U.S. patent application number 12/093437 was filed with the patent office on 2009-09-17 for fgf2-related methods for diagnosing and treating depression.
This patent application is currently assigned to Prtzker Neuropsychiatric Research Fund LLC. Invention is credited to Devin Absher, Huda Akil, Rene Bernard, Michael Boehnke, William E. Bunney, JR., Margit Burmeister, Prabhakara V. Choudary, Simon J. Evans, Edward G. Jones, Ilan Kerman, Jun Li, Fan Meng, Richard M. Myers, Alan F. Schatzberg, Laura J. Scott, Robert C. Thompson, Hiroaki Tomita, Cortney Turner, Marquis P. Vawter, Stanley J. Watson.
Application Number | 20090233842 12/093437 |
Document ID | / |
Family ID | 38049212 |
Filed Date | 2009-09-17 |
United States Patent
Application |
20090233842 |
Kind Code |
A1 |
Akil; Huda ; et al. |
September 17, 2009 |
FGF2-RELATED METHODS FOR DIAGNOSING AND TREATING DEPRESSION
Abstract
The present application relates to the treatment and diagnosis
of mood disorders, including bipolar disorder, major depression
disorder and schizophrenia. The invention provides novel diagnostic
markers and assays, as well as research tools for the development
and discovery of agents and compounds which are useful for treating
patients who suffer from mental illness.
Inventors: |
Akil; Huda; (Ann Arbor,
MI) ; Watson; Stanley J.; (Ann Arbor, MI) ;
Evans; Simon J.; (Milan, MI) ; Turner; Cortney;
(Belleville, MI) ; Bernard; Rene; (Ann Arbor,
MI) ; Kerman; Ilan; (Ann Arbor, MI) ;
Thompson; Robert C.; (Ann Arbor, MI) ; Burmeister;
Margit; (Ann Arbor, MI) ; Scott; Laura J.;
(Ann Arbor, MI) ; Meng; Fan; (Ann Arbor, MI)
; Boehnke; Michael; (Ann Arbor, MI) ; Bunney, JR.;
William E.; (Laguna Hills, CA) ; Vawter; Marquis
P.; (Laguna Nigel, CA) ; Jones; Edward G.;
(Winters, CA) ; Choudary; Prabhakara V.; (Davis,
CA) ; Myers; Richard M.; (Stanford, CA) ;
Schatzberg; Alan F.; (Los Altos, CA) ; Li; Jun;
(Palo Alto, CA) ; Absher; Devin; (San Francisco,
CA) ; Tomita; Hiroaki; (Irvine, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Prtzker Neuropsychiatric Research
Fund LLC
Chicago
IL
|
Family ID: |
38049212 |
Appl. No.: |
12/093437 |
Filed: |
November 13, 2006 |
PCT Filed: |
November 13, 2006 |
PCT NO: |
PCT/US06/44057 |
371 Date: |
April 8, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60736526 |
Nov 12, 2005 |
|
|
|
60829516 |
Oct 13, 2006 |
|
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|
Current U.S.
Class: |
514/1.1 ;
435/6.16 |
Current CPC
Class: |
A61K 38/1825 20130101;
A61P 25/18 20180101; C12Q 2600/106 20130101; C12Q 2600/158
20130101; A61K 38/03 20130101; A61P 43/00 20180101; A61P 25/22
20180101; A61K 38/1774 20130101; G01N 33/5023 20130101; A61K 38/10
20130101; C12Q 1/6883 20130101; A61P 25/24 20180101; G01N 2800/304
20130101; C12Q 2600/136 20130101; A61P 25/30 20180101 |
Class at
Publication: |
514/2 ;
435/6 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C12Q 1/68 20060101 C12Q001/68 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Certain embodiments of the invention described herein were
developed with the support of the United States government (Conte
Center grant (NIMH) L99 MH60398). Accordingly, the U.S. government
may have rights to certain embodiments of the invention.
