Gastric digestion of the sweet-tasting plant protein thaumatin releases bitter peptides that reduce H. pylori induced pro-inflammatory IL-17A release via the TAS2R16 bitter taste receptor.

About half of the world's population is infected with the bacterium Helicobacter pylori. For colonization, the bacterium neutralizes the low gastric pH and recruits immune cells to the stomach. The immune cells secrete cytokines, i.e., the pro-inflammatory IL-17A, which directly or indirectly damage surface epithelial cells. Since (I) dietary proteins are known to be digested into bitter tasting peptides in the gastric lumen, and (II) bitter tasting compounds have been demonstrated to reduce the release of pro-inflammatory cytokines through functional involvement of bitter taste receptors (TAS2Rs), we hypothesized that the sweet-tasting plant protein thaumatin would be cleaved into anti-inflammatory bitter peptides during gastric digestion. Using immortalized human parietal cells (HGT-1 cells), we demonstrated a bitter taste receptor TAS2R16-dependent reduction of a H. pylori-evoked IL-17A release by up to 89.7 ± 21.9% (p ≤ 0.01). Functional involvement of TAS2R16 was demonstrated by the study of specific antagonists and siRNA knock-down experiments.

Gene-edited pigs: a translational model for human food allergy against alpha-Gal and anaphylaxis.

The prevalence of food allergy is rising and is estimated to approach 10%. Red meat allergy is the first known food allergy elicited by immunoglobulin E (IgE) antibodies recognizing a carbohydrate. Due to the loss of function of the alpha-1,3-galactosyltransferase (GGTA1) gene in humans, the disaccharide galactose-α-1,3-galactose (α-Gal) cannot be synthesized and therefore became immunogenic. IgE sensitization is elicited through the skin by repetitive tick bites transmitting α-Gal. The underlying mechanisms regarding innate and adaptive immune cell activation, including the B-cell isotype switch to IgE, are poorly understood, requiring further research and physiologically relevant animal models. Here, we describe a new animal model of red meat allergy using percutaneous α-Gal sensitization of gene-edited GGTA1-deficient pigs. Total and α-Gal-specific IgG, IgG1, IgG2, IgG4, and IgE levels were tracked. Further key factors associated with allergic skin inflammation, type 2 immunity, and allergy development were measured in PBMCs and skin samples. Significant increases in α-Gal-specific IgG1 and IgE levels indicated successful sensitization to the allergen α-Gal. Intracutaneous sensitizations with α-Gal recruited lymphocytes to the skin, including elevated numbers of T helper 2 (Th2) cells. Finally, α-Gal-sensitized pigs not only recognized α-Gal as non-self-antigen following α-Gal exposure through the skin but also developed anaphylaxis upon antigen challenge. Based on the similarities between the porcine and human skin, this new large animal model for α-Gal allergy should help to unveil the consecutive steps of cutaneous sensitization and aid the development of prophylactic and treatment interventions.

The Young Investigator Committee of the International Xenotransplantation Association-Perspective of advancements in the field in 2023.

The 2023 IXA conference, hosted in San Diego, CA, brimmed with excitement against the backdrop of recent innovations in both the pre-clinical and clinical realms with several first-in-human applications of xenotransplantation. The theme, "Pigs are flying," alluded to the adage that xenotransplantation would only become a clinical reality "when pigs fly," suggesting a day that might never come. The event witnessed significant attendance, with 600 participants-the highest in the history of an IXA-IPITA joint congress. Among the attendees were members of the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and corporate sponsors deeply engaged in the field. We summarize the latest topics from the congress, ranging from the pros/cons of decedent models of xenotransplantation and genetic engineering of porcine heart valves, solid organs, and cells for clinical translation and their regulatory and ethical landscape.

IgE and anaphylaxis specific to the carbohydrate alpha-gal depend on IL-4.

