Galactosyltransferase Knockout Research Articles

Comparative immunogenicity of decellularized wild type and alpha 1,3 galactosyltransferase knockout pig lungs

Decellularized pig lungs recellularized with human lung cells offer a novel approach for organ transplantation. However, the potential immunogenicity of decellularized pig lungs following exposure to human tissues has not been assessed. We found that exposure of native lungs from wildtype and transgenic pigs lacking alpha (1,3)-galactosyltransferase (α-gal KO) to sera from normal healthy human volunteers demonstrated similar robust IgM and IgG immunoreactivity, comparably decreased in decellularized lungs. Similar results were observed with sera from patients who had previously undergone transcutaneous porcine aortic valve replacement (TAVR) or from patients with increased circulating anti-α-gal IgE antibodies (α-gal syndrome). Depleting anti-α-gal antibodies from the sera demonstrated both specificity of α-gal immunoreactivity and also residual immunoreactivity similar between wildtype and α-gal KO pig lungs. Exposure of human monocytes and macrophages to native wildtype lungs demonstrated greater induction of M2 phenotype than native α-gal KO pig lungs, which was less marked with decellularized lungs of either type. Overall, these results demonstrate that native wildtype and α-gal KO pig lungs provoke similar immune responses that are comparably decreased following decellularization. This provides a further platform for potential use of decellularized pig lungs in tissue engineering approaches and subsequent transplantation schemes but no obvious overall immunologic advantage of utilizing lungs obtained from α-gal KO pigs.

Immunological response in cynomolgus macaques to porcine α-1,3 galactosyltransferase knockout viable skin xenotransplants-A pre-clinical study.

Allogeneic skin recovered from human deceased donors (HDD) has been a mainstay interim treatment for severe burns, but unfortunately risk of infectious disease and availability limitations exist. Genetically engineered ɑ-1,3 galactosyltransferase knockout (GalT-KO) porcine source animals for viable skin xenotransplants may provide a promising clinical alternative. Four cynomolgus macaque recipients received full-thickness surgical wounds to model the defects arising from excision of full-thickness burn injury and were treated with biologically active skin xenotransplants derived from GalT-KO, Designated Pathogen Free (DPF) miniature swine. Evaluations were conducted for safety, tolerability, and recipient immunological response. All skin xenotransplants demonstrated prolonged survival, vascularity, and persistent dermal adhesion until the study endpoint at post-operative day 30. No adverse outcomes were observed during the study. Varying levels of epidermolysis coincided with histologic detection of CD4+ and CD8+T cells, and other cellular infiltrates in the epidermis. Recipient sera IgM and IgG demonstrated significant antibody immune response to non-α-1,3-galactose porcine xenoantigens. Separately, specific wound healing mediators were quantified. Neither porcine cell migration nor PERV were detected in circulation or any visceral organs. These results provide a detailed analysis of vital skin xenotransplants utilizing a non-human primate model to predict the anticipated immunological response of human patients. The lack of adverse rejection even in the presence of elevated Ig indicates this is a prospective therapeutic option. The findings reported here directly supported regulatory clearance for a first-in-man, Phase I xenotransplantation clinical trial.

Extended Pig-to-Baboon Lung Xenograft Survival

In baboons, lungs from galactosyl transferase knock-out (GTKO) pigs expressing human complement regulatory proteins (hCPRP: hCD46, hCD55) exhibit elevated pulmonary vascular resistance and loss of vascular barrier function within hours. Here we summarize efforts over ten years to understand and prevent these phenomena. Orthotopic left single lung transplantation was performed using GTKO.hCPRP lungs with additional expression of one or more thromboregulatory (hEPCR, hTBM, hTFPI, hCD39) and anti-inflammatory genes (HO-1, HLA-E, hCD47), and β4Gal carbohydrate xenoantigen knock-down (KD), in various combinations. Anti-inflammatory drugs (steroids, sC1Inh, anti-histamine, anti-thromboxane, A1AT, sTNFR, αIL-6, αIL-8, PSGL-1, αMac1), antibody adsorption (AbAds) using donor pig kidneys, and donor pretreatment with chlodronate liposomes were progressively added in recent years. Transient life-supporting GTKO.hCD46 lung function was consistently observed with either hEPCR (alone [n=6] or with hCD55 [6], HO-1 [2], or hCD47 [1]); with hTBM (alone [3] or with HO-1 [1] or CD47 [1]); or with hCD55.hTFPI.CD47 [11]. Although recipient survival was occasionally extended to 2 [n=6] or even 8 days [1], loss of xenograft vascular barrier function and systemic inflammation typically occurred within 24-48 hours. Consistent recipient survival beyond 12 hours was only observed with hEPCR and hTBM co-expression [n=12] in combination with one or more additional genetic modifications (HO-1 [2], CD47.HO-1 [3], hCD55 [2], hCD55.hCD39 [3], or β4GalKD.hCD47 [2]), including 7 and 9 day survivals with selectin/integrin blockade, A1AT, and liposomes. β4GalKD.hCD47, liposome pretreatment, recipient splenectomy, and AbAds were associated with prolonged recipient survival to 31 days in one instance. Graft demise beyond 2 days was association with elaboration of anti-non-Gal antibody whether using conventional or αCD40-based immunosuppression. Consistent life-supporting lung xenograft function and increasingly frequent recipient survival >2d reflect significant progress in understanding the remaining barriers to clinical translation. We identify a pivotal role for dysregulated coagulation and multiple proinflammatory mechanisms in the loss of vascular barrier function which is associated with lung xenograft failure, and the need for more effective immunosuppression.