Claims
1. A method for facilitating the diagnosis of a mood disorder in a
subject, comprising the steps of: (i) measuring the level of
expression of a gene, wherein said gene is selected from the group
consisting of the genes listed in FIG. 5, FIG. 6, FIG. 7, FIG. 8;
Table 2B, Table 3, and Table 4; (ii) determining whether said gene
is dysregulated relative to a control, wherein dysregulation of
said gene indicates an increased likelihood that said subject
suffers from a mood disorder; and (iii) recording or reporting any
finding with respect to said increased likelihood.
2. The method of claim 1, wherein said mood disorder is bipolar
disorder, and said gene is selected from the group consisting of
the genes listed in FIG. 5, FIG. 7, FIG. 8, Table 2B, and Table
4.
3. The method of claim 1, wherein said mood disorder is major
depression disorder and said gene is selected from the group
consisting of the genes listed in FIG. 6, FIG. 8, Table 1 and Table
3.
4. The method of claim 1, wherein said gene dysregulation occurs in
said subject's brain tissue.
5. (canceled)
6. The method of claim 1, wherein said gene expression is assayed
by detecting messenger RNA transcribed from said gene.
7. The method of claim 1, wherein said gene expression is assayed
by selectively detecting the protein product of said gene.
8. (canceled)
9. The method of claim 1, wherein the level of expression of said
gene is higher than a level associated with humans without a mood
disorder.
10. The method of claim 1, wherein the level of expression of said
gene is lower than a level associated with humans without a mood
disorder.
11. The method of claim 1, wherein the level of expression of said
gene is detected using a microarray assay, and wherein said gene is
one of at least two genes on the microarray.
12. The method of claim 1, wherein said gene is selected from Table
1 and wherein said mood disorder is suicidal.
13. The method of claim 12, wherein said subject was previously
diagnosed with a mood disorder associated with an increased
likelihood of suicidal activity.
14. The method of claim 12, wherein said subject was previously
diagnosed with a mood disorder selected from the group consisting
of major depression, bipolar disorder, and schizophrenia.
15. The method of claim 12, further comprising prescribing a
treatment for said subject which reduces the likelihood of a
suicide attempt by said subject.
16. The method of claim 1, wherein said subject has symptoms of
both bipolar disorder and major depressive disorder, and wherein
said gene is differently expressed in bipolar subjects versus major
depression disorder subjects, thereby facilitating a diagnosis of
bipolar disorder or major depressive disorder in said subject.
17. The method of claim 1, wherein said gene is dysregulated in
substance-abusing MDD subjects versus MDD subjects who are not
substance abusers, and wherein said gene is selected from the
dysregulated genes listed in Table 1C, thereby facilitating a
diagnosis of MDD versus MDD coupled with substance abuse.
18. (canceled)
19. (canceled)
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. A method of treating a subject prone to suicide, the method
comprising the step of administering to the subject a
therapeutically effective amount of a polypeptide, the polypeptide
encoded by a polynucleotide corresponding to a gene listed in Table
1 or Table 2.
25. A method of treating symptoms of anxiety in a subject,
comprising the step of administering a sufficient amount of FGF2
peptide to the subject after the subject has been diagnosed with
anxiety or an illness associated with anxiety.
26. The method of claim 25, wherein said subject is a human.
27. The method of claim 25, wherein said sufficient amount is a
dose administered at least twice weekly over a period at least one
week in length.
28. The method of claim 25, wherein said illness is Major
Depression or Major Depressive Disorder.
29. A method for identifying a human suffering from chronic stress
comprising a) obtaining a nucleic acid sample from the subject; and
b) determining the exon IIIb:IIIc splice variant ratio of the
expressed gene selected from the group consisting of FGFR2 and
FGFR3, wherein a ratio less than approximately 10 is associated
with an increased likelihood that said human is suffering from
chronic stress.
30. The method of claim 29, wherein said gene is FGFR2.
31. The method of claim 29, wherein said gene is FGFR3.
32. The method of claim 29, wherein said method further comprises
administering a pharmacological treatment in response to said
identification.
