U.S. patent application number 10/797289 was filed with the patent office on 2004-08-05 for gene-targeted non-human mammal with human fad presenilin mutation and generational offspring.
This patent application is currently assigned to Cephalon, Inc.. Invention is credited to Dorfman, Karen, Reaume, Andrew G., Scott, Richard W..
Application Number | 20040154047 10/797289 |
Document ID | / |
Family ID | 26717896 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040154047 |
Kind Code |
A1 |
Scott, Richard W. ; et
al. |
August 5, 2004 |
Gene-targeted non-human mammal with human FAD presenilin mutation
and generational offspring
Abstract
The present invention provides a gene-targeted, non-human mammal
having a gene encoding a mutant protein product of a mutated FAD
presenilin-1 (PS-1) gene, a human FAD Swedish mutation, and a
humanized A.beta. mutation, and generational offspring thereof and
a gene-targeted, non-human mammal having a gene encoding a mutant
protein product of a mutated FAD PS-1 gene and a human Swedish
APP695 mutation, and generational offspring thereof, as well as
methods of identifying compounds useful in treating Alzheimer's
disease, and to methods of treating Alzheimer's disease.
Inventors: |
Scott, Richard W.; (West
Chester, PA) ; Reaume, Andrew G.; (Waterford, CT)
; Dorfman, Karen; (Waterford, CT) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
Cephalon, Inc.
|
Family ID: |
26717896 |
Appl. No.: |
10/797289 |
Filed: |
March 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10797289 |
Mar 10, 2004 |
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09621897 |
Jul 20, 2000 |
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6734336 |
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09621897 |
Jul 20, 2000 |
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09041185 |
Mar 10, 1998 |
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6284944 |
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60057069 |
Aug 29, 1997 |
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Current U.S.
Class: |
800/12 ;
800/18 |
Current CPC
Class: |
C07K 14/4711 20130101;
A01K 67/0278 20130101; A01K 2217/00 20130101; A01K 2217/072
20130101; A01K 2217/30 20130101; A01K 2267/0312 20130101; C12N
2800/30 20130101; A01K 2217/05 20130101; A01K 67/0275 20130101;
C12N 15/8509 20130101; G01N 33/6896 20130101; G01N 33/5082
20130101; A01K 2227/105 20130101; A01K 2207/15 20130101; A01K
2217/075 20130101 |
Class at
Publication: |
800/012 ;
800/018 |
International
Class: |
A01K 067/027 |
Claims
What is claimed is:
1. A gene-targeted, non-human mammal heterozygous for a human
Familial Alzheimer's Disease (FAD) mutation comprising a human
mutation of the presenilin-1 (PS-1 gene), a human FAD Swedish
mutation, and a humanized A.beta. gene.
2. A gene-targeted, non-human mammal homozygous for a human
Familial Alzheimer's Disease (FAD) mutation comprising a human
mutation of the presenilin-1 (PS-1 gene), a human FAD Swedish
mutation, and a humanized A.beta. gene.
3. The mammal of claim 1 wherein said mutation of said PS-1 gene is
P264L.
4. The mammal of claim 2 wherein said mutation of said PS-1 gene is
P264L.
5. The mammal of claim 1 wherein said mammal is a rodent.
6. The mammal of claim 5 wherein said mammal is a mouse.
7. The mammal of claim 2 wherein said mammal is a rodent.
8. The mammal of claim 7 wherein said mammal is a mouse.
9. Generational offspring of the mammal of claim 1 wherein said
mutant PS-1 gene is expressed.
10. Generational offspring of the mammal of claim 2 wherein said
mutant PS-1 gene is expressed.
11. A method for screening chemical compounds for the ability to
decrease in vivo levels of A.beta. peptide, said method comprising
the steps of: a) administering said chemical compound to the mammal
of claim 1; and b) measuring the amount of A.beta. peptide in a
tissue sample from said mammal, wherein a decrease in the amount of
A.beta. peptide in said tissue sample is indicative of a chemical
compound that has the ability to decrease in vivo levels of said
A.beta. peptide.
12. A method for screening chemical compounds for the ability to
decrease in vivo levels of the A.beta. peptide, said method
comprising the steps of: a) administering said chemical compound to
the mammal of claim 2; and b) measuring the amount of A.beta.
peptide in a tissue sample from said mammal, wherein a decrease in
the amount of A.beta. peptide in said tissue sample is indicative
of a chemical compound that has the ability to decrease in vivo
levels of said A.beta. peptide.
13. A method for screening chemical compounds for the ability to
decrease in vivo levels of the A.beta. peptide, said method
comprising the steps of: a) administering said chemical compound to
the mammal of claim 9; and b) measuring the amount of A.beta.
peptide in a tissue sample from said mammal, wherein a decrease in
the amount of A.beta. peptide in said tissue sample is indicative
of a chemical compound that has the ability to decrease in vivo
levels of said A.beta. peptide.
14. A method for screening chemical compounds for the ability to
decrease in vivo levels of the A.beta. peptide, said method
comprising the steps of: a) administering said chemical compound to
the mammal of claim 10; and b) measuring the amount of A.beta.
peptide in a tissue sample from said mammal, wherein a decrease in
the amount of A.beta. peptide in said tissue sample is indicative
of a chemical compound that has the ability to decrease in vivo
levels of said A.beta. peptide.
15. The method of claim 11 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
16. The method of claim 12 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
17. The method of claim 13 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
18. The method of claim 14 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
19. A method for identifying a compound for treating Alzheimer's
disease comprising the steps of: a) administering a compound to the
mammal of claim 1; and b) measuring the amount of A.beta. peptide
in a tissue sample from said mammal, wherein a decrease in the
amount of A.beta. peptide in said tissue sample is indicative of a
compound that can be used to treat Alzheimer's disease.
20. A method for identifiing a compound for treating Alzheimer's
disease comprising the steps of: a) administering a compound to the
mammal of claim 2; and b) measuring the amount of A.beta. peptide
in a tissue sample from said mammal, wherein a decrease in the
amount of A.beta. peptide in said tissue sample is indicative of a
compound that can be used to treat Alzheimer's disease.
21. A method for identifying a compound for treating Alzheimer's
disease comprising the steps of: a) administering a compound to the
mammal of claim 9; and b) measuring the amount of A.beta. peptide
in a tissue sample from said mammal, wherein a decrease in the
amount of A.beta. peptide in said tissue sample is indicative of a
compound that can be used to treat Alzheimer's disease.
22. A method for identifying a compound for treating Alzheimer's
disease comprising the steps of: a) administering a compound to the
mammal of claim 10; and b) measuring the amount of A.beta. peptide
in a tissue sample from said mammal, wherein a decrease in the
amount of A.beta. peptide in said tissue sample is indicative of a
compound that can be used to treat Alzheimer's disease.
23. The method of claim 19 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
24. The method of claim 20 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
25. The method of claim 21 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
26. The method of claim 22 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
27. A method of treating an individual suspected of having
Alzheimer's disease comprising administering to said individual an
effective Alzheimer's disease treatment amount of a compound
identified by the method of claim 19.
28. A method of treating an individual suspected of having
Alzheimer's disease comprising administering to said individual an
effective Alzheimer's disease treatment amount of a compound
identified by the method of claim 20.
29. A method of treating an individual suspected of having
Alzheimer's disease comprising administering to said individual an
effective Alzheimer's disease treatment amount of a compound
identified by the method of claim 21.
30. A method of treating an individual suspected of having
Alzheimer's disease comprising administering to said individual an
effective Alzheimer's disease treatment amount of a compound
identified by the method of claim 22.
31. A compound identified by the method of claim 11.
32. A compound identified by the method of claim 12.
33. A compound identified by the method of claim 13.
34. A compound identified by the method of claim 14.
35. A compound identified by the method of claim 19.
36. A compound identified by the method of claim 20.
37. A compound identified by the method of claim 21.
38. A compound identified by the method of claim 22.
39. A gene-targeted, non-human mammal heterozygous for a human
Familial Alzheimer's Disease (FAD) mutation comprising a human
mutation of the presenilin-1 (PS-1 gene), and a human transgenic
for Swedish APP695.
40. A gene-targeted, non-human mammal homozygous for a human
Familial Alzheimer's Disease (FAD) mutation comprising a human
mutation of the presenilin-1 (PS-1 gene), and a human transgenic
for Swedish APP695.
41. The mammal of claim 39 wherein said mutation of said PS-1 gene
is P264L.
42. The mammal of claim 40 wherein said mutation of said PS-1 gene
is P264L
43. The mammal of claim 39 wherein said mammal is a rodent.
44. The mammal of claim 43 wherein said mammal is a mouse.
45. The mammal of claim 40 wherein said mammal is a rodent.
46. The mammal of claim 45 wherein said mammal is a mouse.
47. Generational offspring of the mammal of claim 39 wherein said
mutant PS-1 gene is expressed.
48. Generational offspring of the mammal of claim 40 wherein said
mutant PS-1 gene is expressed.
49. A method for screening chemical compounds for the ability to
decrease in vivo levels of the A.beta. peptide, said method
comprising the steps of: a) administering said chemical compound to
the mammal of claim 39; and b) measuring the amount of A.beta.
peptide in a tissue sample from said mammal, wherein a decrease in
the amount of A.beta. peptide in said tissue sample is indicative
of a chemical compound that has the ability to decrease in vivo
levels of said A.beta. peptide.
50. A method for screening chemical compounds for the ability to
decrease in vivo levels of the A.beta. peptide, said method
comprising the steps of: a) administering said chemical compound to
the mammal of claim 40; and b) measuring the amount of A.beta.
peptide in a tissue sample from said mammal, wherein a decrease in
the amount of A.beta. peptide in said tissue sample is indicative
of a chemical compound that has the ability to decrease in vivo
levels of said A.beta. peptide.
51. A method for screening chemical compounds for the ability to
decrease in vivo levels of the A.beta. peptide, said method
comprising the steps of: a) administering said chemical compound to
the mammal of claim 47; and b) measuring the amount of A.beta.
peptide in a tissue sample from said mammal, wherein a decrease in
the amount of A.beta. peptide in said tissue sample is indicative
of a chemical compound that has the ability to decrease in vivo
levels of said A.beta. peptide.
52. A method for screening chemical compounds for the ability to
decrease in vivo levels of the A.beta. peptide, said method
comprising the steps of: a) administering said chemical compound to
the mammal of claim 48; and b) measuring the amount of A.beta.
peptide in a tissue sample from said mammal, wherein a decrease in
the amount of A.beta. peptide in said tissue sample is indicative
of a chemical compound that has the ability to decrease in vivo
levels of said A.beta. peptide.
53. The method of claim 49 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
54. The method of claim 50 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
55. The method of claim 51 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
56. The method of claim 52 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
57. A method for identifying a compound for treating Alzheimer's
disease comprising the steps of: a) administering a compound to the
mammal of claim 39; and b) measuring the amount of A.beta. peptide
in a tissue sample from said mammal, wherein a decrease in the
amount of A.beta. peptide in said tissue sample is indicative of a
compound that can be used to treat Alzheimer's disease.
58. A method for identifying a compound for treating Alzheimer's
disease comprising the steps of: a) administering a compound to the
mammal of claim 40; and b) measuring the amount of A.beta. peptide
in a tissue sample from said mammal, wherein a decrease in the
amount of A.beta. peptide in said tissue sample is indicative of a
compound that can be used to treat Alzheimer's disease.
59. A method for identifying a compound for treating Atzheimer's
disease comprising the steps of: a) administering a compound to the
mammal of claim 47; and b) measuring the amount of A.beta. peptide
in a tissue sample from said mammal, wherein a decrease in the
amount of A.beta. peptide in said tissue sample is indicative of a
compound that can be used to treat Alzheimer's disease.
60. A method for identifying a compound for treating Alzheimer's
disease comprising the steps of: a) administering a compound to the
mammal of claim 48; and b) measuring the amount of A.beta. peptide
in a tissue sample from said mammal, wherein a decrease in the
amount of A.beta. peptide in said tissue sample is indicative of a
compound that can be used to treat Alzheimer's disease.
61. The method of claim 57 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
62. The method of claim 58 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
63. The method of claim 59 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
64. The method of claim 60 wherein said tissue sample is selected
from the group consisting of brain tissue, non-brain tissue and
body fluids.
65. A method of treating an individual suspected of having
Alzheimer's disease comprising administering to said individual an
effective Alzheimer's disease treatment amount of a compound
identified by the method of claim 57.
66. A method of treating an individual suspected of having
Alzheimer's disease comprising administering to said individual an
effective Alzheimer's disease treatment amount of a compound
identified by the method of claim 58.
67. A method of treating an individual suspected of having
Alzheimer's disease comprising administering to said individual an
effective Alzheimer's disease treatment amount of a compound
identified by the method of claim 59.
68. A method of treating an individual suspected of having
Alzheimer's disease comprising administering to said individual an
effective Alzheimer's disease treatment amount of a compound
identified by the method of claim 60.
69. A compound identified by the method of claim 49.
70. A compound identified by the method of claim 50.
71. A compound identified by the method of claim 51.
72. A compound identified by the method of claim 52.
73. A compound identified by the method of claim 57.
74. A compound identified by the method of claim 58.
75. A compound identified by the method of claim 59.
76. A compound identified by the method of claim 60.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part and claims
priority under 35 U.S.C. .sctn. 120 to U.S. Ser. No. 09/041,185
filed Mar. 10, 1998, which claims priority under 35 U.S.C. .sctn.
119(e) to Provisional Serial No. 60/057,069 filed Aug. 29, 1997,
each of which are incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to gene-targeted, non-human
mammals comprising a human mutation in the non-human mammalian
presenilin 1 (PS-1) FAD gene, methods of identifying compounds for
treating Alzheimer's disease, and to methods of treating
Alzheimer's disease.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) is an age-dependent
neurodegenerative disorder that leads to profound behavioral
changes and dementia. Hallmark pathologies include the atrophy of
brain gray matter as a result of the massive loss of neurons and
synapses, and protein deposition in the form of both intraneuronal
neurofibrillary tangles and extracellular amyloid plaques within
the brain parenchyma. In addition, affected areas of the AD brain
exhibit a reactive gliosis that appears to be a response to brain
injury. Surviving neurons from vulnerable populations in AD show
signs of metabolic compromise as indicated by alterations in the
cytoskeleton (Wang et al., Nature Med., 1996, 2, 871-875), Golgi
complex (Salehi et al., J. Neuropath. Exp. Neurol., 1995, 54,
704-709) and the endosomal-lysosomal system (Cataldo et al.,
Neuron, 1995, 14, 671-680).
