U.S. patent application number 11/449216 was filed with the patent office on 2006-10-12 for gene-targeted animal model of apolipoprotein e4 domain interaction and uses thereof.
Invention is credited to Li-Ming Dong, Robert V. Farese, Robert Raffai, Karl H. Weisgraber.
Application Number | 20060230466 11/449216 |
Document ID | / |
Family ID | 26690226 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060230466 |
Kind Code |
A1 |
Weisgraber; Karl H. ; et
al. |
October 12, 2006 |
Gene-targeted animal model of apolipoprotein E4 domain interaction
and uses thereof
Abstract
The invention provides gene-targeted non-human animals
comprising a genetically modified apoE gene encodes a recombinant
apoE polypeptide displaying domain interaction. The invention
further provides cells isolated from the gene-targeted animals,
which cells produce a recombinant apoE polypeptide displaying
domain interaction. The invention further provides methods of
identifying agents that reduce apoE4 domain interaction, and which
are useful to treat apoE4-related neurological and cardiovascular
disorders.
Inventors: |
Weisgraber; Karl H.; (Walnut
Creek, CA) ; Farese; Robert V.; (San Francisco,
CA) ; Raffai; Robert; (San Francisco, CA) ;
Dong; Li-Ming; (Palo Alto, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
26690226 |
Appl. No.: |
11/449216 |
Filed: |
June 7, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10017718 |
Dec 14, 2001 |
7081561 |
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11449216 |
Jun 7, 2006 |
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60276861 |
Mar 16, 2001 |
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Current U.S.
Class: |
800/18 ;
435/354 |
Current CPC
Class: |
A01K 2217/072 20130101;
C12N 15/8509 20130101; A01K 2267/0362 20130101; A01K 2207/15
20130101; G01N 2800/044 20130101; C07K 14/775 20130101; A01K
2227/105 20130101; A01K 2267/0375 20130101; A01K 2217/00 20130101;
G01N 33/6896 20130101; G01N 33/92 20130101; A01K 2267/0312
20130101; A01K 67/0276 20130101; G01N 2500/10 20130101; C12N
2517/02 20130101; A01K 67/0278 20130101; A01K 2267/0356 20130101;
C12N 2800/30 20130101 |
Class at
Publication: |
800/018 ;
435/354 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12N 5/06 20060101 C12N005/06 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with government support under grant
number 2PO1 HL47660 from the National Institutes of Health. The
United States Government may have certain rights in this invention.
Claims
1. A gene-targeted non-human animal comprising a modified
endogenous apolipoprotein E (apoE) allele, wherein said modified
allele comprises an apoE-encoding nucleic acid under
transcriptional control of endogenous regulatory sequences, and
wherein the modified allele encodes a modified apoE that exhibits
domain interaction characteristic of human apolipoprotein E4
(apoE4).
2. The non-human animal of claim 1, wherein the modified apoE
comprises a Thr.fwdarw.Arg substitution at a position equivalent to
amino acid 61 of human apoE4.
3. The non-human animal of claim 1, wherein the gene-targeted
non-human animal is homozygous for the modified apoE allele.
4. The non-human animal of claim 1, wherein the gene-targeted
animal is a mouse.
5. An isolated non-human cell comprising a modified endogenous
apolipoprotein E (apoE) allele, wherein said modified endogenous
allele is under transcriptional control of endogenous regulatory
sequences, and wherein the modified allele encodes a modified apoE
that exhibits domain interaction characteristic of human
apolipoprotein E4 (apoE4).
6. The non-human cell of claim 5, wherein the modified apoE
comprises a Thr.fwdarw.Arg substitution at a position equivalent to
amino acid 61 of human apoE4.
7. The non-human cell of claim 5, wherein the cell is homozygous
for the modified apoE allele.
8. The non-human cell of claim 5, wherein the cell is a mouse
cell.
9. An isolated nucleic acid molecule comprising a nucleotide
sequence derived from a non-human apolipoprotein E (apoE) gene,
which nucleotide sequence is modified such that it encodes a
protein comprising a Thr.fwdarw.Arg substitution at a position
equivalent to amino acid 61 of human apoE4.
10. A recombinant vector comprising the nucleic acid of claim
9.
11. A recombinant host cell comprising the vector of claim 10.
12. A recombinant apolipoprotein E (apoE) protein encoded by a
nucleic acid comprising a nucleotide sequence derived from a
non-human apoE gene, which nucleotide sequence is modified such
that it encodes a protein that exhibits domain interaction
characteristic of human apolipoprotein E4 (apoE4).
13. The recombinant protein of claim 12, wherein the recombinant
protein comprises a Thr.fwdarw.Arg substitution at a position
equivalent to amino acid 61 of human apoE4.
14. A method of identifying an agent that reduces a phenomenon
associated with Alzheimer's disease (AD), the method comprising: a)
contacting the gene-targeted non-human animal of claim 1 with a
test agent; and b) determining the effect of the test agent on a
phenomenon associated with AD.
15. The method of claim 15, wherein the phenomenon associated with
AD is selected from the group consisting of amyloid deposits,
neuronal cell loss, and neurofibrillary tangles.
16. A method for identifying an agent that reduces apolipoprotein
E4 domain interaction, the method comprising: a) contacting the
recombinant protein of claim 12 with a test agent; and b)
determining the effect of the test agent on domain interaction.
17. The method of claim 16, wherein said determining comprises
determining binding of the recombinant apoE to tau.
18. The method of claim 16, wherein said determining comprises
determining the effect of the agent on binding to VLDL.
19. A method of identifying an agent that reduces the risk of heart
disease, comprising: a) contacting the non-human animal of claim 1
with a test agent; and b) determining the effect, if any, on apoE
activity.
Description
CROSS REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/276,861, filed Mar. 16, 2001, which
is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates generally to the field of genetically
altered mice, altered with respect to the expression of
apolipoprotein E (apoE) and to assays for determining the effects
of compounds on apoE4 domain interaction.
BACKGROUND OF THE INVENTION
[0004] Alzheimer's Disease (AD) is a progressive, neurodegenerative
disease characterized by cognitive and behavioral changes which
include: (a) memory loss; (b) language deterioration; (c) impaired
visual-spatial skills; (d) poor judgment; (e) indifferent attitude
and (f) aimless, unpredictable behavior. Although AD usually begins
after age 60, its onset may occur as early as age 40. AD first
appears as memory decline. As the disease progresses over several
years, cognition, personality, and the ability to function are all
impaired or destroyed. Confusion and restlessness may also occur.
The type, severity, sequence, and progression of mental changes
vary widely among AD patients. Some people have the disease for
only the last 5 years of life, while others may have it for as many
as 20 years. The most common cause of death in AD patients is
infection.
[0005] There is no known etiology or cure for AD and no way to slow
the progression of the disease. For some people in the early or
middle stages of the disease, medication such as tacrine may
alleviate some cognitive symptoms. Also, some medications may help
control behavioral symptoms such as sleeplessness, agitation,
wandering, anxiety, and depression. These treatments are aimed
solely at making the patient more comfortable and do nothing to
slow the progression of the underlying disease.
[0006] As such, there is much ongoing research that is aimed at the
identification and development of new therapeutic agents which can
at least slow, if not reverse, the progression of AD. An important
step in the identification of therapeutic agents for AD would be
the development of a non-human animal model of the disease, as such
an animal model would serve as an invaluable tool for screening and
testing potential therapeutic agents.
[0007] The only non-human animals that naturally develop AD
pathological features are aged non-human primates. However,
primates are expensive, difficult to use, and require a significant
period of time prior to developing AD. These factors make the use
of primates as AD animal models prohibitive.
[0008] Apolipoprotein E (apoE) is a 34,000 molecular weight protein
which is the product of a single gene on chromosome 19. Human ApoE
exists in three major isoforms designated apoE2, apoE3 and apoE4.
The different isoforms result from amino acid substitutions at
amino acid residue positions 112 and 158. The common isoform,
apoE3, has a cysteine residue at position 112 and an arginine
residue at position 158. The apoE4 isoform differs from apoE3 only
at position 112, which is an arginine residue. The apoE4 isoform
has been associated with neurological disorders such as AD, with
poor outcome following stroke or traumatic head injury, accelerated
progression of multiple sclerosis, and with certain cardiovascular
disorders.
[0009] Human apoE4 exhibits domain interaction due to the presence
of an Arg-112, together with an Arg-61 and a Glu-255, the latter
two amino acids forming a salt bridge. ApoE4 domain interaction is
predicted to represent a key factor responsible for its association
with both heart disease and neurological disorders. Like many
non-human animals, mouse apoE contains the equivalent of Arg-112
and Glu-255, but lacks the critical Arg-61 required for domain
interaction; instead, mouse apoE, as well as apoE of at least nine
other species, contains Thr-61. Weisgraber (1994) Adv. Protein
Chem. 45:249-302.
[0010] ApoE contains two structural domains: an amino-terminal and
a carboxyl-terminal domain. Weisgraber (1994) Adv. Protein Chem.
45:249-302. Each domain is associated with a specific function. The
amino terminal domain contains the lipoprotein receptor binding
region and the carboxy-terminal domain contains the major
lipid-binding elements. The two domains interact with each other in
an isoform-specific manner such that amino acid substitutions in
one domain influence the function of the other domain, a phenomenon
referred to as domain interaction.
[0011] Domain interaction is responsible for the preference of
apoE4 for very low density lipoproteins (VLDL) contrasted with the
preference of apoE3 for high density lipoproteins (HDL). The
specific amino acid residues in apoE4 that are involved in this
interaction have been identified: arginine-61 in the amino-terminal
domain and glutamic acid-255 in the carboxyl-terminal domain. Dong
et al. (1994) J. Biol. Chem. 269:22358-22365; and Dong and
Weisgraber (1996) J. Biol. Chem. 271:19053-19057. In addition to
differences in lipoprotein preference, apoE4 differs from apoE3 in
its interaction with other proteins. For example, tau, a protein
found in neurofibrillary tangles, interacts in vitro with apoE3,
but not with apoE4.
[0012] ApoE4 transgenic animals expressing human apoE isoforms have
been reported, including transgenic mice in which the endogenous
mouse apoE gene is ablated or inactivated, and gene-targeted mice
in which a human apoE coding sequences is stably integrated into
the mouse apoE locus. See, e.g., U.S. Pat. Nos. 6,046,381; and
5,767,337. In the transgenic animal models, the human apoE-encoding
transgene is under transcriptional control of control elements that
are part of the transgene. Furthermore, the transgene inserts at
essentially random sites in the genome. Although in the
gene-targeted mice, expression of apoE is under the control of
endogenous control elements that normally control apoE gene
expression, such as tissue-specific elements and promoter elements,
there is a potential for species effects.
[0013] In view of the foregoing, there is a need in the art for
improved animal models of human apoE4 activity. The present
invention addresses this need.
Literature
[0014] U.S. Pat. No. 5,767,337 and U.S. Pat. No. 6,046,381 relate
to transgenic mice expressing human apoE4. U.S. patents disclosing
other transgenic AD animal models include: U.S. Pat. Nos.
5,777,194; 5,720,936; 5,672,805; 5,612,486; 5,602,309; and
5,387,742. Dong and Weisgraber (1996) J. Biol. Chem.
271:19053-19057; and Dong et al. (1994) J. Biol. Chem.
269:22358-22365 relate to apoE4 domain interaction. Baum et al.
(2000) Microsc. Res. Tech. 50(4):278-281 review apoE isoforms in
Alzheimer's disease pathology and etiology.
SUMMARY OF THE INVENTION
[0015] The present invention provides non-human gene-targeted
animal models for the study of apolipoprotein E4-associated
pathologies, wherein the endogenous apoE gene of the gene-targeted
animal is genetically altered such that the encoded recombinant
apoE polypeptide exhibits domain interaction. Since domain
interaction is a hallmark of human apoE4, the non-human
gene-targeted animals of the instant invention serve as models for
human apoE4 domain interaction. The invention further provides
cells isolated from a non-human gene-targeted animal of the
invention, in particular, cells that produce recombinant apoE
exhibiting domain interaction. The invention further provides
isolated recombinant apoE protein produced by a cell of the
invention. The non-human gene-targeted animals of the invention, as
well as cells isolated from such animals and recombinant apoE
protein produced by such cells, are useful for screening candidate
agents for their ability to reduce apoE domain interaction in the
recombinant apoE polypeptide. Such agents are useful for reducing
apoE4 domain interaction in human cells producing apoE4, and are
therefore useful for treating apoE4-related neurological and
cardiovascular disorders.
[0016] The invention further provides methods of identifying agents
that reduce apoE4 domain interaction. The methods generally
comprise contacting a non-human gene-targeted animal (or a cell or
a recombinant apoE protein) of the invention with a test agent, and
determining the effect, if any, of the test agent on domain
interaction, or a phenomenon associated with apoE4 domain
interaction. In some embodiments, phenomena associated with apoE4
domain interaction are associated with neurological disorders. The
phenomena measured include behavioral phenomena and physiological
phenomena. Behavioral phenomena may include cognitive function,
such as spatial learning. Physiological phenomena may include the
presence of neurofibrillary tangles and A.beta. deposits.
