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Genetic engineering of source pigs for xenotransplantation: Progress and prospects
... It was therefore determined that the pig should be genetically engineered to protect it from these coagulation discrepancies [41]. As with CRPs, efforts were made to introduce transgenes of human coagulation regulatory proteins (eg thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor, CD39, CD73) [42][43][44][45]. ...
... Multi-gene transgenic pigs have been produced that express not only GTKO and CRPs but also multiple anticoagulant transgenes, including various combinations of thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor and CD39 [41]. The expression of one or more human coagulation-regulatory proteins increases graft survival further. ...
... Ten years ago, developing a pig with a single genetic modification was a prolonged process, involving either pronuclear injection to add genes or homologous recombination for gene knockout. Techniques by which this can be achieved more rapidly, including the addition of co-expressed strings of transgenes using multi-cistronic 2A technology, have been introduced [41]. ...
There is a critical shortage in the number of deceased human organs that become available for purposes of clinical transplantation. This problem might be resolved by the transplantation or organs from pigs genetically-engineered to protect them from the human immune response. The pathobiological barriers to successful pig organ transplantation in primates include activation of the innate and adaptive immune systems, coagulation dysregulation, and inflammation. Genetic engineering of the pig as an organ source has increased the survival of the transplanted pig heart, kidney, islet and corneal graft in nonhuman primates (NHP) from minutes to months or occasionally years. Genetic engineering may also contribute to any physiological barriers that might be identified as well as to reducing the risks of transfer of a potentially infectious micro-organism with the organ. There are now an estimated 40 or more genetic alterations that have been carried out in pigs, with some pigs expressing 5 or 6 manipulations. With the new technology now available, it will become increasingly common for a pig to express even more genetic manipulations, and these could be tested in the pig-to-NHP models to assess their efficacy and benefit. It is therefore likely that clinical trials of pig kidney, heart, and islet transplantation will become feasible in the near future.
... The CRISPR/Cas9 system technology allows the rapid production of pigs with multiple genetic modifications [366][367][368][369][370][371], which is having an impact on developing new pigs for xenotransplantation [280,372]. Worldwide, there are currently at least 40 different genetic modifications expressed in pigs, with some pigs expressing six of these (Table 2) [157,315,373]. ...
The immunologic barriers to successful xenotransplantation are related to the presence of natural anti-pig antibodies in humans and nonhuman primates that bind to antigens expressed on the transplanted pig organ (the most important of which is galactose-α1,3-galactose [Gal]) and activate the complement cascade, which results in rapid destruction of the graft, a process known as hyperacute rejection. High levels of elicited anti-pig IgG may develop if the adaptive immune response is not prevented by adequate immunosuppressive therapy, resulting in activation and injury of the vascular endothelium. The transplantation of organs and cells from pigs that do not express the important Gal antigen (α1,3-galactosyltransferase gene-knockout [GTKO] pigs) and express one or more human complement-regulatory proteins (hCRP, e.g., CD46, CD55), when combined with an effective costimulation blockade-based immunosuppressive regimen, prevents early antibody-mediated and cellular rejection. However, low levels of anti-nonGal antibody and innate immune cells and/or platelets may initiate the development of a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. This pathogenic process is accentuated by the dysregulation of the coagulation–anticoagulation systems between pigs and primates. The expression on GTKO/hCRP pigs of a human coagulation-regulatory protein, e.g., thrombomodulin, is increasingly being associated with prolonged pig graft survival in nonhuman primates.
... These genetic manipulations, [19][20][21][22][23][24][25] coupled with improvements in the immunosuppressive regimens used, resulted in further progress in pig-to-NHP organ transplant models. It is this progress that we review here. ...
There has recently been considerable progress in the results of pig organ transplantation in nonhuman primates (NHPs), largely associated with the availability of (i) pigs genetically-engineered to overcome coagulation dysregulation, and (ii) novel immunosuppressive agents. The barriers of thrombotic microangiopathy and/or consumptive coagulation were believed to be associated with (i) activation of the graft vascular endothelial cells (VECs) by a low level of anti-pig antibody binding and/or complement deposition and/or innate immune cell activity, and (ii) molecular incompatibilities between the NHP and pig coagulation-anticoagulation systems. The introduction of a human coagulation-regulatory transgene, eg, thrombomodulin, endothelial protein C receptor, into the pig VECs has contributed to preventing a procoagulant state from developing, resulting in a considerable increase in graft survival. In the heterotopic (non-life-supporting) heart transplant model, graft survival has increased from a maximum of 179 days in 2005 to 945 days. After life-supporting kidney transplantation, survival has been extended from 90 days in 2004 to 499 days. In view of the more complex coagulation dysfunction seen after pig liver and, particularly, lung transplantation, progress has been less dramatic, but the maximum survival of a pig liver has been increased from 7 days in 2010 to 29 days, and of a pig lung from 4 days in 2007 to 9 days. There is a realistic prospect that the transplantation of a kidney or heart, in combination with a conventional immunosuppressive regimen, will enable long-term recipient survival.
