One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis.

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

Production of Human Endothelial Cells Free from Soluble Xenogeneic Antigens for Bioartificial Small Diameter Vascular Graft Endothelization.

Arterial bypass graft implantation remains the primary therapy for patients with advanced cardiovascular disease, but most lack adequate saphenous vein or other conduits for bypass procedures and would benefit from a bioartificial conduit. This study aimed to produce human endothelial cells (hECs) in large scale, free from xenogeneic antigens, to develop a small diameter, compatible vessel for potential use as a vascular graft. Human adipose-derived stromal cells (hASCs) were isolated, cultured, and differentiated in the presence of human serum and used for the reendothelization of a decellularized rat aorta. hASC derived ECs (hASC-ECs) expressed VEGFR2, vWf and CD31 endothelial cell markers, the latter in higher levels than hASCs and HUVECs, and were shown to be functional. Decellularization protocol yielded aortas devoid of cell nuclei, with preserved structure, including a preserved basement membrane. When seeded with hASC-ECs, the decellularized aorta was completely reendothelized, and the hASC-ECs maintained their phenotype in this new condition. hASCs can be differentiated into functional hECs without the use of animal supplements and are capable of reendothelizing a decellularized rat aorta while maintaining their phenotype. The preservation of the basement membrane following decellularization supported the complete reendothelization of the scaffold with no cell migration towards other layers. This approach is potentially useful for rapid obtention of compatible, xenogeneic-free conduit.

Different sites of xenoantigen delivery lead to a virally induced late-onset hepatitis in mice through molecular mimicry.

BACKGROUND: Epidemiological and laboratory evidences led to the hypothesis that molecular mimicry between viruses and self-proteins could be linked to the onset of autoimmune hepatitis (AIH). Hepatotropic viruses could be good candidates, as a pro-inflammatory environment may facilitate the development of AIH. AIMS: The aims of this study were to test a virus ability to induce an AIH through molecular mimicry and the influence of hepatic inflammation in this process. METHODS: C57BL/6 mice were injected i.v. or i.m. with recombinant adenoviral vectors (RecAdV) encoding for human type 2 AIH antigens to target xenoantigens expression in the liver and to create a transient hepatitis (i.v.) or for 'peripheral' xenoantigens expression (i.m.). Liver injury and B-cell response were evaluated. RESULTS: Late-onset hepatitis was observed 8 months after i.v. or i.m. RecAdV injections, despite presence or absence of an initial transient hepatitis. Intensity of B-cell response was similar for both type of injections, but the Ig isotypes produced were different. B-cell autoimmune response spread to several liver proteins. CONCLUSIONS: Liver autoimmune response can be initiated using molecular mimicry over a long period of time, validating the hit-and-run hypothesis. Initial liver inflammatory injury is neither necessary, nor detrimental to the development of AIH. These results highlight the significance of initial events on the pathogenesis of autoimmune liver injury.

Cardiac xenotransplantation technology provides materials for improved bioprosthetic heart valves.

