Transgenic rodent comprising APP-Swedish

Details Transgenic rodent comprising APP-Swedish Transgenic rodent comprising APP-Swedish

1998-12-10

2001-06-12

Crouch, Deborah (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

The nonhuman animal is a model for human disease

C800S003000, C800S014000, C800S018000, C800S022000

Reexamination Certificate

active

06245964

ABSTRACT:

TECHNICAL FIELD
The invention provides transgenic non-human animals and transgenic non-human mammalian cells harboring a transgene encoding an amyloid precursor protein (APP) comprising the Swedish mutation (lysine
595
-methionine
596
mutated to asparagine
595
-leucine
596
); the invention also provides non-human animals and cells comprising a transgene encoding an APP comprising the Swedish mutation and further comprising functionally disrupted endogenous APP gene loci, transgenes and targeting constructs used to produce such transgenic cells and animals, transgenes encoding human Swedish mutation APP polypeptide sequences, and methods for using the transgenic animals in pharmaceutical screening and as commercial research animals for modeling neurodegenerative diseases (e.g., Alzheimer's disease) and APP biochemistry in vivo.
BACKGROUND OF THE INVENTION
Alzheimer's disease (AD) is a progressive disease known generally as senile dementia. Broadly speaking the disease falls into two categories, namely late onset and early onset. Late onset, which occurs in old age (65+ years), may be caused by the natural atrophy of the brain occurring at a faster rate and to a more severe degree than normal. Early onset AD is much more infrequent but shows a pathologically identical dementia with brain atrophy which develops well before the senile period, i.e., between the ages of 35 and 60 years.
Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates) present in the brains of AD patients, particularly in those regions involved with memory and cognition. While in the past there was significant scientific debate over whether the plaques and tangles are a cause or are merely the result of AD, recent discoveries indicate that amyloid plaque is a causative precursor or factor. In particular, it has been discovered that the production of &bgr;-amyloid peptide, a major constituent of the amyloid plaque, can result from mutations in the gene encoding amyloid precursor protein, a protein which when normally processed will not produce the &bgr;-amyloid peptide. It is presently believed that a normal (non-pathogenic) processing of the &bgr;-amyloid precursor protein occurs via cleavage by a putative “&agr;-secretase” which cleaves between amino acids 16 and 17 of the protein. It is further believed that pathogenic processing occurs via a putative “&bgr;-secretase” at the amino-terminus of the &bgr;-amyloid peptide within the precursor protein. Moreover, &bgr;-amyloid peptide appears to be toxic to brain neurons, and neuronal cell death is associated with the disease.
&bgr;-amyloid peptide (also referred to as A4, &bgr;AP, A&bgr;, or A&bgr;P; see, U.S. Pat. No. 4,666,829 and Glenner and Wong (1984)
Biochem. Biophys. Res. Commun.
120: 1131) is derived from &bgr;-amyloid precursor protein (&bgr;APP), which is expressed in differently spliced forms of 695, 751, and 770 amino acids. See, Kang et al. (1987) Nature 325: 773; Ponte et al. (1988)
Nature
331: 525; and Kitaguchi et al. (1988)
Nature
331: 530. Normal processing of amyloid precursor protein involves proteolytic cleavage at a site between residues Lys
16
and Leu
17
(as numbered for the &ngr;AP region where Asp
597
is residue 1 in Kang et al. (1987)), supra, near the transmembrane domain, resulting in the constitutive secretion of an extracellular domain which retains the remaining portion of the &bgr;-amyloid peptide sequence (Esch et al. (1990) Science 248:1122-1124). This pathway appears to be widely conserved among species and present in many cell types. See, Weidemann et al. (1989) Cell 57:115-126 and Oltersdorf et al. (1990) J. Biol. Chem. 265:4492-4497. This normal pathway cleaves within the region of the precursor protein which corresponds to the &bgr;-amyloid peptide, thus apparently precluding its formation. Another constitutively secreted form of &bgr;APP has been noted (Robakis et al. Soc. Neurosci. Oct. 26, 1993, Abstract No. 15.4, Anaheim, Calif.) which contains more of the &bgr; AP sequence carboxy terminal to that form described by Esch et al. supra.
