CA2101774C - Test and model for alzheimer's disease - Google Patents

Test and model for alzheimer's disease Download PDF

Info

Publication number
CA2101774C
CA2101774C CA2101774A CA2101774A CA2101774C CA 2101774 C CA2101774 C CA 2101774C CA 2101774 A CA2101774 A CA 2101774A CA 2101774 A CA2101774 A CA 2101774A CA 2101774 C CA2101774 C CA 2101774C
Authority
CA
Canada
Prior art keywords
codon
app
thr
glu
ile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
CA2101774A
Other languages
French (fr)
Other versions
CA2101774A1 (en
Inventor
John A. Hardy
Marie-Christine Chartier-Harlin
Alison M. Goate
Michael J. Owen
Michael J. Mullan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Elan Pharmaceuticals LLC
Original Assignee
Elan Pharmaceuticals LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=26298303&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=CA2101774(C) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority claimed from GB919101307A external-priority patent/GB9101307D0/en
Priority claimed from GB919118445A external-priority patent/GB9118445D0/en
Application filed by Elan Pharmaceuticals LLC filed Critical Elan Pharmaceuticals LLC
Priority to CA002372251A priority Critical patent/CA2372251A1/en
Publication of CA2101774A1 publication Critical patent/CA2101774A1/en
Application granted granted Critical
Publication of CA2101774C publication Critical patent/CA2101774C/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027New breeds of vertebrates
    • A01K67/0275Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278Humanized animals, e.g. knockin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/28Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711Alzheimer's disease; Amyloid plaque core protein
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575Hormones
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00Modified animals
    • A01K2207/15Humanized animals
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/05Animals comprising random inserted nucleic acids (transgenic)
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/07Animals genetically altered by homologous recombination
    • A01K2217/072Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00Animals characterised by species
    • A01K2227/10Mammal
    • A01K2227/105Murine
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/03Animal model, e.g. for test or diseases
    • A01K2267/0306Animal model for genetic diseases
    • A01K2267/0312Animal model for Alzheimer's disease
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/008Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/172Haplotypes

Abstract

Model systems of Alzheimer's disease comprise a DNA sequence encoding an amy-loid precursor protein (APP) isoform or fragment that has an amino acid substitution. The substituted amino acid may be other than valine at the amino acid position corresponding to amino acid residue position 717 of APP770. Methods of determining genetic predisposi-tion to Alzheimer's disease are also disclosed.

Description

TEST AND MQDEL FOR ALZHEIMER'S DISEASE

BACKGROUND OF THE INVENTION
Alzheimer's disease 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 Alzheimer's disease is much more infrequent but shows a pathologically identical dementia with diffuse brain atrophy 10, which develops well before the senile period, i.e., between the ages of 35 and 60 years. There is evidence that one form of this type of Alzheimer's disease shows a tendency to run in families and is therefore known as familial Alzheimer's disease (FAD).
In both types of Alzheimer's disease the pathology is the same but the abnormalities tend to be more severe and more widespread in cases beginning at an earlier age. The disease is characterized by two types of lesions in the brain, these are senile plaques and neurofibrillary tangles.
Senile plaques are areas of disorganized neuropil up to 15014m across with extracellular amyloid deposits at the center. Neurofibrillary tangles are intracellular deposits of amyloid protein consisting of two filaments twisted about each other in pairs.
The major protein subunit, S-amyloid protein, of the amyloid filaments of the senile plaque is a highly aggregating small polypeptide of approximate relative molecular mass 4,500. This protein is a cleavage product of a much larger precursor protein called amyloid precursor protein (APP).
At present there is no known effective therapy for the various forms of Alzheimer's disease (AD). However, there are several other forms of dementia for which treatment is available and which give rise to progressive intellectual deterioration closely resembling the dementia associated with Alzheimer's disease.
A diagnostic test for AD would therefore provide a valuable tool in the diagnosis and treatment of these other conditions, by way of being able to exclude Alzheimer's disease. It will also be of value when a suitable therapy does become available.
Also important is the development of experimental models of Alzheimer's disease that can be used to define further the underlying biochemical events involved in AD pathogenesis. 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.

SUMMARY OF THE INVENTION
The present invention provides model systems of Alzheimer's disease, wherein the model system comprises a DNA sequence encoding an amyloid precursor protein (APP) isoform or fragment that has an amino acid other than valine at the amino acid position corresponding to amino acid residue position 717 of APP770.
In a first embodiment, the present invention provides an isolated DNA sequence that encodes an amyloid precursor protein (APP) isoform or fragment that has an amino acid other than valine at the amino acid position corresponding to amino acid residue position 717 of APP770.
In a second embodiment, the present invention provides a transgenic nonhuman animal that harbors at least one integrated copy of a human DNA
sequence that encodes an amyloid precursor protein (APP) isoform or fragment that has an amino acid other than valine at the amino acid position corresponding e i tr,i to amino acid residue position 717 of APP770.
In a third embodiment, the present invention provides a transgenic nonhuman animal wherein at least one of the endogenous nonhuman APP alleles has been completely or partially replaced by all or a portion of a human APP
gene that includes a codon 717 that does not encode valine.
In a fourth embodiment, the present invention provides cells, typically mammalian cells and preferably mammalian cells of the neural, glial, or astrocytic lineage, that have been transformed or transfected with a heterologous DNA sequence, or have been derived from a transgenic nonhuman animal, wherein the cells express an amyloid precursor protein (APP) isoform or fragment that has an amino acid other than valine at the amino acid position corresponding to amino acid residue position 717 of APP770. In accordance with standard protocols, cultured human cells, either primary cultures or immortalized cell lines, may be transfected, either transiently or stably, with a mutant APP allele so that the cultured human cell expresses a mutant APP polypeptide.
In a fifth embodiment, the present invention provides a method of producing transgenic nonhuman animals and transformed cells that contain a DNA
sequence encoding an amyloid precursor protein (APP),isoform or fragment that has an amino acid other than valine at the amino acid position corresponding to amino acid residue position 717 of APP770.
In a sixth embodiment, the present invention provides a method of producing, free from other human proteins, a human amyloid precursor protein (APP) isoform or fragment that has an amino acid other than valine at the amino acid position corresponding to amino acid residue position 717 of APP770.
In a seventh embodiment, the present invention provides a human amyloid precursor protein (APP) isoform or fragment, free from other human proteins, that has an amino acid other than valine at the amino acid position corresponding to amino acid residue position 717 of APP770.
In an eighth embodiment, the invention provides a method for detecting an APP allele that is linked (i.e., cosegregates with) a genetic predisposition to Alzheimer's disease, particularly early onset AD, wherein such a pathognomonic APP allele is detected by determining that codon 717 of the allele does not encode valine. Preferably, a pathognomonic APP allele is detected when codon 717 is determined to encode either isoleucine, glycine, or phenylalanine.
Thus, methods for locating the presence of genetic alterations associated with Alzheimer's disease are provided. This diagnostic method may be used to predict the development of the disease prior to onset, for genetic screening, or to detect a specific mutation in an experimental nonhuman animal or a cell.
In a ninth embodiment, the invention provides a human variant APP
polypeptide free of other human proteins, typically present in a cell of a nonhuman animal. The invention also relates to an isolated nucleic acid encoding such a polypeptide and to uses and applications of such nucleic acid as are described above in relation to the specific embodiment of the invention which involves an amino acid substitution at position 717 (as defined in relation to APP770).
According to one aspect of the invention there is provided a method for detecting the presence, in a nucleic acid or other sample removed from a subject, of the gene for Alzheimer's disease comprising identifying a genetic alteration in a gene sequence coding for APP. Such genetic alterations may include mutations, insertions or deletions.

BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 illustrates a first pedigree in which early onset AD is apparently inherited as an autosomal dominant disorder. The average age of onset in this family is 57 5 years. Black symbols denote affected individuals and oblique lines indicate individuals who are deceased. Females are denoted by circles and males by squares. Triangles are used in the present generation to preserve anonymity. In generation 11 the spouses of the two affected brothers were sisters. Samples were available from the 13 individuals whose haplotypes are illustrated, from a further 19 children and spouses of these individuals and from 7 more distantly related unaffected individuals. Beneath the pedigree are ideograms of the two chromosomes 21 in each individual of the third generation 5 at four loci on the long arm of the chromosome. The linkage data suggest that the black chromosomes were inherited from the affected fathers.
Fig. 2 shows an autoradiograph of a sequencing gel from part of exon 17 of the APP gene in a normal and an affected individual from the Fig. 1 pedigree showing a single base pair change at base pair 2149 in the affected individual. This C to T transition leads to an amino acid substitution of a valine by an isoleucine at codon 717.
Fig. 3 shows part of the amino acid sequence encoded by exons 16 and 17 of the APP gene showing the mutation valine to isoleucine (V to I) within the transmembrane domain and the mutation causing HCHWA-D (E to Q) in the extracellular domain. The shaded region of the transmembrane domain and the boxed amino acids of the extracellular domain represent the sequence of the deposited (3-amyloid peptide. Adapted from Kang et al. (1987) Nature 325:733.
Fig. 4 shows BcII digests of the exon 17 PCR product from unaffected and affected individuals in an early onset AD family showing co-segregation of the restriction site and the disease.
Fig. 5 shows the pedigree of family F19, together with D21S210 data.
Fig. 6 shows APP exon 17 sequences in an affected and unaffected member of F 19. In the affected member there is a G- > T transition at position 2150.
Fig. 7 shows the sequence of APP695.
Fig. 8 shows the sequence of APP751.
Fig. 9 shows the sequence of APP770.

