Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1998-12-16
2002-09-03
Priebe, Scott D. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S018000, C800S025000, C435S455000, C435S463000, C435S320100, C435S325000
Reexamination Certificate
active
06444870
ABSTRACT:
BACKGROUND
Calcineurin, also known as protein phosphatase 2B, was first identified in the bovine brain. It represents a small family of calcium and calmodulin dependent serine/threonine protein phosphatases. It is expressed in all mammalian tissues examined, and is most abundant in the brain. In lymphocytes, calcineurin is the major soluble calmodulin-binding protein. Calcineurin is a heterodimer consisting of a catalytic subunit (A; 61 kD) and a regulatory subunit (B; 19 kD). The A subunit contains a catalytic domain, a carboxyl-terminal inhibitory domain, a B subunit binding site, and a camodulin binding site. The phosphatase activity of the A subunit is regulated by CA
2+
through both calmodulin and the B subunit. The B subunit has only a Ca
2+
dependent regulatory activity and does not have any phosphatase activity. There are two genes encoding closely related (about 80% identical) A subunit isoforms, A&agr; and A&bgr;, in the mouse, human, and rat genomes. The &agr; isoform is the predominant isoform found in brain, thymus, and T cells. The A&agr; and A&bgr; isoforms have distinct cellular distribution in the brain, with A&agr; most abundant in the hippocampus, cerebral cortex, cerebellum, and striatum. The differential distributions of the two isozymes suggest they may each have specific functions in modulating neuronal activities. The physiologic functions of the different calcineurin A isoforms are not yet defined.
SUMMARY OF THE INVENTION
The present invention relates to a method of identifying drugs or agents which have immuno-suppressive effects through or as a result of their effect on calcineurin, including drugs which affect the calcineurin A&agr; (CNA&agr;) subunit or the calcineurin A&bgr; (CNA&bgr;) subunit. It particularly relates to methods of identifying drugs which inhibit the phosphatase activity of calcineurin. The present invention further relates to a method of identifying drugs which overcome, prevent or reduce (partially or totally) the neurotoxic or other adverse effects of immuno-suppressant drugs, such as cyclosporin A (CsA) and FK506, which exert their effects by inhibiting calcineurin phosphatase activity.
In addition, the present invention relates to a method of identifying drugs which reduce (partially or totally) phosphorylation of the microtubule-associated protein tau, in the nervous system of a mammal; a method of identifying drugs which reduce (partially or totally) paired helical filament formation in the nervous system of a mammal; and a method of identifying drugs which reduce (partially or totally) formation of paired helical filaments, amyloid deposits or both. Such drugs are useful in reducing the extent to which Alzheimer's disease occurs, reducing the rate at which Alzheimer's disease progresses or preventing its occurrence.
The present invention also relates to transgenic non-human mammals, such as rodents and particularly mice, which lack a functional calcineurin gene and, thus, have disrupted calcineurin expression. In one embodiment, transgenic non-human mammals of the present invention lack a functional calcineurin A&agr; (CNA&agr;) subunit gene, a functional calcineurin A&bgr; (CNA&bgr;) subunit gene or both CNA&agr; and CNA&bgr; subunit genes. In a further embodiment, transgenic non-human mammals (e.g., rodents such as mice and rats) lack a functional calcineurin gene (e.g., calcineurin subunit A&agr; gene, calcineurin subunit A&bgr; gene) and express human tau protein. In such transgenic mammals, hyperphosphorylation of human tau protein is expressed and polymerizes, resulting in formation of paired helical filaments that make up neurofibrillary tangles in the brain. A third type of transgenic non-human mammal (e.g., rodents, such as mice and rats) lacks a functional calcineurin gene, expresses human tau protein and overexpresses human amyloid precursor protein and human Alzheimer A&bgr; protein. Such transgenic mammals exhibit both of the pathological lesions of Alzheimer's disease—amyloid deposits and paired helical filaments (which make up the neurofibrillary tangles that accumulate in brain neurons in Alzheimer's disease)—and serve as an improved model for Alzheimer's disease in which to identify drugs or agents which will reduce (partially or totally) the pathological lesions.
DETAILED DESCRIPTION OF THE INVENTION
As described herein, a transgenic non-human mammal which lacks a functional calcineurin (CN) gene produces greatly increased amounts of hyperphosphorylated tau protein. The transgenic non-human mammal of the present invention can be used to identify drugs or agents which have immuno-suppressive effects through or as a result of their effect on CN, including drugs or agents which affect the calcineurin A&agr; (CNA&agr;) subunit or the calcineurin A&bgr; (CNA&bgr;) subunit. In addition, further transgenic mammals of the present invention, described herein, can be used to identify agents which are useful in reducing phosphorylation of tau protein and production of pathological lesions characteristic of Alzheimer's Disease.
In one embodiment, the present invention relates to a method of identifying an agent that reduces the phosphorylation of tau protein in the nervous system of a mammal, comprising the steps of a) administering to a transgenic non-human mammal which lacks a functional CN gene, an agent to be assessed for its ability to reduce phosphorylation of tau protein; b) determining the extent to which phosphorylation of tau protein occurs in the nervous system of the transgenic non-human mammal to which the agent is administered; and c) comparing the extent determined in b) to the extent to which phosphorylation occurs in the nervous system of an appropriate control. If phosphorylation occurs to a lesser extent in the nervous system of the transgenic non-human mammal to which the agent is administered than in the nervous system of the control, the agent reduces phosphorylation of tau protein.
In another embodiment, the present invention relates to a method of identifying an agent which reduces paired helical filament (PHF) formation in the nervous system of a mammal, comprising the steps of: a) administering to a transgenic non-human mammal which lacks a functional CN gene and expresses human tau protein, an agent to be assessed for its ability to reduce PHF formation; b) determining the extent to which PHF formation occurs in the nervous system of the transgenic non-human mammal to which the agent is administered; and c) comparing the extent determined in b) to the extent to which PHF formation occurs in the nervous system of an appropriate control, wherein if PHF formation occurs to a lesser extent in the nervous system of the transgenic non-human mammal to which the agent is administered than in the nervous system of the control, the agent reduces PHF formation. In another embodiment, the present invention relates to a method of identifying an agent which reduces a lesion characteristic of Alzheimer's disease in the nervous system of a mammal comprising the steps of: a) administering to a transgenic non-human mammal which lacks a functional CN gene, expresses a human tau protein, and overexpresses the human amyloid precursor protein and the human Alzheimer A&bgr; protein, an agent to be assessed for its ability to reduce a lesion characteristic of Alzheimer's disease; b) determining the extent to which the lesion occurs in the nervous system of the transgenic non-human mammal to which the agent is administered; and c) comparing the extent determined in b) to the extent to which the lesion occurs in the nervous system of an appropriate control; wherein if the lesion occurs to a lesser extent in the nervous system of the transgenic non-human mammal to which the agent is administered than in the nervous system of the control, the agent reduces a lesion characteristic of Alzheimer's disease.
The pathological lesions characteristic of Alzheimer's Disease which can be reduced in a mammal using agents identified by the method of the present inven
Kagyali Usamah S.
Potter Huntington
Seidman Jonathan G.
Zhang Wei
Hamilton Brook Smith & Reynolds P.C.
Paras, JR. Peter
President and Fellows of Harvard College
Priebe Scott D.
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