BACKGROUND: Alpha-gal (Galα1-3Galß1-4GlcNAc) is a carbohydrate with the potential to elicit fatal allergic reactions to mammalian meat and drugs of mammalian origin. This type of allergy is induced by tick bites, and therapeutic options for this skin-driven food allergy are limited to the avoidance of the allergen and treatment of symptoms. Thus, a better understanding of the immune mechanisms resulting in sensitization through the skin is crucial, especially in the case of a carbohydrate allergen for which underlying immune responses are poorly understood. OBJECTIVE: We aimed to establish a mouse model of alpha-gal allergy for in-depth immunologic analyses. METHODS: Alpha-galactosyltransferase 1-deficient mice devoid of alpha-gal glycosylations were sensitized with the alpha-gal-carrying self-protein mouse serum albumin by repetitive intracutaneous injections in combination with the adjuvant aluminum hydroxide. The role of basophils and IL-4 in sensitization was investigated by antibody-mediated depletion. RESULTS: Alpha-gal-sensitized mice displayed increased levels of alpha-gal-specific IgE and IgG1 and developed systemic anaphylaxis on challenge with both alpha-gal-containing glycoproteins and glycolipids. In accordance with alpha-gal-allergic patients, we detected elevated numbers of basophils at the site of sensitization as well as increased numbers of alpha-gal-specific B cells, germinal center B cells, and B cells of IgE and IgG1 isotypes in skin-draining lymph nodes. By depleting IL-4 during sensitization, we demonstrated for the first time that sensitization and elicitation of allergy to alpha-gal and correspondingly to a carbohydrate allergen is dependent on IL-4. CONCLUSION: These findings establish IL-4 as a potential target to interfere with alpha-gal allergy elicited by tick bites.

How genome editing changed the world of large animal research.

The first genetically modified large animals were developed in 1985 by microinjection to increase the growth of agricultural livestock such as pigs. Since then, it has been a difficult trail due to the lack of genetic tools. Although methods and technologies were developed quickly for the main experimental mammal, the mouse, e.g., efficient pronuclear microinjection, gene targeting in embryonic stem cells, and omics data, most of it was-and in part still is-lacking when it comes to livestock. Over the next few decades, progress in genetic engineering of large animals was driven less by research for agriculture but more for biomedical applications, such as the production of pharmaceutical proteins in the milk of sheep, goats, or cows, xeno-organ transplantation, and modeling human diseases. Available technologies determined if a desired animal model could be realized, and efficiencies were generally low. Presented here is a short review of how genome editing tools, specifically CRISPR/Cas, have impacted the large animal field in recent years. Although there will be a focus on genome engineering of pigs for biomedical applications, the general principles and experimental approaches also apply to other livestock species or applications.

Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans.

BACKGROUND: Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. METHODS: The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection. RESULTS: We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in ß(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. CONCLUSION: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.

TNF ΔARE Pigs: A Translational Crohn's Disease Model.

BACKGROUND AND AIMS: Crohn's disease [CD] is a major subtype of inflammatory bowel diseases [IBD] with increasing incidence and prevalence. Results of studies using available small and large animal models are often poorly translatable to patients, and few CD models show small intestinal pathology. Due to its similarities to humans, the pig has emerged as a highly suitable translational disease model, particularly for testing novel nutritional and technological interventions. Our goal was to develop a physiologically relevant porcine CD model to facilitate translation of findings and interventions towards the clinic. METHODS: We generated pigs bearing a 93-bp deletion of the adenosine-uracil-rich element [ARE] and a constitutive-decay element within the 3' untranslated region of the TNF gene. Comparative analysis of physiological, molecular, histological and microbial characteristics was performed between wild-type, TNFΔARE/+ and TNFΔARE/ΔARE animals. Alterations in the microbiome were compared to the TNFΔARE mouse model and IBD patients. RESULTS: TNF ΔARE pigs recapitulate major characteristics of human CD, including ulcerative transmural ileocolitis, increased abundance of proinflammatory cytokines, immune cell infiltration and dysbiotic microbial communities. 16S rRNA gene amplicon sequencing revealed enrichment in members belonging to Megasphaera, Campylobacter, Desulfovibrio, Alistipes and Lachnoclostridum in faecal or mucosa-associated bacteria compared to wild-type littermates. Principal components analysis clustering with a subset of TNFΔARE/+ mice and human IBD patients suggests microbial similarity based on disease severity. CONCLUSIONS: We demonstrate that the TNFΔARE pig resembles a CD-like ileocolitis pathophenotype recapitulating human disease. The ability to conduct long-term studies and test novel surgical procedures and dietary interventions in a physiologically relevant model will benefit future translational IBD research studies.