0% 41 1, ti 1 i 1 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The accumulation of 13-amyloid protein (A4) in particular brain regions is one of the main pathologic characteristics of Alzheimer's disease.
The S-amyloid protein is an approximately 4 kD. protein (39 to 42 amino acids) which is derived, as an internal cleavage product, from one or more isoforms of a larger amyloid precursor protein (APP). There are at least five distinct isoforms of APP
containing 563, 695, 714, 751, and 770 amino acids, respectively (Wirak et al.
(1991) Science 253:323). These isoforms of APP are generated by alternative splicing of primary transcripts of a single gene, designated the APP gene, which is located on human chromosome 21. It is known that most of the APP isoforms are glycosylated transmembrane proteins (Goldgaber et al. (1987) Science 235:877), and that four of the isoforms, AA563, APP714, APP751 and APP770, have a protease inhibitor domain that is homologous to the Kunitz type of serine protease inhibitors. The $-amyloid (A4) segment comprises approximately half of the transmembrane domain and approximately the first 28 amino acids of the extracellular domain of an APP isoform.
Proteolytic processing of APP in vivo is a normal physiological process. Carboxy-terminal truncated forms of APP695, APP751, and APP770 are present in brain and cerebrospinal fluid (Palmert et al. (1989) Proc. Natl.
Acad.
Sci. U.S.A. 86:6338; Weidemann et al. (1989) Cell 57:115) and result from cleavage of the APP isoform at a constitutive cleavage site within the A4 peptide domain of an APP isoform (Esch et al. (1990) Science 248:1122). Normal proteolytic cleavage at the constitutive cleavage site yields a large (approximately 100 kD) soluble, N-terminal fragment that contains the protease inhibitor domain in some isoforms, and a 9-10 kD membrane-bound, C-terminal fragment that includes most of the A4 domain.
Generation of pathogenic 13-amyloid (A4) protein appears to be the result of aberrant or alternative proteolytic processing of APP, such that normal cleavage at the constitutive site within the A4 domain does not occur, but rather WO 92/13069 PCt'/G B92/00123 cleavage occurs at two specific sites which flank the A4 domain. One of these aberrant cleavage sites is in the transmembrane domain and the other aberrant cleavage site is located approximately at the N-terminus of the first 28 amino acids of the extracellular domain (see Fig. 3). Such aberrant proteolytic cleavage produces the $-amyloid A4 polypeptide which is prone to forming dense amyloidogenic aggregates that are resistant to proteolytic degradation and removal.
The resultant 0-amyloid aggregates presumably are involved in the formation of the abundant amyloid plaques and cerebrovascular amyloid that are the neuropathological hallmarks of Alzheimer's disease. However, the exact aberrant cleavage sites are not always precise; /3-amyloid molecules isolated from the brain of a patient with AD show N- and C- terminal heterogeneity. Therefore, the aberrant cleavage pathway may involve either sequence-specific proteolysis followed by exopeptidase activity (creating end-heterogeneity), or may not be sequence-specific.
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 (Hyslop et al. (1987) Science 235:885) close to the APP gene.
Recombinants between the APP gene and the AD locus have been previously reported (Schellenberg et al. (1988) Science 241:1507). The data appeared to exclude the APP gene as the site of any mutation that might cause FAD (Van Broekhoven et al. (1987) Nature 329:153; Tanzi et al. (1987) Nature 329:156).
Recombinant DNA technology provides several techniques for analyzing genes to locate possible mutations. For example, the polymerase chain reaction (Bell (1989) Immunology Today, 10:351) may be used to amplify specific sequences using intronic primers, which can then be analyzed by direct sequencing.
Researchers working in the area of the hereditary cerebral haemorrhage with amyloidosis of the Dutch type ("HCHWA-D") (Levy et al.
(1990) Science 248:11224) found a substitution of Glu to Gln at residue 618 (using the APP695 numbering system) in APP which is thought to result in the deposition of O-amyloid in the cerebral vessels of these patients. The present inventors have identified a single base substitution, a C to T transition at base pair 2149, has been found in part of the sequence of the APP gene in some cases of familial Alzheimer's disease. This base pair transition leads to an amino acid substitution, i.e., valine to isoleucine at amino acid 717 (APP 0) (see Yoshikai et al.
(1990) Gene L7:257), close to the C-terminus of the 9-amyloid protein. This suggests that some cases of Alzheimer's disease are caused by mutations in the APP gene, and specifically mutations that change codon 717 such that it encodes an amino acid other than valine.
Additionally, a further single base substitution, a T to G transition at adjacent base pair 2150, has been found in part of the sequence of the APP
gene in other cases of familial Alzheimer's disease. This base pair transition leads to a different amino acid substitution, namely valine to glycine, at amino acid 717, thereby strengthening the argument that some cases of Alzheimer's disease are caused by mutations in the APP gene, specifically at codon 717.
It is now clear that a mutation in the APP gene locus that results in a substitution of isoleucine for valine at codon 717 (residue 642 in APP695) gives rise to AD in some families (Goate et al. (1991) Nature 349:704). A second APP
allelic variant wherein glycine is substituted for valine at codon 717 is now identified, and is so closely linked to the AD phenotype as to indicate that allelic variants at codon 717 of the APP gene, particularly those encoding an amino acid other than valine, and more particularly those encoding a isoleucine, glycine, or phenylalanine, are pathogenic and/or pathognomonic alleles (Chartier-Harlin et al.
(1991) Nature 353:844).
Proteolysis on either side of the S-amyloid (A4) region of APP may be enhanced or qualitatively altered by the specific mutations at codon 717, increasing the rate of 0-amyloid deposition and aggregation. Such codon 717 mutations may increase /3-amyloid formation by providing a poorer substrate for the main proteolytic pathway (cleavage at the constitutive site) or a better substrate for a competing, alternative cleavage pathway (at aberrant cleavage sites).
DEFINITIONS
A number of terms and expressions are used throughout the specification and, to facilitate the understanding thereof, the following definitions are provided:
As used herein, "exon" refers to any segment of an interrupted gene that is represented in the mature RNA product.
As used herein, "intron" refers to a segment of an interrupted gene that is not represented in the mature RNA product. Introns are part of the primary nuclear transcript but are spliced out to produce mRNA, which is then transported to the cytoplasm.
As used herein, the phrase "gene sequence coding for amyloid is protein precursor" may be interpreted to mean the DNA and cDNA sequence as detailed by Yoshikai et al. (1990) St= $7_:257 and Kang et al, loc. cit., together with the promoter DNA sequence as described by Salbaum et al. (1988) EMBO
712:2807.
As used herein, the terms "label" or "labeled" refers to incorporation of a detectable marker (e.g., by incorporation of a radiolabeled nucleotide or by end-labeling with a terminal radiolabeled phosphate). DNA or RNA is typically labeled by incorporation of a radiolabeled nucleotide (H3, C14, S35, P32) or a biotinylated nucleotide that can be detected by marked avidin (e.g., avidin containing a fluorescent marker or enzymatic activity) or digoxygeninylated nucleotide that can be detected by marked specific antibody.
As used herein, "isoform", "APP", and "APP isoform" refer to a polypeptide that is encoded by at least one exon of the APP gene (Kitaguchi et al.
(1988) Nature 331:530; Ponte et al., ibid., p.525; R.E. Tanzi, i id , p.528;
de Sauvage and Octave (1989) Science 2:651; Golde et al. (1990) Neuron 4:253).

An APP isoform may be encoded by an APP allele (or exon thereof) that is associated with a form of Alzheimer's disease or that is not associated with an AD
disease phenotype.
The term "13-amyloid gene" is used herein as a synonym for the APP
5 gene, as fl-amyloid is a protein product produced by a po3t-translational cleavage of an APP gene product.
As used herein, "fragment" refers to a polypeptide of at least about 9 amino acids, typically 50 to 75, or more, wherein the polypeptide contains an amino acid core sequence (listed in order from amino- to carboxy-terminal 10 direction):
-Ile-Ala-Thr-Val-Ile-X-Ile-Thr-Leu- [SEQ ID NO:6]
where X is any of the twenty conventional amino acids except valine, and particularly where X is isoleucine, glycine, or phenylalanine. A fragment may be a truncated APP isoform, modified APP isoform (as by amino acid substitutions, deletions, or additions outside of the core sequence), or other variant polypeptide sequence, but is n a naturally-occurring APP isoform or fl-amyloid polypeptide that is present in a human individual, whether affected by AD or not. If desired, the fragment may be fused at either terminus to additional amino acids, which may number from 1 to 20, typically 50 to 100, but up to 250 to 500 or more.
As used herein, "APP75 1" and "APP770" refer, respectively, to the 751 and 770 amino acid residue long polypeptides encoded by the human APP
gene (Ponte et al. loc. cit.; Kitaguchi et al. loc, cit.; Tanzi et al. loc, cit.).
As used herein, "codon 717" refers to the codon (i.e., the trinucleotide sequence) that encodes the 717th amino acid position in APP770, or the amino acid position in an APP isoform or fragment that corresponds to the 717th position in APP770. For example but not limitation, a 670 residue long fragment that is produced by truncating APP770 by removing the 100 N-terminal amino acids has its 617th amino acid position corresponding to codon 717. In fact, as used herein, codon 717 refers to the codon that encodes the 698th amino acid f , residue of APP751 [SEQ ID NO:21 and the 642nd amino acid residue of APP695 [SEQ ID NO:I].
As used herein, "human APP isoform or fragment" refers to an APP
isoform or fragment that contains a sequence of at least 9 consecutive amino acids that is identical to a sequence in a human APP770, APP751, or APP695 protein that occurs naturally in a human individual, and wherein an identical sequence is not present in an APP protein that occurs naturally in a nonhuman species.
A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a to promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
The term "corresponds to" is used herein to mean that a sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA".
The term "transcriptional enhancement" is used herein to refer to functional property of producing an increase in the rate of transcription of linked sequences that contain a functional promoter.
The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents are evaluated for potential biological activity by inclusion in screening assays described hereinbelow.
As used herein, the term "mutant" refers to APP alleles having missense mutations that are pathognomonic for a genetic predisposition for developing AD; specifically a mutation at codon 717 (as referenced by the amino acid sequence in APP770) of the APP gene, such that codon 717 encodes one of the nineteen amino acids that are not valine (i.e., glycine, methionine, alanine, serine, isoleucine, leucine, threonine, proline, histidine, cysteine, tyrosine, phenylalanine, glutamic acid, tryptophan, arginine, aspartic acid, asparagine, lysine, and glutamine), but preferably isoleucine, glycine, or phenylalanine.
Thus a mutant APP770 polypeptide is an APP770 polypeptide that has an amino acid residue at position 717 that is not valine. Other mutant APP isoforms comprise a non-valine amino acid at the amino acid residue position that corresponds to codon 717 (i.e., that is encoded by codon 717). Similarly, a mutant APP allele or a variant APP codon 717 allele is an APP allele that encodes an amino acid other than valine at codon 717 (referenced to the human APP770 deduced translation as described in the "codon 717" definition, supra), preferably isoleucine, glycine, or phenylalanine. Hence, an APP allele that encodes valine at codon 717 is a "wild-type" APP allele.
It is apparent to one of skill in the art that nucleotide substitutions, deletions, and additions may be incorporated into the polynucleotides of the invention. However, such nucleotide substitutions, deletions, and additions should not substantially disrupt the ability of the polynucleotide to hybridize to one of the polynucleotide sequences shown in Figs. 5 and 6 under hybridization conditions that are sufficiently stringent to result in specific hybridization.
"Specific hybridization" is defined herein as the formation of hybrids between a probe polynucleotide (e.g., a polynucleotide of the invention which may include substitutions, deletion, and/or additions) and a specific target polynucleotide (e.g., a polynucleotide having the sequence ), wherein the probe preferentially hybridizes to the specific target such that, for example, a band corresponding to a variant APP allele or restriction fragment thereof, can be identified on a Southern blot, whereas a corresponding wild-type APP allele (i,L, one that encodes valine at codon 717) is not identified or can be discriminated from a variant APP allele on the basis of signal intensity. Hybridization probes capable of specific hybridization to detect a single-base mismatch may be designed according to methods known in the art and described in Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989), Cold Spring Harbor, N.Y. and Berger and Kimmel, Methods in Enzymology. Volume 152. Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, CA; Gibbs et al.
(1989) Nucleic Acids Res. 17:2437; Kwok et al. (1990) Nucleic Acids Res.
18:999; Miyada et al. (1987) Methods Enzymol. 154:94, each of which is incorporated herein by reference. The T. for oligonucleotides is calculated under standard conditions (1 M NaCl) to be [4 C x (G+C) + 2 C x (A+T)]. While the conditions of PCR differ from the standard conditions, this Tm is used as a guide for the expected relative stabilities of oligonucleotides. Allele-specific primers are typically 13-15 nucleotides long, sometimes 16-21 nucleotides long, or longer; when short primers are used, such as a 14 nucleotide long primer, low annealing temperatures are used, typically 44 to 50 C, occasionally somewhat higher or lower depending on the base composition of the sequence(s).

DESCRIPTION OF THE PREFERRED EMBODIMENTS
Detection of Mutant Codon 717 APP Alleles In an embodiment of the invention, the method involves identifying a genetic alteration at amino acid 717, which may cause the consensus Val to be changed, for example, to another hydrophobic residue. This will generally be performed on a specimen removed from the subject. Hydrophobic residues include Leu, Ala, Ile and Gly, the first three of which have aliphatic side chains.
Phe also has a hydrophobic residue which may be appropriate. As indicated above, preferred residues include Ile, Gly, and Phe (Murrell et al, (1991) Science 254:97).

I) 17 The fact that these mutations discussed above are at the same codon may be a coincidence, but this seems unlikely on statistical grounds. There are two possibilities that may explain these data. First, substitution of the valine residue at codon 717 may result in increased beta-amyloid deposition due to changes in APP metabolism. Secondly, the variation in the sequence around this position may result in increased translation of APP mRNAs and thus cause AD by a route analogous to that by which AD is believed to be caused in Down Syndrome (Tanzi and Hyman (1991) Nature 350:564 and Rumble et al. (1989) N. Engl. J.
Med. 320:1446). In situ hybridization studies have shown that APP 717 mutations do not alter APP expression (Harrison et al. (1991) Neurorep. 2:152).
The V717I (APP 717 Val- > Ile), V717G (APP 717 Val->Gly) and V717F (APP 717 Val->Phe) mutations would destabilise a putative stem loop structure and destroy a possible iron-responsive element between base pairs 2131 and 2156 (Tanzi and Hyman, loc. cit. ). There are several other possible mutations which could also disrupt this structure, many of which would be silent at the protein level; yet these mutations specifically referred to have involved a change to the same amino acid, and no silent changes or changes to other amino acids have been reported prior to the work described herein.
Examination of sequence data from 10 other mammalian species (Johnstone et al.
(1991) Mol. Brain Res. 10:299) shows that while the valine residue at codon is conserved in all of them, the putative stem loop structure postulated from the human sequence (Tanzi and Hyman loc. cit.) would not be predicted to occur in either cattle or sheep; and in pig and mouse the consensus sequence for the iron-responsive elements is not present. Finally, such stem loop structures are believed to modulate gene translation by altering mRNA stability (Klausner and Harford (1989) Science 246:870); however, Harrison and colleagues (Harrison et al. loc. cit.) have shown by in situ hybridization that APP mRNAs are not grossly altered in the brain of an individual with the V717I mutation. For these reasons, it is believed likely that alterations in the rate of APP translation caused by the specific mutations identified are not likely to be the key to their pathogenicity.
The fact that the specific mutations discussed involve different changes (Val-> Ile, Val-> Gly, and Val-> Phe) suggests that neither side-chain hydrophobicity nor side-chain bulk is the crucial issue. All examples of APP
5 alleles that encode an amino acid other than valine at codcn 717, cosegregate with FAD; suggesting that the valine that occurs at position 717 in wild-type or APP751 is a critical amino acid residue for non-pathogenic APP proteolytic processing (i.e., by the constitutive cleavage pathway).
The major metabolic pathway for the APP molecule involves 10 cleavage within the beta-amyloid fragment (Esch et al. loc. cit.). To generate beta-amyloid, there must be a second pathway in which APP is cleaved outside this sequence. Such a cleavage would be likely to leave a stub of the APP molecule containing the beta-amyloid fragment embedded in the membrane. Possibly, the beta-amyloid-containing fragment which is generated by the second pathway is 15 degraded by peptidase action; the reported mutations may be pathogenic because peptides which contain them may be more resistant to the actions of this peptidase.
Therefore, genetic alterations in the APP gene which result in altered (generally reduced) degradative properties are particularly important in the application of the invention. There are several methodologies available from recombinant DNA
technology which may be used for detecting and identifying a genetic mutation responsible for Alzheimer's disease. These include direct probing, polymerase chain reaction (PCR) methodology, restriction fragment length polymorphism (RFLP) analysis and single strand conformational analysis (SSCA).
Detection of point mutations using direct probing involves the use oligonucleotide probes which may be prepared synthetically or by nick translation.
The DNA probes may be suitably labelled using, for example, a radiolabel, enzyme label, fluorescent label, biotin-avidin label and the like for subsequent visualization in for example a Southern blot hybridization procedure. The labelled probe is reacted with the sample DNA bound to a nitrocellulose or Nylon 66 WO 92/13069 11'' ff PCT/GB92/00123 substrate. The areas that carry DNA sequences complementary to the labelled DNA probe become labelled themselves as a consequence of the reannealling reaction. The areas of the filter that exhibit such labelling may then be visualized, for example, by autoradiography.
Alternative probing techniques, such as ligase chain reaction (LCR) involve the use of mismatch probes, g:,, probes which have full complementarity with the target except at the point of the mutation. The target sequence is then allowed to hybridize both with oligonucleotides having full complementarity and oligonucleotides containing a mismatch, under conditions which will distinguish between the two. By manipulating the reaction conditions it is possible to obtain hybridization only where there is full complementarity. If a mismatch is present then there is significantly reduced hybridization.
The polymerise chain reaction (PCR) is a technique that amplifies specific DNA sequences with remarkable efficiency. Repeated cycles of denaturation, primer annealing and extension carried out with a heat stable enzyme Taq polymerase leads to exponential increases in the concentration of desired DNA
sequences.
Given a knowledge of the nucleotide sequence encoding the precursor of amyloid protein of AD (Kang et al. loc. cit., and Yoshikai, above) it may be possible to prepare synthetic oligonucleotides complementary to sequences which flank the DNA of interest. Each oligonucleotide is complementary to one of the two strands. The DNA is then denatured at high temperatures (e.g., 95 C) and then reannealed in the presence of a large molar excess of oligonucleotides. The oligonucleotides, oriented with their 3' ends pointing towards each other, hybridize to opposite strands of the target sequence and prime enzymatic extension along the nucleic acid template in the presence of the four deoxyribonucleotide triphosphates. The end product is then denatured again for another cycle. After this three-step cycle has been repeated several times, amplification of a DNA segment by more than one million fold can be achieved.

