Note: Descriptions are shown in the official language in which they were submitted.
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PERLECAN TRANSGENIC ANIMALS AND METHODS OF
IDENTIFYING COMPOUNDS FOR THE TREATMENT OF AMYLOIDOSES
The invention relates to the overproduction of
perlecan in transgenic animals and transfected animal
cells as screening tools to identify lead therapeutics
for the amyloid diseases.
BACKGROUND OF THE INVENTION
The Amyloid Diseases
The "amyloid diseases" consist of a group of
clinically and generally unrelated human diseases which
all demonstrate a marked accumulation in tissues of an
insoluble extracellular substance known as "amyloid", and
usually in an amount sufficient to impair normal organ
function. Rokitansky in 1842 (Rokitansky, "Handbuch der
pathologischen Anatomie", Vol. 3, Braumuller and Seidel,
Vienna) was the first to observe waxy and amorphous
looking tissue deposits in a number of tissues from
different patients. However, it wasn't until 1854 when
Virchow (Virchow, Arch. Path. Anat. 8:416 (1854)) termed
these deposits as "amyloid" meaning "starch-like" since
they gave a positive staining with the sulfuric acid-
iodine reaction, which was used in the 1850's for
demonstrating cellulose. Although cellulose is not a
constituent of amyloid, nonetheless, the staining that
Virchow observed was probably due to the present of
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proteoglycans (PGs) which appear to be associated with
all types of amyloid deposits. The name amyloid has
remained despite the fact that Friederich and Kekule in
1859 discovered the protein nature of amyloid (Friedrich
and Kekule, Arch. Path. Anat. Physiol. 16:50 (1859)).
For many years, based on the fact that all amyloids have
the same staining and structural properties, lead to the
postulate that a single pathogenetic mechanism was
involved in amyloid deposition, and that amyloid deposits
were thought to be composed of a single set of
constituents. Current research has clearly shown that
amyloid is not a uniform deposit and that amyloids may
consist of different proteins which are totally unrelated
(Glenner, N. England J. Med. 302:1283-1292 (1980)).
Although the nature of the amyloid itself has
been found to consist of completely different and
unrelated proteins, all amyloids appear similar when
viewed under the microscope due to amyloid's underlying
protein structure to adapt into a fibrillar structure.
All amyloids regardless of the nature of the underlying
protein 1) stain characteristically with the Congo red
dye and display a classic red/green birefringence when
viewed under polarized light (Puchtler et al, J.
Histochem Cytochem. 10:355-364 (1962)), 2)
ultrastructurally consists of fibrils with a diameter of
7-10 nanometers and of indefinite length, 3) adopt a
predominant beta-pleated sheet secondary structure.
Thus, amyloid fibrils viewed under an electron microscope
(30,000 times magnification) from the post-mortem brain
of an Alzheimer's disease patient would look nearly
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identical to the appearance of amyloid present in a
biopsied kidney from a rheumatoid arthritic patient.
Both these amyloids would demonstrate a similar fibril
diameter of 7-10 nanometers.
In the mid to late 1970's amyloid was
clinically classified into 4 groups, primary amyloid,
secondary amyloid, familial amyloid and isolated amyloid.
Primary amyloid, is amyloid appearing de novo, without
any preceding disorder. In 25-4011 of these cases,
primary amyloid was the antecedent of plasma cell
dysfunction such as the development of multiple myeloma
or other B-cell type malignancies. Here the amyloid
appears before rather than after the overt malignancy.
Secondary amyloid, appeared as a complication of a
previously existing disorder. 10-15% of patients with
multiple myeloma eventually develop amyloid (Hanada et
al., J. Histochem. Cytochem. 19:1-15 (1971)). Patients
with rheumatoid arthritis, osteoarthritis, ankylosing
spondylitis can develop secondary amyloidosis as with
patients with tuberculosis, lung abscesses and
osteomyelitis (Benson and Cohen, Arth. Rheum. 22:36-42
(1979); Kamei et al., Acta Path. Jpn. 32:123-133 (1982);
McAdam et al., Lancet 2:572-575 (1975)). Intravenous
drug users who self-administer and who then develop
chronic skin abscesses can also develop secondary amyloid
(Novick, Mt. Sin. J. Med. 46:163-167 (1979)). Secondary
amyloid is also seen in patients with specific
malignancies such as Hodgkin's disease and renal cell
carcinoma (Husby et al., Cancer Res. 42:1600-1603
(1982)). Although these were all initially classified as
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secondary amyloid, once the amyloid proteins were
isolated and sequenced many of these turned out to
contain different amyloid proteins.
The familial forms of amyloid also showed no
uniformity in terms of the peptide responsible for the
amyloid fibril deposited. Several geographic populations
have now been identified with genetically inherited forms
of amyloid. One group is found in Israel and this
disorder is called Familial Mediterranean Fever and is
characterized by amyloid deposition, along with recurrent
inflammation and high fever (Mataxas, Kidney 20:676-685
(1981)). Another form of inherited amyloid is Familial
Amyloidotic Polyneuropathy, and has been found in Swedish
(Skinner and Cohen, Biochem Biophvs Res. Comm. 99:1326-
1332 (1981)), Portuguese (Saraiva et al., J. Lab. Clin.
Med. 102:590-603 (1983); J. Clin. Invest. 74:104-119
(1984)) and Japanese (Tawara et al., J. Lab. Clin. Med.
98:811-822 (1981)) nationalities. Amyloid deposition in
this disease occurs predominantly in the peripheral and
autonomic nerves. Hereditary amyloid angiopathy of
Icelandic origin is an autosomal dominant form of amyloid
deposition primarily affecting the vessels in the brain,
and has been identified in a group of families found in
Western Iceland (Jennson et al., Clin. Genet. 36:368-377
(1989)). These patients clinically have massive cerebral
hemorrhages in early life which usually causes death
before the age of 40.
The primary, secondary and familial forms of
amyloid described above tend to involve many organs of
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the body including heart, kidney, liver, spleen,
gastrointestinal tract, skin, pancreas, and adrenal
glands. These amyloid diseases are also referred to as
"systemic amyloids" since so many organs within the body
demonstrate amyloid accumulation. For most of these
amyloidoses, there is no apparent cure or effective
treatment and the consequences of amyloid deposition can
be detrimental to the patient. For example, amyloid
deposition in kidney may lead to renal failure, whereas
amyloid deposition in heart may lead to heart failure.
For these patients, amyloid accumulation in systemic
organs leads to eventual death generally within 3 to 5
years.
Isolated forms of amyloid, on the other hand,
tend to involve a single organ system. Isolated amyloid
deposits have been found in the lung, and heart (Wright
et al., Lab. Invest. 30:767-773 (1974); Pitkanen et al.,
Am. J. Path. 117:391-399 (1984)). Up to 900 of type II
diabetic patients (non-insulin dependent form of
diabetes) have isolated amyloid deposits in the pancreas
restricted to the beta cells in the islets of Langerhans
(Johnson et al., New Eng1. J. Med. 321:513-518 (1989);
Lab Invest 66:522-535 (1992)). Isolated forms of amyloid
have also been found in endocrine tumors which secrete
polypeptide hormones such as in medullary carcinoma of
the thyroid (Butler and Khan, Arch. Path. Lab. M.d_
110:647-649 (1986); Berger et al., Virch. Arch. A Path
Anat. Hist. 412:543-551 (1988)). A serious complication
of long term hemodialysis is amyloid deposited in the
medial nerve and clinically associated with carpal tunnel
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syndrome (Gejyo et al., Biochem. Biophys. Res. Comm.
129:701-706 (1985); Kidney Int. 30:385-390 (1986)). By
far, the most common type and clinically relevant type of
organ-specific amyloid, and amyloid in general, is that
found in the brains of patients with Alzheimer's disease
(see U.S. Patent No. 4,666,829 and Glenner and Wong,
Biochem Biophys Res Comm 120:885-890 (1984); Masters
et al., Proc. Natl. Acad. Sci. USA 82:4245-4249 (1985)).
In this disorder, amyloid is predominantly restricted to
the central nervous system. Similar deposition of
amyloid in the brain occurs in Down's syndrome patients
once they reach the age of 35 years (Rumble et al., New
England J. Med. 320:1446-1452 (1989); Mann et al.,
Neurobiol. Aaing 10:397-399 (1989)). Other types of
central nervous system amyloid deposition include rare
but highly infectious disorders known as the prion
diseases which include Creutzfeldt-Jakob disease,
Gerstmann-Straussler syndrome, and kuru (Gajdusek et al.,
Science 197:943-960 (1977); Prusiner et al., Cell 38:127-
134 (1984); Prusiner, Scientific American 251:50-59
(1984); Prusiner et al., Micr. Sc. 2:33-39 (1985);
Tateishi et al., Ann. Neurol. 24:35-40 (1988)).
It was misleading to group the various
amyloidotic disorders strictly on the basis of their
clinical features, since when the major proteins involved
were isolated and sequenced, they turned out to be
different. For example, amyloid seen in rheumatoid
arthritis and osteoarthritis, now known as AA amyloid,
was the same amyloid protein identified in patients with
the familial form of amyloid known as Familial
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Mediterranean Fever. Not to confuse the issue, it was
decided that the best classification of amyloid should be
according to the major protein found, once it was
isolated, sequenced and identified.
Thus, amyloid today is classified according to
the specific amyloid protein deposited. The amyloid
diseases include, but are not limited to, the amyloid
associated with Alzheimer's disease, Down's syndrome and
hereditary cerebral hemorrhage with amyloidosis of the
Dutch type (wherein the specific amyloid is now known as
the beta-amyloid protein or Ai3), the amyloid associated
with chronic inflammation, various forms of malignancy
and Familial Mediterranean Fever (AA amyloid or
inflammation-associated amyloidosis), the amyloid
associated with multiple myelorna and other B-cell
abnormalities (AL amyloid), the amyloid associated with
type II diabetes (amylin or islet amyloid), the amyloid
associated with the prion diseases including Creutzfeldt-
Jakob disease, Gerstmann-Straussler syndrome, kuru and
animal scrapie (PrP amyloid), the amyloid associated with
long-term hemodialysis and carpal tunnel syndrome (beta2-
microglobulin amyloid), the amyloid associated with
senile cardiac amyloid and Familial Amyloidotic
Polyneuropathy (prealbumin or transthyretin amyloid), and
the amyloid associated with endocrine tumors such as
medullary carcinoma of the thyroid (variants of
procalcitonin).
Although there are many different types of
amyloid diseases as described above, there are only a few
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animal models for these diseases to develop new
therapeutic agents. Inflammation-associated or AA
amyloidosis has a well-defined experimental mouse model
which is used to induce amyloid deposition in systemic
organs such as spleen, liver and kidney (Snow and
Kisilevsky, Lab. Invest. 53:37-44 (1985)). In addition,
only some of the Alzheimer's disease neuropathology is
observed in current animal models of the disease
(described below). Thus, there is a need for the
development of new transgenic animal models for the
amyloid diseases including Alzheimer's disease. In
addition, there is a need for new cell culture models to
rapidly screen and identify new lead therapeutics for
each of the amyloid diseases.
SiTMMARY OF THE INVENTION
The invention provides a transgenic non-human
animal expressing a perlecan encoding transgene. Also
provided is a double-transgenic non-human animal
expressing a perlecan and a amyloid encoding transgene.
A method of screening for a compound which alters the
rate or extent of amyloid deposition is additionally
provided. The method consists of: (a) constructing a
perlecan transgenic animal; (b) administering an
effective amount of a test compound to said perlecan
transgenic animal; and (c) determining whether said test
compound alters the extent or rate of amyloid deposition.
Finally, the invention provides a method of screening for
a compound which alters the rate or extent of amyloid
deposition. The method consists of: (a) constructing a
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perlecan/amyloid double-transgenic animal; (b) administering
an effective amount of a test compound to said perlecan/
amyloid double-transgenic animal; and (c) determining
whether said test compound alters the extent or rate of
amyloid deposition.
Thus, in one aspect the present invention provides
a transgenic mammalian cell expressing a perlecan encoding
transgene comprising DNA sequence encoding one or more of
perlecan Domain I, perlecan Domain II, perlecan Domain III,
perlecan Domain IV, perlecan Domain V, and fragments
thereof.
In another aspect, the present invention provides
a double-transgenic mammalian cell expressing: (a) a
perlecan encoding transgene comprising DNA sequence encoding
one or more of perlecan Domain I, perlecan Domain II,
perlecan Domain III, perlecan Domain IV, perlecan Domain V,
and fragments thereof, and (b) a beta-amyloid precursor
protein encoding transgene.
In another aspect, the present invention provides
a double-transgenic mammalian cell expressing (a) a perlecan
transgene comprising DNA sequence encoding one or more of
perlecan Domain I, perlecan Domain II, perlecan Domain III,
perlecan Domain IV, perlecan Domain V, and fragments
thereof; and (b) a transgene which encodes for a protein
selected from the group consisting of laminin, type IV
collagen, glypican, syndecan, syndecan-3, neurocan,
phosphacan, aggrecan, decorin, biglycan, hyaluronan, amyloid
P component, alpha-antichymotrypsin, cathepsin D,
cathepsin G, cathepsin B, neuronal thread protein, nicotine
receptors, 5-HT2 receptor, NMDA receptor, alpha 2-adrenergic
receptor, synaptophysin, p65, glutamine synthetase, glucose
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transporter, PPI kinase, GAP43, TGF-beta, presenilin I,
presenilin II, NAC (non-amyloid R-protein component),
cytochrome kinase, hemo oxygenase, calbindin, adenosine
Al receptors, choline acetyltransferase,
acetylcholinesterase, glial fibrillary acidic protein,
alphal-antitrypsin C-reactive protein, alpha2-macroglobulin,
interleukin-lalpha, interleukin-1R, TNF-alpha,
interleukin-6, HLA-DR, HLA-A, HLA-D, HLA-C, CR3 receptor,
MHC I, MHC II, CD 31, CR4, CD45, CD64, CD4, spectrin, tau
protein, ubiquitin, MAP-2, apolipoprotein E, apolipoprotein
E4, apolipoprotein E2, apolipoprotein E3, nerve growth
factor, brain-derived neurotrophic factor, advanced
glycosylation end products, receptor for advanced
glycosylation end products, COX-2, CD18, C3 fibroblast
growth factor, CD44, IcAM-1, lactotransferrin, Clq, C3d,
C4d, C5b-9, gamma R11 Pc gamma RII, CD8, CDS9, vitronectin,
vitronectin receptor, beta-3 integrin, Apo J, clusterin,
type 2 plasminogen activator inhibitor, midline, macrophage
colony stimulating factor receptor, MRP14, 27E10,
interferon-alpha, S100aR, cPLA2, c-jun, c-fos, HSP27, HSP70,
MAP5, membrane lipid peroxidase, protein carbonyl formation,
junB, junD, fosB, fral, cyclin Dl, p53, NGFI-A, NGFI-B,
Ikappa-B, NF-kappa-B, interleukin-8, MCP-1, MIP-lalpha,
matrix metaloproteinases, and 4-hydroxynonenal-protein
conjugates.
In another aspect, the present invention provides
a double-transgenic mammalian cell that is engineered (a) to
express a perlecan transgene comprising DNA sequence
encoding one or more of perlecan Domain I, perlecan
Domain II, perlecan Domain III, perlecan Domain IV, perlecan
Domain V, and fragments thereof; and (b) to knock out
expression of a gene which encodes for a protein selected
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from the group consisting of laminin, type IV collagen,
glypican, syndecan, syndecan-3, neurocan, phosphacan,
aggrecan, decorin, biglycan, hyaluronan, amyloid P
component, alphal-antichymotrypsin D, cathepsin G, cathepsin
B, neuronal thread protein, nicotine receptors, 5-HT2
receptor, NMDA receptor, alpha2-adrenergic receptor,
synaptophysin, p65, glutamine synthetase, glucose
transporter, PPI kinase, GAP43, TGF-beta, presenilin,I,
presenilin II, NAC (non-amyloid R-protein component),
cytochrome kinase, hemo oxygenase, calbindin, adenosine
Al receptors, choline acetyltransferase,
acetylcholinesterase, glial fibrillary acidic protein,
alphal-antitrypsin, C-reactive protein, alph2-macroglobulin,
interleukin-lalpha, interleukin-1R, TNF-alpha,
inlerleukin-6, HLA-DR, HLA-A, HLA-D, HLA-C, CR3 receptor,
MHC I, MHC II, CD 31, CR4, CD45, CD64, CD4, spectrin, tau
protein, ubiquitin, MAP-2, apolipoprotein E, apolipoprotein
E4, apolipoprotein E2, apolipoprotein E3, nerve growth
factor, brain-derived neurotrophic factor, advanced
glycosylation end products, receptor for advanced
glycosylation end products, COX-2, CD18, C3, fibroblast
growth factor, CD44, ICAM-1, lactotransferrin, Clq, C3d,
C4d, C5b-9, gamma R1, Fc gamma RII, CD8, CD59, vitronectin,
vitronectin receptor, beta-3 integrin, Apo J, clusterin,
type 2 plasminogen activator inhibitor, midline, macrophage
colony stimulating factor receptor, MRP14, 27E10,
interferon-alpha, S100R, cPLA2, c-jun, c-fos, HSP27, HSP70,
MAP5, membrane lipid peroxidase, protein carbonyl formation,
junB, junD, fosB, fral, cyclin Dl, p53, NGFI-A, NGFI-B,
I-kappa-B, NF-kappa-B, interleukin-B, MCP-1, MIP-lalpha,
matrix metaloproteinases, and 4-hydroxynonenal-protein
conjugates.
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In another aspect, the present invention provides
a double-transgenic mammalian cell expressing: (a) a
perlecan encoding transgene comprising DNA sequence encoding
one or more of perlecan Domain I, perlecan Domain II,
perlecan Domain III, perlecan Domain IV, perlecan Domain V,
and fragments thereof; and (b) an amyloid encoding
transgene.
In another aspect, the present invention provides
a method of identifying a compound that modulates activity
or expression of an Alzheimer's disease marker, said method
comprising: (a) providing a test compound; (b) contacting
said test compound with the transgenic mammalian cell of the
invention or the double-transgenic cell of the invention,
and (c) determining whether the activity or expression of
the Alzheimer's disease marker is modulated in the presence
of said test compound.
In another aspect, the present invention provides
a method for making a transgenic non-human mammal, the
method comprising the steps of selecting appropriate
restriction sites for ligation of cDNA clones, and ligating
together 7 overlapping cDNA clones to produce a ligated
cDNA clone that encodes for perlecan's -400 kilodalton core
protein.
In another aspect, the present invention provides
a transgenic mammalian cell expressing a perlecan transgene,
wherein the perlecan transgene comprises one or more
sequences each coding for one or more fragments of perlecan,
and wherein the perlecan fragments are each homologous to a
portion of a perlecan domain selected from the group of
perlecan domains consisting of domains I - V and splice
variants of domains I - V.
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In another aspect, the present invention provides
a method for making a transfected mammalian cell, the method
comprising the steps of selecting appropriate restriction
sites for ligation of cDNA clones, and ligating together 7
overlapping cDNA clones to produce a ligated cDNA clone in
the transfected cell that encodes for perlecan's -400
kilodalton core protein.
In another aspect, the present invention provides
a transfected mammalian cell expressing a perlecan encoding
transgene, wherein the perlecan transgene comprises one or
more sequences each coding for one or more fragments of
perlecan, and wherein the perlecan fragments are each
homologous to a portion of a perlecan domain selected from
the group of perlecan domains consisting of domains I - V
and splice variants of domains I - V.
In another aspect, the present invention provides
a transgenic mouse cell that is a somatic cell or a germ
cell, wherein said cell comprises a transgene operatively
linked to a promoter, wherein the transgene encodes mouse
perlecan, and wherein the transgene is further operatively
linked to an enhancer, whereby expression of the transgene
results in overexpression of mouse perlecan in said cell.
In another aspect, the present invention provides
a transgenic mouse cell containing a genome that comprises a
transgene encoding a mouse perlecan domains I-V, wherein the
transgene is operatively linked to a chick beta-actin
promoter and a cytomegalovirus (CMV) enhancer, whereby
expression of the transgene results in over expression of
mouse perlecan, as compared to a non-transgenic mouse cell.
