U.S. patent application number 10/357935 was filed with the patent office on 2003-09-04 for test and model for alzheimer's disease.
This patent application is currently assigned to Elan Pharmaceuticals. Invention is credited to Chartier-Harlin, Marie-Christine, Goate, Alison Mary, Hardy, John Anthony, Mullan, Michael John, Owen, Michael John.
Application Number | 20030165958 10/357935 |
Document ID | / |
Family ID | 26298303 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165958 |
Kind Code |
A1 |
Hardy, John Anthony ; et
al. |
September 4, 2003 |
Test and model for Alzheimer's disease
Abstract
Model systems of Alzheimer's disease comprise a DNA sequence
encoding an amyloid 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 predisposition to Alzheimer's disease are also
disclosed.
Inventors: |
Hardy, John Anthony; (Tampa,
FL) ; Chartier-Harlin, Marie-Christine; (Villeneuve
d'Ascq, FR) ; Goate, Alison Mary; (St. Louis, MO)
; Owen, Michael John; (South Glamorgan, GB) ;
Mullan, Michael John; (Tampa, FL) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Elan Pharmaceuticals
South San Francisco
CA
|
Family ID: |
26298303 |
Appl. No.: |
10/357935 |
Filed: |
February 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10357935 |
Feb 3, 2003 |
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09520581 |
Mar 8, 2000 |
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09520581 |
Mar 8, 2000 |
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08464250 |
Jun 5, 1995 |
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6300540 |
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08464250 |
Jun 5, 1995 |
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08104165 |
Jan 21, 1992 |
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5877015 |
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08104165 |
Jan 21, 1992 |
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PCT/GB92/00123 |
Jan 21, 1992 |
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Current U.S.
Class: |
435/6.16 ;
435/226; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
A01K 2217/072 20130101;
C12Q 1/6883 20130101; A01K 2207/15 20130101; C12N 15/8509 20130101;
A01K 2217/05 20130101; C07K 14/575 20130101; C12Q 2600/172
20130101; A01K 2227/105 20130101; C12Q 2600/156 20130101; C07K
14/4711 20130101; A01K 2267/0312 20130101; A01K 67/0278 20130101;
A61P 25/28 20180101; C12N 2830/008 20130101; A01K 2217/00
20130101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/226; 435/320.1; 435/325; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/64; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 28, 1991 |
GB |
9118445.7 |
Jan 21, 1991 |
GB |
9101307.8 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a nucleic acid sequence
encoding a codon 717 mutant of human amyloid precursor protein 770
(APP770), or an isoform or fragment of APP770 having a mutant amino
acid residue at the position encoded by codon 717.
2. An isolated polynucleotide of claim 1, wherein the amino acid at
the position encoded by codon 717 is an isoleucine, glycine, or
phenylalanine.
3. An isolated polynucleotide of claim 1, wherein the nucleic acid
sequence is a cDNA.
4. A composition comprising a polynucleotide probe capable of
specifically hybridizing to an amyloid precursor protein 770
(APP770) allele exhibiting a mutation at codon 717.
5. A composition of claim 4, wherein codon 717 of the mutant allele
encodes an isoleucine, phenylalanine, or glycine.
6. A composition of claim 4, wherein the probe is labeled.
7. A composition of claim 4, wherein the probe comprises at least
about 10 nucleotides spanning amino acid 717 of the APP770
allele.
8. A transgenic host comprising a nucleic acid segment encoding a
position 717 mutant of human amyloid precursor protein 770
(APP770), or an APP770 isoform or fragment of APP770 having the
mutation.
9. A host of claim 8, which is a primary or immortalized eukaryotic
cell line.
10. A host of claim 8, which is a bacterium.
11. A host of claim 8, wherein the segment is integrated into the
host genome.
12. A host of claim 8, which is a non-human animal having the DNA
segment incorporated into its germline and which is capable of
expressing the mutant APP770 protein.
13. A host of claim 12, wherein the mutant APP770 protein is the
sole APP770 protein produced by the animal.
