Gene-knockout Pigs Research Articles

Phenotypic features of dystrophin gene knockout pigs harboring a human artificial chromosome containing the entire dystrophin gene

Mammalian artificial chromosomes have enabled the introduction of extremely large amounts of genetic information into animal cells in an autonomously replicating, nonintegrating format. However, the evaluation of human artificial chromosomes (HACs) as novel tools for curing intractable hereditary disorders has been hindered by the limited efficacy of the delivery system. We generated dystrophin gene knockout (DMD-KO) pigs harboring the HAC bearing the entire human DMD via asomatic cell cloning procedure (DYS-HAC-cloned pig). Restored human dystrophin expression was confirmed by immunofluorescence staining in the skeletal muscle of the DYS-HAC-cloned pigs. Viability at the first month postpartum of the DYS-HAC-cloned pigs, including motor function in the hind leg and serum creatinine kinase level, was improved significantly when compared with that in the original DMD-KO pigs. However, decrease in systemic retention of the DYS-HAC vector and limited production of the DMD protein might have caused severe respiratory impairment with general prostration by 3months postpartum. The results demonstrate that the use of transchromosomic cloned pigs permitted a straightforward estimation of the efficacy of the DYS-HAC carried in affected tissues/organs in a large-animal disease model, providing novel insights into the therapeutic application of exogenous mammalian artificial chromosomes.

Production of MSTN knockout porcine cells using adenine base-editing-mediated exon skipping

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9 and cytosine base editing (CBE) technologies, adenine base editing (ABE) shows better safety and accuracy in gene modification. However, because of the characteristics of gene sequences, the ABE system cannot be widely used in gene knockout. Alternative splicing of mRNA is an important biological mechanism in eukaryotes for the formation of proteins with different functional activities. The splicing apparatus recognizes conserved sequences of the 5' end splice donor and 3' end splice acceptor motifs of introns in pre-mRNA that can trigger exon skipping, leading to the production of new functional proteins, or causing gene inactivation through frameshift mutations. This study aimed to construct a MSTN knockout pig by inducing exon skipping with the aid of the ABE system to expand the application of the ABE system for the preparation of knockout pigs. In this study, first, we constructed ABEmaxAW and ABE8eV106W plasmid vectors and found that their editing efficiencies at the targets were at least sixfold and even 260-fold higher than that of ABEmaxAW by contrasting the editing efficiencies at the gene targets of endogenous CD163, IGF2, and MSTN in pigs. Subsequently, we used the ABE8eV106W system to realize adenine base (the base of the antisense strand is thymine) editing of the conserved splice donor sequence (5'-GT) of intron 2 of the porcine MSTN gene. A porcine single-cell clone carrying a homozygous mutation (5'-GC) in the conserved sequence (5'-GT) of the intron 2 splice donor of the MSTN gene was successfully generated after drug selection. Unfortunately, the MSTN gene was not expressed and, therefore, could not be characterized at this level. No detectable genomic off-target edits were identified by Sanger sequencing. In this study, we verified that the ABE8eV106W vector had higher editing efficiency and could expand the editing scope of ABE. Additionally, we successfully achieved the precise modification of the alternative splice acceptor of intron 2 of the porcine MSTN gene, which may provide a new strategy for gene knockout in pigs.

Genome Editing of Pig.

Pigs have anatomical and physiological characteristics similar to humans; therefore, genetically modified pigs have the potential to become a valuable bioresource in biomedical research. In fact, considering the increasing need for translational research, pigs are useful for studying intractable diseases, organ transplantation, and regenerative medicine as large-scale experimental animals with excellent potential for extrapolation to humans. With the advent of zinc finger nucleases (ZFNs), breakthroughs in genome editing tools such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) have facilitated the efficient generation of genetically modified pigs. Genome editing has been used in pigs for more than 10 years; now, along with knockout pigs, knock-in pigs are also gaining increasing importance. In this chapter, we describe the establishment of gene-modified cells (nuclear donor cells), which are necessary for gene knockout and production of knock-in pigs via somatic cell nuclear transplantation, as well as the production of gene knockout pigs using a simple cytoplasmic injection method.