In another aspect, the present invention provides
a transgenic mouse cell that is a somatic cell or a germ
cell, wherein said cell comprises a transgene encoding a
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mouse perlecan domains I-V, wherein the transgene is
operatively linked to a chick beta-actin promoter and a
cytomegalovirus (CMV) enhancer, whereby expression of the
transgene results in over expression of mouse perlecan in
all cells, as compared to a corresponding non-transgenic
murine somatic cell or germ cell.
In another aspect, the present invention provides
use, in the obtention of progeny, of a transgenic non-human
mammal having a transgene which comprises DNA sequence
encoding one or more of perlecan Domain I, perlecan
Domain II, perlecan Domain III, perlecan Domain IV, perlecan
Domain V, and fragments thereof in operable linkage with a
promoter.
In another aspect, the present invention provides
a method, comprising the steps of: mating a transgenic non-
human mammal as defined above with a second non-human mammal
to produce progeny; and, recovering said progeny.
In another aspect, the present invention provides
a method for identifying a compound that modulates an
Alzheimer's disease marker, said method comprising:
(a) providing a test compound; (b) administering said test
compound to the transgenic non-human mammal as defined
above; and (c) determining whether the Alzheimer's disease
marker is modulated in the presence of said test compound.
In another aspect, the present invention provides
a transgenic mammalian cell cultured from the transgenic
non-human mammal as defined above.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic demonstrating the five
structural domains of perlecan.
Figures 2A-2G are schematics showing the
construction strategy of the full-length cDNA for perlecan
core protein using plasmid clones containing cDNAs for
overlapping parts of perlecan.
Figures 3A-C shows the construction strategy for
the cytomegalovirus enhancer/chick (3-actin promoter linked
to the expression vector pCA-DI-V used to overexpress mouse
perlecan in transgenic mice and in transfected cells.
Figure 4 is a black and white photograph of
Western blots from the cell lysates of non-transfected
versus transfected COS cells. Overexpression of perlecan in
the cell layer of transfected COS cells is demonstrated.
Figure 5 is a black and white photograph of
Western blots from the media of non-transfected versus
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transfected COS cells. Overexpression of perlecan in the
media of transfected COS cells is demonstrated.
Figure 6 is a black and white photograph of
Western blots from the media and cell lysates of non-
transfected versus transfected P19 cells. Overexpression
of perlecan in the cell layer and media is demonstrated.
Figure 7 is a black and white photograph of
Western blots detecting SPP from the media and cell
lysates of P19 cells overexpressing both perlecan and
SPP-695, versus only fSPP-695.
Figure 8 is a black and white photograph of
Western blots detecting secreted Af3 in the media from P19
cells overexpressing both perlecan and l3PP-695, versus
only i3PP-695.
Figure 9 shows bar graphs indicating normalized
levels of secreted Af3 in the media from P19 cells
overexpressing both perlecan and i3PP-695, versus only
f3PP-695. A marked 8-10 fold increase in the levels of Ai3
in cells overexpressing both perlecan and i3PP-695 is
demonstrated.
Figure 10 shows bar graphs comparing the number
of surviving neurons in P19 cells overexpressing perlecan
only, SPP-695 plus perlecan, SPP-695 only, and control
parental cells (P19 only). Overexpression of perlecan
only or perlecan plus SPP-695 leads to decreased neuronal
survival.
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Figure 11 is a black and white photograph of
Southern blots of DNA taken from transgenic mice
overexpressing mouse perlecan (domains I-V). Chromosomal
integration of the perlecan transgene is demonstrated in
Southern blots of DNA taken from transgenic mice, and
four perlecan transgenic founder mice are shown.
Figure 12 is a black and white photograph of
Northern blots of RNA isolated from tissues of perlecan
transgenic mice versus control litter-mates.
Overexpression of perlecan mRNA in several tissues is
demonstrated.
Figure 13 is a black and white photograph of
Western blots of protein isolated from tissues of
perlecan transgenic mice versus control litter-mates.
Overproduction of perlecan proteoglycan in various organs
of transgenic mice is demonstrated.
Figure 14 are black and white microphotographs
from brain and kidney tissues of 5-month old perlecan
transgenic mice versus control litter-mates. The tissues
were immunostained with an antibody specific to perlecan,
followed by counterstaining with hematoxylin.
Accumulation of overproduced perlecan in tissues of
transgenic mice is demonstrated.
Figures 15A and 15B demonstrate the
construction strategy for the cytomegalovirus
enhancer/chick 9-actin promoter linked to the signal
peptide and to the C-terminal 99 amino acids of the beta-
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amyloid precursor protein as an example to produce beta-
amyloid precursor protein transgenic mice (CA-SfSC).
Figure 16 is a black and white photograph of
Southern blots taken from mouse tail DNA of double
transgenic mice produced by mating perlecan transgenic
mice with transgenic mice that overproduce the C-terminal
99 amino acids of the beta-amyloid precursor protein.
Pup #552 was found to incorporate the genes for both
perlecan and the C-terminal 99 amino acids of the beta-
amyloid precursor protein.
DETAILED DESCRITION OF THE INVENTION
As stated previously, perlecan is a specific
heparan sulfate proteoglycan and a common constituent of
all amyloid deposits regardless of the specific amyloid
protein involved. Perlecan is believed to play a primary
role in the pathogenesis of amyloidosis and contributes
to the formation, deposition, accumulation and/or
persistence of amyloid in a variety of tissues and
different clinical settings. Previous animal models
overexpressing a specific amyloid protein only rarely
produce some of the pathology associated with different
amyloid diseases, or produce fibrillar amyloid in a
different location than that observed clinically in
humans, making it extremely difficult to screen in vivo
for potential therapeutics for the various amyloid
diseases. Additionally, perlecan is an extremely
difficult macromolecule to isolate in pure and
substantial quantities, and a transfected cell line that
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overexpresses perlecan would enable one to isolate
perlecan for purification in sufficient quantities for
use in a number of different biological assays. Since
perlecan is a proteoglycan which contains a large core
protein (-400 kilodaltons), previous attempts to
successfully produce transgenic mice which overexpress
the entire perlecan core protein have not been achieved.
It appears that for the successful overexpression of the
entire perlecan core protein the strategies for ligating
overlapping cDNA clones which cover the entire 12 kb
message of murine perlecan core protein (or the entire 14
kb message of human perlecan core protein), as well as
the choice of specific promoter and enhancers for use in
the construction strategy of perlecan transgenic mice is
essential. In the present irivention, unique restriction
sites were used to ligate together 7 overlapping cDNA
clones to produce a single 12 kb cDNA clone that encodes
for mouse perlecan's -400 kDa core protein. A novel
construct, designated pCA-DI-V, which utilizes a
cytomegalovirus enhancer and chick 9-actin promoter, has
led to successful overexpression of mouse perlecan
(domains I-V) in transfected cells and in transgenic
mice. Overproduction of perlecan was achieved in both
COS cells and P19 cells (embryonic carcinoma cells which
differentiate into neuron-like cells following retinoic
acid treatment). Overexpression of perlecan in P19 cells
led to an 8-10-fold increase in secreted beta-amyloid
protein (Af3) levels, and contributed to a marked decrease
in neuronal survival.These latter studies suggest that
perlecan overexpression in animal cells or non-human
transgenic animals may be sufficient to cause the
i i
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14
accumulation of Alzheimer's-type amyloid leading to
neuropathological and behavioral consequences.
Production of perlecan transgenic mice, and the mating of
these mice with transgenic animals which overexpress a
given amyloid protein or its precursor protein, and/or
other components implicated in amyloid pathogenesis (i.e.
double transgenic mice) will produce progeny that develop
much or all of the phenotypic pathology of a given
amyloid disease. As an example for screening methods for
Alzheimer's disease, perlecan transgenic animals, cells
derived from perlecan transgenic animals, or perlecan-
transfected animal cells, either alone or in combination
with other amyloid disease implicated components, are
used to screen for compounds altering the pathological
course of Alzheimer's disease as measured by their effect
on beta-amyloid precursor proteins (f3PPs), Ai3, and
numerous other Alzheimer's disease markers in animals,
the neuropathology of the animals, as well as behavioral
alterations in the animals. As an example of production
of double transgenic mice, successful mating of perlecan
transgenic animals with transgenic mice which overproduce
the C-terminal 99 amino acids of i3PP, has been achieved
and led to the birth of progeny which carry both the
perlecan and i3PP genes. The production of new transgenic
animal models, and animal cells of amyloid diseases may
be used as in vivo and in vitro screening tools to aid in
the identification of lead therapeutics for the
amyloidoses and for the treatment of clinical
manifestations associated with these diseases. The
successful overproduction of perlecan in transfected
cells also serves as a new means to isolate perlecan
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which will meet the increasing demands for use of
perlecan for a variety of in vitro and in vivo assays.
"Amyloid" as a Therapeutic Target in Alzheimer's Disease
The most common form of amyloidosis is found in
the brains of patients with Alzheimer's disease.
Alzheimer's disease is the most common cause of dementia
in middle and late life, and is manifested by progressive
impairment of memory, language, visuospatial perceptions
and behavior (A Guide to the Understanding of Alzheimer's
Disease and Related Disorders, edited by Jorm, New York
University Press, New York (1987)). A diagnosis of
probable Alzheimer's disease can be made on clinical
criteria (usually by the exclusion of other diseases,
memory tests etc.), but a definite diagnosis requires the
histological examination of specific abnormalities in the
brain tissue usually obtained at autopsy.
In Alzheimer's disease, the parts of the brain
essential for cognitive processes such as memory,
attention, language, and reasoning degenerate, robbing
victims of much that makes us human, including
independence. In some inherited forms of Alzheimer's
disease, onset is in middle age, but more commonly,
symptoms appear from the mid-60's onward. Alzheimer's
disease is characterized by the deposition and
accumulation of a 39-43 amino acid peptide termed the
beta-amyloid protein, AS or S/A4 (Glenner and Wong,
Rincham Biophvs. Res. Comm. 120:885-890 (1984); Masters
et al., proc Natl. Acad. Sci. USA 82:4245-4249 (1985);
Husby et al., Bull. WHO 71:105-108 (1993)). This small
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peptide is a major component which makes up the amyloid
deposits of neuritic "plaques" and in the walls of blood
vessels (known as cerebrovascular amyloid deposits) in
the brains of patients with Alzheimer's disease. In
addition, Alzheimer's disease is characterized by the
presence of numerous neurofibrillary "tangles", consisting
of paired helical filaments which abnormally accumulate
in the neuronal cytoplasm (Grundke-Iqbal et al., Proc.
Narl Acad Sci USA 83:4913-4917 (1986); Kosik et al.,
Proc Natl. Acad. Sci. USA 83:4044-4048 (1986); Lee et
al., Science 251:675-678 (1991)). The pathological
hallmarks of Alzheimer's disease is therefore the
presence of "plaques" and "tangles", with amyloid being
deposited in the central core of plaques and within the
blood vessel walls. It is important to note that a so-
called "normal aged brain" has some amyloid plaques and
neurofibrillary tangles present. However, in comparison,
an Alzheimer's disease brain shows an over abundance of
plaques and tangles. Therefore, differentiation of an
Alzheimer's disease brain from a normal brain from a
diagnostic point of view is primarily based on
quantitative assessment of "plaques" and "tangles".
In an Alzheimer's disease brain, are usually
thousands of neuritic plaques. The neuritic plaques are
made up of extracellular deposits consisting of an
amyloid core usually surrounded by enlarged axons and
synaptic terminals, known as neurites, and abnormal
dendritic processes, as well as variable numbers of
infiltrating microglia and surrounding astrocytes. The
neurofibrillary tangles present in the Alzheimer's
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17
disease brain mainly consist of tau protein, which is a
microtubule-associated protein (Grundke-Iqbal et al.,
Prnr Natl Acad. Sci. USA 83:4913-4917 (1986); Kosik et
al., Proc Natl Acad Sci USA 83:4044-4048 (1986); Lee
et al., Science 251:675-678 (1991)). At the
ultrastructural level, the tangle consists of paired
helical filaments twisting like a ribbon, with a specific
crossing over periodicity of 80 nanometers. In many
instances within a neurofibrillary tangle, there are both
paired helical filaments and straight filaments. In
addition, many times the nerve cell will die, leaving the
filaments behind. These tangles are known as "ghost
tangles" since they are the filamentous remnants of the
dead neuron.
The other major type of lesion found in the
brain of an Alzheimer's disease patient is the
accumulation of amyloid in the walls of blood vessels,
both within the brain parenchyma and in the walls of the
larger meningeal vessels which lie outside the brain.
The amyloid deposits localized to the walls of blood
vessels are referred to as cerebrovascular amyloid or
congophilic angiopathy (Mandybur, J. Neurogath. Exy).
Neurol. 45:79-90 (1986); Pardridge et al., J. Neurochem.
49:1394-1401 (1987)).
In addition, Alzheimer's disease patients
demonstrate neuronal loss and synaptic loss.
Furthermore, these patients also exhibit loss of
neurotransmitters such as acetylcholine. Tacrine, the
first FDA approved drug for Alzheimer's disease is a
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18
cholinesterase inhibitor (Cutler and Sramek, New Engl. J.
Med. 328:808-810 (1993)). However, this dV-ug has showed
limited success, if any, in the cognitive improvement in
Alzheimer's disease patients and initially had major side
effects such as liver toxicity.
For many years there has been an ongoing
scientific debate as to the importance of "amyloid" in
Alzheimer's disease and whether the "plaques" and
"tangles" characteristic of this disease, were a cause or
merely the consequences of the disease. Recent studies
during the last few years have now implicated that
amyloid is indeed a causative factor for Alzheimer's
disease and not merely an innocent bystander. The
Alzheimer's disease major protein known as beta-amyloid
protein (Ai3), in cell culture has been shown to cause
degeneration of nerve cells within short periods of time
(Pike et al., Br. Res. 563:311-314 (1991); J. Neurochem.
64:253-265 (1994)). Studies suggest that it is the
fibrillar structure, a characteristic of all amyloids,
that is responsible for the neurotoxic effects. The Ai3
has also been found to be neurotoxic in slice cultures of
hippocampus (the major memory region affected in
Alzheimer's)(Harrigan et al., Neurobiol. Aqing 16:779-789
(1995)) and induces nerve cel7. death in transgenic mice
(Games et al., Nature 373:523-527 (1995); Hsiao et al.,
Neuron 15:1203-1218 (1995)). In addition,'injection of
the Alzheimer's Af3 into rat brain causes memory
impairment and neuronal dysfunction (Flood et al., Proc.
Natl. Acad. Sci. 88:3363-3366 (1991); Br. Res. 663:271-
276 (1994)), two hallmarks of Alzheimer's disease.
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Probably, the most convincing evidence that amyloid (i.e.
beta-amyloid protein) is directly involved in the
pathogenesis of Alzheimer's disease comes from recent
genetic studies. It has been discovered that the
production of Af3 can result from mutations in the gene
encoding, its precursor, known as the beta-amyloid
precursor protein (Van Broeckhoven et al., Science
248:1120-1122 (1990); Europ. Neurol. 35:8-19 (1995);
Murrell et al., Science 254:97-99 (1991); Haass et al.,
Nature Med 1:1291-1296 (1995)). This precursor protein
when normally processed only usually produces very little
of the toxic Af3. The identification of mutations in the
amyloid precursor protein gene which causes familial,
early onset Alzheimer's disease is the strongest argument
that amyloid is central to the pathogenetic process
underlying this disease. Four reported disease-causing
mutations have now been discovered which demonstrate the
importance of the beta-amyloid protein in causing
familial Alzheimer's disease (reviewed in Hardy, Nature
Genet. 1:233-234 (1992)). These studies suggest that
providing a drug to reduce, eliminate or prevent
fibrillar beta-amyloid protein formation, deposition,
accumulation and/or persistence in the brains of human
patients should be considered an effective therapeutic.
Proteoglycans
Proteoglycans (PGs) are a group of complex
macromolecules which are found in all organs and tissues,
intracellularly in a variety of different cell types, or
extracellularly in the matrix where they are exported for
a variety of functions. Proteoglycans consist of a
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linear protein core backbone to which one or more
glycosaminoglycan (GAG) chains are covalently linked
(Hascall and Hascall, in Cell Biology of the
Extracellular Matrix, Hay editor, New York, Plenum Press,
pp. 39, (1981); Hassell et al., Ann. Rev. Biochem.
55:539-567 (1986)). The highly anionic GAG chains
consist of repeating disaccharide units, containing 1)
hexosamine (either D-glucosamine or D-galactosamine), and
hexuronic acid (either D-glucuronic acid or L-iduronic
acid)(Muir, Am. J. Med. 47:673-690 (1969)). The PGs are
traditionally named according to the identification of
the primary GAG present and several major GAGs have been
identified. These are hyaluronic acid, heparan sulfate,
heparin, chondroitin-4-sulfate, chondroitin-6-sulfate,
dermatan sulfate and keratan sulfate. Usually the
linkage between the GAG chains and the protein core
backbone consists of a xylose-galactose-galactose
attachment region with the xylose molecule covalently
linked to the hydroxyl groups of a serine residue on the
protein core (Roden and Armand, J. Biol. Chem. 241:65-70
(1966)). The exception is hyaluronic acid which has a
backbone consisting of alternating D-glucuronic acid and
D-glucosamine units with no protein component. Keratan
sulfate os the one PG which lacks the typical xylose-
serine linkage. It is linked to protein either via a
N-acetylgalactosamine residue linked to either serine or
threonine (in cartilage) or via a N-acetylglucosamine
residue attached directly to an asparagine residue (in
cornea)(Hascall and Hascall, in Cell Biology of the
Extracellular Matrix, Hay editor, New York, Plenum Press,
pp. 39, (1981); Muir, Am. J. Med. 47:673-690 (1969)).
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Heparan Sulfate Proteoglycans: A Common Component of All
Amyloids
It is clear from the research literature that
finding novel therapeutics for amyloid formation,
deposition, accumulation and persistence is today
considered a relevant strategy for Alzheimer's disease
and other amyloid diseases. The major question that
persisted in amyloid research was: why do all amyloids
containing unrelated proteins all form an amyloid fibril
with similar characteristics (i.e. all consist of fibrils
of 7-10 nm and contain a predominant beta-pleated sheet
secondary structure)? Is there a common component that
may play a similar role in the pathogenesis of all
amyloids?
The answer to this central and important
question in understanding the mechanisms involved in
amyloid diseases. Early studies demonstrated that highly
sulfated GAGs (later determined to be specific heparan
sulfate PGs) were concurrently deposited with
inflammation-associated amyloid (i.e. AA amyloid) in a
well-defined experimental mouse model (Snow et al., Lab.
Invest. 56:665-675 (1987)). Later studies demonstrated
that heparan sulfate PGs were temporally and structurally
associated with the deposition and accumulation of AA
amyloid in a variety of different tissues (Snow et al.,
J. Histochem. Cytochem 39:1321-1330 (1991)). Specific
staining techniques and immunohistochemical methods then
determined that highly sulfated PGs were a common feature
of most, if not all, amyloids, independent of the
specific amyloid protein involved, the stage of the
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22
amyloid disease, and the tissue site of amyloid
deposition (Snow et al., Lab. Invest. 56:120-123 (1987);
Am. J. Path. 133:456-463 (1988); Acta Neuropath. 77:337-
342, (1980); Lab. Invest. 63:601-611 (1990)).
Studies were then pursued to further try to
understand the potential involvement of specific PGs in
Alzheimer's disease amyloidosis. In initial studies
using specific immunohistochemical probes it was first
determined that heparan sulfate PGs were an important
constituent of amyloid in neuritic plaques and
cerebrovascular amyloid deposits (Snow et al., Am. J.
Path= 133:456-463 (1988)). It was later revealed that
the antibodies employed for this initial study were in
fact those that specifically recognized the core protein
of a large heparan sulfate PG, known as "perlecan".
Heparan sulfate PGs (and specifically perlecan) were also
co-localized to prion protein (PrP) amyloid plaques in
Gerstmann-Straussler syndrome, Creutzfeldt-Jakob disease,
kuru and animal scrapie (Snow et al., Lab. Invest.
