Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1997-05-29
2001-12-11
Kunz, Gary L. (Department: 1647)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C435S320100
Reexamination Certificate
active
06329566
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to methods and reagents for diagnosing, treating, and preventing neurodegeneration.
Loss of neurons by a degenerative process is a major pathological feature of many human neurological disorders. Neuronal cell death can occur as a result of a variety of conditions including traumatic injury, ischemia, neurodegenerative diseases (e.g., Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), stroke, or trauma), or as a normal part of tissue development and maintenance. Several inherited disorders produce late onset neuron loss, each of which is highly specific for particular neural cell types. Nine genes have been cloned that are associated with susceptibility to these various neurological disorders (e.g., Huntington's disease, ataxin, and ALS); however, only in the case of Kennedy's syndrome is the biochemical function of the affected gene, the androgen receptor, understood (La Spada et al., Nature 352: 77-79, 1991). Epileptic seizures and stroke also produce neurodegeneration in humans and rodents.
SUMMARY OF THE INVENTION
In general, the invention features methods for the detection, treatment, and prevention of disorders involving neurodegeneration.
In a first aspect, the invention features a method for identifying a compound to treat or prevent the onset of a neurodegenerative disorder. The method involves contacting a cell that includes a reporter gene operably linked to a cAMP regulatory gene or promoter with a candidate compound and measuring the expression of the reporter gene, where a change in reporter gene expression in response to the candidate compound identifies a compound that is useful to treat or prevent the onset of a neurodegenerative disorder.
In various preferred embodiments of the first aspect of the invention, the cAMP regulatory gene may be an acy-1 gene, an eat-4 gene, an unc-36 gene, or a glutamate receptor-encoding gene. In another preferred embodiment, the change in reporter gene expression is a decrease in expression.
In a second aspect, the invention features a cell for identifying a compound to treat or prevent the onset of a neurodegenerative disorder that includes a reporter gene operably linked to a cAMP regulatory gene or promoter.
In various embodiments of the above aspects, the cell is present in an animal, which may be a nematode (e.g.,
C. elegans
) or a mammal (e.g., a rodent).
In a third aspect, the invention features a method for treating or preventing the onset of a neurodegenerative disorder in a mammal that includes administering to the mammal a therapeutically effective amount of a compound that decreases a neuronal cAMP level. In a preferred embodiment of this aspect of the invention, the mammal is a human.
In a fourth aspect, the invention features a method for identifying a mammal (for example, a human) having or likely to develop a neurodegenerative disorder which includes determining whether the mammal has an increased level of cellular cAMP in a neuron, where an increased level indicates that the mammal has or is likely to develop a neurodegenerative disorder.
In a fifth aspect, the invention features a method for identifying a mammal (for example, a human) having or likely to develop a neurodegenerative disorder which involves determining whether the mammal has a mutation in a cAMP regulatory gene. In various preferred embodiments of this aspect, the mutation is in an adenylyl cyclase gene (e.g., the acy-1 gene), or in an unc-36 or eat-4 gene. In other preferred embodiments, the mutation is in a gene encoding a G&agr;
s
subunit; and the mutation results in an increase in a neuronal cAMP level.
In a preferred embodiment of various aspects of the invention, the neurodegenerative disorder is Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, stroke, or epilepsy.
In a sixth aspect, the invention features a method for identifying a gene involved in neurodegeneration that involves providing a nematode (for example,
C. elegans
) that includes an expression construct that includes a promoter derived from a cAMP regulatory gene operably linked to a reporter gene, isolating a mutant of the nematode exhibiting an altered level of reporter gene expression, and identifying the gene comprising the mutation, wherein the gene is involved in neurodegeneration.
In a seventh aspect, the invention features a method for identifying a gene involved in neurodegeneration that involves providing a nematode (for example,
C. elegans
) that includes a glutamate receptor (GluR) promoter operably linked to a gene encoding a GTP-ase defective G&agr;
s
subunit, isolating a mutant of the nematode exhibiting a decreased level of paralysis and neurodegeneration, and identifying the gene that includes the mutation, wherein the gene is involved in neurodegeneration.
In an eighth aspect, the invention features a mammalian (for example, a human) EAT-4 polypeptide, and a vector and cell containing the nucleic acid.
In a final aspect, the invention provides a method for identifying a gene involved in neurodegeneration involving the steps of a) providing a cell that includes a cAMP regulatory gene promoter operably linked to a reporter gene; b) introducing into the cell a candidate gene capable of expressing a candidate protein; and c) measuring reporter gene expression in the cell, where an increase in reporter gene expression in the presence of the candidate protein indicates that the candidate gene is involved in neurodegeneration.
In preferred embodiments, the cell is yeast; and the cAMP regulatory gene is an acy-1 gene, an eat-4 gene, an unc-36 gene, or a glutamate receptor-encoding gene.
As used herein, by “protein” or “polypeptide” is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
By “neurodegenerative disorder” is meant a disorder which is characterized by the death or loss of function of neuronal cells, also known as neurons. Neuronal death or loss of function can be associated with a number of diseases and syndromes including, without limitation, stroke, epilepsy, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer's disease.
By “G&agr;
s
-induced toxicity” is meant the neurodegeneration resulting from expression of the GTP-ase defective G&agr;
s
protein.
By “reporter gene” is meant any gene which encodes a product whose expression is detectable. A reporter gene product may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ), toxicity (e.g., HER-1), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably labelled antibody).
By “cAMP regulatory gene” is meant any gene whose product regulates or is regulated by cAMP. Exemplary gene products include ACY-1, UNC-36, and EAT-4. Other preferred cAMP regulatory gene products include the ionotropic (cation) glutamate receptors (iGluRs), the Cl
−
ionotropic glutamate receptors (GluCls), and the metabotropic glutamate receptors (mGluRs).
By “operably linked” is meant that a gene and a regulatory sequence are connected in such a way as to permit expression of the gene product under the control of the regulatory sequence.
By “purified nucleic acid” is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autosomally replicating plasmid or virus; or into the genomic DNA or a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding
Hart Anne C.
Kaplan Joshua M.
Oppenheimer Allison J.
Clark & Elbing LLP
Gucker Stephen
Kunz Gary L.
The General Hospital Corporation
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