... Table 1).9 Both pig donors were CMV negative.T A B L E 1 Genetic modifications of donor pigs and kidney graft survival in baboons EPCR and tissue factor pathway inhibitor [TFPI]) as well as CD47. ...
Background:
Genetically engineered pigs could provide a source of kidneys for clinical transplantation. The two longest kidney graft survivals reported to date have been 136 and 310 days, but graft survival >30 days has been unusual until recently.
Methods:
Donor pigs (n=4) were on an α1,3-galactosyltransferase gene-knockout (GTKO)/human complement regulatory protein (CD46) background (GTKO/CD46). In addition, the pigs were transgenic for at least one human coagulation regulatory protein. Two baboons received a kidney from a six-gene pig (GroupA) and two from a three-gene pig (GroupB). Immunosuppressive therapy was identical in all four cases and consisted of anti-thymoglobulin (ATG)+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R mAbs were administered to reduce the inflammatory response. Baboons were followed by clinical/laboratory monitoring of immune/coagulation/inflammatory/physiological parameters. At biopsy or euthanasia, the grafts were examined by microscopy.
Results:
The two GroupA baboons remained healthy with normal renal function >7 and >8 months, respectively, but then developed infectious complications. However, no features of a consumptive coagulopathy, eg, thrombocytopenia and reduction of fibrinogen, or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response, and histology of biopsies taken at approximately 4, 6, and 7 months and at necropsy showed no significant abnormalities. In contrast, both GroupB baboons developed features of a consumptive coagulopathy and required euthanasia on day 12.
Conclusions:
The combination of (i) a graft from a specific six-gene genetically modified pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory therapy prevented immune injury, a protein-losing nephropathy, and coagulation dysfunction for >7 months. Although the number of experiments is very limited, our impression is that expression of human endothelial protein C receptor (±CD55) in the graft is important if coagulation dysregulation is to be avoided.
... Genetically engineered pigs (Revivicor, Blacksburg, VA, USA), weighing 7 to 30 kg, all of non-A (O) blood group, served as sources of hearts, livers, or carotid artery patch grafts (Table 1) [15][16][17][18][19]. Four baboons received heterotopic hearts from a1,3-galactosyltransferase gene-knockout (GTKO) pigs expressing two human complement regulatory proteins (CD46/CD55), and five from GTKO/ CD46 pigs expressing one or three human coagulation regulatory proteins (GTKO/CD46/thrombomodulin [TBM] [n = 3] or GTKO/CD46/CD55/ TBM/endothelial cell protein C receptor [EPCR]/ CD39 [n = 2]) [18,20] (Table 1). ...
Background:
It has been well documented that the level of serum/plasma free triiodothyronine (fT3) falls rapidly following brain death or during certain surgical procedures, for example, heart surgery carried out on cardiopulmonary bypass. The level in patients following cardiopulmonary bypass usually recovers within 2 days.
Methods:
We have measured serum fT3 in healthy naïve baboons (n = 31), healthy naïve monkeys (n = 5), and after pig-to-baboon heterotopic heart xenotransplantation (xenoTx) (Group 1, n = 9), orthotopic liver xenoTx (Group 2, n = 10), artery patch xenoTx (Group 3, n = 9), and in monkey-to-monkey heterotopic heart alloTx (Group 4, n = 5).
Results:
The mean level of fT3 in healthy naïve baboons was 3.1 ± 0.9 pg/ml and in healthy naïve monkeys was 2.6 ± 0.3 pg/ml. Following pig heart, liver, and artery patch xenoTx and monkey heart alloTx, there was an immediate rapid fall in fT3 level. Recovery of fT3 was more rapid in Groups 3 and 4 than in Groups 1 and 2. In Group 1, within 4 days fT3 had recovered, but only to the lower limit of normal range, where it remained throughout follow-up (for up to 42 days). In Group 2, no recovery was seen during the 7 days of follow-up. In immunosuppressed baboons with pig patch grafts that received IL-6R blockade (n = 2), the fT3 tended to rise higher than in those that received no IL-6R blockade (n = 6).
Conclusions:
Following operative procedures, there is a dramatic fall in serum fT3 levels. The persistent low level of fT3 after pig heart and liver xenoTx may be associated with a continuing inflammatory state. We suggest that consideration should be given to the replacement of T3 therapy to maintain normal fT3 levels, particularly in nonhuman primates undergoing orthotopic pig heart or liver xenoTx.
... The CRISPR/Cas9 system technology allows the rapid production of pigs with multiple genetic modifications [366][367][368][369][370][371], which is having an impact on developing new pigs for xenotransplantation [280,372]. Worldwide, there are currently at least 40 different genetic modifications expressed in pigs, with some pigs expressing six of these (Table 2) [157,315,373]. ...