63:601-611 (1990)). It was initially postulated that
specific heparan sulfate PGs play important roles in
amyloidosis by 1) influencing amyloidogenic proteins to
adapt predominantly beta-pleated sheet structures (i.e.
indicative of amyloid), 2) determining the anatomical
location of amyloid deposition, and 3) contributing to
the stability of amyloid and its inaccessibility to
proteolytic degradation in tissues, thus not allowing the
body to properly degrade and remove unwanted amyloid
deposits (Snow and Wight, Neurobiol. Aaing 10:481-497
(1989)).
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Perlecan and/or heparan sulfate PG accumulation
in conjunction with a variety of different amyloid
proteins is an early event, and does not merely represent
secondary and non-specific deposition. In experimental
inflammation-associated amyloidosis, perlecan expression
actually precedes AA amyloid deposition (Ailles et al.,
Tab Invest. 69:443-447 (1993)) suggesting that up-
regulation of specific PGs may be an initiating event
leading to eventual amyloid formation and/or deposition.
In a previous study (Snow et al., Am. J. Path. 137:1253-
1270 (1990)), the brains of Down's syndrome patients
(aged 1 day to 51 years) were examined to determine the
possible sequence of events leading to beta-amyloid
protein (Ab) and PG deposition. Down's syndrome
patients, who were completely devoid of any Ai3
immunoreactivity, demonstrated prominent heparan sulfate
immunoreactivity in neurons as early as 1 day after
birth, which was not observed in similar aged-matched
non-Down's syndrome brains. In older patients, aged 18
and 24 years, diffuse Ai3 immunoreactivity (which was
Congo red negative and therefore suggestive of non-
fibrillar deposits) in the extracellular matrix was
accompanied by co-localized heparan sulfate deposition.
In patients, over the age of 35 years, fibrillar AB
deposits in neuritic plaques and cerebrovascular amyloid
accumulation were also observed with co-localized heparan
sulfate immunoreactivity. This important study suggested
that heparan sulfate accumulation within neurons may be a
primary event eventually leading to the co-accumulation
of heparan sulfate and Ai3 in the extracellular matrix.
It is feasible that once the interaction between heparan
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sulfate and Ai3 (or its precursor protein) takes place, a
cascade of events occurs which lead to fibril formation,
deposition and eventual persistence.
The Importance of Heparan Sulfate Proteoglycans in
Alzheimer's Disease
To date at least four different classes of
PGs/GAGs have now been shown to be present in the Af3-
containing deposits (i.e. neuritic plaques,
cerebrovascular amyloid) of Alzheimer's disease. In
1988, it was discovered that heparan sulfate PGs were
specifically present in the amyloid deposits and
neurofibrillary tangles of Alzheimer's disease (Snow et
al., Am. J. Path. 133:456-463 (1988)). In later years,
the particular type of heparan sulfate PG found was a
large PG with a total molecular weight of -800,000 known
as "perlecan". In 1992, a second PG present in the
periphery of neuritic plaques and within neurofibrillary
tangles was discovered, which was a small dermatan
sulfate PG known as decorin (Snow et al., J. Histochem.
Cvtochem. 40:105-113 (1992)). In 1993, a third class of
PGs were discovered, namely chondroitin sulfate, which
were present in the periphery of neuritic plaques and
within neurofibrillary tangles of Alzheimer's disease
brain (DeWitt et al., Exõb. Neurol. 121:149-152 (1993)).
The specific type of chondroitin sulfate PG present to
this day has yet to be identified. In 1996, a keratan
sulfate PG, known as SV2PG, was discovered which was
localized primarily to synaptic vesicles in the periphery
of neuritic plaques (Snow et al., Exp. Neurol. 138:305-
317 (1996)).
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Of all the different specific PGs/GAGs
mentioned above, it has becoine exceedingly clear that the
heparan sulfate PGs may be the most important class of PG
implicated in Alzheimer's disease. This is due to the
fact that heparan sulfate PGs still remain the only class
of PG that has been immunolocalized to 1) all three
characteristic lesions of Alzheimer's disease (i.e.
neuritic plaque, neurofibrillary tangles, and
cerebrovascular amyloid deposits), and 2) specifically to
the Ai3-containing amyloid fibrils in both neuritic
plaques and cerebrovascular amyloid deposits. The data
suggests that perlecan is a major heparan sulfate PG
found within the amyloid core of neuritic plaques and
appears to be present in most, if not all, central
nervous system and systemic amyloids (reviewed in Snow
and Wight, NPurobiol. Agina 10:481-497 (1989)).
Overproduction of perlecan in a transgenic animal or
transfected cell line has, as yet, not been achieved, and
is needed to develop new models for developing
therapeutics for the amyloid diseases.
Perlecan Production by Different Cell Types and its
Postulated Roles in the Pathogenesis of Amyloid Diseases
Perlecan is present on all basement membranes
(Dziadek et al., EMBO J. 4:905-912 (1985); Kato et al.,
J. Cell Biol. 106:2203-2210 (1988); Murdoch et al.,
Histochem. Cytochem. 42:239-249 (1994)) and was
previously cloned from both human (Murdoch et al.,
Biol. Chem. 267:8544-8557 (1992); Kallunki and
Tryggvason, J. Cell. Biol. 116:559-571 (1992)) and mouse
(Noonan et al., J. Biol. Chem. 266:22939-22947 (1991)).
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26
Perlecan is known to be produced by different cell types
including endothelial cells (Kinsella and Wight, Biochem.
27:2136-2144 (1988); Saku and Furthmayr, J. Biol. Chem,
264:3514-3523 (1989); Rescan et al., Am. J. Path.
142:199-208 (1993)), smooth muscle cells (Nikkari et al.,
Am. J. Path, 144:1348-1356 (1994)), fibroblasts (Murdoch
et al., J. Histochem. Cytochem. 42:239-249 (1994);
Heremans et al., J. Cell Biol. 109:3199-3211 (1989)),
epithelial cells (Morris et al., In Vitro Cell D v. Biol.
30:120-128 (1994); Ohji et al., Invest. Opth. Vis. Sci.
35:479-485 (1994); Van Det et al., Biochem. J. 307:759-
768 (1995)), and synovial cells (Dodge et al., Lab.
Invest. 73:649-657 (1995)). Perlecan is also synthesized
by bone marrow derived cells (Grassel et al., Mol. Cell
Biochem, 145:61-68 (1995)) and is present in cancerous
tissue including metastatic melanomas (Cohen et al.,
Cancer Res. 54:5771-5774 (1994)), human breast tumors
(Guelstein et al., Int. J. Cancer 53:269-277 (1993)), and
liver tumors (Kovalsky et al.,. Acta Biomed. Ateneo
Parmense 64:157-163 (1993)). Both F9 embryonal carcinoma
cells (which form parietal endoderm) and P19 embryonal
carcinoma cells (which form cholinergic neurons) also
demonstrate marked increased perlecan expression and
synthesis upon differentiation (Chakravarti et al., Dev.
Dvn. 197:107-114 (1993); Sekiguchi et al., J. Neurosc.
Res. 38:670-686 (1994)).
Perlecan is postulated to play a primary role
in the pathogenesis of Alzheimer's disease (AD)
amyloidosis, as well as in other types of central nervous
system and systemic amyloidoses (reviewed in Snow and
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27
Wight, NPurobiol Aging 10:481-497 (1989) ). Only the
heparan sulfate class of PGs have been found to be
immunolocalized to all three major lesions (i.e. neuritic
plaques, neurofibrillary tangles and cerebrovascular
amyloid deposits) in Alzheimer's disease brain and
specifically to the beta-amyloid protein (Ai3)-containing
amyloid fibrils in both amyloid plaques and congophilic
angiopathy (Snow et al., Am. J. Path. 133:456-463 (1988);
Snow and Wight, Neurobiol. Aginq 10:481-497 (1989);
Perlmutter and Chui, Brain Res. Bull. 24:677-686 (1990);
Snow et al., Am. J. Path. 137:1253-1270 (1990); Su et
al., Neuroscience 51:801-813 (1992); Van Gool et al,
Dementia 4:308-314 (1993)). Accumulating evidence
suggests that perlecan is a major heparan sulfate PG
present within the Af3-containina amyloid deposits in
Alzheimer's disease (Snow et al., Am. J. Path. 133:456-
463 (1988); Snow and Wight, Neurobiol. Aging 10:481-497
(1989); Snow et al., Am. J. Path. 137:1253-1270 (1990);
Snow et al., Am. J. Path. 144:337-347 (1994)) and may
play a primary role in Af3 fibril formation, deposition,
accumulation and persistence. The consistent co-
localization of perlecan to Ai3 deposits which exist in
both a fibrillar and non-fibrillar form (Snow et al., Am.
J. Path. 144:337-347 (1994)) is probably due to
perlecan's high affinity interactions with AfS (Snow et
al., J. Neuropath. Exip. Neurol. 48:352 (1989) Abstract;
Buee et al., Brain Res. 601:154-163 (1993); Buee et al.,
Brain Res. 627:199-204 (1993); Snow et al., Arch.
RinchPm. Biophys. 320:84-95 (1995)) and with beta-amyloid
precursor proteins (Narindrasorasak et al., J. Biol.
Chem. 266:12878-12883 (1991)). Residues 13-16 of A13 have
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-28
been identified as a perlecan binding site (Snow et al.,
J. Neuropath. Exp. Neurol. 48:352 (1989) Abstract;
Brunden et al., J. Neurochem. 61:2147-2154 (1993); Snow
et al., Arch. Biochem. Biophys. 320:84-95 (1995)). This
region contains a heparin/heparan sulfate binding
consensus sequence (Cardin and Weintraub, Arterioscl.
9:21-32 (1989)), and is adjacent to the postulated alpha-
secretase cleavage site on AS (at Lys-16). Once bound,
perlecan is believed to influence the secondary structure
and/or aggregation properties of AS and/or beta-amyloid
precursor proteins (Fraser et al., J. Neurochem. 59:1531-
1540 (1992)). Perlecan also appears to play a role in
stabilizing fibrillar AS amyloid when deposited
in vivo (Snow et al., Neuron 12:219-234 (1994); Snow et
al., Soc. Naurosc. Abst. 21:1292 (1995) Abstract), and
protects A!3 from degradation by proteases as demonstrated
in vitro (Gupta-Bansal et al., J. Biol. Chem. 270:18666-
18671 (1995)). The combined results described above
suggest that perlecan is an important macromolecule that
has now been implicated at several key steps in the
pathogenesis of AS amyloidosis in Alzheimer's disease.
However, due to perlecan's large size and complex
structure (described below), production of transgenic
animals or transfected cells which overproduce perlecan
has, as yet, not been achieved.
DNA Sequences and the Structure of Perlecan
The DNA sequence for human perlecan encodes for
a protein core with a molecular weight of approximately
466.564 kDa (Murdoch et al., J. Biol. Chem. 267:8544-8557
(1992)) whereas the DNA sequence for mouse perlecan
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29
encodes for a protein core with a molecular weight of
approximately 396 kDa (Noonan et al., J. Biol. Chem.
266:22939-22947 (1991)). A schematic demonstrating the
five structural domains of perlecan is shown in Figure 1.
The genes for human (Murdoch et al., J. Biol. Chem.
267:8544-8557 (1992); Kallunki and Tryggvason, Cell Biol.
116:559-571 (1992)) and mouse (Noonan et al., J. Biol.
Chem. 266:22939-22947 (1991)) perlecan have been cloned
and the predicted core protein consists of five distinct
domains (Figure 1). Domain I contains the proposed
heparan sulfate GAG attachment sites and is unique to
perlecan showing no similarity to other known protein
sequences. The location of the three Ser-Gly consensus
heparan sulfate GAG attachment sites at the N-terminus
corresponds with the number and position of known GAG
chains (Kokenyesi and Silbert, Biochem. Biophys. Res.
Comm. 211:262-267 (1995)). Domain II is homologous to
the LDL binding domain present in the LDL-receptor,
whereas Domain III has homology to the globule-rod
regions of the laminin short arms. Domain IV is a highly
repetitive region with numerous immunoglobulin-like
repeats that show the highest similarity to neural cell
adhesion molecule (N-CAM). Domain V has three globular
repeats very similar to the domain G repeats in the
laminin A chain and the equivalent segment of the A chain
homologue, merosin, and two epidermal growth factor-like
regions (Noonan and Hassell, Kidney Int. 43:53-60
(1993)). The perlecan core protein is therefore a unique
and large macromolecule with homology to a number of
other well known proteins.
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Transgenic Models Trying to Mimic the Neuropathology of
Alzheimer's Disease
A number of transgenic animal models have been
produced to try to mimic some or all of the
neuropathology of AD (reviewed in Greenberg et al.,
NPurobiol. Aging 17:153-171 (1996)). The rationale for
most of these models was that overproduction of the beta-
amyloid precursor protein (f3PP) transgene (containing all
or part of the l3PP sequence) could lead to the eventual
development of Ai3 deposits in mouse brain and subsequent
plaque and tangle formation. =n some of the first i3PP
transgenic animal model studies, (Wirak et al., Science
253:323-325 (1991)) generated transgenic mouse lines
containing the Af3 sequence under the control of the human
l3PP promoter. After 1 year, these mice developed Ai3
deposits within hippocampal neurons and formed aggregates
of amyloid-like fibrils. Quon et al (Nature 352:239-241
(1991)) used a full length ZPP-751 sequence linked to a
neuron-specific enolase promoter. Transgenic mice with
this construct displayed extracellular Ag immunoreactive
deposits, which were infrequently stained with Thioflavin
S, but not by Congo red, suggesting a pre-amyloid like
composition. Kawabata et al., (Nature 354:476-478
(1991)) developed transgenic mouse lines massively
overexpressing a construct encoding the C-terminal 100
amino acids of ZPP under control of a Thy-1 element.
These mice displayed pathology remarkably similar to that
observed in AD including amyloid plaques, neurofibrillary
tangles and neurodegeneration in hippocampus, neocortex
and even cerebellum. While extremely promising, the
report by Kawabata et al., (Nature 354:476-478 (1991))
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31
was retracted whereas the study by Wirak et al., (Science
253:323-325 (1991)) was questioned (Jucker et al.,
Science 255:1443-1445 (1992)).
Recently, two transgenic animal models which
probably produce the most advanced neuropathology
observed in animals to date, have been described. Games
et al., (Nature 373:523-527 (1995)) generated transgenic
mice using platelet-derived growth factor (PDGF)-i3
driving a human i3PP minigene encoding the f3PP-717
mutation (valine at residue 717 substituted by
phenylalanine) associated with familial AD. These mice
progressively developed some of the neuropathological
hallmarks of AD including Thioflavin S positive AfS
deposits, neuritic plaques, syriaptic loss, astrocytosis
and microgliosis. This transgenic animal model was
studied more closely by Masliah et al., (J. Neurosc.
16:5795-5811 (1996)) who demonstrated the ultrastructure
of the neuritic plaques formed in the brain parenchyma of
these mice. No cerebrovascular amyloid deposition nor
neurofibrillary tangles were observed to date in these
mice.
Another transgenic animal model was recently
reported (Hsiao et al., Science 274:99-102 (1996)) in
which the 695-amino acid isoform of the human QPP
containing the double mutation (Lys670 to Asn, Met671 to
Leu), which was found in a large Swedish family with
early onset Alzheimer's Disease, was inserted into a
hamster prion protein cosmid vector. These mice
demonstrated a 5-fold increased in Ai3 (1-40) and a
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14-fold increase in AS (1-42/43) accompanied by numerous
amyloid plaques stained with Congo red in cortical and
limbic structures. These transgenic animals had normal
learning and memory in spatial reference and alternating
tasks at 3 months of age but showed impairment by 9-10
months of age. In these mice, no cerebrovascular amyloid
deposition nor neurofibrillary tangles were observed.
The most recent transgenic models described
above may one day lead to fruitful insights into the
pathogenesis of AD and may ultimately be used to screen
and identify new therapeutics to treat AD. However,
these transgenic models do have a major shortcoming.
These models have yet to show convincing cerebrovascular
amyloid deposition and neurofibrillary tangle formation
in brain. This indicates that these transgenic models
are missing two of the three major neuropathological
hallmarks of AD (i.e. the other being the neuritic
plaque). Is there a missing factor that these transgenic
models have yet to take into account?
Heparan Sulfate Containing-Proteoglycans May be the
Additional Factor Necessary for Amyloid Plaque and
Neurofibrillary Tangle Formation in Alzheimer's Disease
Two very recent studies (Goedert et al., Nature
383:550-553 (1996); Perez et al., J. Neurochem. 67:1183-
1190 (1996)) implicate that heparan sulfate containing-
PGs may be necessary additional factors which may play
roles in the formation of neurofibrillary tangles. In
one study, Goedert et al., (Nature 383:550-553 (1996))
found that highly sulfated GAGs such as heparin and
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heparan sulfate stimulated different recombinant isoforms
of tau protein to form paired helical filaments in vitro.
The paired helical filament formation was found to be
GAG-dependent, and phosphorylation-independent indicating
that the presence of heparan sulfate GAGs was a key
element in neurofibrillary tangle formation. Highly
sulfated GAGs such as heparin and heparan sulfate were
potent inducers of paired helical filament formation,
whereas less sulfated GAGs such as chondroitin sulfate
and dermatan sulfate were much less effective. Non-
sulfated GAGs such as hyaluronic acid did not induce any
paired helical filament formation indicating that
sulfation was an important factor. Goedert et al.,
(Nature 383:550-553 (1996)) also confirmed some earlier
work (Snow et al., Am. J. Path. 137:1253-1270 (1990))
that heparan sulfate accumulates in the cytoplasm of
neurons prior to the appearance of neurofibrillary
tangles. Heparan sulfate and chondroitin sulfate have
also been detected in tangle-bearing neurons in
Alzheimer's Disease (Perry et al., J. Neurosc. 11:3679-
3683 (1991)) and in neurons bearing tangles in other non-
Alzheimer's diseases such as progressive supranuclear
palsy (DeWitt et al., Br. Res. 656:205-209 (1994)).
Perez et al., (J. Neurochem.. 67:1183-1190 (1996)) also
found heparin to induce different tau protein fragments
to assemble into paired helical filaments in vitro. The
induction of paired helical filaments by heparin/ heparan
sulfate suggests that PGs containing this particular
class of GAGs may the missing factor essential for the
induction of both amyloid plaques and neurofibrillary
tangles in transgenic animals. Overexpression of
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perlecan (a specific heparan sulfate containing-PG) in
transgenic animals may therefore lead to both amyloid
plaque and neurofibrillary tangle formation, and
ultimately serve as a new animal model of Alzheimer's
disease.
Other Co-Components Present in Alzheimer's and in Other
Amyloid Diseases
Alzheimer's disease is characterized by
numerous changes in the expression levels of various
proteins, the biochemical activity and histopathology of
brian tissue, as well as cognitive changes in affected
individuals. Such characteristic changes associated with
Alzheimer's disease have been well documented. The most
prominent change, as discussed herein, is the deposition
of Ai3 into amyloid plaques (Haass and Selkoe, Cell
75:1039-1042 (1993)). Besides specific PGs, a variety of
other molecules have also been known to be important
components of Alzheimer's disease amyloid or
neurofibrillary tangles and include tau protein (Grundke-
Iqbal et al., Proc. Natl. Acad. Sci. USA 83:4913-4917
(1986); Kosik et al., Proc. Natl. Acad. Sci. USA 83:4044-
4048 (1986); Lee et al., Science 251:675-678 (1991)),
apolipoprotein E (Corder et al., Science 261:921-923
(1993); Strittmatter et al., Proc. Natl. Acad Sci USA
90:8098-8102 (1993)), alphal-antichymotrypsin (Abraham et
al., Cell 52:487-501 (1988)), amyloid P component (Coria
et al., Lab. Invest. 58:454-458 (1988)), ubiquitin (Mori
et al., Science 235:1641-1644 (1987)), cytokines (McGeer
et al., Can. J. Neurol. Sci. 16:516-527 (1989); reviewed
in Rogers, CNS Drugs 4:241-244 (1994)), growth factors
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(Hefti and Weiner, Ann. Neurol. 20:275-281 (1986); Hefti
et al., Neurobiol. AcTincr 10:515-533 (1989) ; Kato et al.,
Neurosc. 122:33-36 (1991); Tooyama et al., Neurosc. Lett.
121:155-158 (1991)), and complement factors (Eikenbloom
et al., Virch Arch B Cell Pathol. 56:259-262 (1989)).