14. A transgenic non-human animal with germ cells or somatic cells
comprising a heterologous gene encoding a position 717 mutant
amyloid precursor protein 770 (APP770), which gene upon expression
promotes neuropathological characteristics of Alzheimer's disease
in the animal.
15. A cultured human primary or immortalized cell, comprising a
nucleic acid segment encoding a position 717 mutant of human
amyloid precursor protein 770 (APP770), or an APP770 isoform or
fragment of APP770 having the mutation.
16. A method of screening for an agent capable of treating
Alzheimer's disease, comprising: contacting a host of claim 8 with
the agent; and monitoring expression or processing of proteins
encoded by the mutant APP770 gene.
17. 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), an isoform or fragment thereof, wherein said allele has a
sequence polymorphism at a position 5 corresponding to codon 717 of
APP770.
18. A method of claim 17, wherein said sequence polymorphism is a
nucleotide substitution, whereby an isoleucine or glycine is
substituted at codon 717 of APP770.
19. A method of claim 17, wherein said sequence polymorphism is a
single nucleotide substitution.
20. A method according to claim 17, wherein the detecting step
comprises sequencing a genomic DNA segment from chromosome 21 of
the subject.
21. A method according to claim 17, 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 reaction and (ii) monitoring
the reaction to determine the presence of the gene allele in the
sample, thereby indicating whether the subject is at risk for
Alzheimer's disease.
22. A method according to claim 20, wherein one probe is a
polynucleotide comprising a sequence of at least about 10
nucleotides spanning codon 717 of an APP770.
23. A method according to claim 22, wherein the probes are
oligonucleotides capable of priming polynucleotide synthesis in a
polymerase chain reaction, wherein a reaction product comprises a
sequence of at least 25 contiguous nucleotides from exon 17 of the
APP gene.
24. A method according to claim 21, wherein at least one
oligonucleotide specifically hybridizes to a sequence present in an
intron or flanking region of an APP770 gene.
25. A method according to claim 21, wherein the monitoring step
comprises analyzing sequencing gel reaction products from the PCR
reaction.
26. A method according to claim 21, wherein the monitoring step
comprises analyzing an autoradiograph of a BclI digest of reaction
products from the PCR reaction.
27. A method according to claim 17, wherein the detecting step
comprises (i) mixing in an immunological assay an APP770 or isoform
protein sample from the subject with an antibody reagent specific
for the allele and (ii) monitoring the assay to determine specific
binding between the antibody reagent and the protein sample,
thereby indicating whether the subject is at risk for Alzheimer's
disease.
28. A method according to claim 27, wherein the antibody reagent is
a monoclonal antibody specifically reactive with an antigenic
determinant specific for an allele.
29. 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 APP770 gene of an amyloid precursor
protein (APP) gene in the subject.
30. A method according to claim 29, wherein the polymorphism is
detected by digesting genomic DNA from the subject with at least
one restriction endonuclease and hybridizing resulting fragments
with a detecting probe
31. A composition comprising 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 any of
the twenty conventional amino acids except valine.
32. A transgenic nonhuman animal containing a polypeptide of claim
31.
33. A transgenic nonhuman animal of claim 32, wherein the
polypeptide is present in the brain.
34. An isolated polynucleotide, comprising a nucleic acid sequence
encoding a mutant human APP allele that cosegregates with a genetic
predisposition to Alzheimer's disease.
35. An isolated polypeptide of claim 34, wherein said mutant human
APP allele comprises a codon 717 mutant.
36. 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 an allele of a gene encoding amyloid
precursor protein (APP).
37. A method as claimed in claim 36, wherein the step of detection
is carried out on material removed from, and not returned to, the
subject's body.
38. A method as claimed in claim 36 or 37, wherein the allele of
the gene encodes a substitution mutant of APP.
39. A method as claimed in claim 38, wherein a single amino acid is
substituted for another.
Description
BACKGROUND OF THE INVENTION
[0001] 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 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).
[0002] 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.
[0003] Senile plaques are areas of disorganized neuropil up to 150
.mu.m 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.
[0004] The major protein subunit, .beta.-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).
[0005] 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.
[0006] 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
[0007] 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.
