U.S. patent application number 11/669123 was filed with the patent office on 2007-11-15 for ldl receptor-related proteins 1 and 2 and treatment of bone or cartilage conditions.
This patent application is currently assigned to Fonterra Corporate Research and Development Ltd. Invention is credited to Jillian Cornish, Andrew Bevis Grey, Dorit Naot, Kay Patricia Palmano, Ian Reginald Reid.
Application Number | 20070265188 11/669123 |
Document ID | / |
Family ID | 29423699 |
Filed Date | 2007-11-15 |
United States Patent
Application |
20070265188 |
Kind Code |
A1 |
Reid; Ian Reginald ; et
al. |
November 15, 2007 |
LDL Receptor-Related Proteins 1 and 2 and Treatment of Bone or
Cartilage Conditions
Abstract
LDL receptor-related proteins 1 and 2 (LRP-1 and LRP-2) and
interaction between lactoferrin and LRP-1, LRP-2, or p42/44 MAP
kinase in diagnosis and treatment of disorders such as bone or
cartilage disorders. Also disclosed are methods of screening for
related therapeutic compounds.
Inventors: |
Reid; Ian Reginald; (Mount
Albert, NZ) ; Cornish; Jillian; (Newmarket, NZ)
; Grey; Andrew Bevis; (Remuera, NZ) ; Naot;
Dorit; (Green Bay, NZ) ; Palmano; Kay Patricia;
(Palmerston North, NZ) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Fonterra Corporate Research and
Development Ltd,
NZMP + Health and Nutrition Unit
Auckland UniServices Ltd, New Zealand corporations
|
Family ID: |
29423699 |
Appl. No.: |
11/669123 |
Filed: |
January 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10436806 |
May 13, 2003 |
|
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11669123 |
Jan 30, 2007 |
|
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60380227 |
May 13, 2002 |
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60463419 |
Apr 16, 2003 |
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Current U.S.
Class: |
435/6.16 ; 435/4;
514/16.7; 514/17.1; 514/7.4 |
Current CPC
Class: |
A61P 19/00 20180101;
A61P 43/00 20180101; G01N 2500/10 20130101; A61P 3/12 20180101;
A61P 3/14 20180101; A61P 11/00 20180101; A61P 1/16 20180101; A61P
1/18 20180101; A61P 5/18 20180101; A61P 25/08 20180101; A61P 5/46
20180101; G01N 33/92 20130101; A61P 29/00 20180101; A61P 35/00
20180101; A61P 19/10 20180101; C07K 14/705 20130101; A61P 21/02
20180101; A61P 13/12 20180101; A61P 19/08 20180101 |
Class at
Publication: |
514/002 ;
435/004; 435/006 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C12Q 1/00 20060101 C12Q001/00; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of determining whether a patient is suffering from or
at risk for developing a bone condition, the method comprising:
providing a test sample from a patient suspected of suffering from
or being at risk for developing a bone condition, and quantifying
an expression level of an LRP-1 gene or quantifying an activity of
an LRP-1 protein, wherein the expression level of the LRP-1 gene or
the activity of the LRP-1 protein in the test sample, if different
from that in a normal sample, indicates that the patient is
suffering from or at risk for developing a bone condition.
2. The method of claim 1, wherein the test sample is prepared from
a bone tissue.
3. A method of treating a bone condition, the method comprising
modulating a level of an LRP-1 protein in bone cells or modulating
an activity of an LRP-1 protein in bone cells.
4. The method of claim 3, wherein the level of the LRP-1 protein is
modulated by providing and expressing a nucleic acid encoding an
LRP-1 protein.
5. The method of claim 4, wherein the nucleic acid is expressed in
the bone cells.
6. The method of claim 3, wherein the level of the LRP-1 protein is
modulated by providing an LRP-1 protein.
7. The method of claim 6, wherein the LRP-1 protein is introduced
into the bone cells.
8. The method of claim 3, wherein the level of the LRP-1 protein is
modulated by providing and expressing a nucleic acid complementary
to a sequence encoding an LRP-1 protein.
9. The method of claim 8, wherein the nucleic acid is expressed in
the bone cells.
10. (canceled)
11. The method of claim 3, wherein the activity of the LRP-1
protein is modulated by providing an agonist of the LRP-1
protein.
12. The method of claim 10, wherein the agonist is introduced into
the bone cells.
13-20. (canceled)
21. The method of claim 3, wherein the activity of the LRP-1
protein is modulated by providing an agonist of the
LRP-1protein.
22. The method of claim 21, wherein the agonist is introduced into
the bone cells.
23-24. (canceled)
25. The method of claim 3, wherein the activity of the LRP-1
protein is modulated by providing an antibody against the LRP-1
protein.
26. The method of claim 25, wherein the antibody is introduced into
the bone cells.
27-70. (canceled)
71. A method of treating a bone disorder, the method comprising
administering an effective amount of a lactoferrin N-lobe
polypeptide or a lactoferricin to a subject suffering from the bone
disorder.
72. The method of claim 71, wherein the subject is in need of
increased bone formation.
73. The method of claim 71, wherein the subject is in need of
decreased bone resorption.
74. The method of claim 71, wherein the lactoferrin N-lobe
polypeptide contains a metal ion.
75. The method of claim 71, wherein the lactoferrin N-lobe
polypeptide contains an iron ion, copper ion, chromium ion, cobalt
ion, manganese ion, zinc ion, or magnesium ion.
76. The method of claim 71, wherein the bone disorder is
osteoporosis, rheumatoid or osteo-arthritis, hepatic
osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica,
renal osteodystrophy, osteosclerosis, osteopenia,
fibrogenesis-imperfecta ossium, secondary hyperparathyrodism,
hypoparathyroidism, hyperparathyroidism, chronic renal disease,
sarcoidosis, glucocorticoid-induced osteoporosis, idiopathic
hypercalcemia, Paget's disease, or osteogenesis imperfecta.
77. The method of claim 71, wherein the lactoferrin N-lobe
polypeptide is administered locally.
78. The method of claim 71, wherein the lactoferrin N-lobe
polypeptide is from human lactoferrin or bovine lactoferrin.
79. The method of claim 71, wherein the lactoferrin is from human
lactoferrin or bovine lactoferrin.
Description
RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application No. 60/380,227, filed May 13, 2002, and U.S.
Provisional Application No. 60/463,419, field Apr. 16, 2003, the
contents of which are incorporated herein by reference.
BACKGROUND
[0002] Low-density lipoprotein (LDL) receptors (LDLRs) are cell
surface receptors involved in general intracellular membrane
cycling and endocytosis. The functions of LDLRs include (1) cargo
transport for internalizing extracellular components into the
endosomal compartment, and (2) lipoproteins metabolism for
maintaining cholesterol homeostasis in the body.
[0003] LDLRs are members of a receptor superfamily, which also
includes LDL receptor-related proteins (LRPs). The roles of LRPs
are more restricted than that of LDLRs in lipid metabolism.
However, in addition to cargo transport and lipid metabolism, the
LRPs are also involved in other cellular processes such as signal
transduction that significantly impacts cell metabolism and
physiology.
[0004] Several LRPs have been identified, including LRP-1, LRP-1b,
LRP-2, LRP-5, LRP-6, LRP-7, LRP-8, LRP-9, LRP-10, and LRP-11
(Strickland et al. (2002) Trends in Endocrinology and Metabolism
13(2): 66-74; Herz and Strickland (2001) The Journal of Clinical
Investigation 108(6): 779-784; Herz (2001) Neuron 29: 571-581). All
LRPs, like LDLRs, contain trans-membrane sequences and are
multi-functional proteins. LRPs differ from each other in minor
ways, and have similar structural motifs, including, for example,
EGF-like repeats, YWTD repeats, O-linked sugar domains,
complement-like repeats, and a cytoplasmic domain. LRPs bind to
many intracellular and extracellular molecules. Such intracellular
molecules include adaptor and scaffold proteins (e.g., Dab-1, FE65,
PSD-95) and such extracellular molecules include lipoproteins,
proteinases, bacterial toxins, antibiotics, viruses and proteinase
inhibitor complexes.
[0005] Lactoferrin, an 80 kDa glycoprotein present in milk and
epithelial secretions, is released by inflammatory cells during
immune responses. It circulates at a concentration of 2-7 .mu.g/ml
in plasma, and is believed to be involved in regulation of iron
metabolism, immunity, and embryonic development.
SUMMARY
[0006] The present invention is based on the discovery that LRP-1
and LRP-2 genes are expressed in bone cells.
[0007] One aspect of the invention features a method of diagnosing
a bone condition.
[0008] In one example, the method includes providing a test sample
(e.g., a bone tissue) from a patient suspected of suffering from or
being at risk for developing a bone condition, and quantifying the
expression level of an LRP-1 gene. The expression level of the
LRP-1 gene in the test sample, if different from that in a normal
sample, indicates that the patient is suffering from or at risk for
developing a bone condition.
[0009] In another example, the method includes providing a test
sample from a bone tissue of a patient suspected of suffering from
or being at risk for developing a bone condition, and quantifying
the expression level of an LRP-2 gene. The expression level of the
LRP-2 gene in the test sample, if different from that in a normal
sample, indicates that the patient is suffering from or at risk for
developing a bone condition.
[0010] In another example, the method includes providing a test
sample (e.g., a bone tissue) from a patient suspected of suffering
from or being at risk for developing a bone condition, and
quantifying the activity of an LRP-1 protein. The activity of the
LRP-1 protein in the test sample, if different from that in a
normal sample, indicates that the patient is suffering from or at
risk for developing a bone condition.
[0011] In still another example, the method includes providing a
test sample from a bone tissue of a patient suspected of suffering
from or being at risk for developing a bone condition, and
quantifying the activity of an LRP-2 protein. The activity of the
LRP-2 protein in the test sample, if different from that in a
normal sample, indicates that the patient is suffering from or at
risk for developing a bone condition.
[0012] Another aspect of the invention features a method of
identifying a candidate compound for treating a bone condition.
[0013] In one example, the method includes contacting a compound
with a cell, for example, a bone cell (e.g., an osteoblast cell,
osteoblast-like cell such as SaOS-2 cell, osteocyte, or osteoclast
cell) expressing and LRP-1 gene, and quantifying the expression
level of the LRP-1 gene in the cell. The expression level of the
LRP-1 gene in the presence of the compound, if different from that
in the absence of the compound, indicates that the compound is a
candidate for treating a bone condition.
[0014] In another example, the method includes contacting a
compound with a bone cell (e.g., an osteoblast cell,
osteoblast-like cell such as SaOS-2 cell, osteocyte, or osteoclast
cell) expressing an LRP-2 gene, and quantifying the expression
level of the LRP-2 gene in the cell. The expression level of the
LRP-2 gene in the presence of the compound, if different from that
in the absence of the compound, indicates that the compound is a
candidate for treating a bone condition.
[0015] In another example, the method includes contacting a
compound with a cell expressing and LRP-1 gene encoding an LRP-1
protein, and quantifying the activity of the LRP-1 protein in the
cell. The activity of the LRP-1 protein in the presence of the
compound, if different from that in the absence of the compound,
indicates that the compound is a candidate for treating a bone
condition.
[0016] In still another example, the method includes contacting a
compound with a bone cell expressing an LRP-2 gene encoding an
LRP-2 protein, and quantifying the activity of the LRP-2 protein in
the cell. The activity of the LRP-2 protein in the presence of the
compound, if different from that in the absence of the compound,
indicates that the compound is a candidate for treating a bone
condition.
[0017] Also within the scope of the invention is a method of
treating a bone condition by modulating the level or activity of an
LRP-1 or LRP-2 protein in bone cells.
[0018] In one example, the method includes administering to a
subject in need thereof an effective amount of a pharmaceutical
composition, thereby modulating a level of an LRP-1 or LRP-2
protein in bone cells. The level of the LRP-1 or LRP-2 protein can
be modulated by providing and expressing a nucleic acid encoding
and LRP-1 or LRP-2 protein, by providing an LRP-1 or LRP-2 protein,
or by providing and expressing a nucleic acid complementary to a
sequence encoding an LRP-1 or LRP-2 protein, i.e., an anti-sense
molecule.
[0019] In another example, the method includes administering to a
subject in need thereof an effective amount of a pharmaceutical
composition, thereby modulating the activity of an LRP-1 or LRP-2
protein in bone cells. The activity of the LRP-1 or LRP-2 protein
can be modulated by providing an agonist of the LRP-1 or LRP-2
protein, by providing an antagonist of the LRP-1 or LRP-2 protein,
or by providing an antibody against the LRP-1 or LRP-2 protein.
[0020] The pharmaceutical composition can be directly administered
into the bone cells. It can also be administered to a subject
without being directly introduced into bone cells in order to
modulate the activity of LRP-1 in the bone cells.
[0021] Methods of diagnosing a bone condition, identifying a
candidate compound for treating a bone condition, and treating a
bone condition involving both LRP-1 and LRP-2 are within the scope
of this invention.
[0022] The naturally occurring LRP-1 or LRP-2 protein, fragments
thereof, biologically active portion thereof, and derivatives
thereof are collectively referred to as "LRP-1 or LRP-2
polypeptides or proteins". Nucleic acid molecules encoding such
polypeptides or proteins are collectively referred to as "LRP-1 or
LRP-2 nucleic acids" (e.g., naturally occurring genes or
recombinant genes). LRP-1 or LRP-2 molecules refer to LRP-1 or
LRP-2 nucleic acids, polypeptides, and antibodies.