Each of these components described above may be utilized
for the production of new transgenic animals and/or
transfected cells. Overexpression of perlecan with
another amyloid or neurofibrillary tangle component
described above may also lead to new models of
amyloidosis (for Alzheimer's disease and/or other amyloid
diseases) and/or models of tangle neuropathology.
Alzheimer's Disease Gene Mutations
A) :9PP Mutations
Certain families are genetically predisposed to
Alzheimer's disease, a condition referred to as familial
Alzheimer's disease, through mutations resulting in an
amino acid replacement at position 717 of the full length
protein (Goate et al., gupra (1991); Murrell et al.,
supra (1991)). Another FAD mutation contains a change in
amino acids at positions 670 and 671 of the full length
protein (Mullan et al., supra (1992)). In one form of
this mutation, the lysine at position 670 is replaced by
asparagine and the methionine at position 671 is replaced
by leucine. The effect of this mutation is to increase
the production of Ai3 in cultured cells approximately
7-fold (Citron et al., Nature 360:672-674 (1992); Lai et
al., Science 259:514-516 (1993)). Additional mutations
in l3PP at amino acids 669, 670 and 671 have been shown to
reduce the amount of Ai3 processed from f3PP (Citron et
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al., Neuron 14:661-670 (1995)). The i3PP construct with
Val at amino acid 690 produces an increased amount of a
truncated form of Af3.
9-PP expression clones can be constructed to
bear a mutation at amino acids 669, 670, 671, 690, 692,
or 717 of the full length protein. The mutations from
Lys to Asn and from Met to Leu at amino acids 670 and
671, respectively, are sometimes referred to as the
Swedish mutation. Additional mutations can also be
introduced at amino acids 669, 670 or 671 which either
increase or reduce the amount of AfS processed from i3PP.
Some mutations at amino acid 717 are sometimes referred
to as the Hardy mutation. Such mutations can include
conversion of the wild-type Va1717 codon to a codon of
Ile, Phe, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Thr,
Asn, or Gln, A preferred substitution for Va1717 is Phe.
These mutations predispose individuals expressing the
mutant proteins to develop Alzheimer's disease. It is
believed that the mutations affect the expression and/or
processing of ZPP, shifting the balance towards
Alzheimer's pathology. Mutations at amino acid 669 can
include conversion of the wild-type Va1669 codon to a
codon for Trp, or deletion of the codon. Mutations at
amino acid 670 can include conversion of the wild-type
Lys670 codon to a codon for Asn or Glu, or deletion of
the codon. Mutations at amino acid 671 can include
conversion of the wild-type Met671 codon to a codon for
Leu, Val, Lys, Tyr, Glu or Ile, or deletion of the codon.
A preferred substitution for Lys670 is Asn, and a
preferred substitution for Met671 is Leu. These
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mutations predispose individuals expressing the mutant
proteins to develop Alzheimer's disease.
B) Presenilin 1 and Presenilin 2
In 1992, evidence for a locus causing early-
onset Alzheimer's disease was reported on the long-arm of
chromosome 14 (Schellenberg et al., Science 258:668-671
(1992)). This result was quickly confirmed by several
groups. A positional cloning strategy was then used to
isolate a candidate gene (S182, later re-named presenilin
1 or PSl) that carried coding region mutations in
families multiply affected by early-onset Alzheimer's
disease (Sherrington et al., Nature 375:754-760 (1995)).
Since then more than 35 different missense mutations have
been found in the PSi gene in over 50 families of
different ethnic origins (Van Broeckhoven, Nat. Genet.
11:230-232 (1995); Clark et al., Nat. Genet. 11:219-222
(1996)). All PS1 gene mutations reported except one are
missense mutations.
The mean age of onset of disease in individuals
with PS1 mutations is generally earlier than it is in
individuals with f3PP mutations, but considerable overlap
does exist (the age range for PS1 is 29 to 62 years, and
for i3PP is 43 to 62 years). Several of the PS1 gene
mutations are found in families of different ethnic
origins suggesting that independent mutational events
have occurred at the same nucleotide.
The PS1 gene contains 10 protein-coding exons
and 2 or 3 additional exons encoding the 5'-untranslated
I I
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region. The major RNA transcript of the PS1 gene is
about 3 kilobases and is expressed in various human brian
regions. The PS1 protein has 467 amino acids and is
thought to span the cell membrane 8 times, but its
function is unknown.
Soon after the isolation of the PS1 gene it
became clear from sequence homologies that PS1 was part
of a gene family. Sequences homologous to the PS1 gene
were found to map to human chromosome 1, indicating the
existence of a second presenilin gene, PS2 (also called
the STM2 gene). At about the same time, genetic linkage
between Alzheimer's disease and DNA markers on chromosome
1 was detected in a group of Volga Germans with
Alzheimer's disease (Levy-Lahad et al., Science 269:970-
973 (1995)). Since the PS2 gene and the markers linked
to the Volga German Alzheimer's disease locus were very
close together, the PS2 gene was sequenced in affected
individuals from several Volga German families, and a
missense mutation causing substitution of asparagine by
isoleucine at codon 141 was identified (Levy-Lahad et
al., Science 269:973-977 (1995)). A second mutation,
M239V, has been identified in an Italian pedigree with
early-onset familial Alzheimer's disease (Rogacv et al.,
Nature 376:775-778 (1996)).
The PS2 protein contains 448 amino acids and
shows 67% identity with the PS1 protein. The PS2 gene is
also widely expressed in many tissues, but shows more
extensive alternative splicing than PS1 (Prohar et al.,
Neurorev.. 7:1680-1684 (1996)). However, the overall
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structures of the PS1 and PS2 proteins are similar, and
all AD-causing mutations are in the nucleotides coding
for amino acids that are conserved between the 2 genes,
suggesting that the 2 proteins serve similar functions
and that mutant amino acids are at positions within the
protein critical to function.
As stated previously, perlecan is a specific
heparan sulfate proteoglycan and a common constituent of
all amyloid deposits regardless of the specific amyloid
protein involved. Perlecan is believed to play a primary
role in the pathogenesis of amyloidosis and contributes
to the formation, deposition, accumulation and/or
persistence of amyloid in a variety of tissues and
different clinical settings. Previous animal models or
overexpressing a specific amyloid protein only rarely
produce some of the pathology associated with different
amyloid diseases, or produce fibrillar amyloid in a
different location than that observed clinically in
humans, making it extremely difficult to screen in vivo
for potential therapeutics for the various amyloid
diseases. In the present invention, unique restriction
sites were used to ligate together 7 overlapping cDNA
clones to produce a single 12 kb cDNA clone that encodes
for mouse perlecan's -400 kDa core protein. In a
preferred embodiment, a novel construct, designated pCA-
DI-V, which utilizes a cytomegalovirus enhancer and chick
9-actin promoter, has led to successful overexpression of
mouse perlecan (domains I-V) in transfected cells and in
transgenic mice.
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For these studies, the constructs are
introduced into animal embryos using standard techniques
such as microinjection or embryonic stem cells. Cell
culture based models can also be prepared by two methods.
Cell cultures can be isolated from the transgenic animals
or prepared from established cell cultures using the same
constructs with standard cell transfection techniques.
The specific constructs that are described
preferably employ a chick i3-actin promoter, which causes
high expression of perlecan in all tissues including
brain. However, other promoters may be used which may be
selected from the following: the human EPP gene promoter,
mouse QPP gene promoter, rat f3PP gene promoter,
metallothionein III gene promoter, metallothionein I
promoter, rat neuron specific enolase gene promoter,
mouse neuron specific enolase promoter, human 9 actin
gene promoter, human platelet derived growth factor B
(PDGF-B) chain gene promoter, rat sodium channel gene
promoter, RNA polymerase I promoter, RNA polymerase II
promoter, polypeptide chain elongation factor 1-alpha
promoter, neurofilament M promoter, neurofilament L
promoter, glial fibrillary acidic protein promoter, prion
protein promoter, insulin promoter, low affinity nerve
growth factor receptor (p75) promoter, mouse myelin basic
protein gene promoter, human copper-zinc superoxide
dismutase gene promoter, and mammalian POU-domain
regulatory gene promoter.
The specific constructs that are described
preferably employ a cytomegalovirus enhancer. However,
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other enhancers may be used which may be selected from
the following: immunoglobulin kappa 3'-enhancer, lambda
enhancer, IgH 3'-enhancer, T cell receptor alpha
enhancer, alpha HS-26 enhancer, alpha HS-40 enhancer, and
rat insulin II gene enhancer.
The specific constructs that are described led
to an overproduction of perlecan in both COS cells and
P19 cells (embryonic carcinoma cells which differentiate
into neuron-like cells following retinoic acid
treatment). Overproduction of perlecan in P19 cells led
to a marked increase in secreted Af3 levels and a marked
decrease in neuronal survival. As an example for
screening methods for Alzheimer's disease, perlecan
transgenic animals, or perlecan-transfected animal cells,
either alone or in combination with other amyloid disease
co-components, are used to screen for compounds altering
the pathological course of Alzheimer's disease as
measured by their effect on i3PPs, Ai3, and numerous other
Alzheimer's disease markers in animals, the
neuropathology of the animals, as well as behavioral
alterations in the animals. The production of new
transgenic animal models, and animal cells of amyloid
diseases may be used as in vivo and in vitro screening
tools to aid in the identification of lead therapeutics
for the amyloidoses and for the treatment of clinical
manifestations associated with these diseases. The
successful overproduction of perlecan in transfected
cells also serves as a new means to isolate perlecan
which will meet the increasing demands for use of
perlecan for a variety of in vitro and in vivo assays.
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In one embodiment, it is an object of the
present invention to provide a transgenic animal or
transfected cell line whose cells include a recombinant
DNA sequence coding for ubiquitous or cell type specific
expression of perlecan or analogs thereof. The mating of
perlecan transgenic mice with transgenic mice
overexpressing a specific amyloid protein or its
precursor, or which overproduce another component
implicated in the disease, will produce new transgenic
mice progeny which overexpress both perlecan and a
specific amyloid protein (or its precursor), or both
perlecan and a specific amyloid co-component. These
transgenic animals which overexpress both perlecan and a
specific amyloid protein (or its precursor protein), or
both perlecan and a specific amyloid co-component will
lead to the production of new transgenic animals which
display much or all of the pathology associated with a
particular amyloid disease. In addition, perlecan
overexpressing transgenic animals may be mated with
transgenic animals which underexpress a specific amyloid
protein or its precursor, or which underproduce another
component implicated in the disease. The production of
these new animals will be effective for the study of the
etiology of various amyloidoses and the efficacy of drugs
in treating each of the amyloid diseases. The amyloid
diseases include, but are not limited to, the amyloid
associated with Alzheimer's disease, Down's syndrome and
hereditary cerebral hemorrhage with amyloidosis of the
Dutch type (wherein the specific amyloid is referred to
as beta-amyloid protein or A!3), the amyloid associated
with chronic inflammation, various forms of malignancy
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and Familial Mediterranean Fever (wherein the specific
amyloid is referred to as AA amyloid or inflammation-
associated amyloidosis), the amyloid associated with
multiple myeloma and other B-cell dyscrasias (wherein the
specific amyloid is referred to as AL amyloid), the
amyloid associated with type II diabetes (wherein the
specific amyloid is referred to as amylin or islet
amyloid), the amyloid associated with the prion diseases
including Creutzfeldt-Jakob disease, Gerstmann-Straussler
syndrome, kuru, and animal scrapie (wherein the specific
amyloid is referred to as PrP amyloid), the amyloid
associated with long-term hemodialysis and carpal tunnel
syndrome (wherein the specific amyloid is referred to as
beta2-microglobulin amyloid), the amyloid associated with
senile cardiac amyloid and Fami-lial Amyloidotic
Polyneuropathy (wherein the specific amyloid is referred
to as prealbumin or transthyretin amyloid), and the
amyloid associated with endocrine tumors such as
medullary carcinoma of the thyroid (wherein the specific
amyloid is referred to as variants of procalcitonin).
One of the major problems in the past in trying
to produce perlecan transgenic mice and perlecan
transfected cells was the large size of the perlecan core
protein (-400 kDa). A unique strategy had to be developed
which allowed one to produce a single 12 kb (from mouse)
cDNA clone that encoded for the entire 400 kDa perlecan
core protein. Another object of the present invention is
to develop a construction strategy using unique
restriction sites in overlapping cDNA clones to produce a
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single 12 kb cDNA clone that encodes for the entire
perlecan core protein.
Another object of the present invention is to
provide a method to produce new transgenic animals that
overexpress both perlecan (or portions thereof) and a
specific amyloid protein (or its precursor), or both
perlecan and a specific amyloid co-component. These new
transgenic animals can be used for the studies pertaining
to the etiology of various amyloidoses and the efficacy
of drugs in treating each of the amyloid diseases.
Another object of the present invention is to
provide transgenic animals which have in their cells
unique promoter/coding sequences which can either
ubiquitously express perlecan in all types of tissue, or
which can e?cpress perlecan in specific types of tissue.
These perlecan transgenic animals can be used to assess
the role that perlecan, and/or portions thereof, play in
development and in a number of relevant biological and/or
pathological processes.
A further object of the invention relates to
the synthesis and use of promoter/coding constructs which
express perlecan alone or in combination with specific
amyloid proteins (or precursor proteins), or other
amyloid co-components, in various tissues of transgenic
animals incorporating such constructs in their genome. A
feature of the transgenic animals which would coexpress
both perlecan and a specific amyloid protein, or both
perlecan and a specific amyloid co-component, will be
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that these new animals will provide both prognostic and
diagnostic means for the study of different amyloid
diseases and for determining the efficacy of
pharmaceutical drugs in treating specific amyloidoses in
a human subject. Initially, the transgenic animals may
be used as in vivo screening tools to help identify one
or more candidate compounds capable of disrupting the
formation, deposition, accumulation and/or persistence of
a given amyloid protein which is associated with a
predisposition to a specific arnyloid disease.
Another object of the present invention relates
to providing transgenic non-human mammals which yield
information regarding the mechanisms (leading to
production and deposition) and location of various
amyloids, as well as providing necessary in vivo models
for testing of potential drugs capable of interfering
with or preventing such formation, deposition,
accumulation and/or persistence in specific tissues and
organs.
Yet another object of the present invention
relates to the production of non-human mammals which
overexpress, mouse or human perlecan (and/or any other
species from which perlecan cDNA sequence is known or
will become known).
Yet another object of the present invention is
to provide an animal model for Alzheimer's disease that
is constructed using transgenic technology.
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It is a further object of the present invention
to provide transgenic animals exhibiting one or more
histopathologies similar to those of Alzheimer's disease.
It is a further object of the present invention
to provide transfected cells expressing one or more AS-
containing proteins at high levels in cell culture media.
It is yet a further object of the present invention to
provide transgenic animals expressing one or more Af~-
containing proteins at high levels in brain tissue.
It is a further object of the present invention
to provide a method of screening potential drugs for the
treatment of Alzheimer's disease using transgenic animal
models and transfected cell lines.
Yet another object of the present invention
relates to the production of mouse and/or human (or any
other species from which the perlecan cDNA sequence is
known or will become known) perlecan by transfected
cells. This invention serves as a new means to provide
for cell culture models for testing of potential drugs
capable of interfering with or preventing Af3 or i3PP (and
constituents thereof) formation, deposition, accumulation
and/or persistence. In addition, these cell cultures can
be utilized to isolate perlecan in sufficient quantities
for use in a variety of different in vitro and in vivo
assays.
This invention can be used either alone or in
combination with other transgenic animals or transfected
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animal cells which overproduce or underproduce a given
amyloid protein or its precursor, or which overproduce or
underproduce another component implicated in a given
amyloid disease. As an example, a double transgenic
mouse has been made which carry the transgenes for both
perlecan and the C-terminal 99 amino acids of the beta-
amyloid precursor protein of Alzheimer's disease. These
model systems provide new in vivo and in vitro methods
for the screening and evaluation of potential drugs for
the treatment of Alzheimer's disease and other amyloid
diseases, and for the production of perlecan in culture
for use in biological systems.
Before the processes for making and using such
transgenic mice and transfected cells are described, it
is to be understood that these inventions are not limited
to particular processes and materials described as such
methods and materials, may vary. It is also to be
understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is
not intended to be limiting.
A unique feature of the present invention is
the strategies employed to produce a single 12 kb cDNA
clone that encodes for mouse perlecan's 400 kDa core
protein. The initial cDNA clones to murine perlecan were
isolated from an expression vector library prepared from
Engelbreth-Holm-Swarm (EHS) tumor mRNA by screening the
library with rabbit antibodies to murine perlecan. The
authenticity of the clones was confirmed by demonstrating
an exact match of amino acid sequences from perlecan
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peptides with that of sequence deduced from the cDNA
clones. Additional cDNA clones were obtained by making
primer extension libraries and screening them with
existing clones to produce 7 overlapping clones covering
perlecan's 12 kb message. These overlapping clones were
then ligated together using unique restriction sites in
the overlapping regions to produce a single 12 kb cDNA
clone that encoded for perlecan's -400 kDa core protein.
The single 12-kb perlecan cDNA was cloned into the
NotI/XbaI sites of pBluescript II SK (Stratagene Cloning
System) to generate pBSDI-V.
Another unique feature of the transgenic mice
of the present invention relates to including general or
cell specific promoters in front of sequences which
encode for domains I to V of mouse or human perlecan.
The ability of the transgenic mice to selectively express
perlecan core protein, including any fragments thereof,
distinguishes the present transgenic mice from others.
The cloned recombinant and/or synthetic DNA
sequences used in connection with the present invention
are sequences which encode a biologically active,
refolded proteoglycan, and contains one or more
glycosaminoglycan chains attached to its protein core
backbone.
Construction of Expression Vectors
Preferred cDNA clones used in making the
transgenic mice and transfected cells include coding
sequences which are initially inserted in a suitable
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expression vector for replication and to confirm
production of protein. In a preferred embodiment, a
full-length cDNA of murine perlecan core protein is
constructed from cDNAs for overlapping parts of perlecan
isolated from mouse cDNA libraries (Noonan et al., J_,.
Biol. Chem. 266:22939-22947 (1991)). Such clones are in
the pBluescript I vector (a product of Strategene) and
are illustrated in Figures 2A-2G. All clones constructed
as described below were verified by sequencing and/or
restriction mapping. Firstly, a plasmid (p Front End)
containing cDNA for the domains I, II and III of perlecan
is constructed from clone 19-J (-XbaI), clone 54 and
Clone DIII (Figure 2B and 2C). The 1.4-kilobase Not
I/Bcl I DNA fragment is isolated from clone 19-J (XbaI)
and cloned into the NotI and Bcl I sites of clone 54 to
produce clone 19-J/54 (Figure 2B). The 1.8-kilobase Not
I/Sal I DNA fragment is isolated from clone 19-J/54 and
ligated onto the Sal I site of the 3.3-kilobase Sal I/Xba
I fragment isolated from clone DIII. The resulting 5.1-
kilobase Not I/Xba I fragment is cloned into the Not I
and Xba I site of pBluescript I to produce clone pFront
End (Figure 2C). Secondly, a plasmid clone (p Back End)
containing cDNA for most of domain IV of perlecan and all
of domain V, is constructed from clone DV and clone 12
(Figure 2D). To introduce a Hind III site into clone DV,
the XhoI site in clone DV is replaced with a Hind III
site using Hind III linkers to produce clone DV wH3. The
3.2-kilobase Xba I/Cla I fragment isolated from clone 12
is cloned into the XbaI/Cla I sites of clone DV with H3
from which the smaller Xba I/Cla I fragment is previously
removed. The resulting clone is designated p Back End
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(Figure 2D). Thirdly, the Front and Back Ends are
connected together to produce clone pF+ B that contains
cDNA for the entire core protein of perlecan except for a
small region in the N-terminal part of domain IV (Figure
2E). The 5.0-kilobase Not I/Xba I fragment isolated from
p Front End is cloned into the same restriction enzyme
sites of p Back End to produce p F + B (Figure 2E).
Next, a plasmid (p Missing Link) containing cDNA for the
small region of the N-terminal part of perlecan domain IV
is constructed from clone 72 and clone 7 (Figure 2F).