OBJECTIVES: Human subjects and Old World primates have high levels of antibody to galactose-α-1,3 galactose ß-1,4-N-acetylglucosamine (α-Gal). Commercially available bioprosthetic heart valves of porcine and bovine origin retain the Gal antigen despite current processing techniques. Gal-deficient pigs eliminate this xenoantigen. This study tests whether binding of human anti-Gal antibody effects calcification of wild-type and Gal-deficient glutaraldehyde-fixed porcine pericardium by using a standard subcutaneous implant model. METHODS: Expression of α-Gal was characterized by lectin Griffonia simplicifolia-IB4 staining. Glutaraldehyde-fixed pericardial disks from Gal-positive and Gal-deficient pigs were implanted into 12-day-old Wistar rats and 1.5-kg rabbits with and without prelabeling with affinity-purified human anti-Gal antibody. Calcification of the implants was determined after 3 weeks by using inductively coupled plasma spectroscopy. RESULTS: The α-Gal antigen was detected in wild-type but not Gal-deficient porcine pericardium. Wild-type disks prelabeled with human anti-Gal antibody exhibited significantly greater calcification compared with that seen in antibody-free wild-type samples (mean ± standard error of the mean: 111 ± 8.4 and 74 ± 9.6 mg/g, respectively; P = .01). In the presence of anti-Gal antibody, a significantly greater level of calcification was detected in wild-type compared with GTKO porcine pericardium (111 ± 8.4 and 55 ± 11.8 mg/g, respectively; P = .005). Calcification of Gal-deficient pericardium was not affected by the presence of anti-Gal antibody (51 ± 9.1 and 55 ± 11.8 mg/g). CONCLUSIONS: In this model anti-Gal antibody accelerates calcification of wild-type but not Gal-deficient glutaraldehyde-fixed pericardium. This study suggests that preformed anti-Gal antibody present in all patients might contribute to calcification of currently used bioprosthetic heart valves. Gal-deficient pigs might become the preferred source for new, potentially calcium-resistant bioprosthetic heart valves.

Human monocytes recognize porcine endothelium via the interaction of galectin 3 and alpha-GAL.

Monocytes are one of the key inflammatory cells recruited to xenografts and play an important role in delayed xenograft rejection. Previous studies have demonstrated the ability of monocytes to bind to the major xenoantigen Gal-alpha(1,3)Gal-beta(1,4)GlcNAc-R; however, the receptor that mediates this interaction has yet to be identified. We provide evidence that it is Galectin-3, a approximately 30-kDa lectin that recognizes beta-galactosides (Gal-beta(1-3/4)GlcNAc) and plays diverse roles in many physiological and pathological events. Human monocyte binding is strikingly increased on porcine aortic endothelial cells (PAEC), which express high levels of Gal-alpha(1,3)Gal-beta(1,4)GlcNAc-R, compared with human aortic endothelial cells. Human monocytes obtained from healthy donors bind to Gal-alpha(1,3)Gal-beta(1,4)GlcNAc-R at variable intensities. This variation of binding intensity was consistent and reproducible in individual donors. Galectin-3 is mainly expressed in human monocytes, not lymphocytes. Purified Galectin-3 is able to bind directly to Gal-alpha(1,3)Gal-beta(1,4)GlcNAc-R. Galectin-3 can also be affinity isolated from monocytes (and not lymphocytes) using an Gal-alpha(1,3)Gal-beta(1,4)GlcNAc-R-biotin/streptavidin-bead pull-down system. Soluble Galectin-3 binds preferentially to PAEC vs human aortic endothelial cells, and this binding can be inhibited by lactose, indicating dependence on the carbohydrate recognition domain of Galectin-3. Gal-alpha(1,3)Gal-beta(1,4)GlcNAc-R is at least partly responsible for this phenomenon, as binding decreased after digestion of PAEC with alpha-galactosidase. Furthermore, monocytes pretreated with a blocking anti-Galectin-3 Ab show decreased adhesion to PAEC when compared with isotype control in a parallel plate flow chamber perfusion assay. Thus, we conclude that Galectin-3 expressed in human monocytes is a receptor for the major xenoantigen (Gal-alpha(1,3)Gal-beta(1,4)GlcNAc-R), expressed on porcine endothelial cells.

[Effect of narrow fractions of chromatin non-histone proteins on the expression of membrane tumor-associated antigen MA-50 and phosphorylation of proteins of cultured rat hepatocytes]. / Vliianie uzkikh fraktsii negistonovykh belkov khromatina na ékspressiiu membrannogo opukholevoassotsiirovannogo antigena MA-50 i fosfolirovanie belkov kul'tiviruemykh gepatotsitov krysy.

