Cytidine monophosphate-N-acetylneuraminic Acid Hydroxylase Research Articles

Loss of a newly discovered microRNA in Chinese hamster ovary cells leads to upregulation of N-glycolylneuraminic acid sialylation on monoclonal antibodies.

Chinese hamster ovary (CHO) cells are known not to express appreciable levels of the sialic acid residue N‐glycolylneuraminic acid (NGNA) on monoclonal antibodies. However, we actually have identified a recombinant CHO cell line expressing an IgG with unusually high levels of NGNA sialylation (>30%). Comprehensive multi‐OMICs based experimental analyses unraveled the root cause of this atypical sialylation: (1) expression of the cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase (CMAH) gene was spontaneously switched on, (2) CMAH mRNA showed an anti‐correlated expression to the newly discovered Cricetulus griseus (cgr) specific microRNA cgr‐miR‐111 and exhibits two putative miR‐111 binding sites, (3) miR‐111 expression depends on the transcription of its host gene SDK1, and (4) a single point mutation within the promoter region of the sidekick cell adhesion molecule 1 (SDK1) gene generated a binding site for the transcriptional repressor histone H4 transcription factor HINF‐P. The resulting transcriptional repression of SDK1 led to a downregulation of its co‐expressed miR‐111 and hence to a spontaneous upregulation of CMAH expression finally increasing NGNA protein sialylation.

Phenotypic and Genetic Characterization for Incompatible Cross-Match Cases in the Feline AB Blood Group System.

The feline AB blood group system (blood types A, B, and AB) encoding the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene is the most significant in transfusion medicine and hemolysis of the newborn for cats. Blood typing and cross-matching in pre-transfusion testing are crucial to determining blood compatibility and thus prevent hemolytic transfusion reactions. We here performed serological and genetic investigations to characterize blood samples from cats with discordant results for card agglutination (CARD) and the alloantibody agglutination test for blood typing in two cats (subjects K and R). Subject K showed incompatible cross-matching in pre-transfusion testing. Red blood cells from subjects K and R determined blood type B from the CARD method showed blood type AB by alloanti-A and alloanti-B antibodies in agglutination testing. Genomic DNA sequencing of the coding region (exons 1a to 14) for the cat CMAH gene showed that subject K had four mutations with heterozygosity at c.139C>T, c.179G>T, c.327A>C, and c.364C>T. Similarly, the CMAH gene of subject R carried six mutations with heterozygosity at c.142G>A, c.187A>G, c.268T>A, c.327A>C, c.773G>A and c.1603G>A, representing a new diplotype including a novel synonymous single nucleotide polymorphism (SNP) in exon 7 (c.773 G>A: Arg258Gln). The CMAH diplotype in subjects K and R was different from major diplotype in blood type B cats. This study is the first to report CMAH variants in cats with discordant blood types between CARD and TUBE methods. These results could assist in the classification of feline AB blood types for transfusion medicine to avoid blood incompatibilities.

Human immune reactivity of GGTA1/CMAH/A3GALT2 triple knockout Yucatan miniature pigs

In this study, we investigated the effect of a triple knockout of the genes alpha-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and alpha 1,3-galactosyltransferase 2 (A3GALT2) in Yucatan miniature pigs on human immune reactivity. We used the CRISPR/Cas9 system to create pigs lacking GGTA1 (GTKO) and GGTA1/CMAH/A3GALT2 triple gene knockout (TKO). The expression of all three xenoantigens was absent in TKO pigs, but there was no additional reduction in the level of Galα1,3Gal (αGal) epitopes expression in the A3GALT2 gene KO. Peripheral blood mononuclear cells (PBMCs), aorta endothelial cells (AECs), and cornea endothelial cells (CECs) were isolated from these pigs, and their ability to bind human IgM/IgG and their cytotoxicity in human sera were evaluated. Compared to wild type (WT) pigs, the level of human antibody binding of the PBMCs, AECs, and CECs of the transgenic pigs (GTKO and TKO) was significantly reduced. However, there were significant differences in human antibody binding between GTKO and TKO depending on the cell type. Human antibody binding of TKO pigs was less than that of GTKO on PBMCs but was similar between GTKO and TKO pigs for AECs and CECs. Cytotoxicity of transgenic pig (GTKO and TKO) PBMCs and AECs was significantly reduced compared to that of WT pigs. However, TKO pigs showed a reduction in cytotoxicity compared to GTKO pigs on PBMCs, whereas in AECs from both TKO and GTKO pigs, there was no difference. The cytotoxicity of transgenic pig CECs was significantly decreased from that of WT at 300 min, but there was no significant reduction in TKO pigs from GTKO. Our results indicate that genetic modification of donor pigs for xenotransplantation should be tailored to the target organ and silencing of additional genes such as CMAH or A3GALT2 based on GTKO might not be essential in Yucatan miniature pigs.