[0004] Approximately 10 to 30% of AD cases are inherited in an
autosomal dominant fashion and are referred to as "familial
Alzheimer's disease" or "FAD." Genetic linkage studies have
revealed that FAD is heterogeneous and a majority of the cases have
been linked to gene mutations on chromosomes 1, 14, 19, or 21
(reviewed in Siman and Scott, Curr. Opin. Biotech., 1996, 7,
601-607). Importantly, these individuals have been shown to develop
the classical symptomatic and pathological profiles of the disease
confirming that the mutations are associated with the development
of the disease rather than a related syndrome. The locus on
chromosome 14 is associated with a significant fraction of FAD, and
mutations at the locus have been mapped to a single-copy gene,
termed "S182" or "presenilin 1" (PS-1), that encodes a 467 amino
acid protein (Sherrington et al., Nature, 1995, 375, 754-760; Clark
et al., Nature Genet., 1995, 11, 219-222). A closely related gene,
"STM2" or "presenilin 2" (PS-2), located on chromosome 1, has been
linked to two additional FAD kindreds including the descendants of
German families from the Volga valley of Russia (Levy-Lahad et al.,
Science, 1995, 269, 973-977; Rogaev et al., Nature, 1995, 376,
775-778). PS-1 and PS-2 share an overall 67% amino acid sequence
homology, and primary structure analysis indicates they are
integral membrane proteins with 6 to 8 trans-membrane domains
(Slunt et al., Amyloid-Int. J Exp. Clin. Invest., 1995, 2, 188-190;
Doan et al., Neuron, 1996, 17, 1023-1030). Much of the information
on function of the presenilins stems from the identification of a
presenilin homolog in C. elegans termed "SEL-12," a 6 to 8
trans-membrane protein that appears to participate in an
intracellular signaling pathway mediated by the lin-12/glp-1/Notch
family (Levitan and Greenwald, Nature, 1995, 377, 351-354). PS-1
and SEL-12 proteins share a 49% sequence homology and have similar
membrane orientations. Importantly, both human PS-1 and PS-2 can
rescue the mutant sel-12 phenotype in C. elegans, indicating a role
for the presenilins in Notch signaling (Levitan et al., Proc. Natl.
Acad. Sci. USA, 1996, 93, 14940-14944).
[0005] FAD linked to the presenilins is highly penetrant and the
aggressive nature of the disease suggests that the mutant protein
participates in a seminal pathway of AD pathology. To date, over
seventy FAD mutations have been identified in PS-1, and three FAD
mutations have been found in PS-2. Most of the FAD mutations occur
in conserved positions between the two presenilin proteins,
suggesting that they are affecting functionally or structurally
important amino acid residues. Interestingly, many of the mutated
amino acids are also conserved in SEL-12. All but two of the
presenilin mutations are missense mutations. One exception results
in an aberrant RNA splicing event that eliminates exon 9, creating
an internally-deleted mutant protein (Perez-Tur et al.,
NeuroReport, 1995, 7, 297-301; Sato et al., Hum. Mutat. Suppl.,
1998, 1, S91-94; and Prihar et al., Nature Med., 1999, 5, 1090).
The other results in two deletion transcripts (.DELTA.4 and
.DELTA.4cryptic) and one full-length transcript with the amino acid
Thr inserted between 113 and 114 (DeJonghe et al., Hum. Molec.
Genet., 1999, 8, 1529-1540). The latter transcript leads to the AD
pathophysiology. These latter points, along with the genetic
dominance of the disease, argue that disease pathogenesis in the
presenilin kindreds requires the production of a mutant presenilin
protein having a novel and detrimental function, rather than the
simple loss or reduction of normal presenilin levels. The mutations
do appear to disrupt normal presenilin function however, because
mutant presenilins are not able to rescue or fully rescue the
sel-12 phenotype (Levitan et al., Proc. Natl. Acad. Sci. USA, 1996,
93, 14940-14944).
[0006] Expression profiles of the presenilins have been examined at
a gross level but, so far, these analyses have yielded little
information on the mechanism of disease pathogenesis. Both
presenilin 1 and 2 are widely expressed in the CNS and peripheral
tissues. In brain, expression is enriched in neurons but is
apparent in both AD-vulnerable and resistant areas. Cellular
localization studies indicate that the proteins accumulate
primarily in the Golgi complex and endoplasmic reticulum but no
significant alterations in expression levels or subcellular
distribution have been attributed to the FAD mutations (Kovacs et
al., Nature Med., 1996, 2, 224-229).
[0007] The presenilin proteins are processed proteolytically
through two intracellular pathways. Under normal conditions,
accumulation of 30 kD N-terminal and 20 kD C-terminal proteolytic
fragments occurs in the absence of the full-length protein. This
processing pathway is highly regulated and appears to be relatively
slow, accounting for turnover of only a minor fraction of the
full-length protein. The remaining fraction appears to be rapidly
degraded in a second pathway by the proteasome (Thinakaran et al.,
Neuron, 1996, 17, 181-190; Kim et al., J. Biol. Chem., 1997, 272,
11006-11010). Proteolytic metabolism of PS-1 variants linked to FAD
appears to be different, but the relevance of the change to
pathogenesis is not known (Lee, et al., Nature Med., 1997, 3,
756-760).
[0008] One pathogenic role for the mutant presenilins in FAD
appears to be related to effects on processing of the amyloid
precursor protein (APP) and production of the A.beta. peptide, the
primary proteinaceous component of the extaacellular neuritic
plaque in the AD brain. Elevated serum levels of the longer form of
A.beta. (A.beta.42), considered to be the more pathogenic species
of the A.beta. peptides, have been measured in patients bearing
PS-1 and PS-2 mutations (Scheuner et al., Nature Med., 1996, 2,
864-870). Additionally, FAD brains with PS-1 mutations have large
amounts of A.beta. deposition (Lemere et al., Nature Med., 1996, 2,
1146-1150; Mann et al., Ann. Neurol., 1996, 40, 149-156; Gmez-Isla
et al., Ann. Neurol., 1997, 41, 809-813). Elevated levels of
A.beta.1-42 were also found in cells transfected with mutant PS-1
or PS-2 and in mice expressing mutant PS-1 (Borchelt et al.,
Neuron, 1996, 17, 1005-1013; Duff et al., Nature, 1996, 383,
710-713; Citron et al., Nature Med., 1997, 3, 67-72; Murayama et
al., Prog. Neuro-Psychopharmacol. Biol. Psychiatr., 1999, 23,
905-913; Murayama et al., Neurosci. Lett., 1999, 265, 61-63; Nakano
et al., Eur. J. Neurosci., 1999, 11, 2577-2581). The mechanism by
which the mutant presenilins affect APP processing is not known,
but these results do support a causative role of increased
A.beta.42 production in the development of FAD. Importantly, it is
possible that mutant presenilins influence other AD pathogenic
processes as well, such as presumptive intracellular signaling and
cell death pathways involved directly or indirectly in neuronal
dysfunction and degeneration.
[0009] Genetically-engineered animals have been used extensively to
examine the function of specific gene products in vivo and their
role in the development of disease phenotypes. The genetic
engineering of mice can be accomplished according to at least two
distinct approaches: (1) a transgenic approach where an exogenous
gene is randomly inserted into the host genome, and (2) a
gene-targeting approach where a specific endogenous DNA sequence or
gene is partially or completely removed, or replaced by homologous
recombination. The transgene of a transgenic organism is comprised
generally of a DNA sequence encoding the protein sequence and a
promoter that directs expression of the protein coding sequences. A
transgenic organism expresses the transgene in addition to all
normally-expressed native genes. The targeted gene of a
gene-targeted animal, on the other hand, can be modified in one of
two ways: (1) a functional form where a modified version of the
targeted gene is expressed, or (2) a non-functional or "null" form
where the targeted gene has been disrupted resulting in loss or
reduction of expression. If the targeted gene is a single copy gene
and the animal is homozygous at the targeted locus, then, depending
on the type of modification, the animal either does not express the
targeted gene or expresses only a modified version of the targeted
gene in absence of the normal form.
[0010] Transgenic mice expressing native and mutant forms of the
presenilin proteins have been described (Borchelt et al., Neuron,
1996, 17, 1005-1013; Duff et al., Nature, 1996, 383, 710-713;
Borchelt et al., Neuron, 1997, 19, 939-945; Citron et al., Nature
Med., 1997, 3, 67-72; Chui et al., Nature Med., 1999, 5, 560-564;
and Nakano et al., Eur. J. Neurosci., 1999, 11, 2577-2581).
Although mice bearing mutations in PS-1 had elevated levels of
A.beta.1-42, they have not formed A.beta. deposits characteristic
of AD or shown behavioral deficits associated with AD. Neuronal
loss has been described by one group (Chui et al., Nature Med.,
1999, 5, 560-564). When transgenic mice with PS-1 mutations were
crossed with transgenic mice bearing the Swedish APP mutations,
there was marked acceleration in the formation of A.beta. deposits
(Borchelt et al., Neuron, 1997, 19, 939-945; Holcomb et al., Nature
Med., 1998, 4, 97-100; Lamb et al., Nature Neurosci., 1999, 2,
695-697). Gene-targeted PS-1 null mice lacking one or both
functional alleles of the PS-1 gene have also been described (Wong
et al., Nature, 1997, 387, 288-292, and Shen et al., Cell, 1997,
89, 629-639). Mice in which both PS-1 alleles have been disrupted
resulting in the complete loss of PS-1 expression are not viable
and die shortly after birth. No abnormal phenotypes or changes in
APP processing have been reported in mice lacking only one of the
two PS-1 alleles, but inhibition of APP processing is found in
neurons derived from PS-1 null mice (DeStrooper et al., Nature,
1998, 391, 387-390).
[0011] In the present application, a gene-targeting approach
(Reaume et al., J. Biol. Chem., 1996, 271, 23380-23388, which is
incorporated herein by reference in its entirety) generating AD
models is described. One model employs the Swedish FAD mutation and
"humanized" mouse A.beta. sequence in the APP gene (U.S. Pat. No.
5,777,194, which is incorporated herein by reference in its
entirety). This mouse (APP.sup.NLh/NLh) produced normal levels of
APP, overproduced human A.beta.1-40 and 1-42, but did not deposit
A.beta. (Reaume et al., J. Biol. Chem., 1996, 271, 23380-23388). A
human PS-1 mutation, the P264L mutation in particular, was
introduced into the mouse PS-1 gene. The P264L mutation is a
non-conservative amino acid substitution in the cluster of
mutations in exon 8, causing an onset of FAD in the middle forties
to middle fifties (Campion et al., Hum. Molec. Genet., 1995, 4,
2373-2377; Wasco et al., Nature Med., 1995, 1, 848). Crosses
produced APP.sup.NLh/NLh.times.PS-1.s- up.P264L/P264L double
gene-targeted mice. These mice had elevated levels of A.beta.1-42,
sufficient to cause A.beta. deposition. Mice bearing the
PS-1.sup.P264L mutation were also crossed with Tg2576 mice that
overexpress Swedish APP695 (Hsiao et al., Science, 1996, 274,
99-102; available from the Mayo Clinic, Rochester, Minn.). One
distinct advantage of the present invention is that for
heterozygous and homozygous gene-targeted mice, the fidelity of
expression patterns of proteins is maintained since the expression
is under the endogenous promoter. Further, expression levels of the
holoprotein are not changed.
SUMMARY OF THE INVENTION
[0012] The present invention relates to a gene-targeted, non-human
mammal comprising a gene encoding a mutant protein product of a
mutated FAD presenilin-1 (PS-1) gene, a human FAD Swedish mutation,
and a humanized A.beta. mutation, and generational offspring
thereof. The present invention also relates to a gene-targeted,
non-human mammal comprising a gene encoding a mutant protein
product of a mutated FAD presenilin-1 (PS-1) gene and a human
Swedish APP695 mutation, and generational offspring thereof.
Preferably, the PS-1 gene has been mutated to contain the human
P264L mutation (Wasco et al., Nature Medicine, 1995, 1, 848). In
particular, the present invention relates to a mouse wherein a part
of a mouse presenilin 1 gene encoding presenilin 1 protein has been
replaced with a DNA sequence that results in a mouse presenilin 1
gene that contains a human mutation, most preferably a P264L
mutation. Still more specifically, the base sequence of codon 264
of the mouse presenilin 1 gene is altered from CCG to CTT, which is
the base sequence found to constitute the P264L mutation of humans.
The mutated gene codon encodes leucine in place of proline in amino
acid number 264 of presenilin 1. Additionally, and still more
specifically, a nucleotide base in codon 265 of the mouse
presenilin 1 gene is altered from adenosine to guanosine, but this
change does not result in an amino acid change in the expressed
protein. However, the combined sequence of codons 264 and 265,
after the incorporation of the most preferred changes described
above, results in a restriction enzyme site for the restriction
enzyme AflII.
[0013] Accordingly, in one embodiment, the present invention
features a non-human mammal and generational offspring homozygous
for a targeted mutant PS-1 gene comprising a mutated FAD gene
preferably a mouse presenilin 1 protein-encoding sequence
comprising a human mutation, most preferably a P264L mutation, in
place of the native presenilin 1 protein-encoding sequence. In
another embodiment, the invention features a non-human mammal and
generational offspring heterozygous for a targeted PS-1 gene
comprising a mutated mouse FAD gene, preferably a mouse presenilin
1 protein-encoding sequence containing a human mutation, most
preferably a P264L mutation, in place of the native presenilin 1
protein-encoding sequence.
[0014] The present invention is also directed to methods for
identifying a compound for treating Alzheimer's disease comprising
administering a compound to a mammal heterozygous or homozygous for
a mutation of the PS-1 gene and a human Swedish APP695 mutation, or
generational offspring thereof, or to a mammal heterozygous or
homozygous for a mutation of the PS-1 gene, a human FAD Swedish
mutation, and a humanized A.beta. mutation, and generational
offspring thereof, and measuring the amount of A.beta.42 peptide in
a tissue sample from the mammal.
[0015] The present invention is also directed to methods of
treating an individual suspected of having Alzheimer's disease
comprising administering to the individual an effective Alzheimer's
disease treatment amount of a compound identified by the method
described above.
[0016] The present invention is also directed to compounds
identified by any of the methods described above
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 is a schematic diagram illustrating general
principles of gene targeting.
[0018] FIG. 2 is a set of mouse PS-1 genomic clone maps prepared
using the Flash.TM. Non-radioactive Gene Mapping Kit. Letter
abbreviations for restriction endonucleases are as follows: E,
EcoRl; X, Xbal; H, HindIII; B, BamHI; Xh, Xhol.
