKO-B) survived for 15, 20, 71, 135, 265, and 316 days. Prolonged CD4+ T cell depletion and low anti-pig antibody titers, which were previously reported important for long-term survival of αGal knock-out (GTKO) xenografts, were not always required for long-term survival of TKO-hTG renal xenografts. This study indicates that OWMs such as cynomolgus monkeys can be used as a relevant model for clinical application of xenotransplantation using TKO pigs.

Acetyl-coenzyme A carboxylase inhibition reduces de novo lipogenesis in overweight male subjects: A randomized, double-blind, crossover study.

NDI-010976, an allosteric inhibitor of acetyl-coenzyme A carboxylases (ACC) ACC1 and ACC2, reduces hepatic de novo lipogenesis (DNL) and favorably affects steatosis, inflammation, and fibrosis in animal models of fatty liver disease. This study was a randomized, double-blind, placebo-controlled, crossover trial evaluating the pharmacodynamic effects of a single oral dose of NDI-010976 on hepatic DNL in overweight and/or obese but otherwise healthy adult male subjects. Subjects were randomized to receive either NDI-010976 (20, 50, or 200 mg) or matching placebo in period 1, followed by the alternate treatment in period 2; and hepatic lipogenesis was stimulated with oral fructose administration. Fractional DNL was quantified by infusing a stable isotope tracer, [1-13 C]acetate, and monitoring 13 C incorporation into palmitate of circulating very low-density lipoprotein triglyceride. Single-dose administration of NDI-010976 was well tolerated at doses up to and including 200 mg. Fructose administration over a 10-hour period stimulated hepatic fractional DNL an average of 30.9 ± 6.7% (mean ± standard deviation) above fasting DNL values in placebo-treated subjects. Subjects administered single doses of NDI-010976 at 20, 50, or 200 mg had significant inhibition of DNL compared to placebo (mean inhibition relative to placebo was 70%, 85%, and 104%, respectively). An inverse relationship between fractional DNL and NDI-010976 exposure was observed with >90% inhibition of fractional DNL associated with plasma concentrations of NDI-010976 >4 ng/mL. CONCLUSION: ACC inhibition with a single dose of NDI-010976 is well tolerated and results in a profound dose-dependent inhibition of hepatic DNL in overweight adult male subjects. Therefore, NDI-010976 could contribute considerable value to the treatment algorithm of metabolic disorders characterized by dysregulated fatty acid metabolism, including nonalcoholic steatohepatitis. (Hepatology 2017;66:324-334).

Prolonged xenokidney graft survival in sensitized NHP recipients by expression of multiple human transgenes in a triple knockout pig.

Genetic modification of porcine donors, combined with optimized immunosuppression, has been shown to improve outcomes of experimental xenotransplant. However, little is known about outcomes in sensitized recipients, a population that could potentially benefit the most from the clinical implementation of xenotransplantation. Here, five highly allosensitized rhesus macaques received a porcine kidney from GGTA1 (α1,3-galactosyltransferase) knockout pigs expressing the human CD55 transgene (1KO.1TG) and were maintained on an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen. These recipients developed de novo xenoreactive antibodies and experienced xenograft rejection with evidence of thrombotic microangiopathy and antibody-mediated rejection (AMR). In comparison, three highly allosensitized rhesus macaques receiving a kidney from GGTA1, CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase), and b4GNT2/b4GALNT2 (ß-1,4-N-acetyl-galactosaminyltransferase 2) knockout pigs expressing seven human transgenes including human CD46, CD55, CD47, THBD (thrombomodulin), PROCR (protein C receptor), TNFAIP3 (tumor necrosis factor-α-induced protein 3), and HMOX1 (heme oxygenase 1) (3KO.7TG) experienced significantly prolonged graft survival and reduced AMR, associated with dampened post-transplant humoral responses, early monocyte and neutrophil activation, and T cell repopulation. After withdrawal of all immunosuppression, recipients who received kidneys from 3KO.7TG pigs rejected the xenografts via AMR. These data suggest that allosensitized recipients may be suitable candidates for xenografts from genetically modified porcine donors and could benefit from an optimized immunosuppression regimen designed to target the post-transplant humoral response, thereby avoiding AMR.