The 1.5-kilobase Not I/Hind III fragment isolated from
clone 72 and the 2.2-kb Hind III/Bam HI fragment isolated
from clone 7 are cloned into the Not I and Bam HI sites
of pBluescript I to produce clone 72/7 (Figure 2F). The
190-base pair Ppu MI fragment isolated from clone 7 is
inserted into the same restriction enzyme site of clone
72/7 from which the smaller Ppu MI fragments are
previously removed. The resulting plasmid is designated
as p Missing Link (Figure 2F). Finally, cDNA in p
Missing Link is inserted into pF + B to produce
pBS DI-V that contains a full-length cDNA for perlecan
(Figure 2G). The 2.8-kilobase Nru I/Bss HII fragment
isolated from p Missing Link is cloned into the same
restriction sites of pF + B from which the smaller Nru
I/Bss HII fragment is previously removed. The resulting
plasmid is designated pBS DI-V (Figure 2G).
The expression vector, pCA-DI-V, is then
constructed to overexpress the entire perlecan core
protein under the control of a cytomegalovirus enhancer
and a chick beta-actin promoter (Figures 3A-3C). This
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expression vector contains intron 1 of chick 9-actin gene
and intron 3 of rabbit 9-globulin gene. First, a Hind
III restriction enzyme site is added next to the Sal I
site in pCAGGSnc (Fukuchi et al., Exp. Exp. Neurol.
127:253-264 (1994)) using a polylinker containing XhoI,
Hind III, and Sal I sites (Figure 3A). pCAGGSnc is
linearized by Sal I digestion and treated with calf
intestinal alkaline phosphatase. The polylinker
containing XhoI, Hind III, and Sal I sites is digested
with Xho I and Sal I and ligated onto the Sal I site of
pCAGGSnc to create pCAGGhnc. In a separate reaction,
pBSDI-V which contains full length perlecan cDNA in
pBluescript II SK (Stratagene Cloning System) is digested
with Asn I and a 11.5-kb DNA fragment containing perlecan
cDNA is isolated (Figure 3B). A polylinker containing
Asn I, Nhe I, and SfuI sites is digested with AsnI and
ligated onto the same restriction enzyme site of the
isolated 11.5-kb DNA fragment (Figure 3B). The isolated
fragment is then digested with Not I and Sfu I and
ligated onto the Not I and Cla I sites of pCAGGShnc which
was previously cut with Not I and Cla I (Figure 3C). Cla
I is compatible with Sfu I. The resulting vector is
designated as pCA-DI-DV; pCA-DI-V is used to establish
lines of transgenic mice and cells.
In another preferred embodiment, a full-length
cDNA of human perlecan core protein can be constructed
from cDNAs for overlapping parts of perlecan isolated
from human cDNA libraries. As demonstrated by Murdoch et
al., (J. Biol. Chem. 267:8544-8557 (1992)), commercially
available cDNA libraries such as human colon cDNA library
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(HL103b, a product of Clontech), or cDNA libraries
prepared from a human fibroblast cell line (CRL 1262,
available from American Tissue Culture Collection) and a
human amnion cell line (WISH also available from American
Tissue Culture Collection) can also be used to isolate
overlapping clones covering the entire coding sequence
for human perlecan core protein. These overlapping
clones can be ligated to form a single cDNA using
appropriate restriction sites in the clones in any manner
known to those skilled in the art. The single 14kb cDNA
clone is then cloned into multiple cloning sites of
pCAGGS or pCAGGShnc to create an expression vector for
human perlecan. Expression vectors are not limited to
pCAGGS or pCAGGShnc. Indeed, any other expression
vectors containing the promoters listed below (or any
other promoters that seem appropriate by those skilled in
the art) can be used and the construction of these
expression vectors can be achieved in any manner known to
those skilled in the art.
In another preferred embodiment, a full-length
cDNA of bovine perlecan core protein can be similarly
constructed and can be used to overexpress bovine
perlecan in transgenic mice and in cultured cells. For
example, bovine perlecan cDNA can be isolated by
screening a bovine kidney cDNA library (BL3001b, a
product of Clontech) using several parts of mouse cDNA as
probes. The isolated overlapping clones can be ligated
into a single cDNA coding for the entire bovine perlecan
and then cloned into any expression vectors, in any
manner known to those skilled in the art.
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Promoter/ Perlecan Sequence Fusion Constructs
A) Promoters that Permit Ubiquitous Expression of
Transgenes
The following promoters allow transgenes to
express at high levels in virtually all tissues.
Therefore such ubiquitous promoters can be used to make
any type of amyloid animal models. Such useful promoters
which can be used to create constructs and inserted into
transgenic animals in connection with the present
invention include, but are not limited to:
Beta-actin Promoter
Beta actin is essential and abundantly
expressed in virtually all cells. pCAGGS (Niwa et al.,
Gene 108:193-200 (1991)) that contained the beta-actin
promoter was used to construct expression vectors for
mouse perlecan and beta-amyloid precursor protein as in
Example 10.
RNA Polymerase I Promoter
RNA polymerase I catalyzes synthesis of
ribosomal RNA. Therefore, this promoter permits
transgene expression in virtually all tissues with the
exception of erythrocytes. RNA polymerase I transcripts,
however, are poorly translated as mRNA since they retain
a triphosphate at their 5' termini rather than receiving
a trimethyl G cap. In the absence of a trimethyl G cap,
ribosomes appear not to recognize the transcript as
message and translation initiation is impaired.
Insertion of an internal ribosome entry site (IRES) into
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the 5' leader of the RNA polymerase I transcript allows
high level protein production under control of the RNA
polymerase I promoter (Palmer et al., Nucleic Acid Res.
21:3451-3457 (1993)). Such RNA polymerase promoters with
IRES are available in the plasmid, pMENA (Palmer et al.,
Nucleic Acid Res. 21:3451-3457 (1993)).
RNA Polymerase II Promoter
RNA polymerase catalyzes the synthesis of RNA
transcription from genes and therefore its promoter
permits the ubiquitous expression of transgenes with the
exception of erythrocytes. The RNA polymerase II
promoter is cloned in pHBII CATm (Ahearn et al., J. Biol.
Chem. 262:10695-10705 (1987)).
Polypeptide Chain Elongation Factor 1-alpha promoter
Polypeptide chain elongation factor 1-alpha
(EF-1-alpha) promotes the GTP-dependent binding of an
aminoacyl-tRNA to ribosomes. EF-1-alpha is one of the
most abundant proteins in eukaryotic cells and expressed
in all kinds of mammalian cells. An expression vector,
pEF-BOS, containing EF-1-alpha promoter has been
constructed and demonstrated to be a strong ubiquitous
promoter (Mizushima and Nagata, Nucleic Acids Res.
18:5322 (1990)).
B) Promoters that Restrict Expression to Specific Tissues
Other useful promoters may also be used to
produce perlecan transgenic animals which may prove
useful for the ultimate development of new transgenic
animals that mimic the amyloid diseases. Some useful
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promoters which can also be used to create constructs and
inserted into transgenic animals in connection with the
present invention include, but are not limited to:
Platelet-Derived Growth Factor (PDGF) Promoter
This promoter has recently been used to produce
transgenic mice that overexpress the beta-amyloid
precursor protein and that deposit fibrillar beta-amyloid
protein deposits in an extracellular location (Games et
al., Nature 373:523-527 (1995)). It is possible that
when perlecan expression is driven by a PDGF promoter,
these resulting transgenic mice may prove more useful for
the ultimate development of a transgenic mouse model for
Alzheimer's disease and Down's syndrome (i.e. beta-
amyloid protein) amyloidosis. A plasmid, psisCAT6a, that
contains the PDGF-9 chain promoter is available (Sasahara
et al., Cell 64:217 (1991)).
Neurofilament M or L Promoters
These promoters demonstrate a high level of
expression and are found in connection with the most
abundant neural protein. They are characterized by
central nervous system/peripheral nervous system
neuronal-specific expression. The mouse gene for this
promoter is a published sequence and the isolation of the
promoter region is necessary in order to use the promoter
in connection with the present invention. The
neurofilament L promoters (Begemann et al., Proc. Natl.
gcad Sci. USA 87:9042-9046 (1990)) and M promoters
(Begemann et al., Mol. Brain Res. 15:99-107 (1992)) have
been characterized and are available.
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Glial Fibrillary Acidic Protein (GFAP) Promoters
Such promoters are characterized by murine
specificity and CNS/PNS glial-specific expression. The
promoter has been characterized and is available
(Balcarek et al., Nucleic Acids Res. 13:5527-5543
(1985)).
Methallothionein III (MT-Ill) Promoters
MT-III is a third member of the
methallothionein gene family and its expression appears
to be restricted to brain in mice. MT-III is abundant in
human brain and is immunohistochemically localized to
astrocytes in the grey matter (Uchida et al., Neuron
7:337-347 (1991)). In the Alzheimer's disease cortex,
the number of MT-III positive astrocytes are drastically
reduced and this reduction is highly correlated with the
abundance of neurofibrillary tangles and curly fibers
(Uchida et al., Neuron 7:337-347 (1991)). The
involvement of astrocytes in beta-amyloid protein
amyloidosis as observed in Alzheimer's disease has been
previously suggested (Potter et al., Prog. Brain Res.
94:447-458 (1992)). The MT-III promoter has been cloned
and characterized (Palmiter et al., Proc. Natl. Acad.
Sci. USA 89:6333-6337 (1992)).
Prion Protein Promoter.
This promoter has been shown to drive position-
independent copy number-dependent transgene product
expression in the mouse brain (Scott et al., Cell 59:847-
857 (1989); Prusiner et al., Cell 63:673-686 (1990)).
This promoter was also used to create transgenic mice
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that overexpress the beta-amyloid precursor protein
(Hsiao et al., Neuron 15:1203-1218 (1995)).
insulin Promoter.
The insulin promoter can be used for the
development of a transgenic mouse model to mimic islet
amyloidosis as observed in patients with type II diabetes
(Verchere et al., Proc. Natl. Acad. Sci. USA vol. 93, pp. 3492-3496
(1996)). The insulin pramter can be used to achieve the
specific expression of transgene in beta-cells
(Sarvetnick et al., Cr,11 52:773-782 (1988)). The human
insulin promoter has been characterized and is available.
Neuron-Specific Eaolase Promoter
This promoter directs the neural-specific
expression of a reporter gene in transgenic mice (Fross-
Petter et al., N~~Qn 5:187-197 (1990)) and has been used
to make tranagenic mice which overexpress the beta-
amyloid precursor protein (Quon et al., NatuXe 352:239-
241 (1991)).
Low-affinity Nerve Growth Factor Receptor (p75) Promoter
p75 is mainly expressed in central and
peripheral cholinergic neurons. The activity of p75
promoter has been tested in cultured neurons (Taiji et
al., Mol. Ce1Z. Bi 1. 12:2193-2202 (1992)). Since loss
of cholinergic neurons in brain represents an important
feature of Alzheimer's disease, this promoter can be
useful to create such models.
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Methallothionein I Promoter
This promoter was used to create transgenic
mice that produce a mutant form of human transthyretin as
a potential model for type I familial amyloidotic
polyneuropathy (Yi et al., Am. J. Path. 138:403-412
(1991))= These mice develop amyloid deposits in the
gastrointestinal tract, cardiovascular system, and
kidney, but not in the peripheral nervous system where
amyloid deposition occurs in humans. Therefore, this
promoter may be effective for producing perlecan
transgenic mice and/or transgenic mice which overexpress
both perlecan and transthyretin, for a new animal model
of prealbumin/transthyretin amyloidosis.
Other known promoters that are tissue specific
may also be used to produce animal models of amyloidoses
affecting specific organ systems. Such promoters can be
used by those skilled in the art. In addition, new
promoters may become available which may be also used by
those skilled in the art to drive the expression within
desired tissues or organs for the ultimate production of
amyloid transgenic mice.
It is understood that modifications which do
not substantially affect the activity of various
embodiments of this invention are also included within
the definition of the invention provided herein.
Accordingly, the following examples are intended to
illustrate but not limit the present invention.
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EXAM1e 1
Expression of Domains I-V of Murine Perlecan
in COS and P19 Ce11s
To facilitate the expression of murine perlecan
in mammalian cells, the expression vector, designated
pCA-DI-V was transfected into an African green monkey
kidney cell line (COS), and into embryonic carcinoma
cells (P19 cells) which upon stimulation with the
appropriate amount of retinoic acid differentiate into
neuron-like cells. This transfection was done by
liposome-mediated gene transfer (Lipofectamine* a product
of Life technologies) according to the manufacturer's
protocol. The neomycin-resistance gene that confers
G418-resistance was also transfected together with the
pCA-DI-V vector. By screening the transfected cells with
G418, 20 clones for each cell line (COS and P19 cells)
were isolated and subjected to Western blot analysis to
determine the levels of perlecan expression. More
precisely, COS-7 cells (African green monkey kidney cell
line; available from American Tissue Culture Collection,
ATCC# CRL-1651) and P19 cells (Rudnicki and McBurny in
Teratocarcinoma and Embrvonic Stem C 1 a: A Pra-ri_al
Anoroach, IRL Press, Washington, D.C., pp. 19-50,(1987))
were cultured in Dulbecco's modified Eagle's medium
(DMEM) containing 10t fetal bovine serum, 100 g/ml
streptomycin and 100 units/ml penicillin (regular
medium). 1-2 x 105 cells/60 mm dish were inoculated one
day before tran=sfection of DNA. The cells were washed
*
with Opti-MEM I Reduced-Serum Medium (a product of Life
Technologies) prior to transfection to remove serum from
*Trade-mark
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the culture. Three ug of pCA-DI-V, 0.1 /.cg of pMAMneo (a
product of Clontech Laboratories Inc.) and 20 41 of
Lipofectamine were mixed, incubated for 15 minutes at
room temperature and added to the cells cultured in
Optim-MEM I Reduced Serum Medium. The COS-7 and P19
cells were incubated at 37 C in a humidified atmosphere of
5o COz for 18 and 6 hours, respectively, and Optim-MEM I
Reduced-Serum medium was replaced with regular medium.
Two days after transfection, the cells were treated with
0.05o trypsin and 530 ,uM EDTA and replaced at a
concentration of 10,000 cells/100 mm dish with selection
medium (regular medium with 800 /.cg/ml G418). Two to
three weeks after transfection, emerging resistant
colonies were isolated using clonal rings and propagated
for storage and Western blot analysis. For each cell
line, approximately 20 clones were isolated.
Example 2
Up-Regulation of Perlecan Levels in Transfected COS
and P19 Cells as Revealed by Western Blot Analyses
To determine the potential differences in
perlecan levels in transfected versus non-transfected COS
and P19 cells, the media and cell layers were analyzed by
Western blotting with a specific monoclonal antibody (HK-
102) against perlecan core protein. From each cell
culture dish, the media was collected for analysis as
described below, making sure that equal volumes of media
were analyzed from either transfected or non-transfected
cells. The cell layers were harvested by scraping into
urea buffer (urea buffer contained 7 M urea, 0.2 M NaCl,
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0.1t (w/v) CHAPS, 50 mM Tris-HC1, pH 8.0, containing a
protease inhibitor cocktail including 10 mM EDTA, 10 mM
NEM, 10 mM 6-aminohexanoic acid, 5.0 mM benzamidine-HC1,
and 1 mM PMSF).
For COS cells, PGs from cell layer and media
extracts were then purified by DEAE-Sephacel* ion-exchange
chromatography. Briefly, cell layer and media extracts
were pooled separately, and supplemented with 0.5t Triton
*
X-100 (v/v) and applied to a 1 ml DEAE-Sephacel column
packed in a plastic syringe equilibrated with urea buffer
(urea buffer contained 7 M urea, 0.2 M NaCl, 0.1k (w/v)
CHAPS, 50 mM Tris-HC1, pH 8.0, containing a protease
cocktail including 10 mM EDTA, 10 mM NEM, 10 mM
6-aminohexanoic acid, 5.0 mM benzamidine-HC1, and 1 mM
PMSF). Proteins and non-PGs were removed by first
washing the column with 5 column volumes of urea buffer
(described above) containing 1%- Triton X-100 and 0.25M
NaCl. Bound PGs were then eluted with 5 column volumes
of urea buffer containing 3M NaCl. Equal proportions of
each sample were then precipitated by adding 3.5 volumes
of 95t ethanol, 1.5$ potassium acetate (w/v), cooled on
dry ice for 1 hour and centrifuged (using an Eppendorf*
5415C desktop centrifuge) at 14,000Xg for 20 minutes.
Samples were then left either undigested, or digested
with heparinase/heparitinase and/or chondroitinase ABC.
For heparinase/heparitinase digestion, samples were
suspended in 50 IA1 of 0.2M Tris-HC1, 5 mM calcium acetate
(pH 7.0) and digested with a mixture of heparinase I, II,
and III (i.e. heparitinase)(Sigma Chemical Co.) used at
lU each (approximately 10 mU/ g protein) in 30 Al of
*Trade-mark
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glycerol:distilled water (1:1). Following addition of 10
Fcl of a 10X protease inhibitor cocktail (as described
above), heparinase/heparitinase containing samples were
incubated overnight at 41 C and ethanol precipitated (as
described above), prior to separation by SDS-PAGE.
Samples treated with chondroitinase ABC were digested
with 100 mU of chondroitin ABC lyase (ICN Biochemicals)
in Tris-buffered solution (50 mM Tris, 50 mM sodium
acetate, 10 mM EDTA, and protease inhibitor cocktail as
described above, pH 7.5) at 37 C for 2 hours. Each sample
was then heated for 5 minutes in a boiling water bath,
loaded onto SDS-PAGE and electrophoresed at 100V for 45
minutes along with pre-stained molecular weight protein
standards (a product of Bio-Rad). SDS-PAGE were
performed according to the procedure of Laemmli (Nature
227:680-685 (1970)) using a Mini-Protean II
electrophoresis system with precast 4-15Ir polyacrylamide
gels. Samples were run under non-reducing conditions for
use with a monoclonal antibody (HK-102) against perlecan
core protein. Following SDS-PAGE, separated proteins
were transferred to nitrocellulose using a Mini transblot
electrophoresis transfer cell. Electrotransfer was
performed at 10oV for 2 hours. Following transfer,
membranes were rinsed in water and blocked overnight with
0.15V (w/v) bovine serum albumin, It (v/v) normal goat
serum, 100 mM Tris-HC1, and 3mM NaN. (pH 7.4). Blots were
probed with a monoclonal antibody (HK-102) which
recognizes the perlecan core protein, diluted 1:3000 in
TBS containing 100 mM Tris-HC1, 50 mM NaCl, 0.05t Tween-
20* and 3 mM NaN3 (pH 7.4)(TTBS). Blots were incubated
with primary antibody for 3 hours, washed with TTBS three
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times (10 minutes each), followed by a 1 hour incubation
in biotinylated goat-anti-rat Ig's (IgG and IgM) (Jackson
Immunoresearch, West Groven, PA) diluted 1:1000 with
TTBS. The membranes were then rinsed three times (10
minutes each) with TTBS, probed for 30 minutes with
avidin alkaline phosphatase conjugate (Vectastain),
rinsed again (as described above), followed by the
addition of an alkaline phosphatase substrate solution
(Vectastain). Following color development, the reaction
was stopped by flushing the membranes with double-
distilled water. Immunodetection of perlecan core
protein using the monoclonal (HK-102) antibody was
employed in at least three different cell culture
experiments in order to determine reproducibility. For
comparisons perlecan was isolated from the Engelbreth-
Holm-Swarm tumor as previously described (Castillo et
al., J. Biochem. 120:433-444 (1996)) and digested with
heparinase I, II and III (as described above) prior to
Western blotting.