aUl I

The resulting DNA may then be directly sequenced in order to locate any genetic alteration. Alternatively, it may be possible to prepare oligonucleotides that will only bind to altered DNA, so that PCR will only result in multiplication of the DNA if the mutation is present. Following PCR, allele-specific oligonucleotide hybridization (Dihella et al. (1988) Lancet 1:497) may be used to detect the AD
point mutation. Alternatively an adaptation of PCR called amplification of specific alleles (PASA) can be employed; this uses differential amplification for rapid and reliable distinction between alleles that differ at a single base pair.
In yet another method PCR may be followed by restriction endonuclease digestion with subsequent analysis of the resultant products. The substitution of T for C at base pair 2149, found as a result of sequencing exon 17, creates a Bc1I restriction site. The creation of this restriction endonuclease recognition site facilitates the detection of the AD mutation using RFLP
analysis or by detection of the presence or absence of a polymorphic Bc1I site in a PCR
product that spans codon 717.
For RFLP analysis, DNA is obtained, for example, from the blood of the subject suspected of having AD and from a normal subject is digested with the restriction endonuclease Bc1I and subsequently separated on the basis of size using agarose gel electrophoresis. The Southern blot technique can then be used to detect, by hybridization with labeled probes, the products of endonuclease digestion. The patterns obtained from the Southern blot can then be compared.
Using such an approach, DNA spanning an Alzheimer's mutation that creates or removes a restriction site at codon 717, such as the BclI site, is detected by determining the number of bands detected and comps' ng this number to a reference allele that has a codon 717 allele that encodes valine.
Correspondingly, the substitution of G for T at base pair 2150 creates a SfaNI restriction site (GCATC), which may be exploited in a manner similar to that described above, mutatis mutandis. Similar creation of additional restriction sites by nucleotide substitutions within codon 717, wherein the codon 717 encodes an amino acid other than valine, can be readily calculated by reference to the genetic code and a list of nucleotide sequences recognized by restriction endonucleases romega Protocols and Applications Guide, (1991) Promega Corporation, Madison, Wisconsin).
Single strand conformational analysis (SSCA) (Orita et al. (1989) Genomics 5:874 and Orita et al. (1990) Genomics 6:271) offers a relatively quick method of detecting sequence changes which may be appropriate in at least some instances.
PCR amplification of specific alleles (PASA) is a rapid method of detecting single-base mutations or polymorphisms (Newton et al. (1989) Nucleic Acids Res. 17:2503; Nichols et al. (1989) n i 1:535; Okayama et al. (1989) J. Lab. Clin. Med..114:105; Sarkar et al. (1990) Anal. Biochem. x$¾:64; Sommer et al. (1989) Mayo Clin. Proc. b4:1361; Wu (1989) Proc. Natl. Acad. Sci.
U.S.A.
$¾:2757; and Dutton et al. (1991) Biotechniques 11:700). PASA (also known as allele specific amplification) involves amplification with two oligonucleotide primers such that one is allele-specific. The desired allele is efficiently amplified, while the other allele(s) is poorly amplified because it mismatches with a base at or near the 3' end of the allele-specific primer. Thus, PASA or the related method of PAMSA may be used to specifically amplify one or more variant APP codon 717 alleles. Where such amplification is done on genetic material (or RNA) obtained from an individual, it can serve as a method of detecting the presence of one or more variant APP codon 717 alleles in an individual.
Similarly, a method known a ligase chain reaction (LCR) has been used to successfully detect a single-base substitution in a hemoglobin allele that causes sickle cell anemia (Barany et al. (1991) Proc. Natl. Acad. Sci. U.S.A.
$$:189; Weiss (1991) Science 254:1292). LCR probes may be combined, or multiplexed for simultaneously screening for multiple different mutations.
Thus, one method of screening for variant APP colon 717 alleles is to multiplex at least two, and preferably all, LCR probes that will detect an APP allele having a codon J1 i a 717 that does not encode valine, but that does encode an amino acid. The universal genetic code provides the degenerate sequences of all the encoded non-valine amino acids, thus LCR probe design for detecting any particular variant codon allele is straightforward, and multiplexed pools of such LCR probes may be selected in the discretion of a practitioner for his particular desired use.
In performing diagnosis using any of the above techniques or variations thereof, it is preferable that several individuals are examined.
These may include an unaffected parent, an affected parent, an affected sibling, an unaffected sibling as well as other perhaps more distant family members.
Model Animals and Cell Lines Having identified specific mutations in codon 717 of the $-amyloid gene as a cause of familial Alzheimer's disease (FAD), it is possible, using genetic manipulation, to develop transgenic model systems and/or whole cell systems containing the mutated FAD gene (or a portion thereof) for use, for example, as model systems for screening for drugs and evaluating drug effectiveness.
Additionally, such model systems provide a tool for defining the underlying biochemistry of APP and S-amyloid metabolism, which thereby provides a basis for rational drug design.
One type of cell system can be naturally derived. For this, blood samples from the affected subject must be obtained in order to provide the necessary cells which can be permanently transformed into a lymphoblastoid cell line using, for example, Epstein-Barr virus.
Once established, such cell lines can be grown continuously in suspension culture and may be used for a variety of in vitro experiments to study APP expression and processing.
Since the FAD mutation is dominant, an alternative method for constructing a cell line is to engineer genetically a mutated gene, or a portion thereof spanning codon 717, into an established (either stably or transiently) cell `3'O 92/13069 PCr/GB92/00123 line of choice. Sisodia (1990) Science 248:492) has described the insertion of a normal APP gene, by transfection, into mammalian cells. Oltersdorf et al.
((1990) J. Biol. Chem. 265:4492) describe the insertion of APP into immortalized eukaryotic cell lines.
5 Baculovirus expression systems are useful for high level expression of heterologous genes in eukaryotic cells. Knops et al. (1991) J. Biol. Chem.
266(11):7285 describes the expression of APP using such a system.
In yet a further use of the present method, it may be possible to excise the mutated gene (i.e., a variant APP codon 717 gene) for use in the 10 creation of transgenic animals containing the mutated gene. For example, an entire human variant APP codon 717 allele may be cloned and isolated, either in parts or as a whole, in a cloning vector (e.g., XCharon35, cosmid, or yeast artificial chromosome). The human variant APP codon 717 gene, either in parts or in whole, may be transferred to a host nonhuman animal, such as a mouse. As 15 a result of the transfer, the resultant transgenic nonhuman animal will preferably express one or more variant APP codon 717 polypeptides. Most preferably, a transgenic nonhuman animal of the invention will express one or more variant APP
codon 717 polypeptides in a neuron-specific manner (Wirak et al. (1991) EMBO
j.Q:289). This may be accomplished by transferring substantially the entire human 20 = APP gene (encoding a codon 717 mutant) including the 4.5 kilobase sequence that is adjacent to and upstream of the first major APP transcriptional start site.
Alternatively, one may design minigenes encoding variant APP
codon 717 polypeptides. Such minigenes may contain a CDNA sequence encoding a variant APP codon 717 polypeptide, preferably full-length, a combination of APP
gene exons, or a combination thereof, linked to a downstream polyadenylation signal sequence and an upstream promoter (and preferably enhancer). Such a minigene construct will, when introduced into an appropriate transgenic host (e.g., mouse or rat), express an encoded variant APP codon 717 polypeptide, most preferably a variant APP codon 717 polypeptide that contains either an isoleucine, w.1 U.L ti glycine, or phenylalanine residue at codon 717 of APP770 or the corresponding position in an APP isoform or fragment.
One approach to creating transgenic animals is to target a mutation to the desired gene by homologous recombination in an embryonic stem (ES) cell line in vitro followed by microinjection of the modified ES cell line into a host blastocyst and subsequent incubation in a foster mother (see Frohman and Martin (1989) CkU 5,¾:145). Alternatively, the technique of microinjection of the mutated gene, or a portion thereof, into a one-cell embryo followed by incubation in a foster mother can be used. Various uses of transgenic animals, particularly transgenic animals that express a wild-type APP isoform or fragment, are disclosed in Wirak et al. (1991) EMBO, 10(2):289; Schilling et al. (1991) Gene 98(2):225;
Quon et al. (1991) Nature =:239; Wirak et al. (1991) Science W:323; and Kawabata et al. (1991) Nature 54:476. Additional methods for producing transgenic animals are known in the art.
Alternatively, site-directed mutagenesis and/or gene conversion can be used to mutate a murine (or other nonhuman) APP gene allele, either endogenous or transfected, such that the mutated allele does not encode valine at the codon position in the mouse APP gene that corresponds to codon 717 (of APP770) of the human APP gene (such position is readily identified by homology matching of the murine APP gene or APP protein to the human APP gene or APP770 protein). Preferably, such a mutated murine allele would encode isoleucine or glycine or phenylalanine at the corresponding codon position.
Therapeutics Having detected the genetic mutation in the gene sequence coding for 0-amyloid protein in an individual not yet showing overt signs of familial AD, using the method of the present invention, it may be possible to employ gene therapy, in the form of gene implants, to prevent the development of the disease.
Additional embodiments directed to modulation of the production of i variant APP proteins include methods that employ specific antisense polynucleotides complementary to all or part of a variant APP sequence, or for some embodiments a wild-type APP sequence. Such complementary antisense polynucleotides may include nucleotide substitutions, additions, deletions, or transpositions, so long as specific hybridization to the relevant target sequence, L&,, a variant APP codon 717 sequence, is retained as a property of the polynucleotide. Thus, an antisense polynucleotide must preferentially bind to a variant APP (i.e., codon 717 does not encode valine) sequence as compared to a wild-type APP (i.e., codon 717 does encode valine). It is evident that the antisense polynucleotide must reflect the exact nucleotide sequence of the variant allele (or wild-type allele where desired) and not a degenerate sequence.
Complementary antisense polynucleotides include soluble antisense RNA or DNA oligonucleotides which can hybridize specifically to a variant APP
MRNA species and prevent transcription of the mRNA species and/or translation of the encoded polypeptide (Ching et al. (1989) Proc. Natl. Acad. Sci. U.S.A.
.M:10006; Broder et al. (1990) Ann. Int. Med. M:604; Loreau et al. (1990) FEBS Letters 274:53-56); Holcenberg et al. W091111535; U.S. No. 7,530,165 ("New human CRIPTO gene" - publicly available through Derwent Publications Ltd., Rochdale House, 128 Theobalds Road, London, UK); W091/09865;
W091104753; W090113641; and EP 386563.
The antisense polynucleotides therefore inhibit production of the variant APP polypeptides. Antisense polynucleotides may preferentially inhibit transcription and/or translation of mRNA corresponding to a variant (or wild-type) polypeptides can inhibit T lymphocyte activation.
Antisense polynucleotides may be produced from a heterologous expression cassette in a transfectant cell or transgenic cell or animal, such as a transgenic neural, glial, or astrocytic cell, preferably where the expression cassette contains a sequence that promotes cell-type specific expression (Wirak et al.
loc.
cit.). Alternatively, the antisense polynucleotides may comprise soluble WO 92/13069 ;l r , PCT/GB92/00123 oligonucleotides that are administered to the external milieu, either in the culture medium in vitro or in the circulatory system or interstitial fluid in vivo.
Soluble antisense polynucleotides present in the external milieu have been shown to gain access to the cytoplasm and inhibit translation of specific mRNA species. In some embodiments the antisense polynucleotides comprise methylphosphonate moieties.
For general methods relating to antisense polynucleotides, see Antisense RNA
an d DNA, (1988), D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Mutant APP Antigens and Monoclonal Antibodies In yet another aspect of the invention, having detected a genetic alteration in a gene sequence coding for APP, it may be possible to obtain samples of the altered S-amyloid protein from the same source. This protein may be derived from the brain tissue of a subject diagnosed as suffering from Alzheimer's disease, or more preferably are produced by recombinant DNA methods or are synthesized by direct chemical synthesis on a solid support. Such polypeptides will contain an amino acid sequence of an APP variant allele spanning codon 717.
Examples of such sequences are:
(a) -Ile-Ala-Thr-Val-Ile-Gly-Ile-Thr-Leu- [SEQ ID NO:7]
(b) -Ile-Ala-Thr-Val-Ile-Met-Ile-Thr-Lau- [SEQ ID NO: 8]
(c) -Ile-Ala-Thr-Val-Ile-Ala-Ile-Thr-Leu- [SEQ ID NO:9]
(d) -Ile-Ala-Thr-Val-Ile-Ser-Ile-Thr-Leu- [SEQ ID NO:10]
(e) -Ile-Ala-Thr-Val-Ile-Ile-Ile-Thr-Leu- [SEQ ID NO:11]
(f) -Ile-Ala-Thr-Val-Ile-Leu-Ile-Thr-Leu- [SEQ ID NO:12]
(g) -Ile-Ala-Thr-Val-Ile-Thr-Ile-Thr-Leu- [SEQ ID NO: 13]
(h) -Ile-Ala-Thr-Val-Ile-Pro-Ile-Thr-Leu- [SEQ ID NO: 14]
(i) -Ile-Ala-Thr-Val-Ile-His-Ile-Thr-Leu- [SEQ ID NO: 15]
(j) -Ile-Ala-Thr-Val-Ile-Cys-Ile-Thr-Leu- [SEQ ID NO: 16]
(k) -Ile-Ala-Thr-Val-Ile-Tyr-Ile-Thr-Leu- [SEQ ID NO:17]

