Generation of cloned sheep lacking galactose-α1,3-galactose and Nglycolylneuraminic acid antigens

Livestock have long been regarded as a potential source of donor organs to alleviate the global organ shortage for transplantation. Sheep have a similar physiology and anatomy to humans, providing the standard model for demonstrating biocompatibility and performance of biological heart valves to obtain regulatory approval for their use in transplantation. Like most mammals, sheep cells contain two well-characterized carbohydrate epitopes, galactose-1,3-galactose (-Gal) and N-glycolylneuraminic acid (Neu5Gc), which are absent in humans. Formation of these xenoantigens is catalysed by two enzymes, namely (1,3) galactosyltransferase (GGTA1) and CMP-Neu5Gc hydroxylase (CMAH), respectively. Towards generating new sheep models, we used Cas9-mediated genome editing in both male and female ovine fetal fibroblasts to knockout (KO) both alleles of CMAH and GGTA1. Selected double KO (DKO) fibroblast strains were used for somatic cell transfer cloning to produce blastocysts that were transferred into gestational surrogate ewes. Following transfer of 128 male and 40 female cloned blastocysts, 6 male and 8 female lambs were born, however, none of the males survived. Molecular analyses of cells from the five surviving ewes confirmed their compound heterozygous state, resulting in a functional DKO phenotype and subsequent immune rejection of embryonic tissues during natural breeding with wild-type rams. This new DKO sheep model better mimics the human immune status, offering greater physiological relevance for preclinical testing of biological heart valves, alleviation of red meat allergy syndrome due to the presence of dietary xenoantigens, and an alternative source of donor organs that may be culturally more widely accepted than pigs.