Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1997-09-15
2002-07-02
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S320100, C435S455000, C424S093210
Reexamination Certificate
active
06413769
ABSTRACT:
Donor organ shortages have led to hopes that xenotransplantation could serve as an alternative means of organ availability. Swine, particularly mini-swine, are an attractive alternative to non-human primate donors because of potentially greater availability, the reduced risk of zoonotic infections, appropriate size of organs and the reduced social and ethical concerns (Sachs, D. H. et al. 1976. Transplantation 22:559-567; Auchincloss, H. Jr. 1988. Transplantation 46:1-20). However, one of the major barriers to xenotransplantation is the phenomenon described as hyperacute rejection (Busch et al. 1972. Am. J. Pathology 79:31-57; Auchincloss, H. Jr. 1988. Transplantation 46:1-20). This phenomenon describes a very rapid and severe humoral rejection, which leads to destruction of the graft within minutes or hours of the transplant of the donor organ. Hyperacute rejection is apparently mediated by a complex series of events, including activation of the complement systems, activation of blood coagulation proteins, activation of endothelial cells and release of inflammatory proteins (Busch et al. 1972. Am. J. Pathology 79:31-57; Platt, J. L. 1992. ASAIO Journal 38:8-16). There is an accumulating body of information that implicates a group of pre-formed antibodies, the so-called natural antibodies, to be of fundamental importance in the hyperacute rejection seen in grafts between species. Species combinations in which the recipients of grafts have circulating antibodies that can initiate the hyperacute response to the donor species are described as discordant. Pigs and humans are one such discordant species combination.
The hyperacute rejection process is initiated when the natural antibodies of the recipient bind to cells of the donor organ (Platt et al. 1990. Transplantation 50:870-822; Platt et al. 1990. Immunology Today 11:450-456). It has been suggested that porcine N-linked carbohydrates carrying a terminal Gal&agr;1-3Gal&bgr;1-4GlcNAc structure are the major targets for anti-swine xenoreactive human natural antibodies (Good et al. 1992. Transplantation Proceedings 24:559-562; Sandrin et al. 1993. Proc. Natl. Acad. Sci. USA 90:11391-11395). One major difference between the glycosylation pattern of swine tissues and human tissues is the presence of high levels of a terminal Gal&agr;1-3Gal&bgr;1-4GlcNAc structure on swine cells and tissues. This structure is expressed at high levels in all lower mammals investigated, but is poorly expressed on cells and tissues of Old World monkeys, apes and humans (catarrhines) (Galili, U. and Swanson, K. 1992. Proc. Natl. Acad. Sci. USA 88:7401-7404; Galili et al. 1987. Proc. Natl. Acad. Sci. USA. 84:1369-1373). A specific transferase, UDP-Gal:Gal&bgr;1→4GlcNAc &agr;1→3-galactosyltransferase (EC 2.4.1.151; &agr;(1,3) galactosyltransferase) is responsible for the transfer of a terminal galactose to the terminal galactose residue of N-acetyllactosamine-type carbohydrate chains and lactosaminoglycans according to the reaction:
where R may be a glycoprotein or a glycolipid (Blanken, W. M. and Van den Eijinden, D. H. 1985. J. Biol. Chem. 260:12927-12934). Thus the Gal&agr;1-3Gal&bgr;1-4GlcNAc epitope. Full length CDNA sequences encoding the murine (Larsen et al. 1989. Proc. Natl. Acad. Sci. USA. 86:8227-8231) and bovine (Joziasse et al. 1989. J. Biol. Chem. 264:14290-14297) enzymes have been determined. In addition, the genomic organization of the murine a(1,3) galactosyltransferase gene has been established (Joziasse et al. 1992. J. Biol. Chem. 267:5534-5541). A partial sequence encoding the 3′ region of the porcine &agr;(1,3) galactosyltransferase cDNA gene has been determined (Dabkowski et al. 1993. Transplantation Proceedings. 25:2921) but the full length sequence has not been reported. The absence of the 5′ sequence is significant for the applications described herein. In contrast to the lower mammals, humans do not express the &agr;(1,3) galactosyltransferase. Furthermore, human sequences homologous to the murine sequence correspond to a processed pseudogene on chromosome 12 and an inactivated remnant on chromosome 9 (Shaper et al. 1992. Genomics 12:613-615).