As shown in Figure 4, Western blot analyses of
PGs isolated from the cell layer of non-transfected COS
cells demonstrated no immunoreactivity on Western blots
probed with a monoclonal antibody (HK-102) which
recognizes perlecan core protein (Figure 4, lanes 1 and
3). On the other hand, Western blot analyses of PGs
isolated from the cell layer of transfected COS cells
overexpressing the entire perlecan core protein, revealed
a -400 kDa band using the monoclonal antibody (HK-102)
specific for perlecan core protein (Figure 4, lane 2 and
lane 4). Chondroitinase ABC digestion did not appear to
I I
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cause a shift in the -400 kDa band in the cell layer of
transfected.cells when compared to undigested sample (not
shown), suggesting that little to no chondroitin sulfate
chains were present on the perlecan core protein
produced. On the other hand, a slight shift and/or
tighter -400 kDa band was observed in the cell layer of
transfected cells following heparinase/ heparitinase
digestion (Figure 4, lane 4) when compared to undigested
sample (Figure 4, lane 2), suggesting that the perlecan
core protein produced in the cell layer of transfected
cells contained heparan sulfate chains that were either
shorter or less in number than normal (i.e. perlecan is
usually thought to contain 3 glycosaminoglycan chains
attached to its core protein). The perlecan core protein
band on immunoblots from cell layer of transfected COS
cells (Figure 4, lanes 2 and 4) was similar in position
to that of pure perlecan isolated from the Engelbreth-
Holm-Swarm tumor (Figure 4, lane 5). However, whereas
isolated perlecan gave a characteristic doublet at -400
kDa (Fiure 4, lane 5), the cell layer from transfected
COS cells demonstrated a single major band at -400 kDa
(Figure 4, lane 2 and lane 4).
Similar results were obtained by Western blot
analyses of PGs isolated from the media (Figure 5) of
non-transfected (Figure 5, lanes 1 and 3) and transfected
(Figure 5, lanes 2 and 4 ) COS cells, when probed with
the monoclonal antibody (HK-102) against perlecan core
protein. A marked accumulation of perlecan core protein
immunoreactivity was observed in transfected COS cells
(Figure 5, lanes 2 and 4) in comparison to non-
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transfected cells (Figure 5, lanes 1 and 3).
Chondroitinase ABC digestion did not cause a shift in the
-400 kDa band isolated from the media of transfected
cells (Figure 5, lane 4) when compared to undigested
sample (Figure 5, lane 2), suggesting that little to no
chondroitin sulfate chains were present on the perlecan
core protein secreted. On the other hand, a slight shift
and/or tighter -400 kDa band was observed in the media of
transfected cells following heparinase/heparitinase
digestion when compared to undigested sample from
transfected media (not shown), suggesting that the
perlecan core protein produced in the media of
transfected cells contained heparan sulfate chains that
were either shorter in length or less in number than
normal. Perlecan core protein band on immunoblots from
media of transfected COS cells (Figure 5, lanes 2 and 4)
was similar in position to that obtained from pure
perlecan (Figure 5, lane 5), and a similar characteristic
double band at -400 kDa was observed in media from
transfected COS cells (Figure 5, lanes 2 and 4).
The fact that the perlecan bound to DEAE-
Sephacel for isolation indicated that the perlecan most
likely contained glycosaminoglycan chains. Since
heparinase/heparitinase digestion, and not chondroitinase
ABC caused a slight shift in the position of the -400 kDa
core protein on Western blots of both media and cell
layer from transfected COS cells, it suggested that
heparan sulfate GAG chains (although shorter in length or
less in number than usual) were present on the perlecan
core protein of transfected cells. Nonetheless, this
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study represents the first time that successful
transfection of the entire perlecan core protein has been
achieved, and represented the first important critical
step for the development of perlecan transgenic mice over
expressing the entire -400 kDa core protein.
For P19 cells, following isolation of PGs from
cell layer and media, and SDS-PAGE, as described above,
the proteins were transferred to PVDF membrane (a product
of Millipore) at 260 mA for 18 hours using the Bio-Rad
Transblot System. The membranes were blocked with PBS
containing 5% nonfat dried milk (w/v), 0.02% sodium
azide, and 0.02% Tween 20, then incubated at 40 for 18
hours with primary antibodies (polyclonal or monoclonal
antibodies recognizing perlecan core protein) and
immunostained with an enhanced chemiluminescence system
(a product of Amersham). As found with the
overexpression of perlecan core protein in transfected
COS cells, the western blot analyses of P19 cell layer
and media revealed high levels of perlecan production in
transfected cells, whereas non-transfected cells showed
little to no immunoreactivity (not shown). These studies
confirmed that overexpression of the perlecan core
protein could be achieved in transfected COS and P19
cells by the construction strategy used in the present
invention.
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Example 3
Up-Regulation of Secreted Ai3 in Media of P19 Cells
Overexpressing Both Perlecan and i3PP-695
A P19 derived cell clone had previously been
established and designated P19 695B2, which overexpresses
the 695 amino acid isoform of the human beta-amyloid
precursor protein (ZPP-695)(Fukuchi et al., J. Neurochem.
66:2201-2204 (1996)). In this example, the P19 695B2
cells were transfected with the pCA-DI-V vector, and
stable transformants designated P19 695B2 PCB6 and P19
695B2 PCD3, which overexpress both perlecan and i3PP-695
were established using the methods described in Example
1. Overexpression of l3PP-695 and perlecan was confirmed
by Western blotting as in Example 2.
To demonstrate that overexpression of perlecan
in P19 695B2 D3 led to increased perlecan protein
production, perlecan was isolated and analyzed by Western
blotting in the presence or absence of heparitinase
and/or chondroitinase ABC pretreatment (to degrade
heparan sulfate and/or chondroitin/dermatan sulfate GAGs,
respectively) as described in Example 2. As shown in
Figure 6, P19 cells only overexpressing i3PP-695 (i.e. P19
695 B2) did not demonstrate detectable perlecan in cell
lysates or media. On the other hand, P19 cells
overexpressing both perlecan and i3PP-695 (i.e. P19 695B2
PC D3) demonstrated a marked increase in perlecan protein
in both cell lysates and media as demonstrated by Western
blots using a specific perlecan polyclonal antibody
(Figure 6). Cellular perlecan was identified as a-400
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kDa band or smear using the specific perlecan antibody
(Figure 6). Heparitinase digestion (to remove heparan
sulfate GAG chains) only slightly increased the
immunodetection of cellular perlecan, suggesting that
cellular perlecan may be lacking full length heparan
sulfate GAG chains. On the other hand, the -400 kDa band
of perlecan isolated from media was visualized only after
heparitinase and heparitinase/Chondroitinase ABC
treatments (Figure 6, arrow), indicating that secreted
perlecan produced in these cells had heparan sulfate GAG
chains. Thus, overexpression of perlecan in P19 cells
leads to overproduction of perlecan in media which may be
useful for the isolation of perlecan in relatively large
quantities.
To determine the potential effects of perlecan
overexpression on f3PP and A9 levels in P19 cells, the
media and cell layers of P19 cells overexpressing both
perlecan and 9PP-695 (i.e. P19 695B2 PC B6 and P19 695B2
PC D3) were compared to cells overexpressing i3PP-695 only
(i.e. P19 695 B2 PC), by immunoprecipitation followed by
Western blotting with specific antibodies (6E10 which
recognizes human Af3, and 6561 which recognizes the
C-terminus of i3PP). All cells (P19 695B2, P19 695B2 PC
B6, and P19 695B2 PC D3) were induced to differentiate
into neurons by treatment with retinoic acid as
previously described (Fukuchi et al., J. N ro-h m.
58:1863-1873 (1996)). On the 8th day of differentiation,
the media was collected and centrifuged at 12,000 X g for
1 hour and the supernatant was made to 10 mM Tris, 1%
Nonidet P-40, 0.5% cholic acid, 0.1% sodium dodecyl
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sulfate (SDS), and 5 mM EDTA, 2 g/ml leupeptin and 0:1
g pepstain (pH 8.0). After preabsorption with 50M1 of
protein A-agarose (previously coupled to rabbit antibody
against mouse IgG), the media was incubated with primary
antibodies 6E10 and 40,ul of protein A-agarose (pretreated
as described above) for 16 hours. Precipitates were
washed three times with TBS containing 1t Nonidet P-40, 5
mM EDTA, 2 fc.g/ml leupeptin, 2-mM phenylmethyl
sulfonylfluoride and was boiled in 20 l of 2X laemmli
buffer for 5 minutes before loading onto 7.5V (to detect
higher molecular weight bands) or 16.5% (to detect low
molecular weight bands) SDS polyacrylamide gels. The
cells were lysed by adding 2X Laemmli buffer (1X = 62.5
mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5t
2-mercaptoethanol, 0.001t bromophenol blue), boiled for 5
minutes,.and sheared with a 26-gauge needle. Protein
concentrations were determined by Bio-Rad Protein Assay
*
(a product of Bio-Rad laboratories). Aliquots
corresponding to 30 g of protein were applied to 7.5%
SDS-polyacrylamide gel electrophoresis (PAGE) and Tris-
Tricine 16.5% SDS-PAGE. Following electrophoresis,
proteins were electrotransferred to a polyvinylidine
difluoride (PVDF) membrane (Immobilon-P* a product of
Millipore). The membrane was blocked with phosphate-
buffered saline (PBS) containing 5t nonfat dried milk
(w/v), 0.02t sodium azide, and 0.02% Tween 20*, then
incubated at 4 C for 16 hours with the 6561 antibody
(which recognizes amino acids 681-695 of BPP-695) or 6E10
antibody (which recognizes human Ab) and immunostained
with an enhanced chemilumineacence system (a product of
Amersham Co.). The relative concentration of the protein
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was determined by densitometric scanning (Molecular
Analyst/PC, a product of Bio-Rad).
As shown in Figure 7, f3PP was evident in both
the cell lysates and media of transfected P19 cells
overexpressing i3PP-695 (i.e. P19 695B2) as discrete or
smeared bands at -100 and 130 kilodaltons (arrow)
following Western blots using the 6561 or 6E10
antibodies. On the other hand, a -4-fold increase in SPP
levels in the media was apparent in transfected P19 cells
overexpressing both perlecan and i3PP-695 (i.e. P19 695B2
PC B6 and P19 695B2 PC D3)(Figure 7). The P19 695B2 PC
D3 cell clone also demonstrated a -4-fold increase in SPP
levels in the cell lysates. These studies indicate that
overexpression of both perlecan and SPP-695 leads to a
marked increase in SPP levels in the media and cell layer
of P19 cells, as compared to P19 cells which overexpress
only ZPP-695. Overexpression of perlecan may therefore
contribute to changes in l3PP metabolism and levels.
Besides an effect on increasing levels of f3PP
in both media and cell, perlecan overexpression also led
to a marked increase in the secretion of AA into the
media. As shown in Figure 8, Western blot analysis
utilizing the 6E10 antibody barely detected AfS (Figure 8)
in media from cells overexpressing i3PP-695 only (i.e. P19
695B2). On the other hand, overexpression of both
perlecan and i3PP-695 (i.e. P19 695B2 PC B6 and P19 695B2
PC D3) led to a marked increase in Ai3 levels in the media
(Figure 8, arrow). When secreted Af3 levels were
normalized to levels of cellular and secreted i3PP, an 8-
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l0-fold increase was demonstrated in P19 cells which
overexpress both perlecan and f3PP-695 (Figure 9). These
results indicate that overexpression of perlecan leads to
an increase in secreted AS levels.
Examnle 4
Overexpression of Perlecan Leads
to Degeneration of P19-Derived Neurons
To study the effects of perlecan overexpression
on neuronal survival, P19 cells overexpressing perlecan
only (designated P19 PC C2 and P19 PC D2) were also
induced to differentiate into neurons. These additional
P19 cell lines were isolated by stable transfection of
pCA-DI-V and perlecan overexpression in these two clones
was confirmed by Western blot analysis as described in
Example 1 and Example 2. Transformed cell clones
overexpressing perlecan only (P19 PC C2, P19 PC D2),
perlecan and f3PP-695 (P19 695B2 PC B6 and P19 695B2 PC
D3), ZPP-695 only (P19 695B2) and their parental cells
(P19) were induced to differentiate into neuronal cells
by treatment with retinoic acid. Proliferating non-
neuronal cells were eliminated from the cultures by
treatment with 10 M of cytosine fS-D-arabinofuranoside
(Ara C). Eight days following initial treatment with
retinoic acid, surviving neurons were quantified by
measuring produced formazan from MTS according to the
manufacturer's protocol (Cell Titer 96 Aqueous Assay, a
product of Promega). For each cell clone 5 independent
cultures were prepared and subjected to statistical
analysis. The results are shown in Figure 10. Cell
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survival of the clones overexpressing perlecan only (i.e.
P19 PC C2, P19 PC D2) were significantly lower than
control P19 cells (i.e. P19), and in comparison to cells
overexpressing EPP-695 only (i.e. P19 695B2). The P19
695B2 PC D3 line which overexpresses both perlecan and
f3PP-695 also showed a decrease in neuronal survival,
especially when compared to cells which only overexpress
f3PP-695 (i.e. P19 695B2). All of the cells of the P19
695B2 PCB6, which overexpress both perlecan and ZPP-695
died out. These studies therefore demonstrated that
overexpression of perlecan also has an effect on neuronal
cell survival. Therefore, cells which overexpress
perlecan only, and perlecan and ZPP may be utilized a
screening tools for the development of new therapeutic
and preventive agents for Alzheimer's disease. In a
preferred embodiment, anti-Alzheimer's compounds can be
tested and will be deemed effective if they cause a
lowering of secreted Af3 levels in the media, and/or
increase neuronal survival.
Example 5
Construction of Perlecan Transgenic Expression Plasmids
The expression vector, pCA-DI-V (Figure 3C),
was used to prepare DNA for microinjection into mouse
fertilized eggs. pCA-DI-V was digested with Hind III
enzyme and electrophoresed on agarose to separate the
bacterial DNA sequences from the DNA construct to be used
for microinjection. The DNA construct that contained the
cytomegalovirus enhancer/beta-actin promoter, cDNA for
the entire perlecan core protein, and a polyadenylation
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signal were electrophoresed onto a DEAE-cellulose
membrane. The DNA on the membrane was eluted by a high
salt buffer (50 mM Tris-HC1, pH 8.0, 1M NaCl, and 10 mM
EDTA, pH 8.0) and extracted with phenol and chloroform.
After precipitating the DNA by ethanol, the DNA was
resuspended in Tris-EDTA (10 mM Tris-HC1, pH 7.5, 0.25mM
EDTA) and used for microinjection into fertilized eggs.
Exanmle 6
Collection and injecting the Eggs with the DNA Construct
The transgenic organisms of the invention all
include within a plurality of their cells a cloned
recombinant or synthetic DNA sequence which is believed
to relate to the pathogenesis of the amyloid diseases.
More s,pec,ifically, the transgenic organisms contain
specific sequences of exogenous genetic material which
are comprised of a specific promoter sequence which
allows for production of perlecan core protein. since it
is possible to produce transgenic organisms of the
invention, a general descriptidn will be given of the
production of transgenic organisms by referring generally
to exogenous genetic material. This general description
can be adapted by those skilled in the art in order to
incorporate the above-described specific DNA sequences
into organisms and obtain expression of those sequences
utilizing the materials and methods described below. For
more information regarding the production of transgenic
mice refer to U.S. Patent No. 4,873,191 issued Oct. 10, 1989,
and to the scientific publications referred to and cited
therein.
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One-cell stage embryos (F2 of C57BL/6J X SJL)
were collected from the oviducts of Fl female mice of
C57BL/6J X SJL/J that had been already mated with F1 male
mice of C57BL/6J X SJL/J. These embryos should be early
enough to distinguish male pronuclei from female
pronuclei. Cumulus cells surrounding oocytes were
removed by hyaluronidase treatment, washed properly and
incubated at 37 C in an atmosphere of 5% CO2 for a certain
time prior to DNA injection. Any method which allows for
the addition of the exogenous genetic material can be
utilized as long as it is not destructive to the cell,
nuclear membrane or other existing cellular or genetic
structures. The exogenous genetic material is preferably
inserted into the nucleic genetic material by
microinjection. Microinjection of cells and cellular
structures is known and is used in the art. About 1-2
picoliters (pl) of the DNA construct prepared in Example
(about 100-200 copies of the transgene per pl) was
microinjected into a male pronucleus. Approximately 100
embryos were injected with the DNA construct described in
Example S. The injected embryos were incubated from
several hours to one day and then transplanted to the
oviducts of pseudopregnant ICR foster mothers of Day 1 of
pregnancy. The day when a vaginal plug was recognized
was recorded as Day 1. The transplanted foster mothers
were fed until a delivery of the fetus. After delivery,
neonates (25 pups) were nursed by the foster mothers for
4 weeks until weaning. At the time of weaning, the tails
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of the neonates were cut for Southern blot analysis to'
determine the integration of transgenes into mouse
chromosomes. The founder mice, judged as transgenic by
Southern blot analysis, were back-crossed with C57BL/6J
mice to establish lines of transgenic mice with C57BL/6J
genetic background.
Exaamle 7
Determination of Tranegene Copy Number'
Pups produced from microinjection of the DNA
construct (pCA-DI-V) were grown to 4 to 6 weeks old and
then the tails of the pups were cut. The DNA was
isolated from the tails using QlAamp tissue kit (a
product of Qiagen Inc.). 10 oug of DNA isolated from
mouse tails were digested with Sca I restriction enzyme
and separated by electrophoresis on a 0.9%- agarose gel.
Additionally, pCA-DI-V was digested with Sca I
restriction enzyme and 0.045 ng (1 copy), 0.225 ng (5
copies) and 0.45 ng (10 copies) of the digested pCA-DI-V
were also separated on the same agarose gel as a
reference positive control. The separated DNA was then
blotted onto a nylon membrane. The membrane was
prehybridized for 6 hours at 42 C in 50t formamide, 5X
saline sodium citrate (SSC; 1XSSC=0.15M NaCl and 0.015 M
sodium citrate, pH 7.0), 5X Denhardt's solution (1X
Denhardt's solution= 0.02% each of Ficoll~,
polyvinylpyrrolidone, and bovine serum albumin), 0.1V
sodium dodecyl sulfate (SDS), and 100 mg/ml of denatured
herring sperm DNA and then hybridized in the above
solution containing 10V dextran sulfate and _10' cpm of a
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radiolabeled probe at 42 C for 18 hours. The radiolabeled
probe was the 1.7-kb Sca I fragment of mouse perlecan
cDNA (bp 5523-7215 of perlecan cDNA)(Noonan et al., y'[,
Biol. Chem. 266:22939-22947 (1991)). The membrane was
then washed at 65 C in 0.2 X SSC and 0.5% SDS. The
*
membrane was exposed to Kodak XAR film with an enhancer
screen, at -80 C for 4 days. As shown in Figure 11, out
of approximately 25 pups, the integrated transgene in the
mouse genome was identified as a 1.7-kb fragment on
Southern blot in four founder mice (designated #3595,
3697, 3763 and 3694 in Figure 11). Founder mouse #3595
was found to have 5-10 copies of pCA-DI-V whereas founder
mouse #3697, 3763 and 3694 had approximately one copy of
pCA-DI-V.
E]CatnDle B
Confirmation of Perlecan overexpreesion
in Perlecan Tranagenic Mice
The founder mice (#3595, 3697, 3763 and 3694)
were mated with C57BL/GJ mice. Each of the male founders
(#3694 and 3595) were housed in one cage together with
two C57BL/6J female mice, and each of the female founders
(# 3697 and 3763) were housed in one cage together with
one C57BL/6J male mouse. Every female mated gave birth
to 8-12 mice indicating that the founders were fertile.
The mouse tails from the progeny are cut to perform
Southern blot analysis as described above to demonstrate
the tranagene segregation. Mice determined as transgenic
by Southern blot analysis are mated with C57BL/6J mice
for propagation.
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Levels of transgene expression in the
transgenic mice were determined by Northern blotting for
mRNA, Western blotting and immunostaining for protein.
Most of the techniques which are used to perform Northern
and Western blot analysis are widely practiced in the art
and most practitioners are familiar with the standard
resource materials as well as specific conditions used in
the procedures.