[0019] FIG. 3 is a representative restriction map used to
illustrate a Flash.TM. restriction mapping method.
[0020] FIG. 4 is a diagram illustrating the strategy for placing
exons 7 and 8 on the restriction map of PS-1.
[0021] FIG. 5 is a pair of genetic maps illustrating the
relationship between Exon 8 of PS-1 and the pPS1-8-TV replacement
vector. Letter abbreviations for restriction endonucleases are as
follows: E, EcoRl; X, Xbal; H, HindIII; B, BamHl; Xh, Xhol, N,
NotI.
[0022] FIG. 6 is a schematic diagram illustrating the construction
of plasmid pPNTIox.sup.2.
[0023] FIG. 7 is a schematic diagram illustrating the construction
of plasmid pPS1-XH16.
[0024] FIG. 8 is a schematic diagram illustrating the construction
of plasmid pPS1-XB1.
[0025] FIG. 9 is a schematic diagram illustrating the construction
of plasmids pPS1-X15 and pPS1-X2.
[0026] FIG. 10 is a schematic diagram illustrating the construction
of plasmid pPS1-X319.
[0027] FIG. 11 is a schematic diagram illustrating the restriction
mapping of the 5' Arm of Homology from plasmids pPS1-X15 and
pPS1-X2.
[0028] FIG. 12 is a pair of restriction maps for the PS1 3' and 5'
arms of homology.
[0029] FIG. 13 is a partial sequence of exon 8 of PS-1 illustrating
the base changes to effect the P264L mutation and the addition of
the AflII restriction endonuclease site of this invention.
[0030] FIG. 14 is a schematic diagram illustrating the construction
of plasmid pPS1-XB85.
[0031] FIG. 15 is a schematic diagram illustrating the construction
of plasmid pPS1-206.
[0032] FIG. 16 is a schematic diagram illustrating the construction
of plasmid pPS1-360.
[0033] FIG. 17 is a schematic diagram illustrating the construction
of plasmid pPNT3'413.
[0034] FIG. 18 is a schematic diagram illustrating the construction
of plasmid pPS1-8-TV.
[0035] FIG. 19 is a schematic diagram illustrating the strategy to
detect homologous recombination within mouse PS1. Letter
abbreviations for restriction endonucleases are as follows: E,
EcoRI; X, XbaI; N NotI; H, HindIII; B, BamHI; A, Apal; Af, AflII;
Sc, Scal; K, KpnI; and Hc, HincII.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0036] The present invention relates to a gene-targeted, non-human
mammal (and generational offspring of such mammal) that contains in
the non-human mammal's endogenous (i.e., native) genome presenilin
1 gene that comprises a human mutation, most preferably a human
P264L mutation. The non-human mammal can also comprise a human FAD
Swedish mutation and a humanized A.beta. mutation. A non-human
mammal can also comprise, in addition to the human PS-1 mutation, a
human Swedish APP695 mutation. Most preferably, the gene-targeted,
non-human mammal produces a mutated presenilin 1 protein instead of
the presenilin 1 protein normally produced by the non-human mammal.
Gene-targeted, non-human mammals homozygous for a presenilin 1 gene
containing a human mutation, such as the human P264L mutation,
produce the mutated presenilin 1 protein exclusively.
Gene-targeted, non-human mammals heterozygous for a resenilin 1
gene containing a human mutation, such as the human P264L mutation,
produce both the mutated presenilin 1 protein and the native
presenilin 1 protein. Preferably, the gene-targeted, non-human
mammal of this invention is a rodent, and more specifically a
mouse.
[0037] Importantly, because the non-human mammal of this invention
is generated by gene targeting, as opposed to transgenic
techniques, the mammal produces the mutated presenilin 1 protein
exclusively by normal endogenous presenilin 1 protein expression
mechanisms. Advantageously, and unlike expression resulting from
trarsgenic approaches, the presenilin 1 protein is expressed from
genes having the normal copy number, and under the control of the
endogenous presenilin 1 gene expression control mechanisms. As a
result, the presenilin 1 protein in the non-human animal of this
invention is produced with the same developmental timing, same
tissue specificity, and same rates of synthesis normally associated
with native presenilin 1 protein in the wild-type, non-human
mammal.
[0038] The gene-targeted, non-human mammals of this invention may
be used as tools or models to elucidate the role of PS-1 comprising
a human mutation, preferably the human P264L mutation, in the
pathology and symptomatology of AD. They may be used to elucidate
the manner in which the human mutation, preferably the P264L
mutation, increases the production of the amyloid protein
A.beta.42. As used herein, the term "increase" when used in the
foregoing context, means that the levels of A.beta.42 produced by
the non-human mammals disclosed herein are elevated relative to
wild-type controls.
[0039] The non-human mammals of this invention and generational
offspring also may be used as assay systems to screen for in vivo
inhibitors and for discovering and testing the efficacy and
suitability of putative chemical compounds for their ability to
inhibit the formation, presence and deposition of excessive amounts
of A.beta. peptide in the brain tissues, other tissues and body
fluids (e.g., blood; plasma, and cerebrospinal fluid), said method
comprising the steps of: (a) administering said chemical compounds
to a non-human mammal homozygous or heterozygous for a targeted
mutant PS-1 gene comprising a human mutation, preferably the human
P264L mutation, comprising: a mouse PS-1 peptide encoding sequence
containing a human mutation, preferably the P264L mutation, in
place of the native PS-1 peptide encoding sequence and (b)
measuring the amounts of A.beta. peptide in brain tissues, other
tissues and body fluids (or some combination thereof) of said
non-human mammal, at an appropriate interval of time after the
administration of said chemical compounds.
[0040] As used in this disclosure, the following terms and phrases
have the following indicated definitions.
[0041] As used herein, "A.beta. peptide" means either A.beta.40 or
A.beta.42 or fragments thereof.
[0042] As used herein, "arms of homology" means nucleotide DNA
sequences in a targeting vector: (1) which have sufficient length
and homology to provide for site-specific integration of part of
the targeting vector into the target gene by homologous
recombination; (2) in which, or between which are located one or
more mutations to be introduced into a target gene; and (3) which
flank a positive selectable marker.
[0043] As used herein, "homologous recombination" means
rearrangement of DNA segments, at a sequence-specific site (or
sites), within or between DNA molecules, through base-pairing
mechanisms.
[0044] As used herein, "human mutation in the non-human mammalian
presenilin 1 (PS-1) FAD gene" means any mutation of the PS-1 gene
in a non-human mammal that results in the non-human mammal having a
nucleotide or nucleotides that correspond to the human PS-1 gene at
the corresponding position of the nucleotide or nucleotides. A
human mutation in the non-human mammalian presenilin 1 (PS-1) FAD
gene includes, but is not limited to, the following: A79V, V82L,
V96F, Y115C, E120D, E120K, M139I, M139T, M139V, I143F, I143T,
M146I, M146L (AT), H163Y, G209V, A231T, A231V, M233T, L235P, L250S,
A260V, L262F, C263R, P264L, P267S, R269H, R278T, E280A, E280G,
A285V, E318G, G378E, G384A, and L392V, each of which is disclosed
in Cruts et al., Human Mutat., 1998, 11, 183-190, which is
incorporated herein by reference in its entirety; M146L (AC) which
is disclosed in Cruts et al., Human Mutat., 1998, 11, 183-190, Duff
et al., Nature, 1996, 383, 710-713, Citron et al., Nature Med.,
1997, 3, 67-72, Lee et al., Nature Med., 1997, 3, 756-760, and Lamb
et al., Nature Neurosci., 1999, 2, 695-697, each of which is
incorporated herein by reference in its entirety; M146V, which is
disclosed in Cruts et al., Human Mutat., 1998, 11, 183-190, Duff et
al., Nature, 1996, 383, 710-713, and Guo et al., Nature Med., 1999,
5, 101-106, each of which is incorporated herein by reference in
its entirety; H163R, which is disclosed in Cruts et al., Human
Mutat., 1998, 11, 183-190, Lamb et al., Nature Neurosci., 1999, 2,
695-697, and Chui et al., Nature Med., 1999, 5, 560-564, each of
which is incorporated herein by reference in its entirety; I213T,
which is disclosed in Cruts et al., Human Mutat., 1998, 11, 183-190
and Nakano et al., Eur. J. Neurosci., 1999, 11, 2577-2581, each of
which is incorporated herein by reference in its entirety; L286V,
which is disclosed in Cruts et al., Human Mutat., 1998, 11,
183-190, Citron et al., Nature Med., 1997, 3, 67-72, and Chui et
al., Nature Med., 1999, 5, 560-564, each of which is incorporated
herein by reference in its entirety; A246E, which is disclosed in
Cruts et al., Human Mutat., 1998, 11, 183-190, Lee et al., Nature
Med., 1997, 3, 756-760, and Qian et al., Neuron, 1998, 20, 611-617,
each of which is incorporated herein by reference in its entirety;
Y115H, which is disclosed in Citron et al., Nature Med., 1997, 3,
67-72, which is incorporated herein by reference in its entirety;
T116N, which is disclosed in Romero et al., Neuroreport., 1999, 10,
2255-2260, which is incorporated herein by reference in its
entirety; P117L and L171P, both of which are disclosed in St.
George Hyslop, Biol. Psychiatr., 2000, 47, 183-199, which is
incorporated herein by reference in its entirety; E123L, which is
disclosed in Yasuda et al., Arch. Neurol., 1999, 56, 65-69, which
is incorporated herein by reference in its entirety; N135D, C410Y,
A426P and P436S, each of which is disclosed in Hardy et al., Trends
Neurosci., 1997, 20, 154-159, which is incorporated herein by
reference in its entirety; M139K, which is disclosed in Dumanchin
et al., J. Med. Genet., 1998, 35, 672-673, which is incorporated
herein by reference in its entirety; T147I, W165C, L173W, and
S390I, each of which is disclosed in Campion et al., Am. J. Human
Genet., 1999, 65, 664-670, which is incorporated herein by
reference in its entirety; L166R, which is disclosed in Ezquerra et
al., Arch. Neurol., 2000, 57, 485-488, which is incorporated herein
by reference in its entirety; S169L and P436Q, each of which is
disclosed in Taddei et al., Neuroreport., 1998, 9, 3335-3339, which
is incorporated herein by reference in its entirety; S169P, which
is disclosed in Ezquerra et al., Neurol., 1999, 52, 566-570, which
is incorporated herein by reference in its entirety; E184D, which
is disclosed in Yasuda et al., Neurosci. Lett., 1997, 232, 29-32,
which is incorporated herein by reference in its entirety; G209R,
which is disclosed in Sugiyarna et al., Online Human Mutat., 1999,
14, 90, which is incorporated herein by reference in its entirety;
L219P, which is disclosed in Smith et al., Neuroreport., 1999, 10,
503-507, which is incorporated herein by reference in its entirety;
M233L and A409T, both of which are disclosed in Aldudo et al.,
Human Mutat., 1999, 14, 433-439, which is incorporated herein by
reference in its entirety; E273A, which is disclosed in Kamimura et
al., J. Neurol. Sci., 1998, 160, 76-81, which is incorporated
herein by reference in its entirety; L282R, which is disclosed in
Aldudo et al., Neurosci. Lett., 1998, 240, 174-176, which is
incorporated herein by reference in its entirety; G378A, which is
disclosed in Besancon et al., Human Mutat., 1998, 11, 481, which is
incorporated herein by reference in its entirety; N405S, which is
disclosed in Yasuda et al., J. Neurol. Neurosurg. Psychiatr., 2000,
68, 220-223, which is incorporated herein by reference in its
entirety; A409T, which is disclosed in Sugiyama et al., Online
Human Mutat., 1999, 14, 90, which is incorporated herein by
reference in its entirety; L424R, which is disclosed in Kowalska et
al., Folia Neuropath., 1999, 37, 57-61, which is incorporated
herein by reference in its entirety; a Delta exon 9 splice acceptor
site deletion mutation (GT with S290C), which is disclosed in Hardy
et al., Trends Neurosci., 1997, 20, 154-159 and Lee et al., Nature
Med., 1997, 3, 756-760, each of which is incorporated herein by
reference in its entirety; a Delta exon 9 splice acceptor site
deletion mutation (GA with S290C), which is disclosed in Sato et
al., Human Mutat. Supp., 1998, 1, S91-94, which is incorporated
herein by reference in its entirety; a Delta exon 9 Finn 4,555
basepair deletion, which is disclosed in Prihar et al., Nature
Med., 1999, 5, 1090, which is incorporated herein by reference in
its entirety; a Delta intron 4 splice donor consensus sequence G
deletion, which is disclosed in DeJonghe et al., Human Molec.
Genet., 1999, 8, 1529-1540, which is incorporated herein by
reference in its entirety; and a CT mutation at position -48 in the
5' promoter, a CG mutation at position -280 in the 5' promoter, and
an AG mutation at position -2818 in the 5' promoter, each of which
is disclosed in Theuns et al., Human Molec. Genet., 2000, 9,
325-331, which is incorporated herein by reference in its entirety.
Although the application exemplifies the P264L mutation in
particular, all aspects of the invention can be applied to each and
every human mutation recited above.
[0045] As used herein, "human P264L mutation" means the following:
the nucleotide sequence of codon 264 of the presenilin 1 gene is
changed from CCG to a sequence selected from the group consisting
of: CTT; CTC; CTA; CTG; TTA; TTG; and most preferably changed from
CCG to CTT. Additionally, the nucleotide sequence of codon 265 of
the presenilin 1 gene optionally, but preferably, is changed from
AAA to AAG. The above described most preferable change of base
sequences in codon 264 constitute the human P264L mutation. The
optional, but preferred, change of the base sequence of codon 265
adds an AflII cleavage site to the gene.
[0046] As used herein, "target gene" or "targeted gene" means a
gene in a cell, which gene is to be modified by homologous
recombination with a targeting vector.
[0047] As used herein, "gene-targeted, non-human mammal" means a
non-human mammal comprising one or more targeted genes via a
gene-targeting, as opposed to transgenic, approach.
[0048] As used herein, "generational offspring" in relationship to
"gene-targeted, non-human mammal" means an animal whose genome
includes the same gene-targeted manipulation as the parent(s) of
that offspring. For example, and not limitation, where a mammal
whose genome has been manipulated by gene-targeting techniques to
include a human mutation is then used for breeding purposes, all
subsequent generations derived from that first mammal(s) are
considered "generational offspring" so long as the genome(s) of
such subsequent generational offspring comprises the gene-targeted
manipulation as the original mammal; by design, this definition
does not exclude other genomic-manipulations which may also be
present in such generational offspring, nor does this definition
require that such generational offspring be derived solely by
cross-breeding techniques between a male and female mammal.