[0008] 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.
[0009] 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 to amino acid
residue position 717 of APP770.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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).
[0017] 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
[0018] 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 II 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 at four loci on the long arm of the chromosome. The
linkage data suggest that the black chromosomes were inherited from
the affected fathers.
[0019] 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.
[0020] 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
.beta.-amyloid peptide. Adapted from Kang et al. (1987) Nature
325:733.
[0021] FIG. 4 shows BclI 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.
[0022] FIG. 5 shows the pedigree of family F19, together with
D21S210 data.
[0023] FIG. 6 shows APP exon 17 sequences in an affected and
unaffected member of F19. In the affected member there is a
G.fwdarw.T transition at position 2150.
[0024] FIG. 7 shows the sequence of APP695.
[0025] FIG. 8 shows the sequence of APP751.
[0026] FIG. 9 shows the sequence of APP770.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0027] The accumulation of .beta.-amyloid protein (A4) in
particular brain regions is one of the main pathologic
characteristics of Alzheimer's disease. The .beta.-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 containg 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 .beta.-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.
[0028] Proteolytic processing of APP in vivo is a normal
physiological process. Carboxy-terminal truncated forms of APP695,
APP75 1, 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.
[0029] Generation of pathogenic .beta.-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 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 .beta.-amyloid
A4 polypeptide which is prone to forming dense amyloidogenic
aggregates that are resistant to proteolytic degradation and
removal. The resultant .beta.-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; .beta.-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.
[0030] 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).
[0031] 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.
[0032] 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 .beta.-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.sub.770) (see Yoshikai et al.
(1990) Gene 7:257), close to the C-terminus of the .beta.-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.
[0033] 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.
[0034] 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).
[0035] Proteolysis on either side of the .beta.-amyloid (A4) region
of APP may be enhanced or qualitatively altered by the specific
mutations at codon 717, increasing the rate of .beta.-amyloid
deposition and aggregation. Such codon 717 mutations may increase
.beta.-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).
[0036] Definitions
[0037] A number of terms and expressions are used throughout the
specification and, to facilitate the understanding thereof, the
following definitions are provided:
[0038] As used herein, "exon" refers to any segment of an
interrupted gene that is represented in the mature RNA product.
[0039] 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.
[0040] As used herein, the phrase "gene sequence coding for amyloid
protein precursor" may be interpreted to mean the DNA and cDNA
sequence as detailed by Yoshikai et al. (1990) Gene 87:257 and Kang
et al, loc. cit., together with the promoter DNA sequence as
described by Salbaum et al. (1988) EMBO 7(9):2807.
[0041] 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 (H.sup.3, C.sup.14,
S.sup.35, P.sup.32) 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.
[0042] 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, ibid., p.528; de Sauvage and Octave. (1989)
Science 245:651; 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.
[0043] The term ".beta.-amyloid gene" is used herein as a synonym
for the APP gene, as .beta.-amyloid is a protein product produced
by a post-translational cleavage of an APP gene product.
[0044] 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 direction):
[0045] Ile-Ala-Thr-Val-Ile-X-Ile-Thr-Leu-[SEQ ID NO:6]
[0046] 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 not a naturally-occurring APP isoform
or .beta.-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.
[0047] As used herein, "APP751" 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.).
[0048] 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 residue of APP751 [SEQ ID NO:2] and the 642nd
amino acid residue of APP695 [SEQ ID NO: 1].
[0049] 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.
[0050] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a 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.
[0051] 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".
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] "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.e., 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, Calif.; 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.sub.m for oligonucleotides
is calculated under standard conditions (1 M NaCl) to be [4.degree.
C..times.(G+C)+2.degree. C..times.(A+T)]. While the conditions of
PCR differ from the standard conditions, this T.sub.m 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.degree. C., occasionally
somewhat higher or lower depending on the base composition of the
sequence(s).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0057] Detection of Mutant Codon 717 APP Alleles
[0058] 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).
[0059] 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).
[0060] The V717I (APP 717 Val.fwdarw.Ile), V717G (APP 717
Val.fwdarw.Gly) and V717F (APP 717 Val.fwdarw.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 717 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 V7171 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.