Alternatively, or in conjunction, the phenomena examined may be
examining phenotypes such as neurodegeneration, including
neurodegeneration that is age-dependent. In other embodiments,
phenomena and/or disorders associated with apoE4 domain interaction
are cardiovascular disorders. In these embodiments, phenomena
include hyperlipidemia and associated disorders such as coronary
artery disease and atherosclerosis.
[0017] A primary object of the invention is to provide a method of
using a gene-targeted animal model for identifying candidate agents
(e.g., a small molecule drug or an endogenous factor) that reduce
apoE4 domain interaction and, in doing so, treat disorders related
to the presence of apoE4, e.g., neurodegenerative disorders and
cardiovascular disorders. Such methods are useful for screening
candidate agents for use in treating or relieving the symptoms of
apoE4-related pathologies. The cells derived therefrom are also
useful for screening biologically active agents that reduce apoE4
domain interaction.
[0018] A feature of the invention is that a gene-targeted mouse of
the invention expresses a form of apoE that differs from the
endogenous apoE in that it exhibits domain interaction. The
genetically modified apoE gene of gene-targeted non-human animals
of the invention is in its normal genomic environment, and
therefore is under control and regulation of the same enhancer and
tissue-specific elements that direct expression of the wild-type
apoE gene. Thus, an advantage of a gene-targeted non-human animal
of the instant invention is that the genetically modified apoE gene
is under control of the same elements that control the apoE gene in
a wild-type animal of the same species, such that the genetically
altered endogenous apoE exhibits wild-type expression patterns.
Thus, gene-targeted animals of the invention provide animal models
that are representative of human apoE4 in its native
environment.
Features of the Invention
[0019] In one aspect, the invention provides a gene-targeted
non-human animal comprising a modified endogenous apoE allele,
wherein said modified endogenous apoE allele is under
transcriptional control of endogenous regulatory elements, and
wherein the modified apoE allele encodes a recombinant apoE
polypeptide that exhibits domain interaction characteristic of
human apoE4.
[0020] In another aspect, the invention provides a gene-targeted
non-human animal comprising a modified endogenous apoE allele,
wherein said modified endogenous apoE allele is under
transcriptional control of endogenous regulatory elements, and
wherein the modified apoE allele comprises a Thr.fwdarw.Arg
substitution at a position equivalent to amino acid 61 of human
apoE4.
[0021] In another aspect, the invention provides a gene-targeted
mouse comprising a modified endogenous mouse apoE allele, wherein
said modified endogenous mouse apoE allele is under transcriptional
control of endogenous regulatory elements, and wherein the modified
mouse apoE allele comprises a Thr.fwdarw.Arg substitution at a
position equivalent to amino acid 61 of human apoE4. In some
aspects, the gene-targeted mouse is homozygous for the modified
endogenous apoE allelc.
[0022] In another aspect, the invention provides an isolated
non-human cell comprising a modified endogenous apoE allele,
wherein said modified endogenous apoE allele is under
transcriptional control of endogenous regulatory elements, and
wherein the modified endogenous apoE allele encodes a recombinant
apoE polypeptide that exhibits domain interaction characteristic of
human apoE4.
[0023] In another aspect, the invention provides an isolated
non-human cell comprising a modified endogenous apoE allele,
wherein said modified endogenous apoE allele is under
transcriptional control of endogenous regulatory elements, and
wherein the modified endogenous apoE allele comprises a
Thr.fwdarw.Arg substitution at a position equivalent to amino acid
61 of human apoE4.
[0024] In another aspect, the invention provides an isolated mouse
cell comprising a modified endogenous mouse apoE nucleic acid
molecule, wherein said modified endogenous mouse apoE nucleic acid
molecule is under transcriptional control of endogenous regulatory
elements, and wherein the modified mouse apoE nucleic acid
comprises a Thr.fwdarw.Arg substitution at a position equivalent to
amino acid 61 of human apoE4.
[0025] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence derived from
a non-human apoE gene, which nucleotide sequence is modified such
that it encodes an apoE protein that exhibits domain interaction
characteristic of human apoE4. In another aspect, the invention
provides a recombinant vector comprising such a nucleic acid
molecule. In another aspect, the invention provides a recombinant
host cell comprising such a recombinant vector.
[0026] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence derived from
a non-human apoE gene, which nucleotide sequence is modified such
that it comprises a Thr.fwdarw.Arg substitution at a position
equivalent to amino acid 61 of human apoE4, and encodes an apoE
protein that exhibits domain interaction characteristic of human
apoE4. In another aspect, the invention provides a recombinant
vector comprising such a nucleic acid molecule. In another aspect,
the invention provides a recombinant host cell comprising such a
recombinant vector.
[0027] In another aspect, the invention provides a recombinant apoE
protein encoded by a nucleic acid molecule comprising a nucleotide
sequence derived from a non-human apoE gene, which nucleotide
sequence is modified such that it encodes an apoE protein that
exhibits domain interaction characteristic of human apoE4.
[0028] In another aspect, the invention provides a method of
identifying an agent for treating an apoE-associated neurological
disorder, comprising: contacting the gene-targeted mouse of the
invention with a test agent; and b) determining the effect of the
test agent on apoE4 activity. In some aspects, the apoE4 activity
tested is apoE4 domain interaction.
[0029] In another aspect, the invention provides a method of
identifying an agent that reduces a phenomenon associated with
Alzheimer's disease (AD), comprising: contacting the gene-targeted
mouse of the invention with a test agent; and b) determining the
effect of the test agent on a phenomenon associated with AD. In
some of these aspects, the phenomenon associated with AD is
selected from the group consisting of amyloid deposits, neuronal
cell loss, and neurofibrillary tangles.
[0030] In another aspect, the invention provides a method of
identifying an agent that reduces serum cholesterol levels in an
individual, comprising: contacting the gene-targeted mouse of the
invention with a test agent; and b) determining the effect of the
test agent on a serum cholesterol level in the mouse.
[0031] In another aspect, the invention provides a method of
identifying an agent for reducing the risk of coronary artery
disease, comprising: contacting the gene-targeted mouse of the
invention with a test agent; and determining the effect, if any, on
plaque deposition on a wall of a coronary artery.
[0032] In another aspect, the invention provides method of
identifying an agent that reduces apoE4 domain interaction,
comprising: contacting a recombinant apoE protein of the invention
with a test agent; and determining the effect, if any, on an
apoE4-associated activity. In some of these aspects, the
determination is by an emulsion binding assay.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 is a schematic depiction of the gene-targeting vector
used to generate an Arg-61 gene-targeted mouse.
[0034] FIG. 2 depicts plasma levels of wild-type mouse apoE and
mutant Arg-61 mouse apoE in plasma (left panel) and cerebrospinal
fluid (right panel) of wild type (wt/wt), homozygous Arg-61 apoE
(Arg-61/Arg-61) and heterozygous Arg-61 (wt/Arg-61) mice.
[0035] FIG. 3A depicts the FPLC profile of plasma cholesterol
distribution, and FIG. 3B depicts distribution of wild-type and
Arg-61 apoE in plasma from a heterozygous targeted mouse on a
high-cholesterol diet.
[0036] FIGS. 4A and 4B are graphs depicting the ability of human
(FIG. 4A), and wild-type and Arg-61 mouse apoE (FIG. 4B) isoforms
to bind to dimyristoylphosphatidylcholine (DMPC).
DETAILED DESCRIPTION OF THE INVENTION
[0037] Non-human gene-targeted animal models useful for screening
drugs or candidate drugs are provided. The animals have a
genetically altered endogenous apolipoprotein E gene, such that the
encoded recombinant apoE exhibits domain interaction as does human
apoE4. In contrast to previous gene-targeted animal models, the
genetically altered apoE gene in gene-targeted animals of the
present invention is under endogenous transcriptional control and
tissue-specific expression. Thus, the genetically altered
endogenous apoE gene is expressed in a normal manner, i.e.,
developmental, tissue-specific, and temporal (e.g., age-dependent)
expression are the same as with the wild-type apoE gene, and any
species-specific effects seen with previous apoE gene-targeted
animals are avoided.
[0038] The subject animals are useful for identifying agents that
reduce apoE4 domain interaction, and therefore modulate human apoE4
activity. Completely selective compounds will interact with apoE4
selectively, and thus will affect only the apoE4 isoform. In
addition, these animals provide a useful model for the behavioral
testing of candidate compounds. The animals are useful for testing
the efficacy of drugs that reduce apoE4 interaction in treating
apoE4-related neurodegenerative disorders and apoE4-associated
cardiovascular disorders.
[0039] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, 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, since the scope of the present invention will be
limited only by the appended claims.
[0040] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0041] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a non-human gene-targeted animal" includes a
plurality of such animals and reference to "the apoE gene" includes
reference to one or more apoE genes and equivalents thereof known
to those skilled in the art, and so forth.
[0042] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Definitions
[0043] As used herein, the term "endogenous apoE gene" refers to
the endogenous apoE gene of the non-human animal which is to be
genetically modified; the term "genetically modified endogenous
apoE gene" refers to the endogenous apoE gene in the non-human
gene-targeted animal which gene is genetically modified to contain
an Arg-61 such that the apoE protein encoded thereby exhibits
domain interaction; and the term "recombinant apoE polypeptide"
refers to the apoE polypeptide encoded by the genetically modified
apoE gene, which polypeptide exhibits domain interaction.
[0044] The term "transgene" is used herein to describe genetic
material which has been or is about to be artificially inserted
into the genome of a cell, particularly a mammalian cell for
implantation into a living animal.
[0045] By "Alzheimer's disease" (abbreviated herein as "AD") is
meant a condition associated with formation of neuritic plaques
comprising amyloid A.beta. protein primarily in the hippocampus and
cerebral cortex, as well as impairment in both learning and memory.
"AD" as used herein is meant to encompass both AD as well as
AD-type pathologies, i.e., diseases of the central nervous system
with symptoms similar to AD.
[0046] By "symptoms similar to AD" and "phenomenon associated with
AD" is meant a structural, molecular, or functional event
associated with AD, particularly such an event that is readily
assessable in an animal model. Such events include, but are not
limited to, amyloid deposition, neuropathological developments,
neurofibrillary tangles, learning and memory deficits, and other
AD-associated characteristics.
[0047] By "A.beta. amyloid deposit" is meant a deposit in the brain
composed of A.beta. amyloid as well as other substances.
[0048] By "transformation" is meant a permanent or transient
genetic change, preferably a permanent genetic change, induced in a
cell following incorporation of new DNA (i.e., DNA exogenous to the
cell). Where the cell is a mammalian cell, a permanent genetic
change is generally achieved by introduction of the DNA into the
genome of the cell.
[0049] By "gene-targeted animal" is meant a non-human animal,
usually a mammal (e.g., mouse, rat, rabbit, hamster, non-human
primate, etc.), having a genetically altered endogenous apoE in the
same genomic location as the wild-type (not genetically altered)
apoE gene, i.e., the counterpart endogenous apoE gene in a
wild-type animal of the same species. Heterologous nucleic acid
(i.e., nucleic acid not normally associated with an apoE gene of
the species of animal into which the heterologous nucleic acid is
introduced) replaces a portion of the endogenous apoE gene in the
germ line of such gene-targeted animals by genetic manipulation of,
for example, embryos or embryonic stem cells of the host
animal.
[0050] As used herein, an "apoE4-associated disorder" is any
disorder that is caused by the presence of apoE4 in a cell, in the
serum, in the interstitial fluid, in the cerebrospinal fluid, or in
any other bodily fluid of an individual; any physiological process
or metabolic event that is influenced by apoE4 domain interaction;
any disorder that is characterized by the presence of apoE4; a
symptom of a disorder that is caused by the presence of apoE4 in a
cell or in a bodily fluid; a phenomenon associated with a disorder
caused by the presence in a cell or in a bodily fluid of apoE4; and
the sequelae of any disorder that is caused by the presence of
apoE4. ApoE4-associated disorders include apoE4-associated
neurological disorders and disorders related to high serum lipid
levels. ApoE4-associated neurological disorders include, but are
not limited to, sporadic Alzheimer's disease; familial Alzheimer's
disease; poor outcome following a stroke; poor outcome following
traumatic head injury; and cerebral ischemia. Phenomena associated
with apoE4-associated neurological disorders include, but are not
limited to, neurofibrillary tangles; amyloid deposits; memory loss;
and a reduction in cognitive function. ApoE4-related disorders
associated with high serum lipid levels include, but are not
limited to, atherosclerosis, and coronary artery disease. Phenomena
associated with such apoE4-associated disorders include high serum
cholesterol levels.
[0051] As used herein, an "apoE4-associated activity" is any
activity that is associated with human apoE4, but not with other
human apoE isoforms.
[0052] As used herein, the terms "determining," "measuring," and
"assessing," and "assaying" are used interchangeably and include
both quantitative and qualitative determinations.
[0053] The terms "polynucleotide" and "nucleic acid molecule" are
used interchangeably herein to refer to polymeric forms of
nucleotides of any length. The polynucleotides may contain
deoxyribonucleotides, ribonucleotides, and/or their analogs.
Nucleotides may have any three-dimensional structure, and may
perform any function, known or unknown. The term "polynucleotide"
includes single-, double-stranded and triple helical molecules.
