Neu5Gc and α1-3 GAL Xenoantigen Knockout Does Not Affect Glycemia Homeostasis and Insulin Secretion in Pigs

Abstract

Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited antibodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and -glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc mice exhibit glycemic dysregulations and pancreatic β-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate--acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities, and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies.

In vitro characterization of neonatal, juvenile, and adult porcine islet oxygen demand, β‐cell function, and transcriptomes

“…69 CMAH and GGTA1 double knockout pigs do not appear to have altered insulin secretion. 70 Consistent with previous studies, our data indicate a higher number of GGTA1 transcripts in NPI versus API, but also a higher number of CMAH transcripts. 29 However, VWF has significantly higher expression in API and has been linked with graft dysfunction due to platelet activation.…”

Evolution of sialic acids: Implications in xenotransplant biology

“…104,136,142,145 Currently, it seems that both the αGal and Neu5Gc-glycans limit the potential of xenotransplantation, as recently reviewed elsewhere, 103,104,[146][147][148] and generation of double-knockout animals deficient in both the CMAH and GGTA1 genes seems to be promising. 139,147,[149][150][151]…”

Gene Editing, Gene Therapy, and Cell Xenotransplantation: Cell Transplantation Across Species

“…More recently, double knockout of GGTA1 and of CMAH (cytidine monophosphate-N-acetylneuraminic hydroxylase) responsible for Neu5Gc (N-glycolylneuraminic acid) synthesis was shown to further decrease porcine islet antigenicity compared to GGTA1 knockout alone [22]. A recent paper by Salama et al showed that such a double knockout in porcine beta cells is possible without affecting islet function both in vivo and in vitro [23]. Another porcine carbohydrate antigen produced by beta-1, 4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2) was identified by screening cDNA from GGTA1…”