Codon optimized membrane cofactor protein expression in α 1, 3 galactosyltransferase knockout pig cells improve protection against cytotoxicity of monkey serum.

In this study, we attempted to upgrade GT -MCP/-MCP pig genetically to express MCP at a higher level and additionally thrombomodulin (TBM), which have respective roles as a complement regulatory protein and a coagulation inhibitor. We constructed a dicistronic cassette consisting of codon-optimized MCP (mMCP) and TBM (m-pI2), designed for ubiquitous expression of MCP and endothelium specific expression of TBM. The cassette was confirmed to allow extremely increased MCP expression compared with non-modified MCP, and an endothelial-specific TBM expression. We thus transfected m-pI2 into ear-skin fibroblasts isolated from a GT -MCP/-MCP pig. By twice selection using magnetically activated cell sorting (MACS), and single-cell culture, we were able to obtain clones over 90% expressing MCP. The cells of a clone were provided as a donor for nuclear transfer resulting in the generation of a GT -MCP/-MCP /mMCP/TBM pig, which was confirmed to be carrying cells expressing MCP and functioning as an inhibitor against the cytotoxic effect of normal monkey serum, comparable with donor cells. Collectively, these results demonstrated an effective approach for upgrading transgenic pig, and we assumed that upgraded pig would increase graft survival.

Vital, Porcine, Gal-Knockout Skin Transplants Provide Efficacious Temporary Closure of Full-Thickness Wounds: Good Laboratory Practice-Compliant Studies in Nonhuman Primates.

Vital, genetically engineered porcine skin transplants have long been regarded as a promising treatment option for severe burn wounds. The objective of this two-part, preclinical study was to evaluate the ability of vital, split-thickness skin xenotransplants derived from designated pathogen-free, alpha 1,3 galactosyltransferase knockout miniature swine to provide temporary wound closure of full-thickness wound defects intended to model severe and extensive, deep partial- and full-thickness burn wounds. In part 1 of the study, four full-thickness wound defects were introduced in four cynomolgus macaques recipients and, then engrafted with two xenografts and two allografts to achieve temporary wound closure. On POD-15, autografts were used to achieve definitive wound closure and were observed until POD-22. In part 2 of the study, four additional subjects each received two full-thickness wound defects, followed by two xenografts to achieve temporary wound closure, and were observed postoperatively for 30 days without further intervention. All grafts were assessed for signs of adherence to the wound bed, vascularity, and signs of immune rejection via gross clinical and histological methods. Xenograft and allograft comparators were equivalent in part 1, and later autografts were otherwise indistinguishable. In part 2, all xenotransplants demonstrated adherence, vascularity, and survival until POD-30. These were unexpected results that exceed previously published findings in similar models. Furthermore, the ensuing GLP-study report directly supported regulatory clearance, permitting a phase I clinical trial. This solution holds great promise as an alternative to human cadaver allograft, the current standard of care for the treatment of severe burns.