"Oligonucleotide" generally refers to polynucleotides of between
about 5 and about 100 nucleotides of single- or double-stranded
DNA. However, for the purposes of this disclosure, there is no
upper limit to the length of an oligonucleotide. Oligonucleotides
are also known as oligomers or oligos and may be isolated from
genes, or chemically synthesized by methods known in the art.
[0054] The following are non-limiting embodiments of
polynucleotides: a gene or gene fragment, exons, introns, mRNA,
tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA of any sequence, nucleic acid probes, and primers. A
nucleic acid molecule may also comprise modified nucleic acid
molecules, such as methylated nucleic acid molecules and nucleic
acid molecule analogs. Analogs of purines and pyrimidines are known
in the art. Nucleic acids may be naturally occurring, e.g. DNA or
RNA, or may be synthetic analogs, as known in the art. Such analogs
may be preferred for use as probes because of superior stability
under assay conditions. Modifications in the native structure,
including alterations in the backbone, sugars or heterocyclic
bases, have been shown to increase intracellular stability and
binding affinity.
[0055] Hybridization reactions can be performed under conditions of
different "stringency". Conditions that increase stringency of a
hybridization reaction of widely known and published in the art.
See, for example, Sambrook et al. (1989). Examples of relevant
conditions include (in order of increasing stringency): incubation
temperatures of 25.degree. C., 37.degree. C., 50.degree. C. and
68.degree. C.; buffer concentrations of 10.times.SSC, 6.times.SSC,
1.times.SSC, 0.1.times.SSC (where SSC is 0.15 M NaCl and 15 mM
citrate buffer) and their equivalents using other buffer systems;
formamide concentrations of 0%, 25%, 50%, and 75%; incubation times
from 5 minutes to 24 hours; 1, 2, or more washing steps; wash
incubation times of 1, 2, or 15 minutes; and wash solutions of
6.times.SSC, 1.times.SSC, 0.1.times.SSC, or deionized water.
Examples of stringent conditions are hybridization and washing at
50.quadrature.C or higher and in 0.1.times.SSC (9 mM NaCl/0.9 mM
sodium citrate).
[0056] "T.sub.m" is the temperature in degrees Celsius at which 50%
of a polynucleotide duplex made of complementary strands hydrogen
bonded in anti-parallel direction by Watson-Crick base pairing
dissociates into single strands under conditions of the experiment.
T.sub.m may be predicted according to a standard formula, such as:
T.sub.m=81.5+16.6 log [X.sup.+]+0.41 (% G/C)-0.61 (% F)-600/L
[0057] where [X.sup.+] is the cation concentration (usually sodium
ion, Na.sup.+) in mol/L; (% G/C) is the number of G and C residues
as a percentage of total residues in the duplex; (% F) is the
percent formamide in solution (wt/vol); and L is the number of
nucleotides in each strand of the duplex.
[0058] A polynucleotide or polypeptide has a certain percent
"sequence identity" to another polynucleotide or polypeptide,
meaning that, when aligned, that percentage of bases or amino acids
are the same when comparing the two sequences. Sequence similarity
can be determined in a number of different manners. To determine
sequence identity, sequences can be aligned using the methods and
computer programs, including BLAST, available over the world wide
web at http://ww.ncbi.nlm.nih.gov/BLAST/. Another alignment
algorithm is FASTA, available in the Genetics Computing Group (GCG)
package, from Madison, Wis., USA, a wholly owned subsidiary of
Oxford Molecular Group, Inc. Other techniques for alignment are
described in Methods in Enzymology, vol. 266: Computer Methods for
Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic
Press, Inc., a division of Harcourt Brace & Co., San Diego,
Calif., USA. Of particular interest are alignment programs that
permit gaps in the sequence. The Smith-Waterman is one type of
algorithm that permits gaps in sequence alignments. See Meth. Mol.
Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman
and Wunsch alignment method can be utilized to align sequences. See
J. Mol. Biol. 48: 443-453 (1970)
[0059] Of interest is the BestFit program using the local homology
algorithm of Smith Waterman (Advances in Applied Mathematics 2:
482-489 (1981) to determine sequence identity. The gap generation
penalty will generally range from 1 to 5, usually 2 to 4 and in
many embodiments will be 3. The gap extension penalty will
generally range from about 0.01 to 0.20 and in many instances will
be 0.10. The program has default parameters determined by the
sequences inputted to be compared. Preferably, the sequence
identity is determined using the default parameters determined by
the program. This program is available also from Genetics Computing
Group (GCG) package, from Madison, Wis., USA.
[0060] The terms "polypeptide" and "protein", used interchangebly
herein, refer to a polymeric form of amino acids of any length,
which can include coded and non-coded amino acids, chemically or
biochemically modified or derivatized amino acids, and polypeptides
having modified peptide backbones. The term includes fusion
proteins, including, but not limited to, fusion proteins with a
heterologous amino acid sequence, fusions with heterologous and
homologous leader sequences, with or without N-terminal methionine
residues; immunologically tagged proteins; and the like.
[0061] A "substantially isolated" or "isolated" polynucleotide is
one that is substantially free of the sequences with which it is
associated in nature. By substantially free is meant at least 50%,
preferably at least 70%, more preferably at least 80%, and even
more preferably at least 90% free of the materials with which it is
associated in nature. As used herein, an "isolated" polynucleotide
also refers to recombinant polynucleotides, which, by virtue of
origin or manipulation: (1) are not associated with all or a
portion of a polynucleotide with which it is associated in nature,
(2) are linked to a polynucleotide other than that to which it is
linked in nature, or (3) does not occur in nature.
[0062] The term "host cell" includes an individual cell or cell
culture which can be or has been a recipient of any recombinant
vector(s) or isolated polynucleotide of the invention. Host cells
include progeny of a single host cell, and the progeny may not
necessarily be completely identical (in morphology or in total DNA
complement) to the original parent cell due to natural, accidental,
or deliberate mutation and/or change. A host cell includes cells
tranfected or infected in vivo or in vitro with a recombinant
vector or a polynucleotide of the invention. A host cell which
comprises a recombinant vector of the invention is a "recombinant
host cell."
[0063] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as polynucleotides. The term "biological sample"
encompasses a clinical sample, and also includes cells in culture,
cell supernatants, cell lysates, serum, plasma, biological fluid,
and tissue samples.
[0064] As used herein, the terms "treatment", "treating", and the
like, refer to obtaining a desired pharmacologic and/or physiologic
effect. The effect may be prophylactic in terms of completely or
partially preventing a disease or symptom thereof and/or may be
therapeutic in terms of a partial or complete cure for a disease
and/or adverse affect attributable to the disease. "Treatment", as
used herein, covers any treatment of a disease in a mammal,
particularly in a human, and includes: (a) preventing the disease
from occurring in a subject which may be predisposed to the disease
but has not yet been diagnosed as having it; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease.
[0065] The terms "individual," "subject," and "patient," used
interchangeably herein, refer to a mammal, including, but not
limited to, murines, simians, humans, mammalian farm animals,
mammalian sport animals, and mammalian pets.
Gene-Targeted Non-Human Animals
[0066] The present invention provides gene-targeted non-human
animals that comprise a genetically altered endogenous apoE gene,
wherein the genetically altered apoE gene encodes a recombinant
apoE protein that displays domain interaction characteristic of
human apoE4. Typically, the recombinant apoE polypeptide encoded by
the genetically modified endogenous apoE gene displays domain
interaction such that a salt bridge is formed between an arginine
at about amino acid 61 and a glutamic acid at about amino acid
255.
[0067] The minimum essential elements for domain interaction are
the presence of Arg-112, Arg-61, and Glu-255, or the functional
equivalent of the foregoing. Thus, any genetic modification of the
endogenous apoE gene that results in formation of a salt bridge
between Arg-61 and Glu-255 is suitable for generating a
gene-targeted non-human animal of the present invention. The amino
acid sequence of apoE from a variety of species examined to date
contain a threonine at a position that is equivalent to Arg-61 in
humans. Weisgraber (1994) Adv. Protein Chem. 45:249-302. Thus,
mutation of the Thr-61 to Arg-61 results in a recombinant apoE
that, like human apoE4, exhibits domain interaction.
[0068] In generating a non-human gene-targeted animal of the
invention, a transgene is used to transform a cell, meaning that a
permanent or transient genetic change, preferably a permanent
genetic change, is induced in a cell following incorporation of
exogenous DNA. A permanent genetic change is generally achieved by
introduction of the DNA into the genome of the cell. Vectors for
stable integration include plasmids, retroviruses and other animal
viruses, YACs, and the like.
[0069] Gene-targeted animals comprise a genetically altered
endogenous apoE gene that comprises a heterologous nucleic acid
sequence that replaces a portion of the endogenous apoE gene, which
heterologous nucleic acid is stably integrated in all or a portion
of the cells of the animal, especially in germ cells. Unless
otherwise indicated, it will be assumed that a gene-targeted animal
comprises stable changes to the germline sequence. During the
initial construction of the animal, "chimeras" or "chimeric
animals" are generated, in which only a subset of cells have the
altered genome. Chimeras are primarily used for breeding purposes
in order to generate the desired gene-targeted animal. Animals
having a heterozygous alteration are generated by breeding of
chimeras. Male and female heterozygotes are typically bred to
generate homozygous animals. Of interest are gene-targeted mammals,
e.g. cows, pigs, goats, non-human primates, horses, etc., and
particularly rodents, e.g. rats; mice, etc.
[0070] In the present invention, gene-targeted animals comprise a
genetic modification in one or both alleles of the endogenous apoE
gene, i.e., gene-targeted animals of the invention include both
heterozygotes and homozygotes for the genetically modified apoE
gene.
[0071] The endogenous apoE gene is a wild-type gene, and is under
transcriptional control of endogenous control elements, i.e.,
control elements that are normally associated with an endogenous
apoE gene in a wild-type animal of the same species. Endogenous
control elements include enhancers, elements that provide for
tissue-specific expression of the endogenous apoE gene, promoter
elements, and the like.
Targeting Constructs
[0072] The introduced heterologous nucleic acid molecule undergoes
homologous recombination with the endogenous apoE gene of the
species, and converts the endogenous apoE gene to a recombinant
apoE gene that encodes a recombinant apoE polypeptide which
exhibits domain interaction. Any modification that results in a
recombinant endogenous apoE gene that encodes a recombinant apoE
protein that exhibits domain interaction is suitable. In some
embodiments, the targeting construct converts the
threonine-encoding codon in the endogenous gene that is equivalent
to Arg-61 of human apoE4 (referred to herein as the "Thr-61 codon")
to a codon encoding arginine. In these embodiments, a targeting
construct comprises the exon containing the codon for Thr-61,
wherein the codon encoding Thr-61 is mutated to encode arginine,
and sufficient flanking sequences to allow homologous recombination
to occur with the endogenous apoE gene. The mutated codon may be
flanked (i.e., may have additional genomic DNA on the 5' and the 3'
side of) by from about 100 nucleotide (nt) to about 10 kb, from
about 200 nt to about 8 kb, from about 400 nt to about 4 kb, or
from about 500 nt to about 2 kb of genomic DNA. The genomic
structure and sequence of the apoE gene of a number of species are
known. The equivalent of Arg-61 in the human apoE gene in a number
of species is known. See, e.g., Weisgraber (1994) Adv. Protein
Chem. 45:249-302.
[0073] The apoE gene of most species encodes an apoE polypeptide
having a threonine at a position equivalent to Arg-61 in human
apoE4. Weisgraber (1994) Adv. Protein Chem. 45:249-302. As noted
above, in addition to Arg-61, an apoE polypeptide should also have
an arginine at a position corresponding to Arg-112 in human apoE4,
and a glutamic acid at a position corresponding to Glu-255 in human
apoE4. If an apoE gene in the species does not contain the
equivalents of Arg-112 and Glu-255, the targeting construct can
comprise additional modifications of the endogenous gene sequence
such that, after homologous recombination, the endogenous apoE gene
is modified to comprise codons for the equivalent of Arg-112 and
Glu-255. (See Weisgraber, (1994)). If codon(s) in addition to the
Thr-61 codon are mutated, sufficient flanking sequences to allow
homologous recombination are included in the targeting
construct.
[0074] Methods for generating mutations are well known in the art;
any known method can be used to generate an Arg-61 targeting
construct. Examples of protocols for site specific mutagenesis may
be found in Gustin et al. (1993) Biotechniques 14:22; Barany (1985)
Gene 37:111-23; Colicelli et al. (1985), Mol. Gen. Genet.
199:537-9; and Prentki et al. (1984), Gene 29:303-13. Methods for
site specific mutagenesis can be found in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.
15.3-15.108; Weiner, et al., (1993) Gene 126:35-41; Sayers et al.,
(1992) Biotechniques 13:592-6; Jones, et al., (1992) Biotechniques
12:528-30; Barton et al., (1990) Nucleic Acids Res 18:7349-55;
Marotti, et al., (1989) Gene Anal. Tech. 6:67-70; and Zhu (1989)
Anal Biochem 177:120-4.