[0049] As used herein, "transgenic non-human mammal" means a
non-human mammal in which a foreign ("transgene") gene sequence has
been inserted randomly in a non-human mammal's genome and is
therefore expressed in addition to all normally expressed native
genes (unless the inserted transgene has interrupted a gene thus
preventing its expression).
[0050] As used herein, "targeting vector" or "replacement vector"
means a DNA molecule that includes arms of homology, the nucleotide
sequence (located within or between the arms of homology) to be
incorporated into the target gene, and one or more selectable
markers.
[0051] As used herein, "wild-type control animal" means a
non-gene-targeted, non-human mammal of the same species as, and
otherwise comparable to (e.g., similar age), a gene-targeted
non-human mammal as disclosed herein. A wild-type control animal
can be used as the basis for comparison, in assessing results
associated with a particular genotype.
[0052] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
[0053] The first step in producing a gene-targeted non-human mammal
of this invention is to prepare a DNA targeting vector. The
targeting vector is designed to replace, via homologous
recombination, part of the endogenous presenilin 1 gene sequence of
a non-human mammal, so as to introduce the human mutation,
preferably the P264L human mutation. The targeting vector is used
to transfect a non-human mammalian cell, e.g., a pluripotent,
murine embryo-derived stem ("ES") cell. In this cell, homologous
recombination (i.e., the gene-targeting event) takes place between
the targeting vector and the target gene. The mutant cell is then
used to produce intact non-human mammals (e.g., by aggregation of
murine ES cells to mouse embryos) to generate germ-line chimeras.
The germline chimeras are used to produce siblings heterozygous for
the mutated targeted gene. Finally, interbreeding of heterozygous
siblings yields non-human mammals (e.g., mice) homozygous for the
mutated target gene.
[0054] Targeting vectors for the practice of this invention can be
constructed using materials, information and processes known in the
art. A general description of the targeting vector used in this
invention follows.
[0055] A targeting vector or replacement vector for use in this
invention has two essential functions: (1) to integrate
specifically (and stably) at the endogenous presenilin 1 target
gene; and (2) to replace a portion of an exon of the endogenous
presenilin 1 gene, thereby introducing the human mutation, and the
mutation that introduces a new cleavage site in the gene. In a
preferred embodiment, a portion of exon 8 is replaced so as to
introduce the P264L mutation. Those two essential functions depend
on two basic structural features of the targeting vector.
[0056] The first basic structural feature of the targeting vector
is a pair of regions, known as "arms of homology," which are
homologous to selected regions of the endogenous presenilin 1 gene
or regions flanking the presenilin 1 gene. This homology causes at
least part of the targeting vector to integrate into the
chromosome, replacing part (or all) of the presenilin 1 target
gene, by homologous recombination.
[0057] Homologous recombination, in general, is the rearrangement
of DNA segments, at a sequence-specific site (or sites), within or
between DNA molecules, through base-pairing mechanisms. The present
invention relates to a particular form of homologous recombination
sometimes known as "gene targeting."
[0058] The second basic structural feature of the targeting vector
consists of the actual base changes (mutation(s)) to be introduced
into the target gene. In the present invention, the base changes in
codon 264 of exon 8, for example, resulted in an amino acid change
in amino acid 264 from proline to leucine when the mutated gene was
expressed to make protein. Other base changes can be made, as
desired, to introduce any of the human mutations listed above into
the mammalian genome. The mutation(s) to be introduced into the
presenilin 1 target gene is located within the "arms of
homology."
[0059] Gene targeting, which affects the structure of a specific
gene already in a cell, is to be distinguished from other forms of
stable transformation, wherein integration of exogenous DNA for
expression in a transformed cell is not site-specific, and thus
does not predictably affect the structure of any particular gene
already in the transformed cell. Furthermore, with the type of
targeting vector preferred in the practice of this invention (e.g.,
the one disclosed below), a reciprocal exchange of genomic DNA
takes place (between the "arms of homology" and the target gene),
and chromosomal insertion of the entire vector is advantageously
avoided.
[0060] The examples of this patent disclosure set forth the
construction of a presenilin 1 gene targeting vector (and its use)
to mutate the murine presenilin 1 protein encoding sequence so that
it encodes the murine presenilin 1 protein, containing the human
P264L mutation, or any of the other human mutations recited above,
and an additional cleavage site. One of ordinary skill in the art
will recognize that numerous other targeting vectors can be
designed to introduce the same mutations, using the principles of
homologous recombination. Gene-targeted, non-human- mammals
produced using such other targeting vectors are within the scope of
the present invention. A discussion of targeting vector
considerations follows.
[0061] The length of the arms of homology that flank the
replacement sequence can vary considerably without significant
effect on the practice of the invention. The arms of homology must
be of sufficient length for effective heteroduplex formation
between one strand of the targeting vector and one strand of a
transfected cell's chromosome, at the presenilin 1 target gene
locus. Increasing the length of the arms of homology promotes
heteroduplex formation and thus targeting efficiency. However, it
will be appreciated that the incremental targeting efficiency
accruing per additional homologous base pair eventually diminishes
and is offset by practical difficulties in target vector
construction, as arms of homology exceed several thousand base
pairs. A preferred length for each arm of homology is 50 to 10,000
base pairs.
[0062] There is considerable latitude in the choice of which
regions of the presenilin 1 target gene, i.e., chromosomal regions
flanking the presenilin 1 target gene, are represented in the
targeting vector's arms of homology. The basic constraint is that
the base pairs to be changed in the presenilin 1 target gene must
lie within the sequence that constitutes the arms of homology. The
arms of homology may lie within the presenilin 1 target gene, but
it is not necessary that they do so and they may flank the
presenilin 1 target gene.
[0063] Preferably, the targeting vector will comprise, between the
arms of homology, a positive selection marker. The positive
selection marker should be placed within an intron of the target
gene, so that it will be spliced out of mRNA and avoid the
expression of a target/marker fusion protein. More preferably the
targeting vector will comprise two selection markers; a positive
selection marker, located between the arms of homology, and a
negative selection marker, located outside the arms of homology.
The negative selection marker is a means of identifying and
eliminating clones in which the targeting vector has been
integrated into the genome by random insertion instead of by
homologous recombination. Exemplary positive selection markers are
neomycin phosphotransferase and hygromycin .beta.
phosphotransferase genes. Exemplary negative selection markers are
Herpes simplex thymidine kinase and diphtheria toxin genes.
[0064] To eliminate potential interference on expression of the
target protein, the positive selection marker can be flanked by
short loxP recombination sites isolated from bacteriophage P1 DNA.
Recombination between the two loxP sites at the targeted gene locus
can be induced by introduction of cre recombinase to the cells.
This results in the elimination of the positive selection marker,
leaving only one of the two short loxP sites. (See, U.S. Pat. No.
4,959,317, which is herein incorporated by reference in its
entirety). Excision of the positive selectable marker from intron 8
of the mutated presenilin 1 gene can thus be effected.
[0065] FIG. 1 illustrates the general principles of gene-targeting
for introducing mutations into a mammalian genome using homologous
recombination (reviewed in Capecchi, M. R, Trends Genet., 1989, 5,
70-76; Koller and Smithies, Ann. Rec. Immunol:, 1992, 10, 705-730).
A length of genomic DNA is first depicted by organizing it into
regions (numbered 0-5 in FIG. 1a). In FIG. 1, several base pair
changes (from 1 -10) are to be incorporated into the cellular DNA
around region 3. Homologous recombination using a gene targeting
vector is utilized. The type of gene targeting vector used to
incorporate these changes is termed a replacement vector.
[0066] As defined previously, a "replacement vector" herein refers
to a vector that includes one or more selectable marker sequences
and two contiguous sequences of ES cell genomic DNA that flank a
selectable marker. These flanking sequences are termed "arms of
homology." In FIG. 1b, the arms of homology are represented by
regions 1-2 and 3-4. The use of DNA derived from the ES cells
(isogenic DNA) helps assure high efficiency recombination with the
target sequences (te Riele et al., Proc. Natl. Acad. Sci. USA,
1992, 89, 5128-5132). The arms of homology are placed in the vector
on either side of a DNA sequence encoding resistance to a drug
toxic to the ES cells (positive selection marker). A gene encoding
susceptibility to an otherwise nontoxic drug (negative selection
marker) is placed outside the region of homology. In the
replacement vector used in this invention, the positive selection
marker is neo.sup.r, a gene that encodes resistance to the neomycin
analog G418, and the negative selection marker is the herpes
simplex virus thymidine kinase gene (HSV-tk) that encodes
susceptibility to gancyclovir. When this replacement vector is
introduced into ES cells via transfection and its DNA undergoes
homologous recombination with ES cellular DNA, the positive
selection marker is inserted into the genome between regions 2 and
3 in this example (making the transformed cells resistant to G418)
while the negative selection markers is excluded (making the cells
resistant to gancyclovir). Thus, to enrich for homologous
recombinants, transfected ES cells are grown in culture medium
containing G418 to select for the presence of the neo.sup.r gene
and gancyclovir to select for the absence of the HSV-TK gene.
Preferably, the positive selection marker is eliminated by using,
for example, cre/lox technology once the mammal is crossed with
another mammal.
[0067] If base pair changes (mutations) are introduced into one of
the arms of homology it is possible for these changes to be
incorporated into the cellular gene as a result of homologous
recombination. Whether or not the mutations are incorporated into
cellular DNA as a result of homologous recombination depends on
where the crossover event takes place in the arm of homology
bearing the changes. For example, as depicted by scenario "A" in
FIG. 1, the crossover in the arm occurs proximal to the mutations
and so they are not incorporated into cellular DNA. In scenario
"B", the crossover takes place distal to the position of the
mutations and they are incorporated into cellular DNA. Because the
location of the crossover event is random, the frequency of
homologous recombination events that include the mutations is
increased if they are placed closer to the positive selection
marker.
[0068] By the above method, the skilled artisan can achieve the
incorporation of the selectable marker at a preselected location in
the gene of interest flanked by specific base pair changes.
Presumably, the artisan would preferably choose to have the
selectable marker incorporated within the intron of the gene of
interest so as not to interfere with endogenous gene expression
while the mutations would be included in adjacent coding sequence
so as to make desired changes in the protein product of interest
(FIG. 1), (Askew et al., Mol. Cell. Biol., 1993, 13, 4115-4124,
Fiering et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 8469-8473;
Rubinstein et al., Nuc. Acid Res., 1993, 21, 2613-2617, Gu et al.,
Cell, 1993, 73, 1155-1164, and Gu et al., Science, 1994, 265,
103-106).
[0069] Thus, in the manner described above, a gene-targeted,
non-human mammal comprising a human PS-1 mutation is prepared. The
mammal can be heterozygous (contains one copy of the human PS-1
mutation) or homozygous (contains two copies of the human PS-1
mutation). In a preferred embodiment, a mouse is prepared which is
PS-1.sup.P264L/+ (heterozygous) or PS-1.sup.P264L/P264L
(homozygous).
[0070] The gene-targeted, non-human mammals comprising a human PS-1
mutation described above can be crossed with mammals having a
Swedish FAD mutation and "humanized" A.beta. sequence in the APP
gene (e.g., APP.sup.NLh/NLh mouse) to produce mammals referred to
as APP.sup.Nlh/NLh.times.PS-1.sup.P264L/P264L,
APP.sup.NLh/+.times.PS-1.sup.- P264L/P264L,
APP.sup.NLh/+.times.PS-1.sup.P264L/+ or
APP.sup.NLh/NLh.times.PS-1.sup.P264L/+. In addition, the
gene-targeted, non-hurman mammals comprising a human PS-1 mutation
described above can be crossed-with mammals having a Swedish APP695
mutation (e.g., Tg2576 mouse). Prior to crossing such mammals,
however, it is preferred to remove the positive selection marker,
such as neo.sup.r, using cre/lox technology.
[0071] The present invention is also directed to a method for
identifiing a compound for treating Alzheimer's disease. A compound
is administered to a mammal that is heterozygous or homozygous for
a mutation of the PS-1 gene and also contains a human Swedish
APP695 mutation, or generational offspring thereof, or to a mammal
heterozygous or homozygous for a mutation of the PS-1 gene, a human
FAD Swedish mutation, and a humanized A.beta. mutation, and
generational offspring thereof. Any compound to be tested can be
administered in a variety of amounts by any variety of routes
including, but not limited to, intravenously, orally, direct
injection in the brain, and the like. A tissue sample from the
mammal including, but not limited to, brain tissue, non-brain
tissue and body fluids (e.g. blood and plasma) is obtained and the
amount of A.beta. peptide in the tissue sample is measured. A
decrease in the amount of A.beta. peptide in the tissue sample is
indicative of a compound that can be used to treat Alzheimer's
disease.
[0072] The present invention is also directed to a method of
treating an individual suspected of having Alzheimer's disease. An
individual suspected of having Alzheimer's disease is any human
having been examined by a physician and diagnosed as having
Alzheimer's disease or symptoms thereof. A compound identified by
the methods described above relating to a mammal that is
heterozygous or homozygous for a mutation of the PS-1 gene and also
contains a human Swedish APP695 mutation, or generational offspring
thereof, or to a mammal that is heterozygous or homozygous for a
mutation of the PS-1 gene, a human FAD Swedish mutation, and a
humanized A.beta. mutation, and generational offspring thereof, is
administered to the individual in an amount effective to decrease
the amount of A.beta. peptide in the brain of the individual. An
amount effective to decrease the amount of A.beta. peptide can be
determined from the identification process of the compound using a
mammal that is heterozygous or homozygous for a mutation of the
PS-1 gene and also contains a human Swedish APP695 mutation, or
generational offspring thereof, or using a mammal that is
heterozygous or homozygous for a mutation of the PS-1 gene, a human
FAD Swedish mutation, and a humanized A.beta. mutation, or
generational offspring thereof, as a starting amount and scaling up
for use in humans as is well known to those skilled in the art. An
effective Alzheimer's disease treatment amount is an amount of a
compound that measurably reduces the physiological pathology of
Alzheimer's disease or an amount that reduces the physical
manifestations or symptoms of Alzheimer's disease. One skilled in
the art can, for example, begin with an amount of a compound that
decreases the amount of A.beta. peptide in the brain, as described
above, and can scale up or down the amount depending on the desired
effect and the effect achieved in a particular individual.
[0073] The present invention is also directed to compounds that are
identified by the screening methods described above. The compounds
can be any identifiable chemical or molecule, including, but not
limited to, a small molecule, a peptide, a protein, a sugar, a
nucleotide, or a nucleic acid, and such compound can be natural or
synthetic.