As earlier reported, the main component of narrow fractions of chromosomal non-histone proteins (NHP) of kidney and of Zaidel hepatoma cells has its own protein kinase activity, and is identified as a heteroorgan NHP-antigen, which is intrinsic to the definite renal tissue and absent in the liver. Effects of narrow fractions of kidney and Zaidel hepatoma NHP on biosynthetic processes and sizes of hepatocytes were studied in vitro. It has been shown that as a result of a 5 h incubation of rat hepatocytes with a narrow fraction of renal NHP the proportion of small hepatocytes increases approximately by 12% as compared with that of cells cultivated without NHP. Besides, binding of organ-specific anti-kidney immune serum with a small hepatocyte population rises by more than 20%, which results from the expression of tumor-associated heteroorgan kidney-specific antigen on the hepatocyte surface. According to immunoprecipitation and subsequent electrophoresis, the molecular mass of a membrane heteroorgan antigen on the surface of hepatocytes amounts approximately to 65 kDa, and an active phosphorylation of cellular proteins takes place. The same effect on hepatocytes is produced by a narrow NHP fraction of chromatin of Zaidel hepatoma cells, whereas no phosphorylation is observed in the presence of liver NHP as well as in the absence of NHP. It is suggested that the heteroorgan NHP-antigen induces biosynthetic processes including synthesis of membrane tumorassociated antigen on the surface of hepatocytes cultivated in vitro by activation of cellular protein phosphorylation, which can lead to changes in size of cultivated cells.

Peach profilin: cloning, heterologous expression and cross-reactivity with Bet v 2.

BACKGROUND: Peach is among the main foods causing allergic reactions in the Mediterranean adult population. Only a single peach allergen, named Pru p 3, has been characterized. However, a potential role of profilin has also been suggested in grass pollen-associated allergy to peach. METHODS: Complementary DNA clones for two different peach profilin isoforms were obtained by reverse transcriptase polymerase chain reaction using non-degenerated primers. Expression of recombinant peach profilin was performed in Escherichia coli, and confirmed using rabbit polyclonal antibodies to sunflower pollen profilin. Twenty-nine individual sera from patients with peach allergy proved by double-blind, placebo-controlled food challenges (DBPCFC), either with (n = 15) or without (n = 14) specific IgE to Bet v 2, were used in immunodetection assays to test recombinant peach profilin reactivity. RESULTS: Each peach profilin cDNA included an open reading frame coding for a 131 amino acid protein. The peach profilin isoforms, designated Pru p 4.01 and Pru p 4.02, showed 80% of amino acid sequence identity, and were very similar (>70% identity) to allergenic profilins from plant foods and pollens. Recombinant Pru p 4.01 was expressed in E. coli as a nonfusion protein, displaying the expected molecular size and reacting with anti-profilin antibodies. rPru p 4.01 was recognized by all sera (15 of 15) with specific IgE to Bet v 2, whereas no sera (zero of 14) without IgE to this birch allergen reacted with rPru p 4.01. CONCLUSIONS: Peach profilin Pru p 4 is very closed to other allergenic profilins from plant foods and pollens. A complete correlation between reactivity to rPru p 4 and rBet v 2 has been found in sera from peach allergic patients.

[Antigen expression and the biomechanical characteristics of the biologic blood vessel matrix].

To explore the changes of the antigen expression and the biomechanical characteristics of blood vessel in Banna little ear pig before and after trypsin treatment, and provide data for xenotransplantation and pig vessel using for tissue engineering. Geometric morphology and microstructure of pig cartoid artery were stuided quantitatively by histologic method and computer image analysis. The relationship between pressure and diameter was observed at different period of time before and after trypsin treatment. Affinity-immunohistochemistry assay was conducted to detect the expression of xenoantigens (alpha-Gal). The results showed that alpha-Gal antigen is only expressed in vascular endothelial cellsouly. There is no significant difference in blood vessel compliance. These demonstrate that the antigenicity of pig carotid artery is significantly reduced, however, the mechanical characteristics did not change significantly. We suppose that pig vessels treated by trypsin can be used as the substrate material for vascular tissue engineering.