[0075] If desired, the introduced mutation can, in addition to
changing the codon for Thr-61 to a codon for arginine, provide for
a restriction endonuclease recognition sequence not present in the
endogenous apoE gene. Such a restriction site can be used to
determine whether a cell comprises a genetically modified apoE
gene.
[0076] In one particular embodiment, a construct as described in
Example 1 is used. This construct comprises mouse exons 1-4, which
were altered to change the codon for Thr-61 to a codon for
arginine. When this construct is used to replace the corresponding
portion of the endogenous mouse apoE gene, the Thr-61 of mouse apoE
is replaced with Arg-61, and apoE that exhibits apoE4 domain
interaction is produced.
[0077] Whether a given construct will replace a portion of an
endogenous apoE gene such that the apoE polypeptide encoded thereby
exhibits domain interaction can be determined before making a
gene-targeted animal with the construct. This can be achieved by
first introducing the construct into a cell line and selecting for
cells in which homologous recombination has occurred. Whether the
recombinant apoE produced by these cells exhibits domain
interaction can be determined using any known assay, including,
e.g., an emulsion binding assay, as described in more detail
below
[0078] DNA constructs for homologous recombination will comprise at
least a portion of the apoE gene with the desired genetic
modification, and will include regions of homology to the target
endogenous apoE locus. Conveniently, markers for positive and
negative selection are included. Methods for generating cells
having targeted gene modifications through homologous recombination
are known in the art. For various techniques for transfecting
mammalian cells, see Keown et al. Methods in Enzymology 185:527-537
(1990).
Generating a Gene-Targeted Animal
[0079] For embryonic stem (ES) cells, an ES cell line may be
employed, or embryonic cells may be obtained freshly from a host,
e.g. mouse, rat, guinea pig, etc. Such cells are grown on an
appropriate fibroblast-feeder layer or grown in the presence of
appropriate growth factors, such as leukemia inhibiting factor
(LIF). When ES cells have been transformed, they may be used to
produce gene-targeted animals. See U.S. Pat. Nos. 5,387,742;
4,736,866; and 5,565,186; and Larson et al. (2000) Mol. Ther.
2:631-639 for methods of making gene-targeted animals. After
transformation, the cells are plated onto a feeder layer in an
appropriate medium. Cells containing the construct may be detected
by employing a selective medium. After sufficient time for colonies
to grow, they are picked and analyzed for the occurrence of
homologous recombination or integration of the construct. Those
colonies that are positive may then be used for embryo manipulation
and blastocyst injection. Blastocysts are obtained from 4 to 6 week
old superovulated females. The ES cells are trypsinized, and the
modified cells are injected into the blastocoel of the blastocyst.
After injection, the blastocysts are returned to each uterine horn
of pseudopregnant females. Females are then allowed to go to term
and the resulting litters screened for mutant cells having the
construct. By providing for a different phenotype of the blastocyst
and the ES cells, chimeric progeny can be readily detected.
[0080] The chimeric animals are screened for the presence of the
modified apoE gene and males and females having the modification
are mated to produce homozygous progeny. If the gene alterations
cause lethality at some point in development, tissues or organs can
be maintained as allogeneic or congenic grafts or transplants, or
in in vitro culture.
Isolated Cells
[0081] The invention further provides cells isolated from a
gene-targeted animal of the invention, particularly cells that
synthesize recombinant apoE that exhibits domain interaction. The
isolated cells are useful for testing agents for their ability to
reduce apoE4 domain interaction. Cells are isolated using standard
procedures. Cell lines may be derived from such isolated cells, and
immortalized using standard techniques, e.g., through use of
viruses. Of particular interest are cells derived from a
gene-targeted animal of the invention that synthesize the
recombinant apoE protein. In some embodiments, of particular
interest are astrocytes and microglial cells that produce the
recombinant apoE protein. In other embodiments, cells derived from
a gene-targeted animal of the invention are cells that take up
recombinant apoE from their environment and which display a
physiological change in response to the recombinant apoE. Such
cells include, but are not limited to, neuronal cells, e.g.,
neuronal cells that display apoE4-mediated neurite outgrowth
inhibition.
Recombinant ApoE Protein
[0082] The invention further provides recombinant apoE proteins,
and compositions comprising a recombinant apoE protein. A
recombinant apoE protein of the invention is a non-human apoE
protein that has been modified such the recombinant apoE protein
exhibits domain interaction characteristic of human apoE4 protein.
Recombinant apoE protein of the invention can be used in screening
assays to identify agents that disrupt domain interaction in the
recombinant apoE polypeptide, which agents are thus useful for
treating apoE4-associated disorders in humans.
[0083] Recombinant apoE proteins comprise one or more amino acid
substitutions compared to the apoE polypeptide encoded by the
endogenous apoE gene of the gene-targeted animal from which they
are derived. Thus, a recombinant apoE polypeptide comprises the
equivalents of human apoE Arg-61, Arg-112, and Glu-255, and, as a
result, exhibits domain interaction.
[0084] The recombinant apoE protein of the subject invention is
typically separated from its source, e.g., the gene-targeted
animal, or the cell derived therefrom, that synthesizes the
recombinant protein. In certain embodiments, the subject proteins
are present in a composition that is enriched for subject protein
as compared to its source. For example, purified recombinant apoE
proteins are provided, where by purified is meant that the
recombinant apoE protein is present in a composition that is
substantially free of non-recombinant apoE proteins, where by
substantially free is meant that less than 90%, usually less than
60% and more usually less than 50% of the composition is made up of
recombinant apoE proteins. The proteins of the subject invention
may also be present as an isolate, by which is meant that the
protein is substantially free of other proteins and other naturally
occurring biologic molecules, such as oligosaccharides,
polynucleotides and fragments thereof, and the like, where
substantially free in this instance means that less than 70%,
usually less than 60% and more usually less than 50% of the
composition containing the isolated protein is some other naturally
occurring biological molecule. In certain embodiments, the proteins
are present in substantially pure form, where by substantially pure
form is meant at least 95%, usually at least 97% and more usually
at least 99% pure.
[0085] A recombinant apoE protein that exhibits domain interaction
may have one or more amino acid substitutions, insertions, and
deletions compared to the wild-type non-human apoE protein, as long
as the protein comprises the equivalents of human apoE Arg-61,
Arg-112, and Glu-255, and, as a result, exhibits domain
interaction. Accordingly, also provided are recombinant apoE
proteins that are substantially identical to the sequence of
wild-type non-human apoE polypeptide, where by substantially
identical is meant that the protein has an amino acid sequence
identity to the sequence of wild-type non-human apoE polypeptide of
at least about 75%, at least about 80% at least about 85%, at least
about 90%, at least about 95%, or at least about 98%.
[0086] The subject proteins and polypeptides may be obtained from a
gene-targeted animal of the invention, a cell derived from a
gene-targeted animal of the invention, or synthetically produced.
The subject proteins may also be derived from synthetic means, e.g.
by expressing a recombinant gene encoding protein of interest in a
suitable host, as described in greater detail below. Any convenient
protein purification procedures may be employed, where suitable
protein purification methodologies are described in Guide to
Protein Purification, (Deuthser ed.) (Academic Press, 1990). For
example, a lysate may prepared from the original source and
purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, and the like.
Nucleic Acid Molecules and Host Cells
[0087] The invention further provides nucleic acid molecules
comprising a nucleotide sequence that encodes a recombinant apoE
protein, as well as host cells comprising the nucleic acid
molecules. The subject nucleic acid molecules may be part of a
vector ("construct") for use in generating a gene-targeted animal
of the invention, as described above. In addition, a nucleic acid
molecule of the invention may encode all or part of a recombinant
apoE polypeptide of the invention, and as such is useful, as part
of an expression vector, in producing recombinant apoE
polypeptide.
[0088] The instant invention provides a nucleic acid molecule
comprising one or more exons and one or more introns of an apoE
gene, wherein the apoE gene is a non-human apoE gene, and the apoE
gene is modified such that the apoE protein encoded thereby
exhibits domain interaction characteristic of human apoE4. In some
embodiments, a subject nucleic acid molecule comprises a non-human
apoE gene, wherein an exon comprising a codon for Thr-61 (or its
equivalent in the apoE gene of the animal to be modified) is
modified to encode arginine. The sequences of the apoE gene from a
number of species are known and are publicly available. Mouse apoE
genomic organization is shown in FIG. 1. The sequence of the mouse
apoE gene is found under Genbank accession number D00466. Various
primate apoE gene sequences are found under GenBank accession
numbers AF200508, AF200507, AF200506, and AH009953 (Hylobates lar,
or gibbon); AH009952, AF200503, AF200504, and AF200505 (Pongo
pygmaeus, or orangutan); AH009951, AF200500, AG200501, and AF200502
(Gorilla gorilla); AH009950, AF200497, AF200498, AF200499 (Pan
troglodytes, or chimpanzee). Any of these sequences can be modified
such that the encoded recombinant apoE polypeptide exhibits domain
interaction, e.g., by modifying the codon for Thr-61 to encode
arginine.
[0089] In some embodiments, nucleic acids of the invention include
the open reading frame encoding all or part of the recombinant apoE
polypeptide, one or more introns, may further include adjacent 5'
and 3' non-coding nucleotide sequences involved in the regulation
of expression, and are generally up to about 10 kb in total length,
but possibly longer. The DNA sequences encoding all or part of the
recombinant apoE are genomic DNA or a fragment thereof. The
recombinant apoE gene may be introduced into an appropriate vector
for extrachromosomal maintenance or for integration into the
host.
[0090] A genomic sequence of interest comprises the nucleic acid
present between the initiation codon and the stop codon, as defined
in the listed sequences, including all of the introns that are
normally present in a native chromosome. It may further include the
3' and 5' untranslated regions found in the mature mRNA. It may
further include specific transcriptional and translational
regulatory sequences, such as promoters, enhancers, etc., including
about 1 kb, but possibly more, of flanking genomic DNA at either
the 5' or 3' end of the transcribed region. The genomic DNA may be
isolated as a fragment of 100 kbp or smaller; and substantially
free of flanking chromosomal sequence.
[0091] The sequence of this 5' region, and further 5' upstream
sequences and 3' downstream sequences, may be utilized for promoter
elements, including enhancer binding sites, that provide for
expression in tissues where apoE is expressed. The tissue specific
expression is useful for determining the pattern of expression, and
for providing promoters that mimic the native pattern of
expression. Naturally occurring polymorphisms in the promoter
region are useful for determining natural variations in expression,
particularly those that may be associated with disease.
Alternatively, mutations may be introduced into the promoter region
to determine the effect of altering expression in experimentally
defined systems. Methods for the identification of specific DNA
motifs involved in the binding of transcriptional factors are known
in the art, e.g. sequence similarity to known binding motifs, gel
retardation studies, etc. For examples, see Blackwell et al Mol Med
1:194-205 (1995); Mortlock et al. Genome Res. 6:327-33 (1996); and
Joulin and Richard-Foy Eur J Biochem 232:620-626 (1995).
[0092] The regulatory sequences may be used to identify cis acting
sequences required for transcriptional or translational regulation
of apoE expression, especially in different tissues or stages of
development, and to identify cis acting sequences and trans acting
factors that regulate or mediate expression. Such transcription or
translational control regions may be operably linked to an apoE
gene in order to promote expression of wild type or altered apoE or
other proteins of interest in cultured cells, or in embryonic,
fetal or adult tissues; and for gene therapy.
[0093] In other embodiments, a nucleic acid molecule of the
invention comprises a cDNA comprising sequences that encode all or
part of a recombinant apoE protein of the invention. The nucleic
acid compositions used in the subject invention may encode all or a
part of the apoE polypeptides as appropriate. Fragments may be
obtained of the DNA sequence by chemically synthesizing
oligonucleotides in accordance with conventional methods, by
restriction enzyme digestion, by PCR amplification, etc. For the
most part, DNA fragments will be of at least 15 nt, usually at
least 18 nt, more usually at least about 50 nt. Such small DNA
fragments are useful as primers for PCR, hybridization screening,
etc. Larger DNA fragments, i.e. greater than 100 nt are useful for
production of the encoded polypeptide. For use in amplification
reactions, such as PCR, a pair of primers will be used.
[0094] In some embodiments, a nucleic acid molecule of the
invention comprises nucleotide sequences of a mouse genomic apoE
gene, modified as described above such that the encoded apoE
protein exhibits domain interaction characteristic of human apoE4.
In other embodiments, a nucleic acid molecule of the invention
comprises the coding regions of a mouse apoE gene, modified as
described above such that the encoded apoE protein exhibits domain
interaction characteristic of human apoE4 (e.g., a cDNA
molecule).
[0095] The invention further provides nucleic acid molecules that
comprise a nucleotide sequence that encodes a recombinant apoE
protein that exhibits domain interaction, wherein the nucleic acid
molecules hybridize under stringent hybridization conditions to one
of a mouse genomic apoE gene, modified such that the encoded apoE
protein exhibits domain interaction characteristic of human apoE4;
and the coding region of a mouse apoE gene, modified as described
above such that the encoded apoE protein exhibits domain
interaction characteristic of human apoE4. Examples of stringent
conditions are hybridization and washing at 50.degree. C. or higher
and in 0.1.times.SSC (9 mM NaCl/0.9 mM sodium citrate).