[0023] As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g.,
an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be
synthesized from nucleotide analogs. The nucleic acid molecule can
be single-stranded or double-stranded.
[0024] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules which include at least an open
reading frame encoding an LRP-1 or LRP-2 protein. The gene can
optionally further include non-coding sequences, e.g., regulatory
sequences and introns.
[0025] As used herein, a "biologically active portion" of an LRP-1
or LRP-2 protein includes a fragment of an LRP-1 or LRP-2 protein
which participates in an interaction, e.g., an intramolecular or an
intermolecular interaction. An intermolecular interaction can be a
specific binding interaction or an enzymatic interaction (e.g., the
interaction can be transient and one in which a covalent bond is
formed or broken). An intermolecular interaction can be between an
LRP-1 or LRP-2 molecule and a non-LRP-1 or LRP-2 molecule or
between a first LRP-1 or LRP-2 molecule and a second LRP-1 or LRP-2
molecule. Biologically active portions of an LRP-1 or LRP-2 protein
include peptides comprising amino acid sequences sufficiently
homologous to or derived from the amino acid sequence of the LRP-1
or LRP-2 protein, which include less amino acids than the full
length LRP-1 or LRP-2 proteins, and exhibit at least one activity
of an LRP-1 or LRP-2 protein. Typically, biologically active
portions contain a domain or motif with at least one activity of
the LRP-1 or LRP-2 protein, e.g., cargo transport, lipid
metabolism, and signal transduction. A biologically active portion
of an LRP-1 or LRP-2 protein can be a polypeptide which is, for
example, 10, 25, 50, 100, 200 or more amino acids in length.
Biologically active portions of an LRP-1 or LRP-2 protein can be
used as targets for developing agents which modulate an LRP-1 or
LRP-2-mediated activity, e.g., cargo transport, lipid metabolism,
and signal transduction.
[0026] As used herein, and "LRP-1 or LRP-2 activity," "biological
activity of LRP-1 or LRP-2," or "functional activity of LRP-1 or
LRP-2," refers to an activity exerted by an LRP-1 or LRP-2
polypeptide, protein, or nucleic acid molecule. For example, an
LRP-1 or LRP-2 activity can be an activity exerted by LRP-1 or
LRP-2 in a physiological milieu on, e.g., an LRP-1 or
LRP-2-responsive cell or an LRP-1 or LRP-2 substrate such as a
protein substrate. An LRP-1 or LRP-2 activity can be determined in
vivo or in vitro. In one example, an LRP-1 or LRP-2 activity is a
direct activity, such as an association with an LRP-1 or LRP-2
target molecule. A "target molecule" or "binding partner" is a
molecule with which an LRP-1 or LRP-2 protein binds or interacts in
nature.
[0027] An LRP-1 or LRP-2 activity can also be an indirect activity,
e.g., cellular signaling activity mediated by interaction of the
LRP-1 or LRP-2 protein with an LRP-1 or LRP-2 ligand.
[0028] Aberrant expression or activity of LRP-1 or LRP-2 or both
molecules can mediate disorders associated with bone metabolism.
"Bone metabolism" refers to direct or indirect effects in the
formation or degeneration of bone structures, e.g., bone formation,
bone resorption, and the balance between the two metabolic
processes. This term also includes activities mediated by LRP-1 or
LRP-2 molecules in bone cells, e.g., osteoclasts and osteoblasts,
that in turn result in bone formation and degeneration. For
example, LRP-1 or LRP-2 molecules can support different activities
of bone resorbing osteoclasts such as the stimulation of
differentiation of monocytes and mononuclear phagocytes into
osteoclasts. Accordingly LRP-1 or LRP-2 molecules that modulate the
production of bone cells can influence bone formation and
degeneration, and thus be used to treat bone disorders. Examples of
such disorders include, but are not limited to, osteoporosis,
osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica,
renal osteodystrophy, osteosclerosis, anti-convulsant treatment,
osteopenia, fibrogenesis-imperfecta ossium, secondary
hyperparathyrodism, hypoparathyroidism, hyperparathyroidism,
cirrhosis, obstructive jaundice, drug induced metabolism, medullary
carcinoma, chronic renal disease, rickets, sarcoidosis,
glucocorticoid antagonism, malabsorption syndrome, steatorrhea,
tropical sprue, idiopathic hypercalcemia, milk fever, Paget's
disease, and osteogenesis imperfecta.
[0029] "Misexpression or aberrant expression", as used herein,
refers to a non-wildtype pattern of gene expression at the RNA or
protein level. In includes: expression at non-wild type levels,
i.e., over- or under-expression; a pattern of expression that
differs from wild type in terms of the time or stage at which the
gene is expressed, e.g., increased or decreased expression (as
compared with wild type) at a predetermined developmental period or
stage; a pattern of expression that differs from wild type in terms
of altered, e.g., increased or decreased, expression (as compared
with wild type) in a predetermined cell type or tissue type; a
pattern of expression that differs from wild type in terms of the
splicing size, translated amino acid sequence, post-transitional
modification, or biological activity of the expressed polypeptide;
a pattern of expression that differs from wild type in terms of the
effect of an environmental stimulus or extracellular stimulus on
expression of the gene, e.g., a pattern of increased or decreased
expression (as compared with wild type) in the presence of an
increase or decrease in the strength of the stimulus.
[0030] As used herein, a "compound" includes, e.g., nucleic acids,
proteins, peptides, peptidomimetics, peptoids, or small molecules.
An "agonist" refers to a compound that enhances the activity of the
LRP-1 or LRP-2 protein; it can be a naturally occurring ligand for
LRP-1 or LRP-2. An "antagonist" refers to a compound that inhibits
the activity of the LRP-1 or LRP-2 protein.
[0031] As used herein, a "subject" refers to a human or a non-human
animal.
[0032] This invention also relates to diagnosing and treating bone
or cartilage conditions by determining and modulating interaction
between lactoferrin and a low-density lipoprotein receptor-related
protein (LRP-1 or LRP-2) or MAP kinase in bone or cartilage
cells.
[0033] In one aspect, the invention features a method of
determining whether a subject is suffering from or at risk for
developing a bone or cartilage condition. The method involves
providing a test sample (e.g., a bone or cartilage tissue sample)
from a subject, and determining the level of interaction between a
lactoferrin polypeptide and an LRP-1 protein, an LRP-2 protein, or
a p42/44 MAP kinase in the test sample. If the level of such
interaction in the test sample is different from that in a normal
sample, it indicates that the subject is suffering from or at risk
for developing a bone or cartilage condition. Interaction between
lactoferrin and LRP-1, LRP-2, or p42/44 MAP kinase can be
determined, e.g., by measuring binding of lactoferrin to LRP-1 or
LRP-2, endocytosis of lactoferrin mediated by LRP-1 or LRP-2, or
phosphorylation of p42/44 MAP kinase.
[0034] In another aspect, the invention features a method of
identifying a candidate compound for treating a bone or cartilage
condition. The method involves introducing a compound to a system
containing a lactoferrin polypeptide and an LRP-1 protein, an LRP-2
protein, or a p42/44 MAP kinase, and determining the level of
interaction between the lactoferrin polypeptide and the LRP-1
protein, the LRP-2 protein, or the p42/44 MAP kinase. The system
can be a cell system, e.g., containing osteoblastic, osteoclastic
or fibroblastic cells or chondrocytes, or a cell-free system. If
the level of the interaction between lactoferrin and LRP-1, LRP-2,
or p42/44 MAP kinase in the presence of the compound is different
from that in the absence of the compound, it indicates that the
compound is a candidate for treating a bone or cartilage
condition.
[0035] In still another aspect, the invention features a method of
treating a bone or cartilage condition by modulating interaction
between a lactoferrin polypeptide and an LRP-1 protein, an LRP-2
protein, or a p42/44 MAP kinase in bone or cartilage cells. In one
example, the interaction is modulated by providing an agonist of
LRP-1, LRP-2, or p42/44 MAP kinase, e.g., an exogenous lactoferrin
polypeptide. In another example, the interaction is modulated by
providing an antagonist of LRP-1 or LRP-2 (e.g., a
receptor-associated protein), or an antagonist of p42/44 MAP kinase
(e.g., a p42/44 MAP kinase inhibitor, i.e., a compound that
inhibits expression, phosphorylation, or activity of a p42/44 MAP
kinase such as PD98059 and U-0126). The agonist or antagonist of
LRP-1, LRP-2, or p42/44 MAP kinase can be introduced directly into
bone or cartilage cells. These agonists or antagonists can be used
individually or in combination, sequentially or concurrently, with
or without other bone- or cartilage-healing therapies.
[0036] A "lactoferrin polypeptide" can be a naturally occurring
polypeptide, a recombinant polypeptide, or a synthetic polypeptide.
Variants of a wild-type lactoferrin polypeptide (e.g., a fragment
of the wild-type lactoferrin polypeptide containing at least 2
(e.g., 4, 6, 8, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700)
amino acids, or a recombinant protein containing a lactoferrin
polypeptide sequence that maintain the biological activity of a
wild-type lactoferrin polypeptide) are within the scope of the
invention. The lactoferrin polypeptide can be of a mammalian
origin, e.g., from human or bovine milk. The metal ion bound to the
polypeptide can be an iron ion (as in a naturally occurring
lactoferrin polypeptide), a copper ion, a chromium ion, a cobalt
ion, a manganese ion, a zinc ion, or a magnesium ion, or any other
co-ordinating metal ion such as scandium or bismuth.
[0037] The details of one or more embodiments of the invention are
set forth in the accompanying description below. Other features,
objects, and advantages of the invention will be apparent from the
detailed description, and from the claims.
DETAILED DESCRIPTION
[0038] The LRP-1 and LRP-2 nucleic acid molecules, proteins,
protein homologues, agonists, antagonists and antibodies can be
used in one or more of the following methods: a) screening assays;
b) predictive medicine (e.g., diagnostic assays, prognostic assays,
monitoring clinical trials, and pharmacogenetics); and c) methods
of treatment (e.g., therapeutic and prophylactic).
[0039] The LRP-1 and LRP-2 nucleic acid molecules can be used, for
example, to express an LRP-1 or LRP-2 protein (e.g., via a
recombinant expression vector in a host cell in gene therapy
applications), to detect an LRP-1 or LRP-2 mRNA (e.g., in a
biological sample) or a genetic alteration in an LRP-1 or LRP-2
gene, and to modulate LRP-1 or LRP-2 activity, as described further
below. The LRP-1 or LRP-2 proteins can be used to treat disorders
characterized by insufficient or excessive production of an LRP-1
or LRP-2 substrate or production of LRP-1 or LRP-2 inhibitors. In
addition, the LRP-1 or LRP-2 proteins can be used to screen for
naturally occurring LRP-1 or LRP-2 substrates, to screen for drugs
or compounds which modulate LRP-1 or LRP-2 activity, as well as to
treat disorders characterized by insufficient or excessive
production of LRP-1 or LRP-2 protein or production of LRP-1 or
LRP-2 protein forms which have decreased, aberrant or unwanted
activity compared to LRP-1 or LRP-2 wild type protein (e.g., bone
disorders such as osteoporosis, Paget's disease, and osteogenesis
imperfecta). Moreover, the anti-LRP-1 or LRP-2 antibodies can be
used to detect and isolate LRP-1 or LRP-2 proteins, regulate the
bioavailability of LRP-1 or LRP-2 proteins, and modulate LRP-1 or
LRP-2 activity.
[0040] A method of evaluating a compound for the ability to
interact with, e.g., bind, a subject LRP-1 or LRP-2 polypeptide is
provided. The method includes: contacting the compound with the
subject LRP-1 or LRP-2 polypeptide; and evaluating ability of the
compound to interact with, e.g., to bind or form a complex with the
subject LRP-1 or LRP-2 polypeptide. This method can be performed in
vitro, e.g., in a cell free system, or in vivo, e.g., in a
two-hybrid interaction trap assay. This method can be used to
identify naturally occurring molecules that interact with subject
LRP-1 or LRP-2 polypeptide. It can also be used to find natural or
synthetic inhibitors of subject LRP-1 or LRP-2 polypeptide.
Screening methods are discussed in more detail below.
Screening Assays
[0041] The invention provides methods (also referred to herein as
"screening assays") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., proteins, peptides,
peptidomimetics, peptoids, small molecules or other drugs) which
bind to LRP-1 or LRP-2 proteins, have a stimulatory or inhibitory
effect on, for example, LRP-1 or LRP-2 expression or LRP-1 or LRP-2
activity, or have a stimulatory or inhibitory effect on, for
example, the expression or activity of an LRP-1 or LRP-2 substrate.
Compounds thus identified can be used to modulate the activity of
target gene products (e.g., LRP-1 or LRP-2 genes) in a therapeutic
protocol, to elaborate the biological function of the target gene
product, or to identify compounds that disrupt normal target gene
interactions.
[0042] In one embodiment, the invention provides assays for
screening candidate or test compounds which are substrates of an
LRP-1 or LRP-2 protein or polypeptide or a biologically active
portion thereof. In another embodiment, the invention provides
assays for screening candidate or test compounds that bind to or
modulate an activity of an LRP-1 or LRP-2 protein or polypeptide or
a biologically active portion thereof.