Detection of porcine cytomegalovirus, a roseolovirus, in pig ovaries and follicular fluid: implications for somatic cells nuclear transfer, cloning and xenotransplantation.

BACKGROUND: Porcine cytomegalovirus (PCMV) is a porcine roseolovirus (PCMV/PRV) which is widely distributed in pigs. Transmission of PCMV/PRV in preclinical xenotransplantations was shown to significantly reduce the survival time of the pig transplants in non-human primates. PCMV/PRV was also transmitted in the first transplantation of a pig heart into a human patient. To analyze how PCMV/PRV could be introduced into pig breeds, especially considering cloned transgenic pigs, and subsequently spread in breeding facilities, we screened ovaries and derived materials which are used to perform somatic cell nuclear transfer (SCNT). METHODS: DNA was isolated from ovarian tissues, follicular fluids, oocytes with cumulus cells, denuded oocytes and parthenotes. A real-time PCR with PCMV/PRV-specific primers and a probe was performed to detect PCMV/PRV. Furthermore, a Western blot assay using a recombinant fragment of the gB protein of PCMV/PRV was performed to screen for virus-specific antibodies in the follicular fluids. RESULTS: PCMV/PRV was found by real-time PCR in ovarian tissues, in the follicular fluid and in oocytes. In parthenotes the virus could not be detected, most-likely due to the low amount of DNA used. By Western blot assay specific antibodies against PCMV/PRV were found in 19 of 20 analyzed follicular fluids. CONCLUSION: PCMV/PRV was found in ovarian tissues, in the follicular fluids and also in denuded oocytes, indicating that the virus is present in the animals of which the oocytes were taken from. Despite several washing steps of the denuded oocytes, which are subsequently used for microinjection or SCNT, the virus could still be detected. Therefore, the virus could infect oocytes during genetic modifications or stay attached to the surface of the oocytes, potentially infecting SCNT recipient animals.

Bitter Peptides YFYPEL, VAPFPEVF, and YQEPVLGPVRGPFPIIV, Released during Gastric Digestion of Casein, Stimulate Mechanisms of Gastric Acid Secretion via Bitter Taste Receptors TAS2R16 and TAS2R38.

Eating satiating, protein-rich foods is one of the key aspects of modern diet, although a bitter off-taste often limits the application of some proteins and protein hydrolysates, especially in processed foods. Previous studies of our group demonstrated that bitter-tasting food constituents, such as caffeine, stimulate mechanisms of gastric acid secretion as a signal of gastric satiation and a key process of gastric protein digestion via activation of bitter taste receptors (TAS2Rs). Here, we tried to elucidate whether dietary non-bitter-tasting casein is intra-gastrically degraded into bitter peptides that stimulate mechanisms of gastric acid secretion in physiologically achievable concentrations. An in vitro model of gastric digestion was verified by casein-fed pigs, and the peptides resulting from gastric digestion were identified by liquid chromatography-time-of-flight-mass spectrometry. The bitterness of five selected casein-derived peptides was validated by sensory analyses and by an in vitro screening approach based on human gastric parietal cells (HGT-1). For three of these peptides (YFYPEL, VAPFPEVF, and YQEPVLGPVRGPFPIIV), an upregulation of gene expression of TAS2R16 and TAS2R38 was observed. The functional involvement of these TAS2Rs was verified by siRNA knock-down (kd) experiments in HGT-1 cells. This resulted in a reduction of the mean proton secretion promoted by the peptides by up to 86.3 ± 9.9% for TAS2R16kd (p < 0.0001) cells and by up to 62.8 ± 7.0% for TAS2R38kd (p < 0.0001) cells compared with mock-transfected cells.

A novel monoclonal IgG1 antibody specific for Galactose-alpha-1,3-galactose questions alpha-Gal epitope expression by bacteria.