Repair of bone defects in rhesus monkeys with α1,3-galactosyltransferase-knockout pig cancellous bone.

Introduction: Since xenografts offer a wide range of incomparable advantages, they can be a better option than allografts but only if the possibility of immunological rejection can be eliminated. In this study, we investigated the ability of α1,3-galactosyltransferase (α1,3-GT) gene knockout (GTKO) pig cancellous bone to promote the repair of a femoral condyle bone defect and its influence on heterologous immune rejection. Materials and methods: Cylindrical bone defects created in a rhesus monkey model were transplanted with GTKO bone, WT bone or left empty. For immunological evaluation, T lymphocyte subsets CD4+ and CD8+ in peripheral blood were assayed by flow cytometry, and the IL-2 and IFN-γ contents of peripheral blood serum were analyzed by ELISA at 2, 5, 7, 10, and 14 days post-surgery. Micro-CT scans and histological assessment were conducted at 4 and 8 weeks after implantation. Results: Compared with WT-pig bone, the heterologous immunogenicity of GTKO-pig bone was reduced. The defect filled with fresh GTKO-pig bone was tightly integrated with the graft. Histological analysis showed that GTKO-pig cancellous bone showed better osseointegration and an appropriate rate of resorption. Osteoblast phenotype progression in the GTKO group was not affected, which revealed that GTKO-pig bone could not only fill and maintain the bone defect, but also promote new bone formation. Conclusion: GTKO-pig cancellous bone decreased the ratio of CD4+ to CD8+ T cells and cytokines (IFN-γ and IL-2) to inhibit xenotransplant rejection. Moreover, GTKO group increased more bone formation by micro-CT analysis and osteoblastic markers (Runx2, OSX and OCN). Together, GTKO-pig cancellous bone showed better bone repair than WT-pig cancellous bone.

Transplantation of human cells into Interleukin-2 receptor gamma gene knockout pigs under several conditions.

IntroductionPreviously, we performed gene knockout (KO) of interleukin-2 receptor gamma (IL2RG) in porcine fetal fibroblasts using zinc finger nuclease-encoding mRNAs, subsequently generating IL2RG KO pigs using these cells through somatic cell nuclear transfer. The IL2RG KO pigs lacked a thymus and were deficient in T lymphocytes and natural killer cells, similar to human X-linked severe combined immunodeficiency (SCID) patients. The present study aimed to evaluate whether pigs can support the growth of xenografted human cells and have the potential to be an effective animal model.MethodsThe IL2RG XKOY pigs used in this study were obtained by mating IL2RG XKOX females with wild-type boars. This permitted the routine production of IL2RG KO pigs via natural breeding without complicated somatic cell cloning procedures; therefore, a sufficient number of pigs could be prepared. We transplanted human HeLa S3 cells expressing the tandem dimer tomato into the ears and pancreas of IL2RG KO pigs. Additionally, a newly developed method for the aseptic rearing of SCID pigs was used in case of necessity.ResultsTumors from the transplanted cells quickly developed in all pigs and were verified by histology and immunohistochemistry. We also transplanted these cells into the pancreas of designated pathogen-free pigs housed in novel biocontainment facilities, and large tumors were confirmed.ConclusionsIL2RG KO pigs have the potential to become useful animal models in a variety of translational biology fields.

Proteomics Analysis of Aqueous Humor and Rejected Graft in Pig-to-Non-Human Primate Corneal Xenotransplantation.