(1) -Ile-Ala-Thr-Val-Ile-Phe-Ile-Thr-Leu- [SEQ ID NO:18]
(m) -Ile-Ala-Thr-Val-Ile-Glu-Ile-Thr-Leu- [SEQ ID NO: 19]
(n) -Ile-Ala Thr-Val-Ile-Trp-Ile-Thr-Leu- [SEQ ID NO:20]
(o) -Ile-Ala-Thr-Val-Ile-Arg-Ile-Thr-Leu- [SEQ ID NO:21]
(p) -Ile-Ala-Thr-Val-Ile-Asp-Ile-Thr-Leu- [SEQ ID NO:22]
(q) -Ile-Ala-Thr-Val-Ile-Asn-Ile-Thr-Leu- [SEQ ID NO:23]
(r) -Ile-Ala-Thr-Val-Ile-Lys-Ile-Thr-Leu- [SEQ ID NO:24]
(s) -Ile-Ala Thr-Val-Ile-Gln-Ile-Thr-Leu- [SEQ ID NO:25]

Using such polypeptide material it may then be possible to prepare antisera and monoclonal antibodies using, for example, the method of Kohler and Milstein ((1975) Nature 256:495). Such monoclonal antibodies could then form the basis of a diagnostic test.
Such variant APP polypeptides may be used to immunize an animal for the production of specific antibodies. These antibodies may comprise a polyclonal antiserum or may comprise a monoclonal antibody produced by hybridoma cells. For general methods to prepare antibodies, see Antibodies: A
Laboratory Manual, (1988) E. Harlow and D. Lane, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
For example but not for limitation, a recombinantly produced fragment of a variant APP codon 717 polypeptide can be injected into a mouse along with an adjuvant so as to generate an immune response. Murine immunoglobulins which bind the recombinant fragment with a binding affinity of at least 1 x W M-1 can be harvested from the immunized mouse as an antiserum, and may be further purified by affinity chromatography or other means.
Additionally, spleen cells are harvested from the mouse and fused to myeloma cells to produce a bank of antibody-secreting hybridoma cells. The bank of hybridomas can be screened for clones that secrete immunoglobulins which bind the recombinantly produced fragment with an affinity of at least 1 x 106 M-1. More i specifically, immunoglobulins that bind to the variant APP codon 717 polypeptide but have limited crossreactivity with a wild-type (i.e., codon 717 encodes valine) APP polypeptide are selected, either by preabsorption with wild-type APP or by screening of hybridoma cell lines for specific idiotypes that preferentially bind the 5 variant as compared to the wild-type.
The nucleic acid sequences of the present invention capable of ultimately expressing the desired variant APP polypeptides can be formed from a variety of different polynucleotides (genomic or cDNA, RNA, synthetic oligonucleotides, etc.) as well as by a variety of different techniques.
10 As stated previously, the DNA sequences will be expressed in hosts after the sequences have been operably linked to (,gõ positioned to ensure the functioning of) an expression control sequence. These expression vectors are typically, replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain 15 selection markers, gam, tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired DNA
sequences (, g,, U.S. Patent 4,704,362) .

Polynucleotides encoding a variant APP codon 717 polypeptide may 20 include sequences that facilitate transcription (expression sequences) and translation of the coding sequences, such that the encoded polypeptide product is produced.
Construction of such polynucleotides is well known in the art and is described further in Maniatis et al. Molecular Cloning: A Laboratory Manual, 2nd Ed.
(1989), Cold Spring Harbor, N. Y. For example, but not for limitation, such 25 polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts, and, optionally, sequences necessary for replication of a vector.

i . j is one prokaryotic host useful particularly for cloning the DNA sequences of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Ste, Serrati , and various Pseudomonas species. In these prokaryotic hosts, one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (g,g,, an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
Other microbes, such as yeast, may also be used for expression.
Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
In addition to microorganisms, mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention (, Winnacker, "From Genes to Clones," VCH Publishers, N.Y., N.Y. (1987)) .
Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al. (1986) Immunol. Rev.
$2:49) and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are I

promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, and the like. The vectors containing the DNA segments of interest (LL, polypeptides encoding a variant APP polypeptide) can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (, generally, Maniatis, et al. Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Press, (1982)) Alternatively, homologous recombination may be used to insert an APP mutant sequence into a host genome at a specific site, for example, at a host APP locus. In one type of homologous recombination, one or more host sequence(s) are replaced; for example, a host APP allele (or portion thereof) is replaced with a mutant APP allele (or portion thereof). In addition to such gene replacement methods, homologous recombination may be used to target a mutant APP allele to a specific site other than a host APP locus. Homologous recombination may be used to produce transgenic non-human animals and/or cells that incorporate mutant APP alleles.
The method lends itself readily to the formulation of test kits which can be utilized in diagnosis. Such a kit would comprise a carrier being compartmentalized to receive in close confinement one or more containers wherein a first container may contain suitably labelled DNA probes. Other containers may contain reagents useful in the localization of the labelled probes, such as enzyme substrates. Still other containers may contain a restriction enzyme (such as BcII), buffers and the like, together with instructions for use.

EXPERIMENTAL EXAMPLES
The following examples are provided for illustration and are not intended to limit the invention to the specific example provided.

i EXAMPLE 1 - Detection of a Val-> Ile mutation in the 6-amyloid (APP) gene The segregation of AD and markers along the long arm of chromosome 21 in a single family with autopsy-confirmed Alzheimer's disease (see Fig. 1) were examined. DNA samples were available from a total of six affected and 33 unaffected and at risk individuals.
The APP gene in an affected family member was analyzed by polymerase chain reaction (PCR) direct sequencing using intronic primers (Gyllensten, U. in PCR Technology, Ed. Erlich, H.A., Stockton Press, 45-60, 1989; Yoshikai et al. (1990) Gene 87:257). (Fig. 2). The primers were made according to the manufacturer's protocol using a Gene Assembler Plus (Pharmacia LKB).
PCR was carried out using the following intronic primers in order to amplify exon 17 of the APP gene:

[A] 5'-CCTCATCCAAATGTCCCCGTCATr-3' [SEQ ID NO:26] AND
[B] 5'-GCCTAATTCTCTCATAGTCTTAATTCCCAC-3' [SEQ ID NO 27]
PCR conditions were 94"C for 10min to denature; then 35 cycles of 600C for 1min, 72 C for 3min, 94 C for 1.5min; and a single cycle of 72 C for 10min.
The reaction was carried out using 10mM tris-HC1 pH 8.3, 50mM potassium chloride, 0.01% gelatin, 1.5mM magnesium chloride, 200 M of dNTPs, 50 pmoles of each PCR primer and 1 unit of Taq polymerise. The total final reaction volume was 25 l.
A second PCR reaction was then performed with a final concentration of 50 pmol of primer [A] and 0.5 pmol of primer [B]. The PCR
product was purified on a centricon 100 microconcentrator (Amicon) and used directly for sequencing with the SEQUENASE kit (version 2.0, United States Biochemical Corp.; the word SEQUENASE is a trade mark) following the manufacturer's protocol.

*Trade-mark WO 92/13069 f =~ 1l PCT/GB92/00123 `~

Exon 17 was sequenced first because it encodes part of the ~-amyloid peptide and is the site of the mutation (at APP693) leading to Hereditary Cerebral Haemorrhage with Amyloidosis-Dutch Type (HCHWA-D).
Sequencing of exon 17 revealed a C to T transition at base pair 2149, causing a valine to isoleucine change at amino acid 717 (Fig. 2 and Fig.
3).
This C to T transition creates a BclI restriction site enabling detection within the PCR product (Fig. 4). BcII digests were carried out at 50 C
for 2-4 hours, as recommended by the manufacturer, then electrophoresed in 3%
agarose.
Screening by PCR of 100 unrelated, normal individuals and 14 cases (9 families) of familial late onset disease failed to demonstrate this substitution.
Screening of 11 (9 families) cases of early onset familial disease revealed the BclI
restriction site in two affected individuals from an unrelated family. The genetic data show that the disease loci are linked to the missense mutation. Also, failure to detect this polymorphism in 200 normal chromosomes supports the contention that it is a pathogenic mutation.
The valine to isoleucine substitution occurs within the transmembrane domain two residues from the C-terminus of the $-amyloid peptide.
Computer analysis predicts that the substitution makes the transmembrane more hydrophobic and might thus anchor the protein more firmly within the membrane.
The position of the substitution, two residues from the C-terminus of the $-amyloid peptide may be of significance to the origin of the deposited peptide. This finding links Alzheimer's disease to HCHWA-D, a disease in which amyloid deposition is due to a mutation closer to the N-terminus but withir the O-amyloid peptide (Levy et al. loc. cit.).

Ui 30 EXAMPLE 2 - Preparation of a cell line containing a defective 8-amyloid (APP) gene 10mi of fresh blood are collected from each individual suffering from familial Alzheimer's disease. Lymphocytes are purified from the blood on a Percoll gradient and mixed with Epstein-Barr virus (EBV). The cells are then plated out in medium supplemented with 10% foetal calf serum, antibiotics, glutamine and Cyclosporin A to kill the T lymphocytes. B lymphocytes which are infected by EBV become immortalized and establish a permanent cell line derived from the B cells of the patient.
A lymphoblastoid cell line, AC21, has been deposited with the European Collection of Animal Cell Cultures, Porton Down.

EXAMPLE 3 - Detection of a Val-> Gly mutation in the 8-amyloid (APP) gene A pedigree, designated F19 and shown in Fig. 5, which has autopsy-confirmed AD with an onset age of 59.4 years was identified by observing that an allele of the highly polymorphic dinucleotide repeat marker GT12 (D21S210), which is located close to the APP gene, co-segregated with the disease. Linkage analysis gave a peak lod score between the marker and the disease of 3.02 at a recombination fraction of zero, as the following table shows:
Theta 0 0.01 0.05 0.1 0.2 0.3 0.4 Lod 3.02 2.97 2.75 2.47 1.86 1.22 0.6 Lod scores were calculated with seven liability classes modelling age-dependent penetrances from 0.01 to 0.95 with a phenocopy rate of 0.001 and a gene frequency of 0.001 using MLINK from the LINKAGE package (Lathrop et al. (1984) Proc. Natl. Acad. Sci. USA 81:3443).
APP exon 17 sequences in an affected and an unaffected member of F19 were determined. In the affected member, there was a G->T transition at position 2150, as can be seen from Fig. 6.

N A t A+ I!