N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus.

The first step in influenza virus infection is the binding of hemagglutinin to sialic acid-containing glycans present on the cell surface. Over 50 different sialic acid modifications are known, of which N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two main species. Animal models with α2,6 linked Neu5Ac in the upper respiratory tract, similar to humans, are preferred to enable and mimic infection with unadapted human influenza A viruses. Animal models that are currently most often used to study human influenza are mice and ferrets. Additionally, guinea pigs, cotton rats, Syrian hamsters, tree shrews, domestic swine, and non-human primates (macaques and marmosets) are discussed. The presence of NeuGc and the distribution of sialic acid linkages in the most commonly used models is summarized and experimentally determined. We also evaluated the role of Neu5Gc in infection using Neu5Gc binding viruses and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH)−/− knockout mice, which lack Neu5Gc and concluded that Neu5Gc is unlikely to be a decoy receptor. This article provides a base for choosing an appropriate animal model. Although mice are one of the most favored models, they are hardly naturally susceptible to infection with human influenza viruses, possibly because they express mainly α2,3 linked sialic acids with both Neu5Ac and Neu5Gc modifications. We suggest using ferrets, which resemble humans closely in the sialic acid content, both in the linkages and the lack of Neu5Gc, lung organization, susceptibility, and disease pathogenesis.

Knockout of cytidine monophosphate-N-acetylneuraminic acid hydroxylase in Chinese hamster ovary cells by CRISPR/Cas9-based gene-editing technology

Chinese hamster ovary (CHO) cells are mainly used mammalian host to produce therapeutic recombinant glycoproteins which are close to the human-like glycosylation of glycoproteins. However, cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) in CHO cells can produce N-glycolylneuraminic acids (Neu5Gc) which elicits the immune response in the human body. Therefore, it is vital to silence the expression of CMAH in CHO cells using gene-editing technology. CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9-based gene editing system was performed to knock out the expression of CMAH in CHO cells. Plasmids containing Cas9, single guide (sg) RNA and reporter sequences of CMAH gene were constructed and transformed into HEK293 T cells to screen the sgRNA with high cleavage efficiency. Then the plasmids were co-transfected into the CHO cells. Eight CHO monoclonal cells were obtained by flow cytometry sorting and expanded. Base deletion mutations were identified by PCR and Sanger sequencing. qRT-PCR and MTT assay were confirmed the CMAH gene mutation in the CHO cells. Moreover, the recombinant human erythropoietin (rEPO) protein was also expressed in the mutant CHO cells, and the N-Glycosylation of rEPO showed that G0F, G1F-GN increased and G0F-GN, G0, Man5 were decreased. CRISPR/Cas9 system is a very convenient system for editing CMAH gene in CHO cells. The mutant CHO cells can be used as host to express the recombinant glycoprotein.

Molecular characterization of blood type A, B, and C (AB) in domestic cats and a CMAH genotyping scheme.

In domestic cats, the AB blood group system consists of the three types A, B, and C (usually called AB), which vary in frequency among breeds and geographic regions. Mismatches cause acute hemolytic transfusion reactions and hemolysis of the newborn due to the presence of naturally occurring anti-A alloantibodies. Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) converts N-acetylneuraminic acid (type B) to N-glycolylneuraminic acid (type A), and type C erythrocytes express both antigens. We examined the feline CMAH coding regions and genotyped cats to characterize type A, B, and C animals. Of 421 phenotypically typed cats, 60% were A, 35% B and 5% C. Among the 70 cats for which the CMAH coding region was sequenced, 13 new variants were identified in addition to 16 of the previously reported 18 variants. The CMAH variant c.268T>A is seen in type B cats of most breeds, and the variant c.179G>T results in type B in Turkish breeds. The variants c.1322delT and c.933delA cause frameshifts with early stop codons and thereby type B in some Ragdolls and domestic shorthair cats, respectively. Protein modeling with PROVEAN affirmed their deleterious effects. No type A and C cats had more than one allele with one of the above variants. Variant analysis of three SNVs (c.142G>A, c.268T>A and Δ-53) and blood typing of an additional 351 typed cats showed complete phenotype-genotype concordance. In conclusion, the three CMAH variants c.179G>T, c.268T>A and c.1322delT are the main reasons for the defective NeuGc synthesis causing blood type B in domestic purebred and non-pedigreed cats. Together with the variant c.364C>T for type C in Ragdolls they offer a molecular screening scheme for clinical diagnostics to assure blood type compatibility.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.

