David Ayares

Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs

Galactose-α1,3-galactose (α1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig α1,3-galactosyltransferase (α1,3GT) by homologous recombination is a means to completely remove the α1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig α1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig α1,3GT allele.

Long‐Term Controlled Normoglycemia in Diabetic Non‐Human Primates After Transplantation with hCD46 Transgenic Porcine Islets

Xenotransplantation of porcine islets into diabetic non‐human primates is characterized by (i) an initial massive graft loss possibly due to the instant blood‐mediated inflammatory reaction and (ii) the requirement of intensive, clinically unfriendly immunosuppressive therapy. We investigated whether the transgenic expression of a human complement‐regulatory protein (hCD46) on porcine islets would improve the outcome of islet xenotransplantation in streptozotocin‐induced diabetic Cynomolgus monkeys. Immunosuppression consisted of thymoglobulin, anti‐CD154 mAb for costimulation blockade, and mycophenolate mofetil. Following the transplantation of islets from wild‐type pigs (n = 2) or from 1,3‐galactosyltransferase gene‐knockout pigs (n = 2), islets survived for a maximum of only 46 days, as evidenced by return to hyperglycemia and the need for exogenous insulin therapy. The transplantation of islets from hCD46 pigs resulted in graft survival and insulin‐independent normoglycemia in four of five monkeys for the 3 months follow‐up of the experiment. One normalized recipient, selected at random, was followed for >12 months. Inhibition of complement activation by the expression of hCD46 on the pig islets did not substantially reduce the initial loss of islet mass, rather was effective in limiting antibody‐mediated rejection. This resulted in a reduced need for immunosuppression to preserve a sufficient islet mass to maintain normoglycemia long‐term.

A Porcine-Derived Acellular Dermal Scaffold That Supports Soft Tissue Regeneration: Removal of Terminal Galactose-α-(1,3)-Galactose and Retention of Matrix Structure

Sub-optimal clinical outcomes after implantation of animal-derived tissue matrices may be attributed to the nature of the processing of the material or to an immune response elicited in response to xenogeneic epitopes. The ability to produce a porcine-derived graft that retains the structural integrity of the extracellular matrix and minimizes potential antigenic response to galactose-alpha-(1,3)-galactose terminal disaccharide (alpha-Gal) may allow the scaffold to support regeneration of native tissue. Dermal tissue from wild-type (WT-porcine-derived acellular dermal matrix [PADM]) or Gal-deficient (Gal(-/-) PADM) pigs was processed to remove cells and DNA while preserving the structural integrity of the extracellular matrix. In addition, the WT tissue was subjected to an enzymatic treatment to minimize the presence of alpha-Gal (Gal-reduced PADM). Extracellular matrix composition and integrity was assessed by histological, immunohistochemical (IHC), and ultrastructural analysis. In vivo performance was evaluated by implantation into the abdominal wall of Old World primates in an exisional repair model. Anti-alpha-Gal activity in the serum of monkeys implanted subcutaneously was assessed by ELISA. Minimal modification to the extracellular matrix was assessed by evaluation of intact structure as demonstrated by staining patterns for type I and type VII collagens, laminin, and fibronectin similar to native porcine skin tissues. Explants from the abdominal wall showed evidence of remodeling, notably fibroblast cell repopulation and revascularization, as early as 1 month. Serum ELISA revealed an initial anti-alpha-Gal induction that decreased to baseline levels over time in the primates implanted with WT-PADM, whereas no or minimal anti-Gal activity was detected in the primates implanted with Gal(-/-) PADM or Gal-reduced PADM. The combination of a nondamaging process, successful removal of cells, and reduction of xenogeneic alpha-Gal antigens from the porcine dermal matrix are critical for producing a material with the ability to remodel and integrate into host tissue and ultimately support soft tissue regeneration.