[Expression and distribution of xenoantigen alpha-Gal in intervertebral disk of Chinese banna minipig inbred line].

OBJECTIVE: To investigate the expression and distribution of xenoantigen in intervertebral disk of Chinese banna minipig inbred line, and to study the availability of xenograft transplantation of intervertebral disk. METHODS: Samples of intervertebral disk were collected from six Banna pigs of 8 to 11-month-old. The fixation, embedment and slice were performed. alpha-Gal specific binding lection (BSI-B4) were used as affinity reagents and affinity-immunohistochemistry assays (SABC methods and DAB stain) were conducted to detect the expression and distribution of xenoantigen (alpha-Gal). RESULTS: alpha-Gal was found in chondrocyte cell and chondrocyte-like cell in intervertebral disk which have the positive yellow-stained particulate aggradation. There was no stain in the matrix, elastic fiber and collagen fiber. CONCLUSIONS: The distribution of xenoantigen is locally in the tissue of intervertebral disk and its expression is weak. This suggests that the intervertebral disk of Banna pig may be alternative donor for xenotransplantation.

Antigen expression in xenotransplantation: how low must it go?

BACKGROUND: Acute vascular rejection (AVR) is an important immunological barrier to xenotransplantation. Thought to be initiated by xenoreactive antibodies, acute vascular rejection might, in principle, be avoided by engineering animals to express low levels of antigen. The extent to which antigen expression would have to be decreased to achieve such a goal is unknown. METHODS: We estimated the decrease in expression of a xenogeneic antigen, Galalpha1-3Gal, which might be needed to avert acute vascular rejection of xenotransplants based on the decrease in antibody binding to endothelium that would prevent tissue damage. RESULTS: The level of decrease needed in Galalpha1-3Gal expression needed to avoid acute vascular rejection was estimated to exceed 96% of baseline. The extent of the decrease needed reflected, in part, a substantial "excess" of Galalpha1-3Gal on porcine endothelial cell surfaces. CONCLUSIONS: Although the change in antigen expression required to avoid acute vascular rejection might be conditioned by various factors, the very large magnitude of this change necessitates application of highly efficient approaches to antigen modification.

Total synthesis of the natural antigen involved in the hyperacute rejection response to xenotransplants.

The major glycosphingolipid in pig vascular endothelium is the ceramide pentasaccharide Gal alpha(1 --> 3)Gal beta(1 --> 4)GlcNAc beta(1 --> 3)Gal beta(1 --> 4)Glc beta(1 --> 0)Cer (1), which binds specifically to human anti-Gal antibody and is involved in the hyperacute rejection response in xenotransplantation from pig to man. The synthesis of 1 and its methyl glycoside 2 is described.

Xenotransplantation: the importance of the Galalpha1,3Gal epitope in hyperacute vascular rejection.

The transplantation of organs from other species into humans is considered to be a potential solution to the shortage of human donor organs. Organ transplantation from pig to human, however, results in hyperacute rejection, initiated by the binding of human natural antidonor antibody and complement. The major target antigen of this natural antibody is the terminal disaccharide Galalphal,3Gal, which is synthesized by Galbeta1,4GlcNAc alpha1,3-galactosyltransferase. Here we review our current knowledge of this key enzyme. A better understanding of structure, enzyme properties, and expression pattern of alpha1,3-galactosyltransferase has opened up several novel therapeutic approaches to prevent hyperacute vascular rejection. Cloning, and expression in vitro of the corresponding cDNA, has allowed to develop strategies to induce immune tolerance, and deplete or neutralize the natural xenoreactive antibody. Elucidation of the genomic structure has led to the production of transgenic animals that are lacking alpha1,3-galactosyltransferase activity. A detailed knowledge of the enzyme properties has formed the basis of approaches to modify donor organ glycosylation by intracellular competition. Study of the expression pattern of alpha1,3-galactosyltransferase has helped to understand the mechanism of hyperacute rejection in discordant xenotransplantation, and that of complement-mediated, natural immunity against interspecies transmission of retroviruses.

