The unavailability of acceptable human donor organs, the low rate of long term success due to host versus graft rejection, and the serious risks of infection and cancer are serious challenges now facing the field of tissue and organ transplantation. Because the demand for acceptable organs exceeds the supply, many people die each year while waiting for organs to become available. To help meet this demand, research has been focused on developing alternatives to allogenic transplantation. For example, dialysis has been available to patients suffering from kidney failure, artificial heart models have been tested, and other mechanical systems have been developed to assist or replace failing organs. Such approaches, however, are quite expensive, and the need for frequent and periodic access to such machines greatly limits the freedom and quality of life of patients undergoing such therapy.
Xenograft transplantation represents a potentially attractive alternative to artificial organs for human transplantation. The potential pool of nonhuman organs is virtually limitless, and successful xenograft transplantation would not render the patient virtually tethered to machines as is the case with artificial organ technology. Pigs are considered the most likely source of xenograft organs. The supply of pigs is plentiful, breeding programs are well established, and their size and physiology are compatible with humans. Therefore, xenotransplantation with pig organs offers a solution to the shortage of organs available for clinical transplantation.
Host rejection of such cross-species tissue, however, remains a major concern in this area, and the success of xenotransplantion depends on avoiding rejection of the foreign species organ. The immunological barriers to xenotransplantation have been, and remain, formidable. The first immunological hurdle is “hyperacute rejection” (HAR). HAR can be defined by the ubiquitous presence of high titers of pre-formed natural antibodies binding to the foreign tissue. The binding of these natural antibodies to target epitopes on the donor organ endothelium is believed to be the initiating event in HAR. This binding, within minutes of perfusion of the donor organ with the recipient blood, is followed by complement activation, platelet and fibrin deposition, and ultimately by interstitial edema and hemorrhage in the donor organ, all of which cause failure of the organ in the recipient (Strahan et al. (1996) Frontiers in Bioscience 1, e34-41).
Glycoproteins and glycolipids are present on virtually all mammalian cell membranes, and play important roles in the structure and physiology of the cell (Kolter T and Sandhoff K, (1998) Brain Pathol 8:79-100). Glycolipids that contain the Forssman antigen (pentaglycosylceramide) (GalNAcα(1,3)GalNAcβ(1,3)Galα(1,4)Galβ(1,4)Galβ(1,1)Cer) are found on the cells of many mammals, including pigs (Copper et al. (1993) Transplant Immunol 1:198-205). This antigen is chemically related to the human A, B, and 0 blood antigens. However, the glycolipids of Old World monkeys, apes, and humans do not normally contain FSM antigens, although certain malignancies in humans have been shown to express this particular antigen (Hansson G C et al. (1984) FEBS Lett. 170:15-18; —Stromberg N et al. (1988) FEBS Lett. 232:193-198). Although humans do express the FSM antigen precursor—globotriaosylceramide (Xu H et. al. (1999) 274(41):29390-29398), it is not converted to the FSM antigen. In other mammals, the modification of this FSM antigen precursor with the addition of an N-acetylgalactosamine via the FSM synthetase enzyme creates the Forssman antigen.
Because humans lack the FSM antigen, exposure to discordant cells, tissues or organs containing the antigen can lead to the development of anti-FSM antigen antibodies. This antibody development can ultimately play a role in the rejection of FSM antigen containing xenografts. Because pig cells express FSM antigen (see, for example, Cooper M A. et al. (1993) Transplant Immunol 1:198-205), the use of pig organs in a xenotransplant strategy can be compromised due to the potential of organ rejection induced by the FSM antigen.
To date, much research has focused on the reduction of immunogenic cell surface carbohydrate epitopes expressed in discordant xenograft organs. For example, the alpha galactosyltransferase (α(1,3)GT) enzyme is one of the molecules that mediates the formation of Galα(1,3)Gal moieties, a highly immunogenic molecule in humans. Research has focused on the modulation of this particular enzyme to reduce or eliminate the expression of Galα(1,3)Gal moieties on the cell surface. The elimination of the α(1,3)GT gene from porcine has long been considered one of the most significant hurdles to accomplishing xenotransplantation from pigs to humans. Recently, this has been accomplished (Dai et al., Science January 17; 299(5605): 411-4 (2003)).
Haslam D B et al. (Biochemistry 93:10697-10702 (1996) describes a cDNA sequence that encodes for canine Forssman synthetase isolated from a canine kidney cDNA library.
Xu H et al. (J. Bio. Chem. 274(41):29390-29398 (1999) describe a cDNA sequence that encodes for human Forssman synthetase isolated from human brain and kidney cDNA libraries.
U.S. Pat. No. 6,607,723 to the Alberta Research Council and Integris Baptist Medical Center describes removing preformed antibodies to various identified carbohydrate xenoantigens, including the FSM antigen, from a recipient's circulation prior to transplantation by extracorporeal perfusion of the recipient's blood over a biocompatible solid support to which the xenoantigens are bound and/or parenterally administering a xenoantibody-inhibiting amount of an identified xenoantigen to the recipient shortly before graft revascularization.
U.S. Pat. No. 6,331,658 to Integris Baptist Medical Center and Oklahoma Medical Research Foundation describes methods for making a non-human tissue or organ less susceptible to antibody-mediated rejection by human serum by genetically engineering the genome of a non-human mammal to stably include a nucleotide sequence encoding a sialyltransferase or a fucosyltransferase in operable linkage with a promoter, wherein the mammal lacks, or has reduced amounts of, on the surface of its organ cells, carbohydrate structures including Forssman saccharides.
U.S. Patent Publication No. 2003/0153044 to Liljedahl et al. discloses a partial cDNA sequence, including portions of exons 4, 5, 6, and 7, of the porcine Forssman synthetase gene.
It is an object of the present invention to provide genomic and regulatory sequences of the porcine Forssman synthetase gene.
It is an additional object of the present invention to provide cDNA, as well as novel variants, of the porcine Forssman synthetase gene.
It is another object of this invention to provide novel nucleic acid and amino acid sequences that encode the Forssman synthetase protein.
It is yet a further object of the present invention to provide cells, tissues and/or organs deficient in the porcine FSM synthetase gene.
It is another object of the present invention to generate animals, particularly pigs, lacking a functional porcine FSM synthetase gene.