Carol Phelps

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.

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 …

Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft

Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

PIG-TO-MONKEY ISLET XENOTRANSPLANTATION USING MULTI-TRANSGENIC PIGS

The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3‐galactosyltransferase gene‐knockout background with ubiquitous expression of human CD46, with islet beta cell‐specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4‐lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin‐diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti‐CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi‐transgenic islet grafts did not demonstrate consistency in regard to long‐term success, with only two of five demonstrating function beyond 5 months.

Pig kidney graft survival in a baboon for 136 days: longest life‐supporting organ graft survival to date

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.

The role of genetically engineered pigs in xenotransplantation research

There is a critical shortage in the number of deceased human organs that become available for the purposes of clinical transplantation. This problem might be resolved by the transplantation of 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 non‐human primates (NHPs) 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 five or six 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. Copyright

Hepatic Function After Genetically Engineered Pig Liver Transplantation in Baboons

Background. If “bridging” to allo-transplantation (Tx) is to be achieved by a pig liver xenograft, adequate hepatic function needs to be assured. Methods. We have studied hepatic function in baboons after Tx of livers from &agr;1,3-galactosyltransferase gene-knockout (GTKO, n=1) or GTKO pigs transgenic for CD46 (GTKO/CD46, n=5). Monitoring was by liver function tests and coagulation parameters. Pig-specific proteins in the baboon serum/plasma were identified by Western blot. In four baboons, coagulation factors were measured. The results were compared with values from healthy humans, baboons, and pigs. Results. Recipient baboons died or were euthanized after 4 to 7 days after internal bleeding associated with profound thrombocytopenia. However, parameters of liver function, including coagulation, remained in the near-normal range, except for some cholestasis. Western blot demonstrated that pig proteins (albumin, fibrinogen, haptoglobin, and plasminogen) were produced by the liver from day 1. Production of several pig coagulation factors was confirmed. Conclusions. After the Tx of genetically engineered pig livers into baboons (1) many parameters of hepatic function, including coagulation, were normal or near normal; (2) there was evidence for production of pig proteins, including coagulation factors; and (3) these appeared to function adequately in baboons although interspecies compatibility of such proteins remains to be confirmed.

Clinical lung xenotransplantation--what donor genetic modifications may be necessary?

Cooper DKC, Ekser B, Burlak C, Ezzelarab M, Hara H, Paris L, Tector AJ, Phelps C, Azimzadeh AM, Ayares D, Robson SC, Pierson RN III. Clinical lung xenotransplantation – what donor genetic modifications may be necessary? Xenotransplantation 2012; 19: 144–158.

Human dominant‐negative class II transactivator transgenic pigs – effect on the human anti‐pig T‐cell immune response and immune status

Swine leucocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4+ T cells. A human dominant‐negative mutant class II transactivator (CIITA‐DN) transgene under a CAG promoter with an endothelium‐specific Tie2 enhancer was constructed. CIITA‐DN transgenic pigs were produced by nuclear transfer/embryo transfer. CIITA‐DN pig cells were evaluated for expression of SLA class II with/without activation, and the human CD4+ T‐cell response to cells from CIITA‐DN and wild‐type (WT) pigs was compared. Lymphocyte subset numbers and T‐cell function in CIITA‐DN pigs were compared with those in WT pigs. The expression of SLA class II on antigen‐presenting cells from CIITA‐DN pigs was significantly reduced (40–50% reduction compared with WT; P < 0·01), and was completely suppressed on aortic endothelial cells (AECs) even after activation (100% suppression; P < 0·01). The human CD4+ T‐cell response to CIITA‐DN pAECs was significantly weaker than to WT pAECs (60–80% suppression; P < 0·01). Although there was a significantly lower frequency of CD4+ cells in the PBMCs from CIITA‐DN (20%) than from WT (30%) pigs (P < 0·01), T‐cell proliferation was similar, suggesting no significant immunological compromise. Organs and cells from CIITA‐DN pigs should be partially protected from the human cellular immune response.

Generation of antibody- and B cell-deficient pigs by targeted disruption of the J-region gene segment of the heavy chain locus

A poly(A)-trap gene targeting strategy was used to disrupt the single functional heavy chain (HC) joining region (JH) of swine in primary fibroblasts. Genetically modified piglets were then generated via somatic cell nuclear transfer (SCNT) and bred to yield litters comprising JH wild-type littermate (+/+), JH heterozygous knockout (±) and JH homozygous knockout (−/−) piglets in the expected Mendelian ratio of 1:2:1. There are only two other targeted loci previously published in swine, and this is the first successful poly(A)-trap strategy ever published in a livestock species. In either blood or secondary lymphoid tissues, flow cytometry, RT-PCR and ELISA detected no circulating IgM+ B cells, and no transcription or secretion of immunoglobulin (Ig) isotypes, respectively in JH −/− pigs. Histochemical and immunohistochemical (IHC) studies failed to detect lymph node (LN) follicles or CD79α+ B cells, respectively in JH −/− pigs. T cell receptor (TCR)β transcription and T cells were detected in JH −/− pigs. When reared conventionally, JH −/− pigs succumbed to bacterial infections after weaning. These antibody (Ab)- and B cell-deficient pigs have significant value as models for both veterinary and human research to discriminate cellular and humoral protective immunity to infectious agents. Thus, these pigs may aid in vaccine development for infectious agents such as the pandemic porcine reproductive and respiratory syndrome virus (PRRSV) and H1N1 swine flu. These pigs are also a first significant step towards generating a pig that expresses fully human, antigen-specific polyclonal Ab to target numerous incurable infectious diseases with high unmet clinical need.