[0096] The invention further provides nucleic acid molecules that
comprise a nucleotide sequence that encodes a recombinant apoE
protein that exhibits domain interaction, wherein the nucleic acid
molecules have at least about 75%, at least about 80%, at least
about 85%, at least about 90%, at least about 95%, or higher,
nucleotide sequence identity with one of a mouse genomic apoE gene,
modified such that the encoded apoE protein exhibits domain
interaction characteristic of human apoE4; and the coding region of
a mouse apoE gene, modified as described above such that the
encoded apoE protein exhibits domain interaction characteristic of
human apoE4.
[0097] Subject nucleic acid molecules may comprise other,
non-recombinant apoE nucleic acid molecules ("heterologous nucleic
acid molecules") of any length. For example, the subject nucleic
acid molecules may be flanked on the 5' and/or 3' ends by
heterologous nucleic acid molecules of from about 1 nt to about 10
nt, from about 10 nt to about 20 nt, from about 20 nt to about 50
nt, from about 50 nt to about 100 nt, from about 100 nt to about
250 nt, from about 250 nt to about 500 nt, or from about 500 nt to
about 1000 nt, or more in length. For example, when used as a probe
to detect nucleic acid molecules capable of hybridizing with the
subject nucleic acids, the subject nucleic acid molecules may be
flanked by heterologous sequences of any length.
[0098] The subject nucleic acid molecules may also be provided as
part of a vector, a wide variety of which are known in the art and
need not be elaborated upon herein. Vectors include, but are not
limited to, plasmids; cosmids; viral vectors; artificial
chromosomes (YAC's, BAC's, etc.); mini-chromosomes; and the like.
Vectors are amply described in numerous publications well known to
those in the art, including, e.g., Short Protocols in Molecular
Biology, (1999) F. Ausubel, et al., eds., Wiley & Sons. Vectors
may provide for expression of the subject nucleic acids, may
provide for propagating the subject nucleic acids, or both.
[0099] The subject nucleic acid molecules are isolated and obtained
in substantial purity, generally as other than an intact
chromosome. Usually, the DNA will be obtained substantially free of
other nucleic acid sequences that do not include a sequence or
fragment thereof of the subject genes, generally being at least
about 50%, usually at least about 90% pure and are typically
"recombinant", i.e. flanked by one or more nucleotides with which
it is not normally associated on a naturally occurring
chromosome.
[0100] The subject nucleic acid compositions find use in the
preparation of all or a portion of the recombinant apoE
polypeptides of the invention, as described above. For expression,
an expression cassette may be employed. The expression vector will
provide a transcriptional and translational initiation region,
which may be inducible or constitutive, where the coding region is
operably linked under the transcriptional control of the
transcriptional initiation region, and a transcriptional and
translational termination region. These control regions may be
native to a gene encoding the subject peptides, or may be derived
from exogenous sources.
[0101] Expression vectors generally have convenient restriction
sites located near the promoter sequence to provide for the
insertion of nucleic acid sequences encoding heterologous proteins.
A selectable marker operative in the expression host may be
present. Expression vectors may be used for the production of
fusion proteins, where the exogenous fusion peptide provides
additional functionality, i.e. increased protein synthesis,
stability, reactivity with defined antisera, an enzyme marker, e.g.
.beta.-galactosidase, etc.
[0102] Expression cassettes may be prepared comprising a
transcription initiation region, the gene or fragment thereof, and
a transcriptional termination region. Of particular interest is the
use of sequences that allow for the expression of functional
epitopes or domains, usually at least about 8 amino acids in
length, more usually at least about 15 amino acids in length, to
about 25 amino acids, or any of the above-described fragment, and
up to the complete open reading frame of the gene. After
introduction of the DNA, the cells containing the construct may be
selected by means of a selectable marker, the cells expanded and
then used for expression.
[0103] Proteins and polypeptides may be expressed in prokaryotes or
eukaryotes in accordance with conventional ways, depending upon the
purpose for expression. For large scale production of the protein,
a unicellular organism, such as E. coli, B. subtilis, S.
cerevisiae, insect cells in combination with baculovirus vectors,
or cells of a higher organism such as vertebrates, particularly
mammals, e.g. COS 7 cells, Neuro-2A cells, may be used as the
expression host cells. In some situations, it is desirable to
express the gene in eukaryotic cells, where the encoded protein
will benefit from native folding and post-translational
modifications. Small peptides can also be synthesized in the
laboratory. Polypeptides that are subsets of the complete sequences
of the subject proteins may be used to identify and investigate
parts of the protein important for function.
[0104] Specific expression systems of interest include bacterial,
yeast, insect cell and mammalian cell derived expression systems. A
wide variety of such systems are known to those skilled in the
art.
[0105] Recombinant host cells comprising a subject nucleic acid
molecule may serve as a source of recombinant apoE protein of the
invention. They may also serve in drug screening assays to identify
agents that reduce apoE4 domain interaction.
[0106] In some embodiments, of particular interest are mammalian
cells that normally produce apoE, and cells that normally take up
apoE from their environment. Examples of such cells include
neuronal cells, microglial cells, and astrocytes. Immortalized
neuronal cells, microglial cells, and astrocytes are also of
interest.
Drug Screening Assays
[0107] The present invention provides methods for identifying
agents that reduce apoE4 domain interaction, using a gene-targeted
animal, or isolated cell, or isolated recombinant apoE protein of
the invention. In some embodiments, the methods identify agents
that reduce a phenomenon associated with an apoE4-associated
neurological disorder. In other embodiments, the methods identify
agents that treat an apoE4-associated cardiovascular disorder. The
methods generally comprise contacting a gene-targeted animal or
isolated cell of the invention with a test agent, and determining
the effect of the agent on the gene-targeted animal, the isolated
cell, or recombinant apoE produced by an isolated cell. Agents that
reduce apoE4 domain interaction are identified by a change in an
activity associated with apoE4; a phenomenon associated with an
apoE4-related neurological disorder; or a phenomenon associated
with an apoE4-related cardiovascular disorder. ApoE4-associated
activities include, but are not limited to, binding preference of
the apolipoprotein for a particular class of lipoprotein; binding
to tau protein in vitro and/or in vivo; and binding to A.beta.
protein.
[0108] In some embodiments, the invention provides methods of
identifying an agent that reduces apoE domain interaction,
comprising contacting a non-human gene-targeted animal of the
invention with a test agent, and determining the effect, if any, on
an activity or disorder associated with apoE4. In other
embodiments, the invention provides methods for identifying an
agent useful for treating an apoE4-associated neurological
disorder, comprising contacting a non-human gene-targeted animal of
the invention with a test agent, and determining the effect, if
any, on a phenomenon associated with an apoE4-related neurological
disorder.
[0109] An effect on apoE4 domain interaction, or any associated
phenomenon (e.g., a neurological disorder, a symptom of a
neurological disorder, a cardiovascular disorder, a symptom of a
cardiovascular disorder), or any apoE4-associated activity, can be
determined in comparison to a suitable control. Suitable controls
for assays using a gene-targeted animal of the invention include a
wild-type animal of the same species contacted with the test agent;
and a gene-targeted animal not contacted with the test agent.
Suitable controls for assays using a cell isolated from a
gene-targeted animal of the invention include a cell not contacted
with the test agent; and a cell of the same cell type from a
wild-type animal of the same species, which cell is contacted with
the test agent. Controls for specificity of the test agent on
recombinant apoE polypeptide include controls which assay the
effect of the test agent on another human apoE isoform, e.g., human
apoE3; and controls which assay an activity of the recombinant apoE
protein in the absence of the test agent.
[0110] A wide variety of assays may be used for this purpose,
including in vivo behavioral studies, physiological analyses (e.g.,
to measure plaque formation on arterial walls; to measure a
parameter associated with cardiac function), isoelectric
focusing/western blot assays (as described in the Examples), assays
to measure serum lipid levels, emulsion binding assays,
immunoassays for protein binding (e.g., binding to tau protein),
and the like. Depending on the particular assay, whole animals may
be used, or cells derived therefrom, or isolated recombinant apoE
protein. Cells may be freshly isolated from an animal, or may be
immortalized in culture. Any cell that produces recombinant apoE
can be used, or that takes up apoE4 from its environment, e.g.,
neuronal cells, microglial cells, and astrocytes. Cells of
particular interest include neural and brain tissue of
gene-targeted animals of the invention. The assays may also measure
response to acute injury, i.e. examining the neurite growth, repair
and remodelling Compounds which stimulate neurite extension in vivo
are likely to promote nerve regeneration or the formation of
synaptic connections during neuronal remodeling in both the central
and peripheral nervous system.
[0111] The term "agent" as used herein describes any molecule, e.g.
protein or non-protein organic pharmaceutical, with the capability
of affecting any of the biological actions of apoE4. Agents of
particular interest are those that reduce apoE4 domain interaction.
Generally a plurality of assay mixtures are run in parallel with
different agent concentrations to obtain a differential response to
the various concentrations. Typically, one of these concentrations
serves as a negative control, i.e. at zero concentration or below
the level of detection.
[0112] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, e.g., small organic compounds
having a molecular weight of more than 50 and less than about 2,500
daltons. Candidate agents comprise functional groups necessary for
structural interaction with proteins, e.g., van der Waals
interactions and hydrogen bonding, and may include at least an
amine, carbonyl, hydroxyl or carboxyl group, and may include at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Candidate agents are also found among
biomolecules including, but not limited to: peptides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives,
structural analogs or combinations thereof.
[0113] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligopeptides. Alternatively, libraries of
natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means, and may be used to produce combinatorial
libraries. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification, etc. to produce
structural analogs. New potential therapeutic agents may also be
created using methods such as rational drug design or computer
modeling.
[0114] Screening may be directed to known pharmacologically active
compounds and chemical analogs thereof, or to new agents with
unknown properties such as those created through rational drug
design. Candidate agents for arresting and/or reversing dementia,
neuron remodeling, or recovery from acute insults to the nervous
system can be screened for their ability to modulate apoE4 function
or phenotypes associated with apoE4. Efficacious candidates can be
identified by phenotype, i.e. an arrest or reversal of particular
cognitive behaviors in comparison with wild-type animals and a
gene-targeted animal of the invention.
[0115] Agents that have an effect in an assay method of the
invention may be subjected to directed or random and/or directed
chemical modifications, such as acylation, alkylation,
esterification, amidification, etc. to produce structural analogs.
Such structural analogs include those that increase
bioavailability, and/or reduced cytotoxicity. Those skilled in the
art can readily envision and generate a wide variety of structural
analogs, and test them for desired properties such as increased
bioavailability and/or reduced cytotoxicity and/or ability to cross
the blood-brain barriers.
[0116] Screening may also determine if an agent for a different use
has an unintended adverse effect on apoE-related functions,
including but not limited to neuronal remodeling, repair and
recovery from acute insults. For example, certain classes of
pharmaceutical agents widely used to treat behavior problems in
people with dementia may actually worsen their mental decline.
Neuroleptic agents such as chlorpromazine, haloperidol and
thioridazine are widely used to treat behavior problems in patients
with various forms of dementia, including AD. Recent studies
suggest that these drugs may in fact worsen the cognitive or any
other function of people treated with these agents. Therapeutic
agents such as neuroleptic drugs can be subjected to the methods of
the present invention to determine if they are in fact having a
detrimental effect on cognitive or any other function by modulation
of apolipoprotein E4. This screening can be used for any agent
predicted to affect cognitive function to determine if the agent
may inadvertently have an unintended effect on apolipoprotein E4
function.
[0117] Where the screening assay is a binding assay, one or more of
the molecules may be joined to a label, where the label can
directly or indirectly provide a detectable signal. Various labels
include radioisotopes, fluorescers, chemiluminescers, enzymes,
specific binding molecules, particles, e.g. magnetic particles, and
the like. Specific binding molecules include pairs, such as biotin
and streptavidin, digoxin and antidigoxin etc. For the specific
binding members, the complementary member would normally be labeled
with a molecule that provides for detection, in accordance with
known procedures.
[0118] A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc that are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Reagents that improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors, anti-microbial
agents, etc. may be used. The mixture of components are added in
any order that provides for the requisite binding. Incubations are
performed at any suitable temperature, typically between 4 and
40.degree. C. Incubation periods are selected for optimum activity,
but may also be optimized to facilitate rapid high-throughput
screening. Typically between 0.1 and 1 hours will be
sufficient.
[0119] Samples derived from a gene-targeted animal of the invention
may also be used in assays. Samples, as used herein, include
biological fluids such as tracheal lavage, blood, cerebrospinal
fluid, tears, saliva, lymph, dialysis fluid and the like; organ or
tissue culture derived fluids; and fluids extracted from
physiological tissues. Also included in the term are derivatives
and fractions of such fluids. The number of cells in a sample will
generally be at least about 10.sup.3, usually at least 10.sup.4
more usually at least about 10.sup.5. The cells may be dissociated,
in the case of solid tissues, or tissue sections may be analyzed.
Alternatively a lysate of the cells may be prepared.
