Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-16
2001-01-23
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C514S04400A
Reexamination Certificate
active
06177246
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the design and synthesis of oligonucleotides that are complementary to and specifically hybridizable with nucleic acids encoding &bgr;-amyloid protein. The oligonucleotides of the invention are useful in assays for &bgr;-amyloid and as diagnostic reagents for the detection of diseases associated with abnormal accumulation of &bgr;-amyloid. The oligonucleotides of this invention may also be used either prophylactically or therapeutically to reduce the severity of disease states resulting from abnormal accumulation of &bgr;-amyloid in the cells and tissues of a human patient.
BACKGROUND OF THE INVENTION
Abnormal deposition of &bgr;-amyloid in the cells and tissues of a human patient is a prominent feature associated with several brain disorders in humans. These disorders include Alzheimer's disease, Alzheimer disease changes associated with Down's syndrome, hereditary cerebral hemorrhage with amyloidosis of Dutch origin (HCHWA-D), Parkinson-dementia of Guam, sporadic cerebral amyloid angiopathy (SCAA) and dementia pugilistica. Abnormal accumulation of &bgr;-amyloid is also implicated in severe and progressive muscle diseases such as sporadic inclusion body myositis (IBM) and hereditary inclusion body myopathy (hIBM). Collectively, these diseases are referred to as &bgr;-amyloidoses.
Alzheimer's disease is the most common cause of dementia in aging humans in many developed countries. The disease is characterized by gradual loss of memory, emotional stability and judgment. Death usually occurs between four and twelve years after the onset of symptoms. Patients with Alzheimer's disease require constant supervision and eventual total custodial care due to their severely debilitated condition. The cost of diagnosing and managing Alzheimer's patients is currently estimated at more than $80 billion a year in the U.S. alone [Selkoe, D. J. (1991)
Scientific American
265:68-78].
Two types of brain lesions, senile plaques and neurofibrillary tangles, were originally described by Dr. Alois Alzheimer in patients with dementia. In Alzheimer's disease patients, senile plaques occur in great numbers in the areas of the brain responsible for cognitive function, particularly the cerebral cortex, hippocampus and amygdala. These spherical plaques consist of altered neurites (axons and dendrites), which are the long tapering portions of neurons, surrounding an extracellular mass of filaments. It is presently believed that degeneration of neurons in Alzheimer's disease patients is the result of their entanglement in this filament matrix. There is currently no treatment for the prevention or retardation of the progression of Alzheimer's disease.
At approximately 40-50 years of age, individuals with Down's syndrome acquire brain lesions which are characteristic of Alzheimer's disease. The behavior as well as the mental ability of these patients begins to deteriorate at about the same time. The cerebrovascular plaques which are observed in Down's syndrome patients over forty years of age are known to comprise &bgr;-amyloid. Also, recent studies suggest that women under age 35 who have borne a child with Down's syndrome are themselves more likely to develop Alzheimer's disease earlier in life than women who bear children with Down's syndrome at or above age 35.
Hereditary cerebral hemorrhage with amyloidosis of Dutch type (HCHWA-D) is an autosomal dominant disease characterized by extensive &bgr;-amyloid plaques in the cerebral cortex. The senile plaques are widespread and have a fibrillar appearance. Also, amyloid fibrils are known to infiltrate small arteries, arterioles and veins in these individuals. Patients with HCHWA-D suffer recurrent intracerebral hemorrhages leading to early death at the age of 50 to 60 years old. In almost 50% of the patients the first cerebral hemorrhage leads to death.
Parkinson-dementia of Guam is caused by extensive deposition of &bgr;-amyloid in the brain, and is characterized by parkinsonism and slowly progressive dementia. This disease also exhibits the formation of neurofibrillary tangles similar to those observed in Alzheimer's disease patients.
Sporadic cerebral amyloid angiopathy (SCAA) is characterized by an intense and extensive &bgr;-amyloid deposition within cerebral arteries, veins, arterioles and capillaries. SCAA is observed in 50% of the autopsies performed on individuals over 50 years of age. Symptoms include dementia, cerebral hemorrhages and ischemic strokes.
Severe head injury in humans may lead to a chronic degenerative condition referred to as dementia pugilistica. This disorder is a consequence of abnormal deposition of &bgr;-amyloid in the form of diffuse plaques in the neurons and neurites of the patient's brain, and is characterized by a cavum septum, neuronal loss, cerebellar scarring and intense neurofibrillary tangle formation in the cortex.
Muscle diseases such as sporadic inclusion body myositis (IBM) and hereditary inclusion body myopathy (hIBM) are characterized by abnormal accumulation of &bgr;-amyloid in vacuolated muscle fibers, and are most common in patients of who are fifty-five years of age or older. This is a progressive disease resulting in severe disability, whose symptoms include proximal and distal muscle weakness, thinning of the forearms, male predominance and either the absence of or a poor response to immunosuppressive treatment.
There is a long felt need for effective diagnosis, prophylaxis and treatment of these and other conditions whose common feature includes abnormal &bgr;-amyloid synthesis and accumulation.
SUMMARY OF THE INVENTION
&bgr;-Amyloid is a small protein approximately 40 amino acids in length, which is derived from the carboxyl terminus of a much longer (770 amino acids) precursor protein called &bgr;-amyloid precursor protein (&bgr;APP). The gene which encodes RAPP therefore also encodes &bgr;-amyloid.
In accordance with the present invention, oligonucleotides are provided that are specifically hybridizable with DNA or RNA encoding abnormally expressed &bgr;-amyloid. The oligonucleotides comprise nucleotide units sufficient in identity and number to effect such specific hybridization. Abnormally expressed &bgr;-amyloid includes overexpressed &bgr;-amyloid, &bgr;-amyloid with reduced solubility, &bgr;-amyloid which is longer than wild type &bgr;-amyloid, preferably comprising at least 42 amino acid residues, and mutated &bgr;-amyloid. In accordance with another preferred embodiment, oligonucleotides that specifically hybridize with codon 717 of the gene encoding &bgr;-amyloid are provided. In another such embodiment, oligonucleotides are provided that specifically hybridize preferentially with codon 717 of the gene encoding a mutated &bgr;-amyloid, preferably comprising a sequence GAT, GAA or GCC. In yet another embodiment, oligonucleotides that specifically hybridize with codons 670 and 671 of the gene encoding &bgr;-amyloid are provided. In another such embodiment, oligonucleotides are provided that specifically hybridize preferentially with codons 670 and 671 of the gene encoding a mutated &bgr;-amyloid. Such oligonucleotides may be conveniently and desirably presented in a pharmaceutically acceptable carrier.
In accordance with other preferred embodiments, the oligonucleotides are formulated such that at least one of the linking groups between nucleotide units of the oligonucleotide comprises a sulfur-containing species such as a phosphorothioate moiety.
Other aspects of the invention are directed to methods for modulating the expression of &bgr;-amyloid in cells or tissues and for specifically modulating the expression of mutated &bgr;-amyloid in cells or tissues suspected of harboring such a mutation. Additional aspects of the invention are directed to methods of detecting the gene encoding &bgr;-amyloid in cells or tissues and specific detection of the gene encoding mutated &bgr;-amyloid in cells or tissues suspected of harboring the mutated gene. Such methods co
Ecker David J.
Freier Susan M.
Monia Brett P.
Campbell Eggerton A.
ISIS Pharmaceuticals Inc.
Woodcock Washburn Kurtz Mackiewicz & Norris LLP
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