[0061] The fact that the specific mutations discussed involve
different changes (Val.fwdarw.Ile, Val.fwdarw.Gly, and
Val.fwdarw.Phe) suggests that neither side-chain hydrophobicity nor
side-chain bulk is the crucial issue. All examples of APP alleles
that encode an amino acid other than valine at codon 717,
cosegregate with FAD; suggesting that the valine that occurs at
position 717 in wild-type APP770 or APP751 is a critical amino acid
residue for non-pathogenic APP proteolytic processing (i.e., by the
constitutive cleavage pathway).
[0062] The major metabolic pathway for the APP molecule involves
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 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).
[0063] 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 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.
[0064] Alternative probing techniques, such as ligase chain
reaction (LCR) involve the use of mismatch probes, i.e., 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.
[0065] The polymerase 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.
[0066] 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.degree. 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. 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.
[0067] 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 BclI 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 BclI site
in a PCR product that spans codon 717.
[0068] 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 BclI 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 comparing this number to a reference
allele that has a codon 717 allele that encodes valine.
[0069] 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 (Promega Protocols and
Applications Guide, (1991) Promega Corporation, Madison, Wis.).
[0070] 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.
[0071] 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)
Genomics 5:535; Okayama et al. (1989) J. Lab. Clin. Med. 114: 105;
Sarkar et al. (1990) Anal. Biochem. 186:64; Sommer et al. (1989)
Mayo Clin. Proc. 64: 1361; Wu (1989) Proc. Natl. Acad. Sci. U.S.A.
86:2757; 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.
[0072] 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. 88:189; 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 codon 717 alleles is to
multiplex at least two, and preferably all, LCR probes that will
detect an APP allele having a codon 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 717 allele is straightforward, and
multiplexed pools of such LCR probes may be selected in the
discretion of a practitioner for his particular desired use.
[0073] 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.
[0074] Model Animals and Cell Lines
[0075] Having identified specific mutations in codon 717 of the
.beta.-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.
[0076] Additionally, such model systems provide a tool for defining
the underlying biochemistry of APP and .beta.-amyloid metabolism,
which thereby provides a basis for rational drug design.
[0077] 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.
[0078] 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.
[0079] 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 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.
Bio. Chem. 265:4492) describe the insertion of APP into
immortalized eukaryotic cell lines.
[0080] 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.
[0081] 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 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., .lambda.Charon35, 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 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 10:289). This may be accomplished by transferring
substantially the entire human 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.
[0082] 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, glycine, or phenylalanine
residue at codon 717 of APP770 or the corresponding position in an
APP isoform or fragment.
[0083] 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) Cell
56:145). 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 352:239; Wirak et al. (1991) Science
253:323; and Kawabata et al. (1991) Nature 354:476. Additional
methods for producing transgenic animals are known in the art.
[0084] 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.
[0085] Therapeutics
[0086] Having detected the genetic mutation in the gene sequence
coding for .beta.-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.
[0087] Additional embodiments directed to modulation of the
production of 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,
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.
[0088] 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.
86:10006; Broder et al. (1990) Ann. Int. Med. 113:604; Loreau et
al. (1990) FEBS Letters 274:53-56); Holcenberg et al. WO91/11535;
U.S. Pat. No. 7,530,165 ("New human CRIPTO gene"--publicly
available through Derwent Publications Ltd., Rochdale House, 128
Theobalds Road, London, UK); WO91/09865; WO91/04753; WO90/13641;
and EP 386563, each of which is incorporated herein by reference).
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.
[0089] 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 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 and DNA, (1988), D.
A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.).