[0043] The test compounds of the present invention can be obtained
using any of the numerous approaches in combinatorial library
methods known in the art, including: biological libraries; peptoid
libraries (libraries of molecules having the functionalities of
peptides, but with a novel, non-peptide backbone which are
resistant to enzymatic degradation but which nevertheless remain
bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med. Chem.
37:2678-85); spatially addressable parallel solid phase or solution
phase libraries; synthetic library methods requiring deconvolution;
the `one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library and peptoid library approaches are limited to peptide
libraries, while the other four approaches are applicable to
peptide, non-peptide oligomer or small molecule libraries of
compounds (Lam (1997) Anticancer Drug Des. 12:145).
[0044] Examples of method for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994), J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0045] Libraries of compounds can be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S.
Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad
Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol.
Biol. 222:301-310; Ladner supra.).
[0046] In one embodiment, an assay is a cell-based assay in which a
cell which expresses an LRP-1 or LRP-2 protein or biologically
active portion thereof is contacted with a test compound, and the
ability of the test compound to modulate LRP-1 or LRP-2 activity is
determined. Determining the ability of the test compound to
modulate LRP-1 or LRP-2 activity can be accomplished by monitoring,
for example, cargo transport, lipid metabolism, signal transduction
or some other aspect of cell activity, e.g., cell proliferation,
mineral deposition or dissolution, or protein synthesis. The cell,
for example, can be of mammalian origin, e.g., human.
[0047] The ability of the test compound to modulate LRP-1 or LRP-2
binding to a compound, e.g., an LRP-1 or LRP-2 substrate, or to
bind to LRP-1 or LRP-2 can also be evaluated. This can be
accomplished, for example, by coupling the compound, e.g., the
substrate, with a radioisotope or enzymatic label such that binding
of the compound, e.g., substrate, to LRP-1 or LRP-2 can be
determined by detecting the labeled compound, e.g., substrate, in a
complex. Alternatively, LRP-1 or LRP-2 could be coupled with a
radioisotope or enzymatic label to monitor the ability of a test
compound to modulate LRP-1 or LRP-2 binding to an LRP-1 or LRP-2
substrate in a complex. For example, compounds (e.g., LRP-1 or
LRP-2 substrates) can be labeled with .sup.125I, .sup.35S,
.sup.14C, or .sup.3H, either directly or indirectly, and the
radioisotope detected by direct counting of radioemmission or by
scintillation counting. Alternatively, compounds can be
enzymatically labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product.
[0048] The ability of a compound (e.g., an LRP-1 or LRP-2
substrate) to interact with LRP-1 or LRP-2 with or without the
labeling of any of the interactants can be evaluated. For example,
a microphysiometer can be used to detect the interaction of a
compound with LRP-1 or LRP-2 without the labeling of either the
compound or the LRP-1 or LRP-2 (McConnell, H. M. et al. (1992)
Science 257:1906-1912). As used herein, a "microphysiometer" (e.g.,
Cytosensor) is an analytical instrument that measures the rate at
which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate
can be used as an indicator of the interaction between a compound
and LRP-1 or LRP-2.
[0049] In yet another embodiment, a cell-free assay is provided in
which an LRP-1 or LRP-2 protein or biologically active portion
thereof is contacted with a test compound and the ability of the
test compound to bind to the LRP-1 or LRP-2 protein or biologically
active portion thereof is evaluated. Preferred biologically active
portions of the LRP-1 or LRP-2 proteins to be used in assays of the
present invention include fragments which participate in
interactions with non-LRP-1 or LRP-2 molecules, e.g., fragments
with high surface probability scores.
[0050] Soluble or membrane-bound forms of isolated proteins (e.g.,
LRP-1 or LRP-2 proteins or biologically active portions thereof)
can be used in the cell-free assays of the invention. When
membrane-bound forms of the protein are used, it is desirable to
utilize a solubilizing agent. Examples of such solubilizing agents
include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.n,
3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0051] Cell-free assays involve preparing a reaction mixture of the
target gene protein and the test compound under conditions and for
a time sufficient to allow the two components to interact and bind,
thus forming a complex that can be removed or detected.
[0052] The interaction between two molecules can also be detected,
e.g., using fluorescence energy transfer (FET) (see, for example,
Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al.,
U.S. Pat. No. 4,868,103). A fluorophore label on the first, `donor`
molecule is selected such that its emitted fluorescent energy will
be absorbed by a fluorescent label on a second, `acceptor`
molecule, which in turn is able to fluoresce due to the absorbed
energy. Alternately, the `donor` protein molecule can simply
utilize the natural fluorescent energy of tryptophan residues.
Labels are chosen that emit different wavelengths of light, such
that the `acceptor` molecule label can be differentiated from that
of the `donor`. Since the efficiency of energy transfer between the
labels is related to the distance separating the molecules, the
spatial relationship between the molecules can be assessed. In a
situation in which binding occurs between the molecules, the
fluorescent emission of the `acceptor` molecule label in the assay
should be maximal. An FET binding event can be conveniently
measured through standard fluorometric detection means well known
in the art (e.g., using a fluorimeter).
[0053] In another embodiment, determining the ability of the LRP-1
or LRP-2 protein to bind to a target molecule can be accomplished
using real-time Biomolecular Interaction Analysis (BIA) (see, e.g.,
Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345
and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705).
"Surface plasmon resonance" or "BIA" detect biospecifc interactions
in real time, without labeling any of the interactants (e.g.,
BIAcore). Changes in the mass at the binding surface (indicative of
a binding event) result in alterations of the refractive index of
light near the surface (the optical phenomenon of surface plasmon
resonance (SPR)), resulting in a detectable signal which can be
used as an indication of real-time reactions between biological
molecules.
[0054] In one embodiment, the target gene product or the test
substance is anchored onto a solid phase. The target gene
product/test compound complexes anchored on the solid phase can be
detected at the end of the reaction. Preferably, the target gene
product can be anchored onto a solid surface, and the test
compound, (which is not anchored), can be labeled, either directly
or indirectly, with detectable labels discussed herein.
[0055] It is desirable to immobilize either LRP-1 or LRP-2, an
anti-LRP-1 or LRP-2 antibody or its target molecule to facilitate
separation of complexed from uncomplexed forms of one or both of
the proteins, as well as to accommodate automation of the assay.
Binding of a test compound to an LRP-1 or LRP-2 protein, or
interaction of an LRP-1 or LRP-2 protein with a target molecule in
the presence and absence of a candidate compound, can be
accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and
micro-centrifuge tubes. In one embodiment, a fusion protein can be
provided which adds a domain that allows one or both of the
proteins to be bound to a matrix. For example,
glutathione-S-transferase/LRP-1 or LRP-2 fusion proteins or
glutathione-S-transferase/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtiter plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or LRP-1 or LRP-2 protein, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads or microtiter plate wells are washed to
remove any unbound components, the matrix immobilized in the case
of beads, the complex so formed determined either directly or
indirectly, for example, as described above. Alternatively, the
complexes can be dissociated from the matrix, and the level of
LRP-1 or LRP-2 binding or activity determined using standard
techniques.
[0056] Other techniques for immobilizing either an LRP-1 or LRP-2
protein or a target molecule on matrices include using conjugation
of biotin and streptavidin. Biotinylated LRP-1 or LRP-2 protein or
target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques known in the art (e.g.,
biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical).
[0057] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the immobilized component (the
antibody, in turn, can be directly labeled or indirectly labeled
with, e.g., a labeled anti-Ig antibody.
[0058] In one embodiment, this assay is performed utilizing
antibodies reactive with LRP-1 or LRP-2 protein or target molecules
but which do not interfere with binding of the LRP-1 or LRP-2
protein to its target molecule. Such antibodies can be derivatized
to the wells of the plate, and unbound target or LRP-1 or LRP-2
protein trapped in the wells by antibody conjugation. Methods for
detecting such complexes, in addition to those described above for
the GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the LRP-1 or LRP-2 protein or target
molecule, as well as enzyme-linked assays which rely on detecting
an enzymatic activity associated with the LRP-1 or LRP-2 protein or
target molecule.
[0059] Alternatively, cell free assays can be conducted in a liquid
phase. In such an assay, the reaction products are separated from
unreacted components, by any of a number of standard techniques,
including but not limited to: differential centrifugation (see, for
example, Rivas, G., and Minton, A. P., (1993) Trends Biochem Sci
18:284-7); chromatography (gel filtration chromatography,
ion-exchange chromatography); electrophoresis (see, e.g., Ausubel,
F. et al., eds. Current Protocols in Molecular Biology 1999, J.
Wiley: New York); and immunoprecipitation (see, for example,
Ausubel, F. et al., eds. (1999) Current Protocols in Molecular
Biology, J. Wiley: New York). Such resins and chromatographic
techniques are known to one skilled in the art (see, e.g.,
Heegaard, N. H., (1998) J Mol Recognit 11:141-8; Hage, D. S., and
Tweed, S. A. (1997) J Chromatogr B Biomed Sci Appl. 699:499-525).
Further, fluorescence energy transfer can also be conveniently
utilized, as described herein, to detect binding without further
purification of the complex from solution.
[0060] In a preferred embodiment, the assay includes contacting the
LRP-1 or LRP-2 protein or biologically active portion thereof with
a known compound which binds LRP-1 or LRP-2 to form an assay
mixture, contacting the assay mixture with a test compound, and
determining the ability of the test compound to interact with an
LRP-1 or LRP-2 protein, wherein determining the ability of the test
compound to interact with an LRP-1 or LRP-2 protein includes
determining the ability of the test compound to preferentially bind
to LRP-1 or LRP-2 or biologically active portion thereof, or to
modulate the activity of a target molecule, as compared to the
known compound.
[0061] The target gene products can, in vivo, interact with one or
more cellular or extracellular macromolecules, such as proteins.
For the purposes of this discussion, such cellular and
extracellular macromolecules are referred to herein as "binding
partners." Compounds that disrupt such interactions can be useful
in regulating the activity of the target gene product. Such
compounds can include, but are not limited to molecules such as
antibodies, peptides, and small molecules. The preferred target
genes/products for use in this embodiment are the LRP-1 or LRP-2
genes herein identified. In an alternative embodiment, the
invention provides methods for determining the ability of the test
compound to modulate the activity of an LRP-1 or LRP-2 protein
through modulation of the activity of a downstream effector of an
LRP-1 or LRP-2 target molecule. For example, the activity of the
effector molecule on an appropriate target can be determined, or
the binding of the effector to an appropriate target can be
determined, as previously described.
[0062] To identify compounds that interfere with the interaction
between the target gene product and its cellular or extracellular
binding partner(s), a reaction mixture containing the target gene
product and the binding partner is prepared, under conditions and
for a time sufficient, to allow the two products to form complex.
In order to test an inhibitory agent, the reaction mixture is
provided in the presence and absence of the test compound. The test
compound can be initially included in the reaction mixture, or can
be added at a time subsequent to the addition of the target gene
and its cellular or extracellular binding partner. Control reaction
mixtures are incubated without the test compound or with a placebo.
The formation of any complexes between the target gene product and
the cellular or extracellular binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the target gene product
and the interactive binding partner. Additionally, complex
formation within reaction mixtures containing the test compound and
normal target gene product can also be compared to complex
formation within reaction mixtures containing the test compound and
mutant target gene product. This comparison can be important in
those cases wherein it is desirable to identify compounds that
disrupt interactions of mutant but not normal target gene
products.
[0063] These assays can be conducted in a heterogeneous or
homogeneous format. Heterogeneous assays involve anchoring either
the target gene product or the binding partner onto a solid phase,
and detecting complexes anchored on the solid phase at the end of
the reaction. In homogeneous assays, the entire reaction is carried
out in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction between the target gene products and the
binding partners, e.g., by competition, can be identified by
conducting the reaction in the presence of the test substance.
Alternatively, test compounds that disrupt preformed complexes,
e.g., compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are briefly described below.
[0064] In a heterogeneous assay system, either the target gene
product or the interactive cellular or extracellular binding
partner, is anchored onto a solid surface (e.g., a microtiter
plate), while the non-anchored species is labeled, either directly
or indirectly. The anchored species can be immobilized by
non-covalent or covalent attachments. Alternatively, an immobilized
antibody specific for the species to be anchored can be used to
anchor the species to the solid surface.
[0065] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface.
Where the non-immobilized species is pre-labeled, the detection of
label immobilized on the surface indicates that complexes were
formed. Where the non-immobilized species is not pre-labeled, an
indirect label can be used to detect complexes anchored on the
surface; e.g., using a labeled antibody specific for the initially
non-immobilized species (the antibody, in turn, can be directly
labeled or indirectly labeled with, e.g., a labeled anti-Ig
antibody). Depending upon the order of addition of reaction
components, test compounds that inhibit complex formation or that
disrupt preformed complexes can be detected.
[0066] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds that inhibit complex
or that disrupt preformed complexes can be identified.
[0067] In an alternate embodiment of the invention, a homogeneous
assay can be used. For example, a preformed complex of the target
gene product and the interactive cellular or extracellular binding
partner product is prepared in that either the target gene products
or their binding partners are labeled, but the signal generated by
the label is quenched due to complex formation (see, e.g., U.S.
Pat. No. 4,109,496 that utilizes this approach for immunoassays).
The addition of a test substance that competes with and displaces
one of the species from the preformed complex will result in the
generation of a signal above background. In this way, test
substances that disrupt target gene product-binding partner
interaction can be identified.