[0120] For example, detection may utilize staining of cells or
histological sections, performed in accordance with conventional
methods. The antibodies of interest are added to the cell sample,
and incubated for a period of time sufficient to allow binding to
the epitope, usually at least about 10 minutes. The antibody may be
labeled with radioisotopes, enzymes, fluorescers, chemiluminescers,
or other labels for direct detection. Alternatively, a second stage
antibody or reagent is used to amplify the signal. Such reagents
are well known in the art. For example, the primary antibody may be
conjugated to biotin, with horseradish peroxidase-conjugated avidin
added as a second stage reagent. Final detection uses a substrate
that undergoes a color change in the presence of the peroxidase.
The absence or presence of antibody binding may be determined by
various methods, including flow cytometry of dissociated cells,
microscopy, radiography, scintillation counting, etc.
[0121] An alternative method depends on the in vitro detection of
binding between antibodies and apoE4 in a lysate. Measuring the
concentration of binding in a sample or fraction thereof may be
accomplished by a variety of specific assays. A conventional
sandwich type assay may be used. For example, a sandwich assay may
first attach specific antibodies to an insoluble surface or
support. The particular manner of binding is not crucial so long as
it is compatible with the reagents and overall methods of the
invention. They may be bound to the plates covalently or
non-covalently, preferably non-covalently.
[0122] The insoluble supports may be any compositions to which
polypeptides can be bound, which is readily separated from soluble
material, and which is otherwise compatible with the overall
method. The surface of such supports may be solid or porous and of
any convenient shape. Examples of suitable insoluble supports to
which the receptor is bound include beads, e.g. magnetic beads,
membranes and microtiter plates. These are typically made of glass,
plastic (e.g. polystyrene), polysaccharides, nylon or
nitrocellulose. Microtiter plates are especially convenient because
a large number of assays can be carried out simultaneously, using
small amounts of reagents and samples.
[0123] A number of assays are known in the art for determining the
effect of a drug on animal behavior. Behavioral abnormalities and
recovery from acute or chronic injury in animal models are useful
for testing the effect, interactions, and specificity of a
candidate biologically active agent. Some examples are provided,
although it will be understood by one of skill in the art that many
other assays may also be used. The subject animals may be used by
themselves, or in combination with control animals.
[0124] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligopeptides. Alternatively, libraries of
natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means, and may be used to produce combinatorial
libraries. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification, etc. to produce
structural analogs.
[0125] Detection may utilize staining of cells or histological
sections, performed in accordance with conventional methods. The
antibodies of interest are added to the cell sample, and incubated
for a period of time sufficient to allow binding to the epitope,
usually at least about 10 minutes. The antibody may be labeled with
radioisotopes, enzymes, fluorescers, chemiluminescers, or other
labels for direct detection. Alternatively, a second stage antibody
or reagent is used to amplify the signal. Such reagents are well
known in the art. For example, the primary antibody may be
conjugated to biotin, with horseradish peroxidase-conjugated avidin
added as a second stage reagent. Final detection uses a substrate
that undergoes a color change in the presence of the peroxidase.
The absence or presence of antibody binding may be determined by
various methods, including flow cytometry of dissociated cells,
microscopy, radiography, scintillation counting, etc.
[0126] A number of assays are known in the art for determining the
effect of a drug on animal behavior and other phenomena associated
with neurodegeneration or impairment of cognitive abilities. Some
examples are provided, although it will be understood by one of
skill in the art that many other assays may also be used. The
subject animals may be used by themselves, or in combination with
control animals.
[0127] The screen using the gene-targeted animals of the invention
can employ any phenomena associated learning impairment, dementia
or cognitive disorders that can be readily assessed in an animal
model. The screening can include assessment of phenomena including,
but not limited to: 1) analysis of molecular markers (e.g., levels
of expression of recombinant apoE gene products in brain tissue;
presence/absence in brain tissue of recombinant apoE; and formation
of neurite plaques; formation of A.beta. deposits); 2) assessment
behavioral symptoms associated with memory and learning; 3)
detection of neurodegeneration characterized by progressive and
irreversible deafferentation of the limbic system, association
neocortex, and basal forebrain (neurodegeneration can be measured
by, for example, detection of synaptophysin expression in brain
tissue) (see, e.g., Games et al. Nature 373:523-7 (1995)). These
phenomena may be assessed in the screening assays either singly or
in any combination.
[0128] Preferably, the screen will include control values (e.g.,
the level of amyloid production in the test animal in the absence
of test compound(s)). Test substances which are considered
positive, i.e., likely to be beneficial in the treatment of
apoE4-associated disorders, will be those which have a substantial
effect upon an apoE4-associated phenomenon (e.g., test agents that
are able to rescue behavioral disorders caused by altered
expression of apoE).
[0129] Methods for assessing these phenomena, and the effects
expected of a candidate agent for treatment of apoE4-associated
disorders, are known in the art. For example, methods for using
gene-targeted animals in various screening assays for, for example,
testing compounds for an effect on AD, are found in WO 9640896,
published Dec. 19, 1996; WO 9640895, published Dec. 19, 1996; WO
9511994, published May 4, 1995 (describing methods and compositions
for in vivo monitoring of A.beta.; each of which is incorporated
herein by reference with respect to disclosure of methods and
compositions for such screening assays and techniques). Examples of
assessment of these phenomena are provided below, but are not meant
to be limiting.
ApoE4-Associated Activity
[0130] Assays include assays for apoE4-associated activity, and
include, but are not limited to, a binding preference of apoE4 to
particular class of lipoprotein, and binding to tau protein. Such
assays typically involve contacting a recombinant apoE protein with
a test agent, to form a sample, and determining the effect, if any,
of the agent on an apoE4-associated activity.
[0131] ApoE4 has a binding preference for VLDL, while apoE3 has a
binding preference for HDL. Typically, when plasma lipoproteins are
allowed to bind to labeled apoE4 and apoE3, the bound proteins
fractionated, and the amount of apoE4 and apoE3 in each fraction
measured, the amount of apoE4 in the VLDL, IDL/LDL, and HDL
fractions is about 35%, about 23%, about 42%, respectively, while
the amount of apoE3 in each of these fractions is about 20%, about
20%, about 60%, respectively. Thus, in some embodiments, an agent
that reduces apoE4 domain interaction causes apoE4 to have a
binding preference for HDL. Whether apoE4, when contacted with an
agent that reduces apoE4 domain interaction, has a binding
preference for HDL over VLDL can be determined using any known
assay. As one non-limiting example, an assay as described in Dong
et al. (1994) J. Biol. Chem. 269:22358-22365. For example, samples
comprising detectably labeled apoE4 and apoE3 (e.g., labeled with
.sup.125I), are mixed with plasma at about 37.degree. C. for about
2 hours, after which time the samples are fractionated into various
lipoprotein classes (e.g., by chromatography), and the amount of
label in each fraction is determined.
Emulsion Binding Assays
[0132] A non-limiting example of an emulsion binding assay to
assess domain interaction is as follows. Triolein (160 mg) and
L-alpha-Phosphatidylcholine (40 mg) are combined and dried under
nitrogen. After the addition of 8 mls of buffer (10 mM Tris, 100 mM
KCl, 1 mM EDTA, pH 8.0), the mixture is sonicated in a water bath
to obtain a heterogeneous mix of emulsion particles. The particles
are harvested by ultracentrifugation (TLA 100.2 rotor, 30,000 rpm
for 30 minutes) and the subsequent lipid cake is removed by tube
slicing and resuspended in 100 .mu.l 20 mM Phosphate Buffer (PB).
Triolein and phospholipid content are measured and total emulsion
particle concentration is determined. Freshly denatured and
renatured Apolipoprotein E3 and E4 are radiolabelled using
Bolton-Hunter Reagent [.sup.125I] (ICN). Specific Activity is
determined using Lowry method and Gamma 8000 counter. The binding
affinity of apoE3 and apoE4 to emulsion particles may be determined
as follows. In glass tubes, 25 .mu.g of protein (with iodinated
tracer) are reduced with 1% .beta.-mercaptoethanol. Two hundred
fifty .mu.g of emulsion particles and 2.5 .mu.l of compound (10 mM
stock) are added and the final mixture is brought up to 250 .mu.l
with 20 mM phosphate buffer (PB). The reaction mixture is then
incubated in a 37.degree. C. water bath for 2 hours before being
transferred to 1.5 ml ultracentrifuge tubes. Finally, 50 .mu.l of
60% sucrose is mixed with the sample and 400 .mu.l 20 mM PB is
carefully layered on top. Using a TLA 100.2 rotor, the tube is spun
at 30,000 rpm for 30 minutes and subsequently cut to separate the
floating emulsion particle layer from the free protein at the
bottom of the tube. These fractions are then combined with the
respective half of the actual tube and counted using a Gamma-8000.
From these results, total emulsion-bound protein is compared to
total free protein. Control binding assays are conducted without
the addition of test compounds to determine recovery and apoE3 and
apoE4 respective affinity for emulsion particles.
Binding to Tau Protein
[0133] ApoE3 interacts with tau in vitro, while apoE4 does not. In
some embodiments, an agent that reduces apoE4 domain interaction
causes apoE4 to bind tau in vitro and/or in vivo. Whether a protein
binds tau in vitro, e.g., in the presence of an agent that reduces
apoE4 domain interaction, can be determined using standard assays
for measuring or detecting protein-protein interaction. A
non-limiting example of an assay is provided in Strittmatter et al.
(1994) Exp. Neurol. 125:163-171.
Pathological Studies
[0134] After exposure to the candidate agent, the animals are
sacrificed and analyzed by immunohistology for either: 1) neuritic
plaques and neurofibrillary tangles (NFTs) in the brain and/or 2)
levels of recombinant apoE. The brain tissue is fixed (e.g. in 4%
paraformladehyde) and sectioned; the sections are stained with
antibodies reactive with apoE. Secondary antibodies conjugated with
fluorescein, rhodamine, horse radish peroxidase, or alkaline
phosphatase are used to detect the primary antibody. These
experiments permit identification of amyloid plaques and the
regionalization of these plaques to specific areas of the
brain.
[0135] Sections can also be stained with other diagnostic
antibodies recognizing antigens such as Alz-50, tau, A2B5,
neurofilaments, neuron-specific enolase, and others that are
characteristic of neurodegeneration. Staining with thioflavins and
congo red can also be carried out to analyze co-localization of
A.beta. deposits within the neuritic plaques and NFTs.
[0136] To assess the effect of a test agent on apoE4-related
cardiovascular disorders and phenomena associated with such
disorders, a gene-targeted animal can be fed a high-cholesterol
diet. A gene-targeted animal of the invention fed a
high-cholesterol diet can be treated with a test agent, and various
parameters measured, including, but not limited to, serum
cholesterol levels, total plasma cholesterol levels, total plasma
lipoproteins levels, levels of specific plasma lipoproteins (e.g.,
HDL, LDL, VLDL), and atherosclerosis. Standard assays to measure
such parameters are known to those skilled in the art, and any
known assay can be used. Serum cholesterol levels in response to
the test agent can be measured using standard assays. Development
of arterial plaques can be measured using standard assays.
Analysis of Recombinant ApoE Expression
[0137] 1) mRNA: mRNA can be isolated by the acid guanidinium
thiocyanate phenol:chloroform extraction method (Chomczynski et
al., Anal Biochem 162:156-159 (1987)) from cell lines and tissues
of gene-targeted animals to determine expression levels by Northern
blots. Whether a recombinant apoE gene is expressed can be
determined using, e.g., a labeled oligonucleotide probe that spans
the Thr-61 to Arg-61 mutation. The labeled oligonucleotide may
comprise the mutation that led to the Arg-61 substitution, in which
case expression of the genetically modified gene is identified by
hybridization with the labeled probe.
[0138] 2) In situ Hybridizations: Radioactive or enzymatically
labeled probes can be used to detect mRNA in situ. The probes are
degraded approximately to 100 nucleotides in length for better
penetration of cells. The procedure of Chou et al. J Psychiatr Res
24:27-50 (1990) for fixed and paraffin embedded samples is briefly
described below although similar procedures can be employed with
samples sectioned as frozen material.
[0139] Paraffin slides for in situ hybridization are dewaxed in
xylene and rehydrated in a graded series of ethanols and finally
rinsed in phosphate buffered saline (PBS). The sections are
postfixed in fresh 4% paraformaldehyde. The slides are washed with
PBS twice for 5 minutes to remove paraformaldehyde. Then the
sections are permeabilized by treatment with a 20 mu g/ml
proteinase K solution. The sections are refixed in 4%
paraformaldehyde, and basic molecules that could give rise to
background probe binding are acetylated in a 0.1 M triethanolamine,
0.3M acetic anhydride solution for 10 minutes. The slides are
washed in PBS, then dehydrated in a graded series of ethanols and
air dried. Sections are hybridized with antisense probe, using
sense probe as a control. After appropriate washing, bound
radioactive probes are detected by autoradiography or enzymatically
labeled probes are detected through reaction with the appropriate
chromogenic substrates.
[0140] 3) Western Blot Analysis: Protein fractions can be isolated
from tissue homogenenates and cell lysates and subjected to Western
blot analysis as described by Harlow et al., Antibodies: A
laboratory manual, Cold Spring Harbor, N.Y., (1988); Brown et al.,
J. Neurochem 40:299-308 (1983); and Tate-Ostroff et al., Proc Natl
Acad Sci 86:745-749 (1989)). Only a brief description is given
below.