In accordance with the invention, swine organs or tissues or cells that do not express &agr;(1, 3) galactosyltransferase will not produce carbohydrate moieties containing the distinctive terminal Gal&agr;1-3Gal&bgr;1-4GlcNAc epitope that is a significant factor in xenogeneic, particularly human, transplant rejection of swine grafts. Further in accordance with the invention, is the aspect of diminishing the production of &agr;(1,3) galactosyltransferase to an extent sufficient to prevent the amount produced from providing carbohydrates with the Gal&agr;1-3Gal&bgr;1-4GlcNAc epitope from being presented to the cell surface thereby rendering the transgenic animal, organ, tissue, cell or cell culture immunogenically tolerable to the intended recipient without requiring complete &agr;(1,3) galactosyltransferase gene suppression.
One principal aspect of the present invention is that the inventors have isolated the entire porcine &agr;(1,3) galactosyltransferase CDNA gene (SEQ. ID NO. 1). The identification, isolation and sequencing of the entire cDNA gene, now particularly providing the sequence of the 5′ end is an important advance because, as described in Example 2, this region has been identified as the most efficient for antisense targeting. Moreover, as compared with mouse and bovine homologous sequences (FIG.
2
), this region of the &agr;(1,3) galactosyltransferase MRNA appears to deviate extensively between these species making it extremely unlikely that a use of “cross-species” antisense constructs would be successful.
Another principle aspect of this invention related to genetically altered animals, more specifically transgenic, chimeric or mosaic swine in which the expression of biologically active &agr;(1,3) galactosyltransferase is prevented in at least one organ, tissue or cell type. Transgenic animals carry a gene which has been introduced into the germline of the animal, or an ancestor of the animal, at an early developmental stage. The genetic alteration in transgenic animals is stably incorporated into the genome as a result of intentional experimental intervention. Typically, this results from the addition of exogenous foreign DNA or novel constructs (Palmiter et al. 1986. Ann. Rev. Genet. 20:465). With the advent of embryonic stem (ES) cells and specific gene targeting, the definition of transgenesis now includes specific modification of endogenous gene sequences by direct experimental manipulation and by stable incorporation of DNA that codes for effector molecules that modulate the expression of endogenous genes (Gossler et al. 1986. Proc. Natl. Acad. Sci. USA. 83:9065; Schwarzberg et al. 1989. Science 246:799; Joyner et al. 1989. Nature 338:153).
One preferred approach for generating a transgenic animal involves micro-injection of naked DNA into a cell, preferentially into a pronucleus of an animal at an early embryonic stage (usually the zygote/one-cell stage). DNA injected as described integrates into the native genetic material of the embryo, and will faithfully be replicated together with the chromosomal DNA of the host organism. This allows the transgene to be passed to all cells of the developing organism including the germ line. Transgene DNA that is transmitted to the germ line gives rise to transgenic offspring. If transmitted in a Mendelian fashion, half of the offspring will be transgenic. All transgenic animals derived from one founder animal are referred to as a transgenic line. If the injected transgene DNA integrates into chromosomal DNA at a stage later than the one cell embryo not all cells of the organism will be transgenic, and the animal is referred to as being genetically mosaic. Genetically mosaic animals can be either germ line transmitters or non-transmitters. The general approach of microinjection of heterologous DNA constructs into early embryonic cells is usually restricted to the generation of dominant effects, i.e.,
Baetscher Manfred W.
Gustafsson Kenth T.
Sachs David H.
Crouch Deborah
Lillie Raymond J.
Olstein Elliot M.
University of London
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