Although pig-to-non-human primate (NHP) corneal xenotransplantation has shown long-term graft survival, xenogeneic antigen-related immune responses are still stronger than allogeneic antigen-associated responses. Therefore, there is an unmet need to investigate major rejection pathways in corneal xenotransplantation, even with immunosuppression. This study aimed to identify biomarkers in aqueous humor for predicting rejection and to investigate rejection-related pathways in grafts from NHPs transplanted with porcine corneas following the administration of steroids combined with tacrolimus/rituximab. NHPs who had received corneas from wild-type (WT) or α-1,3-galactosyltransferase gene-knockout (GTKO) pigs were divided into groups with or without rejection according to clinical examinations. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the proteomes of corneal tissues or aqueous humor. The biological functions of differentially expressed proteins (DEPs) were assessed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for pathways and protein–protein interaction network analysis. Among the 66 DEPs in aqueous humor, complement proteins (C3, C5, and C9) and cholesterol metabolic proteins (APOA1 and APOA2) were related to xenogeneic rejection as biomarkers, and alternative pathways of the complement system seemed to be important in xenogeneic graft rejection. Among the 416 DEPs of the cornea, NF-κB1 and proteosomes (PSMD7, PSMA5, and PSMD3) seemed to be related to xenogeneic graft rejection. Additionally, oxidative phosphorylation and leukocyte activation-related pathways are involved in rejection. Overall, our proteomic approach highlights the important role of NF-κB1, proteosomes, oxidative phosphorylation, and leukocyte activation-related inflammation in the cornea and the relevance of complement pathways of the aqueous humor as a predictive biomarker of xenogeneic rejection.

Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Genetically engineered pig kidney transplantation in a brain-dead human subject.

In September 2021, a kidney (with donor-specific thymic tissue) from an α1, 3-galactosyltransferase gene-knockout (GTKO) pig was transplanted into the groin (with anastomoses to the femoral vessels) of a brain-dead subject by a surgical team at New York University Langone Health (NYU). It was reported to function immediately, passing urine and excreting creatinine. The experiment was terminated after 54h and, during this period, the kidney did not show macroscopic features of rejection. Does this experiment provide information not available to us previously and does it move the field forward to clinical trials? The information provided was very limited, but the following points are worthy of note. (i) Numerous in vivo studies in nonhuman primates have predicted that the pig kidney would function immediately. (ii) Numerous in vitro studies have predicted that a GTKO pig kidney would not be rejected within the first few days after transplantation into a human subject. (iii) GTKO kidneys are not optimal for clinical transplantation, and the transplantation of a triple-knockout (TKO) pig kidney would have been more relevant. (iv) There was no purpose in transplanting a "thymokidney" without pre-transplant conditioning therapy and follow-up for several months. (v) Because the native kidneys were retained, it is difficult to determine whether the function of the graft was sufficient to support life. (vi) The experiment was announced to the media rather than published in a peer-reviewed medical journal (although hopefully this will follow), suggesting that it was primarily carried out to gain attention to the great potential of xenotransplantation (and/or possibly to NYU). In this respect the experiment was successful. Because of the very limited period of time for which a brain-dead subject can be maintained in a metabolically and hemodynamically stable state, the value of experiments in such subjects will remain very limited. It is hoped that any future similar experiments will be planned to be more relevant to the clinical situation. Nevertheless, the report has stimulated public attention towards xenotransplantation which, unless there is an adverse response to what some might consider to be a bizarre experiment, should be of significant benefit to future progress.

Skeletal muscle-secreted myokine interleukin-6 induces white adipose tissue conversion into beige adipose tissue in myostatin gene knockout pigs

Myostatin (MSTN) is primarily expressed in skeletal muscle and plays an important role in the regulation of muscle growth and development as well as fat deposition; however, little is known about the molecular mechanism through which MSTN regulates body fat deposition. Therefore, in this study, we sought to identify the signaling pathways through which MSTN regulates fat accumulation in pigs. MSTN knockout (MSTN−/−) pigs showed increased muscle mass, decreased fat mass, and a leaner body composition. In this study, we found that the adipose tissue of MSTN−/− pigs exhibits the characteristics of beige adipose tissue, and the mRNA expression levels of beige adipose marker genes, including UCP3, Cidea, and CD137, were significantly increased. Remarkably, the observed beige phenotype was not adipocyte autonomous but rather caused by muscle-secreted myokine interleukin (IL)-6. This occurrence results in increased AMP-activated protein kinase (AMPK) phosphorylation in adipose tissue, which subsequently activates peroxisome proliferator-activated receptor gamma coactivator 1α and the conversion of white adipocytes to beige in pigs. Therefore, we concluded that MSTN deficiency leads to increased IL-6 secretion in skeletal muscle and activates AMPK in adipocytes, thereby increasing the beige adipose tissue in MSTN−/− pigs.