[0141] The protein fractions can be denatured in Laemmli sample
buffer and electrophoresed on SDS-polyacrylamide gels. The proteins
are be then transferred to nitrocellulose filters by
electroblotting. The filters are blocked, incubated with primary
antibodies, and finally reacted with enzyme conjugated secondary
antibodies. Subsequent incubation with the appropriate chromogenic
substrate reveals the position of apoE proteins. An antibody that
distinguishes between an apoE polypeptide having Thr-61 from one
having Arg-61 may be employed.
[0142] 4) Isoelectric focusing combined with western blot analysis.
As a result of the arginine for threonine substitution, a
recombinant apoE protein may have a different charge than the
wild-type protein. Therefore, isoelectric focusing can be used to
distinguish between wild-type and recombinant apoE proteins.
Protein fractions are isolated as described above, and subjected to
isoelectric focusing, as described in the Examples, using standard
techniques. Isoelectric focusing is generally performed using
ampholines, typically in a pH range of 4-7, on polyacrylamide gels
(or other suitable matrix), in the presence of 6 M urea.
Behavioral Studies of Gene-Targeted Mice and Rats
[0143] Behavioral tests designed to assess learning and memory
deficits can be employed. An example of such as test is the Morris
Water maze (Morris Learn Motivat 12:239-260 (1981)). In this
procedure, the animal is placed in a circular pool filled with
water, with an escape platform submerged just below the surface of
the water. A visible marker is placed on the platform so that the
animal can find it by navigating toward a proximal visual cue.
Alternatively, a more complex form of the test in which there are
no formal cues to mark the platform's location will be given to the
animals. In this form, the animal must learn the platform's
location relative to distal visual cues. Alternatively, or in
addition, memory and learning deficits can be studied using a 3
runway panel for working memory impairment (attempts to pass
through two incorrect panels of the three panel-gates at four
choice points) (Ohno et al. Pharmacol Biochem Behav 57:257-261
(1997)).
Studies of Animal Models of Neuronal Damage
[0144] Rodent models of neuronal damage, for example neuronal
damage caused by cerebral ischemia, may be examined to determine
the effect on an agent on apoE4 domain interaction in the extent of
neuronal damage caused by traumatic events as well as their role in
neuronal remodeling, repair and recovery from such insults. Rodent
models of cerebral ischemia, both global ischemia and focal
ischemia, are useful for studying mechanisms controlling the
occurrence of cerebral ischemia and potential therapeutic
strategies for treatment of injury caused by ischemic events.
Animal models of global ischemia, which is usually transient, have
widely affected brain areas but typically give rise to neuronal
alterations in selectively vulnerable brain regions. Examples of
such models include, but are not limited to, the two vessel
occlusion model of forebrain ischemia, the four vessel occlusion
model of forebrain ischemia, and ischemia models involving elevated
cerebrospinal fluid pressure. See Ginsberg and Busto, Stroke,
20:1627-1642 (1989), which is herein incorporated by reference.
Models of focal ischemia may mimic ischemic stroke injury, and
typically give rise to localized brain infarction. Examples of
models of focal ischemia include, but are not limited to, middle
cerebral artery occlusion, photochemically induced focal cerebral
thrombosis, blood clot embolization, microsphere embolization and
the like. See McAuley, Cerebrovasc. Brain Metab. Review, 7:153-180
(1995) which is herein incorporated by reference.
[0145] Any of these models may be used in the gene-targeted animals
of the present invention to examine the effect of an agent on apoE4
domain interaction, both during traumatic brain injury and in
neuronal remodeling and repair following a traumatic ischemic
insult to the brain. For example, to examine the effect of a test
agent in a rodent stroke model, the rate and nature of injury
following cerebral ischemia may be examined using the gene-targeted
animals of the present invention. In such rodent models, the
trangenic animals can be subjected to an ischemic injury, and the
animals monitored for extent of damage and/or recovery following
injury. In addition, the effect of the agent may be examined in the
neuronal remodeling and recovery of these animal models. The effect
of the test agent in these processes may be examined using
biochemical, pathological, physiological or behavioral methods, as
described in the preceding sections.
[0146] Moreover, the effects of different therapies, including the
use of therapeutic agents, may be examined to determine potential
therapeutic strategies for mitigating and/or reversing the neuronal
damage in these animal models. For instance, in a rodent model of
focal cerebral ischemia using the animals of the present invention,
different candidate therapuetic agents may be administered prior to
the induction of the trauma to examine the preventive effect of
agents in specific brain regions, and whether that preventive
effect is apoE-isoform-dependent. Alternatively, a candidate
therapeutic agent may be administered following the induction of
the injury to determine the mitigating or recovery effects of that
agent, whether the agent is specific to certain brain regions, and
if the effect or the specificity of the agent is apoE-isoform
dependent.
ApoE4-Related Cardiovascular Disorders
[0147] Gene-targeted animals of the invention can be used to screen
agents for their ability to treat hyperlipidemia and other
apoE4-related cardiovascular disorders.
[0148] A gene-targeted animal of the invention can be used to test
agents for an effect on serum cholesterol levels. In these
embodiments, the methods comprise contacting a gene-targeted animal
of the invention with a test agent, and determining the effect, if
any, on serum cholesterol level. A reduction in serum cholesterol
level, when compared with an effect of the agent on a control
animal, indicates that the agent is effective in reducing serum
cholesterol levels.
[0149] Gene-targeted animals of the invention can also be used to
test agents for an effect on a disease associated with
hyperlipidemia, e.g., coronary artery disease and atherosclerosis.
A gene-targeted animal treated with a test agent can be analyzed
for the presence of arterial plaques using standard methods.
Therapeutic Agents
[0150] The invention provides agents identified using the methods
described herein. Agents that reduce apoE4 domain interaction are
used to treat apoE4-related disorders. An effective amount of the
active agent is administered to the host, where "effective amount"
means a dosage sufficient to produce a desired result. Generally,
the desired result is at least a reduction in enzymatic activity of
a subject therapeutic agent as compared to a control.
Formulations, Dosages, and Routes of Administration
[0151] The invention provides formulations, including
pharmaceutical formulations, comprising an agent that reduces apoE4
domain interaction. In general, a formulation comprises an
effective amount of an agent that reduces apoE4 domain interaction.
An "effective amount" means a dosage sufficient to produce a
desired result, e.g., reduction in apoE4 domain interaction, an
increase in neurite outgrowth, a reduction in serum lipid levels, a
reduced risk of heart disease, etc. Generally, the desired result
is at least a reduction in apoE4 domain interaction as compared to
a control. An agent that reduces apoE4 domain interaction may
delivered in such a manner as to avoid the blood-brain barrier, as
described in more detail below. An agent that reduces apoE4 domain
interaction may be formulated and/or modified to enable the agent
to cross the blood-brain barrier, as described in more detail
below.
Formulations
[0152] In the subject methods, the active agent(s) may be
administered to the host using any convenient means capable of
resulting in the desired inhibition of apoE4 domain interaction,
reduction in any apoE4-associated neurological disorder,
apoE4-associated cardiovascular disorder, reduction in an
apoE4-associated activity, etc.
[0153] Thus, the agent can be incorporated into a variety of
formulations for therapeutic administration. More particularly, the
agents of the present invention can be formulated into
pharmaceutical compositions by combination with appropriate,
pharmaceutically acceptable carriers or diluents, and may be
formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants and
aerosols.
[0154] In pharmaceutical dosage forms, the agents may be
administered in the form of their pharmaceutically acceptable
salts, or they may also be used alone or in appropriate
association, as well as in combination, with other pharmaceutically
active compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0155] For oral preparations, the agents can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0156] The agents can be formulated into preparations for injection
by dissolving, suspending or emulsifying them in an aqueous or
nonaqueous solvent, such as vegetable or other similar oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic
acids or propylene glycol; and if desired, with conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives.
[0157] The agents can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0158] Furthermore, the agents can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0159] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more inhibitors. Similarly, unit dosage forms for
injection or intravenous administration may comprise the
inhibitor(s) in a composition as a solution in sterile water,
normal saline or another pharmaceutically acceptable carrier.
[0160] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0161] Other modes of administration will also find use with the
subject invention. For instance, an agent of the invention can be
formulated in suppositories and, in some cases, aerosol and
intranasal compositions. For suppositories, the vehicle composition
will include traditional binders and carriers such as, polyalkylene
glycols, or triglycerides. Such suppositories may be formed from
mixtures containing the active ingredient in the range of about
0.5% to about 10% (w/w), preferably about 1% to about 2%.
[0162] Intranasal formulations will usually include vehicles that
neither cause irritation to the nasal mucosa nor significantly
disturb ciliary function. Diluents such as water, aqueous saline or
other known substances can be employed with the subject invention.
The nasal formulations may also contain preservatives such as, but
not limited to, chlorobutanol and benzalkonium chloride. A
surfactant may be present to enhance absorption of the subject
proteins by the nasal mucosa.
[0163] An agent of the invention can be administered as
injectables. Typically, injectable compositions are prepared as
liquid solutions or suspensions; solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection may also
be prepared. The preparation may also be emulsified or the active
ingredient encapsulated in liposome vehicles.
[0164] Suitable excipient vehicles are, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof.
In addition, if desired, the vehicle may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents or pH
buffering agents. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in the art. See, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 17th edition, 1985. The composition or formulation to
be administered will, in any event, contain a quantity of the agent
adequate to achieve the desired state in the subject being
treated.
[0165] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
Dosages
[0166] Although the dosage used will vary depending on the clinical
goals to be achieved, a suitable dosage range is one which provides
up to about 1 .mu.g to about 1,000 .mu.g or about 10,000 .mu.g of
an agent that reduces apoE4 domain interaction and can be
administered in a single dose. Alternatively, a target dosage of an
agent that reduces apoE4 domain interaction can be considered to be
about in the range of about 0.1-1000 .mu.M, about 0.5-500 .mu.M,
about 1-100 .mu.M, or about 5-50 .mu.M in a sample of host blood
drawn within the first 24-48 hours after administration of the
agent.
[0167] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Preferred dosages for a given compound are readily determinable by
those of skill in the art by a variety of means.
Routes of Administration
[0168] An agent that reduces apoE4 domain interaction is
administered to an individual using any available method and route
suitable for drug delivery, including in vivo and ex vivo methods,
as well as systemic and localized routes of administration.
[0169] Conventional and pharmaceutically acceptable routes of
administration include intranasal, intramuscular, intratracheal,
intratumoral, subcutaneous, intradermal, topical application,
intravenous, rectal, nasal, oral and other parenteral routes of
administration. Routes of administration may be combined, if
desired, or adjusted depending upon the agent and/or the desired
effect. The composition can be administered in a single dose or in
multiple doses.
[0170] The agent can be administered to a host using any available
conventional methods and routes suitable for delivery of
conventional drugs, including systemic or localized routes. In
general, routes of administration contemplated by the invention
include, but are not necessarily limited to, enteral, parenteral,
or inhalational routes.
[0171] Parenteral routes of administration other than inhalation
administration include, but are not necessarily limited to,
topical, transdermal, subcutaneous, intramuscular, intraorbital,
intracapsular, intraspinal, intrasternal, and intravenous routes,
i.e., any route of administration other than through the alimentary
canal. Parenteral administration can be carried to effect systemic
or local delivery of the agent. Where systemic delivery is desired,
administration typically involves invasive or systemically absorbed
topical or mucosal administration of pharmaceutical
preparations.
[0172] The agent can also be delivered to the subject by enteral
administration. Enteral routes of administration include, but are
not necessarily limited to, oral and rectal (e.g., using a
suppository) delivery.
[0173] Methods of administration of the agent through the skin or
mucosa include, but are not necessarily limited to, topical
application of a suitable pharmaceutical preparation, transdermal
transmission, injection and epidermal administration. For
transdermal transmission, absorption promoters or iontophoresis are
suitable methods. Iontophoretic transmission may be accomplished
using commercially available "patches" which deliver their product
continuously via electric pulses through unbroken skin for periods
of several days or more.
[0174] By treatment is meant at least an amelioration of the
symptoms associated with the pathological condition afflicting the
host, where amelioration is used in a broad sense to refer to at
least a reduction in the magnitude of a parameter, e.g. symptom,
associated with the pathological condition being treated, such as
an apoE4-associated neurological disorder and pain associated
therewith. As such, treatment also includes situations where the
pathological condition, or at least symptoms associated therewith,
are completely inhibited, e.g. prevented from happening, or
stopped, e.g. terminated, such that the host no longer suffers from
the pathological condition, or at least the symptoms that
characterize the pathological condition.
[0175] A variety of hosts (wherein the term "host" is used
interchangeably herein with the terms "subject" and "patient") are
treatable according to the subject methods. Generally such hosts
are "mammals" or "mammalian," where these terms are used broadly to
describe organisms which are within the class mammalia, including
the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice,
guinea pigs, and rats), and primates (e.g., humans, chimpanzees,
and monkeys). In many embodiments, the hosts will be humans.