The alpha-Gal epitope (α-Gal) with the determining element galactose-α1,3-galactose can lead to clinically relevant allergic reactions and rejections in xenotransplantation. These immune reactions can develop because humans are devoid of this carbohydrate due to evolutionary loss of the enzyme α1,3-galactosyltransferase (GGTA1). In addition, up to 1% of human IgG antibodies are directed against α-Gal, but the stimulus for the induction of anti-α-Gal antibodies is still unclear. Commensal bacteria have been suggested as a causal factor for this induction as α-Gal binding tools such as lectins were found to stain cultivated bacteria isolated from the intestinal tract. Currently available tools for the detection of the definite α-Gal epitope, however, are cross-reactive, or have limited affinity and, hence, offer restricted possibilities for application. In this study, we describe a novel monoclonal IgG1 antibody (27H8) specific for the α-Gal epitope. The 27H8 antibody was generated by immunization of Ggta1 knockout mice and displays a high affinity towards synthetic and naturally occurring α-Gal in various applications. Using this novel tool, we found that intestinal bacteria reported to be α-Gal positive cannot be stained with 27H8 questioning whether commensal bacteria express the native α-Gal epitope at all.

Xenotransplantation becoming reality.

To bridge the gap between organ demand and supply, xenotransplantation has long been considered as a realistic option for end-stage organ failure. Early this year this promise became reality for David Bennett Sr., the first patient whose own failing heart was replaced with a xeno-pig heart. To get here has been a rollercoaster ride of physiological hurdles seemingly impossible to overcome, technological breakthroughs and ethical and safety concerns. It started in 1984, with Stephanie Fae Beauclair, also known as baby Fae, receiving a baboon heart, which allowed her to survive for another 30 days. For ethical reasons primate work was soon abandoned in favour of the pig. But increased phylogenetic distance also brought with it an increased immunological incompatibility. It has been the development of ever more sophisticated genetic engineering tools, which brought down the physiological barriers, enabled humanisation of porcine organs and helped addressing safety concerns. This renewed the confidence in xenotransplantation, brought new funding opportunities and resulted finally in the first in human trial.

Molecular Genetic Investigation of Digital Melanoma in Dogs.

Canine digital melanoma, in contrast to canine oral melanoma, is still largely unexplored at the molecular genetic level. The aim of this study was to detect mutant genes in digital melanoma. Paraffin-embedded samples from 86 canine digital melanomas were examined for the BRAF V595E variant by digital droplet PCR (ddPCR), and for exon 11 mutations in c-kit. Furthermore, exons 2 and 3 of KRAS and NRAS were analysed by Sanger sequencing. Copy number variations (CNV) of KITLG in genomic DNA were analysed from nine dogs. The BRAF V595E variant was absent and in c-kit, a single nucleotide polymorphism was found in 16/70 tumours (23%). The number of copies of KITLG varied between 4 and 6. KRAS exon 2 codons 12 and 13 were mutated in 22/86 (25.6%) of the melanomas examined. Other mutually exclusive RAS mutations were found within the hotspot loci, i.e., KRAS exon 3 codon 61: 2/55 (3.6%); NRAS exon 2 codons 12 and 13: 2/83 (2.4%); and NRAS exon 3 codon 61: 9/86 (10.5%). However, no correlation could be established between histological malignancy criteria, survival times and the presence of RAS mutations. In summary, canine digital melanoma differs from molecular genetic data of canine oral melanoma and human melanoma, especially regarding the proportion of RAS mutations.

New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as a Promising Cell Source.