Evidence that sensitization to triple-knockout pig cells will not be detrimental to subsequent allotransplantation.

The current evidence is that sensitization to a pig xenograft does not result in the development of antibodies that cross-react with alloantigens, and therefore, sensitization to a pig xenograft would not be detrimental to the outcome of a subsequent allograft. This evidence relates almost entirely to the transplantation of cells or organs from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. However, it is not known whether recipients of triple-knockout (TKO) pig grafts who become sensitized to TKO pig antigens develop antibodies that cross-react with alloantigens and thus be detrimental to a subsequent organ allotransplant. We identified a single baboon (B1317) in which no (or minimal) serum anti-TKO pig antibodies could be measured-in our experience unique among baboons. We sensitized it by repeated subcutaneous injections of TKO pig peripheral blood mononuclear cells (PBMCs) in the absence of any immunosuppressive therapy. After TKO pig PBMC injection, there was a transient increase in anti-TKO pig IgM, followed by a sustained increase in IgG binding to TKO cells. In contrast, there was no serum IgM or IgG binding to PBMCs from any of a panel of baboon PBMCs (n=8). We conclude that sensitization to TKO pig PBMCs in the baboon did not result in the development of antibodies that also bound to baboon cells, suggesting that there would be no detrimental effect of sensitization on a subsequent organ allotransplant.

MICROCAPSULE AGAINST HYPOXIA SHOWED LONGER GRAFT SURVIVAL THAN MICROCAPSULE AGAINST FIBROSIS IN DIABETIC MICE RECEIVING GALT-KO PORCINE ISLET TRANSPLANTATION

Introduction: Fibrosis is one of the most important barrier to improve encapsulated islet graft survival. Hypoxia is another major obstacle for encapsulated islet transplantation. Previously, we have developed several alginate-based microcapsules improving fibrosis (alginate-chitosan) or hypoxia (alginate-perfluorodecalin) and proved their superior effects compared with alginate microcapsule. In this study, we compared long-term graft survival of encapsulated islet with microcapsule against fibrosis or hypoxia in diabetic mice model, using 1,3-galactosyltransferase gene-knockout (GalT-KO) pigs. Materials and Methods: Islets were isolated form GalT-KO pigs and then encapsulated with alginate-chitosan or alginate-perfluorodecalin microcapsule. To magnify graft survival gap between different microcapsules, marginal doses of islets were transplanted into streoptozotocin-induced diabetic mice intraperitoneally. Blood glucose level were measured for a year. Intraperitoneal glucose tolerance test (IPGTT) was performed at 6 and 12 months after transplantation. Results: In the alginate-chitosan group, the blood glucose level of diabetic mice decreased after encapsulated islet transplantation. However, blood glucose level increased again within two months in 3 out of 4 mice. In the alginate-perfluorodecalin group, the blood glucose level of diabetic mice decreased after transplantation and maintained normoglycemia more than 6 months. Although blood glucose level tended to increase 6 months after transplantation, it remained below 300 mg/dL by one year. The area under curve (AUC) of IPGTT was similar between two groups at baseline. At 6 months after transplantation, AUC of IPGTT in alginate-perflourodecalin group lower than AUC of alginate-chitosan group. At one year after transplantation, microcapsules were retrieved. Dithizone stain was positive in most retrieved islet and fibrosis was minimal in both groups. Conclusion: In conclusion, microcapsule against hypoxia showed a longer xenograft survival than microcapsule against fibrosis in diabetic mice. This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ0134532019)” Rural Development Administration, Republic of Korea.

