Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2000-04-05
2004-11-30
Paras, Peter (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S017000, C800S018000
Reexamination Certificate
active
06825395
ABSTRACT:
TECHNICAL FIELD
This invention provides transgenic mammals. Particularly, the invention provides the nonhuman transgenic mammals carrying the human complement-inhibitor (hDAF/CD55) gene. More particularly, the invention provides domestic and laboratory animals carrying the hDAF gene.
BACKGROUND OF THE INVENTION
Recently, studies on animal-to-man organ transplantation (xenotransplantation) have been carried out mainly in European countries and the United States. Because of close relation to human beings, apes may be desirable donors, but the use of their organs may be infeasible because of the shortage of these animals and their high intelligence. However, domestic animals, particularly pigs, have advantages of their organ sizes and shapes similar to those of man, easy supply due to mass rearing and established basic technology. Consequently, organ transplantation from the pig to man has mainly been studied.
If a porcine organ is transplanted to man, it will immediately (within minutes) and severely be rejected (hyperacute rejection), resulting in loss of its functions.
These phenomena are thought to be caused by a series of reactions: (1) Human blood contains endogenous antibodies against porcine cells (termed natural antibodies). If a porcine organ is transplanted to man, such antibodies recognize the porcine organ and form antigen-antibody complexes. (2) The antigen-antibody complexes activate complement in human serum and trigger the complement cascade reaction. The attachment of C1 to the antigen-antibody complexes triggers reactions of C4 and C2. resulting in formation of C3 convertase, which activates C3 and cleaves it to C3b and C3a. The attachment of C3b to the cell surface of the porcine organ results in formation of C5 convertase, which activates C5 and cleaves it to C5b and C5a. The attachment of C5b to the cell surface results in sequential attachments of C6, C7, C8 and C9. (3) In consequence of the complement cascade reaction, the membrane attack complex (MAC) is formed (termed the classical complement pathway). MAC attaches the transplanted organ and causes thrombosis. (4) The alternative complement pathway is known to cause also the same cascade reaction as described above after the C3 step and finally to form MAC.
Miyagawa, S. et al. (Transplantation, Vol. 46(6), 825-830, 1988) reported the following: (1) the complement cascade reaction triggered hyperacute rejection of xenografts via the classical and/or alternative pathway; (2) no hyperacute rejection occurred, if the recipients had previously been treated with CVF (cobra venom factor) to cause deprivation of C3. From such findings, it has long been desired to generate transgenic animals expressing membrane-bound DAF and/or MCP, especially those homologous to recipient species, which can inhibit the cascade reaction at the C3 step.
It has been tried to generate transgenic pigs expressing a complement inhibitor hDAF (CD55) to decompose human C3 convertase in the porcine organs (Rosengard, A. M. et al., Transplantation, Vol. 59(9). 1325-1333, 1995: G. Byrne et al., Transplantation Proceedings, Vol. 28(2), 759, 1996).
However, it has never been explained whether these transgenic pigs completely suppresses hyperacute rejection. Therefore, questions like the following should be answered: 1) Do these transgenic pigs express sufficient amounts of hDAF in target organs? 2) Is it necessary to co-express some other complement inhibitors? 3) Isn't it necessary to express sugar-transferase gene in order to reduce the antigen (sugar-chain antigen), which Is expressed on the porcine cells and to which human natural antibodies bind? 4) Isn't it necessary to co-express the above-described gene and other genes encoding the thrombosis-preventing protein and the like? Thus, many problems are left unsolved to overcome the hyperacute rejection.
To solve these problems, it is urgent to generate pigs and/or other small-sized laboratory animals that can be handled more easily than pigs and to examine these animals from various viewpoints. Particularly, in order to carry out studies in this field and/or to develop clinical application, it is valuable to generate transgenic pigs and/or small-sized easy-to-handle laboratory animals, of which tissues and organs express hDAF of at least the same amounts as or larger amounts than those expressed in man.
Therefore, it has been tried to generate transgenic pigs expressing the human complement inhibitors as described above. Expression was examined by such methods as the following; (1) in vitro immunohistological examination, (2) ex vivo examination by allowing the transgenic pig tissues to contact directly with human blood, or (3) in vivo examination by transplanting the transgenic pig tissues to primates. It was confirmed that the tissues from the transgenic pigs survived and functioned longer than those from nontransgenic pigs in ex vivo and in vitro examinations.
However, it was not necessarily explained whether the amounts of the human complement inhibitors expressed in the transgenic pig tissues were at least equivalent to or larger than those expressed in man.
To generate transgenic pigs expressing the human complement inhibitors, the following have been reported as the promoter genes of transgenes: (1) the promoter genes from nonporcine sources (G. A. Langford et al., Transplant. Proc., 26, 1400, 1994; W. L. Fodor et al., Proc. Natl. Acad. Sci. USA., 91, 11153-11157, 1994; G. W. Byrne et al., Transplantation, 63, 149-155, 1997) and/or (2) the promoter genes relating to molecules distributed throughout the whole bodies of animals (e.g., beta-actin, H2K
b
).
Transgenic mice expressing hDAF have also been generated (N. Cary et al., Transplant. Proc. Vol. 25(1), 400-401, 1993; D. Kagan et al., Transplant. Proc. Vol.26(3), 1242, 1994). The loci and amounts of hDAF expressed in these transgenic mice, however, varied from report to report. Strictly speaking, no transgenic mouse expressing the human complement inhibitor in the due organ to develop it (particularly, vascular endothelial cells) in an amount larger than that expressed in human organ has ever been generated.
To solve the above problems, the present inventors studied to generate transgenic animals, particularly those other than man, expressing complement inhibitor(s) in the due organs, tissues and cells, particularly the vascular endothelial cells, in which the complement inhibitors should essentially be expressed. The inventors succeeded in generating transgenic animals fulfilling the purposes with the promoter gene of the porcine complement inhibitor (pMCP) previously invented by the inventors (see Japanese Patent Application No. 142961/1997), by introducing the transgene designed to express the complement inhibitor(s) in the due organs, tissues and cells, particularly in the vascular endothelial cells, in which the complement inhibitors should essentially be expressed, into animals' fertilized eggs, by implanting the eggs in the uteri of recipient animals and by obtaining their youngs.
The examples described below show that the transgenic mice of this invention expressed hDAF in various organs, tissues, endothelial cells, erythrocytes, and central and peripheral nerves in amounts larger than those expressed in human cells. Furthermore, the expression of hDAF was confirmed in their erythrocytes and nerves of the transgenic pigs of the invention.
This invention was accomplished on the basis of such findings. The purpose of the invention was to provide transgenic animals useful in the medical and pharmacological fields.
DISCLOSURE OF THE INVENTION
This invention is related to nonhuman mammals carrying the human complement inhibitor (DAF/CD55) gene and expressing the inhibitor in their organs and tissues. Furthermore, the invention is related to transgenic mammals expressing the human complement inhibitor (DAF/CD55) in their vascular endothelial cells, particularly in those of all the organs and tissues.
It is favorable that the transgenic mammals of the invention are carrying the promoter ge
Fujimura Tatsuya
Murakami Hiroshi
Shigehisa Tamotsu
Takahagi Yoichi
Toyomura Koji
Nippon Meat Packers, Inc.
Paras Peter
Sughrue & Mion, PLLC
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