AGI-134: a fully synthetic \u03b1-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models

BackgroundTreatments that generate T cell-mediated immunity to a patient’s unique neoantigens are the current holy grail of cancer immunotherapy. In particular, treatments that do not require cumbersome and individualized ex vivo processing or manufacturing processes are especially sought after. Here we report that AGI-134, a glycolipid-like small molecule, can be used for coating tumor cells with the xenoantigen Galα1-3Galβ1-4GlcNAc (α-Gal) in situ leading to opsonization with pre-existing natural anti-α-Gal antibodies (in short anti-Gal), which triggers immune cascades resulting in T cell mediated anti-tumor immunity.MethodsVarious immunological effects of coating tumor cells with α-Gal via AGI-134 in vitro were measured by flow cytometry: (1) opsonization with anti-Gal and complement, (2) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, and (3) phagocytosis and antigen cross-presentation by antigen presenting cells (APCs). A viability kit was used to test AGI-134 mediated complement dependent cytotoxicity (CDC) in cancer cells. The anti-tumoral activity of AGI-134 alone or in combination with an anti-programmed death-1 (anti-PD-1) antibody was tested in melanoma models in anti-Gal expressing galactosyltransferase knockout (α1,3GT−/−) mice. CDC and phagocytosis data were analyzed by one-way ANOVA, ADCC results by paired t-test, distal tumor growth by Mantel–Cox test, C5a data by Mann–Whitney test, and single tumor regression by repeated measures analysis.ResultsIn vitro, α-Gal labelling of tumor cells via AGI-134 incorporation into the cell membrane leads to anti-Gal binding and complement activation. Through the effects of complement and ADCC, tumor cells are lysed and tumor antigen uptake by APCs increased. Antigen associated with lysed cells is cross-presented by CD8α+ dendritic cells leading to activation of antigen-specific CD8+ T cells. In B16-F10 or JB/RH melanoma models in α1,3GT−/− mice, intratumoral AGI-134 administration leads to primary tumor regression and has a robust abscopal effect, i.e., it protects from the development of distal, uninjected lesions. Combinations of AGI-134 and anti-PD-1 antibody shows a synergistic benefit in protection from secondary tumor growth.ConclusionsWe have identified AGI-134 as an immunotherapeutic drug candidate, which could be an excellent combination partner for anti-PD-1 therapy, by facilitating tumor antigen processing and increasing the repertoire of tumor-specific T cells prior to anti-PD-1 treatment.

Topical Alpha-Gal Nanoparticles Enhances Wound Healing in Radiated Tissue

BACKGROUND: Radiation is a common primary, adjuvant, and neoadjuvant therapy in oncologic patients. It is well known that surgery on radiated tissues is associated with significantly higher complication rates due to permanently compromised wound healing. It is thought that one cause of impaired wound healing is the aberrant inflammatory response that occurs in radiated tissues. Previous work has demonstrated that the topical application of naturally occurring antigen α-gal (Galα1-3Galb1-[3]4GlcNAc-R) nanoparticles (AGNs) onto wound surface accelerates macrophage recruitment. As we have already observed accelerated wound healing in both normal and diabetic wounds treated with topical AGN, we hypothesized that application of this natural antigen would similarly enhance healing of the wounds in irradiated tissue. METHODS: To simulate human physiology, α-1,3galactosyltransferase knockout mice (KO), which do not produce the antigen and therefore can be stimulated to produce antibodies against it, were used. KO were exposed to the antigen to produce anti α-gal antibodies at titers comparable to those seen in humans. Ten days before wounding, dorsal skin was isolated using a low-pressure clamp as previously described and was irradiated with one session of 40 Gy. Bilateral 6-mm dorsal splinted full-thickness wounds were created and treated with AGN in a 2% carboxymethyl cellulose carrier, immediately after wounding and again on postoperative day 1. Control knocked out group underwent similar irradiation and wounding protocols but were treated with phosphate buffered saline (PBS) in 2% carboxymethyl cellulose. Wild-type mice, which are indolent to the antigen, went through the same radiation and wounding to eliminate confounding factors other than immunogenic response to AGN. Wounds were harvested from all animals up to 21 days after the wounding for histologic and immunohistochemistry measures. The extent of keratinocyte migration, neovascularization, and macrophage recruitment was assessed. RESULTS: Full closure of all wounds by day 9 in the nonradiated control compared to no completely closed wounds in the radiated group confirmed the known inhibitory effects of irradiation on wound healing. In addition, histologic changes such as increased epidermal thickness in the skin surrounding the wound further confirmed the effects of irradiation on the skin. Histologic analysis demonstrated enhanced keratinocyte migration in the AGN-treated KO wounds, which was significantly improved in comparison to PBS-treated KO wounds noted by day 15 and until the end of the study (P < 0.01). On day 21, ≈63% of all α-gal–treated wounds were completely healed as opposed to only ≈17% in the PBS-treated group. In wild-type mice, treatment with AGN showed no improvement in keratinocyte migration or time to full closure. CONCLUSIONS: Topical application of AGN onto radiated wounds significantly ameliorate the delayed wound healing in radiated tissue resulting in faster wound closure. We believe that this naturally occurring agent has great promise for clinical translation as it has demonstrated efficacy in not only normal wounds but pathologic (diabetic, radiated) ones as well.