[0074] In order that the invention disclosed herein may be more
efficiently understood, examples are provided below. It should be
understood that these examples are for illustrative purposes only
and are not to be construed as limiting to the invention in any
manner. Throughout these examples, molecular cloning reactions, and
other standard recombinant DNA techniques, were carried out
according to methods described in Maniatis et al., Molecular
Cloning-A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Press
(1989) (hereinafter, "Maniatis et al."), using commercially
available enzymes, except where otherwise noted.
EXAMPLES
Example 1
Cloning of Mouse PS-1 Exon 8 Region
[0075] The mouse PS-1 genomic DNA was cloned from a bacteriophage
library created from 129/Sv mouse DNA partially digested with Sau3A
and into the BamHI site of Lambda DASH.TM. II (Reaume et al.,
Science, 1995, 267, 1831-1833, which is incorporated herein by
reference in its entirety). Using standard molecular biology
techniques (Maniatis et al.), approximately 1.2.times.10.sup.6
recombinant bacteriophages were screened for the presence of PS-1
sequences by hybridization with a small, radiolabeled PS-1 specific
DNA probe. This 477 base pair PS-1 probe was generated by
polymerase chain reaction (PCR) amplification (Mullis et al.,
Methods Enzymol., 1987, 155, 335-350) of mouse genomic DNA using
primers R892 (CTC ATC TTG GCT GTG ATT TCA; SEQ ID NO:1) and R885
(GTT GTG TTC CAG TCT CCA; SEQ ID NO:2) which hybridize to the 3'
end of exon 7 and the 5' end of exon 11 respectively (FIG. 2). The
amplified fragment was separated from other components of the
reaction by electrophoresis on a 1.0% agarose gel, and purified
using GeneClean.RTM.II (Bio 101, Inc., La Jolla, Calif.). Purified
probe DNA was radioactively labeled with .sup.32P-dCTP by the
random primer method using materials and methods supplied by the
kit manufacturer (Multiprime DNA Labeling System; Amersham Life
Sciences, Arlington Heights, Ill.).
[0076] From this screen, 13 clones were identified that hybridized
to the PS-1 probe. The clones were identified as: .lambda.PS1-4,
.lambda.PS1-5, .lambda.PS1-6, .lambda.PS1-10, .lambda.PS1-11,
.lambda.PS1-17, .lambda.PS1-19, .lambda.PS1-20, .lambda.PS1-22,
.lambda.PS1-24, .lambda.PS1-28, .lambda.PS1-31,and .lambda.PS1-35.
These clones were purified by limiting dilution and plaque
hybridization with the PS-1 probe (Maniatis et al.).
[0077] From each clone, DNA was prepared from bacteriophage
particles purified on a CsCl gradient (Maniatis et al.).
Restriction maps were then generated for each of the cloned inserts
using the FLASH.TM. Non-radioactive Gene Mapping Kit
(Stratagene.RTM. Inc., La Jolla, Calif.). A typical restriction map
generated by this method is illustrated in FIG. 3. This method of
restriction enzyme mapping involves first completely digesting 10
.mu.g of the bacteriophage DNA with the restriction enzyme Notl
using standard restriction enzyme digest conditions (Maniatis et
aL) . Notl cuts all clones in the vector DNA at either end of the
cloned insert so as to leave a T3 bacteriophage promoter attached
to one end of the insert and a T7 bacteriophage promoter attached
to the other end. The Notl digested DNA is then partially digested
with the enzyme EcoRl, as an example, using limiting amounts of
enzyme (0.2 units/.mu.g DNA) in an 84 .mu.l reaction volume at
37.degree. C. Aliquots (26 .mu.l) were removed after 3 minutes, 12
minutes and 40 minutes and the digest reaction was stopped by the
addition of 1 .mu.l of 0.5 M EDTA. DNA from all three time points
was resolved on a 0.7% agarose gel, visualized by ethidium bromide
staining, and then transferred to a GeneScreen Plus.RTM. membrane
(NEN.RTM. Research Products, Boston, Mass.) by capillary transfer
(Maniatis et al., supra). The membrane was hybridized with an
alkaline phosphatase labeled oligonucleotide that was specific for
the T3 promoter (supplied with the FLASH.TM. kit) using reagents
and methods supplied by the kit manufacturer. After hybridization,
the membrane was washed and developed with a
chemiluminescent-yielding substrate and then exposed to X-ray film
in the dark for approximately 60 minutes.
[0078] The oligonucleotide probes effectively label one end of the
insert. By determining the positions of the bands on the X-ray film
and calculating the DNA size to which they corresponded, it was
possible to determine the position of the EcoRl sites relative to
the T3 end of the insert (FIG. 3). These results could then be
complemented by stripping the probe off of the membrane, and
rehybridizing with a T7-specific oligonucleotide in order to
determine the positions of the EcoRl sites relative to the T7 end
of the insert. This process was repeated using the enzymes HindIII
and XbaI. By comparing the restriction enzyme maps of the different
overlapping clones a composite map was assembled. Of the 13
original clones isolated, 6 independent clones were identified
(FIG. 2).
[0079] Exon 8 was located on the restriction map hybridizing
exon-specific probes to complete digests of each of the 6 different
lambda genomic clones. Initially, 3 .mu.g of DNA from each of the 6
different clones was completely digested with the restriction
enzymes EcoRl and Xbal. The digested DNA was resolved on a 0.8%
agarose gel, visualized by means of ethidium bromide staining and
transferred to a GeneScreen Plus.RTM. membrane by capillary
transfer. The membrane was then hybridized with a DNA probe that
specifically hybridized to sequences from mouse PS-1 exon 8. This
probe was generated by PCR using oligonucleotides FEX8 (ATT TAG TGG
CTG TTT TGT G; SEQ ID NO:3) and REX8 (AGG AGT AAA TGA GAG CTG GA;
SEQ ID NO:4) which hybridize to the 5' and 3' ends of exon 8,
respectively. After hybridization, the membrane was washed and
exposed to X-ray film (FIG. 4). This experiment revealed that all
clones contained a 2.5 kb fragment that hybridized to the exon 8
probe. By combining this information with the restriction map data
for each lambda clone, exon 8 was identified on the map (position
11.5 to 14 on the summary map, FIG. 2).
[0080] A similar procedure was used to identify the position of
exon 7 on our composite map using exon 7-specific probe and
utilizing the restriction enzymes Xbal and EcoRl. The exon
7-specific probe was generated using PCR primers F892 (TGA AAT CAC
AGC CAA GAT GAG; SEQ ID NO:5) and PS1-1 (GCA CTC CTG ATC TGG AAT
TTT G; SEQ ID NO:6). Exon 7 was localized to the 2 kb Xbal-EcoRl
fragment of all clones except .lambda.PS1-22 which allowed for the
determination that exon 7 is located between positions 7.0 and 9.0
on the summary map (FIG. 2).
[0081] Exon-specific probes were also used to obtain additional
restriction map information using additional restriction enzymes.
For example, when .lambda.PS1-22 was digested with Notl and BamHI,
resolved on an agarose gel, transferred to a Genescreen Plus.RTM.
membrane and probed with the exon 8-specific probe, a 700 bp
fragment was identified. This information, when combined with the
information from the other bacteriophage clones, allowed placement
of the BamHI at position 11.7 on the composite map (FIG. 2). This
process was repeated for the restriction enzyme Xhol.
[0082] Cloning of additional regions of the mouse PS-1 gene can
also be accomplished, as desired, in order to prepare additional
vectors comprising other human mutations.
Example 2
Construction of a Replacement Vector
[0083] A 4.7 kb XbaI-BamHI fragment (which also contains two
internal XbaI fragments) located at positions 7.0 to 11.7 on the
summary map (FIG. 2), was chosen as the 5' arm that included the
necessary mutations and a 4.1 kb BamHI-EcoRI fragment (which also
contains an internal EcoRI site) located at positions 11.7 to 15.8
on the summary map (FIG. 2), as a 3' arm. These fragments were
isolated first and cloned into pBlueScript.RTM. SK+ (Stratagene
Cloning Systems, LaJolla, Calif.) and then further subcloned into
the plasmid pPNTIox.sup.2 (described below) that contained a
neo.sup.r gene, an HSV-TK gene and linker sequences to produce a
replacement vector (pPS1-8-TV, FIG. 5) with the same general
structure as that discussed above.
[0084] (a) Construction of the Intermediate Plasmid
pPNTlox.sup.2
[0085] pPSI-8-TV was created from pPNT (Tybulewicz et al., Cell,
1991, 65, 1153-1163; obtained from Dr. Richard Mulligan, MIT) by
first inserting two oligonucleotide linkers on each side of the
neo.sup.r cassette creating the intermediate called pPNTIox.sup.2
(FIG. 6). A double-stranded 79 base pair 5' linker was created by
annealing two single-stranded oligonucleotides that overlap at
their 3' ends and then filling in the remaining single-stranded
regions with the Klenow fragment of DNA polymerase I. The
oligonucleotides PNT Not (GGA AAG AAT GCG GCC GCT GTC GAC GTT AAC
ATG CAT ATA ACT TCG TAT; SEQ ID NO:7) and PNT Xho (GCT CTC GAG ATA
ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA TAT GC; SEQ ID NO:8) (150
ng of each) were combined in a 30 .mu.l reaction mixture containing
5 U of Klenow polymerase, Klenow polymerase buffer and 2 mM dNTPs
(dATP, dCTP, dGTP, and dTTP). After incubating for 1 hour at
37.degree. C., a portion (5 .mu.l) of this reaction mixture was
simultaneously digested with the restriction enzymes NotI and XhoI
to liberate the restriction enzyme sites at each end of the linker.
In addition, 200 ng of pPNT was digested with NotI and XhoI. The
digested plasmid was resolved on a 0.8% agarose gel, purified from
the gel, and treated with calf intestinal phosphatase according to
standard methods (Maniatis et al.). A quantity (66 ng) of the
double digested linker was ligated to the double-digested and
phosphatase-treated pPNT DNA (Maniatis et al.). Following DNA
transformation of competent WM 1100 E. coli (Dower, Nucleic Acids
Res., 1988, 16, 6127-6145), plasmid DNA was isolated from
ampicillin-resistant bacteria (Holmes et al., Anal. Biochem., 1981,
114, 193-197) and analyzed by restriction enzyme analysis. The
proper recombinant plasmids were identified as having acquired
SalI, HpaI and NsiI sites (present in the linker) while still
retaining the NotI and XhoI sites of the starting plasmid. One such
recombinant plasmid with a 79 bp linker sequence was identified and
called pXN-4 (FIG. 6).
[0086] A similar approach was used to insert a 3' linker between
the XbaI and BamHI sites of pXN-4. The oligonucleotides used to
synthesize the linker were PNT Xba (CGT TCT AGA ATA ACT TCG TAT AAT
GTA TGC TAT; SEQ ID NO:9) and PNT Bam (CGT GGA TCC ATA ACT TCG TAT
AGC ATA CAT TAT; SEQ ID NO:10). In this case, pXN-4 and the
double-stranded linker DNA were digested with XbaI and BamHI. The
purified fragments were joined by DNA ligation and transformed into
competent WM1100 bacteria. Plasmid DNA was digested with XbaI and
BamHI, end-labeled with .sup.32P-dCTP and Klenow polymerase, and
resolved on an 8% acrylamide gel (Maniatis et al.). The gel was
dried and exposed to X-ray film. Proper recombinant clones were
identified by the presence of a 40 base pair band liberated by the
XbaI-BamHI double digest. The resulting plasmid was designated
"pPNTIox.sup.2" (FIG. 6).
[0087] To confirm the sequences of the inserted linkers, a fragment
containing both linkers was isolated from pPNTIox.sup.2 using NotI
and EcoRI and cloned into pBlueScript.RTM. SK+, a vector that was
more amenable to nucleotide sequencing. Identity of the linkers was
confirmed by direct nucleotide sequencing (Sanger, Proc. Natl.
Acad. Sci. USA, 1977, 74, 5463-5467) using T3 and T7 sequencing
primers (Stratagene.RTM. Inc., La Jolla, Calif.) and Sequenase
Version 2.0 DNA Sequencing Kit (United States Biochemical,
Cleveland, Ohio).
[0088] (b) Subcloning Arms of Homology.
[0089] An XbaI-HindIII fragment (positions 11.5 to 15.9 on the
summary map, FIG. 2) containing the 3' arm of homology and the
fragment used for in vitro mutagenesis was first isolated from
.lambda.PS1-22 by digesting 30 .mu.l of the phage DNA with XbaI and
HindIII, resolving the digested DNA on a 0.8 agarose gel,
visualizing the DNA with ethidium bromide staining and then
excising the 4.4 kb fragment from the gel. DNA was purified from
the gel using GeneClean.RTM. II (Bio101 Inc., La Jolla, Calif.).
Simultaneously, 1 .mu.g of pBlueScript.RTM. SK+ was digested with
XbaI and HindIII and subsequently purified by the same procedure.
Approximately 400 ng of the purified lambda DNA and 100 ng of the
purified plasmid DNA were combined in a 10 .mu.l ligation reaction.
Following taansformation of competent WM100 E. coli, plasmid DNA
was isolated from ampicillin-resistant bacteria and analyzed by
restriction enzyme analysis to identify the resultant plasmids
(FIG. 7). In this case, plasmid DNA from transformed bacteria was
first analyzed by digesting it with XbaI and HindIII in order to
determine whether the plasmid DNA had acquired the 4.4 kb PS-1
fragment. This plasmid was designated "pPS I-XH16."
[0090] Similar procedures were used to isolate a 200 bp XbaI-BamHI
fragment from pPS1-XH16 and subclone it into pBlueScript.RTM. SK+.
The resulting plasmid was designated "pPS1-XB1" (FIG. 8).
[0091] One of the fragments in the 5' arm of homology (a 4.2 kb
XbaI fragment at positions 7.0 to 11.2 on summary map; FIG. 2) was
similarly subcloned from .lambda.PSI-6 into pBlueScript.RTM. SK+
and designated "pPS1-X15" (FIG. 9). Because this insert could be
positioned in the plasmid in either of two orientations, plasmid
DNA was further screened by digesting it with the enzyme EcoRI. In
this way, it was determined that the clone pPS1-X15 had the PS-1
insert oriented such that the 5' end was closest to the T3 promoter
while in pPS1-X2 the 5' end was adjacent to the T7 promoter (FIG.
9).
[0092] The 300 bp XbaI fragment in the 5' arm (position 11.2 to
11.5 on summary map; FIG. 2) was also similarly cloned into
pBlueScript.RTM. SK+ from .lambda.PS1-20 and named pPS1-X319 (FIG.
