Michael J. Martin

Human complement regulatory proteins protect swine-to-primate cardiac xenografts from humoral injury

The susceptibility of xenografts to hyperacute rejection is postulated to reflect in part failure of complement regulatory proteins (CRPs) to control activation of heterologous complement on graft endothelium. To test this concept, transgenic swine expressing the human CRP decay accelerating factor and CD59 were developed using a novel expression system involving transfer of the proteins from erythrocytes to endothelial cells. Hearts from transgenic swine transplanted into baboons had markedly less vascular injury and functioned for prolonged periods compared to hearts from nontransgenic swine. These results indicate that expression of human CRPs in xenogeneic organs may contribute to successful xenografting and suggest that intercellular protein transfer might be a useful approach for expression of heterologous proteins in endothelial cells.

Transgenic pigs expressing human CD59 and decay-accelerating factor produce an intrinsic barrier to complement-mediated damage.

We characterize a line of transgenic pigs that express the human complement-regulatory proteins human CD59 and human decay-accelerating factor. These genes, under the control of heterologous promoters, are expressed in a variety of organs, including the vasculature of the heart, kidney, and liver. We demonstrate that moderate levels of these gene products are sufficient to protect peripheral blood cells from human or baboon complement. Using pig to baboon heterotopic heart transplants, we show that expression of these proteins is sufficient to block the complement-mediated damage that is the hallmark of such xenografts, when nontransgenic organs are used. These results indicate that there is significant species specificity of intrinsic complement regulatory protein function. This specificity is evident in transgenic organs in which low levels of human CD59 and human decay-accelerating factor expression significantly effect the humoral immune response that causes xenograft rejection. This result suggests that transgenic organs with high levels of human complement-regulatory protein expression will be sufficient to alleviate the humoral immunological barriers that currently block the use of xenogeneic organs for human transplantation.

A human CD46 transgenic pig model system for the study of discordant xenotransplantation.

Background. Thechronic shortage in the supply of human organs available for allotransplantation has turned attention toward the use of animals as potential donors, with pigs as the most likely species under consideration. Hyperacute rejection, the initial and immediate barrier to a pig-to-primate xenograft, has been addressed by generation of transgenic pigs that express the human membrane-bound complement-regulatory proteins CD59 and/or CD55. Difficulty has been encountered in generation of transgenic animals that express a third membrane-bound complement-regulatory protein, CD46. Methods. We have generated transgenic animals by using a large genomic construct that encompasses the entire human CD46 gene. Results. We report the first description of transgenic mice and pigs that express high levels of human CD46 in a cell and tissue type-specific manner, resembling patterns of endogenous CD46 expression observed in human tissues. Furthermore, when human CD46 transgenic porcine hearts were transplanted into baboons, the grafts did not succumb to hyperacute rejection, and survival extended for up to 23 days. Under the same conditions, nontransgenic grafts underwent hyperacute rejection within 90 min. Conclusions. This is the first report to describe generation of transgenic pigs that express human CD46, and the first in vivo demonstration of the ability of human CD46 expressed on pig organs to regulate complement activation and overcome hyperacute rejection upon transplantation of a vascularized organ into nonhuman primates.

Characterization of transgenic pigs expressing functionally active human CD59 on cardiac endothelium

The critical shortage of human donor organs has generated interest in the potential for porcine to human xenotransplantation. The initial immunological barrier to xenotransplantation is hyperacute rejection, which is mediated by xenoreactive antibodies and complement, and results in rapid and irreversible tissue destruction. While endogenous complement regulatory proteins (CRPs) protect cells from injury caused by autologous complement, they are relatively species specific and most likely ineffectual in this setting. This has led to the hypothesis that expression of human CRPs in transgenic pigs may affect susceptibility to complement-mediated tissue injury in a porcine-to-human xenograft. Using specific lines of transgenic pigs that express low levels of human CD59, a CRP that acts at the terminal stage of the complement cascade, we present evidence that shows that the human CD59 protein inhibits membrane attack complex assembly and reduces tissue damage when the heart is transplanted to a baboon. Examination by immunohistochemistry of transgenic porcine hearts after transplantation revealed markedly reduced deposition of C5b and MAC, but a similar level of C3 deposition as compared with transplanted control hearts. This finding supports the concept that the species specific function of CRPs contributes to the humoral barrier to xenotransplantation and, given the low level of human CD59 protein expression in the porcine heart, argues that the human protein contributes a unique rather than an additive function in regulation of complement in a xenogeneic setting.

Pig cells that lack the gene for alpha1-3 galactosyltransferase express low levels of the gal antigen.

