Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1998-02-18
2001-07-17
Hauda, Karen M. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S022000, C800S025000
Reexamination Certificate
active
06262337
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a transgenic animal containing recombinant DNA having a modified nucleotide sequence from the vascular endothelial growth factor B (VEGF-B) gene and cells derived from the animal. The transgenic animal and cells are useful for research and development in the fields of angiogenesis and tumor development, and particularly for assaying substances for VEGF-B-like activity.
BACKGROUND OF THE INVENTION
Vascular endothelial growth factor (VEGF), as its name implies, is an endothelial cell-specific mitogen. It has potent angiogenic activity, that is, it promotes the growth of new blood vessels. A vital physiological process, angiogenesis implicates a number of proteins of the blood and of the cells in the blood vessels. During the angiogenic process, mitogenic factors, such as VEGF, play an important role. However, the biochemical details of the role these factors play have not always been forthcoming.
Since the identification and characterization of VEGF, a number of important findings have focused attention on the activity of angiogenic factors and the elucidation of new factors. The early findings showed that angiogenesis is required for normal development and physiology. Processes such as embryogenesis, wound healing, and corpus luteum formation, specifically, all involve angiogenesis and angiogenic factors. During wound healing, for example, VEGF mRNA levels increase suggesting a direct correlation between the expression of VEGF and the healing process. Also, a defect in VEGF regulation might be associated with wound healing disorders. (Frank, S., et al., J. Biol. Chem.,2705: 12607-12613 (1995).)
Other important findings related to angiogenic factors indicate that persistent and unregulated angiogenesis exacerbates and causes many diseases. For example, arthritis involves new capillaries invading the joint and destroying cartilage. In diabetes, new capillaries in the retina invade the vitreous humour, causing bleeding and blindness. (Folkman, J. and Shing, Y., J. Biol. Chem., 267(16): 10931-10934 (1992).) The role of angiogenic factors in these and other diseases has not yet been clearly established.
Another important finding involves the connection between angiogenesis and tumor development. Both tumor growth and metastasis are angiogenesis-dependent processes. (Folkman, J. and Shing, Y., J. Biol. Chem., 267(16): 10931-10934 (1992).) For example, when tumor cells are introduced into an animal, the expression pattern of VEGF mRNA reveals expression at the highest level in cells at the periphery of necrotic, tumor growth areas. Numerous blood vessels were identified within these areas. The expression of VEGF in these areas suggests that hypoxemia, a state of deficient oxygenation, triggers expression and release of VEGF in the necrotic tumor. The expression of another vascular endothelial cell mitogen, VEGF-B, discussed more fully below, has also been directly correlated with tumor growth, especially in melanomas. (U.S. application Ser. No. 08/609,443, filed Mar. 1, 1996.) VEGF is a member of a family of proteins structurally related to platelet-derived growth factor (PDGF). PDGF is a potent mitogen for smooth muscle cells, glial cells, and several other cell types. In one aspect, the members of the PDGF family are characterized by the presence of eight conserved cysteine residues. In their active, physiological state, the proteins are dimers formed by disulfide bonding, by both inter- and intramolecular bonds, at the eight cysteine residues. In another aspect, the family members are related in their mitogenic actions, especially on endothelial and related cell types.
Vascular endothelial growth factor B (VEGF-B), a non-glycosylated, highly basic growth factor, is a newly defined member of the PDGF family. With close structural similarities to VEGF, PDGF-A, PDGF-B, and PlGF (Placental Growth Factor), VEGF-B plays a role in vascularization of adult and embryonic tissues, and in muscle tissue in particular. VEGF-B acts also as a angiogenic mitogen. VEGF-B is expressed throughout many tissues in mammals but most abundantly in heart, skeletal muscle, and pancreas. The expression pattern of VEGF-B is different from that of VEGF, although both are expressed in many tissues. (Olofsson, B., et al., Proc. Natl. Acad. Sci. USA, 93:2576-2581 (1996).)
Like its related mitogenic proteins, VEGF-B exists as a disulfide bonded dimer in vivo. As a demonstration of the structural similarity, VEGF-B forms heterodimers with VEGF, consistent with the conservation of the eight cysteine residues involved in inter- and intramolecular disulfide bonding of PDGF-like proteins. Furthermore, the co-expression of VEGF-B and VEGF in many tissues suggests that VEGF-B-VEGF heterodimers occur naturally. VEGF also forms heterodimers with PlGF. (DiSalvo, et al, J. Biol. Chem. 270:7717-7723 (1995).) The production of heterodimeric complexes between the members of this family of growth factors could provide a basis for a diverse array of angiogenic or regulatory molecules.
As noted, VEGF-B is expressed in a number of tissues and cell types. For example, co-pending U.S. application Ser. No. 08/649,443, filed Mar. 1, 1996, specifically incorporated herein by reference, details RT-PCR assays demonstrating the presence of VEGF-B mRNA in melanoma, normal skin, and muscle. In addition, Northern blots show mRNA in a variety of mouse and human tissues, including heart, brain, and skeletal muscle.
Transgenic animal models are useful tools to study the functions and physiological activities of proteins, and a variety of such animals have been produced for this purpose. One particular technique for producing transgenic animals involves the process of homologous recombination. In homologous recombination, all or part of a genomic sequence is replaced with another DNA containing homologous sequences. Through transgenic manipulation and homologous recombination, a gene or part of a gene in the cells of an animal can be changed. Changing the gene to encode a protein that no longer functions as the native protein does creates a null mutant or null allele. (See, for example, U.S. Pat. No. 5,557,032.)
To study VEGF function and physiology, transgenic embryos containing a null mutant for the VEGF gene have been reported. (Carmetliet, P., et al., Nature, 380:435-439 (1996).) Some important findings resulted. The formation of blood vessels was abnormal, but not abolished, in embryos heterozygous for the VEGF null mutant. Embryos homozygous for the VEGF null mutant demonstrate an even greater impairment in angiogenesis. The homozygous embryos died in mid-gestation. Similar phenotypes were observed in heterozygous offspring embryos, generated by germ line transmission. However, since the Carmetliet et al. study was confined to embryos, no phenotype or use of animals was reported. Furthermore, the production of the VEGF transgenic embryos has not produced findings on the angiogenic properties or tumor growth modulating properties of all VEGF-like proteins.
Thus, despite the efforts of the art to elucidate the function and physiology of angiogenic factors, these factors are still incompletely understood, and there remains a need for means which can be used to evaluate the activities of angiogenic growth factors such as VEGF-B, as well as their roles in the various disease states discussed above, and/or which are useful to develop and/or evaluate other angiogenic peptides.
SUMMARY OF THE INVENTION
In one of its aspects, the invention involves production of transgenic animals that contain a mutated VEGF-B gene. The VEGF-B gene was mutated through a homologous recombination process. There are a number of uses for these transgenic animals. For example, study of the animals may provide important information on the therapeutic use and administration of VEGF-B polypeptides, fragments thereof, or analogs such as small molecules. In particular, the transgenic animals of the invention may be useful in elucidating the effects of VEGF-B and/or other cytokines on physiological phenomena such as permeabilit
Aase Karin
Betsholtz Christer
Eriksson Ulf
Gebre-Medhin Samuel
Li Xuri
Evenson, McKeown, Edwards & Lenahan P.L.L.C.
Hauda Karen M.
Ludwig Institute for Cancer Research
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