10). In this case, the orientation of the XbaI fragment was not
determined by subsequent restriction mapping.
[0093] (c) Restriction Mapping Arms of Homology.
[0094] Further restriction enzyme mapping was performed on the
pPS1-X315 and pPS1-X2 As an example, each of the two plasmids were
digested with the enzyme HincII, resolved on an agarose gel, and
visualized with ethidium bromide. Because a HincII site is known to
exist in the pBlueScrip.RTM. SK+ plasmid backbone within the
multiple cloning site region near the T7 promoter relative to the
insert position, it was possible to determine the position of the
HincII site in the 4.2 PS-1 fragment by determining the fragment
sizes in each of the two digested samples (FIG. 11).
[0095] Positions of restriction enzymes sites that occurred once or
twice in the 4.2 kb PS-1 fragment were determined by the above
method. If more than two sites of a given enzyme were present, it
became necessary to determine the relative positions by
double-digesting each of the two plasmids with the enzyme in
question as well as an additional enzyme which cut at sites capable
of resolving ambiguities. In many cases, enzymes that cut more than
twice were not resolved in this way but simply noted as having
multiples sites in the 4.2 kb PS-1 fragment. The list of additional
enzymes used to characterize this region include, but are not
limited to, AccI, ApaI, BamHI, EcoRI, HincII, HpaI, KpnI, NsiI,
PstI, SaII, SmaI, SpeI, and XhoI. A summary of these data is
illustrated in FIG. 12. The same procedures were used to create a
restriction enzyme map for the pPS1-XH16 (FIG. 12).
[0096] (d) Mutagenesis of the 3' arm of homology.
[0097] A total of 3 base pair changes were introduced into the exon
8 region using a PCR strategy (for summary of changes, see FIG.
13). The P264L mutation, and an AflII site were introduced. Teri ng
of pPS1-XB1 were included into each of two PCR reactions. The first
reaction contained the primers EXPL2 (TTG TGT CTT AAG GGT CCG CTT
CGT ATG; SEQ ID NO:1 1) and T7 (Stratagene Cloning Systems, La
Jolla, Calif.). This generated a 220 bp band that encompassed the
3' end of exon 8 and clone PS1-XB1. This fragment also included the
P264L mutation and a novel AflII site that resulted as part of the
P264L change.
[0098] The second PCR reaction used the primers EXPL1 (CGG ACC CTT
AAG ACA CAA AAC AGC CAC; SEQ ID NO:12) and T3 (Stratagene Cloning
Systems, La Jolla, Calif.). This generated a 137 bp fragment that
encompassed the 5' end of exon 8 and PS1-XB1. This fragment also
included the P264L change and an AflII site (FIG. 14).
[0099] The product of the first reaction was purified using
Magic.TM. PCR Preps DNA Purification System (Promega Corporation,
Madison, Wis.) and digested with BamHI and AflII in order to
liberate the restriction sites at its ends. Similarly, the product
of the second reaction was purified and digested with AflII and
XbaI. These two fragments, as well as XbaI and BamHI digested
pBlueScript.RTM. SK+ were ligated together and transformed into
HB101 competent E. coli cells. The DNA was isolated and analyzed
from the ampicillin resistant colonies. The clone bearing a
recombinant plasmid in which the two PCR fragments had joined
together at their AflII site and inserted into the BamHI and XbaI
sites of pBlueScript.RTM. SK+ was called pPS1-XB85 (FIG. 14). To
confirm the sequences of the mutagenized exon 8, direct nucleotide
sequencing (Sanger, Proc. Natl. Acad Sci. USA, 1977, 74, 5463-5467)
was performed using T3 and T7 sequencing primers (Stratagene Inc.,
LaJolla, Calif.) and Sequenase Version 2.0 DNA Sequencing Kit
(United States Biochemical, Cleveland, Ohio).
[0100] The 5' arm of homology was assembled in pBlueScript.RTM. SK+
through several cloning steps. First, pPS1-XB15 was partially
digested with XbaI so that only one XbaI site was cut. The
resulting DNA was then digested with BamHI and gel purified (FIG.
15).
[0101] The mutated insert in pPS1-XB85 was released by digesting it
with XbaI and BamHI and gel purifying the resulting mutated insert.
The 200 bp XbaI-BamHI fragment was ligated into the linearized
pPS1-X15 and recombinant plasmids were screened for the proper
orientation of the insert by means of an AflII digest. The
correctly oriented plasmid yielded 1.9 kb and 5.8 kb fragments.
This plasmid was designated "pPS1-206."
[0102] To insert the small 300 bp XbaI fragment 5' relative to the
mutated 200 bp XbaI-BamHI fragment, pPS1-206 was linearized by a
partial XbaI digest (FIG. 16). The XbaI fragment from pPS1-X319 was
isolated and cloned into the linearized pPS1-206 DNA. Orientation
of the 300 bp XbaI fragment was determined by sequencing the
recombinant clone as well as .lambda.PS1-20 with primer EX8PL1
using the Thermo Sequenase radiolabeled terminator cycle sequencing
kit (Amersham Life Science Inc., Cleveland, Ohio). A plasmid clone
that shared sequence identity with .lambda.PS1-20 beyond the XbaI
site had the 300 bp XbaI fragment inserted in the proper
orientation. This plasmid, which contained the assembled 5' arm,
was designated "pPS1 -5'360" (FIG. 16).
[0103] (e) Assembling the Targeting Vector pPS-1-8-TV.
[0104] The plasmid pPNTlox.sup.2 was prepared for receiving the 3'
arm of homology by first digesting plasmid DNA with EcoRI and BamHI
and gel isolating the linear plasmid (FIG. 17). In parallel, the 3'
arm was prepared by partially digesting pPS1-XH16 with EcoRI and
isolating the linear form. This fragment was then digested with
BamHI and the 4.1 bp fragment gel isolated. The 3' arm was ligated
to pPNTlox.sup.2. The resulting plasmid was designated
"pPNT3'413."
[0105] The 5' arm was inserted into pPNT3'413 to give the final
plasmid pPSI-8-TV. The 5' arm was liberated from plasmid DNA by
first digesting with XhoI and NotI. In parallel, pPNT3'413 was
prepared by double digesting with Noti and SalI The two fragments
of DNA were ligated and transformed into competent WM 1100 E. coli
cells (FIG. 18).
[0106] Additional vectors can be prepared in the manner described
above in order to comprise other human mutations.
Example 3
Mutagenesis of the Mouse PS-1 Gene in ES cells
[0107] (a) Cells.
[0108] The R1 line of ES cells derived from 129/Sv.times.129/Sv-CP
FI hybrid mice (Nagy et al., Proc. Natl. Acad. Sci. USA, 1993, 90,
8424-8429) and obtained from Dr. Janet Rossant (Mt. Sinai Hospital,
Toronto, Ontario, Canada) was utilized. These cells were grown in
ES cell medium consisting of Dulbecco's Modification of Eagle's
Medium (with L-glutamine and 4500 mg/L glucose; Mediatech Inc.,
Herndon, Va.) supplemented with 20% fetal bovine serum (FBS;
Hyclone Laboratories Inc., Logan, Utah; cat. # A-1115; Lot #
11152154), 0.1 mM non-essential amino acids (Mediatech 25-025-L1),
2 mM L-glutamine (Mediatech 25-005-L1), 10.sup.-6 M
.beta.-mercaptoethanol (Gibco 21985-023) 1 mM sodium pyruvate
(Mediatech 25-000-L1), 1.times. concentration of a penicillin
streptomycin solution (Mediatech 30-001-L1) and 1000 U/ml of
leukemia inhibitory factor (Gibco BRL 13275-029). The cells were
grown on tissue culture plastic that had been briefly treated with
a solution of 0.1% gelatin (Sigma G9391).
[0109] The cultures were passed every 48 hours or when the cells
became about 80% confluent. For passage, the cells were first
washed with phosphate buffered saline (without Ca.sup.2+ and
Mg.sup.2+) and then treated with a trypsin/EDTA solution (0.05%
trypsin, 0.02% EDTA in PBS without Ca.sup.2+ and Mg.sup.2+). After
all of the cells were in suspension, the trypsin digestion was
stopped by the addition of tissue culture medium. The cells were
collected by centrifugation, resuspended in 5 ml of tissue culture
medium and a 1 ml aliquot of the cell suspension was used to start
a new plate of the same size.
[0110] (b) DNA Transfection of ES Cells.
[0111] pPS1-8-TV DNA (400 .mu.g) was prepared for electroporation
by digesting it with NotI in a 1 ml reaction volume. The DNA was
then precipitated by the addition of ethanol, washed with 70%
ethanol and resuspended in 500 .mu.l of sterile water.
[0112] The NotI-linearized pPS1-8-TV DNA was electroporated into ES
cells using a Bio-Rad Gene Pulser.RTM. System (Bio-Rad
Laboratories, Hercules, Calif.). In each of 10 electroporation
cuvettes, 40 .mu.g of DNA was electroporated into
2.5.times.10.sup.6 cells suspended in ES cell culture medium. The
electroporation conditions were 250 V and 500 .mu.F which typically
result in time constants ranging between 6.0-6.1 seconds. After
electroporation the cells were incubated for 20 minutes at room
temperature in the electroporation cuvettes. All the electroporated
cells were then pooled and distributed equally onto 10 gelatinized
plates. After 24 hours the plates were aspirated and fresh ES cell
medium was added. The next day, the medium in 9 plates was replaced
with ES cell medium supplemented with 150 .mu.g/ml of G418 (Gibco)
and 0.2 .mu.M gancyclovir (Syntex) while one plate received medium
supplemented only with 150 .mu.g/ml of G418. After an additional 8
days, resulting individual ES cell colonies were picked off of the
plates and separately expanded in a well of 24-well plates as
described by Wurst et al., pp 33-61 in Gene Targeting Vol. 126,
1993, Edited by A. L. Joyner, IRL Press, Oxford University Press,
Oxford, England. Comparison of the number of colonies that grew on
the plates supplemented with G418 and gancyclovir versus the number
that grew with only G418 supplementation was used to determine the
efficiency of negative selection.
[0113] (c) Analyses of the ES Cell Transformants.
[0114] When the cell culture in each well of the 24-well plates
became approximately 80% confluent, it was washed and the cells
were dispersed with two drops of trypsin-EDTA. Trypsinization was
stopped by the addition of 1 ml of ES cell medium. An aliquot (0.5
ml) of this suspension was transferred to each of two wells of
separate 24-wel plates. After the cells had grown to near
confluence, one of the plates was used for cryopreservation of the
cell line while the other was used as a source of DNA for each of
the clones.
[0115] For cryopreservation, the cells in a 24-well plate were
first chilled by placing the plate on ice. The medium was then
replaced with fresh ES cell medium supplemented with 10% DMSO and
25% FBS and the plate was cooled at approximately 0.5.degree.
C./min by insulating the plate in a styrofoam box and placing it in
a -70.degree. C. freezer.
[0116] To isolate the DNA from the clones on the other plate, the
medium in each well was replaced with 500 .mu.l of digestion buffer
(100 mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl, 100
.mu.g/ml proteinase K). After overnight incubation at 37.degree.
C., 500 .mu.l of isopropanol was added to each well and the plate
was agitated for 15 minutes on an orbital shaker. The supernatant
was aspirated and replaced with 500 .mu.l of 70% ethanol and the
plate was shaken for an additional 15 minutes. The DNA precipitate
was picked out of the well and dissolved in 50 .mu.l of TE solution
(10 mM Tris-HCl pH 7.5, 1 mM EDTA).
[0117] The primary analysis for mutagenesis of the mouse PS-1 gene
involved a Southern hybridization screen of ApaI digested ES cell
DNA. The probe for this analysis was derived from the 3' end of our
cloned PS-1 region outside of the 3' arm of homology (FIG. 19d). It
was prepared by f isolating the 6 kb XbaI fragment corresponding to
the 3' end of .lambda.PS1-6 (FIG. 2) and subcloning it into XbaI
digested pBlueScript.RTM. SK+. A further digest of this subclone,
called pPS1-X6 with Xhol (an internal site) and HindIII (from the
Bluescript.RTM. S K+ polylinker) yielded the 1000 bp probe.
[0118] For the Southern hybridization screen, an aliquot (10 .mu.l)
of each ES cell clone DNA was digested with ApaI, resolved on a
0.8% agarose gel, and transferred to a GeneScreen Plus.RTM.
membrane. The probe was labelled with .sup.32P-DCTP by random
priming and hybridized overnight to the membrane at 58.degree. C.
(Church et al., Proc. Natl. Acad Sci, USA, 1984, 81, 1991-1995). An
ES cell line in which the PS-1 gene has successfully undergone
homologous recombination yields 9 and 15 kb Apal fragments in this
assay (FIG. 19). This is because homologous recombination
advantageously introduces a novel ApaI site into the region where
the neo.sup.r cassette is incorporated. The 15 kb band represents
the unaltered cellular copy of PS-1 while the 9 kb band is derived
from the PS-1 copy in which the novel ApaI site results in a
shorter fragment. In this first screen, 8 cell lines were
identified as potential targeted cell lines out of 260 cell lines
analyzed.
[0119] All cell lines scored as putative homologous recombinants by
the primary screen were then further screened using a 500 bp
KpnI-ApaI fragment isolated from a 5.5 kb 5' XbaI fragment from
.lambda.PS1-20 on ScaI digested ES cell DNA. In this case, the
normal PS-1 gene yielded a 13.8 kb fragment and the mutant PS-1
gene a 10.5 kb fragment (FIG. 19e). Of the 8 cell lines examined in
this screen, 4 were shown to have undergone homologous
recombination at their 5' end.
[0120] Cell lines that were identified as having undergone
homologous recombination by both screens were considered to have
undergone bonafide homologous recombination (as opposed to
homologous insertion which would give positive results for only 1
of the 2 preceding screens). However, depending on where crossover
takes place when the 5' arm recombines, the mutations that were
included in this arm may or may not have been incorporated into
cellular DNA as a result of proper homologous recombination (FIG.
1). A further Southern hybridization screen aimed at detecting the
novel AflII site created as a result of the P264L mutation was
therefore implemented. For this, a 1.2 kb HindIII-XbaI fragment
isolated from pPS1-X15 as a probe on AflII digested DNA was
utilized. An unaltered PS-1 gene yielded a 6.7 kb band (FIG. 19f).
A PS-1 gene in which proper homologous recombination has taken
place, but which lacks the planned mutations yields a 8.7 kb band
while the inclusion of the planned mutations yields a 2.2 kb band.