Northern Blotting: Three mice were sacrificed
by cervical dislocation under anesthesia; T 213, a 3-
month old progeny from perlecan transgenic founder 3595
(5 copy founder); T 156, a 3-month old progeny from
perlecan transgenic founder 3694 (1 copy founder), and C
196, a 3-month non-transgenic control litter-mate of T
213. Tissues including brain, heart, liver, kidney,
spleen and intestine were quickly removed and homogenized
in a denaturing solution consisting of 4M guanidinium
thiocyanate, 25mM sodium citrate, pH 7.0; 0.5% sarcosyl,
0.1M 2-mercaptoethanol) and passed through a syringe
fitted with a 27 gauge needle. After adding sodium
acetate, the RNA was extracted with phenol/chloroform and
precipitated with isopropanol. The precipitated RNA was
resuspended again with the above denaturing solution and
precipitated with isopropanol. After rinsing the RNA
with 75% ethanol, the RNA was resuspended with sterile
double distilled water. 20 ).cg of total RNA from each
tissue was electrophoresed through a 1% agarose-
formaldehyde gel, followed by capillary transfer to a
nylon membrane. A perlecan probe (mouse perlecan cDNA,
bp 5523-7215 of perlecan cDNA as described in Noonan et
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al., J_ Biol. Chem_ 266:22939-22947 (1991)) was
radiolabeled with 12P dCTP to a specific activity of 4 X
lo= cpm/ g using a random primed labeling kit (a product
of Boehringer Mannheim). The membrane was hybridized for
20 hours at 42 C in 50% formamide 5X SSC (1X SSC= 0.15M
NaCl, 0.015M sodium citrate, pH 7.0), 0.1V SDS and 100
g/ml of denatured herring sperm DNA. Following
hybridization, the membrane was washed twice at 65 C in
0.2 X SSC and 0.2k SDS for 30 minutes each_ The membrane
*
was then exposed to Kodak XAR film with enhancer screens
at -700C for 6 hours. The results are shown in Figure 12.
High levels of expression of the transgene mRNA with the
expected 11.6 kb size, were observed in brain, heart,
kidney, spleen and intestine of T 213 (progeny of founder
with 5 copies of tranagene). Levels of perlecan mRNA in
T 156.(progeny of founder with 1 copy of transgene) were
much lower'than those in T 213 (Figure 12). No
endogenous mRNA for perlecan was detected in any of the C
196 tissues. This study demonstrated that perlecan
overexpression was particularly evident in brain and in
other tissues of tranagenic mice carrying -5 copies of
the transgene.
Western 8lotting: Levels of the protein
products from the transgene in the tranagenic mice were
determined by Western blot analysis. The tissues (i.e.
brain, heart, liver, kidney, spleen, intestine and
pancreas) isolated from transgenic T 213 and control C
196 were homogenized in 2X Laemmli buffer (1X= 62.5 mM
Tris-HC1, pH 6.8, 2t SDS, 10t glycerol, 5%
2-mercaptoethanol, 0.001% bromophenol blue), boiled for 5
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min, and sheared with a 26-gauge needle. Protein
*
concentration was determined by Bio-Rad Protein Assay (a
product of Bio-Rad). Aliquots corresponding to 30 jcg of
protein were applied to 4-203c gradient gels for SDS-PAGE.
Following electrophoresis, proteins were
electrotransfered to a polyvinylidine difluoride (PVDF)
membrane (Immobilon-F;' Millipore). The membrane was
blocked with phosphate-buffered saline containing 5t
nonfat dried milk (w/v), 0.02t sodium azide, and 0.02t
Tween 20, then incubated at 4 C for 16 hours with perlecan
monoclonal or polyclonal antibodies which recognize
perlecan core protein, and immunostained with an enhanced
chemiluminescence system (a product of Amersham). The
results are shown in Figure 13. Substantial increased
amounts of transgene protein product (i.e. perlecan
detect,ed as a -400 kDa band or smear) were observed in
brain, heart, liver, kidney, and pancreas of T 213 in
comparison to control (i.e. C196). This study
demonstrated that perlecan overproduction successfully
occurred in brain and in other tissues, and was apparent
in a 3-month old perlecan transgenic mouse.
Iamunostaining: For immunostaining studies,
animals were first sacrificed by deep anesthesia. The
tissues of two 5-month old transgenic progenies from the
3595 (5 copy founder) and two 5-month old non-transgenic
litter-mates were subjected to immunostaining. The
tissues including brain, heart, intestine, kidney and
pancreas were removed, fixed by 10t formalin and embedded
in paraffin by procedures known to those skilled in the
art. 10 km sections were then cut and subjected to the
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avidin-biotin immunoperoxidase method to detect perleCan
using perlecan specific antibodies and the Vectastain ABC
kit (a product of Vector Laboratories) as described
below. Endogenous peroxidase was eliminated by treatment
with 3% hydrogen peroxide for 30 minutes after
deparaffinization of the sections. After rinsing with
distilled water, the sections were blocked with 15t goat
serum in Tris-buffered saline (TBS) for 60 minutes at
room temperature and incubated with a perlecan primary
polyclonal antibody (which specifically recognizes the
core protein of perlecan; antibody of Dr. J. Hassell) in
0.1M TBS containing 15t goat serum for 16 hours at 4 C.
The sections were then rinsed in 0.1 M TBS containing it
goat serum and incubated with biotinylated goat anti-
rabbit IgG secondary antibody for 1 hour at room
temperature. After washing, the sections were incubated
with Vectastain reagent for 60 minutes at room
temperature. Peroxidase activity was then detected by
treatment with 3,3'-diaminobenzidine. Strong
immunoreactivity for perlecan was evident in transgenic
mice but not in control liter-mates, especially in the
epithelium of the choroid plexus, the myofibrils of the
heart, the acidophil cells of the gastric gland, the
glomeruli of the kidney, and the cells in the islets of
Langerhans in pancreas. In addition, transgenic mice,
but not control litter-mates demonstrated moderate
perlecan immunostaining in the C3A neuronal layer of the
hippocampus, the Purkinje cell layer of the cerebellum
and the large neurons of the brain stem. Figure 14
demonstrates strong perlecan immunostaining in kidney
glomeruli (Figure 14A, arrows), and in the C3A neuronal
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layer of the hippocampus (Figure 14C, arrows) of a
5-month old perlecan transgenic mouse. In comparison,
control litter-mates demonstrate weak to no staining for
perlecan in kidney glomeruli (Figure 14B) and hippocampal
neurons (Figure 14D). These results confirm that
perlecan overexpression leads to perlecan protein
accumulation in specific sites in brain and in systemic
organs.
Examtile 9
Production of Beta-Amyloid Precursor Protein Mice
as a First Step in Producing a New Animal Model
of Alzheimer's Disease Amyloidosis
Four founder lines of: transgenic mice that
constitutively overproduce the signal sequence and the 99
amino acid C-terminal region of the human beta-amyloid
precursor protein were established. The cDNA for the
signal sequence and the 99 amino acid C-terminal region
(the construct referred to as SQC) of the human beta-
amyloid precursor protein (hereinafter called i3PP) was
placed under the control of a cytomegalovirus enhancer
and a chick beta-actin promoter in pCAGGS (Figure
15A)(Niwa et al., Gene 108:193-200 (1991)). To
overexpress SQC under control of a cytomegalovirus
enhancer and a chick 8 actin promoter, an expression
vector, pCA-SZC was constructed. Using the LMG2 human
brain stem lambda gtll cDNA library (a product of ATCC,
catalog #ATCC37432), a partial cDNA of the human f3PP,
extending from base pairs 901-2851 (Kang et al., Nature
325:733-736 (1987)) was isolated. The EcoRI/CIaI
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fragment (bp 1796-2473; Kang et al., Nature 325:733-736
(1987)) of the cDNA was subcloned into pBluescript*KS (a
product of Stratagene) at the same restriction enzyme
sites to create pKS-BC (Figure 15A). In a separate
reaction the signal peptide coding sequence for i3PP and
the sequence for the first 3 amino acids of the beta-
amyloid protein (BamHI site + bp-4-51 + bp 1789-1795 +
EcoRI site; bps are in Kang et al., Nature 325:733-736
(1987)) was chemically synthesized (Figure 15A). This
synthetic fragment was also ligated to the EcoRI/ClaI
fragment (bp 1796-2473) of EPP in pKS-8C using BamHI and
EcoRI sites to create the pKS-SSC plasmid. This plasmid
was digested with BamHI and SaII and the 0.8-kb fragment
coding for Si3C of EPP was isolated. The SaII site of the
0.8-kb fragment was ligated onto one of the XhoI sites of
the expression vector, pCAGGS (Niwa et al., Gena 108:193-
200 (1991)), which was previously digested with Xhol
(Figure 15B). The other ends were ligated after blunt
end formation with the Klenow fill-in reaction to create
pCA-SBC (Figure 15B). Fertilized eggs were injected into
pCA-SBC after removing bacterial sequence from pCA-SBC.
Four independent lines of tranagenic mice were
established. To determine the levels of mRNA expression
from the transgene, the total RNA from transgenic mice
was isolated and analyzed by Northern blot using a
radiolabeled human cDNA probe (bp 1795-2851; Kang et al.,
Nature 325:733-736 (1987)). The high levels of
expression of mRNA with the expected size, 1.06-kb from
the transgene were observed in virtually all tissues
examined including the brain (Fukuchi et al., Am J Path
149:219-227 (1996)). Native mRNA with a size of 3.2 kb
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was also observed in the brain and kidney. On Western
blots, -11-17 kDa protein products from the transgene
were observed in lung, muscle, intestine, liver and
kidney in transgenic mice, whereas no corresponding
fragments were observed in Western blots from the same
tissues of non-transgenic mice. A 3-5 fold increase in
the C-terminal fragments of SPP was found in the brains
of transgenic mice compared to controls. The amount of
the C-terminal fragments in the intestine was at least 50
times greater than that observed in the brain of
transgenic mice, and at least 3 times greater than that
observed in the heart of transgenic animals (Fukuchi et
al., Am. J. Path. 149:219-227 (1996)).
Six-month, 9-month, 12-month, 14-month and 16-
month-old transgenic mice bearing CA-Si3C were sacrificed
for immunohistochemical and histopathological analysis.
Nontransgenic siblings, ranging from 12-16 months were
used as controls. By immunohistochemical analysis, using
antibodies 1282 (a rabbit polvclonal antiserum raised
against beta-amyloid protein; Tamaoka et al., Proc. Natl.
Acad. Sci. USA 89:1345-1349 (1992)) and 10D5 (directed
against amino acids 1-16 of beta-amyloid protein; Hyman
et al., J. Neuropath. Exp. Neurol. 51:76-83 (1992)),
large immunoreactive deposits were found in the lamina
propria of the mucosa of the small intestine from
transgenic mice >12 months of age, but not in younger
mice. These deposits were positive for Congo red
(indicative of amyloid) demonstrating a red/green
birefringence as viewed under polarized light and
consisted of fibrils 7-10 nm in diameter as observed by
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electron microscopy. Surprisingly, these beta-amyloid
deposits were also found to be positive for perlecan
using perlecan specific core protein antibodies. The
accumulation of both beta-amyloid and perlecan in these
deposits suggests that both components are necessary for
amyloid deposition and serves as further evidence for the
basis of the present invention. Although fibrillar beta-
amyloid deposits were observed in the small intestine in
these transgenic mice, no pathological changes were
observed in the brains of transgenic mice indicating that
other co-factors, such as perlecan, are probably
necessary for the ultimate formation, deposition and
persistence of fibrillar beta-amyloid protein in brain.
Example 10
Production of Perlecan-Amyloid
Protein Double Transgenic Animals
Perlecan-amyloid protein transgenic animals are
produced by the mating of perlecan transgenic mice with,
for example, beta-amyloid precursor protein transgenic
animals. Double transgenic mice carrying both transgenes
(CA-Si3C and perlecan) were created by mating the two
lines of transgenic mice. Generally, 25% of emerging
pups from mating heterozygous parents for each transgene
should carry both transgenes, and 50 s of emerging pups
from mating heterozygous mice for one transgene with
homozygous mice for the other transgene should carry both
transgenes. Homozygous mice for CA-Si3C transgenes were
established by mating heterozygous mice for CA-Si3C. The
homozygotes for CA-Si3C was confirmed by crossing the
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homozygous mice for CA-SEC to C57BL/6J non-transgenic
mice. A homozygous male for CA-SJ3C was crossed to a
heterozygous female (a progeny from founder 3595) for the
perlecan transgene. Eight pups were produced by the
mating and the tails from the pups were cut to extract
the DNA for Southern blot analysis. The DNA was
hybridized with radiolabeled perlecan (bp 5523-7215 of
perlecan cDNA in Noonan et al., J. Biol. Chem. 266:22939-
22947 (1991)) and beta-amyloid (bp 1796-2473 in Kang et
al., Nature 325:733-736 (1987)) probes. The results are
shown in Figure 16. Pup #552 was found to positive for
both perlecan and beta-amyloid precursor protein (C-
terminal portion which contains the Af3 region)(shown in
Figure 16) and verified the successful production of a
double transgenic mouse which carried transgenes for both
perlecan and beta-amyloid.
Example 11
Screening of Compounds for Treatment of Alzheimer's
and Other Amyloid Diseases
The transgenic animals, transfected cells, or
animal cells derived from transgenic animals, can be used
to screen compounds for a potential effect in the
treatment of Alzheimer's disease and other amyloidoses
using standard methodology. In such screening assays,
the compound is administered to the animals, or
introduced into the culture media of transfected cells,
or cells derived from these animals, over a period of
time and in various dosages. Then the animals or animal
cells are examined for alteration in amyloid protein or
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amyloid precursor protein processing, expression levels,
or localization of other Alzheimer's disease or other
amyloid disease markers, and/or histopathology. In the
case of animals utilized for Alzheimer's disease,
behavior tests are implemented utilizing standard known
memory tests known to those skilled in the art. In
general, any improvement in behavioral tests, alteration
in Alzheimer's disease-associated markers, reduction in
the severity of Alzheimer's disease-related
histopathology, reduction in the expression of Ai3 or i3PP
cleavage products, and/or changes in the presence,
absence or levels or other compounds that are correlated
with Alzheimer's disease which are observed in treated
animals, relative to untreated animals, is indicative of
a compound useful for treating Alzheimer's disease. The
specific proteins, and/or histopathology of those
proteins, that are associated with and characteristic of
Alzheimer's disease are referred to herein as markers.
Expression or localization of these markers
characteristic of Alzheimer's disease has either been
detected, or is expected to be present, in the disclosed
transgenic animals. These markers can be measured or
detected, and those measurements compared between treated
and untreated transgenic animals to determine the effect
of a tested compound.
Markers useful for Alzheimer's disease
screening assays are selected based on detectable changes
in these markers that are associated with Alzheimer's
disease. Many such markers have been identified in
Alzheimer's disease and are expected to be present in the
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disclosed animals. These markers fall into several
categories based on their nature, location or function.
Preferred examples of markers useful in Alzheimer's
disease screening assays are described below, grouped as
Ai3-related markers, plaque-related markers, cytoskeletal
and neuritic markers, inflammatory markers, and neuronal
and neurotransmitter-related markers.
A. AS-Related Markers
Expression of the various forms of i3PP and Ai3
can be directly measured in treated and untreated
transgenic animals both by immunocytochemistry and by
quantitative ELISA measurements. Currently, it is known
that two forms of i3PP products are found, QPP and Ai3
(Haass and Selkoe, Cell 75:1039-1042 (1993)). They have
been shown to be intrinsically associated with the
pathology of Alzheimer's disease in a time dependent
manner. Therefore, preferred assays compare age-related
changes in f3PP and Ai3 expression in the transgenic mice.
Generally, specific forms of Ai3 can be assayed, either
quantitatively or qualitatively using specific
antibodies. When referring to amino acid positions in
forms of Ai3, the positions correspond to the Ai3 region of
i3PP. Amino acid 1 of Ai3 corresponds to amino acid 672 of
i3PP, and amino acid 42 of AS corresponds to amino acid
714 of f3PP.
Also preferred as targets for assay measurement
are f3PP markers. For example, different forms of
secreted EPP (termed f3PP-alpha and f3PP-i3) can also be
measured (Seubert et al., Nature 361:260-263 (1993)).
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f3PP forms are also preferred targets for assays to assess
the potential for compounds to affect Alzheimer's
disease. The absolute level of SPP and QPP transcripts,
the relative levels of the different i3PP forms and their
cleavage products, and localization of f3PP expression or
processing are all markers associated with Alzheimer's
disease that can be used to measure the effect of
treatment with potential therapeutic compounds. The
localization of EPP to plaques and neuritic tissue is an
especially preferred target for these assays.
Quantitative measurement can be accomplished
using many standard assays. For example, transcript
levels can be measured using RT-PCR and hybridization
methods including RNase protection assays, Northern blot
analysis, and R-dot analysis. i3PP and Ai3 levels can be
assayed by ELISA, Western blot analysis, and be
comparison of immunohistochemically stained tissue
sections. Immunohistochemical staining can also be used
to assay localization of i3PP and Af3 to particular tissues
and cell types.
B. Plaque-Related Markers
A variety of other molecules are also present
in plaques of individuals with Alzheimer's disease, and
their presence in plaques and neuritic tissue can be
detected. Preferred plaque markers are tau protein
(Grundke-Iqbal et al., Pror. Natl. Acad. Sci. USA
83:4913-4917 (1986); Kosik et al., Proc. Natl. Acad. Sci.
UEA 83:4044-4048 (1986); Lee et al., Science 251:675-678
(1991)), apolipoprotein E (Corder et al., Science
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261:921-923 (1993); Strittmatter et al., Proc. Natl.
Acad. Sci. USA 90:8098-8102 (1993)), alphal-
antichymotrypsin (Abraham et al., Cell 52:487-501
(1988)), amyloid P component (Coria et al., Lab. Investi
58:454-458 (1988)), ubiquitin (Mori et al., Science
235:1641-1644 (1987)), cytokines (McGeer et al., Can. J.
Neurol, Sci. 16:516-527 (1989) ; reviewed in Rogers, CNS
Dras 4:241-244 (1994)), growth factors (Hefti and
Weiner, Ann. Neurol, 20:275-281 (1986); Hefti et al.,
Neurobiol. Aginq 10:515-533 (1989); Kato et al., Neurosc.
122:33-36 (1991); Tooyama et al., Neurosc. Lett. 121:155-
158 (1991))), complement factors (Eikenbloom et al.,
Slirch. Arch. B Cell Pathol. 56:259-262 (1989)), advanced
glycosylation end products (Smith et al., Proc. Natl.
Acad. Sci. USA 91:5710 (1994)), receptor for advanced
glycosylation products, growth inhibitory factor, as well
as heparan sulfate PGs (Snow et al., Am. J. Path.
133:456-463 (1988)), chondroitin sulfate PGs (DeWitt et
al., Ex8. Neurol, 121:149-152 (1993)), dermatan sulfate
PGs (Snow et al., J. Histochem. Cytochem. 40:105-113
(1992)) and keratan sulfate PGs (Snow et al., Ep.
Neurol, 138:305-317 (1996)). While the above markers can
be used to detect specific components of plaques and
neuritic tissue, the location and extent of plaques can
also be determined by using well known histochemical
stains, such as Congo red and Thioflavin S.
C. Cytoskeletal and Neuritic Markers
Changes in cytoskeletal markers can also be
used in Alzheimer's disease screening assays to determine
the effect of compounds on Alzheimer's disease. Many of
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the changes in cytoskeletal markers occur in the
neurofibrillary tangles or dystrophic neurites associated
with plaques. The following are preferred cytoskeletal
and neuritic markers that exhibit changes in and/or an
association with Alzheimer's disease. These markers can
be detected, and changes can be determined, to measure
the effect of compounds on the disclosed transgenic
animals, or transgenic animals formed by mating with the
disclosed animals. Spectrin exhibits increased breakdown
in Alzheimer's disease. Tau and neurofilaments display
an increase in hyperphosphorylation in Alzheimer's
disease, and levels of ubiquitin increase in Alzheimer's.
Tau protein, ubiquitin, MAP-2, neurofilaments, heparan
sulfate PGs, dermatan sulfate PGs, chondroitin sulfate
GAGs and keratan sulfate PGs are localized to plaques and
neuritic tissues in Alzheimer's disease and in general
change from their normal localization. GAP43 levels are
decreased in the hippocampus in Alzheimer's disease.
D. Tnflaawnatory Markers
Alzheimer's disease is also known to stimulate
an inflammatory response, with a corresponding increase
in inflammatory markers (McGeer et al., Can. J. Neurol.
Sci. 16:516-527 (1989); Rogers, CNS Drugs 4:241-244
(1994)). The following are preferred inflammatory
markers that exhibit changes in and/or an association
with Alzheimer's disease. Detection of changes in these
markers are useful in Alzheimer's disease screening
assays. Acute phase proteins and glial markers such as
alphal-antitrypsin, C-reactive protein, alpha2-
macroglobulin (Tooyama et al., Molecular and Chem.