Low Immunogenicity Pericardia from Gene Knockout Pigs as Biological Valve Materials

The immunogenicity of heterogeneous pericardium is a major factor causing the failure of commercial biological valves. Both decellularization and glutaraldehyde crosslinking can reduce the immunogenicity of pericardium, but it is difficult to completely remove it. Getting gene knockout pigs is considered a highly efficient method to obtain low-immunogenic biological tissues, including the pericardium. Our results showed that repeated cycles of decellularization could obviously reduce the residual DNA in tissues for pigs without gene knockout, but at the same time they could also break down the components of pericardia, resulting in a decline in mechanical properties. We compared the mechanical and biological properties of the glutaraldehyde crosslinked pericardia of GGTA1 knockout pig, GGTA1/CMAH knockout pig, GGTA1/β4GalNT2 knockout pig and wild type (WT) pig. The mechanical properties of the pericardia of GGTA1 knockout pig and GGTA1/CMAH knockout pig were similar to those of control pig (WT), and the experimental results of subdermal implantation in rats showed significantly reduced immune rejection and calcification. Pulsatile flow and fatigue in vitro tests showed that the fabricated artificial aortic valve met the hydrodynamic requirements after 2×10^8 accelerated fatigue cycles. Gene knockout pigs have great potential in the application of artificial biological valve products.

Scaffold derived from GGTA1 and CMAH double knockout pigs elicits only slight inflammation in a gene-edited pig model

Acellular extracellular matrix (ECM) contains α-Gal and N-glycolylneuraminic acid (Neu5Gc) antigens that cause clinical side effects. We prepared porcine cornea and small intestinal submucosa ECM (SIS-ECM) from either wild-type (WT) or knockout (KO) pigs, including GGTA1/CMAH double KO (dKO) and CMAH KO (CMAHKO), through gene editing to eliminate α-Gal and/or Neu5Gc antigens. The quality of the SIS-ECM was confirmed by assessing residual DNA, glycosaminoglycan content and histology. The effects of histopathological and IL-6 responses of SIS-ECM from WT, CMAHKO and dKO pigs were compared to those shown by implanting SIS-ECM into the longissimus of WT and dKO pigs. In the histopathological study, WT and dKO SIS-ECM were simultaneously implanted into WT or dKO pigs. Results indicated granulocyte infiltration but no significant difference between the implanted WT and dKO SIS-ECM, however, multinuclear giant cells were significantly more abundant around WT than dKO SIS-ECM if implanted into dKO pigs. In the IL-6 response study, its concentration was significantly higher in WT SIS-ECM than in dKO SIS-ECM; meanwhile, a moderate level of IL-6 was observed in CMAHKO or WT SIS-ECM implanted into dKO or CMAHKO pigs, respectively. Since dKO SIS-ECM elicits only slight inflammation, we conclude that dKO pigs lacking α-Gal and Neu5Gc can serve as biocompatible SIS-ECM donors or as animal models for testing anthropomorphic immune responses to biomedical devices. Statement of SignificanceXenograft‐derived scaffolds, even acellular scaffolds, encounter the challenges of α-Gal and Neu5Gc nonprotein antigens present in the ECM, including the SIS- and corneal-ECM. Gene-edited pigs with depleted α-Gal and nullified Neu5Gc may represent a valuable source for the procurement of tissues for biomaterial manufacturing. This value is indicated by the evidence that the SIS-ECM prepared from these pigs and not wild-type (WT) animals elicited only slight inflammation histopathologically and serologically if implanted into a double gene-knockout (dKO) pig model with α-Gal and Neu5Gc absent. The ECM prepared from dKO pigs provides a further improvement in biocompatibility, and those animals could be used as a testing model and for precisely evaluating Gal and non-Gal antigen immune responses, regardless of biomaterial or biomedical devices.