Antigen-Mediated, Macrophage-Stimulated, Accelerated Wound Healing Using α-Gal Nanoparticles.

Macrophages are known to be crucial to timely and efficacious wound healing. They have been shown to modulate inflammation and the migration and proliferation of regenerative cells, promoting tissue deposition and wound closure. This study explored the use of the natural antigen Galα1-3Galβ1-4GlcNAc-R (α-gal), present in lower mammals yet absent in Old World primates and humans, to induce a transiently enhanced macrophage response and thereby direct accelerated wound closure and healing in a standard murine model. α1,3galactosyltransferase knockout mice were stimulated to produce anti-Gal antibodies at levels comparable with humans. α-Gal-containing micelle nanoparticles were generated and applied to full-thickness splinted wounds on the mice. At 1, 2, 3, 6, and 9 days postoperatively, mice were killed, and wounds were analyzed histologically for macrophage invasion, epithelialization, vascularization, and granulation tissue deposition. Flow cytometry of wound tissue was performed to quantify relative levels of proinflammatory M1 to anti-inflammatory M2 macrophage subtypes. Treatment of splinted full-thickness murine wounds with α-gal-containing nanoparticles led to accelerated wound healing and closure as demonstrated by accelerated rates of keratinization, vascular growth, and wound tissue deposition. Furthermore, treated wounds demonstrated early and enhanced macrophage invasion, as well as a lower M1-M2 ratio. Application of α-gal-containing nanoparticles to wounds stimulated a transiently increased inflammatory response, accelerating the rate of wound healing. Use of α-gal may be a simple and effective way to stimulate the wound healing response in both normal and pathologic wound beds.

Comparative Decellularization and Recellularization of Wild-Type and Alpha 1,3 Galactosyltransferase Knockout Pig Lungs: A Model for Ex Vivo Xenogeneic Lung Bioengineering and Transplantation.

A novel potential approach for lung transplantation could be to utilize xenogeneic decellularized pig lung scaffolds that are recellularized with human lung cells. However, pig tissues express several immunogenic proteins, notably galactosylated cell surface glycoproteins resulting from alpha 1,3 galactosyltransferase (α-gal) activity, that could conceivably prevent effective use. Use of lungs from α-gal knock out (α-gal KO) pigs presents a potential alternative and thus comparative de- and recellularization of wild-type and α-gal KO pig lungs was assessed. Decellularized lungs were compared by histologic, immunohistochemical, and mass spectrometric techniques. Recellularization was assessed following compartmental inoculation of human lung bronchial epithelial cells, human lung fibroblasts, human bone marrow-derived mesenchymal stromal cells (all via airway inoculation), and human pulmonary vascular endothelial cells (CBF) (vascular inoculation). No obvious differences in histologic structure was observed but an approximate 25% difference in retention of residual proteins was determined between decellularized wild-type and α-gal KO pig lungs, including retention of α-galactosylated epitopes in acellular wild-type pig lungs. However, robust initial recellularization and subsequent growth and proliferation was observed for all cell types with no obvious differences between cells seeded into wild-type versus α-gal KO lungs. These proof of concept studies demonstrate that decellularized wild-type and α-gal KO pig lungs can be comparably decellularized and comparably support initial growth of human lung cells, despite some differences in retained proteins. α-Gal KO pig lungs are a suitable platform for further studies of xenogeneic lung regeneration.

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.

Inhibition of complement component C5 prevents clotting in an ex vivo model of xenogeneic activation of coagulation.