The amplification of exon 17 was performed as described in Example 1 above and Chartier-Harlin et al. (1991) Neurosci. Letts. 129:134, with the following modifications: (a) the amplification primer sequences were:
ATA-ACC-TCA-TCC-AAA-TGT-CCC-C [SEQ ID NO:28] and GTA-ACC-CAA-GCA-TCA-TGG-AAG-C [SEQ ID NO:29]; and (b) the PCR conditions were 94 C/10 minutes then 35 cycles of 60 C/l minute, 72 C/i minute, 94 C/1 minute, followed by 72 C/5 minutes.
50 pmol of the second primer were used to generate single stranded product, which was then purified (Chartier-Harlin er al. loc. cit.). The purified product was sequenced with the SEQUENASE kit (2.0) (Trade mark; USB) using a primer of sequence:
AAA-TGA-AAT-TCT-TCT-AAT-TGC-G [SEQ ID NO:30].
The presence of the T- > C transition creates gel artefacts which were resolved by the inclusion of inosine (SEQUENASE kit) in the sequencing reaction.

Direct sequencing of exons 7 and 16 from affected individuals from F19 (Chartier-Harlin et al. loc. cit.) shows that these were of normal sequence and SSCA
(Orita et al. loc. cit.) and Orita et al.) failed to identify changes in exons 2, 3, 7, 9, 12, 13 or 15. SSCA of exon 17 detects both APP693 (Levy, et al. loc. cit. and Hardy et al. (1991) Lancet 337:1342-1343) and APP717 Val->Ile under standard screening conditions and, when modified APP717 Val-> Gly.

EXAMPLE 4 - Production of Transgenic Animals with Mutant APP Allele Generation of the constructs: The vector plink was constructed by cloning polylinker between the PvuII and EcoRI sites of pBR322 such that the HindIIl end of the polylinker was adjacent to the PvuII site. The ligation destroyed both the EcoRl and PvuII sites associated with the pBR322 segments.
The 700bp HpaI to EcoRl fragment of pSV2neo (Southern and Berg (1982) L

i oi. Appi. Genet. 1:327) that contains the SV40 polyadenylation signal was cloned into the Hpal to EcoRl sites of plink to generate pNotSV. The 200 bp Xhol to Pstl fragment of pL2 containing the SV40 16S/lgS splice site (Okayama and Berg (1983) Mol. Cell Biol.1:280) was isolated, blunted with Kienow, then cloned into the HpaI site of pNotSV to generate pSplice. The 2.3kb Nrul to Spel fragment of pAPP695 containing the coding region of the cDNA for APP (Tani et al. (1987) Science 235:880) was cloned into the Nrul to Spel site of pSplice to generate pd695. The same strategy was used to generate pd751 using the cDNA
for APP751 (Tanzi et al. (1988) Nature 331:528). A variety of promoters have been inserted into the pd695 and pd751 vectors by using the unique NruI or the IiindlII and Nrul sites.

Generation of pshAPP695 & pshAPP751: The construct pAmyproBam was generated by cloning the 1.5kb BamHI fragment of the APP
cDNA into the BarHI site of puc19 xHamy. The 700 bp Hindi to Asp718 fragment of the pAmyproBam (similar to the 700 bp BamHI to Asp718 fragment described in Salbaum et al. (1988) EMBO 7:2807) was cloned into the HindlU to Asp718 sites of pd695 and pd751 to yield pshAPP695 and pshAPP751.

pAPP695 and RAPP751: The pAPP695 and pAPP751 vectors were generated by a three-way ligation of the 3.0 kb EcoRI to Xhol fragment of pAmyProBam, the 1.5kb Xhol to Spel fragment of APP751cDNA, and the Spel to EcoRI site of pd751.

Generation of pNSE751(+47): The pNSE751 (+47) was constructed using a three-way ligation of the HindIII to KpnI fragment of pNSE6 (Forss-Petter et al. (1990) Neuron 5:187) . The KpnI to BstY 1 fragment of pNSE6 and a partial BamH1 (-47nt relative to the ATG) to Hindif fragment of pAPP751.
This resulted in the generation of a Kpnl fragment that was cloned into the KpnI
*Trade-mark sites of pNSE751(+47). The BstYl/Barn fusion results in the loss of both sites.
Generation of pNSE751: This vector was generated using a four primer two-step PCR protocol (Higuchi et al. (1988) Nucl. Acids Res. 16:7351) that resulted in a direct fusion of the NSE initiation codon to the APP coding region. Oligonucleotides C2, 1072, 1073, and A2 (see Nucleotide Sequences, infra.) were used to generate a PCR product. The Kpnl fragment was generated by digestion with the restriction enzyme. The KpnI fragment was used to replace a similar fragment in pNSE751(+47).

Generation of pNSE751-Hardy and pNSE751-Dutch: The Hardy (APP642 Val-> Ile of APP695) and Dutch (APP618 Gln-> Glu of APP695) mutations were introduced using a four primer two-PCR protocol. Both sets of reactions used the same "outside primers" with the "inside primers" containing the appropriate mutations. This resulted in the generation of Bg1II to Spel fragment after digestion, that contained either the Dutch or the Hardy mutation. The Bg1II
to Spel fragment of pNSE751 was replaced by the mutated fragment to generate the appropriate vector. The presence of the mutation was conformed by sequence analysis of the vectors.

Generation of pNSE751-Hardand pNSE751-Dutch: The Hardy VI
(APP642 V to I), Hardy VG (APP642 V to G), and Dutch (APP618 E to Q) mutations were introduced using the four primer two-step PCR protocol (Higuchi et al. (1988)). The Hardy VI mutant was generated using primers 117/738, 922, 923, and 785; Hardy VG mutant was generated using primers 117/738, 1105, 1106, and 785; Dutch mutant was generated using primers 117/738, 1010, 1011, and 785. In all these mutations the 700 bp BgIII to Spel fragment was isolated by digestion of the PCR product with the restriction enzymes, then cloned into the same sites of pNSE751. The mutations were confirmed by sequence analysis.

WO 92/13069 r; - a PCT/GB92/00123 1. 'v t 1 Generation of pNFH751: The human NFH gene (Lees et al. (1988) EMBO 7(7):1947) was isolated from a genomic library using a rat NFH cDNA as a probe (Lieberburg et al. (1989) Proc. Nat!. Acids. Res. USA 86:2463). An SstI
fragment was subcloned into the pSK vector. A pair of PCR primers was generated to place a Nrul site at the 3' end of the 150 bp amplified fragment immediately upstream of the initiation codon of the NFH gene. The 5' end contains a KpnI site 150nt upstream of the initiation codon. The final construction of pNFH751 was generated by a three-way ligation of the 5.5b Hindif to KpnI
fragment of pNFH8.8, the KpnI to NruI PCR generated fragment, and the HindIII
to NruI fragment of pd751. The sequence surrounding the PCR generated fusion at the initiation codon was confirmed by sequence analysis. The Dutch and Hardy variants of pNFH751 were generated by substitution of the 600 bp BgIll to Spel fragment from a sequence confirmed mutated vector for the same fragment of pNFH751. The presence of the mutation was confirmed by hybridization with the mutated oligomer or by sequence analysis.

Generation of pThv751: The pThy751 vector was generated by a three-way ligation. The HindlII to BamHI fragment of pThy8.2 which was isolated from a human genomic library (Chang et al. (1985) Proc. Natl. Acad.
Sci.
USA 82:3819), the synthetic fragment ThyAPP, and the HindIll to NruI fragment of pd751.
ThyAPP:
CAGACTGAGATCCCAGAACCCTAGGTCTGACTCTAGGGTCTTGG[SEQ
ID NO:31]
Generation of 12ThvC100: This pThyC100 construct was generated by a three-way ligation. The 3.6kb Hindlll to BamH1 fragment of pThy8.2, the synthetic fragment ThyAPP2, and the HindIll to BglII fragment of pd751 or pNSE751 Dutch or pNSE751 Hardy were ligated to yield pThyClOO.

i ThyAPP2:
CAGACTGAGATCCCAGAACCGATCCTAGGTCTGACTCTAGGGTCTTGG
[SEQ ID NO:32]
The region around the initiation codon was confirmed by sequence 5 analysis.

EMRaration of DNA for injection: The transgene for injection was isolated from the corresponding vector of interest for digestion with Nod and gel electrophoresis. The transgene was purified by using the Gene Clean kit (Biol0l), 10 then further purified on an Elutip or HPLC purified on a Nucleogen 4000 column.
Microinjection: The transgene was injected at 2-20 mcg/ml into the most convenient pronucleus (usually the male pronucleus) of FVB or B6D2F2 one-cell embryos (Manipulating the Mouse Embryo, B. Hogan, F. Constantin, E.
15 Lacy, Cold Spring Harbor, 1986). The injected embryos were cultured overnight.
Embryos that split to the two-cell stage were implanted into pseudo-pregnant female CD 1 mice. The mice were weaned at approximately 21 days. Samples of DNA obtained from tail biopsy were analyzed by Southern blot using a transgene specific probe (usually the SV40 3's splice and polyadenylation signal sequences).
20 Transgenic mice harboring at least one copy of the transgene were identified.
Use of Transgenic Mice: A mouse that expresses the hAPP gene or its variants can be used to test the pathogenesis of amyloid deposition and therapeutic intervention designed to modulate amyloid deposition.
Biochemical analysis of the transgenic mice reveals possible 25 intermediates in the catabolism of APP that are likely precursors to beta amyloid.
This analysis can be carried out in the animal or in primary tissue culture of the expressing cells.
The animal can be used to test potential therapeutic agents. The test group of mice is treated with the test compound administered in an appropriate *Trade-mark fashion for a set period. At the conclusion of the test period, the animals are assessed behaviourally, biochemically, and histologically for any possible effects of the test compound. The exact protocol depends on the anticipated mechanism of action of the test compound. Compounds that may have utility in treating AD
can be identified using this approach.

W() 92/13069 PCT/GB92/OG123 SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: Imperial College of Science, Technology &
Medicine (not US) (B) STREET: Sherfield Building, Exhibition Road, (C) CITY: London (E) COUNTRY: GB
(F) POSTAL CODE (ZIP): SW7 2AZ

(A) NAME: HARDY, John Anthony (US only) (B) STREET: 187 Drakefell Road (C) CITY: London (E) COUNTRY: GB
(F) POSTAL CODE (ZIP): SE4 (A) NAME: GOATE, Alison Mary (US only) (B) STREET: 100 High Street, Hampton Wick, (C) CITY: Kingston-on-Thames (D) STATE: Surrey (E) COUNTRY: GB
(F) POSTAL CODE (ZIP): KT1 4DQ

(A) NAME: MULLAN, Michael John (US only) (B) STREET: Suncoast Gerontology Ctr, 12901 Bruce B.
Downs Blvd. MDC 50, (C) CITY: Tampa (D) STATE: Florida (E) COUNTRY: US
(F) POSTAL CODE (ZIP): 33612 (A) NAME: CHARTIER-HARLIN, Marie-Christine (US only) (B) STREET: 63 Francis Road (C) CITY: London (E) COUNTRY: GB
(F) POSTAL CODE (ZIP): E10 6PN

(A) NAME: OWEN, Michael John (US only) (B) STREET: Four Hedges, Castlehill, LLanblethian, (C) CITY: Cowbridge (D) STATE: South Glamorgan (E) COUNTRY: GB

(ii) TITLE OF INVENTION: Test and Model for Alzheimer's Disease (iii) NUMBER OF SEQUENCES: 44 (iv) COMPUTER READABLE FORM:
Not Applicable (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: WO PCT/GB92/_`

0 -1.