2289-PUB: Comparison of GalT-KO Porcine Islet Xenograft Survival between Microcapsule against Hypoxia and Microcapsule against Fibrosis in Diabetic Mice Model

Hypoxia and fibrosis are one of the most important obstacles for encapsulated islet transplantation. Previously, we have developed alginate-based microcapsules improving fibrosis (alginate-chitosan) or hypoxia (alginate-perfluorodecalin) and proved their effects. In this study, we compared long-term graft survival of encapsulated islet with microcapsule against fibrosis or hypoxia in diabetic mice model, using 1,3-galactosyltransferase gene-knockout (GalT-KO) pigs. Islets were isolated form GalT-KO pigs and then encapsulated with alginate-chitosan or alginate-perfluorodecalin microcapsule. To magnify graft survival gap between different microcapsules, marginal dose of islets were transplanted into streoptozotocin-induced diabetic mice intraperitoneally. Blood glucose level were measured for a year. Intraperitoneal glucose tolerance test (IPGTT) was performed at 6 and 12 months after transplantation. In the alginate-chitosan group, the blood glucose level of diabetic mice decreased after encapsulated islet transplantation. However, blood glucose level increased again within two months in 3 out of 4 mice. In the alginate-perfluorodecalin group, the blood glucose level of diabetic mice decreased after transplantation and maintained normoglycemia more than 6 months. Although blood glucose level tended to increase 6 months after transplantation, it remained below 300 mg/dL by one year. The area under curve (AUC) of IPGTT was similar between two groups at baseline. At 6 months after transplantation, AUC of IPGTT in alginate-perflourodecalin group lower than AUC of alginate-chitosan group. At one year after transplantation, microcapsules were retrieved. Dithizone stain was positive in most retrieved islet and fibrosis was minimal in both groups. In conclusion, microcapsule against hypoxia showed a longer xenograft survival than microcapsule against fibrosis in diabetic mice. Disclosure E. Lee: None. Y. Yang: None. Y. Cho: None. S. Lee: None. J. Cho: None. K. Yoon: None. Funding Cooperative Research Program for Agriculture Science and Technology Development (PJ01345301); Rural Development Administration, Republic of Korea

The Scaffold Derived from GGTA1 and CMAH Gene Knockout Pigs Provoked Little Inflammatory Responses in a Humanized Pig Model

The attempt to produce a porcine small intestinal submucosa extracellular matrix (SIS-ECM) that is fully biocompatible was made. Samples of SIS-ECM were prepared from either wild-type (WT) or double-gene knockout (dKO) pigs without α-Gal and N-glycolylneuraminic acid (Neu5Gc) antigens. The scaffolds, which were sutured into cuts made in the longissimus muscle of the dKO pigs, were expected to exhibit xeno-reactions through natural antibodies as in a human response. A process was established to manufacture ameliorating acellular porcine SIS-ECM implants with consistent quality characteristics, which were assured by analyzing the level of residual DNA, the glycosaminoglycan content, and histochemical stains. Once implanted, the acellular SIS-ECM from the WT pigs caused a significant increase in serum IL-6 levels in the dKO recipient pigs, indicating a host defense through immune reactions. The levels remained unchanged when preparations from the dKO pigs were used. The results together with pathological evidences suggested that dKO pigs without antigenic response to α-Gal or Neu5Gc can serve as biocompatible SIS-ECM donors or as animal models for testing anthropomorphic immune responses to biomedical devices.

Intra-bone Bone Marrow Transplantation in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers.

