Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – The nonhuman animal is a model for human disease
Reexamination Certificate
2000-07-19
2003-08-26
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
The nonhuman animal is a model for human disease
C800S003000, C800S004000, C800S008000, C800S013000, C800S018000, C800S021000
Reexamination Certificate
active
06610905
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to transgenic animals and their use as a disease model system to study Kaposi's sarcoma and the effects of various therapeutic agents on disease onset and progression. The invention also relates to the use of transgenic animals as a molecular model in the study of vGPCR and molecules affected by vGPCR's action or which affect vGPCR's function.
BACKGROUND OF THE INVENTION
Kaposi's sarcoma was considered to be a rare tumor, but its incidence has dramatically increased with the AIDS epidemic and is at present the most common tumor associated with HIV infection. In 10 to 20% of HIV-1-infected patients, Kaposi's sarcoma has led to a significant morbidity from edema, lymphatic obstruction, organ infiltration and pulmonary dysfunction. Up to 30% of patients have died from complications of Kaposi's sarcoma.
Human herpesvirus 8 (“HHV8”), a &ggr;-2 lymphotropic herpesvirus, appears to be the etiological agent associated with Kaposi's sarcoma (Ganem, 1997
, Cell
91:157-160; Greenblatt, 1998
Infect. Dis. Clin. North Am
. 12:63-82), but its role in Kaposi's sarcoma pathogenesis has remained an enigma (Gallo, 1998
Science
282:1837-1839). The present invention provides a key link between HHV8 and Kaposi's sarcoma pathogenesis. HHV8 infects malignant and progenitor cells of Kaposi's sarcoma, it encodes putative oncogenes and genes that may cause Kaposi's sarcoma pathogenesis by stimulating angiogenesis. The G-protein-coupled receptor (vGPCR) encoded by an open reading frame (ORF 74) of HHV8 is expressed in Kaposi's sarcoma lesions and stimulates signaling pathways constitutively linked to cell proliferation and cell death.
Understanding the role of vGPCR in HHV-8 replication and its overall function in the development of Kaposi's sarcoma has been hampered by the lack of an in vivo model. It is therefore an object of the present invention to generate a transgenic animal expressing vGPCR. This transgenic animal represents and important new tool to further dissect the pathogenesis of Kaposi's sarcoma.
SUMMARY OF THE INVENTION
The present invention relates to the identification of a model system to study Kaposi's sarcoma and other similar diseases.
A transgenic non-human animal model for disease and molecular mechanism is provided, together with methods and compositions for preparation of the animal model and methods for using it. The invention provides a transgenic non-human animal whose genome comprises a constitutively active G-coupled receptor protein transgene comprising regulatory sequences from a promoter operably linked to a coding sequence, wherein expression of said transgene produces Kaposi's sarcoma-like symptoms.
The invention also provides a transgenic non-human animal embryo whose somatic and germ cells contain a gene encoding a constitutively active G-protein coupled receptor.
The invention further provides a method for producing a non-human animal having somatic and germ cells that contain an HHV8 gene encoding a constitutively active G-protein coupled receptor which comprises the steps of introducing multiple copies of an expression cassette into the non-human mammal at an embryonic stage, and developing the embryo to term in a pseudo-pregnant foster female. The expression cassette comprises a human herpesvirus 8 gene encoding a chemokine receptor sequence operably joined to regulatory sequences for expression of the coding sequence in hematopoietic cells. The resulting transgenic non-human mammals develop Kaposi's sarcoma-like lesions in multiple organs, which are characterized by one or more of the symptoms including intense angiogenic activity, presence of spindle and inflammatory cells, and expression of vGPCR, CD34 and VEGF.
The transgenic animals are useful, for example, in screening protocols for agents which can be used for treatment and/or prevention of Kaposi's sarcoma and diseases/conditions associated with vGPCR, CD34 or VEGF.
DETAILED DESCRIPTION OF THE INVENTION
In order that the invention described herein may be more fully understood, the following detailed description is set forth. All references cited herein are incorporated in their entirety by reference.
The present invention provides a non-human transgenic animal which is heterozygous for a functional HHV8 gene encoding a chemokine receptor, including a constitutively active G-protein coupled receptor (vGPCR) that binds several CC and CXC chemokines or homologues thereof. As used herein, functional is used to describe a gene or protein that, when present in a cell or in vitro system, performs normally as if in a native or unaltered condition or environment.
The animals of this invention are useful for the study of the tissue and temporal specific expression or activity of vGPCR in animals having a functional copy of the gene. The animals are also useful for studying the ability of a variety of compounds to act as modulators of vGPCR activity or expression in vivo or, by providing cells for culture, in vitro. As used herein, a modulator is a compound that causes a change in the expression or activity of vGPCR, or causes a change in the effect of the interaction of vGPCR with its ligand(s), or other protein(s).
In reference to the transgenic animals of this invention, “transgenes” or “genes” will be referred to. As used herein, a transgene is a genetic construct including a gene. The transgene is integrated into one or more chromosomes in the cells in an animal by methods know in the art. Once integrated, the transgene is carried in at least one place in the chromosomes of the transgenic animal. A gene is a nucleotide sequence that encodes a protein. The gene and/or transgene may also include genetic regulatory elements and/or structural elements known in the art.
Another aspect of the invention is a non-human animal embryo carrying the gene encoding vGPCR. This embryo is useful in studying the effects of Kaposi's sarcoma in the developing animal. In particular embodiments, the animal is a mouse. The animal embryo is also a source of cells carrying a functional vGPCR gene.
The present invention is further directed to a transgenic non-human eukaryotic animal, preferably a rodent, such as a mouse or other animal capable of developing detectable characteristics from the expression of vGPCR. The animal expresses vGPCR sequence in hematopoietic cells such that the animal develops Kaposi's sarcoma-like lesions in one or more organs, including for example, intense angiogenic activity, presence of spindle and inflammatory cells, teleangiectasia, erythematous maculae, plaques or tumors, or expression of vGPCR, CD34 or VEGF within a short period of time from birth, generally within a year from birth, preferably within 1 to 4 months from birth. The vGPCR sequence is introduced into the animal, or an ancestor of the animal, at an embryonic stage, preferably the one cell, or fertilized oocyte stage and generally not later than about the 8-cell stage. The zygote or embryo is then developed to term in a pseudo-pregnant foster female. The vGPCR genes are introduced into an animal embryo so as to be chromosomally incorporated in a state which results in super-endogenous expression of the vGPCR protein and the development of a Kaposi's sarcoma-like disease. The proliferative vascular lesions in affected transgenic animals are indicative of a Kaposi's sarcoma-like disease.
The present invention offers several advantages over existing models for progressive vascular lesions, characterized in Kaposi's sarcoma-like disease. The vascular lesions are often multicentric and seen in the skin, heart, skeletal muscle and submucosa and tunica muscularic of the small and large intestine. These lesions often consisted of dilated thin-walled blood vessels lined by normal or plump endothelial cells and spindle-shaped cells surrounding irregular vascular channels containing erythrocytes. Cellular pleomorphism may also be seen in more extensive lesions, but mitotic figures ar
Lira Sergio A.
Yang Tong-Yuan
Biro Michael G.
Crouch Deborah
McLaughlin Jaye P.
Schering Corporation
Thampoe Immac J.
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