As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.

Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs.

Antipig antibodies are a barrier to clinical xenotransplantation. We evaluated antibody binding of waitlisted renal transplant patients to 3 glycan knockout (KO) pig cells and class I swine leukocyte antigens (SLA). Peripheral blood mononuclear cells from SLA identical wild type (WT), α1, 3-galactosyltransferase (GGTA1) KO, GGTA1/ cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) KO, and GGTA1/ CMAH /b1,4 N-acetylgalactosaminyl transferase (B4GalNT2) KO pigs were screened for human antibody binding using flow cytometric crossmatch (FCXM). Sera from 820 patients were screened on GGTA1/CMAH/B4GalNT2 KO cells and a subset with elevated binding was evaluated further. FCXM was performed on SLA intact cells and GGTA1/SLA class I KO cells after depletion with WT pig RBCs to remove cell surface reactive antibodies, but leave SLA antibodies. Lastly, human and pig reactive antibodies were eluted and tested for cross-species binding and reactivity to single-antigen HLA beads. Sequential glycan KO modifications significantly reduce antibody binding of waitlisted patients. Sera exhibiting elevated binding without reduction after depletion with WT RBCs demonstrate reduced binding to SLA class I KO cells. Human IgG, eluted from human and pig peripheral blood mononuclear cells, interacted across species and bound single-antigen HLA beads in common epitope-restricted patterns. Many waitlisted patients have minimal xenoreactive antibody binding to the triple KO pig, but some HLA antibodies in sensitized patients cross-react with class I SLA. SLA class I is a target for genome editing in xenotransplantation.

Molecular Characterization of the Cytidine Monophosphate-N-Acetylneuraminic Acid Hydroxylase (CMAH) Gene Associated with the Feline AB Blood Group System.

Cat’s AB blood group system (blood types A, B, and AB) is of major importance in feline transfusion medicine. Type A and type B antigens are Neu5Gc and Neu5Ac, respectively, and the enzyme CMAH participating in the synthesis of Neu5Gc from Neu5Ac is associated with this cat blood group system. Rare type AB erythrocytes express both Neu5Gc and Neu5Ac. Cat serum contains naturally occurring antibodies against antigens occurring in the other blood types. To understand the molecular genetic basis of this blood group system, we investigated the distribution of AB blood group antigens, CMAH gene structure, mutation, diplotypes, and haplotypes of the cat CMAH genes. Blood-typing revealed that 734 of the cats analyzed type A (95.1%), 38 cats were type B (4.9%), and none were type AB. A family of three Ragdoll cats including two type AB cats and one type A was also used in this study. CMAH sequence analyses showed that the CMAH protein was generated from two mRNA isoforms differing in exon 1. Analyses of the nucleotide sequences of the 16 exons including the coding region of CMAH examined in the 34 type B cats and in the family of type AB cats carried the CMAH variants, and revealed multiple novel diplotypes comprising several polymorphisms. Haplotype inference, which was focused on non-synonymous SNPs revealed that eight haplotypes carried one to four mutations in CMAH, and all cats with type B (n = 34) and AB (n = 2) blood carried two alleles derived from the mutated CMAH gene. These results suggested that double haploids selected from multiple recessive alleles in the cat CMAH loci were highly associated with the expression of the Neu5Ac on erythrocyte membrane in types B and AB of the feline AB blood group system.

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

Streptococcus pneumoniae Senses a Human-like Sialic Acid Profile via the Response Regulator CiaR.

Streptococcus pneumoniae is a human-adapted pathogen that encounters terminally sialylated glycoconjugates and free sialic acid (Sia) in the airways. Upon scavenging by the bacterial sialidase NanA, Sias serve as carbon sources for the bacteria. Unlikemost animals in which cytidine-monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) converts Sia N-acetylneuraminic acid (Neu5Ac) into N-glycolylneuraminic acid (Neu5Gc), humans have an inactive CMAH, causing an absence of Neu5Gc and excess Neu5Ac. We find that pneumococcal challenge in Cmah(-/-) mice leads to heightened bacterial loads, virulence, and NanA expression. Invitro, NanA is upregulated in response to Neu5Ac compared with Neu5Gc, a process controlled by the two-component response regulator CiaR and requiring Sia uptake by the transporter SatABC. Additionally, compared with Neu5Gc, Neu5Ac increases pneumococcal resistance to antimicrobial reactive oxygen species in aCiaR-dependent manner. Thus, S.pneumoniae senses and responds to Neu5Ac, leading to CiaR activation and increased virulence and potentially explaining the greater susceptibility in humans.

Modifying the sugar icing on the transplantation cake.