Mixed chimerism and thymic tissue transplantation strategies have achieved xenogeneic tolerance in pig-to-mouse models, and both have been extended to pig-to-baboon models. A mixed chimerism strategy has shown promise toward inducing tolerance in allogeneic models in mice, pigs, nonhuman primates (NHP), humans, and a rat-to-mouse small animal xeno-model. However, even though α-1,3-galactosyltransferase gene knockout (GalTKO) pigs have been used as bone marrow (BM) donors, direct intravenous injection of porcine BM cells was detected for only up to 4days (peripheral macro-chimerism) in one case, and the rest lost chimerism within 2days.Recent data in allogeneic models demonstrated that direct injection of donor BM cells into recipient BM spaces (intra-bone bone marrow transplantation: IBBMTx) produces rapid reconstitution and a higher survival rate compared to i.v. injection. In order to minimize the loss of injected porcine BM peripherally before reaching the BM space, Yamada developed a xeno-specific regimen including IBBMTx coated with a collagen gel matrix in a preclinical pig-to-baboon model (Yamada IBBMTx). This strategy aims to achieve improved, persistent macro-chimerism as well as engraftment of BM across a xenogeneic barrier. The initial study published in 2015 demonstrated that this IBBMTx strategy leads to markedly prolonged peripheral macro-chimerism detectable for up to 23days. Furthermore, a more recent study using human CD47-transgenic (Tg) GalTKO pigs as xeno-donors achieved long-lasting macro-chimerism >60days with evidence of reduction of anti-pig natural antibodies (nAb). This is the longest macro-chimerism that has ever been achieved in a preclinical large animal xenotransplant model to date. In this chapter, we introduce a brief summary of our achievements in regard to successful tolerance induction by utilizing our novel strategy of IBBMTx as well as describe the step-by-step methodology of surgical and in vitro procedures that are required for this project.

Profiling natural serum antibodies of non-human primates with a carbohydrate antigen microarray.

Engineering of α-Galactosyltransferase gene-knockout pigs circumvented hyperacute rejection of pig organs after xenotransplantation in non-human primates. Overcoming this hurdle revealed the importance of non-α-Gal carbohydrate antigens in the immunobiology of acute humoral xenograft rejection. This study analyzed serum from seven naïve cynomolgus monkeys (blood type O/B/AB=3/2/2) for the intensity of natural IgM and IgG signals using carbohydrate antigen microarray, which included historically reported α-Gal and non-α-Gal carbohydrate antigens with various modifications. The median (range) of IgM and IgG signals were 12.71 (7.23-16.38) and 9.05 (7.23-15.90), respectively. The highest IgM and IgG signals with narrowest distribution were from mono- and disaccharides, followed by modified structures. Natural anti-α-Gal antibody signals were medium to high in IgM (11.2-15.9) and medium in IgG (8.5-11.6) spectra, and was highest with Lac core structure (Galα1-3Galβ1-4Glc, iGb3) and lowest with LacNAc core structure (Galα1-3Galβ1-4GlcNAc). Similar signal intensities (up to 15.8 in IgM and up to 11.8 in IgG) were observed for historically detected natural non-α-Gal antigens, which included Tn antigen, T antigen, GM2 glycolipid, and Sda antigen. The hierarchical clustering analysis revealed the presence of clusters of anti-A antibodies and was capable of distinguishing between the blood group B and AB non-human primates. The results presented here provide the most comprehensive evaluation of natural antibodies present in cynomolgus monkeys.

Human CTLA4-Ig therapy can give false-positive anti-pig antibody results in primates after xenotransplantation