[0090] Mutant APP Antigens and Monoclonal Antibodies
[0091] 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 .beta.-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:
1 [SEQ ID NO:7] (a) -Ile-Ala-Thr-Val-Ile-Gly-Ile-Thr-Le- u- [SEQ ID
NO:8] (b) -Ile-Ala-Thr-Val-Ile-Met- -Ile-Thr-Leu- [SEQ ID NO:9] (c)
-Ile-Ala-Thr-Val-IIe-Ala-Ile-Thr-Leu- [SEQ ID NO:10] (d)
-Ile-Ala-Thr-Val-Ile-Ser-Ile-Thr-Leu- [SEQ ID NO:11] (e)
-Ile-Ala-Thr-Val-Ile-Ile-Ile-Thr-Leu- [SEQ ID NO:12] (f)
-Ile-Ala-Thr-Val-Ile-Leu-Ile-Thr-Leu- - [SEQ ID NO:13] (g)
-Ile-Ala-Thr-Val-Ile-Thr- -Ile-Thr-Leu- [SEQ ID NO:14] (h)
-Ile-Ala-Thr-Val-Ile-Pro-Ile-Thr-Leu- [SEQ ID NO:15] (i)
-Ile-Ala-Thr-Val-Ile-His-Ile-Thr-Leu- [SEQ ID NO:16] (j)
-Ile-Ala-Thr-Val-Ile-Cys-Ile-Thr-Leu- [SEQ ID NO:17] (k)
-Ile-Ala-Thr-Val-Ile-Tyr-IIe-Thr-Leu- - [SEQ ID NO:18] (l)
-Ile-Ala-Thr-Val-Ile-Phe- -Ile-Thr-Leu- [SEQ ID NO:19] (m)
-Ile-Ala-Thr-Val-Ile-Glu-Ile-Thr-Leu- [SEQ ID NO:20] (n)
-Ile-Ala-Thr-Val-Ile-Trp-Ile-Thr-Leu- [SEQ ID NO:21] (o)
-Ile-Ala-Thr-Val-Ile-Arg-Ile-Thr-Leu- [SEQ ID NO:22] (p)
-Ile-Ala-Thr-Val-Ile-Asp-Ile-Thr-Leu- - [SEQ ID NO:23] (q)
-Ile-Ala-Thr-Val-Ile-Asn- -Ile-Thr-Leu- [SEQ ID NO:24] (r)
-Ile-Ala-Thr-Val-Ile-Lys-Ile-Thr-Leu- [SEQ ID NO:25] (s)
-Ile-Ala-Thr-Val-Ile-Gln-Ile-Thr-Leu-
[0092] 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.
[0093] 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, N.Y., which is incorporated herein by reference.
[0094] 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.times.10.sup.7
M.sup.-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.times.10.sup.7 M.sup.-1.
More 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 variant as compared to the wild-type.
[0095] 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.
[0096] As stated previously, the DNA sequences will be expressed in
hosts after the sequences have been operably linked to (i.e.,
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
selection markers, e.g., tetracycline resistance or hygromycin
resistance, to permit detection and/or selection of those cells
transformed with the desired DNA sequences (see, e.g., U.S. Pat.
No. 4,704,362, which is incorporated herein by reference).
[0097] Polynucleotides encoding a variant APP codon 717 polypeptide
may 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 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.
[0098] E. coli 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 Salmonella, Serratia, 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 (e.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.
[0099] 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.
[0100] In addition to microorganisms, mammalan 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), which is incorporated herein by
reference). 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. :49, which
is incorporated herein by reference), and necessary processing
information sites, such as ribosome binding sites, RNA splice
sites, polyadenylation sites, and transcriptional terminator
sequences. Preferred expression control sequences are promoters
derived from immunoglobulin genes, SV40, Adenovirus, Bovine
Papilloma Virus, and the like. The vectors containing the DNA
segments of interest (e.g., 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. (See,
generally, Maniatis, et al. Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press, (1982), which is incorporated herein by
reference.)
[0101] 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.
[0102] 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 BclI), buffers and the like, together with instructions
for use.
EXPERIMENTAL EXAMPLES
[0103] The following examples are provided for illustration and are
not intended to limit the invention to the specific example
provided.
Example 1
[0104] Detection of a Val.fwdarw.Ile Mutation in the .beta.-amyloid
(APP) Gene
[0105] 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.
[0106] 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).
