Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-12-09
2001-10-30
Schwartzman, Robert A. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S091100, C435S325000, C435S375000, C536S023100, C536S024500, C536S024300, C536S024310, C536S034000
Reexamination Certificate
active
06310048
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to compositions and methods for modulating the expression of amyloid beta protein, and more particularly to antisense oligonucleotides that specifically hybridize with nucleic acids encoding human amyloid precursor protein and modulate the expression of the amyloid beta portion of amyloid precursor protein.
(2) Description of the Related Art
Alzheimer's disease is a neurodegenerative disorder characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles in the brains of affected individuals. (Masters, C. L. et al.,
Proc. Natl. Acad. Sci. USA,
82:4245-4249 (1985)). The senile plaques, found in abundance in Alzheimer's disease-affected brain cells, are composed of a core of extracellular amyloid beta protein (A&bgr;P) surrounded by reactive cells and degenerating neurites. (Lenders, M. B. et al.,
Acta Neurologica Belgica,
89:279-285 (1989); and Perry, G. et al.,
Lancet,
2:746 (1988)). While the plaques form primarily in particular parts of the brain—such as the hippocampus—in some cases they are also found in the walls of cerebral and meningeal blood vessels. (Delacourt, A. et al.,
Virchows Archiv.—A, Pathological Analomy
&
Histopathology,
411:199-204 (1987); and Masters, C. L. et al.,
EMBO Journal,
4:2757-2763 (1985)).
The major protein subunit of the senile plaques, amyloid beta protein (and also referred to in the art as &bgr;-amyloid protein or A4 protein) is a 4 ki) (39-43 amino acid) protein that is a cleavage product of a much larger precursor protein called amyloid precursor protein (APP). Whereas amyloid precursor protein is a transmembrane protein with no known harmful physiological effects, amyloid beta protein is known to be highly aggregating and to deposit and form plaques and to accumulate at high levels in the brain in Alzheimer's disease, Down's syndrome and some normal aged individuals. (Verga, L. et al.,
Neuroscience Letters,
105:294-299 (1989)). Strong evidence that amyloid beta protein deposition plays a critical role in the development of Alzheimer's disease came from the identification of familial Alzheimer's disease kindreds in which the Alzheimer's disease phenotype co-segregates with mutations from the amyloid precursor protein gene. (Younkin, S. G.,
Tohuku J. of Exper. Med.,
174:217-223 (1994); and Matsumura, Y. et al.,
Neurology,
46:1721-1723 (1996)).
Nucleic acid sequences for amyloid precursor protein, amyloid beta protein (A&bgr;P), and related proteins have been reported by Ponte et al., (U.S. Pat. No. 5,220,013), and Greenberg et al., (WO88/03951), among others. Amyloid precursor protein has several isoforms generated by alternative splicing of a 19-exon gene made up of exons 1-13, 13a, and 14-18 (Yoshikai et al.,
Gene,
87:257 (1990)). The predominant transcripts are APP695 (exons 1-6, 9-18, not 13a); APP751 (exons 1-7, 9-18, not 13a); and APP770 (exons 1-18, not 13a). All of these encode multidomain proteins with a single membrane spanning region. The A&bgr;P segment of amyloid precursor protein comprises approximately one-half of the transmembrane domain and approximately the first 28 amino acids of the extracellular domain of an amyloid precursor protein isoform. (U.S. Pat. No. 5,455,169). This structure is illustrated in
FIG. 1
, where the 42 amino acid sequence of the A&bgr;P segment of mouse amyloid precursor protein is shown—having its C-terminal to the left and an N-terminal portion to the right. That part of the A&bgr;P segment that normally resides within the transmembrane domain is enclosed by a dashed oval.
The amyloid precursor protein isoforms differ in that APP751 and APP770, but not APP 695, contain exon 7, which encodes a serine protease inhibitor domain. APP695 is a predominant form in neuronal tissue, whereas APP751 is the predominant variant elsewhere. Beta amyloid protein is derived from that part of the amyloid precursor protein encoded by parts of exons 16 and 17.