THE INNATE IMMUNE RESPONSE AND ACTIVATION OF COAGULATION IN α1,3-GALACTOSYLTRANSFERASE GENE-KNOCKOUT XENOGRAFT RECIPIENTS

Background. The role of the innate immune system in the development of thrombotic microangiopathy (TM) after α1,3-galactosyltransferase gene-knockout (GTKO) pig organ transplantation in primates is uncertain. Methods. Twelve organs (nine hearts, three kidneys) from GTKO pigs were transplanted into baboons that received no immunosuppressive therapy, partial regimens, or a full regimen based on costimulation blockade. After graft failure, histologic and immunohistologic examinations were carried out. Results. Graft survival of less than 1 day was prolonged to 2 to 12 days with partial regimens (acute humoral xenograft rejection) and to 5 and 8 weeks with the full regimen (TM). Clinical or laboratory features of consumptive coagulopathy occurred in 7 of 12 baboons. Immunohistochemistry demonstrated IgM, IgG, and complement deposition in most cases. Histopathology demonstrated neutrophil and macrophage infiltrates, intravascular fibrin deposition, and platelet aggregation (TM). Grafts showed expression of primate tissue factor (TF), with increased mRNA levels, and TF was also expressed on baboon macrophages/monocytes infiltrating the graft. Conclusions. Our data suggest that (1) irrespective of the presence or absence of the adaptive immune response, early or late xenograft rejection is associated with activation of the innate immune system; and (2) porcine endothelial cell activation and primate TF expression by recipient innate immune cells may both contribute to the development of TM.

Carbohydrates in xenotransplantation

The success of allotransplantation has led to an increasing shortage of human organs from deceased donors. This crisis could be resolved by the use of organs from an anatomically suitable animal, such as the pig. The pig and human have, however, been evolving differently for approximately 80 million years, and numerous immunological and physiological barriers have developed that need to be overcome. Differences in carbohydrate epitopes on pig and human cells have been found to play a major role in some of the immunological barriers that have been identified to date. The rejection caused by the presence of galactose‐α1,3‐galactose (Gal) on the pig vascular endothelium and of natural anti‐Gal antibodies in humans has recently been prevented by the breeding of pigs that do not express Gal, achieved by knocking out the gene for the enzyme α1,3‐galactosyltransferase, which was made possible by the introduction of nuclear transfer/embryo transfer techniques. N‐glycolylneuraminic acid (the so‐called Hanganutziu‐Deicher antigen) has been identified as another carbohydrate antigen present in pigs that may need to be deleted if xenotransplantation is to be successful, although some doubt remains regarding its importance. There remain other antipig antibodies against hitherto unidentified antigenic targets that may well be involved in graft destruction; their possible carbohydrate target epitopes are discussed.

Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs.

Abstract:  Background:  The presence of multiple copies of porcine endogenous retrovirus (PERV) within the pig genome, and the demonstration that replication competent PERV, that infect human cells in culture, can be isolated from pig cells, directly impacts the drive towards the development of pigs for xenotransplantation. The development of technology to produce pigs that do not propagate PERV has the potential to facilitate the development of xenotransplantation products for human use, and as such, is the focus of this investigation. The shear number of PERV loci, most of which are defective or pseudogenes, renders conventional gene targeting impractical, if not impossible, to inactivate all PERV provirus within the pig genome, including potential replication competent PERV arising from spontaneous recombination. The recently developed RNA interference (RNAi) technology to knockdown/silence post‐transcriptional gene expression, offers a promising alternative to achieving this goal.

Current status of xenotransplantation and prospects for clinical application

Abstract:  Xenotransplantation is one promising approach to bridge the gap between available human cells, tissues, and organs and the needs of patients with diabetes or end‐stage organ failure. Based on recent progress using genetically modified source pigs, improving results with conventional and experimental immunosuppression, and expanded understanding of residual physiologic hurdles, xenotransplantation appears likely to be evaluated in clinical trials in the near future for some select applications. This review offers a comprehensive overview of known mechanisms of xenograft injury, a contemporary assessment of preclinical progress and residual barriers, and our opinions regarding where breakthroughs are likely to occur.

Clinical islet xenotransplantation: how close are we?