Xenogeneic thrombotic microangiopathy (TMA) and acute vascular rejection (AVR) prevent long-term survival of porcine xenografts after transplantation into non-human primates. Preformed xenoreactive natural antibodies (XNA) cause endothelial damage and activate the complement system. Mechanisms of xenogeneic coagulation and platelet activation are only poorly characterized. A microfluidic flow chamber was used to study platelet activation and thrombus formation of human platelet-rich plasma (PRP) upon perfusion over wild-type (WT) or α-1,3- galactosyltransferase knockout (GTKO) and human CD46 (hCD46) transgenic porcine aortic endothelial cells (PAEC). Activation of plasma coagulation (thrombin-anti-thrombin complex; TAT) and complement (C3a, C5a) was studied in human platelet-free plasma (PFP) after co-incubation with PAEC. The activation of PAEC (E-Selectin, tissue factor, ICAM-1, ICAM-2, VCAM-1) was studied after incubation with human serum. Eculizumab (200 μg/ml) was used to inhibit terminal complement activation in all experiments. WT-PAEC perfused with human PRP showed thrombus formation at different shear rates (3 dyn/cm(2) : 23 ± 10%; 10 dyn/cm(2) : 17 ± 10% of flow chamber viewing field). GTKO/hCD46 PAEC exhibited reduced, but not fully prevented thrombus formation (3 dyn/cm(2) : 12 ± 12%). Porcine PRP caused little or no thrombus formation (3.0 ± 4% and 0.5 ± 0.9%, respectively). Flow cytometry of human platelets after perfusion over WT-PAEC revealed an increase in platelet CD62P expression (29.5 ± 3%), compared to non-perfused PRP (7 ± 2%) or PRP running through empty flow chambers (12.7 ± 0.3%). Incubation of human PFP with PAEC resulted in an increase of TAT that correlated with C5a activation. Specific inhibition of complement by eculizumab prevented thrombus formation (WT-PAEC: 1.6 ± 2% at 3 dyn/cm(2) and 0.24 ± 0.33% at 10 dyn/cm(2) , GTKO/hCD46 PAEC: 0.2 ± 0.3% at 3 dyn/cm(2) ) as well as activation of coagulation and platelets. Induction of endothelial E-Selectin and VCAM-1 in WT-PAEC upon incubation with human serum was significantly reduced by eculizumab. Eculizumab did not reduce thrombin generation capacity of human PRP or normal platelet aggregation. Thrombus formation in this ex vivo model of xenogeneic TMA was closely linked with complement activation. Specific inhibition of complement C5 by eculizumab prevented endothelial cell activation, but also coagulation and platelet activation without compromising thrombin generation capacity of human blood or normal platelet function.

Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs

The pig is an ideal source to provide organs because its organ size and physiology are similar to humans. However, an acute rejection will ensue after pig-to-human xenotransplantation. The α-1,3 galactosyltransferase gene knockout (GTKO) pigs were generated in recent years, and could solve the problem of hyperacute rejection. But due to lack of reporting genes, the rejection status of cells and organs post pig-to-human xenotransplantation cannot be visualized. In this study, we introduced the enhanced green fluorescent protein (EGFP) gene driven by the CAG promoter into GTKO porcine ear fibroblasts. Then we produced transgenic pigs expressing the EGFP gene by nuclear transfer technology. Expression levels of EGFP in different tissues and organs of the cloned pig were investigated by Nightsea DFP-1 Fluorescent Protein Flashlight, fluorescence microscope and quantitative PCR assays. The results showed that the protein and transcript of EGFP were expressed in all tissues and organs of the GTKO pig, but the expression was weak in the liver and central nervous system. In conclusion, we have successfully produced the transgenic GTKO pigs expressing EGFP in all tested tissues and organs, which builds up a good basis to track transplanted cells or tissues.

Clonidine inhibits anti-non-Gal IgM xenoantibody elicited in multiple pig-to-primate models.