As a transplant surgeon, my interest in glycobiology began through my research into ABO-incompatible allotransplantation, and grew when my goal became overcoming the shortage of organs from deceased human donors by the transplantation of pig organs into patients with terminal organ failure (xenotransplantation/cross-species transplantation). The major target for human "natural" (preformed) anti-pig antibodies is galactose-α(1,3)-galactose (the "Gal" epitope), which is expressed on many pig cells, including the vascular endothelium. The binding of human IgM and IgG antibodies to Gal antigens initiates the process of hyperacute rejection, resulting in destruction of the pig graft within minutes or hours. This major barrier has been overcome by the production of pigs in which the gene for the enzyme α(1,3)-galactosyltransferase (GT) has been deleted by genetic engineering, resulting in GT knockout (GTKO) pigs. The two other known carbohydrate antigenic targets on pig cells for human anti-pig antibodies are (i) the product of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene, i.e., N-glycolylneuraminic acid, and (ii) the product of the β1,4 N-acetylgalactosaminyltransferase gene, i.e., the Sd(a) antigen. Expression of these two has also been deleted in pigs. These genetic manipulations, together with others directed to overcoming primate complement and coagulation activation (the latter of which also relates to glycobiology) have contributed to the prolongation of pig graft survival in nonhuman primate recipients to many months rather than a few minutes. Clinical trials of the transplantation of pig cells are already underway and transplantation of pig organs may be expected within the relatively near future.

21 GENERATION OF GGTA1 AND CMAH BIALLELIC KNOCKOUT PORCINE FIBROBLAST CELL LINES BY TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE AND CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9

The use of porcine organs for pig-to-human transplantation is considered the most promising solution to overcome the growing shortage of human grafts for all transplantation. Unfortunately, human antibodies recognise Gala-1, 3-gal (Gal), and N-glycolylneuraminic acid (Neu5Gc) epitopes on the surface of pig cells, causing hyperacute rejection. Transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are 2 powerful tools for producing gene knockouts with high efficiency. Here we generate α-1, 3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene knockout porcine fibroblasts cell lines with TALEN and CRISPR/Cas9. Porcine fibroblasts were treated with TALEN and CRISPR/Cas9 designed against the region coding for the catalytic core of GGTA1 and CMAH. A magnetic separation of these cells was used in somatic cell NT. The cloned fetuses and piglets were characterised by T7E1, fluorescent PCR, and DNA sequencing. Nineteen fetuses were harvested using TALEN; 4 of them were GGTA1 gene mutated, and 11 were CMAH gene mutated. The DNA sequence analysis confirmed 5 of 11 are biallelic disruptions of the CMAH gene. For the CRISPR/Cas9, 6 piglets were obtained, and DNA sequence analysis confirmed 1 of 6 is biallelic disruptions of the GGTA1 and 2 of 6 are mono-allelic disruptions of the CMAH gene. We produced several fetuses or piglets via gene editing technology (TALEN and CRISPR/Cas9) and somatic cell NT technology. Furthermore, GGTA1 and CMAH gene knockout porcine fetal fibroblast cell lines derived from the fetus individuals were successfully established. However, expression of α-Gal and Neu5Gc epitopes is needed to be further examined by flow cytometry and confocal microscopy. Our study will provide the possibility of making GGTA1 and CMAH knockout pigs in the future.

Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors.

Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). Neu5Gc is synthesized from Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid—α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains.

Parallel evolution of a self-signal: humans and new world monkeys independently lost the cell surface sugar Neu5Gc

Human sialic acid biology is unusual and thought to be unique among mammals. Humans lack a functional cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) protein and cannot synthesize the sugar Neu5Gc, an innate mammalian signal of self. Losing this sugar changed how humans interact with some of our deadliest pathogens: malaria, influenza, and streptococcus among others. We show that the New World monkeys, comprising the third of all primate species, have human-like sialic acid biology. They have lost Neu5Gc because of an independent CMAH inactivation ~30 millionyears ago (mya) (compared to ~3mya in hominids). This parallel loss of Neu5Gc opens sialic acid biology to comparative phylogenetic analysis and reveals an unexpected conservation priority. New World monkeys risk infection by human pathogens that can recognize cells in the absence of Neu5Gc. This striking molecular convergence provides a mechanism that could explain the long-standing observation that New World monkeys are susceptible to some human diseases that cannot be transmitted to other primates.

Double knockout pigs deficient in N‐glycolylneuraminic acid and Galactose α‐1,3‐Galactose reduce the humoral barrier to xenotransplantation

Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα-1,3-Gal (Gal) and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. We report the homozygous disruption of the α1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. This technology will accelerate the development of pigs for xenotransplantation research.