The immunologic barriers to successful xenotransplantation are related to the presence of natural anti-pig antibodies in humans and non-human primates that bind to antigens expressed on the transplanted pig organ (the most important of which is galactose-α1,3-galactose [Gal]), and activate the complement cascade, which results in rapid destruction of the graft, a process known as hyperacute rejection. High levels of elicited anti-pig IgG may develop if the adaptive immune response is not prevented by adequate immunosuppressive therapy, resulting in activation and injury of the vascular endothelium. The transplantation of organs and cells from pigs that do not express the important Gal antigen (α1,3-galactosyltransferase gene-knockout [GTKO] pigs) and express one or more human complement-regulatory proteins (hCRP, e.g., CD46, CD55), when combined with an effective costimulation blockade-based immunosuppressive regimen, prevents early antibody-mediated and cellular rejection. However, low levels of anti-non-Gal antibody and innate immune cells and/or platelets may initiate the development of a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. This pathogenic process is accentuated by the dysregulation of the coagulation-anticoagulation systems between pigs and primates. The expression in GTKO/hCRP pigs of a human coagulation-regulatory protein, for example, thrombomodulin, is increasingly being associated with prolonged pig graft survival in non-human primates. Initial clinical trials of islet and corneal xenotransplantation are already underway, and trials of pig kidney or heart transplantation are anticipated within the next few years.
... Genetically engineered pigs (Revivicor, Blacksburg, VA), weighing 7 to 30 kg, all of non-A(O) blood group, served as sources of heart or carotid artery patch grafts (Table 1) [12][13][14][15][16][17]. Four baboons received hearts from a1,3-galactosyltransferase gene-knockout (GTKO) pigs expressing two human complement-regulatory proteins (CD46/ CD55) (Group 1A), and four from GTKO/CD46 pigs expressing at least one human complementregulatory and one human coagulation-regulatory protein (Group 1B). ...
In pig-to-baboon heart/artery patch transplantation models, adequate costimulation blockade prevents a T-cell response. After heart transplantation, coagulation dysfunction (thrombocytopenia, reduced fibrinogen, increased D-dimer) and inflammation (increased C-reactive protein [CRP]) develop. We evaluated whether coagulation dysfunction and/or inflammation can be detected following pig artery patch transplantation.
Baboons received heart (n = 8) or artery patch (n = 16) transplants from genetically engineered pigs and a costimulation blockade-based regimen. Heart grafts functioned for 15-130 days. Artery recipients were euthanized after 28-84 days. Platelet counts, fibrinogen, D-dimer, and CRP were measured.
Thrombocytopenia and reduced fibrinogen developed only in recipients of hearts not expressing a coagulation-regulatory protein (n = 4), but not in other heart or patch recipients. However, in heart recipients (n = 8), there were sustained increases in D-dimer (<0.5 to 1.9 ug/ml [P < 0.01]) and CRP (0.26-2.2 mg/dl [P < 0.01]). In recipients of artery patches, there were also sustained increases in D-dimer (<0.5 to 1.4 ug/ml [P < 0.01]) and CRP (0.26 to 1.5 mg/dl [P < 0.001]). An IL-6R antagonist suppressed the increase in CRP, but not D-dimer.
The pig artery patch model has proved valuable for determining immunosuppressive regimens that prevent sensitization to pig antigens. This model also provides information on the sustained systemic inflammation in xenograft recipients (SIXR). An IL-6R antagonist may help suppress this response.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
... The kidney was taken from an a-1,3-galactosyltransferase gene-knockout pig transgenic for two human complement-regulatory proteins (CD46, CD55) and three human coagulation-regulatory proteins (thrombomodulin [TBM], endothelial protein C receptor [EPCR], CD39), weight 8.5 kg, and blood type O (non-A) (Revivicor, Blacksburg, VA) [4]. The promoters for the transgenes were CD46 (endogenous), CD55 and EPCR (CAG), and TBM and CD39 (porcine ICAM-2). ...
The longest survival of a non-human primate with a life-supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α-1,3-galactosyltransferase gene-knockout pig transgenic for two human complement-regulatory proteins and three human coagulation-regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory agents prevented immune injury and a protein-losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Xenotransplantation may become the highly desired solution to close the gap between the availability of donated organs and number of patients on the waiting list. In recent years, enormous progress has been made in the development of genetically engineered donor pigs. The introduced genetic modifications showed to be efficient in prolonging xenograft survival. In this review, we focus on the type of immune responses that may target xeno‐organs after transplantation and promising immunogenetic modifications that show a beneficial effect in ameliorating or eliminating harmful xenogeneic immune responses. Increasing histocompatibility of xenografts by eliminating genetic discrepancies between species will pave their way into clinical application.
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