Background. The major antigen recognized on pig tissue by primate antibodies is a terminal gal&agr;1-3gal carbohydrate structure (gal antigen) present on glycolipids and glycoproteins. The production of animals from somatic cells allows for the inactivation of specific genes. It is anticipated that the complete inactivation of the gene encoding &agr;1-3 galactosyltransferase, the enzyme that synthesizes the gal&agr;1-3gal linkage, will result in loss of that antigen from pig organs and tissue and will provide a survival benefit in pig-to-primate xenotransplants. Methods. Positive-negative selection was used to produce fetal-pig fibroblasts that were a heterozygous knockout (+/−) of the &agr;1-3 galactosyltransferase gene. Nuclear transfer of these cells generated pig embryos and live born pigs with the appropriate genotype. Using a novel selection method with cells from (+/−) embryos, we produced homozygous (−/−) fetal-pig fibroblast cells. Results. Southern blot analysis of the &agr;1-3 galactosyltransferase gene showed that we had produced (+/−) pig embryos, (+/−) live born pigs, and (−/−) pig-fetal fibroblast cells. Fluorescence-activated cell sorter (FACS) analysis with some, but not all, mouse anti-gal monoclonal antibodies and sensitized human serum showed that (−/−) cells still synthesized the gal antigen at 1 to 2% of the level of control heterozygous cells. Conclusions. Fetal-pig fibroblasts homozygous for the knockout of the &agr;1-3 galactosyltransferase gene appear to express low but detectable levels of the gal antigen.

Protection of xenogeneic cardiac endothelium from human complement by expression of CD59 or DAF in transgenic mice

We investigated the ability of membrane-bound human complement regulatory proteins to control complement-driven humoral immune reactions on murine microvasculature. The human complement regulatory proteins CD59 and DAF were expressed using heterologous promoters in a variety of tissues in transgenic mice. Animals expressing these gene products are healthy and exhibit significant levels of endothelial cell expression of CD59 and DAF in cardiac muscle. Transgenic hearts perfused with human plasma exhibited profound reductions in the level of complement deposition compared with nontransgenic controls. We have also produced transgenic pigs that express these two human genes. Our results indicate that expression of complement regulatory proteins can control activation of complement and suggest that these proteins may have therapeutic applications in some inflammatory diseases and in the development of xenogeneic organs for human transplantation.

Transgenic swine as a recombinant production system for human hemoglobin

Publisher Summary Human hemoglobin, after appropriate modification, may be potentially useful as a red blood cell substitute for transporting oxygen from the lungs to the tissues. A recombinant source of human hemoglobin that is separate from the human donor pool has several potential advantages: unlimited supply, controlled and validated source, and use of genetically modified variants. This chapter describes the steps necessary to produce recombinant human hemoglobin in the red blood cells of pigs and illustrates the separation of human and porcine hemoglobins by ion-exchange chromatography. Although hemoglobin A is the initial molecule, a similar process would be appropriate with hemoglobin variants. There has been extensive research on the expression and regulation of human globin genes in transgenic mice and, although it is not yet known at the same level of detail which of the sequences are necessary for expression in transgenic pigs, several features are likely to be important.

Skin graft survival in genetically identical cloned pigs.

Nuclear transfer technology allows for the reprogramming of somatic cells, and the production of embryonic stem cells and animals that are genetically identical in terms of nuclear DNA to the parental somatic cell. It is assumed that these products of nuclear transfer technology will be immunologically compatible to each other in spite of the fact that there are data that show differences in the expression patterns and phenotypes between animals produced by nuclear transfer. We have produced a series of cloned pigs from embryonic fibroblasts. Microsatellite analysis was used to confirm that the clones were genetically identical. Skin transplants were performed to assess immunological reactivity. Skin transplants between genetically identical cloned pigs were accepted, whereas third party grafts were rejected. Histological analysis of the grafts showed edema and mononuclear cell infiltrates in the recipients skin in rejected grafts and not in grafts that were accepted. Our data supports the notion that genetically identical cloned pigs are immunologically compatible.

Transgenic animals with an altitude

In the summer of 1999, scientists representing academia and industry returned to California’s High Sierra and Tahoe City for the second meeting of the Transgenic Animal Research Conference. Once again the University of California at Davis did an outstanding job coordinating the conference. Everyone left the conference enlarged physically and intellectually. The scientific program presented at this year’s conference covered a wide variety of topics and proved to be every bit as informative as its predecessor. Sessions included discussions on:

Alternative Methods for Transgenesis in Domestic Animal Species

While continued refinement and increased efficiency have led to the adoption of nuclear transfer and other homologous recombination-based techniques as the primary means of creating transgenic farm animal species, genetic modification strategies now considered to be “alternatives” (e.g., pronuclear microinjection, cell- or sperm-mediated transfer, and intracytoplasmic sperm injection (ICSI)) are still in use today. This chapter contains discussions on the history, efficacy, and performance of each of these alternative strategies. Because the majority of these technologies require unfertilized or recently fertilized ova, the chapter also presents step-by-step methodologies for all aspects of ova production including: superovulation, recovery, micromanipulation, culture, and transfer. Finally, appendixes that list specific equipment requirements, semen extender compositions, and genes that have been introduced into swine to date are also included.