Transgenic animal and methods

Details Transgenic animal and methods Transgenic animal and methods

2001-07-13

2004-08-24

Falk, Anne-Marie (Department: 1636)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S008000, C800S012000, C800S003000, C435S004000

Reexamination Certificate

active

06781029

ABSTRACT:

1. FIELD OF THE INVENTION
This invention relates to a transgenic animal model of Alzheimer's disease and related neurological disorders in which the animal harbors a transgene encoding a protease inhibitor such as antichymotrypsin (ACT) protein. The invention further relates to transgenic animal models of Alzheimer's disease and related neurological disorders in which the animal harbors a transgene encoding a protease inhibitor such as antichymotrypsin (ACT) protein and one or more further transgenes affecting said neurological disorders. The invention further relates to cells comprising a transgene encoding an antichymotrypsin (ACT) protein. The invention also relates to drug screening assays using the invented transgenic cells or transgenic animals and progeny thereof.
2. BACKGROUND OF THE INVENTION
Biochemical, genetic, and epidemiological evidence indicates that inflammation is an essential part of the pathogenesis of Alzheimer's disease. For example, several acute phase/inflammatory molecules in the brain, specifically antichymotrypsin (ACT) and apolipoprotein E (apoE) can promote the formation of the neurotoxic amyloid deposits that are the main pathological hallmark of the disease. For further details and background information on Alzheimer's disease and related neurological diseases see, for example, U.S. Pat. Nos. 5,297,562; 6,043,283; 5,986,054; 5,981,208; 5,958,883; 5,854,215; 5,849,560; 5,830,670; 5,817,626; 5,773,220; 5,753,624; 5,731,284; 5,705,401; 5,571,671; 5,535,760; 5,506,097; 5,449,604; 5,434,170; 5,429,947; and 5,276,059 as incorporated herein by way of reference.
Amyloid plaque formation is found in a number of diseases including Alzheimer's Disease (AD), scrapie, bovine spongiform encephalopathy, Gerstmann-Straussler Syndrome and related transmissible spongiform encephalopathies (TSEs). These amyloid plaques comprise protein molecules bound together in a fibrinous matrix. Other disorders, such as Creutzfeldt-Jakob's disease, are characterized by the accumulation of amyloidogenic protein without deposition of amyloid plaques. Together these groups of conditions are referred to hereinafter as “Amyloidogenic Diseases.” The present inventors were the first to discover that the acute phase protein, alpha1-antichymotrypsin (ACT) is a structural component of Alzheimer amyloid deposits. In vitro and in vivo studies have shown that the increased ACT expression in Alzheimer's disease is induced in astrocytes by the inflammatory cytokine IL-1 released from reactive microglial cells in the regions of amyloid deposition (Das, S. & Potter, H. “Expression of the Alzheimer amyloid-promoting factor antichymotrypsin is induced in human astrocytes by IL-1” Neuron 14:447-465,1995).
ACT is an inhibitor of chymotrypsin-like serine proteases and is normally produced in the liver as part of the body's “acute phase response” to inflammation. An important function of the acute phase response is to increase the general level of anti-protease activity in the body so as to reduce the potential damage that inflammation-associated proteases can inflict on normal serum proteins and healthy tissue outside of the immediate area of inflammation. The finding that ACT is overexpressed in astrocytes in affected areas of the Alzheimer brain provides the first clear indication that inflammation and an acute phase response in the brain are part of the disease. Other, independent, biochemical and epidemiological studies have confirmed the likelihood that these processes indeed play important roles in the pathogenesis of Alzheimer's disease. The important conclusion is that A&bgr; does not act alone to cause Alzheimer's disease, but acts in concert with an inflammatory cascade, whose products are required for efficient amyloid formation.
The findings that ACT, together with the A&bgr; peptide, is an integral component of the Alzheimer amyloid filaments, and that the mature amyloid deposits are restricted to the same brain regions in which ACT is overproduced, led to the proposal that ACT contributes directly to amyloid formation (Abraham, C. R., Selkoe, D. J. & Potter, H. “Immunohistochemical identification of the serine protease inhibitor &agr;
1
-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease” Cell 52:487-501,1990). When apolipoprotein E was found to be also present in Alzheimer amyloid, suggesting a similar role, the term “pathological chaperone” was coined to describe the potential function of these two, and possibly other proteins, in amyloid formation (Wisniewski, T. & Frangione, B. “Apolipoprotein E: a pathological chaperone protein in patients with cerebral and systemic amyloid” Neurosci. Lett. 135:235-238, 1992). Indeed, when ACT or apoE are added to preparations of synthetic A&bgr; peptide in vitro, they promote the polymerization of A&bgr; into amyloid filaments (Ma, J., Yee, A. Brewer, H. B. Jr. & Das, J. & Potter, H. “Amyloid-associated proteins &agr;
1
-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer's &bgr;-protein into filament” Nature 372:92-94, 1994; Wisniewski, T., Castano, E. M., Golabek, A., Vogel, T. & Frangione, B. “Acceleration of Alzheimer's fibril formation by apolipoprotein E in vitro” Am. J. Pathol. 145:1030-1035, 1994; Sanan, D. A., Weisgraber, K. H., Russell, S. J., Mahley, R. W., Huang, D., Saunders, A., Schmechel, D., Wisniewski, T., Frangione, B., Roses, A. D. & Strittmatter, W. J. “Apolipoprotein E associates with beta amyloid peptide of Alzheimer's disease to form novel monofibrils. Isoform apoE4 associates more efficiently than apoE3” J. Clin. Invest. 94:860-869, 1994). ApoE4, the isoform of apoE, identified by epidemiological studies as a strong risk factor for inherited Alzheimer's disease (Strittmatter, W. et. al. “Apolipoprotein E: high avidity binding to &bgr;-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer's disease” Proc Natl Acad Sci USA 90, 1977-1981, 1993; Corder, E. et. al. “Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families” Science 261, 921-923, 1993), is a much more active amyloid promoting factor than the non-pathogenic apoE3 or apoE2 isoforms. Furthermore, the greater number and length of the filaments formed under the promoting effect of ACT and apoE4 show increased toxicity to human cortical neurons in culture (Ma, J., Brewer, H. B. Jr., & Potter, H. “Alzheimer A beta neurotoxicity: promotion by antichymotrypsin. ApoE4; inhibition by A beta-related peptides.” Neurobiol Aging 17:773-78, 1996). Together, these results support the hypothesis that Alzheimer's disease involves an inflammation-like reaction and a consequent acute phase response in the brain that is essential for the development of mature amyloid neuropathology and neuronal cell death.
Since demonstration that ACT and apoE, especially apoE4, are amyloid promoters in vitro, many other proteins have been tested for their possible effect on A&bgr; polymerization, such as amyloid P component, acetylcholinesterase and complement factors (for review see Nilsson, L., Rogers, J. & Potter, H. “The essential role of inflammation and induced gene expression in the pathogenic pathway of Alzheimer's disease” Front. Biosci. 16:426-446, 1998). ACT and apoE are the only such molecules for which genetic studies support their involvement in the Alzheimer pathogenic pathway. For example, one of the greatest genetic risk factors for developing Alzheimer's disease is the inheritance of one, or worse, two copies of the apoE4 allele. Furthermore, inheritance of apoE leads to increased numbers of amyloid deposits in both Alzheimer patients and normal aged individuals, suggesting that apoE plays a direct role in amyloid formation, rather than, for instance increasing cells' sensitivity to neurotoxicity.
Genetic support for the involvement of ACT in Alzheimer's disease has been not so obvious as for apoE4. In one study, the inheritance of a specific isoform of ACT (an alani

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