Transplantation of xenogenic porcine chondrocytes could represent a future strategy for the treatment of human articular cartilage defects. Major obstacles are humoral and cellular rejection processes triggered by xenogenic epitopes like α-1,3-Gal and Neu5Gc. Besides knockout (KO) of genes responsible for the biosynthesis of respective epitopes (GGTA1 and CMAH), transgenic expression of human complement inhibitors and anti-apoptotic as well as anti-inflammatory factors (CD46, CD55, CD59, TNFAIP3 and HMOX1) could synergistically prevent hyperacute xenograft rejection. Therefore, chondrocytes from different strains of single- or multi-genetically modified pigs were characterized concerning their protection from xenogeneic complement activation. Articular chondrocytes were isolated from the knee joints of WT, GalTKO, GalT/CMAH-KO, human CD59/CD55//CD46/TNFAIP3/HMOX1-transgenic (TG), GalTKO/TG and GalT/CMAHKO/TG pigs. The tissue-specific effectiveness of the genetic modifications was tested on gene, protein and epitope expression level or by functional assays. After exposure to 20% and 40% normal human serum (NHS), deposition of C3b/iC3b/C3c and formation of the terminal complement complex (TCC, C5b-9) was quantified by specific cell ELISAs, and generation of the anaphylatoxin C5a by ELISA. Chondrocyte lysis was analyzed by Trypan Blue Exclusion Assay. In all respective KO variants, the absence of α -1,3-Gal and Neu5Gc epitope was verified by FACS analysis. In chondrocytes derived from TG animals, expression of CD55 and CD59 could be confirmed on gene and protein level, TNFAIP3 on gene expression level as well as by functional assays and CD46 only on gene expression level whereas transgenic HMOX1 expression was not evident. Complement activation in the presence of NHS indicated mainly effective although incomplete protection against C3b/iC3b/C3c deposition, C5a-generation and C5b-9 formation being lowest in single GalTKO. Chondrocyte viability under exposure to NHS was significantly improved even by single GalTKO and completely preserved by all other variants including TG chondrocytes without KO of xenoepitopes.

Advances in single-cell sequencing: insights from organ transplantation.

Single-cell RNA sequencing (scRNA-seq) is a comprehensive technical tool to analyze intracellular and intercellular interaction data by whole transcriptional profile analysis. Here, we describe the application in biomedical research, focusing on the immune system during organ transplantation and rejection. Unlike conventional transcriptome analysis, this method provides a full map of multiple cell populations in one specific tissue and presents a dynamic and transient unbiased method to explore the progression of allograft dysfunction, starting from the stress response to final graft failure. This promising sequencing technology remarkably improves individualized organ rejection treatment by identifying decisive cellular subgroups and cell-specific interactions.

Porcine model elucidates function of p53 isoform in carcinogenesis and reveals novel circTP53 RNA.

Recent years have seen an increasing number of genetically engineered pig models of human diseases including cancer. We previously generated pigs with a modified TP53 allele that carries a Cre-removable transcriptional stop signal in intron 1, and an oncogenic mutation TP53R167H (orthologous to human TP53R175H) in exon 5. Pigs with the unrecombined mutant allele (flTP53R167H) develop mainly osteosarcoma but also nephroblastomas and lymphomas. This observation suggested that TP53 gene dysfunction is itself the key initiator of bone tumorigenesis, but raises the question which aspects of the TP53 regulation lead to the development of such a narrow tumour spectrum. Molecular analysis of p53 revealed the presence of two internal TP53 promoters (Pint and P2) equivalent to those found in human. Consequently, both pig and human express TP53 isoforms. Data presented here strongly suggest that P2-driven expression of the mutant R167H-Δ152p53 isoform (equivalent to the human R175H-Δ160p53 isoform) and its circular counterpart circTP53 determine the tumour spectrum and play a critical role in the malignant transformation in flTP53R167H pigs. The detection of Δ152p53 isoform mRNA in serum is indicative of tumorigenesis. Furthermore, we showed a tissue-specific p53-dependent deregulation of the p63 and p73 isoforms in these tumours. This study highlights important species-specific differences in the transcriptional regulation of TP53. Considering the similarities of TP53 regulation between pig and human, these observations provide useful pointers for further investigation into isoform function including the novel circTP53 in both the pig model and human patients.

Effect of Dietary Sodium Modulation on Pig Adrenal Steroidogenesis and Transcriptome Profiles.

Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied in rodents despite differences in adrenal physiology with humans. We, therefore, investigated pig adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and zona fasciculata in response to activation of the renin-angiotensin-aldosterone system by dietary sodium restriction. Six-week-old pigs were fed a low- or high-sodium diet for 14 days (3 pigs per group, 0.4 g sodium/kg feed versus 6.8 g sodium/kg). Plasma aldosterone concentrations displayed a 43-fold increase (P=0.011) after 14 days of sodium restriction (day 14 versus day 0). Low dietary sodium caused a 2-fold increase in thickness of the zG (P<0.001) and an almost 3-fold upregulation of CYP11B (P<0.05) compared with high dietary sodium. Strong immunostaining of the KCNJ5 (G protein-activated inward rectifier potassium channel 4), which is frequently mutated in primary aldosteronism, was demonstrated in the zG. mRNA sequencing transcriptome analysis identified significantly altered expression of genes modulated by the renin-angiotensin-aldosterone system in the zG (n=1172) and zona fasciculata (n=280). These genes included many with a known role in the regulation of aldosterone synthesis and adrenal function. The most highly enriched biological pathways in the zG were related to cholesterol biosynthesis, steroid metabolism, cell cycle, and potassium channels. This study provides mechanistic insights into the physiology and pathophysiology of aldosterone production in a species closely related to humans and shows the suitability of pigs as a translational animal model for human adrenal steroidogenesis.

Cytokine-inducible promoters to drive dynamic transgene expression: The "Smart Graft" strategy.

BACKGROUND: Ubiquitous expression of T-cell regulatory transgenes such as the cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or the high-affinity variant LEA29Y improves xeno graft survival. Such donor pigs are however immunocompromised and susceptible to infection. Continous high expression of CTLA4 or LEA29Y in the graft could also compromise the health status of recipients. The novel "Smart Graft" strategy is likely to avoid these problems by controlling the expression of T-cell regulatory transgenes as and when required. METHODS: Candidate promoters inducible by inflammatory cytokines were identified by in silico screening for potential NF-κB binding sites. Basal promoter levels and responsiveness to TNFα and IL1ß were quantified by expression of secreted embryonic alkaline phosphatase in cultured cells. Promoters were modified to increase responsiveness by removing regulatory elements or adding SP-1 or NF-κB binding sites and again tested in vitro. The most promising promoters were then assessed in vivo. Porcine cells expressing inducible Renilla luciferase constructs were transplanted into immunodeficient NOD-Scid-IL2 receptor gammanull (NSG) mice. Following engraftment, the recipient's immune system was reconstituted by splenocyte transfer raising an immune response to the porcine xenograft. The resulting induction of promoter activity was detected by in vivo bioimaging. RESULTS: Three human (hTNFAIP1, hVCAM1 and hCCL2), and one porcine promoter (pA20) were chosen for in vitro tests. In all experiments, the semi-synthetic and inducible ELAM promoter as well as the CAG promoter were used as references. In contrast to hTNFAIP1 and hVCAM1 the ELAM, hCCL2 and pA20 promoters showed significant induction after cytokine challenge. The hCCL2 and pA20 promoters were further optimized, resulting in increased responsiveness to TNFα and IL1ß. Cytokine-dependent upregulation of promoter activity was tested in vivo, where the ELAM and the optimized hCCL2 promoters showed a 2-fold upregulation, while one of the improved A20 promoters showed almost 10-fold upregulation. Our results also revealed more than 4-fold cytokine inducibility of the CAG promoter. CONCLUSION: This is the first in vivo comparison of existing and newly designed cytokine-inducible promoters. Optimization of promoter structure resulted in almost 10-fold inducibility of promoter activity. Such a rapid and dynamically regulated response to inflammation and cell damage could reduce initial graft rejection, making the "Smart Graft" approach a useful means of modulating the expression of immune regulatory transgenes to avoid deleterious effects on porcine and human health. Expressing transgenes in this fashion could provide a safer organ for transplantation.

Xeno-organ donor pigs with multiple genetic modifications - the more the better?

The number of donated human organs and tissues for patients with terminal organ failure falls far short of the need. Alternative sources, such as organs and tissues from animals, are therefore urgently required. During the past few years, major progress has been made in the development of genetically multi-modified donor pigs, and their organs have been shown to be safe and efficacious in life-supporting transplantation models into non-human primates, paving the way to clinical xenotransplantation studies. Here, we summarize recent developments in pig genome engineering and discuss efforts to develop the optimum donor pig for xenotransplantation. In addition, we speculate on how many genetic modifications may be required for initial xenotransplantation clinical trials.

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

BACKGROUND: Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. METHODS: Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. RESULTS: Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. CONCLUSION: We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.