[0107] PCR was carried out using the following intronic primers in
order to amplify exon 17 of the APP gene:
2 [A] 5'-CCTCATCCAAATGTCCCCGTCATT-3' AND [SEQ ID NO:26] [B]
5'-GCCTAATTCTCTCATAGTCTTAATTCCCAC-3' [SEQ ID NO 27]
[0108] PCR conditions were 94.degree. C. for 10 min to denature;
then 35 cycles of 60.degree. C. for 1 min, 72.degree. C. for 3 min,
94.degree. C. for 1.5 min; and a single cycle of 72.degree. C. for
10 min. The reaction was carried out using 10 mM tris-HCl pH 8.3,
50 mM potassium chloride, 0.01% gelatin, 1.5 mM magnesium chloride,
200 .mu.M of dNTPs, 50 pmoles of each PCR primer and 1 unit of Taq
polymerase. The total final reaction volume was 25 .mu.l.
[0109] 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.
[0110] Exon 17 was sequenced first because it encodes part of the
.beta.-amyloid peptide and is the site of the mutation (at APP693)
leading to Hereditary Cerebral Haemorrhage with Amyloidosis-Dutch
Type (HCHWA-D).
[0111] 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).
[0112] This C to T transition creates a BclI restriction site
enabling detection within the PCR product (FIG. 4). BclI digests
were carried out at 50.degree. C. for 2-4 hours, as recommended by
the manufacturer, then electrophoresed in 3% agarose.
[0113] 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.
[0114] The valine to isoleucine substitution occurs within the
transmembrane domain two residues from the C-terminus of the
.beta.-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 .beta.-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 within the .beta.-amyloid peptide
(Levy et al. loc. cit.).
Example 2
[0115] Preparation of a Cell Line Containing a Defective
.beta.-Amyloid (APP) Gene
[0116] 10 ml 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.
[0117] A lymphoblastoid cell line, AC21, has been deposited with
the European Collection of Animal Cell Cultures, Porton Down.
Example 3
[0118] Detection of a Val.fwdarw.Gly Mutation in the .beta.-Amyloid
(APP) Gene
[0119] 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:
3 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
[0120] 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).
[0121] APP exon 17 sequences in an affected and an unaffected
member of F19 were determined. In the affected member, there was a
G.fwdarw.T transition at position 2150, as can be seen from FIG.
6.
[0122] 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:
4 ATA-ACC-TCA-TCC-AAA-TGT-CCC-C and [SEQ ID NO:28]
GTA-ACC-CAA-GCA-TCA-TGG-AAG-C; and [SEQ ID NO:29]
[0123] and (b) the PCR conditions were 94.degree. C./10 minutes
then 35 cycles of 60.degree. C./1 minute, 72.degree. C./1 minute,
94.degree. C./1 minute, followed by 72.degree. C./5 minutes.
[0124] 50 pmol of the second primer were used to generate single
stranded product, which was then purified (Chartier-Harlin et al.
loc. cit.). The purified product was sequenced with the SEQUENASE
kit (2.0) (Trade mark; USB) using a primer of sequence:
5 AAA-TGA-AAT-TCT-TCT-AAT-TGC-G. [SEQ ID NO:30]
[0125] The presence of the T.fwdarw.C transition creates gel
artefacts which were resolved by the inclusion of inosine
(SEQUENASE kit) in the sequencing reaction.
[0126] 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.fwdarw.Ile under standard screening conditions and, when
modified APP717 Val.fwdarw.Gly.
Example 4
[0127] Production of Transgenic Animals with Mutant APP Allele
[0128] Generation of the constructs: The vector plink was
constructed by cloning polyliner between the PvuII and EcoRI sites
of pBR322 such that the HindIII end of the polylinker was adjacent
to the PvuII site. The ligation destroyed both the EcoR1 and PvuII
sites associated with the pBR322 segments. The 700 bp HpaI to EcoR1
fragment of pSV2neo (Southern and Berg (1982) J. Mol. Appl. Genet.