Golde et al. (1992) Science 255:728-730, prepared a series of deletion mutants of amyloid precursor protein and observed a single cleavage site within the &bgr;-amyloid peptide region. Based on this observation, it was postulated that &bgr;-amyloid peptide formation does not involve a secretory pathway. Estus et al. (1992) Science 255:726-728, teaches that the two largest carboxy terminal proteolytic fragments of amyloid precursor protein found in brain cells contain the entire &bgr;-amyloid peptide region.
Recent reports show that soluble &bgr;-amyloid peptide is produced by healthy cells into culture media (Haass et al. (1992) Nature 359:322-325) and in human and animal CSF (Seubert et al. (1992) Nature 359:325-327). Palmert et al. (1989) Biochm. Biophys. Res. Comm. 165:182-188, describes three possible cleavage mechanisms for &bgr;APP and presents evidence that &bgr;APP cleavage does not occur at methionine
596
in the production of soluble derivatives of &bgr;APP. U.S. Pat. No. 5,200,339, discusses the existence of certain proteolytic factor(s) which are putatively capable of cleaving &bgr;APP at a site near the &bgr;APP amino-terminus.
The APP gene is known to be located on human chromosome 21. A locus segregating with familial Alzheimer's disease has been mapped to chromosome 21 (St. George Hyslop et al (1987)
Science
235: 885) close to the APP gene. Recombinants between he APP gene and the AD locus have been previously reported (Schellenberg et al. (1988)
Science
241: 1507; Schellenberg et al. (1991)
Am. J. Hum. Genetics
48: 563; Schellenberg et al. (1991)
Am. J. Hum. Genetics
49: 511, incorporated herein by reference).
The identification of mutations in the amyloid precursor protein gene which cause familial, early onset Alzheimer's disease is evidence that amyloid metabolism is the central event in the pathogenic process underlying the disease. Four reported disease-causing mutations include with respect to the 770 isoform, valine
717
to isoleucine (Goate et al. (1991)
Nature
349: 704), valine
717
to glycine (Chartier Harlan et al. (1991)
Nature
353: 844), valine
7
l
7
to phenylalanine (Murrell et al. (1991)
Science
254: 97) and with respect to the 695 isoform, a double mutation changing lysine
595
-methionine
596
to asparagine
596
-leucine
596
(Mullan et al. (1992)
Nature Genet
1: 345; Citron et al. (1992)
Nature
360: 672) referred to as the Swedish mutation. APP alleles which are positively correlated with AD are termed “disease-associated alleles”.
The development of experimental models of Alzheimer's disease that can be used to define further the underlying biochemical events involved in AD pathogenesis would be highly desirable. Such models could presumably be employed, in one application, to screen for agents that alter the degenerative course of Alzheimer's disease. For example, a model system of Alzheimer's disease could be used to screen for environmental factors that induce or accelerate the pathogenesis of AD. In contradistinction, an experimental model could be used to screen for agents that inhibit, prevent, or reverse the progression of AD. Presumably, such models could be employed to develop pharmaceuticals that are effective in preventing, arresting, or reversing AD.
Unfortunately, only humans and aged non-human primates develop any of the pathological features of AD; the expense and difficulty of using primates and the length of time required for developing the AD pathology makes extensive research on such animals prohibitive. Rodents do not develop AD, even at an extreme age. It has been reported that the injection of &bgr;-amyloid protein (&bgr;AP) or cytotoxic &bgr;AP fragments into rodent brain results in cell loss and induces an antigenic marker for neurofibrillary tangle components (Kowall et al. (1991)
Proc. Natl. Acad. Sci.
(
U.S.A.
) 88: 7247). Mice which carry an extra copy of the APP

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