[0068] In yet another aspect, the LRP-1 or LRP-2 proteins can be
used as "bait proteins" in a two-hybrid assay or three-hybrid assay
(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell
72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054;
Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
(1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify
other proteins, which bind to or interact with LRP-1 or LRP-2
("LRP-1 or LRP-2 binding proteins" or "LRP-1 or LRP-2-bp") and are
involved in LRP-1 or LRP-2 activity. Such LRP-1 or LRP-2-bps can be
activators or inhibitors of signals by the LRP-1 or LRP-2 proteins
or LRP-1 or LRP-2 targets as, for example, downstream elements of
an LRP-1 or LRP-2 mediated signaling pathway.
[0069] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for an LRP-1 or
LRP-2 protein is fused to a gene encoding the DNA binding domain of
a known transcription factor (e.g., GAL-4). In the other construct,
a DNA sequence, from a library of DNA sequences, that encodes an
unidentified protein ("prey" or "sample") is fused to a gene that
codes for the activation domain of the known transcription factor.
(Alternatively the: LRP-1 or LRP-2 protein can be the fused to the
activator domain.) If the "bait" and the "prey" proteins are able
to interact, in vivo, forming an LRP-1 or LRP-2 dependent complex,
the DNA-binding and activation domains of the transcription factor
are brought into close proximity. This proximity allows
transcription of a reporter gene (e.g., lacZ) which is operably
linked to a transcriptional regulatory site responsive to the
transcription factor. Expression of the reporter gene can be
detected and cell colonies containing the functional transcription
factor can be isolated and used to obtain the cloned gene which
encodes the protein which interacts with the LRP-1 or LRP-2
protein.
[0070] In another embodiment, modulators of LRP-1 or LRP-2
expression are identified. For example, a cell or cell free mixture
is contacted with a candidate compound and the expression of LRP-1
or LRP-2 mRNA or protein evaluated relative to the level of
expression of LRP-1 or LRP-2 mRNA or protein in the absence of the
candidate compound. When expression of LRP-1 or LRP-2 mRNA or
protein is greater in the presence of the candidate compound than
in its absence, the candidate compound is identified as a
stimulator of LRP-1 or LRP-2 mRNA or protein expression.
Alteratively, when expression of LRP-1 or LRP-2 mRNA or protein is
less (statistically significantly less) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as an inhibitor of LRP-1 or LRP-2 mRNA or protein
expression. The level of LRP-1 or LRP-2 mRNA or protein expression
can be determined by methods described herein for detecting LRP-1
or LRP-2 mRNA or protein.
[0071] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a cell
free assay, and the ability of the agent to modulate the activity
of an LRP-1 or LRP-2 protein can be confirmed in vivo, e.g., in an
animal such as an animal model for bone disorders such as
osteoporosis, Paget's disease, and osteogenesis imperfecta.
[0072] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein (e.g., an LRP-1 or LRP-2 modulating agent, an
antisense LRP-1 or LRP-2 nucleic acid molecule, an LRP-1 or
LRP-2-specific antibody, or an LRP-1 or LRP-2 binding partner) in
an appropriate animal model to determine the efficacy, toxicity,
side effects, or mechanism of action, of treatment with such an
agent. Furthermore, novel agents identified by the above-described
screening assays can be used for treatments as described
herein.
Predictive Medicine
[0073] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring clinical trials are used for prognostic (predictive)
purposes to thereby treat an individual.
[0074] Generally, the invention provides, a method of determining
if a subject is at risk for a disorder related to a lesion in or
the misexpression of a gene which encodes LRP-1 or LRP-2.
[0075] Such disorders include, e.g., a disorder associated with the
misexpression of LRP-1 or LRP-2 gene; a disorder of bone.
[0076] The method includes one or more of the following:
[0077] detecting, in a tissue of the subject, the presence or
absence of a mutation which affects the expression of the LRP-1 or
LRP-2 gene, or detecting the presence or absence of a mutation in a
region which controls the expression of the gene, e.g., a mutation
in the 5' control region;
[0078] detecting, in a tissue of the subject, the presence or
absence of a mutation which alters the structure of the LRP-1 or
LRP-2 gene;
[0079] detecting, in a tissue of the subject, the misexpression of
the LRP-1 or LRP-2 gene, at the mRNA level, e.g., detecting a
non-wild type level of a mRNA;
[0080] detecting, in a tissue of the subject, the misexpression of
the gene, at the protein level, e.g., detecting a non-wild type
level of an LRP-1 or LRP-2 polypeptide.
[0081] In preferred embodiments the method includes: ascertaining
the existence of at least one of: a deletion of one or more
nucleotides from the LRP-1 or LRP-2 gene; an insertion of one or
more nucleotides into the gene, a point mutation, e.g., a
substitution of one or more nucleotides of the gene, a gross
chromosomal rearrangement of the gene, e.g., a translocation,
inversion, or deletion.
[0082] For example, detecting the genetic lesion can include: (i)
providing a probe/primer including an oligonucleotide containing a
region of nucleotide sequence which hybridizes to a sense or
antisense sequence from naturally occurring mutants thereof or 5'
or 3' flanking sequences naturally associated with the LRP-1 or
LRP-2 gene; (ii) exposing the probe/primer to nucleic acid of the
tissue; and detecting, by hybridization, e.g., in situ
hybridization, of the probe/primer to the nucleic acid, the
presence or absence of the genetic lesion.
[0083] In preferred embodiments detecting the misexpression
includes ascertaining the existence of at least one of: an
alteration in the level of a messenger RNA transcript of the LRP-1
or LRP-2 gene; the presence of a non-wild type splicing pattern of
a messenger RNA transcript of the gene; or a non-wild type level of
LRP-1 or LRP-2.
[0084] Methods of the invention can be used prenatally or to
determine if a subject's offspring will be at risk for a
disorder.
[0085] In preferred embodiments the method includes determining the
structure of an LRP-1 or LRP-2 gene, an abnormal structure being
indicative of risk for the disorder.
[0086] In preferred embodiments the method includes contacting a
sample from the subject with an antibody to the LRP-1 or LRP-2
protein or a nucleic acid, which hybridizes specifically with the
gene. These and other embodiments are discussed below.
Diagnostic and Prognostic Assays
[0087] Diagnostic and prognostic assays of the invention include
method for assessing the expression level of LRP-1 or LRP-2
molecules and for identifying variations and mutations in the
sequence of LRP-1 or LRP-2 molecules.
Expression Monitoring and Profiling
[0088] The presence, level, or absence of LRP-1 or LRP-2 protein or
nucleic acid in a biological sample can be evaluated by obtaining a
biological sample from a test subject and contacting the biological
sample with a compound or an agent capable of detecting LRP-1 or
LRP-2 protein or nucleic acid (e.g., mRNA, genomic DNA) that
encodes LRP-1 or LRP-2 protein such that the presence of LRP-1 or
LRP-2 protein or nucleic acid is detected in the biological sample.
The term "biological sample" includes tissues, cells and biological
fluids isolated from a subject, as well as tissues, cells and
fluids present within a subject. A preferred biological sample is
serum. The level of expression of the LRP-1 LRP-2 gene can be
measured in a number of ways, including, but not limited to:
measuring the mRNA encoded by the LRP-1 or LRP-2 genes; measuring
the amount of protein encoded by the LRP-1 or LRP-2 genes; or
measuring the activity of the protein encoded by the LRP-1 or LRP-2
genes.
[0089] The level of mRNA corresponding to the LRP-1 or LRP-2 gene
in a cell can be determined both by in situ and by in vitro
formats.
[0090] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length LRP-1 or LRP-2 nucleic acid, such as the nucleic acid
of SEQ ID NO:1, or a portion thereof, such as an oligonucleotide of
at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and
sufficient to specifically hybridize under stringent conditions to
LRP-1 or LRP-2 mRNA or genomic DNA. The probe can be disposed on an
address of an array, e.g., an array described below. Other suitable
probes for use in the diagnostic assays are described herein.
[0091] In one format, mRNA (or cDNA) is immobilized on a surface
and contacted with the probes, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probes are immobilized on a surface and the mRNA (or cDNA) is
contacted with the probes, for example, in a two-dimensional gene
chip array described below. A skilled artisan can adapt known mRNA
detection methods for use in detecting the level of mRNA encoded by
the LRP-1 or LRP-2 genes.
[0092] The level of mRNA in a sample that is encoded by one of
LRP-1 or LRP-2 can be evaluated with nucleic acid amplification,
e.g., by rtPCR (Mullis (1987) U.S. Pat. No. 4,683,202), ligase
chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA
88:189-193), self sustained sequence replication (Guatelli et al.,
(1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional
amplification system (Kwoh et al., (1989), Proc. Natl. Acad. Sci.
USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., (1988)
Bio/Technology 6:1197), rolling circle replication (Lizardi et al.,
U.S. Pat. No. 5,854,033) or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques known in the art. As used herein, amplification primers
are defined as being a pair of nucleic acid molecules that can
anneal to 5' or 3' regions of a gene (plus or minus strands,
respectively, or vice-versa) and contain a short region in between.
In general, amplification primers are from about 10 to 30
nucleotides in length and flank a region from about 50 to 200
nucleotides in length. Under appropriate conditions and with
appropriate reagents, such primers permit the amplification of a
nucleic acid molecule comprising the nucleotide sequence flanked by
the primers.
[0093] For in situ methods, a cell or tissue sample can be
prepared/processed and immobilized on a support, typically a glass
slide, and then contacted with a probe that can hybridize to mRNA
that encodes the LRP-1 or LRP-2 gene being analyzed.
[0094] In another embodiment, the methods further contacting a
control sample with a compound or agent capable of detecting LRP-1
or LRP-2 mRNA, or genomic DNA, and comparing the presence of LRP-1
or LRP-2 mRNA or genomic DNA in the control sample with the
presence of LRP-1 or LRP-2 mRNA or genomic DNA in the test sample.
In still another embodiment, serial analysis of gene expression, as
described in U.S. Pat. No. 5,695,937, is used to detect LRP-1 or
LRP-2 transcript levels.
[0095] A variety of methods can be used to determine the level of
protein encoded by LRP-1 or LRP-2. In general, these methods
include contacting an agent that selectively binds to the protein,
such as an antibody with a sample, to evaluate the level of protein
in the sample. In a preferred embodiment, the antibody bears a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab').sub.2) can be used. The term "labeled", with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with a detectable
substance. Examples of detectable substances are provided
herein.
[0096] The detection methods can be used to detect LRP-1 or LRP-2
protein in a biological sample in vitro as well as in vivo. In
vitro techniques for detection of LRP-1 or LRP-2 protein include
enzyme linked immunosorbent assays (ELISAs), immunoprecipitations,
immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay
(RIA), and Western blot analysis. In vivo techniques for detection
of LRP-1 or LRP-2 protein include introducing into a subject a
labeled anti-LRP-1 or LRP-2 antibody. For example, the antibody can
be labeled with a radioactive marker whose presence and location in
a subject can be detected by standard imaging techniques. In
another embodiment, the sample is labeled, e.g., biotinylated and
then contacted to the antibody, e.g., an anti-LRP-1 or LRP-2
antibody positioned on an antibody array (as described below). The
sample can be detected, e.g., with avidin coupled to a fluorescent
label.
[0097] In another embodiment, the methods further include
contacting the control sample with a compound or agent capable of
detecting LRP-1 or LRP-2 protein, and comparing the presence of
LRP-1 or LRP-2 protein in the control sample with the presence of
LRP-1 or LRP-2 protein in the test sample.
[0098] The invention also includes kits for detecting the presence
of LRP-1 or LRP-2 in a biological sample. For example, the kit can
include a compound or agent capable of detecting LRP-1 or LRP-2
protein or mRNA in a biological sample; and a standard. The
compound or agent can be packaged in a suitable container. The kit
can further comprise instructions for using the kit to detect LRP-1
or LRP-2 protein or nucleic acid or an agonist or antagonist of the
LRP-1 or LRP-2 protein.
[0099] For antibody-based kits, the kit can include: (1) a first
antibody (e.g., attached to a solid support) which binds to a
polypeptide corresponding to a marker; and, optionally, (2) a
second, different antibody which binds to either the polypeptide or
the first antibody and is conjugated to a detectable agent.
[0100] For oligonucleotide-based kits, the kit can include: (1) an
oligonucleotide, e.g., a detectably labeled oligonucleotide, which
hybridizes to a nucleic acid sequence encoding a polypeptide
corresponding to a marker or (2) a pair of primers useful for
amplifying a nucleic acid molecule corresponding to a marker. The
kit can also include a buffering agent, a preservative, or a
protein stabilizing agent. The kit can also include components
necessary for detecting the detectable agent (e.g., an enzyme or a
substrate). The kit can also contain a control sample or a series
of control samples which can be assayed and compared to the test
sample contained. Each component of the kit can be enclosed within
an individual container and all of the various containers can be
within a single package, along with instructions for interpreting
the results of the assays performed using the kit.
[0101] The diagnostic methods described herein can identify
subjects having, or at risk of developing, a disease or disorder
associated with misexpressed or aberrant or unwanted LRP-1 or LRP-2
expression or activity. As used herein, the term "unwanted"
includes an unwanted phenomenon involved in a biological response
such as bone disorders such as osteoporosis, Paget's disease, and
osteogenesis imperfecta.