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 695 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Gin Gly Ile Leu Gln Tyr Cys Gin Glu Val Tyr Pro Glu Leu Gln Ile Thr Asn Val Val Glu Ala Asn Gin Pro Val Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gin Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys WO 92/13069 N 9 1 u PCT/GB92/00123 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gin Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gin Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gin Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gin Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr WO 92/13069 vs I f} PCT/G B92/00123 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin Met Gin Asn ti U 1 1 77 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 751 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Leu Pro Gly Leu Ala, Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gin Ile Ala Net Phe Cys Gly Arg Leu Asn Net His Met Asn Val Gln Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu Gin Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gin Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Net Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Giy Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys t. l S. i 1 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Giu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gin Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gin Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gin Lys Asp Arg Gin His Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu 2L 01 j-1 Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Giu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val lle Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn WO 92/13069 + 11 i ~ f 1 PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 770 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Giy Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu Gin Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys WO 92/13069 , ~ PCT/G B92/00123 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gin Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gin Gln Leu Val Glu Thr His Met Ala Arg 1al Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gin Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Sear Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Giy Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2088 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

WkO 92/13069 PCT/GB92/00123 :1i11 i i (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2265 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GAGTCTGTGG AAGAGGTGGT TCGAGAGGTG TGCTCTGAAC AAGCCGAGA: GGGGCCGTGC 900 k 0 92/13069 v ( " PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ile Ala Thr Val Ile Xaa Ile Thr Leu (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ile Ala Thr Val Ile Gly Ile Thr Leu (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ile Ala Thr Val Ile Met Ile Thr Leu uI1 ' (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Ile Ala Thr Val Ile Ala Ile Thr Leu WO 92/13069 ? i 0 I PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ile Ala Thr Val Ile Ser Ile Thr Leu (2) INFORMATION FOR SEQ ID NO:11:

(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ile Ala Thr Val Ile Ile Ile Thr Leu W() 92/13069 1 PCT/GB92/00123 1 ! i (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ile Ala Thr Val Ile Leu Ile Thr Leu .; q n WO 92/13069 i iU i I 1 4 PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Ile Ala Thr Val Ile Thr Ile Thr Leu (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Ile Ala Thr Val Ile Pro Ile Thr Leu -. l i+ 1 f (2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Ile Ala Thr Val Ile His Ile Thr Leu I lJ .i I 1 (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Ile Ala Thr Val Ile Cys Ile Thr Leu (2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ile Ala Thr Val Ile Tyr Ile Thr Leu (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (E3) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Ile Ala Thr Val Ile Phe Ile Thr Leu (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Ile Ala Thr Val Ile Glu Ile Thr Leu a i ".
ire (2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Ile Ala Thr Val Ile Trp Ile Thr Leu i ` PCT/GB92/00123 WO 92/13069 y i u (2) INFORMATION FOR SEQ ID NO:21:

(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (Es) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Ile Ala Thr Val Ile Arg Ile Thr Leu (2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ile Ala Thr Val Ile Asp Ile Thr Leu W() 92/13069 PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Ile Ala Thr Val Ile Asn Ile Thr Leu WO 92/13069 .= PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Ile Ala Thr Val Ile Lys Ile Thr Leu WO 92/13069 U 1 '1 PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Ile Ala Thr Val Ile Gln Ile Thr Leu WO 92/13069 C) a PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Primer) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

f1 1 ('t IB i i 72 (2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Primer) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

WO 92/13069 , r, , -= t PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Primer) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Primer) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear {ii) MOLECULE TYPE: DNA (Primer) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

+Ji (2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (Primer) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

U - , x (2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

~LUi(14 (2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

A i i 82 (2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

61 7 , 1un IT 4 (2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

i ! j S r r ;x i U i S J

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

ACCCACATCT TGTGCAA.AGA ACAC 24 WO 92/13069 "'- V PCT/GB92/00123 (2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

(2) INFORMATION FOR SEQ ID N0:41:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

` i r -(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Claims (26)

CLAIMS:
1. An isolated polynucleotide comprising a nucleic acid sequence encoding a codon 717 mutant of human amyloid precursor protein wherein the amino acid at codon 717 as defined in relation to APP770 is selected from the group consisting of isoleucine, glycine, and phenylalanine.
2. The isolated polynucleotide of claim 1, wherein the nucleic acid sequence is a cDNA.
3. A polynucleotide probe capable of specifically hybridizing to an amyloid precursor protein 770 (APP770) allele exhibiting a mutation at codon 717 under hybridization conditions that distinguish between binding of the mutant allele and binding of the probe to a wildtype allele, wherein the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
4. The polynucleotide probe of claim 3, wherein the probe is labeled.
5. A cultured human primary or immortalized cell, comprising a nucleic acid segment encoding a codon 717 mutant of human amyloid precursor protein (APP), wherein codon 717 is defined in relation to APP770 and wherein the amino acid at codon 717 is a hydrophobic residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
6. A diagnostic method for determining an inherited predisposition to Alzheimer's disease in a subject, comprising detecting in the subject the presence of an allele of amyloid precursor protein (APP), wherein said allele has a sequence polymorphism at a position encoded by codon 717 defined in relation to APP770, and wherein the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
7. The method of claim 6, wherein said sequence polymorphism is a nucleotide substitution at codon 717 of APP770, said nucleotide substitution encoding an isoleucine or glycine.
8. The method of claim 6, wherein said sequence polymorphism is a single nucleotide substitution.
9. The method according to claim 6, wherein the detecting step comprises sequencing a genomic DNA segment from chromosome 21 of the subject.
10. The method according to claim 6, wherein the detecting step comprises (i) mixing a nucleic acid sample from the subject with one or more polynucleotide probes capable of hybridizing selectively to an APP gene allele in a PCR reaction and (ii) monitoring the PCR
reaction to determine the presence of the gene allele in the sample, thereby indicating whether the subject is at risk for Alzheimer's disease.
11. A method for genetic analysis of a human subject which comprises detecting the presence or absence of at least one polymorphism at codon 717 of an amyloid precursor protein (APP) gene in the subject, wherein codon 717 is defined in relation to APP770; and wherein the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
12. The method according to claim 11, wherein the polymorphism is detecting by digesting genomic DNA from the subject with at least one restriction endonuclease and hybridizing resulting fragments with a detecting probe.
13. A polypeptide free from human proteins, comprising a core sequence:
Ile-Ala-Thr-Val-Ile-X-Ile-Thr-Leu- [SEQ ID NO:6]
wherein X is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
14. An isolated polynucleotide, comprising a nucleic acid sequence encoding a mutant human amyloid precursor protein (APP) allele that cosegregates with a genetic predisposition to early onset familial Alzheimer's disease, wherein the amino acid at codon 717 of the mutant human APP allele is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine and codon 717 is defined in relation to APP770.
15. A method of determining a genetic predisposition of a subject to Alzheimer's disease, the method comprising detecting in the subject's DNA the presence of a mutant allele of a gene encoding amyloid precursor protein (APP) that cosegregates with genetic predisposition to early onset familial Alzheimer's disease, wherein codon 717 is defined in relation to APP770, and the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
16. The method as claimed in claim 15, wherein the step of detection is carried out on material removed from, and not returned to, the subject's body.
17. Use of a recombinant nucleic acid sequence encoding a mutant human amyloid precursor protein (APP) allele that cosegregates with genetic predisposition to early onset familial Alzheimer's disease to transform a host organism, which transformed organism has a genetic predisposition to Alzheimer's disease, wherein codon 717 is defined in relation to APP770, and the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
18. Use of a recombinant heterologous gene encoding a mutant amyloid precursor protein (APP) gene that cosegregates with genetic predisposition to early onset familial Alzheimer's disease, which gene upon expression promotes neuropathological characteristics of Alzheimer's disease, to produce a transgenic non human animal with germ cells or somatic cells comprising said gene, wherein codon 717 is defined in relation to APP770, and the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
19. Use of a transgenic non human animal having a mutant amyloid precursor protein (APP) gene at codon 717 that cosegregates with genetic predisposition to early onset familial Alzheimer's disease in screening for a drug capable of treating Alzheimer's disease, wherein codon 717 is defined in relation to APP770, and the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
20. Use of a transgenic non human animal having a mutant amyloid precursor protein (APP) gene at codon 717 that cosegregates with genetic predisposition to early onset familial Alzheimer's disease to derive cells transfected with the mutant amyloid precursor protein gene, wherein codon 717 is defined in relation to APP770, and the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
21. An isolated cell comprising a nucleic acid segment encoding a position 717 mutant of human amyloid precursor protein (APP), wherein the cell naturally contains the nucleic acid or the nucleic acid has been introduced by genetic engineering, wherein codon 717 is defined in relation to APP770 and the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
22. The cell of claim 21, which is a primary or immortalized eukaryotic cell.
23. The cell of claim 21, wherein the mutant APP770 protein is the sole APP770 protein produced by the animal.
24. A method of screening for an agent capable of treating Alzheimer's disease, comprising:
contacting a non-human host organism comprising a recombinant polynucleotide including a nucleic acid sequence encoding a mutant human amyloid precursor (APP) protein allele that cosegregates with a genetic predisposition to early onset familial Alzheimer's disease with the agent; and monitoring expression or processing of proteins encoded by the mutant allele, wherein codon 717 is defined in relation to APP770 (SEQ ID
NO:3), and the amino acid at codon 717 is a residue selected from the group consisting of isoleucine, glycine, and phenylalanine.
25. The method of claim 24, wherein the host organism comprises a polypeptide free of human proteins, comprising a core sequence:
Ile-Ala-Thr-Ile-X-Thr-Leu- [SEQ ID NO:6]
wherein X is phenylalanine, glycine or isoleucine.
26. The method of claim 25, wherein the polypeptide is present in the brain.
CA2101774A 1991-01-21 1992-01-21 Test and model for alzheimer's disease Expired - Lifetime CA2101774C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA002372251A CA2372251A1 (en) 1991-01-21 1992-01-21 Test and model for alzheimer's disease

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB919101307A GB9101307D0 (en) 1991-01-21 1991-01-21 Test for alzheimers disease
GB9101307.8 1991-01-21
GB9118445.7 1991-08-28
GB919118445A GB9118445D0 (en) 1991-08-28 1991-08-28 Test for alzheimer's disease
PCT/GB1992/000123 WO1992013069A1 (en) 1991-01-21 1992-01-21 Test and model for alzheimer's disease

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CA002372251A Division CA2372251A1 (en) 1991-01-21 1992-01-21 Test and model for alzheimer's disease

Publications (2)

Publication Number Publication Date
CA2101774A1 CA2101774A1 (en) 1992-07-22
CA2101774C true CA2101774C (en) 2011-01-04

Family

ID=26298303

Family Applications (2)

Application Number Title Priority Date Filing Date
CA002372251A Abandoned CA2372251A1 (en) 1991-01-21 1992-01-21 Test and model for alzheimer's disease
CA2101774A Expired - Lifetime CA2101774C (en) 1991-01-21 1992-01-21 Test and model for alzheimer's disease

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CA002372251A Abandoned CA2372251A1 (en) 1991-01-21 1992-01-21 Test and model for alzheimer's disease

Country Status (10)

Country Link
US (3) US5877015A (en)
EP (2) EP0568575B2 (en)
JP (3) JP3510244B2 (en)
AT (2) ATE447016T1 (en)
AU (1) AU652997B2 (en)
CA (2) CA2372251A1 (en)
DE (3) DE971033T1 (en)
DK (2) DK0971033T3 (en)
ES (2) ES2335720T3 (en)
WO (1) WO1992013069A1 (en)