Of the 4 bona fide homologous recombinant cell lines examined, all
4 were shown to have incorporated the novel AflII site near the
planned mutations.
[0121] The mutagenized form of the PS-1 gene described here has
been called PS1.sup.nP264L as opposed to the normal PS-1 gene
termed PSI.sup.+. The four ES cell lines bearing one copy of
PS1.sup.nP264L have been called, PS1-87, PS1-175, PS1-176, and
PS1-243. Three of these lines were thawed, cell numbers expanded,
and used to establish PS-1 mutant mice.
[0122] Additional mutagenesis of the mouse PS-1 gene can be
performed in ES cells in the manner described above in order to
comprise other human mutations.
Example 4
Establishment of PS-1 Mutant Mice
[0123] PS-1 mutant ES cells were used to make chimeric mice by
aggregating the mutant ES cells to E2.5 embryos and transferring
the aggregated embryos to pseudopregnant females (Wood et al.,
Nature, 1993, 365, 87-89). ES cells were prepared for aggregation
by limited trypsinization to produce clumps that average 10-15
cells. E2.5 embryos were collected from superovulated CD-1 female
mice by oviduct flushing as described by Hogan et al., Manipulating
the Mouse Embryo: A Laboratory Manual, 1986, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.). The zona pelucida was
removed from the embryos using acidic Tyrode's solution (Sigma
Chemical Co., St. Louis, Mo.). Aggregation wells were created by
pressing a blunt metal instrument (a darning needle) into tissue
culture plastic. Embryos were then placed in a well together with a
clump of approximately 10-15 ES cells in a small drop
(approximately 20 .mu.l) of M16 medium (Sigma Chemical Co., St.
Louis, Mo.) under mineral oil. After an overnight incubation
(37.degree. C., 100% humidity, 5% CO.sub.2 in air) the aggregate
embryos were transferred to the uterine horns of a pseudopregnant
female (Hogan et al., 1986, supra). Contribution of the ES cells to
the offspring was scored by the appearance of pigmented coat color.
Positive mice are termed chimeric founders. Germline contribution
by the ES cells was scored by the appearance of pigmented offspring
from a cross between the chimeric founders and CD-1 females.
Example 5
Chimeras
[0124] Of 3 mutant PS-1 ES cell lines used in embryo aggregations,
one produced a germline chimera:
1 TABLE 1 Number Number Number of Embryo of Chimeric of Germline
Clone Aggregation Founders Chimeras PSI-175 400 5 1 PSI-176 75 4 0
PSI-243 120 0 0
[0125] The germline chimera was then used to establish lines of
mice carrying PS-1.sup.nP264L. The presence of the mutant PS-1
allele in the pigmented offspring was determined using a PCR
strategy aimed at detecting the neo.sup.r cassette, following
substantially the same procedure as set forth in Example 1. PCR
primers were as follows: neo28 (GGA TTG CAC GCA GGT TCT CC; SEQ ID
NO:13); and neo445 (CCG GCT TCC ATC CGA GTA CG; SEQ ID NO:14). The
genomic DNA was prepared from a tail sample (Hogan, 1986, supra).
Of the four pigmented offspring, one female mouse was heterozygous
for PS-1.sup.nP264L (PS-1.sup.nP264L/+), i.e., this mouse was
positive for the neo.sup.r cassette based upon the foregoing PCR
strategy. Subsequent generational offspring which are also
heterozygous for PS-1.sup.nP264L have been developed by mating of
this female with wild-type males.
[0126] Mice heterozygous for PS-1.sup.nP264L (PS-1.sup.nP264L/+)
were genotyped using a PCR-based method. The presence of the
wild-type allele for murine PS-1 was scored using the following
primers: X8F (CCC GTG GAG GAG GTC AGA AGT CAG; SEQ ID NO:15) and
X8R (TTA CGG GTT GAG CCA TGA ATG; SEQ ID NO:16). Scoring with these
primers yields a 142 bp fragment (data not shown). The presence of
the mutant allele was scored using the primers neo28 and neo445,
which yields a 417 bp fragment. Thus, mice which are heterozygous
for the mutation yield both bands; mice which are homozygous for
the mutation yield only the 417 bp band; and mice that are
homozygous for the wild-type allele yield only the 142 bp band.
Tissue samples were derived from animal tails, and the PCR
procedures of Example 1 were utilized for such scoring.
[0127] Mice homozygous for the PS-1.sup.nP264L allele (i.e.,
PS-1.sup.nP264L/nP264L) were generated by cross breeding of
heterozygous mice (PS-1.sup.nP264L/+) with mice which are
homozygous for a humanized APP gene (as disclosed in PCT
Publication Number W096/34097, published Oct. 31, 1996;
incorporated herein fully by reference). The resulting generational
offspring were then determined to be heterozygous for both the
PS-1.sup.nP264L allele and heterozygous for the humanized APP gene
(data not shown); these generational offspring were then utilized
for cross-breeding, with resulting generational offspring
determined (using the PCR procedure outlined above) to be
homozygous for the PS-1.sup.nP264L allele, as well heterozygous for
the humanized APP gene (generational offspring from this liter were
also found to be heterozygous for the PS-1.sup.nP264L
allele/homozygous for the humanized APP gene; and heterozygous for
the PS-1.sup.nP264L allele/heterozygous for the humanized APP
gene--due to the limited number of pups obtained from this liter,
double homozygotes were not found). Subsequent matings produced
PS1.sup.nP264L/nP264L.times.APP.sup.NLh/NLh mice.
[0128] Mice homozygous for the PS-1.sup.nP264L allele were also
generated by cross-breeding of heterozygous mice
(PS-1.sup.nP264L/+). In one set of matings, 6 homozygotes were
found amongst 27 offspring, which is well within the expected 25%
recovery of homozygotes from a heterozygous cross.
[0129] Accordingly, and based upon the various breeding approaches
disclosed above, substantially normal viability and embryonic
survival of the animals is evident.
Example 6
Excision of the PGK-neo Cassette
[0130] pBS185 plasmid DNA (Sauer et al., New Biol., 1990, 2,
441-449, incorporated herein by reference in its entirety) encoding
Cre recombinase was introduced by pronuclear injection into
one-cell embryos generated from a PS-1.sup.nP264L/+.times.CD-1
cross. Since the plasmid was circular, DNA integration into the
genome had a very low frequency of occurrence. Transient expression
of the DNA to produce Cre recombinase excised the PGK-neo cassette
in the early embryos. Injected embryos were transferred to
pseudopregnant females. Excision of the PGK-neo cassette was
confirmied by genotyping of the offspring. These mice were
designated PS-1.sup.P264L/+ and were crossed to generate
PS-1.sup.P264L/P264L mice.
[0131] Because the neomycin-selectable marker reduced transcription
of the PS-1 gene, the PGK-neo gene was excised by recombination at
the flanking loxP sites after transient expression of Cre
recombinase. One-cell embryos (n=154) generated by a
PS-1.sup.nP264L/+.times.CD-1 cross were injected with pBS185
plasmid DNA and implanted into pseudopregnant females. The loss of
the PGK-neo gene was scored in the offspring as a 219-base pair
fragment by PCR using the X8F and X8R primer pairs as described in
Example 5. The mutant PS-1 allele with the neomycin-selectable
marker excised was designated PS-1.sup.P264L. Successful excision
occurred in one founder mouse that generated heterozygous
(PS-1.sup.P264L/+) and homozygous (PS-1.sup.P264L/P264L) lines of
mice. PS-1.sup.P264L/+ mice were crossed with Tg2576 mice and
APP.sup.NLh/NLh mice to fiuther study the effects of the P264L
mutation on A.beta. production and deposition.
Example 7
Northern And Western Blots
[0132] PS-1.sup.+/+, PS-1.sup.nP264L/nP264L, and
PS-1.sup.P264L/P264L mice, aged 2-6 months were used for evaluating
mRNA and protein levels of PS-1. Total RNA was extracted from
one-half brain by homogenization in RNAzol B (Tel-Test,
Friendswood, Tex.). Messenger RNA was selected with Oligotex
columns (Qiagen, Valencia, Calif.). Equal volumes of mRNA were
mixed with loading buffer (NortherMAX-Gly, Ambion, Austin, Tex.)
heated to 50.degree. C. for 30 min, separated on a 0.7% agarose
gel, and transferred to a nylon membrane. PS-1 mRNA was detected
with a .sup.32P-dUTP-labeled riboprobe representing the 3' end of
human PS-1: nucleotides 1083-1428 cloned into a pGEM-T vector
(Promega, Madison, Wis.). The same blot was hybridized with a GAPDH
probe (Ambion) for normalization. To visualize mRNAs, the membrane
was exposed to a phosphor screen, scanned on a Storm 840
PhosphorImager, and densitometry performed with ImageQuaNT software
(Molecular Dynamics, Sunnyvale, Calif.).
[0133] One-half brain was homogenized in 2.5 ml of buffer
containing 10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% SDS, 0.25%
deoxycholate, 0.25% NP-40, and protease inhibitors (5 mM PMSF, 10
.mu.g/ml aprotinin, 10 .mu.g/ml leupeptin, 10 .mu.g/ml pepstatin)
(Lee et al., Nature Med., 1997, 3, 756-760). Protein concentration
was determined by BCA assay (Pierce, Rockford, Ill.). Total brain
lysates were mixed with reducing loading buffer and heated at
37.degree. C. for 45 min. Fifty .mu.g total protein of each sample
was separated by electrophoresis on NuPAGE 10% polyacrylamide gels
(Novex, San Diego, Calif.), and transferred to nitrocellulose.
Membranes were blocked overnight at 4.degree. C. in Tris buffered
saline (TBS) with 0.05% Tween-20 and 5% nonfat dry milk. PS-1 was
detected with rabbit polyclonal antibodies diluted in the same
solution. The C-terminal fragment was detected with antibody B17.2
(De Strooper et al., J. Biol. Chem., 1997, 272, 3590-3598) at
1:2000 dilution. B17.2 was raised against amino acid residues
300-315 (EGDPEAQRRVSKNSKY; SEQ ID NO:17) in the hydrophilic loop
domain of human PS-1. The N-terminal fragment was detected with
CP160 at 1:500. CP160 was generated using a 6X-histidine tagged
N-terminal fragment of human PS-1 (amino acids 1-80), expressed in
bacteria with a pQE-9 plasmid (Qiagen). The synthetic PS-1
N-terminal fragment was purified using Ni-NTA agarose (Qiagen) and
SDS-PAGE. Peptide was cut out of acrylamide gels for injection into
rabbits. The IgG fraction of CP160 was affinity purified and used
for blotting. The primary antibodies were detected with horseradish
peroxidase-conjugated anti-rabbit secondary antibodies (New England
Biolabs, Beverly, Mass.). Blots were reacted with chemiluminescent
reagent (LumiGLO, New England Biolabs) and exposed to Hyperfilm
(Amersham, Arlington Heights, Ill.). Films were scanned and
densitometry performed with RFLP2.1 software (Scanalytics, Fairfax,
Va.).
[0134] Northern blot analysis demonstrated a 3.1 kb band for PS-1
mRNA. PS-1 mRNA levels were normalized for loading differences with
GAPDH mRNA levels. The presence of the PGK-neo gene in the
PS-1.sup.nP264L/nP264L mice resulted in levels of PS-1 mRNA that
were 20% of wild type levels (data not shown). mRNA levels in
PS-1.sup.P264L/P264L mice were 100% of PS-1.sup.+/+ mice (data not
shown). Thus, removal of the PGK-neo cassette returned PS-1 mRNA
levels to normal levels.
[0135] Western blotting demonstrated an N-terminal PS-1 fragment of
.about.30 kDa using antibody CP160 and a C-terminal PS-1 fragment
of .about.20 kDa using antibody B17.2 in all three genotypes.
Blotting with CP160, preabsorbed with antigen, eliminated the
.about.30 kDa N-terminal band (data not shown). Specificity of
B17.2 for the C-terminal PS-1 fragment has been previously
demonstrated (De Strooper et al., J. Biol. Chem., 1997, 272,
3590-3598). Because PS-1 mRNA levels were reduced in the
PS-1.sup.nP264L/nP264L mice, the level of PS-1 protein was also
reduced to approximately 15-20% of normal levels (data not shown).
In spite of normal mRNA expression of mutant PS-1 in the
PS-1.sup.P264L/P264L mice, PS-1 protein levels were reduced by
about 50% (data not shown). Both the N- and C-terminal fragments
were reduced to a similar degree. These data indicate that the
PS-1.sup.P264L mutation affects PS-1 protein levels either via
effects on translation, processing of the full length PS-1 protein,
or stability of the cleaved fragments. Reports have described
variable reductions in the N-terminal fragment and/or accumulation
of holoprotein due to some FAD mutations in PS-1 (Mercken et al.,
FEBS Lett., 1996, 389, 297-303; Murayama et al., Neurosci. Lett.,
1997, 229, 61-64; Levey et al., Ann. Neurol., 1997, 41, 742-753;
Murayama et al., Prog. Neuro-Psychopharmacol. Biol. Psychiatr.,
1999, 23, 905-913; Takahashi et al., Neurosci. Lett., 1999, 260,
121-124). In contrast, a variety of PS-1 FAD mutations were found
to cause no reductions in fragment formation in FAD patients, in
transfected cells, and in transgenic or gene-targeted mice
(Hendriks et al., NeuroReport, 1997, 8, 1717-1721; Lee, et al.,
Nature Med., 1997, 3, 756-760; Podlisny et al., Neurobiol. Dis.,
1997, 3, 325-337; Guo et al., Nature Med., 1999, 5, 101-106;
Lvesque et al., Molec. Med., 1999, 5, 542-554; Vanderhoeven et al.,
Neurosci. Lett., 1999, 274, 183-186; Borchelt et al., Neuron, 1996,
17, 1005-1013; Duff et al., Nature, 1996, 383, 710-713; Citron et
al., Nature Med., 1997, 3, 67-72; Nakano et al., Eur. J. Neurosci.,
1999, 11, 2577-2581). The level of reduction in the N-terminal
fragment for the P264L mutation was to about 35-40% of wild type
PS-1 values in transfected PC12 cells (Murayama et al., Prog.
Neuro-Psychopharmacol. Biol. Psychiatr., 1999, 23, 905-913),
consistent with the degree of reduction that has been seen in the
PS-1.sup.P264L/P264L mice compared with PS-1.sup.+/+ mice.