[0102] In one embodiment, a disease or disorder associated with
aberrant or unwanted LRP-1 or LRP-2 expression or activity is
identified. A test sample is obtained from a subject and LRP-1 or
LRP-2 protein or nucleic acid (e.g., mRNA or genomic DNA) is
evaluated, wherein the level, e.g., the presence or absence, of
LRP-1 or LRP-2 protein or nucleic acid is diagnostic for a subject
having or at risk of developing a disease or disorder associated
with aberrant or unwanted LRP-1 or LRP-2 expression or activity. As
used herein, a "test sample" refers to a biological sample obtained
from a subject of interest, including a biological fluid (e.g.,
serum), cell sample, or tissue.
[0103] The prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., an
agonist, antagonist, peptidomimetic, protein, peptide, nucleic
acid, small molecule, or other drug candidate) to treat a disease
or disorder associated with aberrant or unwanted LRP-1 or LRP-2
expression or activity. For example, such methods can be used to
determine whether a subject can be effectively treated with an
agent for a bone disorder such as osteoporosis, Paget's disease, or
osteogenesis imperfecta.
[0104] In another aspect, the invention features a computer medium
having a plurality of digitally encoded data records. Each data
record includes a value representing the level of expression of
LRP-1 or LRP-2 in a sample, and a descriptor of the sample. The
descriptor of the sample can be an identifier of the sample, a
subject from which the sample was derived (e.g., a patient), a
diagnosis, or a treatment (e.g., a preferred treatment). In a
preferred embodiment, the data record further includes values
representing the level of expression of genes other than LRP-1 or
LRP-2 (e.g., other genes associated with an LRP-1 or
LRP-2-disorder, or other genes on an array). The data record can be
structured as a table, e.g., a table that is part of a database
such as a relational database (e.g., a SQL database of the Oracle
or Sybase database environments).
[0105] Also featured is a method of evaluating a sample. The method
includes providing a sample, e.g., from the subject, and
determining a gene expression profile of the sample, wherein the
profile includes a value representing the level of LRP-1 or LRP-2
expression. The method can further include comparing the value or
the profile (i.e., multiple values) to a reference value or
reference profile. The gene expression profile of the sample can be
obtained by any of the methods described herein (e.g., by providing
a nucleic acid from the sample and contacting the nucleic acid to
an array). The method can be used to diagnose a bone disorders such
as osteoporosis, Paget's disease, and osteogenesis imperfecta in a
subject wherein an increase/decrease in LRP-1 or LRP-2 expression
is an indication that the subject has or is disposed to having a
bone disorders such as osteoporosis, Paget's disease, and
osteogenesis imperfecta. The method can be used to monitor a
treatment for bone disorders such as osteoporosis, Paget's disease,
and osteogenesis imperfecta in a subject. For example, the gene
expression profile can be determined for a sample from a subject
undergoing treatment. The profile can be compared to a reference
profile or to a profile obtained from the subject prior to
treatment or prior to onset of the disorder (see, e.g., Golub et
al. (1999) Science 286:531).
[0106] In yet another aspect, the invention features a method of
evaluating a test compound (see also, "Screening Assays", above).
The method includes providing a cell and a test compound;
contacting the test compound to the cell; obtaining a subject
expression profile for the contacted cell; and comparing the
subject expression profile to one or more reference profiles. The
profiles include a value representing the level of LRP-1 or LRP-2
expression. In a preferred embodiment, the subject expression
profile is compared to a target profile, e.g., a profile for a
normal cell or for desired condition of a cell. The test compound
is evaluated favorably if the subject expression profile is more
similar to the target profile than an expression profile obtained
from an uncontacted cell.
[0107] In another aspect, the invention features, a method of
evaluating a subject. The method includes: a) obtaining a sample
from a subject, e.g., from a caregiver, e.g., a caregiver who
obtains the sample from the subject; b) determining a subject
expression profile for the sample. Optionally, the method further
includes either or both of steps: c) comparing the subject
expression profile to one or more reference expression profiles;
and d) selecting the reference profile most similar to the subject
reference profile. The subject expression profile and the reference
profiles include a value representing the level of LRP-1 or LRP-2
expression. A variety of routine statistical measures can be used
to compare two reference profiles. One possible metric is the
length of the distance vector that is the difference between the
two profiles. Each of the subject and reference profile is
represented as a multi-dimensional vector, wherein each dimension
is a value in the profile.
[0108] The method can further include transmitting a result to a
caregiver. The result can be the subject expression profile, a
result of a comparison of the subject expression profile with
another profile, a most similar reference profile, or a descriptor
of any of the aforementioned. The result can be transmitted across
a computer network, e.g., the result can be in the form of a
computer transmission, e.g., a computer data signal embedded in a
carrier wave.
[0109] Also featured is a computer medium having executable code
for effecting the following steps: receive a subject expression
profile; access a database of reference expression profiles; and
either i) select a matching reference profile most similar to the
subject expression profile or ii) determine at least one comparison
score for the similarity of the subject expression profile to at
least one reference profile. The subject expression profile, and
the reference expression profiles each include a value representing
the level of LRP-1 or LRP-2 expression.
Detection of Variations or Mutations
[0110] The methods of the invention can also be used to detect
genetic alterations in an LRP-1 or LRP-2 gene, thereby determining
if a subject with the altered gene is at risk for a disorder
characterized by misregulation in LRP-1 or LRP-2 protein activity
or nucleic acid expression, such as a bone disorder (e.g.,
osteoporosis, Paget's disease, or osteogenesis imperfecta). In
preferred embodiments, the methods include detecting, in a sample
from the subject, the presence or absence of a genetic alteration
characterized by at least one of an alteration affecting the
integrity of a gene encoding an LRP-1 or LRP-2-protein, or the
misexpression of the LRP-1 or LRP-2 gene. For example, such genetic
alterations can be detected by ascertaining the existence of at
least one of 1) a deletion of one or more nucleotides from an LRP-1
or LRP-2 gene; 2) an addition of one or more nucleotides to an
LRP-1 or LRP-2 gene; 3) a substitution of one or more nucleotides
of an LRP-1 or LRP-2 gene, 4) a chromosomal rearrangement of an
LRP-1 or LRP-2 gene; 5) an alteration in the level of a messenger
RNA transcript of an LRP-1 or LRP-2 gene, 6) aberrant modification
of an LRP-1 or LRP-2 gene, such as of the methylation pattern of
the genomic DNA, 7) the presence of a non-wild type splicing
pattern of a messenger RNA transcript of an LRP-1 or LRP-2 gene, 8)
a non-wild type level of an LRP-1 or LRP-2-protein, 9) allelic loss
of an LRP-1 or LRP-2 gene, and 10) inappropriate post-translational
modification of an LRP-1 or LRP-2-protein.
[0111] An alteration can be detected without a probe/primer in a
polymerase chain reaction, such as anchor PCR or RACE PCR, or,
alternatively, in a ligation chain reaction (LCR), the latter of
which can be particularly useful for detecting point mutations in
the LRP-1 or LRP-2-gene. This method can include the steps of
collecting a sample of cells from a subject, isolating nucleic acid
(e.g., genomic, mRNA or both) from the sample, contacting the
nucleic acid sample with one or more primers which specifically
hybridize to an LRP-1 or LRP-2 gene under conditions such that
hybridization and amplification of the LRP-1 or LRP-2-gene (if
present) occurs, and detecting the presence or absence of an
amplification product, or detecting the size of the amplification
product and comparing the length to a control sample. It is
anticipated the PCR or LCR can be desirable to use as a preliminary
amplification step in conjunction with any of the techniques used
for detecting mutations described herein. Alternatively, other
amplification methods described herein or known in the art can be
used.
[0112] In another embodiment, mutations in an LRP-1 or LRP-2 gene
from a sample cell can be identified by detecting alterations in
restriction enzyme cleavage patterns. For example, sample and
control DNA is isolated, amplified (optionally), digested with one
or more restriction endonucleases, and fragment length sizes are
determined, e.g., by gel electrophoresis and compared. Differences
in fragment length sizes between sample and control DNA indicates
mutations in the sample DNA. Moreover, the use of sequence specific
ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used
to score for the presence of specific mutations by development or
loss of a ribozyme cleavage site.
[0113] In other embodiments, genetic mutations in LRP-1 or LRP-2
can be identified by hybridizing a sample and control nucleic
acids, e.g., DNA or RNA, two-dimensional arrays, e.g., chip based
arrays. Such arrays include a plurality of addresses, each of which
is positionally distinguishable from the other. A different probe
is located at each address of the plurality. A probe can be
complementary to a region of an LRP-1 or LRP-2 nucleic acid or a
putative variant (e.g., allelic variant) thereof. A probe can have
one or more mismatches to a region of an LRP-1 or LRP-2 nucleic
acid (e.g., a destabilizing mismatch). The arrays can have a high
density of addresses, e.g., can contain hundreds or thousands of
oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation
7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759).
For example, genetic mutations in LRP-1 or LRP-2 can be identified
in two-dimensional arrays containing light-generated DNA probes as
described in Cronin, M. T. et al. supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[0114] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
LRP-1 or LRP-2 gene and detect mutations by comparing the sequence
of the sample LRP-1 or LRP-2 with the corresponding wild-type
(control) sequence. Automated sequencing procedures can be utilized
when performing the diagnostic assays ((1995) Biotechniques
19:448), including sequencing by mass spectrometry.
[0115] Other methods for detecting mutations in the LRP-1 or LRP-2
gene include methods in which protection from cleavage agents is
used to detect mismatched bases in RNA/RNA or RNA/DNA
heteroduplexes (Myers et al. (1985) Science 230:1242; Cotton et al.
(1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992)
Methods Enzymol. 217:286-295).
[0116] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in LRP-1
or LRP-2 cDNAs obtained from samples of cells. For example, the
mutY enzyme of E. coli cleaves A at G/A mismatches and the
thymidine DNA glycosylase from HeLa cells cleaves T at G/T
mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662; U.S.
Pat. No. 5,459,039).
[0117] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in LRP-1 or LRP-2
genes. For example, single strand conformation polymorphism (SSCP)
can be used to detect differences in electrophoretic mobility
between mutant and wild type nucleic acids (Orita et al. (1989)
Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat.
Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl.
9:73-79). Single-stranded DNA fragments of sample and control LRP-1
or LRP-2 nucleic acids will be denatured and allowed to renature.
The secondary structure of single-stranded nucleic acids varies
according to sequence, the resulting alteration in electrophoretic
mobility enables the detection of even a single base change. The
DNA fragments can be labeled or detected with labeled probes. The
sensitivity of the assay can be enhanced by using RNA (rather than
DNA), in which the secondary structure is more sensitive to a
change in sequence. In a preferred embodiment, the subject method
utilizes heteroduplex analysis to separate double stranded
heteroduplex molecules on the basis of changes in electrophoretic
mobility (Keen et al. (1991) Trends Genet 7:5).
[0118] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[0119] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989)
Proc. Natl Acad. Sci USA 86:6230). A further method of detecting
point mutations is the chemical ligation of oligonucleotides as
described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent
oligonucleotides, one of which selectively anneals to the query
site, are ligated together if the nucleotide at the query site of
the sample nucleic acid is complementary to the query
oligonucleotide; ligation can be monitored, e.g., by fluroescent
dyes coupled to the oligonucleotides.
[0120] Alternatively, allele specific amplification technology that
depends on selective PCR amplification can be used in conjunction
with the instant invention. Oligonucleotides used as primers for
specific amplification can carry the mutation of interest in the
center of the molecule (so that amplification depends on
differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res.
17:2437-2448) or at the extreme 3' end of one primer where, under
appropriate conditions, mismatch can prevent, or reduce polymerase
extension (Prossner (1993) Tibtech 11:238). In addition it is
desirable to introduce a novel restriction site in the region of
the mutation to create cleavage based detection (Gasparini et al.
(1992) Mol. Cell Probes 6:1). It is anticipated that in certain
embodiments amplification can also be performed using Taq ligase
for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189).
In such cases, ligation will occur only if there is a perfect match
at the 3' end of the 5' sequence making it possible to detect the
presence of a known mutation at a specific site by looking for the
presence of absence of amplification.
[0121] In another aspect, the invention features a set of
oligonucleotides. The set includes a plurality of oligonucleotides,
each of which is at least partially complementary (e.g., at least
50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary)
to an LRP-1 or LRP-2 nucleic acid.
[0122] In a preferred embodiment the set includes a first and a
second oligonucleotide. The first and second oligonucleotide can
hybridize to the same or to different locations of an LRP-1 or
LRP-2 gene sequence or the complement of an LRP-1 or LRP-2 gene
sequence. Different locations can be different but overlapping, or
non-overlapping on the same strand. The first and second
oligonucleotide can hybridize to sites on the same or on different
strands.
[0123] The set can be useful, e.g., for identifying SNP's, or
identifying specific alleles of LRP-1 or LRP-2. In a preferred
embodiment, each oligonucleotide of the set has a different
nucleotide at an interrogation position. In one embodiment, the set
includes two oligonucleotides, each complementary to a different
allele at a locus, e.g., a biallelic or polymorphic locus.