Two major pathways of amyloid precursor protein processing in vivo have been described. Normal processing of amyloid precursor protein in the secretory pathway occurs by proteolytic cleavage within the A&bgr;P sequence of the amyloid precursor protein resulting in the generation of a large (approximately 100 kD) soluble, secreted N-terminal fragment of the protein (Oltersdorf, T.,
Nature,
14.341, 144-147 (1989); and de Sauvage, F., and J. N. Octave,
Science,
11:245, 651-653 (1989)) and a smaller (approximately 9-10 kD), membrane-associated C-terminal fragment (Wolf, D. et al.,
EMBO Journal,
9:2079-2084 (1990); and Ghiso, J. et al.,
Biochemical Journal,
288:1053-1059 (1992).
FIG. 1
illustrates this type of cleavage as occurring at, or near, the position marked at “&agr;-secretase”. Neither of the two protein fragments that result from the cleavage is amyloidogenic (i.e., tends to form senile plaques), because neither of them contains the entire A&bgr;P protein.
However, another pathway of amyloid precursor protein metabolism involves the endosomal-lysosomal system and results in generation of an amyloidogenic C-terminal fragment of amyloid precursor protein. When amyloid precursor protein is processed by the endosomal-lysosomal system, a complex set of —COOH terminal derivatives of amyloid precursor protein is produced that includes potentially amyloidogenic forms having the entire A&bgr;P at, or near, their N-terminal. One form of this aberrant cleavage of amyloid precursor protein occurs at, or near, the positions identified in
FIG. 1
as “&bgr; and &ggr; secretases” (Glenner and Wong, Biochem.
Biophys. Res. Commun.,
122:1131-1135 (1984); Volloch,
FEBS Letters,
390:124-128 (1996)) and results in the generation of A&bgr;P that is known to deposit and form plaques. The plaques have been shown to be associated with the clinical severity of Alzheimer's disease. Abundant deposition of A&bgr;P in the brains of patients with Alzheimer's disease has suggested that regulation of amyloid precursor protein expression and metabolism are key pathological events. It is known that some amount of A&bgr;P is constantly produced in the brain, but is continuously cleared. Apparently, the two alternative pathways of amyloid precursor protein metabolism must be precisely balanced in order to avoid the accumulation of A&bgr;P in harmful concentrations.
It is known that the amyloid precursor protein gene in humans is located on chromosome 21. Several different studies have suggested the apparent involvement of several particular sites in the amyloid precursor protein gene in Alzheimer's disease. Three separate mutations in codon 717 of the amyloid precursor protein transcript have been found in familial Alzheimer's disease: val717-to-ile, val717-to-phe, and val717-to-gly. See, Hardy et al., U.S. Pat. No. 5,877,015. The location of these mutations and of the double mutation disclosed by Mullan (U.S. Pat. No. 5,455,169) suggested to Suzuki et al.,
Science,
264:1336-1340 (1994), that they may cause Alzheimer's disease by altering amyloid beta protein processing in a way that is amyloidogenic. They found that the APP717 mutations were consistently associated with a 1.5- to 1.9-fold increase in the percentage of longer peptide fragments generated and that the longer peptide fragments formed insoluble amyloid fibrils more rapidly than did the shorter ones. Alternative splicing of transcripts from the single amyloid precursor protein gene results in at least 10 isoforms of the gene product (Sandbrink et al.,
J. Biol. Chem.,
269: 1510-1517 (1994)), of which APP695 is preferentially expressed in neuronal tissues. In 3 mutations, valine-642 in the transmembrane domain of APP695 is replaced by isoleucine, phenylalanine, or glycine, in association with dominantly inherited familial Alzheimer's disease. According to an earlier numbering system, val642 was numbered 717 and the 3 mutations were V7171, V717F, and V717G, respectively). Yamatsuji et al.,
Embo J.
15: 498-50
Howell & Haferkamp L.C.
Lacourciere Karen A.
Schwartzman Robert A.
St. Louis University
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