Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Rodent cell – per se
Reexamination Certificate
1998-02-20
2003-12-30
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Rodent cell, per se
C435S353000, C435S368000, C435S369000, C435S320100, C536S023500
Reexamination Certificate
active
06670182
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the design, construction and use of eukaryotic cell lines useful in the identification of inhibitors of &bgr;-amyloid processing. More specifically, the invention relates to in vitro assays capable of identifying or quantifying 4.2 kDa &bgr;-amyloid protein. The present invention also provides for DNA and protein molecules for the design, construction and use of eukaryotic cell lines and in vitro assays useful in the identification of inhibitors of &bgr;-amyloid processing.
BACKGROUND OF THE INVENTION
Alzheimer's disease has emerged as a significant health problem. It is estimated that over 5% of the U.S. population and over 15% of the U.S. population over 85 are beset with some form of Alzheimer's disease (Cross,
Eur. J. Pharmacol.
82: 77-80 (1982); Terry, et al.,
Ann. Netirol.
14:497-506 (1983)). Currently, there is no remission in the progression of the disease, nor is there any truly effective pharmaceutical intervention or method for diagnosing the disease (Schehr, Bio/Fechnology 12: 140-144 (1994); Cordell,
Ann. Rev. Pharmacol. Toxicol.
34: 69-89 (1994), herein incorporated by reference). A patient with Alzheimers progresses toward increasing mental and physical incapacitation.
A characteristic feature of Alzheimer's disease is the formation or deposit of &bgr;-amyloid plaques in affected individuals. Mature &bgr;-amyloid plaques are often associated with degenerating neuronal processes. &bgr;-amyloid deposits are not solely associated with persons suffering from Alzheimer's disease but are also associated with persons suffering from other amyloidoses, for example, brain trauma or Downs syndrome (Cordell,
Ann. Rev. Pharmacol. Toxicol.
34: 69-89 (1994); Pierce et al.,
Journal of Neuroscience,
16: 1083-1090 (1996); Roberts,
Lancet
338:1422-1423 (1991); Motte and Williams,
Acta Neuropathol
77: 535-546 (1989); Mann and Esiri,
Journal of the Neurological Sciences
89: 169-179 (1989); Masters,
Proc. Natl. Acad. Sci.,
82: 4245-4249 (1985)).
The &bgr;-amyloid proteins isolated from neuritic plaques are self aggregating moieties termed the 4.2 kDa &bgr;-amyloid protein or alternatively termed one of the following: the A4 protein (Ponte et al.,
Nature
311: 525-527 (1988), herein incorporated by reference); the &bgr;-amyloid peptide (Schehr,
Bio/Technology
12: 140-144 (1994), herein incorporated by reference); 4.2 kDa &bgr;-amyloid polypeptide (Neve et al.,
Neuron
1: 669-677 (1988), herein incorporated by reference); 4K peptide or AP (Haas et al.,
Nature
359: 322-325 (1992), herein incorporated by reference); amyloid B-protein or amyloid A4 (Tanzi et al.,
Nature
331: 528-530 (1988), herein incorporated by reference); 4 kD amyloid 5 protein, &bgr;AP or 4 kD protein (Shoji et al.,
Science
258: 126-129 (1992), herein incorporated by reference); the 4.2-4.5 kd amyloid protein subunit (Wolf et al.,
EMBO
9.7: 2079-2084 (1990), herein incorporated by reference); &bgr; protein (Weidmann et al.,
Cell
57:115-126 (1989); the beta-amyloid core protein (Cordell, U.S. Pat. No. 5,221,607, herein incorporated by reference); a composition of peptides individually referred to as A&bgr;1-39 peptide, A&bgr;1-40 peptide, A&bgr;1-41 peptide and A&bgr;1-42 peptide (described in Glenner and Wong,
Biochem. Biophys. Res. Comm.
120: 885-890 (1984), herein incorporated by reference; (Schehr,
Nature Biotechnology
15: 19-20 (1997), herein incorporated by reference) and A&bgr;1-43 peptide (Shoji et al.,
Science
258: 126-129 (1992); Selkoe,
Science
275: 630-631 (1997), herein incorporated by reference). A&bgr;1-39 peptide, A&bgr;1-40 peptide, A&bgr;1-41 peptide, A&bgr;1-42 peptide and A&bgr;1-43 peptide comprise 39, 40, 41, 42 and 43 amino acids respectively.