[0176] Kits with unit doses of the active agent, e.g. in oral or
injectable doses, are provided. In such kits, in addition to the
containers containing the unit doses will be an informational
package insert describing the use and attendant benefits of the
drugs in treating pathological condition of interest. Preferred
compounds and unit doses are those described herein above.
Crossing the Blood-Brain Barrier
[0177] The blood-brain barrier limits the uptake of many
therapeutic agents into the brain and spinal cord from the general
circulation. Molecules which cross the blood-brain barrier use two
main mechanisms: free diffusion; and facilitated transport. Because
of the presence of the blood-brain barrier, attaining beneficial
concentrations of a given therapeutic agent in the central nervous
system (CNS) may require the use of drug delivery strategies.
Delivery of therapeutic agents to the CNS can be achieved by
several methods.
[0178] One method relies on neurosurgical techniques. In the case
of gravely ill patients such as accident victims or those suffering
from various forms of dementia, surgical intervention is warranted
despite its attendant risks. For instance, therapeutic agents can
be delivered by direct physical introduction into the CNS, such as
intraventricular or intrathecal injection of drugs.
Intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir. Methods of introduction may also be
provided by rechargeable or biodegradable devices. Another approach
is the disruption of the blood-brain barrier by substances which
increase the permeability of the blood-brain barrier. Examples
include intra-arterial infusion of poorly diffusible agents such as
mannitol, pharmaceuticals which increase cerebrovascular
permeability such as etoposide, or vasoactive agents such as
leukotrienes. Neuwelt and Rappoport (1984) Fed. Proc. 43:214-219;
Baba et al. (1991) J. Cereb. Blood Flow Metab. 11:638-643; and
Gennuso et al. (1993) Cancer Invest. 11:638-643.
[0179] Further, it may be desirable to administer the
pharmaceutical agents locally to the area in need of treatment;
this may be achieved by, for example, local infusion during
surgery, by injection, by means of a catheter, or by means of an
implant, said implant being of a porous, non-porous, or gelatinous
material, including membranes, such as silastic membranes, or
fibers.
[0180] Therapeutic compounds can also be delivered by using
pharmacological techniques including chemical modification or
screening for an analog which will cross the blood-brain barrier.
The compound may be modified to increase the hydrophobicity of the
molecule, decrease net charge or molecular weight of the molecule,
or modify the molecule, so that it will resemble one normally
transported across the blood-brain barrier. Levin (1980) J. Med.
Chem. 23:682-684; Pardridge (1991) in: Peptide Drug Delivery to the
Brain; and Kostis et al. (1994) J. Clin. Pharmacol. 34:989-996.
[0181] Encapsulation of the drug in a hydrophobic environment such
as liposomes is also effective in delivering drugs to the CNS. For
example WO 91/04014 describes a liposomal delivery system in which
the drug is encapsulated within liposomes to which molecules have
been added that are normally transported across the blood-brain
barrier.
[0182] Another method of formulating the drug to pass through the
blood-brain barrier is to encapsulate the drug in a cyclodextrin.
Any suitable cyclodextrin which passes through the blood-brain
barrier may be employed, including, but not limited to,
J-cyclodextrin, K-cyclodextrin and derivatives thereof. See
generally, U.S. Pat. Nos. 5,017,566, 5,002,935 and 4,983,586. Such
compositions may also include a glycerol derivative as described by
U.S. Pat. No. 5,153,179.
[0183] Delivery may also be obtained by conjugation of a
therapeutic agent to a transportable agent to yield a new chimeric
transportable therapeutic agent. For example, vasoactive intestinal
peptide analog (VIPa) exerted its vasoactive effects only after
conjugation to a monoclonal antibody (Mab) to the specific carrier
molecule transferrin receptor, which facilitated the uptake of the
VIPa-Mab conjugate through the blood-brain barrier. Pardridge
(1991); and Bickel et al. (1993) Proc. Natl. Acad. Sci. USA
90:2618-2622. Several other specific transport systems have been
identified, these include, but are not limited to, those for
transferring insulin, or insulin-like growth factors I and II.
Other suitable, non-specific carriers include, but are not limited
to, pyridinium, fatty acids, inositol, cholesterol, and glucose
derivatives. Certain prodrugs have been described whereby, upon
entering the central nervous system, the drug is cleaved from the
carrier to release the active drug. U.S. Pat. No. 5,017,566.
Subjects Suitable for Treatment with a Therapeutic Agent of the
Invention
[0184] A variety of subjects are suitable for treatment with an
agent identified by a method of the invention. Suitable subjects
have one or two apoE4 alleles. Suitable subjects include any
individual, particularly a human, who has an apoE4-associated
disorder, who is at risk for developing an apoE4, who has had an
apoE4-associated disorder and is at risk for recurrence of the
apoE4-associated disorder, or who is recovering from an
apoE4-associated disorder.
[0185] Such subjects include, but are not limited to, individuals
who have been diagnosed as having Alzheimer's disease; individuals
who have suffered one or more strokes; individuals who have
suffered traumatic head injury; individuals who have high serum
cholesterol levels; individuals who have A.beta. deposits in brain
tissue; individuals who have had one or more cardiac events;
subjects undergoing cardiac surgery; and subjects with multiple
sclerosis.
EXAMPLES
[0186] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric.
Example 1
Targeting the Mouse ApoE Gene Locus to Introduce Arg-61
[0187] The gene-targeting vector that was designed is shown in FIG.
1. Like human Apoe, the mouse gene contains four exons and three
introns, contained within an 8-kb EcoR1 fragment. The codon for the
Thr-61 equivalent in the mouse gene is located at the end of the
third exon, exactly as it is in the human gene. Mutation of the ACG
threonine codon to a AGG arginine codon resulted in introduction of
a unique Dde1 restriction site, which was used as a diagnostic
marker. A thymidine kinase (TK) gene was used as a negative
selection marker, and the Neo gene was used as a positive selection
marker. The Neo gene was placed within the third intron close to
the mutation site to reduce the probability that it would be
separated from the mutation during recombination. The Neo gene was
flanked with loxP sites (the recognition sequence for the
site-specific DNA recombinase, Cre) to allow its removal by
Cre-mediated recombination by crossing the gene-targeted mice with
Cre transgenic mice (obtained from Dr. Gail Martin, UCSF) carrying
the Nes-Cre1 gene, which is active in the germline. Meyers et al.
(1998) Nat. Genet. 18:136-141. Our strategy was to remove the Neo
gene to avoid any complications of its presence.
[0188] Embryonic stems (ES) 129 SvJ cells (obtained from the
Gladstone Blastocyst Core) were electroporated with the targeting
vector and were selected first against TK incorporation and then
for neomycin resistance. Targeted cells heterozygous for the
recombination event displayed the expected expansion of the mouse
locus to 10 kb for the EcoR1 fragment on southern blots and the
presence of the Dde1 restriction site. The targeted ES cells were
injected into C57B1/6 blastocysts, and the blastocysts were
implanted into pseudopregnant female mice. Three chimeric males
were obtained, all of which displayed germline transmission of the
targeted allele. Southern blot analysis of DNA from heterozygous
and homozygous targeted mice before removal of the Neo gene
displayed the expected expansion of the apoE locus. The Neo gene
was removed by crossing one of the lines with the Nes-Cre1
mice.
[0189] Comparison of apoE mRNA tissue distribution. Comparison of
apoE mRNA levels in various tissues and organs of wild-type (wt)
and homozygous Arg-61 (Neo removed) mice demonstrated that
expression was restored in all apoE-expressing tissues and organs,
including the brain, by Cre-mediated recombination. Furthermore,
the mRNA expression levels in Arg-61 mice were identical to levels
in wt mice. These results demonstrate that the pattern and level of
expression of the Arg-61 mutant gene was identical to that of the
wt gene. Targeted mice have been crossed into the C57B1/6
background to remove the Nes-Cre1 gene.
[0190] Arg-61 mice: Proof that domain interaction occurs in vivo.
Domain interaction in the mouse Arg-61 mutant apoE was demonstrated
using an in vitro lipoprotein binding assay. To demonstrate domain
interaction with this mutation in vivo, we analyzed the effect of
the Arg-61 mutant apoE on plasma lipoprotein metabolism. The
results unequivocally demonstrate that the Arg-61 mice exhibit
evidence of domain interaction with effects on plasma lipoprotein
metabolism that are consistent with the known behavior of apoE4 in
humans. For example, in humans, plasma levels of apoE4 are lower
than those of apoE3 and apoE2. Davignon et al. (1988)
Arteriosclerosis 8:1-21; and Boerwinkle and Utermann (1988) Am. J.
Hum. Genet. 42:104-112. This is clearly illustrated in the plasma
from apoE4/3 heterozygotes using isoelectric focusing. In these
subjects, the apoE4 band is reduced compared with that of apoE3 or
apoE2. This difference may result from the preference of apoE4 for
VLDL, which turns over at a faster rate in plasma than HDL.
[0191] Isoelectric focusing of wt/wt, wt/Arg-61 and Arg-61/Arg-61
plasma revealed that the Arg-61 apoE focused one charge position in
the positive direction as the result of the arginine for threonine
substitution, as shown in FIG. 2. This confirms that mutation of
the mouse Apoe gene results in expression of the mutant Arg-61
protein. In addition, as shown in FIG. 2, the amount of the Arg-61
apoE in the heterozygous mice was reduced (.about.70%) compared to
wt apoE, reproducing the human heterozygous apoE4/3 phenotype. In
contrast, in cerebrospinal fluid (CSF), which does not contain VLDL
or any apoB-containing lipoproteins, there were equal levels of the
Arg-61 and wt apoE, indicating that the residence times of the two
isoforms are very similar in CSF. In further support for the
differential metabolism of the Arg-61 and wt mouse apoE in plasma,
primary hepatocytes were cultured, and the relative amounts of the
two mouse isoforms secreted into the medium were determined by
isoelectric focusing. Equal amounts of each isoform were secreted
by the hepatocytes.
[0192] Since the liver is the primary source of plasma apoE, these
results also support the conclusion that the two isoforms are
metabolized at different rates in plasma, as a result of domain
interaction and the preference of Arg-61 apoE for VLDL and other
rapidly cleared lower density lipoproteins.
[0193] To verify the differential binding of Arg-61 apoE for lower
density lipoproteins in vivo, we examined the distribution in
lipoprotein fractions separated by gel filtration from heterozygous
mouse plasma. Since mice are an HDL species, transporting more than
85% of their cholesterol in HDL, they have much lower plasma
concentrations of apoB-containing VLDL, intermediate density
lipoproteins (IDL), and LDL than humans, where 70-80% of plasma
cholesterol is transported in apoB-containing lipoproteins. To
increase the plasma concentration of apoB-containing lipoproteins
in heterozygous targeted mice, they were fed an atherogenic diet
for 6 days. FIG. 3A shows an FPLC profile of plasma cholesterol
distribution in these mice. As shown in FIG. 3B, relative to wt
apoE the Arg-61 apoE clearly distributed differently, with the
majority of the Arg-61 apoE in the lower density apoB-containing
lipoproteins. Thus, our results establish that apoE4 domain
interaction occurs in vivo in plasma, leading us to the expectation
that it will also occur in the CNS.
[0194] Effect of domain interaction on phospholipid binding
activity. Since apoB-containing lipoproteins, including VLDL, do
not occur in brain or CSF, it was important to establish that a
lipid binding effect resulted from domain interaction in
lipoproteins more relevant to the CSF. A common characteristic of
soluble plasma apolipoproteins is their ability to clear turbid
solutions of phospholipid vesicles to form discoidal lipoprotein
complexes. The discoidal complexes formed by apoE resemble the
apoE-containing discoidal complexes found in CSF. Pitas et al.
(1987) J. Biol. Chem. 262:14352-14360; and Rebeck et al. (1998)
Exp. Neurol. 149:175-182. When we compared the abilities of human
apoE3 and apoE4 to clear turbid solutions of phospholipid vesicles
of dimyristoylphosphatidylcholine (DMPC), apoE4 was more effective
than apoE3, as shown in FIGS. 4A and 4B. Together these results
suggest that domain interaction in apoE4 is responsible for its
efficacy in binding DMPC. Similarly, when the recombinant Arg-61
mouse apoE was compared with mouse wt apoE, the Arg-61 apoE also
bound to DMPC more effectively that wt apoE. These results
demonstrate that domain interaction influences the ability of human
apoE4 to interact with phospholipid and that the Arg-61 mouse apoE
and human apoE4 behave similarly.
[0195] In view of the examples presented above, it is clear that
the instant invention provides a mouse model for human apoE4 domain
interaction, and demonstrates unequivocally that the Arg-61 apoE
mouse model displays the expected physiological effects of domain
interaction on plasma lipoprotein metabolism, and that mouse Arg-61
apoE mirrors the behavior of human apoE4. Our novel mouse model of
human apoE4 provides the opportunity to test the effect of agents
that are capable of interfering with the interaction of Arg-61 and
Glu-255 in apoE4 thereby converting the closed conformation of
apoE4 into an "apoE3-like" open conformation, which agents are
useful to treat both cardiovascular diseases and neurodegenerative
diseases that are associated with human apoE4.
[0196] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *
References