1:327) that contains the SV40 polyadenylation signal was cloned
into the HpaI to EcoR1 sites of plink to generate pNotSV. The 200
bp XhoI to PstI fragment of pL2 containing the SV40 16S/1 gS splice
site (Okayama and Berg (1983) Mol. Cell Biol. 3:280) was isolated,
blunted with Klenow, then cloned into the HpaI site of pNotSV to
generate pSplice. The 2.3 kb Nru1 to SpeI fragment of pAPP695
containing the coding region of the cDNA for APP (Tanzi et al.
(1987) Science 235:880) was cloned into the NruI to SpeI 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 HindIII and NruI
sites.
[0129] Generation of pshAPP695 & pshAPP751: The construct
pAmyproBam was generated by cloning the 1.5 kb BamHI fragment of
the APP cDNA into the BamHI site of puc19 xHamy. The 700 bp HindIII
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 HindIII to Asp718 sites of pd695 and pd751 to
yield pshAPP695 and pshAPP751.
[0130] pAPP695 and pAPP75 1: The pAPP695 and pAPP751 vectors were
generated by a three-way ligation of the 3.0 kb EcoRI to XhoI
fragment of pAmyProBam, the 1.5 kb XhoI to SpeI fragment of
APP751cDNA, and the SpeI to ECORI site of pd751.
[0131] 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 BstY1 fragment of pNSE6 and a partial BamH1 (-47 nt
relative to the ATG) to HindIII fragment of pAPP751. This resulted
in the generation of a KpnI fragment that was cloned into the KpnI
sites of pNSE751(+47). The BstY1/Bam fusion results in the loss of
both sites.
[0132] 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 KpnI fragment was generated by
digestion with the restriction enzyme. The KpnI fragment was used
to replace a similar fragment in pNSE751(+47).
[0133] Generation of pNSE751-Hardy and pNSE751-Dutch: The Hardy
(APP642 Val.fwdarw.Ile of APP695) and Dutch (APP618 Gln.fwdarw.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 BglII to SpeI fragment after
digestion, that contained either the Dutch or the Hardy mutation.
The BglII to SpeI 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.
[0134] Generation of pNSE75 1-Hardy and pNSE75 1-Dutch: The Hardy
VI (APP642 V to 1), 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 BglII to SpeI 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.
[0135] 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. Natl.
Acids. Res. USA 86:2463). An SstI fragment was subcloned into the
pSK vector. A pair of PCR primers was generated to place a NruI
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 50 nt upstream of the initiation codon. The
final construction of pNFH751 was generated by a three-way ligation
of the 5.5 b HindIII 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
BgIII to SpeI fragment from a sequence confirmed mutated vector for
the same fragment of pNFH75 1. The presence of the mutation was
confirmed by hybridization with the mutated oligomer or by sequence
analysis.
[0136] Generation of pThy751: The pThy751 vector was generated by a
three-way ligation. The HindIII 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 HindIII to NruI fragment of pd751.
[0137] ThyAPP:
6 [SEQ ID NO:31] CAGACTGAGATCCCAGAACCCTAGGTCTGACTCTAGGG- TCTTGG
[0138] Generation of pThyC100: This pThyC100 construct was
generated by a three-way ligation. The 3.6 kb HindIII to BamHI
fragment of pThy8.2, the synthetic fragment ThyAPP2, and the
HindIII to BglII fragment of pd751 or pNSE751 Dutch or pNSE751
Hardy were ligated to yield pThyC100.
[0139] ThyAPP2:
7 [SEQ ID NO:32] CAGACTGAGATCCCAGAACCGATCCTAGGTCTGACTCTAGG-
GTCTTGG
[0140] The region around the initiation codon was confirmed by
sequence analysis.
[0141] Preparation of DNA for injection: The transgene for
injection was isolated from the corresponding vector of interest
for digestion with NotI and gel electrophoresis. The transgene was
purified by using the Gene Clean kit (Bio101), then further
purified on an Elutip or HPLC purified on a Nucleogen 4000
column.
[0142] 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. Constantini, E. 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 CD1 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). Transgenic mice harboring at
least one copy of the transgene were identified.
[0143] 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.
[0144] Biochemical analysis of the transgenic mice reveals possible
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.
[0145] 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 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.
Sequence CWU 1
1
* * * * *