[0124] In another embodiment, the set includes four
oligonucleotides, each having a different nucleotide (e.g.,
adenine, guanine, cytosine, or thymidine) at the interrogation
position. The interrogation position can be a SNP or the site of a
mutation. In another preferred embodiment, the oligonucleotides of
the plurality are identical in sequence to one another (except for
differences in length). The oligonucleotides can be provided with
differential labels, such that an oligonucleotide that hybridizes
to one allele provides a signal that is distinguishable from an
oligonucleotide that hybridizes to a second allele. In still
another embodiment, at least one of the oligonucleotides of the set
has a nucleotide change at a position in addition to a query
position, e.g., a destabilizing mutation to decrease the T.sub.m of
the oligonucleotide. In another embodiment, at least one
oligonucleotide of the set has a non-natural nucleotide, e.g.,
inosine. In a preferred embodiment, the oligonucleotides are
attached to a solid support, e.g., to different addresses of an
array or to different beads or nanoparticles.
[0125] In a preferred embodiment the set of oligo nucleotides can
be used to specifically amplify, e.g., by PCR, or detect, an LRP-1
or LRP-2 nucleic acid.
[0126] The methods described herein can be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which can
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving an LRP-1 or LRP-2 gene.
Use of LRP-1 or LRP-2 Molecules as Surrogate Markers
[0127] The LRP-1 or LRP-2 molecules are also useful as markers of
disorders or disease states, as markers for precursors of disease
states, as markers for predisposition of disease states, as markers
of drug activity, or as markers of the pharmacogenomic profile of a
subject. Using the methods described herein, the presence, absence
or quantity of the LRP-1 or LRP-2 molecules can be detected, and
can be correlated with one or more biological states in vivo. For
example, the LRP-1 or LRP-2 molecules can serve as surrogate
markers for one or more disorders or disease states or for
conditions leading up to disease states. As used herein, a
"surrogate marker" is an objective biochemical marker which
correlates with the absence or presence of a disease or disorder,
or with the progression of a disease or disorder (e.g., with the
presence or absence of a tumor). The presence or quantity of such
markers is independent of the disease. Therefore, these markers can
serve to indicate whether a particular course of treatment is
effective in lessening a disease state or disorder. Surrogate
markers are of particular use when the presence or extent of a
disease state or disorder is difficult to assess through standard
methodologies (e.g., early stage tumors), or when an assessment of
disease progression is desired before a potentially dangerous
clinical endpoint is reached (e.g., as assessment of cardiovascular
disease can be made using cholesterol levels as a surrogate marker,
and an analysis of HIV infection can be made using HIV RNA levels
as a surrogate marker, well in advance of the undesirable clinical
outcomes of myocardial infarction or fully-developed AIDS).
Examples of the use of surrogate markers in the art include: Koomen
et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS
Treatment News Archive 209.
[0128] The LRP-1 or LRP-2 molecules are also useful as
pharmacodynamic markers. As used herein, a "pharmacodynamic marker"
is an objective biochemical marker which correlates specifically
with drug effects. The presence or quantity of a pharmacodynamic
marker is not related to the disease state or disorder for which
the drug is being administered; therefore, the presence or quantity
of the marker is indicative of the presence or activity of the drug
in a subject. For example, a pharmacodynamic marker can be
indicative of the concentration of the drug in a biological tissue,
in that the marker is either expressed or transcribed or not
expressed or transcribed in that tissue in relationship to the
level of the drug. In this fashion, the distribution or uptake of
the drug can be monitored by the pharmacodynamic marker. Similarly,
the presence or quantity of the pharmacodynamic marker can be
related to the presence or quantity of the metabolic product of a
drug, such that the presence or quantity of the marker is
indicative of the relative breakdown rate of the drug in vivo.
Pharmacodynamic markers are of particular use in increasing the
sensitivity of detection of drug effects, particularly when the
drug is administered in low doses. Since even a small amount of a
drug can be sufficient to activate multiple rounds of marker (e.g.,
an LRP-1 or LRP-2 marker) transcription or expression, the
amplified marker can be in a quantity which is more readily
detectable than the drug itself. Also, the marker can be more
easily detected due to the nature of the marker itself; for
example, using the methods described herein, anti-LRP-1 or LRP-2
antibodies can be employed in an immune-based detection system for
an LRP-1 or LRP-2 protein marker, or LRP-1 or LRP-2-specific
radiolabeled probes can be used to detect an LRP-1 or LRP-2 mRNA
marker. Furthermore, the use of a pharmacodynamic marker can offer
mechanism-based prediction of risk due to drug treatment beyond the
range of possible direct observations. Examples of the use of
pharmacodynamic markers in the art include: Matsuda et al. U.S.
Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90:
229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3:
S21-S24; and Nicolau (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3:
S16-S20.
[0129] The LRP-1 or LRP-2 molecules are also useful as
pharmacogenomic markers. As used herein, a "pharmacogenomic marker"
is an objective biochemical marker which correlates with a specific
clinical drug response or susceptibility in a subject (see, e.g.,
McLeod et al. (1999) Eur. J. Cancer 35:1650-1652). The presence or
quantity of the pharmacogenomic marker is related to the predicted
response of the subject to a specific drug or class of drugs prior
to administration of the drug. By assessing the presence or
quantity of one or more pharmacogenomic markers in a subject, a
drug therapy which is most appropriate for the subject, or which is
predicted to have a greater degree of success, be selected. For
example, based on the presence or quantity of RNA, or protein
(e.g., LRP-1 or LRP-2 protein or RNA) for specific tumor markers in
a subject, a drug or course of treatment be selected that is
optimized for the treatment of the specific tumor likely to be
present in the subject. Similarly, the presence or absence of a
specific sequence mutation in LRP-1 or LRP-2 DNA correlate LRP-1 or
LRP-2 drug response. The use of pharmacogenomic markers therefore
permits the application of the most appropriate treatment for each
subject without having to administer the therapy.
Methods of Treatment
[0130] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant or unwanted LRP-1 or LRP-2 expression or activity. As used
herein, the term "treatment" is defined as the application or
administration of a therapeutic agent to a patient, or application
or administration of a therapeutic agent to an isolated tissue or
cell line from a patient, who has a disease, a symptom of disease
or a predisposition toward a disease, with the purpose to cure,
heal, alleviate, relieve, alter, remedy, ameliorate, improve or
affect the disease, the symptoms of disease or the predisposition
toward disease. A therapeutic agent includes, but is not limited
to, small molecules, peptides, antibodies, ribozymes and antisense
oligonucleotides.
[0131] With regards to both prophylactic and therapeutic methods of
treatment, such treatments can be specifically tailored or
modified, based on knowledge obtained from the field of
pharmacogenomics. "Pharmacogenomics", as used herein, refers to the
application of genomics technologies such as gene sequencing,
statistical genetics, and gene expression analysis to drugs in
clinical development and on the market. More specifically, the term
refers the study of how a patient's genes determine his or her
response to a drug (e.g., a patient's "drug response phenotype", or
"drug response genotype".) Thus, another aspect of the invention
provides methods for tailoring an individual's prophylactic or
therapeutic treatment with either the LRP-1 or LRP-2 molecules of
the present invention or LRP-1 or LRP-2 modulators according to
that individual's drug response genotype. Pharmacogenomics allows a
clinician or physician to target prophylactic or therapeutic
treatments to patients who will most benefit from the treatment and
to avoid treatment of patients who will experience toxic
drug-related side effects.
[0132] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant or unwanted LRP-1 or LRP-2 expression or activity, by
administering to the subject an LRP-1 or LRP-2 or an agent which
modulates LRP-1 or LRP-2 expression or at least one LRP-1 or LRP-2
activity. Subjects at risk for a disease which is caused or
contributed to by aberrant or unwanted LRP-1 or LRP-2 expression or
activity can be identified by, for example, any or a combination of
diagnostic or prognostic assays as described herein. Administration
of a prophylactic agent can occur prior to the manifestation of
symptoms characteristic of the LRP-1 or LRP-2 aberrance, such that
a disease or disorder is prevented or, alternatively, delayed in
its progression. Depending on the type of LRP-1 or LRP-2 aberrance,
for example, an LRP-1 or LRP-2, LRP-1 or LRP-2 agonist or LRP-1 or
LRP-2 antagonist agent can be used for treating the subject. The
appropriate agent can be determined based on screening assays
described herein.
[0133] It is possible that some LRP-1 or LRP-2-related disorders
can be caused, at least in part, by an abnormal level of gene
product, or by the presence of a gene product exhibiting abnormal
activity. As such, the reduction in the level or activity of such
gene products would bring about the amelioration of disorder
symptoms.
[0134] As discussed, successful treatment of LRP-1 or LRP-2-related
disorders can be brought about by techniques that serve to inhibit
the expression or activity of target gene products. For example,
compounds, e.g., an agent identified using an assays described
above, that proves to exhibit negative modulatory activity, can be
used in accordance with the invention to prevent or ameliorate
symptoms of LRP-1 or LRP-2-related disorders. Such molecules can
include, but are not limited to peptides, phosphopeptides, small
organic or inorganic molecules, or antibodies (including, for
example, polyclonal, monoclonal, humanized, anti-idiotypic,
chimeric or single chain antibodies, and Fab, F(ab').sub.2 and Fab
expression library fragments, scFV molecules, and epitope-binding
fragments thereof).
[0135] Further, antisense and ribozyme molecules that inhibit
expression of the target gene can also be used in accordance with
the invention to reduce the level of target gene expression, thus
effectively reducing the level of target gene activity. Still
further, triple helix molecules can be utilized in reducing the
level of target gene activity. Antisense, ribozyme and triple helix
molecules are discussed above.
[0136] It is possible that the use of antisense, ribozyme, or
triple helix molecules to reduce or inhibit mutant gene expression
can also reduce or inhibit the transcription (triple helix) or
translation (antisense, ribozyme) of mRNA produced by normal target
gene alleles, such that the concentration of normal target gene
product present can be lower than is necessary for a normal
phenotype. In such cases, nucleic acid molecules that encode and
express target gene polypeptides exhibiting normal target gene
activity can be introduced into cells via gene therapy method.
Alternatively, in instances in that the target gene encodes an
extracellular protein, it can be preferable to co-administer normal
target gene protein into the cell or tissue in order to maintain
the requisite level of cellular or tissue target gene activity.
[0137] Another method by which nucleic acid molecules can be
utilized in treating or preventing a disease characterized by LRP-1
or LRP-2 expression is through the use of aptamer molecules
specific for LRP-1 or LRP-2 protein. Aptamers are nucleic acid
molecules having a tertiary structure which permits them to
specifically bind to protein ligands (see, e.g., Osborne, et al.
(1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D. J. (1997) Curr
Opin Chem Biol 1:32-46). Since nucleic acid molecules can in many
cases be more conveniently introduced into target cells than
therapeutic protein molecules can be, aptamers offer a method by
which LRP-1 or LRP-2 protein activity can be specifically decreased
without the introduction of drugs or other molecules which can have
pluripotent effects.
[0138] Antibodies can be generated that are both specific for
target gene product and that reduce target gene product activity.
Such antibodies can, therefore, by administered in instances
whereby negative modulatory techniques are appropriate for the
treatment of LRP-1 or LRP-2-related disorders. For a description of
antibodies, see the Antibody section above.
[0139] In circumstances wherein injection of an animal or a human
subject with an LRP-1 or LRP-2 protein or epitope for stimulating
antibody production is harmful to the subject, it is possible to
generate an immune response against LRP-1 or LRP-2 through the use
of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999)
Ann Med 31:66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A.
(1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody
is introduced into a mammal or human subject, it should stimulate
the production of anti-anti-idiotypic antibodies, which should be
specific to the LRP-1 to LRP-2 protein. Vaccines directed to a
disease characterized by LRP-1 or LRP-2 expression can also be
generated in this fashion.
[0140] In instances where the target antigen is intracellular and
whole antibodies are used, internalizing antibodies can be
preferred. Lipofectin or liposomes can be used to deliver the
antibody or a fragment of the Fab region that binds to the target
antigen into cells. Where fragments of the antibody are used, the
smallest inhibitory fragment that binds to the target antigen is
preferred. For example, peptides having an amino acid sequence
corresponding to the Fv region of the antibody can be used.
Alternatively, single chain neutralizing antibodies that bind to
intracellular target antigens can also be administered. Such single
chain antibodies can be administered, for example, by expressing
nucleotide sequences encoding single-chain antibodies within the
target cell population (see e.g., Marasco et al. (1993) Proc. Natl.
Acad. Sci. USA 9:7889-7893).
[0141] The identified compounds that inhibit target gene
expression, synthesis or activity can be administered to a patient
at therapeutically effective doses to prevent, treat or ameliorate
LRP-1 or LRP-2-related disorders. A therapeutically effective dose
refers to that amount of the compound sufficient to result in
amelioration of symptoms of the disorders. Toxicity and therapeutic
efficacy of such compounds can be determined by standard
pharmaceutical procedures as described above.
[0142] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0143] Another example of determination of effective dose for an
individual is the ability to directly assay levels of "free" and
"bound" compound in the serum of the test subject. Such assays can
utilize antibody mimics or "biosensors" that have been created
through molecular imprinting techniques. The compound which is able
to modulate LRP-1 or LRP-2 activity is used as a template, or
"imprinting molecule", to spatially organize polymerizable monomers
prior to their polymerization with catalytic reagents. The
subsequent removal of the imprinted molecule leaves a polymer
matrix which contains a repeated "negative image" of the compound
and is able to selectively rebind the molecule under biological
assay conditions. A detailed review of this technique can be seen
in Ansell, R. J. et al (1996) Current Opinion in Biotechnology
7:89-94 and in Shea, K. J. (1994) Trends in Polymer Science
2:166-173. Such "imprinted" affinity matrixes are amenable to
ligand-binding assays, whereby the immobilized monoclonal antibody
component is replaced by an appropriately imprinted matrix. An
example of the use of such matrixes in this way can be seen in
Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of
isotope-labeling, the "free" concentration of compound which
modulates the expression or activity of LRP-1 or LRP-2 can be
readily monitored and used in calculations of IC.sub.50.