Families Citing this family (140)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0568575B2 (en) 1991-01-21 2010-11-03 Elan Pharmaceuticals, Inc. Test and model for alzheimer's disease
WO1993010459A1 (en) * 1991-11-12 1993-05-27 The University Of Melbourne A method for assaying and treating alzheimer's disease
AU3249793A (en) 1991-12-24 1993-07-28 Isis Pharmaceuticals, Inc. Compositions and methods for modulating beta -amyloid
US5604131A (en) * 1992-01-07 1997-02-18 Athena Neurosciences, Inc. cDNA-genomic DNA hybrid sequence encoding APP770 containing a genomic DNA insert of the KI and OX-2 regions
DK0620849T3 (en) * 1992-01-07 2003-10-20 Elan Pharm Inc Transgenic animal models for Alzheimer's disease
US5455169A (en) 1992-06-04 1995-10-03 Alzheimer's Institute Of America, Inc. Nucleic acids for diagnosing and modeling Alzheimer's disease
US6610493B1 (en) 1993-06-17 2003-08-26 Brigham And Women's Hospital Screening compounds for the ability to alter the production of amyloid-β peptide
US6328971B1 (en) * 1993-01-22 2001-12-11 Ludwig Institute For Cancer Research MAGE-1 derived nona peptides, and compositions thereof
AU702293B2 (en) * 1993-10-27 1999-02-18 Athena Neurosciences, Inc. Transgenic animals harboring APP allele having Swedish mutation
JPH07132033A (en) * 1993-11-12 1995-05-23 Hoechst Japan Ltd Transgenic animal for alzheimer's disease model
AU1909695A (en) 1994-01-27 1995-08-15 Regents Of The University Of Minnesota Transgenic non-human mammals with progressive neurologic disease
US5877399A (en) * 1994-01-27 1999-03-02 Johns Hopkins University Transgenic mice expressing APP-Swedish mutation develop progressive neurologic disease
US6211428B1 (en) * 1994-09-01 2001-04-03 Merck & Co., Inc. Transgenic mouse expressing a familial form of human amyloid precursor protein
US6187992B1 (en) 1994-12-05 2001-02-13 Merck & Co., Inc. Transgenic mouse having a disrupted amyloid precursor protein gene
US5674681A (en) * 1994-12-06 1997-10-07 Rothenberg; Barry E. Methods to identify hemochromatosis
US6717031B2 (en) 1995-06-07 2004-04-06 Kate Dora Games Method for selecting a transgenic mouse model of alzheimer's disease
JP2001517065A (en) * 1995-06-07 2001-10-02 アセナ ニューロサイエンシーズ,インコーポレイテッド Methods for identifying therapeutic agents for Alzheimer's disease using transgenic animal models
AUPN649395A0 (en) * 1995-11-10 1995-12-07 Ramsay Health Care Pty Ltd A method for diagnosing alzheimer's disease
US7129061B1 (en) * 1996-08-07 2006-10-31 Biogen Idec Ma Inc. Tumor necrosis factor related ligand
CA2183901A1 (en) * 1996-08-22 1998-02-23 Johanna E. Bergmann Targets for therapy and diagnosis of alzheimer's disease and down syndrome in humans
US6933331B2 (en) * 1998-05-22 2005-08-23 Nanoproducts Corporation Nanotechnology for drug delivery, contrast agents and biomedical implants
US5898094A (en) * 1996-10-21 1999-04-27 University Of South Florida Transgenic mice expressing APPK670N,M671L and a mutant presenilin transgenes
ES2128265B1 (en) * 1997-06-04 2000-03-01 Euroespes S A GENETIC KIT FOR THE MOLECULAR CHARACTERIZATION OF ALZHEIMER'S DISEASE.
JP2001521043A (en) * 1997-10-24 2001-11-06 アバンテイス・フアルマ・エス・アー Peptide capable of inhibiting the interaction between presenilin and β-amyloid peptide or its precursor
FR2770217B1 (en) * 1997-10-24 2001-12-07 Rhone Poulenc Rorer Sa PEPTIDES CAPABLE OF INHIBITING THE INTERACTION BETWEEN PRESENILINS AND THE PRECURSOR OF B-AMYLOID PEPTIDE AND / OR B-AMYLOID PEPTIDE
US20080050367A1 (en) 1998-04-07 2008-02-28 Guriq Basi Humanized antibodies that recognize beta amyloid peptide
TWI239847B (en) 1997-12-02 2005-09-21 Elan Pharm Inc N-terminal fragment of Abeta peptide and an adjuvant for preventing and treating amyloidogenic disease
US6710226B1 (en) 1997-12-02 2004-03-23 Neuralab Limited Transgenic mouse assay to determine the effect of Aβ antibodies and Aβ Fragments on alzheimer's disease characteristics
US7790856B2 (en) 1998-04-07 2010-09-07 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize beta amyloid peptide
US6905686B1 (en) 1997-12-02 2005-06-14 Neuralab Limited Active immunization for treatment of alzheimer's disease
US7179892B2 (en) 2000-12-06 2007-02-20 Neuralab Limited Humanized antibodies that recognize beta amyloid peptide
US6787523B1 (en) 1997-12-02 2004-09-07 Neuralab Limited Prevention and treatment of amyloidogenic disease
US6923964B1 (en) 1997-12-02 2005-08-02 Neuralab Limited Active immunization of AScr for prion disorders
US7964192B1 (en) 1997-12-02 2011-06-21 Janssen Alzheimer Immunotherapy Prevention and treatment of amyloidgenic disease
US6913745B1 (en) 1997-12-02 2005-07-05 Neuralab Limited Passive immunization of Alzheimer's disease
US20030147882A1 (en) 1998-05-21 2003-08-07 Alan Solomon Methods for amyloid removal using anti-amyloid antibodies
CN1300320C (en) 1998-09-24 2007-02-14 法玛西雅厄普约翰美国公司 Alzhimer's disease secretase
US6844148B1 (en) * 1998-09-24 2005-01-18 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US20040234976A1 (en) * 1998-09-24 2004-11-25 Gurney Mark E. Alzheimer's disease secretase, app substrates therefor, and uses therefor
US6699671B1 (en) 1998-09-24 2004-03-02 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US7115410B1 (en) * 1999-02-10 2006-10-03 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7456007B1 (en) 1998-12-31 2008-11-25 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
CA2359785A1 (en) 1999-02-10 2000-08-17 John P. Anderson .beta.-secretase enzyme compositions and methods
US6355425B1 (en) * 1999-03-26 2002-03-12 Billups-Rothenberg, Inc. Mutations associated with iron disorders
US7056661B2 (en) * 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US6787637B1 (en) 1999-05-28 2004-09-07 Neuralab Limited N-Terminal amyloid-β antibodies
UA81216C2 (en) 1999-06-01 2007-12-25 Prevention and treatment of amyloid disease
US20090162883A1 (en) * 1999-09-23 2009-06-25 Pharmacia & Upjohn Company Alzheimer's Disease Secretase, APP Substrates Thereof, and Uses Thereof
US7514408B1 (en) 1999-12-02 2009-04-07 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US6310048B1 (en) 1999-12-09 2001-10-30 St. Louis University Antisense modulation of amyloid beta protein expression
CA2395926A1 (en) * 1999-12-28 2001-07-05 Curagen Corporation Nucleic acids containing single nucleotide polymorphisms and methods of use thereof
EP1265849B1 (en) * 2000-03-23 2006-10-25 Elan Pharmaceuticals, Inc. Compounds and methods to treat alzheimer's disease
US6992081B2 (en) 2000-03-23 2006-01-31 Elan Pharmaceuticals, Inc. Compounds to treat Alzheimer's disease
JP2004502665A (en) * 2000-06-30 2004-01-29 イーラン ファーマスーティカルズ、インコーポレイテッド Compounds treating Alzheimer's disease
PE20020276A1 (en) 2000-06-30 2002-04-06 Elan Pharm Inc SUBSTITUTE AMINE COMPOUNDS AS ß-SECRETASE INHIBITORS FOR THE TREATMENT OF ALZHEIMER
US20030096864A1 (en) * 2000-06-30 2003-05-22 Fang Lawrence Y. Compounds to treat alzheimer's disease
US6846813B2 (en) * 2000-06-30 2005-01-25 Pharmacia & Upjohn Company Compounds to treat alzheimer's disease
EP1666452A2 (en) 2000-06-30 2006-06-07 Elan Pharmaceuticals, Inc. Compounds to treat Alzheimer's disease
JP2004502427A (en) * 2000-07-06 2004-01-29 フラームス・インテルウニフェルシタイル・インステイチュート・フォール・ビオテヒノロヒー・ヴェーゼットウェー Novel APP mutation associated with abnormal Alzheimer's disease pathology
PE20020574A1 (en) 2000-12-06 2002-07-02 Wyeth Corp HUMANIZED ANTIBODIES THAT RECOGNIZE THE AMYLOID PEPTIDE BETA
US7700751B2 (en) 2000-12-06 2010-04-20 Janssen Alzheimer Immunotherapy Humanized antibodies that recognize β-amyloid peptide
CA2448834A1 (en) * 2001-06-01 2002-12-12 Elan Pharmaceuticals, Inc. Hydroxy alkyl amine derivatives as beta-secretase inhibitors and their use for the treatment of alzheimer's disease and similar diseases
EP1395251A2 (en) 2001-06-13 2004-03-10 Elan Pharmaceuticals, Inc. Aminediols as agents for the treatment of alzheimer's disease
US6982264B2 (en) * 2001-06-27 2006-01-03 Elan Pharmaceuticals, Inc. Substituted alcohols useful in treatment of Alzheimer's disease
US20070213407A1 (en) * 2001-06-29 2007-09-13 Elan Pharmaceuticals And Pharmacia & Upjohn Company Llc Compounds to treat Alzheimer's disease
BR0211121A (en) * 2001-07-10 2004-10-26 Elan Pharm Inc Compound, methods for treating or preventing disease and for making a compound, intermediate, and use of a compound or salt
WO2003006013A1 (en) * 2001-07-10 2003-01-23 Elan Pharmaceuticals, Inc. Diaminediols for the treatment of alzheimer's disease
US7297553B2 (en) * 2002-05-28 2007-11-20 Nanosphere, Inc. Method for attachment of silylated molecules to glass surfaces
MXPA04003245A (en) 2001-10-04 2004-08-11 Elan Pharm Inc Hydroxypropylamines.
OA12846A (en) * 2001-11-08 2006-09-15 Elan Pharm Inc N,N'-substituted-1,3-diamino-2-hydroxypropane derivatives.
US20050080141A1 (en) * 2001-11-19 2005-04-14 Schostarez Heinrich J. Amino diols useful in the treatment of alzheimer's disease
US20040016008A1 (en) * 2002-01-07 2004-01-22 Brimijoin William Stephen Hybrid transgenic mouse with accelerated onsent of Alzheimer type amyloid plaques in brain
CA2477002A1 (en) * 2002-02-27 2003-09-04 Merck & Co., Inc. Assays to monitor amyloid precursor protein processing
MY139983A (en) 2002-03-12 2009-11-30 Janssen Alzheimer Immunotherap Humanized antibodies that recognize beta amyloid peptide
US7262208B2 (en) * 2002-04-30 2007-08-28 Elan Pharmaceuticals, Inc. Hydroxypropyl amides for the treatment of Alzheimer's disease
UY27967A1 (en) * 2002-09-10 2004-05-31 Pfizer 2-HINDROXI-1,3-DIAMINOALCANE OIL
PE20040762A1 (en) * 2002-11-27 2004-11-06 Elan Pharm Inc SUBSTITUTED UREAS AND CARBAMATES USEFUL FOR THE TREATMENT OF ALZHEIMER'S DISEASE
CA2513722A1 (en) 2003-02-01 2004-08-19 Neuralab Limited Active immunization to generate antibodies to soluble a-beta
AR044043A1 (en) * 2003-04-21 2005-08-24 Elan Pharm Inc FENACILO 2-HIDROXI-3-DIAMINOALCANOS
AR044044A1 (en) * 2003-04-21 2005-08-24 Elan Pharm Inc BENZAMIDA 2-HYDROXI-3-DIAMINOALCANS
TWI374893B (en) 2003-05-30 2012-10-21 Janssen Alzheimer Immunotherap Humanized antibodies that recognize beta amyloid peptide
US20050009110A1 (en) * 2003-07-08 2005-01-13 Xiao-Jia Chang Methods of producing antibodies for diagnostics and therapeutics
WO2005010039A1 (en) * 2003-07-31 2005-02-03 Pfizer Products Inc. Bsep polypeptide variants and uses thereof
WO2005070407A1 (en) * 2004-01-21 2005-08-04 Elan Pharmaceuticals, Inc. Methods of treatment of amyloidosis using aspartyl-protease inihibitors
EP1734961A2 (en) * 2004-03-09 2006-12-27 Elan Pharmaceuticals, Inc. Methods of treatment of amyloidosis using bi-cyclic aspartyl protease inhibitors
CA2558249A1 (en) * 2004-03-09 2005-09-22 Elan Pharmaceuticals, Inc. Substituted urea and carbamate, phenacyl-2-hydroxy-3-diaminoalkane, and benzamide-2-hydroxy-3-diaminoalkane aspartyl-protease inhibitors
JP2007528400A (en) * 2004-03-09 2007-10-11 エラン ファーマシューティカルズ,インコーポレイテッド Substituted hydroxyethylamine aspartic protease inhibitors
WO2005087752A2 (en) * 2004-03-09 2005-09-22 Elan Pharmaceuticals, Inc. Substituted hydroxyethylamine aspartyl protease inhibitors
US7544855B2 (en) * 2004-04-23 2009-06-09 Buck Institute Transgenic mouse whose genome comprises an APP having a mutation at amino acid 664
WO2005110422A2 (en) * 2004-05-19 2005-11-24 Boehringer Ingelheim International Gmbh Treatment of diseases associated with altered level of amyloid beta peptides
CA2572775A1 (en) * 2004-07-09 2006-01-26 Elan Pharmaceuticals, Inc. Oxime derivative hydroxyethylamine aspartyl-protease inhibitors
WO2006010095A2 (en) * 2004-07-09 2006-01-26 Elan Pharmaceuticals Inc. Oxime derivative substituted hydroxyethylamine aspartyl protease inhibitors
US8436006B2 (en) * 2004-08-06 2013-05-07 Jansssen Pharmaceutica N.V. 2-amino-quinazoline derivatives useful as inhibitors of β-secretase (BACE)
US8426429B2 (en) * 2004-08-06 2013-04-23 Jansssen Pharmaceutica N.V. 2-amino-quinazoline derivatives useful as inhibitors of β-secretase (BACE)
US8383637B2 (en) * 2004-08-06 2013-02-26 Jansssen Pharmaceutica N.V. 2-amino-quinazoline derivatives useful as inhibitors of β-secretase (BACE)
CA2577392A1 (en) * 2004-08-27 2006-03-09 Elan Pharmaceuticals, Inc. Methods of treatment of amyloidosis using ethanol cyclicamine derivatives aspartyl protease inhibitors
CN101072591A (en) * 2004-11-05 2007-11-14 惠氏公司 Pet and magnetic resonance for screening alzheimer's diseasetherapeutics
AR052051A1 (en) 2004-12-15 2007-02-28 Neuralab Ltd AB HUMANIZED ANTIBODIES USED TO IMPROVE COGNITION
CA2597945C (en) 2005-02-17 2016-07-12 Biogen Idec Ma Inc. Treating neurological disorders
JP4853892B2 (en) * 2005-04-13 2012-01-11 独立行政法人国立精神・神経医療研究センター Method for evaluating specific RNAi for mutant allele
CA2607697C (en) 2005-05-10 2015-01-06 Biogen Idec Ma Inc. Treating and evaluating inflammatory disorders
GB0511861D0 (en) * 2005-06-13 2005-07-20 Merck Sharp & Dohme Proteins
EP1746092A1 (en) 2005-07-22 2007-01-24 Exonhit Therapeutics SA Compounds and methods for treatment of amyloid-B-peptide related disorders
CA2617294A1 (en) * 2005-08-03 2007-02-08 Boehringer Ingelheim International Gmbh Substituted ethane-1,2-diamines for the treatment of alzheimer's disease ii
CA2618474A1 (en) * 2005-08-11 2007-02-15 Boehringer Ingelheim International Gmbh Compounds for treating alzheimer's disease
CA2618481A1 (en) * 2005-08-11 2007-02-15 Boehringer Ingelheim International Gmbh Compounds for treating alzheimer's disease
CA2618019A1 (en) * 2005-08-11 2007-02-15 Boehringer Ingelheim International Gmbh Compounds for the treatment of alzheimer's disease
US20100298278A1 (en) * 2005-08-11 2010-11-25 Christian Eickmeier Inhibitors of beta-secretase for the treatment of alzheimer's disease
WO2007047306A1 (en) * 2005-10-12 2007-04-26 Elan Pharmaceuticals, Inc. Methods of treating amyloidosis using aryl-cyclopropyl derivative aspartyl protease inhibitors
WO2007047305A1 (en) * 2005-10-12 2007-04-26 Elan Pharmaceuticals, Inc. Methods of treating amyloidosis using cyclopropyl derivative aspartyl protease inhibitors
US7932261B2 (en) * 2006-02-06 2011-04-26 Janssen Pharmaceutica Nv Macrocycle derivatives useful as inhibitors of β-secretase (BACE)
WO2007092854A2 (en) 2006-02-06 2007-08-16 Janssen Pharmaceutica N.V. 2-AMINO-QUINOLINE DERIVATIVES USEFUL AS INHIBITORS OF β-SECRETASE (BACE)
WO2007092846A2 (en) * 2006-02-06 2007-08-16 Janssen Pharmaceutica N.V. 2-AMINO-3,4-DIHYDRO-QUINOLINE DERIVATIVES USEFUL AS INHIBITORS OF β-SECRETASE (BACE)
US8784810B2 (en) 2006-04-18 2014-07-22 Janssen Alzheimer Immunotherapy Treatment of amyloidogenic diseases
CA2653628C (en) 2006-06-01 2015-07-14 Elan Pharmaceuticals, Inc. Neuroactive fragments of app
SI2495307T1 (en) 2006-07-13 2018-05-31 Wyeth Llc Production of coagulation factor IX with improved glycosylation pattern
LT3851447T (en) 2006-10-12 2023-12-27 Bellus Health Inc. Methods, compounds, compositions and vehicles for delivering 3-amino-1-propanesulfonic acid
MX2009004519A (en) 2006-11-03 2009-05-12 Wyeth Corp Glycolysis-inhibiting substances in cell culture.
EP2115126B1 (en) 2007-03-02 2015-04-08 Wyeth LLC Use of copper and glutamate in cell culture for production of polypeptides
US8003097B2 (en) 2007-04-18 2011-08-23 Janssen Alzheimer Immunotherapy Treatment of cerebral amyloid angiopathy
WO2008131431A2 (en) * 2007-04-23 2008-10-30 St. Louis University Modulation of blood brain barrier protein expression
EA200971050A1 (en) 2007-05-11 2010-06-30 Томас Джефферсон Юниверсити METHODS OF TREATMENT AND PREVENTION OF NEURODEGENERATIVE DISEASES AND DISORDERS
US8222264B2 (en) 2007-07-06 2012-07-17 Boehringer Ingelheim International Gmbh Substituted amino-quinazolinones, medicaments comprising said compound, their use and their method of manufacture
JP5889529B2 (en) 2007-07-27 2016-03-22 ヤンセン・サイエンシズ・アイルランド・ユーシー Treatment of amyloidogenic diseases
JO3076B1 (en) 2007-10-17 2017-03-15 Janssen Alzheimer Immunotherap Immunotherapy regimes dependent on apoe status
ES2400173T3 (en) * 2008-01-28 2013-04-08 Janssen Pharmaceutica Nv 6-Substituted thio-2-amino-quinoline derivatives useful as beta-secretase inhibitors (BACE)
JP2011510993A (en) * 2008-01-29 2011-04-07 ジヤンセン・フアーマシユーチカ・ナームローゼ・フエンノートシヤツプ 2-Amino-quinoline derivatives useful as β-secretase (BACE) inhibitors
US20110166197A1 (en) * 2008-02-19 2011-07-07 Edunn Biotechnology, Inc. Antisense Modulation Of Amyloid Beta Protein Expression
US9067981B1 (en) 2008-10-30 2015-06-30 Janssen Sciences Ireland Uc Hybrid amyloid-beta antibodies
US20110023152A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genome editing of cognition related genes in animals
DK2605655T3 (en) 2010-08-19 2019-02-04 Buck Institute For Age Res METHODS OF TREATING SMALL COGNITIVE DISABILITY (MCI) AND RELATED DISORDERS
US10357508B2 (en) 2012-03-19 2019-07-23 Buck Institute For Research On Aging APP specific BACE inhibitors (ASBIs) and uses thereof
ES2651113T3 (en) 2012-06-25 2018-01-24 The Brigham And Women's Hospital, Inc. Targeted therapeutic treatments
CA2899938C (en) 2013-02-12 2021-10-19 Buck Institute For Research On Aging Hydantoins that modulate bace-mediated app processing
WO2014160871A2 (en) 2013-03-27 2014-10-02 The General Hospital Corporation Methods and agents for treating alzheimer's disease
KR101890978B1 (en) 2014-06-17 2018-08-24 서울대학교산학협력단 Transgenic cloned porcine Models for alzheimer's disease and the Use thereof
US11168369B2 (en) 2014-11-25 2021-11-09 The Brigham And Women's Hospital, Inc. Method of identifying and treating a person having a predisposition to or afflicted with a cardiometabolic disease
WO2016085876A1 (en) 2014-11-25 2016-06-02 The Broad Institute Inc. Clonal haematopoiesis
CN108349905A (en) 2015-08-27 2018-07-31 南顿欧洲有限责任公司 For APP selectivity BACE compositions inhibited and application thereof
JP7027341B2 (en) 2016-05-12 2022-03-01 バック・インスティテュート・フォー・リサーチ・オン・エイジング Compounds that promote normal processing of APP