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Neuropath. 18:153-160 (1993)), glial fibrillary acidic
protein, Mac-1, F4/80, and cytokines, such as
interleukin-lalpha and 9, TNF-alpha, interleukin-8, MIP-
lalpha (Kim et al., J. Neuroimmunoloav 56:127-134
(1995)), MCP-1 (Kim et al., J. Neurol. Sci. 128:28-35
(1995); J. Neuroimmunol. 56:127-134 (1995)) and
interleukin-6, all increase in Alzheimer's disease and
are expected to increase in the disclosed animals.
Complement markers such as C3d, Clq, C5, C4d, C4bp, and
C5a-C9 are localized in plaques and neuritic tissue.
Major histocompatibility complex (MHC) glycoproteins,
such as HLA-DR and HLA-A, HLA-D,HLA-C increase in
Alzheimer's disease. Microglial markers, such as CR3
receptor, MHCI, MHCII, CD31, CD11a, CD11b, CD11c, CD68,
CD45RO, CD45RO, CD45RD, CD18, CD59, CR4, CD45, CD64, and
CD44 (Akiyama et al., Br. Res. 632:249-259 (1993))
increase in Alzheimer's disease. Additional inflammatory
markers useful in Alzheimer's disease screening assays
include alpha2-macroglobulin receptor, fibroblast growth
factor (Tooyama et al., Neurosc. Letts. 121:155-158
(1991)), ICAM-1 (Akiyama et al., Acta Neuropath. 85:628-
634 (1993)), lactotransferrin (Kawamata et al., Am. J.
Path. 142:1574-1585 (1993)), Clq, C3d, C4d, C5b-9, Fc
gamma RII, CD8 (McGeer et al., Can. J. Neurol. Sc.
16:516-527 (1989)), LCA (CD45)(Akiyama et al., J.
Neuroimmunol. 50:195-201 (1994)), CD18 (beta-2
integrin)(Akiyama and McGeer, J. Neuroimmunolncry 30:81-93
(1990)), CD59 (McGeer et al., Br. Res. 544:315-319
(1991)), vitronectic (McGeer et al., Can J. Neurnl_ Sri
18:376-379 (1991)), vitronectin receptor, beta-3
integrin, Apo J, clusterin (McGeer et al., Br. Res.
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579:337-341 (1992)), type 2 plasminogen activator
inhibitor (Akiyama et al., Neurosc. Lett. 164:233-235
(1993)), CD44 (Akiyama et al., Br. Res. 633:249-259
(1993)), midkine (Yasuhara et al., Biochem. Biopllys. Res.
Comm. 192:246-251 (1993)), macrophage colony stimulating
factor receptor (Akiyama et al., Br. Res. 639:171-174
(1994)), MRP14, 27E10, and interferon-alpha (Akiyama et
al., J. Neuroimmunol. 50:195-201 (1994)). Additional
markers which are associated with inflammation or
oxidative stress include 4-hydroxynonenal-protein
conjugates (Uchida and Stadtman, Meth. Enz. 233:371-380
(1994)), IkappaB, NFkappaB (Kaltschmidt et al., Mol.
As-pects Med. 14:171-190 (1993)), cPLA2 (Stephenson et
al. , Neurobiol. Dis. 3 : 51-63 (1996)), COX-2 (Chen et al.,
Neurorepor 6:245-248 (1995)), matrix metalloproteinases
(Backstrom et al., J. Neurochem. 58:983-992 (1992)),
membrane lipid peroxidation, protein oxidation (Smith et
al., Proc. Natl. Acad. Sci. USA 88:10540-10543 (1991))
and diminished ATPase activity (Mark et al., J. Neurosc.
15:6239 (1995)). These markers can be detected, and
changes can be determined to measure an effect of
compounds on the disclosed transgenic animals.
E. Neuronal and Neurotransmitter-Related Markers
Changes in neuronal and neurotransmitter
biochemistry have been associated with Alzheimer's
disease. In Alzheimer's, there is profound reduction in
cortical and hippocampal cholinergic innervation. This
is evidenced by the dramatic loss of the synthetic enzyme
choline acetyltransferase and decreased
acetylcholinesterase, synaptosomal choline uptake and
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synthesis and release of acetylcholine (Sims et al., J,
Neurochem. 40:503-509 (1983)), all of which are useful
markers.These markers can be used in Alzheimer's disease
screening assays to determine the effect of compounds on
Alzheimer's disease. There is also a loss of basal
forebrain neurons and the galanin system becomes
hypertrophic in Alzheimer's disease.
In addition to changes in the markers described
above in Alzheimer's disease, there is also atrophy and
loss of basal forebrain cholinergic neurons that project
to the cortex and hippocampus (Whitehouse et al., Science
215:1237-1239 (1982)), as well as alterations of
entorhinal cortex neurons (Van Hoesan et al., Hippocam,pus
1:1-8 (1991)). Based upon these observations measurement
of these enzyme activities, neuronal size, and neuronal
count numbers are expected to decrease in the disclosed
transgenic animals and are therefore useful targets for
detection in Alzheimer's disease screening assays. Basal
forebrain neurons are dependent on nerve growth factor.
Brain-derived neurotrophic factor (BDNF) may also
decrease in the hippocampus and is therefore a useful
target for detection in Alzheimer's disease screening
assays. In addition, screening assays that measure the
effect of compounds on neurotransmitter receptors can
possibly be used to identifying compounds useful in
treating Alzheimer's disease.
In addition to the well-documented changes in
the cholinergic system, dysfunction in other receptor
systems such as the serotinergic, adrenergic, adenosine
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and nicotine receptor systems, has also been documented.
Markers characteristic of these changes, as well as other
neuronal markers that exhibit both metabolic and
structural changes in Alzheimer's disease are listed
below. Changes in the level and/or localization of these
markers can be measured using similar techniques as those
described for measuring and detecting the earlier
markers.
The following are preferred cytoskeletal and
neuritic markers that exhibit changes in and/or an
association with Alzheimer's disease. Levels of
cathepsin D, cathepsin B, cathepsin G, and neuronal
thread protein, and phosphorylation of elongation factor-
2, increase in Alzheimer's. Cathepsin D, cathepsin B,
protein kinase C and NADPH are localized in plaque and
neuritic tissue in Alzheimer's disease. Activity and/or
levels of nicotine receptors, 5-HT2 receptor, NMDA
receptor, alpha2-adrenergic receptor, synaptophysin,
synaptoglycan (SV2PG), p65, glutamine synthetase, glucose
transporter, PPI kinase, drebrin, GAP43, cytochrome
oxidase, heme oxygenase, calbindin, adenosine Al
receptors, mono amine metabolites, choline
acetyltransferase, acetylcholinesterase, and symptosomal
choline are all reduced in Alzheimer's disease.
Additional markers that are associated with
Alzheimer's disease or after treatment of cells with Ai3
include a) cPLAz which is upregulated in Alzheimer's
disease (Stephenson et al., Neurobiol. Dis, 3:51-63
(1996)), b) heme oxygenase-1 (Smith et al., Am. J. Path.
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145:42-47 (1994)), c-jun (Anderson et al., Exp. Neurol.
125:286-295 (1994)), c-fos (Zhang et al., Neurosci. 46:9-
21 (1992)), HSP27 (Renkawek et al., Acta Neuropath.
87:511-519 (1994)), HSP70 (Cisse et al., Acta Neuronath.
85:233-240 (1993)) and MAP5 (Geddes et al., J. Neurosc.
Res. 30:183-191 (1991)), which are induced in Alzheimer's
disease and in cortical cells after Af3 treatment and c)
junB, junD, fosB, fral (Estus et al., J. Cell Biol.
127:1717-1727 (1994)), cyclin Dl (Freeman et al., Neuron
12:343-355 (1994)), NGFI-A (Vaccarino et al., Mol. Br.
Res. 12:233-241 (1992)), and NGFI-B, which are induced in
cortical cells after Af3 treatment.
F. Measuring the Amounts and Localization of Alzheimer's
Disease Markers
Quantitative measurements can be accomplished
using many standard assays. For example, transcript
levels can be measured using RT-PCR and hybridization
methods including RNase protection, Northern analysis,
and R-dot analysis. Protein marker levels can be assayed
by ELISA, Western analysis, and by comparison of
immunohistochemically stained tissue sections.
Immunohistochemical staining can also be used to assay
localization of protein markers to particular tissues and
cell types. The localization and the histopathological
association of Alzheimer's disease markers can be
determined by histochemical detection methods such as
antibody staining, laser scanning confocal imaging, and
electron and immunoelectron microscopy.
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In the case of receptors and enzymatic markers,
activity of the receptors or enzymes can be measured.
For example, the activity of neurotransmitter
metabolizing enzymes such as choline acetyltransferase
and acetylcholine esterase can be measured using standard
radiometric enzyme activity assays.
G. Screening Assays Using Cultured Cells
Screening assays for determining the
therapeutic potential of compounds can also be performed
using cells derived from animals transgenic for the
disclosed perlecan constructs or cell cultures stably
transfected with the disclosed constructs, or from cells
derived from progeny derived from the mating of perlecan
transgenic mice with other transgenics implicated in
Alzheimer's disease. For example, P19 cells which
overproduce perlecan also demonstrate a marked increase
in Ai3 levels in the media. In a preferred embodiment, a
potential therapeutic compound for Alzheimer's disease is
tested on perlecan transfected P19 cells, or other cells
types that also demonstrate increased Af3 production due
to perlecan transfection. For such testing, derived
transgenic cells or transfected cell cultures, following
an overnight incubation at 37 C in an incubator
equilibrated with 5o carbon dioxide, will have the media
removed and replaced with media containing a compound to
be tested for a 2 hour pretreatment period with the cells
then incubated as above. At the end of the pretreatment
period, the media are again removed and replaced with
fresh media containing the compound to be tested (as
described above) and cells are incubated for an
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additional 2 to 16 hours. After treatment, plates are
*
centrifuged in a Beckman GPR at 1200 rpm for 5 minutes at
room temperature to pellet cellular debris from the
conditioned media. From each well, 100 1 of conditioned
media or appropriate dilutions thereof are transferred
into an ELISA plate precoated with antibody 266 (an
antibody directed against amino acids 13 to 28 of Ai3) as
described in International patent Application No.
94/10569 and stored at 4 C overnight. An ELISA assay
employing any labelled antibody against Af3 (such as 4C38
from Senetek) can be run to measure the amount of AS
produced. Different capture and detection antibodies can
also be used. In addition, various cytotoxicity assays,
such as the MTS assay described in Example 6 of the
disclosed invention, can be utilized to determine whether
the compound protect cells from neurodegeneration or
neuronal death.
H. Screening for Compounds Effective in Other Amyloid
Diseases
Use of the perlecan transgenic mice,
tranafected cells, or cells derived from transgenic mice
as disclosed in the present invention, can be used either
alone or in combination with other transgenic mice,
transfected cells, or cells derived from other transgenic
mice implicated in a given amyloid disease, to produce
new animal and cell culture models of amyloid diseases
(other than the Alzheimer's-type of amyloid disease).
For example, the mating of perlecan mice with transgenic
animals which overproduce islet amyloid (amylin) may
produce progeny which exhibit a new animal model
*Trade-mark
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completely mimicking islet amyloid deposition in
pancreas. In such transgenic animals amyloid formation,
deposition, accumulation and/or persistence of an amyloid
protein (other than the Alzheimer's disease Af3) is
occurring in peripheral tissues, making these animals
useful for the screening of anti-amyloid compounds to
determine if said compounds affect or diminish
accumulation of a specific amyloid protein in the
tissues. Initial analysis may include, but is not
limited to, detecting levels and immunolocalization of a
given amyloid protein using specific anti-amyloid
antibodies which are commercially available including
those against AA amyloid. AL amyloid, islet amyloid
(i.e. amylin), beta2-microglobulin, transthyretin/
prealbumin, procalcitonin, and PrP amyloid (brain
amyloidosis), and comparison of compound-treated versus
placebo-treated animals. In addition, qualitative and
quantitative assessment of the extent of amyloid can be
made following detection by specific amyloid stains such
as Congo red (and viewed under polarized light) or
Thioflavin S (viewed under fluorescent light). In such
assessments, a given compound is administered to an
animal at various dosages, for various treatment periods,
and at varying stages of amyloid formation, deposition
accumulation and persistence in tissues. A given
compound is said to be effective by reducing or
eliminating the amyloid by a correlative decrease or
elimination of amyloid protein immunostaining, Congo red
staining (as viewed under polarized light) or Thioflavin
T fluorescence. In addition, electron microscopic
analysis can be used for the detection and localization
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of amyloid fibrils in a given tissue, and qualitative
and quantitative assessments can be made prior-to and
following compound treatments in groups of animals. In
all of the screening assays described above, the
effective compounds will be the ones which diminish or
eliminate amyloid in a particular tissue or animal.
New Amyloid Disease Models
The strategy of crossing perlecan transgenic
mice with transgenic mice that overexpress any other
amyloid protein, or another component implicated in a
given amyloid disease, will also produce new animal
models of different amyloidoses which can be utilized as
screening tools to identify new therapeutics for the
treatment of amyloidoses.
In one preferred embodiment, perlecan
transgenic mice mated with transgenic mice overexpressing
any isoform of beta-amyloid precursor protein (and/or its
portions thereof) produce transgenic progeny that
overexpress both perlecan and f3PP. This new transgenic
animal model can be used as a screening tool to identify
potential therapeutics targeting Af3 amyloidosis as
observed in Alzheimer's disease, Down's syndrome and
cerebral hemorrhage with amyloidosis of the Dutch type.
In another preferred embodiment, perlecan
transgenic mice are mated with transgenic mice wherein
the Ai3-containing protein consists of all or a contiguous
portion of a protein selected from a group consisting of
13PP-770, ZPP-770 bearing a mutation in one or more of the
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amino acids selected from the group consisting of amino
acids 669, 670, 671, 690, 692 and 717, i3PP-751, f3PP-751
bearing a mutation in one or more of the amino acids
selected from the group consisting of amino acid 669,
670, 671, 690, 692 and 717, i3PP-695, and f3PP-695 bearing
a mutation in one or more of the amino acids selected
from the group consisting of amino acid 669, 670, 671,
690, 692 and 717, wherein the Af3-containing protein
includes amino acids 672 to 714 of human i3PP.
In another preferred embodiment, perlecan
transgenic mice are mated with transgenic mice which
overexpress presenilin 1, presenilin 2, or transgenics
which contain mutations to either presenilin 1 or
presenilin 2.
In another preferred embodiment, perlecan
transgenic mice are mated with transgenic mice which
overexpress a protein selected from the group consisting
of laminin, type IV collagen, heparan sulfate PGs,
chondroitin sulfate PGs, dermatan sulfate PGs, keratan
sulfate PGs, glypican, syndecan, syndecan-3, neurocan,
phosphacan, aggrecan, decorin, biglycan, hyaluronan,
amyloid P component, alphal- antichymotrypsin, cathepsin
D, cathepsin G, cathepsin B, neuronal thread protein,
nicotine receptors, 5-HT2 receptor, NMDA receptor, alpha2-
adrenergic receptor, synaptophysin, p65, glutamine
synthetase, glucose transporter, PPI kinase, GAP43,
cytochrome kinase, heme oxygenase, calbindin, adenosine
Al receptors, choline acetyltransferase,
acetylcholinesterase, glial fibrillary acidic protein,
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alphal-antitrypsin, C-reactive protein, alpha2-
macroglobulin, interleukin-lalpha, interleukin-19, TNF-
alpha, interleukin-6, HLA-DR, HLA-A, HLA-D, HLA-C, CR3
receptor, MHC I, MHC II, CD 31, CR4, CD45, CD64, CD4,
spectrin, tau protein, ubiquitin, MAP-2, apolipoprotein
E, apolipoprotein E4, apolipoprotein E2, apolipoprotein
E3, nerve growth factor, brain-derived neurotrophic
factor, advanced glycosylation end products, receptor for
advanced glycosylation end products, COX-2, CD18, C3,
fibroblast growth factor, CD44, ICAM-l, lactotransferrin,
Clq, C3d, C4d, CSb-9, gamma Rl, Fc gamma RII, CD8, CD59,
vitronectin, vitronectin receptor, beta-3 integrin, Apo
J, clusterin, type 2 plasminogen activator inhibitor,
midline, macrophage colony stimulating factor receptor,
MRP14, 27E10, interferon-alpha, S1009, cPLA2, c-jun, c-
fos, HSP27, HSP70, MAP5, membrane lipid peroxidase,
protein carbonyl formation, junB, junD, fosB, fral,
cyclin D1, p53, NGFI-A, NGFI-B, I-kappa-B, NF-kappa-B,
interleukin-8, MCP-1, MIP-lalpha, matrix
metaloproteinases, and 4-hydroxvnonenal-protein
conjugates.
In another preferred embodiment, perlecan
transgenic mice mated with transgenic mice overexpressing
AL amyloid (and/or its portions thereof) produce
transgenic progeny that overexpress both perlecan and AL
amyloid. This new transgenic animal model can be used as
a screening tool to identify potential therapeutics
targeting AL amyloidosis as observed in multiple myeloma
and other i3-cell dyscrasias.
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In another preferred embodiment, perlecan
transgenic mice mated with transgenic mice overexpressing
islet amyloid (amylin) produce transgenic progeny that
overexpress both perlecan and amylin. This new
transgenic animal model can be used as a screening tool
to identify potential therapeutics targeting islet
amyloidosis as observed in type II diabetes.
In another preferred embodiment, perlecan
transgenic mice mated with transgenic mice overexpressing
PrP amyloid produce transgenic progeny that overexpress
both perlecan and PrP protein. This new transgenic
animal model can be used as a screening tool to identify
potential therapeutics targeting PrP amyloidosis as
observed in Creutzfeldt-Jakob disease, Gerstmann-
Straussler syndrome, kuru and animal scrapie.
In yet another preferred embodiment, perlecan
transgenic mice mated with transgenic mice overexpressing
betaz-microglobulin produce transgenic progeny that
overexpress both perlecan and beta2-microglobulin. This
new transgenic animal model can be used as a screening
tool to identify potential therapeutics targeting beta2-
microglobulin amyloidosis as observed in long-term
hemodialysis and carpal tunnel syndrome.
In another preferred embodiment, perlecan
transgenic mice mated with transgenic mice overexpressing
prealbumin/ transthyretin produce transgenic progeny that
overexpress both perlecan and prealbumin/ transthyretin.
This new transgenic animal model can be used as a
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screening tool to identify potential therapeutics
targeting prealbumin/ transthyretin amyloidosis as
observed in senile cardiac amyloid and Familial
Amyloidotic Polyneuropathy.
In yet another preferred embodiment, perlecan
transgenic mice mated with transgenic mice overexpressing
variants of procalcitonin produce transgenic progeny that
overexpress both perlecan and variants of procalcitonin.
This new transgenic animal model can be used as a
screening tool to identify potential therapeutics
targeting the amyloid associated with endocrine tumors
such as in medullary carcinoma of the thyroid.
In yet another preferred embodiment, perlecan
transgenic mice are mated with "knock-out" transgenic mice
which do not express, or express at much reduced levels
than normal, a given amyloid protein or protein
implicated to be important in amyloid disease as listed
above. These new transgenic animal models can be used as
a screening tool to identify potential therapeutics
targeting various amyloid diseases.
In yet another preferred embodiment, transgenic
mice that overexpress both perlecan and any amyloid
protein, (amyloid precursor protein and/or a fragment
thereof), is produced by injection of both perlecan and
amyloid protein DNA constructs into fertilized mouse eggs
(one cell stage embryos). The embryos bearing both the
DNA constructs are transferred to the oviducts of
pseudopregnant foster mothers. The tails from the
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emerging pups are cut for Southern blot analysis or PCP,
to determine the chromosomal integration of both the
transgenes. Pups determined positive for both the
transgenes are crossed with other mice (example:
C57BL/6J) to establish lines of transgenic'mice bearing
both the DNA constructs.
Although the invention has been described with
reference to the disclosed embodiments, those skilled in
the art will readily appreciate that the specific
experiments detailed are only illustrative of the
invention. It should be understood that various
modifications can be made without departing from the
spirit of the invention. Accordingly, the invention is
limited only by the following claims.