[0144] Such "imprinted" affinity matrixes can also be designed to
include fluorescent groups whose photon-emitting properties
measurably change upon local and selective binding of target
compound. These changes can be readily assayed in real time using
appropriate fiberoptic devices, in turn allowing the dose in a test
subject to be quickly optimized based on its individual IC.sub.50.
An rudimentary example of such a "biosensor" is discussed in Kriz,
D. et al (1995) Analytical Chemistry 67:2142-2144.
[0145] Another aspect of the invention pertains to methods of
modulating LRP-1 or LRP-2 expression or activity for therapeutic
purposes. Accordingly, in an exemplary embodiment, the modulatory
method of the invention involves contacting a cell with an LRP-1 or
LRP-2 or agent that modulates one or more of the activities of
LRP-1 or LRP-2 protein activity associated with the cell. An agent
that modulates LRP-1 or LRP-2 protein activity can be an agent as
described herein, such as a nucleic acid or a protein, a
naturally-occurring target molecule of an LRP-1 or LRP-2 protein
(e.g., an LRP-1 or LRP-2 substrate or receptor), an LRP-1 or LRP-2
antibody, an LRP-1 or LRP-2 agonist or antagonist, a peptidomimetic
of an LRP-1 or LRP-2 agonist or antagonist, or other small
molecule.
[0146] In one embodiment, the agent stimulates one or LRP-1 or
LRP-2 activities. Examples of such stimulatory agents include
active LRP-1 or LRP-2 protein and a nucleic acid molecule encoding
LRP-1 or LRP-2. In another embodiment, the agent inhibits one or
more LRP-1 or LRP-2 activities. Examples of such inhibitory agents
include antisense LRP-1 or LRP-2 nucleic acid molecules, anti-LRP-1
or LRP-2 antibodies, and LRP-1 or LRP-2 inhibitors. These modulator
methods can be performed in vitro (e.g., by culturing the cell with
the agent) or, alternatively, in vivo (e.g., by administering the
agent to a subject). As such, the present invention provides
methods of treating an individual afflicted with a disease or
disorder characterized by aberrant or unwanted expression or
activity of an LRP-1 or LRP-2 protein or nucleic acid molecule. In
one embodiment, the method involves administering an agent (e.g.,
an agent identified by a screening assay described herein), or
combination of agents that modulates (e.g., up regulates or down
regulates) LRP-1 or LRP-2 expression or activity. In another
embodiment, the method involves administering an LRP-1 or LRP-2
protein or nucleic acid molecule as therapy to compensate for
reduced, aberrant, or unwanted LRP-1 or LRP-2 expression or
activity.
[0147] Stimulation of LRP-1 or LRP-2 activity is desirable in
situations in which LRP-1 or LRP-2 is abnormally downregulated or
in which increased LRP-1 or LRP-2 activity is likely to have a
beneficial effect. For example, stimulation of LRP-1 or LRP-2
activity is desirable in situations in which an LRP-1 or LRP-2 is
downregulated or in which increased LRP-1 or LRP-2 activity is
likely to have a beneficial effect. Likewise, inhibition of LRP-1
or LRP-2 activity is desirable in situations in which LRP-1 or
LRP-2 is abnormally upregulated or in which decreased LRP-1 or
LRP-2 activity is likely to have a beneficial effect.
Pharmacogenomics
[0148] The LRP-1 or LRP-2 molecules, as well as agents, or
modulators which have a stimulatory or inhibitory effect on LRP-1
or LRP-2 activity (e.g., LRP-1 or LRP-2 gene expression) as
identified by a screening assay described herein can be
administered to individuals to treat (prophylactically or
therapeutically) LRP-1 or LRP-2 associated disorders (e.g., bone
disorders such as osteoporosis, Paget's disease, and osteogenesis
imperfecta) associated with aberrant or unwanted LRP-1 or LRP-2
activity. In conjunction with such treatment, pharmacogenomics
(i.e., the study of the relationship between an individual's
genotype and that individual's response to a foreign compound or
drug) can be considered. Differences in metabolism of therapeutics
can lead to severe toxicity or therapeutic failure by altering the
relation between dose and blood concentration of the
pharmacologically active drug. Thus, a physician or clinician can
consider applying knowledge obtained in relevant pharmacogenomics
studies in determining whether to administer an LRP-1 or LRP-2
molecule or LRP-1 or LRP-2 modulator as well as tailoring the
dosage or therapeutic regimen of treatment with an LRP-1 or LRP-2
molecule or LRP-1 or LRP-2 modulator.
[0149] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, for
example Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol.
23:983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43:254-266.
In general, two types of pharmacogenetic conditions can be
differentiated. Genetic conditions transmitted as a single factor
alternating the way drugs act on the body (altered drug action) or
genetic conditions transmitted as single factors altering the way
the body acts on drugs (altered drug metabolism). These
pharmacogenetic conditions can occur either as rare genetic defects
or as naturally-occurring polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarial,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0150] One pharmacogenomics approach to identifying genes that
predict drug response, known as "a genome-wide association," relies
primarily on a high-resolution map of the human genome consisting
of already known gene-related markers (e.g., a "bi-allelic" gene
marker map which consists of 60,000-100,000 polymorphic or variable
sites on the human genome, each of which has two variants.) Such a
high-resolution genetic map can be compared to a map of the genome
of each of a statistically significant number of patients taking
part in a Phase II/III drug trial to identify markers associated
with a particular observed drug response or side effect.
Alternatively, such a high resolution map can be generated from a
combination of some ten-million known single nucleotide
polymorphisms (SNPs) in the human genome. As used herein, a "SNP"
is a common alteration that occurs in a single nucleotide base in a
stretch of DNA. For example, a SNP can occur once per every 1000
bases of DNA. A SNP can be involved in a disease process, however,
the vast majority is likely not associated with diseases. Given a
genetic map based on the occurrence of such SNPs, individuals can
be grouped into genetic categories depending on a particular
pattern of SNPs in their individual genome. In such a manner,
treatment regimens can be tailored to groups of genetically similar
individuals, taking into account traits that can be common among
such genetically similar individuals.
[0151] Alternatively, a method termed the "candidate gene
approach," can be utilized to identify genes that predict drug
response. According to this method, if a gene that encodes a drug's
target is known (e.g., an LRP-1 or LRP-2 protein of the present
invention), all common variants of that gene can be fairly easily
identified in the population and it can be determined if having one
version of the gene versus another is associated with a particular
drug response.
[0152] Alternatively, a method termed the "gene expression
profiling," can be utilized to identify genes that predict drug
response. For example, the gene expression of an animal dosed with
a drug (e.g., an LRP-1 or LRP-2 molecular or LRP-1 or LRP-2
modulator of the present invention) can give an indication whether
gene pathways related to toxicity have been turned on.
[0153] Information generated from more than one of the above
pharmacogenomics approaches can be used to determine appropriate
dosage and treatment regimens for prophylactic or therapeutic
treatment of an individual. This knowledge, when applied to dosing
or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance therapeutic or prophylactic efficiency
when treating a subject with an LRP-1 or LRP-2 molecule or LRP-1 or
LRP-2 modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0154] The present invention further provides methods for
identifying new agents, or combinations, that are based on
identifying agents that modulate the activity of one or more of the
gene products encoded by one or more of the LRP-1 or LRP-2 genes of
the present invention, wherein these products can be associated
with resistance of the cells to a therapeutic agent. Specifically,
the activity of the proteins encoded by the LRP-1 or LRP-2 genes of
the present invention can be used as a basis for identifying agents
for overcoming agent resistance. By blocking the activity of one or
more of the resistance proteins, target cells, e.g., human cells,
will become sensitive to treatment with an agent that the
unmodified target cells were resistant to.
[0155] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of an LRP-1 or LRP-2 protein can be applied
in clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to increase
LRP-1 or LRP-2 gene expression, protein levels, or upregulate LRP-1
or LRP-2 activity, can be monitored in clinical trials of subjects
exhibiting decreased LRP-1 or LRP-2 gene expression, protein
levels, or downregulated LRP-1 or LRP-2 activity. Alternatively,
the effectiveness of an agent determined by a screening assay to
decrease LRP-1 or LRP-2 gene expression, protein levels, or
downregulated LRP-1 or LRP-2 activity, can be monitored in clinical
trials of subjects exhibiting increased LRP-1 or LRP-2 gene
expression, protein levels, or upregulated LRP-1 or LRP-2 activity.
In such clinical trials, the expression or activity of an LRP-1 or
LRP-2 gene, and preferably, other genes that have been implicated
in, for example, an LRP-1 or LRP-2-associated disorder can be used
as a "read out" or markers of the phenotype of a particular
cell.
[0156] Each of the novel compounds of this invention can be used as
a bio-active component for use in bone implants, transplants,
prostheses or the like. They can also be used as either
neutraceuticals or pharmaceuticals, or both, especially when
clinical management of a bone condition requires both nutritional
and pharmaceutical intervention.
[0157] The methods described above can be used alone or in
combination for diagnosing bone diseases, monitoring bone growth
and development, or following the course of healing and recovery
from bone diseases.
[0158] This invention is also based on the unexpected discovery
that lactoferrin interacts with LRP-1 or LRP-2 and induces
phosphorylation of MAP kinase in bone or cartilage cells. The
interaction between lactoferrin and LRP-1, LRP-2, or MAP kinase in
bone or cartilage cells can be monitored in diagnosing and treating
bone or cartilage conditions, and identifying therapeutic compounds
for treating such conditions.
[0159] A diagnostic method of the invention involves comparing the
level of interaction between a lactoferrin polypeptide and an LRP-1
protein, an LRP-2 protein, or a p42/44 MAP kinase in a sample
(e.g., a bone or cartilage tissue sample) prepared from a test
subject with that in a sample prepared from a normal subject, i.e.,
a subject who does not suffer from a bone or cartilage condition.
Such interaction can be determined, e.g., by measuring binding of
lactoferrin to LRP-1 or LRP-2, endocytosis of lactoferrin mediated
by LRP-1 or LRP-2, or phosphorylation of p42/44 MAP kinase, or by
any other methods known in the art. A higher or lower level of
interaction between lactoferrin and LRP-1, LRP-2, or p42/44 MAP
kinase indicates that the test subject is suffering from or at risk
for developing a bone or cartilage condition. This method can be
used on its own or in conjunction with other procedures to diagnose
bone or cartilage conditions.
[0160] The invention also provides a method for identifying and
manufacturing compounds (e.g., proteins, peptides, peptidomimetics,
peptoids, antibodies, or small molecules) that modulate (increase
or decrease) interaction between lactoferrin and LRP-1, LRP-2, or
p42/44 MAP kinase in a cell (e.g., a bone or cartilage cell).
Compounds thus identified can be used, e.g., for preventing and
treating bone or cartilage conditions.
[0161] The candidate compounds of the present invention can be
obtained using any of the numerous approaches described above. To
identify compounds that modulate interaction between lactoferrin
and LRP-1, LRP-2, or p42/44 MAP kinase, a system (a cell system or
a cell-free system) containing lactoferrin and LRP-1, LRP-2, or
p42/44 MAP kinase is contacted with a candidate compound and the
level of interaction between lactoferrin and LRP-1, LRP-2, or
p42/44 MAP kinase is evaluated relative to that in the absence of
the candidate compound. In a cell system, e.g., a system containing
bone or cartilage cells, the cells can be ones that naturally
express lactoferrin and LRP-1, LRP-2, or p42/44 MAP kinase, or ones
that are modified to express recombinant lactoferrin and LRP-1,
LRP-2, or p42/44 MAP kinase, for example, having lactoferrin and
LRP-1, LRP-2, or p42/44 MAP kinase genes fused to marker genes. The
level of interaction between lactoferrin and LRP-1, LRP-2, or
p42/44 MAP kinase, or the level of interaction between marker
proteins, can be determined by methods described above and any
other methods known in the art. If the level of interaction between
lactoferrin and LRP-1, LRP-2, or p42/44 MAP kinase, or the level of
interaction between marker proteins is higher or lower in the
presence of the candidate compound than that in the absence of the
candidate compound, the candidate compound is identified as being
useful for preventing and treating bone or cartilage
conditions.