Family Cites Families (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4704362A (en) 1977-11-08 1987-11-03 Genentech, Inc. Recombinant cloning vehicle microbial polypeptide expression
US4492760A (en) * 1983-04-19 1985-01-08 The Wistar Institute Of Anatomy And Biology HLA D Typing assay
JPS6147500A (en) * 1984-08-15 1986-03-07 Res Dev Corp Of Japan Chimera monoclonal antibody and its preparation
EP0173494A3 (en) * 1984-08-27 1987-11-25 The Board Of Trustees Of The Leland Stanford Junior University Chimeric receptors by dna splicing and expression
GB8422238D0 (en) * 1984-09-03 1984-10-10 Neuberger M S Chimeric proteins
JPS61134325A (en) * 1984-12-04 1986-06-21 Teijin Ltd Expression of hybrid antibody gene
US4666829A (en) * 1985-05-15 1987-05-19 University Of California Polypeptide marker for Alzheimer's disease and its use for diagnosis
JPS62100291A (en) * 1985-10-28 1987-05-09 Teijin Ltd Gene fragment and plasmid
EP0247091B1 (en) * 1985-11-01 1993-09-29 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
DE3702789A1 (en) 1987-01-30 1988-08-18 Bayer Ag PROCUREMENT PROTEIN OF APC POLYPEPTIDE, FOR CODING DNA AND DIAGNOSTIC USE OF DNA AND PROTEIN
AU3056289A (en) * 1988-01-13 1989-08-11 Mclean Hospital Corporation, The Genetic constructs containing the alzheimer brain amyloid gene
US5134062A (en) * 1988-03-22 1992-07-28 Cornell Research Foundation, Inc. Diagnosis of neuronal disorders and screening potential therapeutic agents therefor
DE3907562A1 (en) 1989-03-09 1990-09-13 Bayer Ag ANTISENSE OLIGONUCLEOTIDS FOR INHIBITING THE TRANSACTIVATOR TARGET SEQUENCE (TAR) AND THE SYNTHESIS OF THE TRANSACTIVATOR PROTEIN (TAT) FROM HIV-1 AND THE USE THEREOF
CA2055435A1 (en) 1989-05-10 1990-11-11 Eli Gilboa Stably transformed eucaryotic cells comprising a foreign transcribable dna under the control of a pol iii promoter
US5234814A (en) * 1989-06-01 1993-08-10 Du Pont Merck Pharmaceutical Company Diagnostic assay for alzheimer's disease
WO1991004753A1 (en) 1989-10-02 1991-04-18 Cetus Corporation Conjugates of antisense oligonucleotides and therapeutic uses thereof
US5180819A (en) 1989-12-22 1993-01-19 The Trustees Of Columbia University In The City Of New York Purified myeloblastin, nucleic acid molecule encoding same, and uses thereof
EP0451700A1 (en) * 1990-04-10 1991-10-16 Miles Inc. Recombinant APP minigenes for expression in transgenic mice as models for Alzheimers's disease
CA2079880A1 (en) * 1990-04-24 1991-10-25 William E. Van Nostrand Purification, detection and methods of use of protease nexin-2
JPH06507782A (en) * 1990-06-15 1994-09-08 サイオス ノバ インコーポレイテッド Non-human recombinant mammals exhibiting amyloid formation symptoms of Alzheimer's disease
WO1992000521A1 (en) * 1990-06-29 1992-01-09 Case Western Reserve University Diagnostic and prognostic methods based on soluble derivatives of the beta amyloid protein precursor
US5200339A (en) * 1990-08-17 1993-04-06 Abraham Carmela R Proteases causing abnormal degradation of amyloid β-protein precursor
BE1003316A5 (en) * 1990-11-27 1992-02-25 Will L F & Cie Sa MONOCLONAL ANTIBODY USEFUL FOR THE DIAGNOSIS OF ALZHEIMER'S DISEASE, SECRETARY HYBRIDOMA OF SUCH A MONOCLONAL ANTIBODY AND PROCESS FOR PREPARING THE SAME.
EP0568575B2 (en) * 1991-01-21 2010-11-03 Elan Pharmaceuticals, Inc. Test and model for alzheimer's disease
AU2765992A (en) * 1991-10-03 1993-05-03 Indiana University Foundation Method for screening for alzheimer's disease
DK0620849T3 (en) * 1992-01-07 2003-10-20 Elan Pharm Inc Transgenic animal models for Alzheimer's disease
US5604102A (en) * 1992-04-15 1997-02-18 Athena Neurosciences, Inc. Methods of screening for β-amyloid peptide production inhibitors
US7371129B2 (en) 2005-04-27 2008-05-13 Samtec, Inc. Elevated height electrical connector

Also Published As

Publication number Publication date
DK0568575T3 (en) 2005-05-23
DE69233468T3 (en) 2011-05-05
WO1992013069A1 (en) 1992-08-06
JP3574432B2 (en) 2004-10-06
ATE447016T1 (en) 2009-11-15
JP2002306195A (en) 2002-10-22
EP0568575B1 (en) 2005-01-12
JP2004261187A (en) 2004-09-24
US20030165958A1 (en) 2003-09-04
DK0568575T4 (en) 2010-12-20
ES2236682T5 (en) 2011-03-31
DK0971033T3 (en) 2009-12-14
JPH06504441A (en) 1994-05-26
AU1169492A (en) 1992-08-27
DE69233468T2 (en) 2005-12-22
DE69233468D1 (en) 2005-02-17
CA2372251A1 (en) 1992-08-06
EP0568575A1 (en) 1993-11-10
EP0568575B2 (en) 2010-11-03
ES2236682T3 (en) 2005-07-16
DE971033T1 (en) 2001-05-03
US5877015A (en) 1999-03-02
ATE286971T1 (en) 2005-01-15
EP0971033A2 (en) 2000-01-12
DE69233774D1 (en) 2009-12-10
ES2335720T3 (en) 2010-03-31
CA2101774A1 (en) 1992-07-22
EP0971033B1 (en) 2009-10-28
US6300540B1 (en) 2001-10-09
JP3510244B2 (en) 2004-03-22
EP0971033A3 (en) 2003-11-12
AU652997B2 (en) 1994-09-15

Similar Documents

Publication Publication Date Title
CA2101774C (en) Test and model for alzheimer's disease
US5455169A (en) Nucleic acids for diagnosing and modeling Alzheimer's disease
JP4368522B2 (en) Mutations in the myostatin gene that cause double massing in mammals
US6037521A (en) Transgenic mouse expressing an β-Amyloid transgene
US6103466A (en) Double-muscling in mammals
US20040076977A1 (en) Marker assisted selection of bovine for improved milk production using diacylglycerol acyltransferase gene dgat1
US5849995A (en) Mouse model for Huntington's Disease and related DNA sequences
US6080550A (en) Isolation and characterization of Agouti: a diabetes/obesity related gene
CA2330944A1 (en) Pathogeniv tau mutations
US20100107265A1 (en) Double-muscling in mammals
CA2040077A1 (en) Recombinant app minigenes for expression in transgenic mice as models for alzheimer's disease

Legal Events

Date Code Title Description
EEER Examination request
MKEX Expiry