Blockade of induced xenoantigen expression prevents rejection after retransplantation of accommodated hamster-to-rat heart xenografts.

BACKGROUND: We have shown previously that a 2-week course of leflunomide (LF) together with a maintenance therapy of cyclosporine (CsA) rendered hamster-to-rat heart xenografts (Xg) resistant against anti-hamster IgM xenoantibody (XAb)-mediated rejection, a state compatible with the notion of accommodation. Our aim in this study was to investigate the mechanism underlying this Xg accommodation. METHODS: "Accommodated" Xgs were retransplanted to CsA-treated naive rats in the presence or absence of additional LF treatment or anti-hamster IgM serum injection. Immunohistopathology and fluorescence-activated cell sorting was performed to detect IgM and complement (C) deposition in Xgs, and endothelial cell (EC) expression of P- and E-selectin, ICAM-1, and VCAM-1 in vivo and in vitro. RESULTS: Retransplanted accommodated Xgs were rejected in CsA-treated naive rats and elicited IgM XAbs. Passive transfer of IgM XAbs provoked hyperacute rejection of both control and retransplanted Xgs. Addition of a 5-day course of LF prevented the rejection of only accommodated Xgs. Adoptively transferred IgM XAbs were deposited in rejected control and accommodated Xgs, but not in accommodated Xgs accepted by LF-treated rats. LF blocked the EC induction of P- and E-selectins in both control fresh and accommodated Xgs. Hence, after retransplantation accommodated Xgs express mainly induced xenoantigens (XAgs), such as P- and E-selectins, that can entirely be suppressed by LF. In contrast, control hamster Xgs express additional XAgs and remain susceptible to XAb-mediated rejection. These findings are in agreement with in vitro studies showing that LF totally suppressed induced EC antigens (e.g., P-selectin and E-selectin), but not constitutively expressed antigens (e.g., ICAM-1). CONCLUSION: Accommodated Xgs show a down-regulation of constitutive XAgs, but may be rejected after retransplantation by a mechanism involving EC expression of inducible XAgs. LF is able to block this latter XAg induction.

HLA-Cw3 expression on porcine endothelial cells protects against xenogeneic cytotoxicity mediated by a subset of human NK cells.

There is increasing evidence that NK cells make an important contribution to human anti-porcine xenogeneic cytotoxicity. Most allogeneic as well as autologous normal cells are not susceptible to NK cell-mediated cytotoxicity because they express inhibitory molecules encoded within the MHC class I loci. The protective signal is delivered to NK cells through killer cell-inhibitory receptors expressing different MHC class I specificities. It has been proposed that xenogeneic target cells may be susceptible to NK cell-mediated lysis because their MHC class I molecules fail to be recognized by human killer cell-inhibitory receptors. To explore this hypothesis, we examined the effect of human MHC class I expression on porcine target cell lysis by human NK cells. An immortalized porcine bone marrow-derived endothelial cell line (2A2) was transfected with three different human MHC class I allelic genes (HLA-A2, -B27, or -Cw3). The cytotoxic activity of several GL183+ NK clones, which lysed untransfected porcine cells effectively, was substantially blocked by the presence of HLA-Cw3. In contrast, HLA-Cw3-positive cells were not protected against lysis by GL183- EB6+ NK clones. The expression of HLA-B27 or HLA-A2 molecules on pig target cells did not provide substantial protection from lysis by any of the NK clones tested. In addition to confirming the hypothetical basis of NK cell-mediated killing of xenogeneic targets, these results have practical implications as an approach to overcoming NK cell-mediated cytotoxicity, which may be an obstacle to pig-to-human xenotransplantation.