[0162] This invention further provides a method for treating a bone
or cartilage condition by administering to a subject in need
thereof an effective amount of a composition, e.g., containing an
agonist or an antagonist of LRP-1, LRP-2, or p42/44 MAP kinase. An
"effective amount" is an amount of the composition that is capable
of producing a medically desirable result (e.g., an increased or
decreased level of interaction between lactoferrin and LRP-1,
LRP-2, or p42/44 MAP kinase) in a treated subject. The term
"treating" is defined as administration of a composition to a
subject, who has a bone or cartilage condition, with the purpose to
cure, alleviate, relieve, remedy, prevent or ameliorate the
condition, the symptom, of the condition, the disease state
secondary to the condition, or the predisposition toward the
condition. A subject can be a person who suffers from a bone or
cartilage condition, who has positive markers but no clinical
evidence of the condition, or who is at risk of the condition for
genetic (e.g., familial), habitual (e.g., smoking), or
environmental (e.g., radon) reasons. Examples of such bone
conditions include, but are not limited to, osteoporosis,
rheumatoid, hepatic osteodystrophy, osteomalacia, rickets, osteitis
fibrosa cystica, renal osteodystrophy, osteosclerosis, osteopenia,
fibrogenesis-imperfecta ossium, secondary hyperparathyrodism,
hypoparathyroidism, hyperparathyroidism, chronic renal disease,
sarcoidosis, glucocorticoid-induced osteoporosis, idiopathic
hypercalcemia, Paget's disease, and osteogenesis imperfecta, bone
transplants, non-healing fractures and bone defects. The
compositions can also be used for stimulating chondrocytes, with
the potential of cartilage repair, to treat osteoarthritis and
other arthritides and facilitate autologous bone and cartilage
transplants in humans, race horses and other animals. Thus, the
composition offers the benefit of concurrent healing of bone and
cartilage injuries.
[0163] Subjects to be treated can be identified, for example, by
determining the level of interaction between lactoferrin and LRP-1,
LRP-2, or p42/44 MAP kinase in a sample prepared from a subject by
methods described above. If the level of such interaction is higher
or lower in the sample from the subject than that in a sample from
a normal subject, the subject is a candidate for treatment with an
effective amount of a compound that decreases or increases the
level of interaction between lactoferrin and LRP-1, LRP-2, or
p42/44 MAP kinase in the subject. The treatment method can be
performed in vivo or ex vivo, alone or in conjunction with other
drugs or therapy.
[0164] In one in vivo approach, a therapeutic composition (e.g., a
composition containing a compound that modulates interaction
between lactoferrin and LRP-1, LRP-2, or p42/44 MAP kinase in a
cell) is administered to the subject. Generally, the compound will
be suspended in pharmaceutically-acceptable carrier (e.g.,
physiological saline) and administered orally or by intravenous
infusion, or injected or implanted subcutaneously, intramuscularly,
intrathecally, intraperitoneally, intrarectally, intravaginally,
intranasally, intragastrically, intratracheally, or
intrapulmonarily. For prevention and treatment of bone or cartilage
conditions, the compound can be delivered directly to a bone or
cartilage lesion or to bone or cartilage cells.
[0165] The dosage required depends on the choice of the route of
administration; the nature of the formulation; the nature of the
subject's illness; the subject's size, weight, surface area, age,
and sex; other drugs being administered; and the judgment of the
attending physician. Suitable dosages are in the range of
0.01-100.0 mg/kg. Wide variations in the needed dosage are to be
expected in view of the variety of compounds available and the
different efficiencies of various routes of administration. For
example, oral administration would be expected to require higher
dosages than administration by intravenous injection. Variations in
these dosage levels can be adjusted using standard empirical
routines for optimization as is well understood in the art.
Encapsulation of the compound in a suitable delivery vehicle (e.g.,
polymeric microparticles or implantable devices) may increase the
efficiency of delivery, particularly for oral delivery.
[0166] Alternatively, a polynucleotide containing a nucleic acid
sequence encoding a lactoferrin polypeptide can be delivered to the
subject, for example, by the use of polymeric, biodegradable
microparticles or microcapsule delivery devices known in the
art.
[0167] Another way to achieve uptake of the nucleic acid is using
liposomes, prepared by standard methods. The vectors can be
incorporated alone into these delivery vehicles or co-incorporated
with tissue-specific antibodies. Alternatively, one can prepare a
molecular conjugate composed of a plasmid or other vector attached
to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine
binds to a ligand that can bind to a receptor on target cells
(Cristiano et al. (1995) J. Mol. Med. 73, 479). Alternatively,
tissue specific targeting can be achieved by the use of
tissue-specific transcriptional regulatory elements (TRE) which are
known in the art. Delivery of "naked DNA" (i.e., without a delivery
vehicle) to an intramuscular, intradermal, or subcutaneous site is
another means to achieve in vivo expression.
[0168] In the relevant polynucleotides (e.g., expression vectors),
the nucleic acid sequence encoding a lactoferrin polypeptide is
operatively linked to a promoter or enhancer-promoter combination.
Enhancers provide expression specificity in terms of time,
location, and level. Unlike a promoter, an enhancer can function
when located at variable distance from the transcription initiation
site, provided a promoter is present. An enhancer can also be
located downstream of the transcription initiation site.
[0169] Suitable expression vectors include plasmids and viral
vectors such as herpes viruses, retroviruses, vaccinia viruses,
attenuated vaccinia viruses, canary pox viruses, adenoviruses and
adeno-associated viruses, among others.
[0170] Polynucleotides can be administered in a pharmaceutically
acceptable carrier. As is well known in the medical arts, the
dosage will vary. A preferred dosage for administration of
polynucleotide is from approximately 10.sup.6 to 10.sup.12 copies
of the polynucleotide molecule. This dose can be repeatedly
administered, as needed. Routes of administration can be any of
those listed above.
[0171] An ex vivo strategy for treating subjects with bone or
cartilage conditions can involve transfecting or transducing cells
obtained from the subject with a polynucleotide encoding a
lactoferrin protein. Alternatively, a cell can be transfected in
vitro with a vector designed to insert, by homologous
recombination, a new, active promoter upstream of the transcription
start site of the naturally occurring endogenous lactoferrin gene
in the cell's genome. Such methods, which "switch on" an otherwise
largely silent gene, are well known in the art. After selection and
expansion of a cell that expresses lactoferrin at a desired level,
the transfected or transduced cells are then returned to the
subject. The cells can be any of a wide range of types including,
without limitation, bone marrow stromal cells, osteoblasts,
chondrocytes, or their precursors. Such cells act as a source of
lactoferrin for as long as they survive in the subject.
[0172] The ex vivo methods include the steps of harvesting cells
from a subject, culturing the cells, transducing them with an
expression vector, and maintaining the cells under conditions
suitable for expression of the lactoferrin gene. These methods are
known in the art of molecular biology. The transduction step is
accomplished by any standard means used for ex vivo gene therapy,
including calcium phosphate, lipofectin, electroporation, viral
infection, and biolistic gene transfer. Alternatively, liposomes or
polymeric microparticles can be used. Cells that have been
successfully transduced can then be selected, for example, for
expression of the lactoferrin gene. The cells may then be injected
or implanted into the subject.
[0173] The specific examples below are to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present invention to its fullest extent. All
publications recited herein are hereby incorporated by reference in
their entirety.
Primary Rat Osteoblast-Like Cell Culture
[0174] Osteoblasts were isolated from 20-day old fetal rat
calvariae as previously described (Cornish et al. (1999) Amer. J.
Physiol-Endocrinol. Metab. 277:E779-E783). Briefly, calvariae were
excised and the frontal and parietal bones, free of suture and
periosteal tissue, were collected. After washing, the calvariae
were treated twice with 3 mL of 1 mg/mL collagenase for 7 minutes
at 37.degree. C. After discarding the supernatants from these two
digestions, the calvariae were treated a further twice with 3 mL of
2 mg/mL collagenase (30 mins, 37.degree. C.). The supernatants of
the latter two digestions were pooled, centrifuged, and the cells
washed. Cells were grown to confluence and the subcultured into 24
well plates. Cells were growth arrested in minimum essential medium
(MEM)/0.1% bovine serum albumin for 24 h before harvest.
SaOS-2 Cell Line Culture
[0175] The human osteoblast-like osteosarcoma cell line SaOS-2 was
maintained in DMEM (GIBCO 12100-046) supplemented with 2.2 g/L
NaHCO.sub.3, 10% FBS, and 10 ml/L penicillin-streptomycin (GIBCO
15140-122). Cells were plated in 75 cm.sup.2 flasks and when
confluent used for total RNA isolation or proliferation assays.
Detection of LRP-1 and LRP-2 Gene Expression
[0176] LRP-1 and LRP-2 gene expression was identified by reverse
transcription polymerase chain reaction (RT-PCR). Total cellular
RNA was purified from cultured primary rat osteoblast cells and
SaOS-2 cells by a modified method of single-step guanidinium
thiocyanate-phenol-chloroform RNA extraction (Chomczynski and
Sacchi (1987) Analyt. Biochem. 162:156-159; and Grey et al. (2001)
Endocrinology 142:1098-1106). RNA concentration and purity were
determined by measuring the optical density using a Gene Quant.TM.
spectrophotometer (Pharmacia, Little Chalfont, UK) and the quality
was determined by electrophoresis on a 1% agarose gel. RNA was
treated with DNase and RT-PCR amplifications were carried out
following the previously published protocol (Grey et al. (2001)
Endocrinology 142:1098-1106). PCR was performed in an automatic DNA
thermal cycler (Mastercycler Personal, Eppendorf, Hamburg,
Germany). After an initial denaturation step of 2 min at 94.degree.
C., 35 cycles of denaturing at 94.degree. C. for 30 seconds,
annealing at 56.degree. C. for 30 seconds, and extension at
72.degree. C. for 1 minute were performed. At the end of each set
of cycles there was a final extension step at 72.degree. C. for 15
minutes. PCR reaction products were visualized on a 1% TBE agarose
gel. The primers used to amplify LRP-1 and LRP-2 were: LRP-2
primers 5' GAG TGT TCC GTG TAT GGC AC and 5' GAT GCC TTG GAT GAT
GGTC; LRP-2 primers 5' GTC ACC AGT TCA CTT GCT CC and 5' CCA CTC
CCA TAA CCA TTCC. PCR products were purified from agarose gels
using QIAquick gel extraction kit (Qiagen, Valencia, Calif.), and
their sequences were determined on an ABI 377 XL DNA Sequencer (PE
Biosystems, Foster City, Calif.). Control RT-PCR amplifications
were carried out without reverse transcriptase.
[0177] Unexpectedly, LRP-1 was found to be expressed in both
primary rat osteoblasts and SaOS-2 cells. LRP-2 was found to be
expressed in primary rat osteoblasts but not in SaOS-2 cells.
Lactoferrin has been identified as an osteoblast growth factor
present in fractionated bovine milk. It stimulates proliferation of
cultured osteoblastic cells of rat, mouse, and human origin
potently and in a dose-dependent manner. Lactoferrin is also
mitogenic to chondrocytes, and is capable of inducing osteoblast
differentiation as well. It was found to significantly increase the
number of mineralized bone nodules in long-term osteoblast
cultures. Lactoferrin also inhibits apoptosis induced by serum
withdrawal in primary rat osteoblasts. Lactoferrin strongly
inhibits osteoclastogenesis in a murine bone marrow culture assay,
but does not affect the bone-resorbing activity of mature
osteoclasts. The ability of lactoferrin to induce osteoblast
anabolism and inhibit osteoclast development in vitro suggests that
it may be anabolic to bone in vivo. Indeed, when lactoferrin was
administered to adult mice by unilateral local hemicalvarial
injection, there were dose-dependent and substantial increases in
indices of bone formation and bone area.
Interactions Between Lactoferrin and LRP-1, LRP-2, and p42/44 MAP
Kinase
[0178] Lactoferrin has been found to bind two endocytic members of
the low-density lipoprotein receptor family, LRP-1 and
LRP-2/megalin. LRP-1 and LRP-2 are both expressed in rodent
osteoblastic cells, but only LRP-1 is expressed in SaOS-2 cells. It
was observed under confocal microscopy that lactoferrin is
endocytosed by primary rat osteoblastic cells, and that the
LRP-1/2-specific inhibitor, receptor-associated protein (RAP),
abrogates this process. Further, lactoferrin activates the p42/44
MAP kinase signaling pathway in osteoblastic cells. It was found
that lactoferrin-induced osteoblast proliferation is inhibited by
specific inhibitors of MAP kinase. Lactoferrin-induced osteoblast
proliferation and MAP kinase phosphorylation are also blocked by
RAP, implicating LRP-1/2 as mediators of the mitogenic effects of
lactoferrin in osteoblasts. Moreover, lactoferrin induces
proliferation in SaOS-2 cells. Lactoferrin-induced proliferation in
LRP-1-null fibroblasts is substantially less marked than that
observed in LRP-1+/+cells. Taken together, these data demonstrate
that lactoferrin is anabolic to bone, and that its growth-factor
like effects on osteoblastic cells are mediated in a large part by
LRP-1. This work provides further evidence for an important role of
the LRP receptor family in regulation of bone growth.
[0179] The three dimensional structure of lactoferrin has revealed
that it is subdivided into an N-lobe and a C-lobe, each binding one
atom of iron. The N-lobe has been shown to have mitogenic effect
near comparable to that of the full-length protein, suggesting that
shorter polypeptides are potentially therapeutic. There is also
possibility that, as lactoferrin is slowly broken down, latent
activities in either lobe may emerge. Lactoferricin, a short
N-terminal peptide of lactoferrin that has been shown to be
effective in bacterial killing, had only a modest osteogenic
effect. Transferrin also was not a potent mitogen on osteoblasts.
When lactoferrin was stripped of iron and re-loaded with chromium
or manganese ions, the osteogenic activity was similar to the
iron-loaded molecule, implying that the iron itself is not crucial
for the mitogenic activity of lactoferrin in osteoblasts.
OTHER EMBODIMENTS
[0180] All of the features disclosed in this specification can be
combined in any combination. Each feature disclosed in this
specification can be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0181] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the scope of the following claims.
* * * * *