Survival of vascularized xenografts is dependent on pre-emptive inhibition of the xenoantibody response against galactosyltransferase knockout (GTKO) porcine organs. Our analysis in multiple GTKO pig-to-primate models of xenotransplantation has demonstrated that the anti-non-gal-α-1,3-gal (anti-non-Gal) xenoantibody response displays limited structural diversity. This allowed our group to identify an experimental compound which selectively inhibited induced anti-non-Gal IgM xenoantibodies. However, because this compound had an unknown safety profile, we extended this line of research to include screening small molecules with known safety profiles allowing rapid advancement to large animal models. The NIH clinical collections of small molecules were screened by ELISA for their ability to inhibit xenoantibody binding to GTKO pig endothelial cells. Serum collected from non-immunosuppressed rhesus monkeys at day 14 post-injection with GTKO pig endothelial cells was utilized as a source of elicited xenoantibody for initial screening. Virtual small molecule screening based on xenoantibody structure was used to assess the likelihood that the identified small molecules bound xenoantibody directly. As a proxy for selectivity, ELISAs against tetanus toxoid and the natural antigens laminin, thyroglobulin, and single-stranded DNA (ssDNA) were utilized to assess the ability of the identified reagents to inhibit additional antibody responses. The identified inhibitory small molecules were further tested for their ability to inhibit xenoantibody elicited in multiple settings, including rhesus monkeys pre-treated with an anti-non-Gal selective anti-idiotypic antibody, non-immunosuppressed rhesus monkeys immunized with wild-type fetal pig isletlike cell clusters, and non-immunosuppressed baboons transplanted with GTKO multiple transgenic pig kidneys. Four clinically relevant small molecules inhibited anti-non-Gal IgM binding to GTKO pig endothelial cells in vitro. Three of these drugs displayed a limited region of structural similarity suggesting they may inhibit xenoantibody by a similar mechanism. One of these, the anti-hypertensive agent clonidine, displayed only minimal inhibition of antibodies elicited by vaccination against tetanus toxoid or pre-existing natural antibodies against laminin, thyroglobulin, or ssDNA. Furthermore, clonidine inhibited elicited anti-non-Gal IgM from all animals that demonstrated a xenoantibody response in each experimental setting. Clinically relevant small molecule drugs with known safety profiles can inhibit xenoantibody elicited against non-Gal antigens in diverse experimental xenotransplantation settings. These molecules are ready to be tested in large animal models. However, it will first be necessary to optimize the timing and dosing required to inhibit xenoantibodies in vivo.

Xenotransplantation Transgenesis. Are we there yet?

The xenotransplantation of pig organs and tissues aimed at overcoming the world-wide shortage that exists for these in humans will require multiple genetic modifications to be incorporated to overcome rejection and associated problems. We have recently produced α1,3 galactosyltransferase knockout (GTKO) pigs expressing human (h) CD55&59, fucosyltransferase (HTF), hCD39 and hTBM This was done by breeding GTKO pigs to animals expressing human hCD55 and hCD59 plus HTF and then transfecting adult fibroblasts obtained from their progeny with a hCD39 plus hTBM construct. Transfected cells were then selected based on hCD39 expression and used for animal cloning to produce these (Figure 1). The generation of these animals represents almost two decades of research focussed on developing the necessary molecular, cellular and reproductive technologies. Having produced GT KO pigs expressing five transgenes, it is an appropriate juncture to reflect on how this was achieved and what progress still needs to be made in terms of transgenesis technology to advance xenotransplantation to the clinic.

Acceleration of wound healing by α-gal nanoparticles interacting with the natural anti-Gal antibody.

Application of α-gal nanoparticles to wounds and burns induces accelerated healing by harnessing the natural anti-Gal antibody which constitutes ~1% of human immunoglobulins. α-gal nanoparticles present multiple α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R), the carbohydrate ligand of anti-Gal. Studied α-gal nanoparticles were comprised of glycolipids with α-gal epitopes, phospholipids, and cholesterol. Binding of anti-Gal to α-gal nanoparticles in wounds activates the complement cascade, resulting in formation of chemotactic complement cleavage peptides that induce rapid recruitment of many macrophages. The Fc/Fcγ receptors interaction between anti-Gal coating α-gal nanoparticles and the recruited macrophages activates macrophages to produce cytokines/growth factors that promote wound healing and recruit stem cells. Studies of wound healing by α-gal nanoparticles were feasible in α1,3galactosyltransferase knockout mice and pigs. In contrast to other nonprimate mammals, these mice and pigs lack the α-gal epitope, and thus they are not immunotolerant to it and produce anti-Gal. Treatment of skin wounds and burns with α-gal nanoparticles resulted in 40–60% decrease in healing time in comparison with control wounds treated with saline. This accelerated healing is associated with increased recruitment of macrophages and extensive angiogenesis in wounds, faster regrowth of epidermis, and regeneration of the dermis. The accelerated healing further decreases and may completely eliminate fibrosis and scar formation in wounds. Since healing of internal injuries is mediated by mechanisms similar to those in external wound healing, it is suggested that α-gal nanoparticles treatment may also improve regeneration and restoration of biological function following internal injuries such as surgical incisions, myocardial ischemia following infarction, and nerve injuries.

Avoiding detrimental human immune response against Mammalian extracellular matrix implants.