Characterization of cDNA encoding the 4.2 kDa &bgr;-amyloid protein showed that the 4.2 kDa &bgr;-amyloid protein was transcribed as part of a larger precursor protein, &bgr;-amyloid precursor protein (APP)(Lemaire et al.,
Nucleic Acid Research
17: 517-22 (1989)). Three major APP protein isoforms, APP
695
, APP
751
and APP
770
, have been characterized (Cordell,
Ann. Rev. Pharmacol. Toxicol.
34: 69-89 (1994)). The isoforms APP
695
, APP
751
and APP
770
, contain respectively, 695, 751 and 770 amino acids (Weidmann et al.,
Cell
57:115-126 (1989), herein incorporated by reference). These isoforms are the result of differential splicing of the primary APP RNA transcript. The APP
695
isoform differs from the longer isoforms in not containing the Kunitz proteinase inhibitor domain (Lemaire, et al.,
Nucleic Acids Research
172: 517-522 (1989); herein incorporated by reference); Mullan, U.S. Pat. No. 5,455,169, herein incorporated by reference); Ponte et al.,
Nature
331: 525-527 (1988), herein incorporated by reference). The mRNA corresponding to the APP
695
isoform also differs from the longer isoforms in that it has been shown to be preferentially expressed in the brain (Neve et al.,
Neuron
1: 669-677 (1988)).
Certain APP mutations have been reported (Ashall and Goate,
Trends in Biochemical Sciences,
19: 42-46 (1994), herein incorporated by reference); Hardy and Allsop,
Trends in Pharmological Sciences
12: 383-388 (1991), herein incorporated by reference). Examples of characterized APP mutants include: the “Swedish FAD double mutant” (Mullan et al.,
Nature Genetics
1: 345-347 (1992), herein incorporated by reference), the “London mutant” (Van Broeckhoven, et al.,
Science
248: 1120-1123 (1990), herein incorporated by reference); Levy,
Science
24: 1124-1126 (1990), herein incorporated by reference; valine
717
→isoleucine mutant (Goate et al.,
Nature
349: 704-706 (1991), herein incorporated by reference); Hardy et al.,
Lancet
337: 1342-1343 (1991), herein incorporated by reference); valine
717
→glycine mutant (Harlan et al.,
Nature
353: 844-846 (1991); and valine
717
→phenylalanine mutant (Murrell et al.,
Science
254: 97-99 (1991), herein incorporated by reference).
In each of the APP isoforms, the 4.2 kDa &bgr;-amyloid protein corresponds to an internal region that begins 99 residues from the carboxyl terminal end of the APP isoforms (Shoji et al.,
Science
258: 126-129 (1992)). Two major pathways of APP processing have been reported (See Higaki et al.,
Neuron
14: 651-659 (1995), herein incorporated by reference). One pathway involves proteolytic cleavages that result in the formation of truncated carboxyl terminal 8-12 kD remnants (Caporaso et al.,
Proc. Natl. Acad. Sci. USA
89: 2252-2256 (1992), herein incorporated by reference). It has been reported that carboxyl terminal 8-12 kD remnants are nonamyloidgenic (Seubert et al.,
Nature
361:260-263 (1993), herein incorporated by reference). It is suggested that the proteolytic processing of APP in this pathway occurs at a number of intracellular and membrane locations (See Higaki et al.,
Neuron
14: 651-659 (1995)). The carboxyl terminal 8-12 kD remnants have been reported to remain cell-associated, possibly due to retention of a transmembrane domain within the carboxyl terminal 8-12 kD remnants (Cordell,
Ann. Rev. Pharmacol. Toxicol.
34: 69-89 (1994)). The second processing pathway has been reported to be associated with the endosomal/lysosomal system and it is this pathway that is reportedly responsible for the production of the 4.2 kDa &bgr;-amyloid protein (See Higaki et al.,
Neuron
14: 651-659 (1995)).
Cell lines capable of producing a 4.2 kDa &bgr;-amyloid protein have been reported (Naidu et al.,
Journal of Biological Chemistry
270: 1369-1374 (1995), herein incorporated by reference; Higaki et al.,
Neuron
14: 651-669 (1995), herein incorporated by reference). Naidu et al. and Higaki et al. report the establishment of the CP-6 cell line by stably transfecting a human cDNA encoding the 695-amino acid isoform of &bgr;-amyloid precursor protein, driven by a &bgr;-actin promoter, into Chinese hamster ovary fibroblasts. Busciglio et al. report transient transfection of a COS cell line (a monkey cell line containing part of the SV 40 early promote
Cordell Barbara
Huggins John
Mischak Ronald P.
Pruss Rebecca
Rautmann Guy
Aventis Pharmaceuticals Inc.
Ketter James
Schmeizer Michael J.
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