Type 1 diabetes (T1D) is a major health problem throughout the world. In the U.S., it is estimated that about 1.5 million people suffer from T1D. Even when well controlled—by frequent monitoring of blood glucose and administration of insulin, the long-term complications of the disease are significant and include cardiovascular disease, nephropathy, retinopathy, and neuropathy (1). Here we review recent progress in preclinical models of pig islet xenotransplantation and discuss the remaining challenges that need to be addressed before the application of this form of therapy can be established in patients with T1D. During the past decade, islet allotransplantation alone (without previous kidney transplantation) using deceased human donor pancreata has been indicated mainly in patients who have had T1D for >5 years with life-threatening hypoglycemic episodes and wide fluctuations in blood glucose levels. Although the initial long-term results were rather disappointing (2), the results of islet allotransplantation have improved significantly in recent years, with 5-year insulin-independent normoglycemia achieved in >50% of patients at experienced centers (3). There is increasing evidence that successful islet allotransplantation greatly reduces the incidence of hypoglycemic episodes (2) and reduces or slows the incidence of late complications of T1D (4). This may extend the indications for islet transplantation to patients with progressive complications. For example, islet transplantation in a patient with preterminal renal failure may prevent disease progression, possibly avoiding the need for hemodialysis and kidney transplantation, provided that nonnephrotoxic immunosuppressive drug therapy is administered. Currently, in the U.S., the median waiting time for a kidney allograft from a deceased human donor is >4 years (5). However, islets from two deceased human donor pancreata are frequently required to achieve normoglycemia in a diabetic patient. Because of the limited number of suitable deceased donor pancreata, the overall number of …

Reduction of early graft loss after intraportal porcine islet transplantation in monkeys

Background. Pig islets constitute a possible resolution to the shortage of human islets for transplantation. After intraportal infusion of porcine islets in primates, many islets are lost through what has been termed the instant blood-mediated inflammatory reaction (IBMIR). We report on our experience with IBMIR. Methods. Ten monkeys underwent intraportal porcine islet transplantation. Immunosuppressive therapy was with conventional agents (n=3) or based on costimulation blockade (n=7). Treatment specific for IBMIR was administered in eight monkeys; two additional monkeys received no such therapy (group 1). Cobra venom factor completely inhibited complement activity in four (group 2) and dextran sulfate provided anticoagulation in four (group 3). Islet graft function was monitored by following blood glucose, insulin requirement, and porcine C-peptide values. Results. In monkeys that received neither cobra venom factor nor dextran sulfate (group 1), there was rapid destruction of islets indicated by severe hypoglycemia and the need for dextrose infusion; C-peptide levels were initially low and further reduction occurred within the first five days. In both groups 2 and 3, there was significantly less destruction of islets and some reversal of diabetes. However, when 40,000 IEQ/kg were infused, normoglycemia was lost within five days, but when 80,000 IEQ/kg were infused in one case, normoglycemia was more persistent. We observed that even when C-peptide levels were in the normal range for healthy nondiabetic pigs, these were not sufficient to maintain normoglycemia in the monkeys. Conclusions. Although pretransplantation complement depletion or anticoagulation reduces porcine islet xenograft loss significantly, neither alone is sufficient to prevent IBMIR.

In vitro investigation of pig cells for resistance to human antibody-mediated rejection.

Although human complement‐dependent cytotoxicity (CDC) of α1,3‐galactosyltransferase gene‐knockout (GTKO) pig cells is significantly weaker than that of wild‐type (WT) cells, successful xenotransplantation will require pigs with multiple genetic modifications. Sera from healthy humans were tested by (i) flow cytometry for binding of IgM/IgG, and (ii) CDC assay against peripheral blood mononuclear cells and porcine aortic endothelial cells from five types of pig – WT, GTKO, GTKO transgenic for H‐transferase (GTKO/HT), WT transgenic for human complement regulatory protein CD46 (CD46) and GTKO/CD46. There was significantly higher mean IgM/IgG binding to WT and CD46 cells than to GTKO, GTKO/HT, and GTKO/CD46, but no difference between GTKO, GTKO/HT, and GTKO/CD46 cells. There was significantly higher mean CDC to WT than to GTKO, GTKO/HT, CD46, and GTKO/CD46 cells, but no difference between GTKO and GTKO/HT. Lysis of GTKO/CD46 cells was significantly lower than that of GTKO or CD46 cells. CD46 expression provided partial protection against serum from a baboon sensitized to a GTKO pig heart. GTKO/CD46 cells were significantly resistant to lysis by human serum and sensitized baboon serum. In conclusion, the greatest protection from CDC was obtained by the combination of an absence of Gal expression and the presence of CD46 expression, but the expression of HT appeared to offer no advantage over GTKO. Organs from GTKO/CD46 pigs are likely to be significantly less susceptible to CDC.