This review describes the antibodies formed against mammalian extracellular matrix (ECM) implants in humans and proposes methods for avoiding the detrimental effects of these antibodies. There are two types of antibodies against ECM implants: (i) The natural anti-Gal antibody constituting ∼1% of immunoglobulins in humans. This antibody binds to a carbohydrate antigen called the α-gal epitope with the structure Galα1-3Galβ1-4GlcNAc-R. The α-gal epitope is abundant in nonprimate mammals, including on ECM proteins and proteoglycans. Moreover, anti-Gal antibody titers increase within 2-4 weeks by 10- to 100-folds in human recipients of mammalian implants or xenografts expressing α-gal epitopes. (ii) Anti-non gal antibodies formed against ECM peptide sequences differing from those in homologous proteins in humans. Most homologous proteins in mammals contain immunogenic peptides that elicit anti-non gal antibody production when introduced into humans. Formation of anti-non gal antibodies is much slower than that of elicited anti-Gal antibodies. Both anti-Gal and anti-non gal antibodies are detrimental to ECM implant regeneration in humans by binding to the ECM and directing extensive macrophage-mediated degradation of the implant. In addition, antibodies binding to ECM proteins/proteoglycans may hinder stem cells interaction with the ECM, which is required for directing stem cell differentiation. The anti-Gal immunological barrier can be avoided by using mammalian ECM implants lacking α-gal epitopes. Such implants can be engineered by enzymatic destruction of α-gal epitopes with recombinant α-galactosidase. Alternatively, implants may be obtained from α1,3galactosyltransferase knockout donor species that lack α-gal epitopes. Since postimplantation production of anti-non gal antibodies is a slow process, the detrimental effects of these antibodies may be avoided by accelerating stem cells recruitment into implants, thus accelerating the regeneration process. Acceleration of stem cell recruitment may be achieved by introducing α-gal nanoparticles into the implant. α-Gal nanoparticles present multiple α-gal epitopes, which bind anti-Gal and induce recruitment of macrophages by generating complement chemotactic factors. Fc/Fcγ receptor interaction between anti-Gal coating α-gal nanoparticles and recruited macrophages activates the macrophages to secrete "pro-healing" cytokines/growth factors that recruit stem cells. These recruited cells are instructed by the implanted ECM to regenerate the implant before anti-non gal antibodies can reach detrimental titers.

Development of a consensus protocol to quantify primate anti-non-Gal xenoreactive antibodies using pig aortic endothelial cells.

Scientists working in the field of xenotransplantation do not employ a uniform method to measure and report natural and induced antibody responses to non-Galα(1,3)Gal (non-Gal) epitopes. Such humoral responses are thought to be particularly pathogenic after transplantation of vascularized GalTKO pig organs and having a more uniform assay and reporting format would greatly facilitate comparisons between laboratories. Flow cytometry allows examination of antibody reactivity to intact antigens in their natural location and conformation on cell membranes. We have established a simple and reproducible flow cytometric assay to detect antibodies specific for non-Gal pig antigens using primary porcine aortic endothelial cells (pAECs) and cell culture-adapted pAEC cell lines generated from wild type and α1,3galactosyl transferase knockout (GalTKO) swine. The consensus protocol we propose here is based on procedures routinely used in four xenotransplantation centers and was independently evaluated at three sites using shared cells and serum samples. Our observation support use of the cell culture-adapted GalTKO pAEC KO:15502 cells as a routine method to determine the reactivity of anti-non-Gal antibodies in human and baboon serum. We have developed an assay that allows the detection of natural and induced non-Gal xenoreactive antibodies present in human or baboon serum in a reliable and consistent manner. This consensus assay and format for reporting the data should be accessible to laboratories and will be useful for assessing experimental results between multiple research centers. Adopting this assay and format for reporting the data should facilitate the detection, monitoring, and detailed characterization of non-Gal antibody responses.

Results of life-supporting galactosyltransferase knockout kidneys in cynomolgus monkeys using two different sources of galactosyltransferase knockout Swine.