Example 8
A.beta.40- and 42-specific ELISAs
[0136] Half brains from predepositing double gene-targeted mice
(APP.sup.NLh/NLh mice at 1-6 months,
APP.sup.NLh/NLh.times.PS-1.sup.P264L- /+ mice at 5 months, and
APP.sup.NLh/NLh.times.PS-1.sup.P264L/P264L mice at 1-2 months) and
predepositing Tg2576 mice (Hsiao et al., Science, 1996, 274,
99-102) at 2-4 months were frozen on dry ice and stored at
-70.degree. C. Additional half brains from predepositing Tg2576
mice crossed with PS-1.sup.P264L/+ mice (Tg2576.times.PS-1.sup.+/+
mice at 2-4 months, Tg2576.times.PS-1.sup.P264L/+ mice at 2 months,
and Tg2576.times.PS-1.sup.P264L/P264L mice at 1 month) were
similarly prepared. Half brains were homogenized in 4 ml of 0.2%
diethylamine and 50 mM NaCl and centrifuged at 100,000.times. g.
The supernatants were neutralized to pH 8 with 2 M Tris-HCl,
assayed for protein concentration by the BCA method (Pierce,
Rockford, Ill.), and diluted 1:1 in 5% fetal clone serun (HyClone,
Logan, Utah) and 1% nonfat dry milk in TBS. The A.beta.42-specific
ELISA was runas previously described (Savage et al., J. Neurosci.,
1998, 18, 1743-1752). The A.beta.40-specific ELISA was modified
(Savage et al., J. Neurosci., 1998, 18, 1743-1752) so that the
capture antibody was 6E10 (Senetek, Napa, Calif.) and the detecting
antibody was selective for A.beta.40 (BioSource International,
Camarillo, Calif.). ELISA signals were reported as nanograms of
A.beta. per milligram of total extracted protein based upon
standard curves generated using A.beta.40 or 42 (Bachem, King of
Prussia, Pa.).
[0137] Table 2 shows the effect of the PS-1.sup.P264L mutation on
A.beta.40 and A.beta.42 levels in the brains of APP.sup.NLh/NLh
mice before the appearance of A.beta. deposition. The
PS-1.sup.P264L mutation did not have a significant effect on the
level of A.beta.40. One copy of the PS-1.sup.P264L mutation
slightly elevated A.beta.42 but the effect of the mutation was
significant only in the APP.sup.NLh/NLh.times.PS-1.sup.P-
264L/P264L mice compared with the
APP.sup.NLh/NLh.times.PS-1.sup.+/+ mice. This increase in A.beta.42
levels caused a significant elevation in the ratio of A.beta.42 to
A.beta.40 in the APP.sup.NLh/NLh.times.PS-1.sup.P26- 4L/P264L mice
relative to APP.sup.NLh/NLh.times.PS-1.sup.+/+ and
APP.sup.NLh/NLh.times.PS-1.sup.P264L/+ mice. Tg2576 mice had
markedly more A.beta.40 and A.beta.42 than the
APP.sup.NLh/NLh.times.PS-1.sup.P264- L/P264L mice but the ratio of
A.beta.42/40 was similar in the Tg2576 and
APP.sup.NLh/NLh.times.PS-1.sup.+/+ mice.
[0138] Table 3 shows the effect of the PS-1.sup.P264L mutation on
A.beta.40 and A,42 levels in the brains of Tg2576 mice before the
appearance of A.beta. deposition. The PS-1.sup.P264L mutation did
not have a significant effect on the level of A.beta.40. One copy
of the PS-1.sup.P264L mutation slightly elevated A.beta.42 but the
effect of the mutation was significant only in the
Tg2576.times.PS-1.sup.P264L/P264L mice compared with the
Tg2576.times.PS-1.sup.+/+ mice. The increase in A.beta.42 levels
caused a significant elevation in the ratio of A.beta.42 to
A.beta.40 in the Tg2576.times.PS-1.sup.P264L/P264L mice relative to
Tg2576.times.PS-1.sup.+/+ mice. Thus, the effect of the
PS-1.sup.P264L mutation on A.beta. levels was similar for the
Tg2576 and APP.sup.NLh/NLh mice.
2TABLE 2 A.beta.40 and 42 Levels in Predepositing APP.sup.NLh/NLh
with PS-1.sup.P264L Mutations and Tg2576 Mice A.beta.40 A.beta.42
PS-1 Genotype Age (days) N (ng/mg protein) (ng/mg protein)
A.beta.42/40 Ratio APP.sup.NLh/NLh Mice with PS-1.sup.P264L
Mutations X PS-1.sup.+/+ 103 8 0.40 .+-. 0.03 0.08 .+-. 0.01 0.18
.+-. 0.03 X PS-1.sup.P264L/+ 138 5 0.47 .+-. 0.08 0.11 .+-. 0.03
0.22 .+-. 0.02 X PS-1.sup.P264L/P264L 54 10 0.40 .+-. 0.03 0.15
.+-. 0.01* 0.37 .+-. 0.01** Tg2576 Mice on C57B6/SJL Background 99
6 1.52 .+-. 0.17 0.29 .+-. 0.05 0.18 .+-. 0.01 .+-.standard error
of the mean *Kruskal-Wallis ANOVA, p = 0.012, PS-1.sup.P264L/P264L
versus PS-1.sup.+/+ **ANOVA, p = 1.64 E-8, PS-1.sup.P264L/P264L
versus PS-1.sup.+/+, PS-1.sup.P264L/+, and Tg2576
[0139]
3TABLE 3 A.beta.40 and 42 Levels in Predepositing Tg2576 Mice with
PS-1.sup.P264L Mutations Tg2576 Mice with PS-1.sup.P264L Mutations
A.beta.40 A.beta.42 PS-1 Genotype Age (days) N (ng/mg protein)
(ng/mg protein) A.beta.42/40 Ratio X PS-1.sup.+/+ 92 6 1.73 .+-.
0.10 0.27 .+-. 0.2 0.16 .+-. 0.01 X PS-1.sup.P264L/+ 62 5 2.15 .+-.
0.19 0.44 .+-. 0.07 0.20 .+-. 0.02 X PS-1.sup.P264L/P264L 31 8 1.72
.+-. 0.22 0.57 .+-. 0.08* 0.33 .+-. 0.01** .+-.standard error of
the mean *Kruskal-Wallis ANOVA, p = 0.0024, PS-1.sup.P264L/P264L
versus PS-1.sup.+/+ **Kruskal-Wallis ANOVA, p = 5.87 E-4,
PS-1.sup.P264L/P264L versus PS-1.sup.+/+
Example 9
Immunohistochemistry and Histology
[0140] PS-1.sup.P264L/P264L mice were examined at 12 months of age.
APP.sup.NLh/NLh mice that were PS-1.sup.+/+, PS-1.sup.P264L/+, or
PS-1.sup.P264L/P264L, aged 3, 6, 9, 12, 15, and 18 months of age
were evaluated. Additional mice examined were Tg2576 and were
PS-1.sup.+/+, PS-1.sup.P264L/+, or PS-1.sup.P264L/P264L, aged 1, 2,
4, 6, 9, 12, 15, and 18 months of age. Other Tg2576 mice maintained
by crossing to C57B6/SJL mice were also examined at 6, 9, 12, 15,
18, and 21 months of age. Mice were perfused with Ringer's solution
and the brains removed and hemisected. One-half of each brain was
immersed in 70% ethanol and 150 mM NaCl for 48 hours, paraffin
embedded and sectioned in the sagittal plane at 10 .mu.m. Sets of
16 sections taken at intervals of 200 .mu.m were stained to
demonstrate A.beta. deposits by immunohistochemistry. Antibodies
used were 1153, a rabbit polyclonal antibody generated against
amino acids 1-28 of human A.beta. (Savage et al., Neuroscience,
1994, 60, 607-619) and monoclonal antibodies 4G8 and 6E10
(Senetek). Sections were pretreated with 80% formic acid for 4G8,
not pretreated for 1153 and 6E10, and were reacted with the primary
antibodies overnight at 1:1,000. Antibodies were complexed using
biotinylated secondary antibodies (1:100), linked using
streptavidin labeled horseradish peroxidase (BioGenex, San Ramon,
Calif.), and visualized using nickel-intensified
3,3'-diaminobenzidene. Non-transgenic mice, as well as pre-absorbed
primary antisera, served as staining controls. Additional sets of
sections were stained using thioflavine S and examined with a
fluorescence microscope.
[0141] Plaque load was quantified in neocortex in one set of 16
sections stained with antibody 1153 using the CastGrid system
(Olympus, Copenhagen, Denmark). Volume of neocortex and percent
volume of neocortex occupied by A.beta. deposits were determined
stereologically by point counting (Weibel et al., (1979)
Stereological methods, vol. 1: practical methods for biological
morphometry, 415 pp. London: Academic Press). Representative
results are shown in Table 4.
4TABLE 4 A.beta. Plaque Load (% Volume Fraction) in Neocortex at 6
Months of Age Genotype % Plaque Load Tg2576 (C57B6/SJL) 0.0018
Tg2576 .times. PS-1.sup.+/+ 0.0016 Tg2576 .times. PS-1.sup.P264L/+
2.92 Tg2576 .times. PS-1.sup.P264L/P264L 9.09 APP.sup.NLh/NLh
.times. PS-1.sup.+/+ 0 APP.sup.NLh/NLh .times. PS-1.sup.P264L/+ 0
APP.sup.NLh/NLh .times. PS-1.sup.P264L/P264L 0.026
[0142] Extracellular A.beta. deposition was markedly accelerated in
Tg2576 mice that were PS-1.sup.P264L/+ or PS-1.sup.P264L/P264L
compared with those that were PS-1.sup.+/+. In
Tg2576.times.PS-1.sup.P264L/+ mice, A.beta. deposition was not
noted at 2 months of age but was present at 4 months of age. In
Tg2576.times.PS-1.sup.P264L/P264L mice, A.beta. deposition was not
present at 1 month of age but was present at 2 months of age.
Tg2576.times.PS-1.sup.+/+ mice did not show A.beta. deposition
until 6 months of age, and the amount was comparable to that seen
in 6-month-old Tg2576 mice maintained on the C57B6/SJL background.
A.beta. plaque load visualized with antibody 1153 increased
dramatically in neocortex of the Tg2576.times.PS-1.sup.P264L/+ and
Tg2576.times.PS-1.sup.P264L/P264L mice at later ages (data not
shown). Tg2576.times.PS-1.sup.P264L/+ mice at 4 months and
Tg2576.times.PS-1.sup.P264L/P264L mice at 2 months had numerous
deposits that stained with 4G8 or thioflavine S, indicating that
the earliest deposits contained compact, fibrillar amyloid.
[0143] In addition to the acceleration in deposition seen in
Tg2576.times.PS-1.sup.P264L/P264L mice, another difference was the
regional distribution of deposition. Comparing 6-month-old
Tg2576.times.PS-1.sup.P264L/P264L mice, 9-month-old
Tg2576.times.PS-1.sup.P264L/+ mice, and 18-month-old Tg2576
(C57B6/SJL background) mice, the density of deposition was similar
in telencephalic structures (data not shown). However, in
subcortical structures of the Tg2576.times.PS-1.sup.P264L/P264L
mice the amount of A.beta. deposition was much greater than in the
Tg2576.times.PS-1.sup.P264L/+ and Tg2576 mice (data not shown).
[0144] A.beta. deposition in the APP.sup.NLh/NLh mice has been
assessed out to 22 months of age. No evidence for deposition was
found. Similarly, no deposition was found in PS-1.sup.P264L/P264L
mice that were wild type for mouse APP at 12 months of age.
Extremely rare A.beta. deposition was noted in the cortex of
APP.sup.NLh/NLh.times.PS-1.sup.P264L/+ mice using both antibody
1153 and thioflavine S at 12 months of age. At 18 months of age
A.beta. deposits in these mice were more numerous and larger.
[0145] Two copies of the PS-1.sup.P264L mutation resulted in an
increase in A.beta.42 in the
APP.sup.NLh/NLh.times.PS-1.sup.P264L/P264L mouse (Table 2) and have
resulted in A.beta. deposition at an early age. A.beta. deposition
was not found at 3 months of age but was present at 6 months in
APP.sup.NLh/NLh.times.PS-1.sup.P264L/P264L mice. Deposition
increased with age in the
APP.sup.NLh/NLh.times.PS-1.sup.P264L/P264L mice.
[0146] The disclosures of each patent, patent application and
publication cited or described in this document are hereby
incorporated herein by reference, in their entirety.
[0147] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims.
Sequence CWU 1
1
17 1 21 DNA Artificial Sequence Oligonucleotide Primer 1 ctcatcttgg
ctgtgatttc a 21 2 18 DNA Artificial Sequence Oligonucleotide Primer
2 gttgtgttcc agtctcca 18 3 19 DNA Artificial Sequence
Oligonucleotide Primer 3 atttagtggc tgttttgtg 19 4 20 DNA
Artificial Sequence Oligonucleotide Primer 4 aggagtaaat gagagctgga
20 5 21 DNA Artificial Sequence Oligonucleotide Primer 5 tgaaatcaca
gccaagatga g 21 6 22 DNA Artificial Sequence Oligonucleotide Primer
6 gcactcctga tctggaattt tg 22 7 48 DNA Artificial Sequence
Oligonucleotide Primer 7 ggaaagaatg cggccgctgt cgacgttaac
atgcatataa cttcgtat 48 8 47 DNA Artificial Sequence Oligonucleotide
Primer 8 gctctcgaga taacttcgta tagcatacat tatacgaagt tatatgc 47 9
33 DNA Artificial Sequence Oligonucleotide Primer 9 cgttctagaa
taacttcgta taatgtatgc tat 33 10 33 DNA Artificial Sequence
Oligonucleotide Primer 10 cgtggatcca taacttcgta tagcatacat tat 33
11 27 DNA Artificial Sequence Oligonucleotide Primer 11 ttgtgtctta
agggtccgct tcgtatg 27 12 27 DNA Artificial Sequence Oligonucleotide
Primer 12 cggaccctta agacacaaaa cagccac 27 13 20 DNA Artificial
Sequence Oligonucleotide Primer 13 ggattgcacg caggttctcc 20 14 20
DNA Artificial Sequence Oligonucleotide Primer 14 ccggcttcca
tccgagtacg 20 15 24 DNA Artificial Sequence Oligonucleotide Primer
15 cccgtggagg aggtcagaag tcag 24 16 21 DNA Artificial Sequence
Oligonucleotide Primer 16 ttacgggttg agccatgaat g 21 17 16 PRT Homo
sapiens 17 Glu Gly Asp Pro Glu Ala Gln Arg Arg Val Ser Lys Asn Ser
Lys Tyr 1 5 10 15
* * * * *