BackgroundMeasurement of serum anti-pig antibodies is an important parameter in immune monitoring after pig-to-nonhuman primate xenotransplantation. Pig aortic endothelial cells (pAECs) are commonly used for this purpose. However, human (h) CTLA4-Ig (abatacept/belatacept) could bind to pCD80/86 on the cells, and a secondary antibody (i.e., anti-human IgG) may recognize hCTLA4-Ig (in the absence of serum anti-pig IgG antibody binding to pAECs), potentially leading to misinterpretation of the results. Our aim was to determine whether hCTLA4-Ig binding to pAECs is associated with false-positive results. MethodsSera were obtained from (i) naïve baboons (n = 3) and (ii) baboons (n = 2) that had undergone pig artery patch transplantation with/without hCTLA4Ig therapy. Serum IgM and IgG binding to (i) AECs, (ii) red blood cells (RBCs), and (iii) CD3+T cells in peripheral blood mononuclear cells (PBMCs) from an α1,3-galactosyltransferase gene-knockout pig expressing human CD46 (GTKO/hCD46) was measured by flow cytometry in the presence or absence of hCTLA-4Ig. Complement-dependent cytotoxicity (CDC) of wild-type (WT) pAECs by hCTLA4Ig was measured by flow cytometry. ResultsSera containing hCTLA4-Ig demonstrated significantly increased IgG (but not IgM) binding to pAECs (relative geometric mean [rGM] = 1.8) compared to sera without hCTLA-4Ig (rGM =1.3) (p < .01). In contrast, there was no increased binding to pRBCs or CD3+T cells. hCTLA4-Ig did not result in cytotoxicity of WT pAECs. ConclusionspAECs might not be an optimal cell to investigate anti-pig IgG binding when hCTLA4-Ig is administered to the recipient, as a false-positive result may result from hCTLA4-Ig binding to the pAECs. CD3+T cells would be preferable targets (compared to pRBCs) because they express both carbohydrate and MHC class I/II antigens.

Downregulation of Gabarapl1 significantly attenuates antibody binding to porcine aortic endothelial cells.

After hyperacute rejection in pig-to-primate xenotransplantation had been overcome by the introduction of α1,3-galactosyltransferase gene-knockout (GTKO) pigs, acute and chronic antibody-mediated rejection became one of the major barriers to long-term graft survival. This was associated with exposure of non-Gal antigens to the recipient's immune system and indicated that further genetic engineering of the pigs would be necessary. We here report that Gabarapl1, a regulator of tumorigenesis, plays a role in the regulation of immunogenicity of porcine aortic endothelial cells (PAECs). Knockdown of Gabarapl1 in PAECs results in a remarkable reduction in binding of serum antibody from PAEC-immunized monkeys, associated with decreased serum cytotoxicity of pig cells. Expression of swine leukocyte antigens (SLA) II DR was downregulated by Gabarapl1 knockdown. However, suppression of expression of SLA II is associated with less reduction of antibody binding than achieved by Gabarapl1 knockdown, suggesting that other Gabarapl1-regulated xenoantigens may be more important. These findings indicate a hitherto unknown relationship between Gabarapl1 and xenoimmunogenicity, suggesting a potential new strategy to reduce rejection initiated by the presence of non-Gal antigens.

Indicators of impending pig kidney and heart xenograft failure: Relevance to clinical organ xenotransplantation - Review article.

In pig-to-baboon organ xenotransplantation, coagulation dysfunction and inflammation have been suggested to be associated with acute humoral xenograft rejection. We have evaluated platelet counts, plasma fibrinogen, and parameters of inflammation as indicators of xenograft failure in baboons with kidney and heart grafts from genetically-engineered pigs. Blood chemistry, hematologic, immune, and inflammatory parameters were measured in recipient baboons (n = 16) with organs from α1,3-galactosyltransferase gene-knockout pigs expressing human complement- and coagulation-regulatory proteins. Thrombocytopenia and reduction of plasma fibrinogen level were observed in baboons developing graft failure, and these correlated with histopathologic findings of glomerular and interstitial thrombosis, and vasculitis in the graft. Not infrequently, in baboons with pig kidney grafts, a consumptive coagulopathy developed prior to a rise in serum creatinine. In contrast, when kidney graft survival was prolonged, no changes were observed in platelet count or fibrinogen. Indicators of the inflammatory response, particularly the serum amyloid A (SAA) assay, increased when graft failure was developing. There were no changes in cellular immune parameters, e.g., T or B cell counts or phenotypes that indicated graft failure. Therefore, in clinical xenotransplantation, noninvasive parameters (e.g., platelet count, fibrinogen level, SAA) might provide more reliable indicators of impending xenograft failure than measurements of immune parameters or even of serum creatinine.