Various durations of survival have been observed in the xenotransplantation of life-supporting α-1,3-galactosyltransferase knockout (GalT-KO) porcine kidneys into nonhuman primates. Although others have demonstrated loss of GalT-KO-transplanted kidneys within 2 weeks, we have reported an average survival of 51 days with the cotransplantation of the kidney and vascularized thymus and an average of 29 days with the kidney alone. To determine the factors responsible for this difference in survival time, we performed xenogeneic kidney transplantations into cynomolgus monkeys with an anti-CD40L-based regimen using two different strains of GalT-KO swine, one derived from MGH miniature swine and the other obtained from Meji University. Eight cynomolgus moneys received GalT-KO kidneys. Three kidney grafts were from Massachusetts General Hospital (MGH)-Nippon Institute for Biological Science (NIBS) GalT-KO pigs and five GalT-KO grafts were from MEIJI GalT-KO swine. All cynomolgus recipients were treated identically. Recipients of kidneys from the MGH GalT-KO kidneys swine, produced by nuclear transfer in Japan, survived an average of 28.7 days, whereas recipients of MEIJI GalT-KO kidneys swine survived an average of 9.2 days. Among the differences between these two groups, one potentially revealing disparity was that the MEIJI swine were positive for porcine cytomegalovirus, whereas the MGH-derived swine were negative. This is the first study comparing renal xenotransplantation from two different sources of GalT-KO swine into nonhuman primates at a single center. The results demonstrate that porcine cytomegalovirus may be responsible for early loss of GalT-KO swine kidney xenografts.

Evaluation of biocompatibility of α-1,3 galactosyltransferase knockout pig bone graft in a rat calvarial defect model

The current study was conducted to evaluate the biocompatibility of α-1,3 galactosyltransferase knockout pig bone graft in a rat calvarial defect model. Porcine cancellous bones were harvested from general and alpha-gal KO pigs and washed with 70% ethanol solution and normal saline. Bone pieces of the alphagal KO pig underwent a chemical treatment process to delipidize and deproteinize the bone. Bone graft particles were freeze-dried and stored at ?70°C until use. Each bone graft was implanted into the rat calvarial defect in a fresh general pig, fresh transgenic pig, and chemical-treated pig bone group. There was no systemic adverse effect on hematology or necropsy findings in all groups at 1 week and 4 weeks. In the microcomputed tomography analysis, bone volume increased significantly in the chemical-treated transgenic pig bone group, whereas bone mineral density decreased significantly in the fresh general pig bone group compared with other groups. Histological evaluation showed cellular infiltration located at the margin of the bone graft particles, especially in the fresh general pig bone group. These results indicate that fresh general pig bone can elicit a greater local inflammatory response than fresh transgenic pig bone. Further, chemical-treated transgenic pig bone graft was less immunogenic than fresh bone graft. In conclusion, transgenic pig bone is a more biocompatible graft material. In addition, chemical treatment can reduce bone graft immunogenicity by delipidizing and deproteinizing bone.

α1,3Galactosyltransferase knockout pigs produce the natural anti‐Gal antibody and simulate the evolutionary appearance of this antibody in primates

Anti-Gal is the most abundant natural antibody in humans and Old World primates (apes and Old World monkeys). Its ligand, the α-gal epitope (Galα1-3Galβ1-4GlcNAc-R), is abundant in nonprimate mammals, prosimians and New World monkeys whereas it is absent in humans and Old World primates as a result of inactivation of the α1,3galactosyltransferase (α1,3GT) gene in ancestral Old World primates, as recent as 20-28 million years ago. Since anti-Gal has been a "forbidden" autoantibody for >140 million years of evolution in mammals producing α-gal epitopes it was of interest to determine whether ancestral Old World primates could produce anti-Gal once α-gal epitopes were eliminated, i.e. did they carry anti-Gal encoding immunoglobulin genes, or did evolutionary selection eliminate these genes that may be detrimental in mammals synthesizing α-gal epitopes. This question was studied by evaluating anti-Gal prodution in α1,3GT knockout (GT-KO) pigs recently generated from wild-type pigs in which the α-gal epitope is a major self-antigen. Anti-Gal antibody activity in pig sera was assessed by ELISA, flow cytometry and complement mediated cytolysis and compared to that in human sera. The study demonstrates abundant production of the natural anti-Gal antibody in GT-KO pigs at titers even higher than in humans. The fine specificity of GT-KO pig anti-Gal is identical to that of human anti-Gal. Pigs and probably other mammals producing α-gal epitopes carry immunoglobulin genes encoding anti-Gal as an autoantibody. Once the α-gal epitope is eliminated in GT-KO pigs, they produce anti-Gal. These findings strongly suggest that similar to GT-KO pigs, inactivation of the α1,3GT gene in ancestral Old World primates enabled the immediate production of anti-Gal, possibly as a protective antibody against detrimental microbial agents carrying α-gal epitopes.