33. (canceled)
34. (canceled)
35. (canceled)
36. A method for detecting global glial alterations in a subject
suffering from MDD, comprising the steps of determining the level
of gene expression in the LC region of a subject, wherein said gene
is a glial marker gene selected from the group of glial marker
genes in Table 3.
37. A method for distinguishing between BP and MDD in a human
subject, comprising a) measuring the level of expression of at
least one MDD- or BP-specific gene in DR tissue of said subject,
wherein said MDD- or BP-specific gene is selected from the MDD- or
BP-specific dysregulated genes listed in Table 4; b) and
identifying an increased likelihood that said subject suffers from
BP versus MDD, wherein downregulation of an MDD-specific gene in
Table 4 correlates with an increased likelihood of MDD in said
subject, and wherein downregulation of a BP-specific gene in Table
4 correlates with an increased likelihood of BP in said
subject.
38. A method for identifying a human subject with an increased risk
of BP or MDD, comprising: (i) measuring the level of expression of
a dysregulated gene selected from the genes listed in Table 3; and
(ii) correlating said measurement with an increased risk of BP or
MDD in said subject.
39. The method of claim 38, further comprising recording or
reporting said risk of developing BP or MDD.
40. The method of claim 38, wherein said risk of developing bipolar
disorder is reported to a physician or said human subject.
41. A method for facilitating the diagnosis of major depression
disorder in a subject, comprising the steps of: (i) measuring the
ratio of expression of FGFR2 exon 5 to FGFR2 exon 11; (ii)
determining whether said ratio is lower than a control, wherein a
lower ratio indicates an increased likelihood that said subject
suffers from major depression disorder; and (iii) recording or
reporting any finding with respect to said increased
likelihood.
42. The method of claim 41, wherein said expression is in said
subject's dorsolatereral prefrontal cortex.
43. A method for facilitating the diagnosis of major depression
disorder in a subject, comprising the steps of: (i) measuring the
expression of FGFR2 exon 9; (ii) determining whether said
expression is lower than a control, wherein a lower level of
expression indicates an increased likelihood that said subject
suffers from major depression disorder; and (iii) recording or
reporting any finding with respect to said increased
likelihood.
44. The method of claim 43, wherein said expression is in said
subject's dorsolatereral prefrontal cortex.
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/736,526, filed Nov. 12, 2005, and
U.S. Provisional Patent Application Ser. No. 60/829,516, filed Oct.
13, 2006.
BACKGROUND OF THE INVENTION
[0003] Clinical depression, including both bipolar disorders and
major depression disorders, is a major public health problem,
affecting an estimated 9.5% of the adult population of the United
States each year. While it has been hypothesized that mental
illness, including mood disorders such as major depression ("MDD")
and bipolar disorder ("BP") as well as psychotic disorders such as
schizophrenia, may have genetic roots, little progress has been
made in identifying gene sequences and gene products that play a
role in causing these disorders, as is true for many diseases with
a complex genetic origin (see, e.g., Burmeister, Biol. Psychiatiy
45:522-532 (1999)).
[0004] The current lack of biomarkers and the ineffectiveness and
reliability of the diagnosis and rates are important issues for the
treatment of mental disorders. For example, around 15% of the
population suffers from MDD while approximately 1% suffers from BP
disorders. Diagnosing bipolar disorder is difficult when, as
sometimes occurs, the patient presents only symptoms of depression
to the clinician. At least 10-15% of BP patients are reported to be
misdiagnosed as MDD. The consequences of such misdiagnosis include
a delay in being introduced to efficacious treatment with mood
stabilizers and a delay in seeking or obtaining counseling specific
to bipolar disorder. Also treatment with antidepressants alone
induces rapid cycling, switching to manic or mixed state, and
consequently increases the risk of suicide. Furthermore, in
addition to a lack of efficacy, long onset of action and side
effects (sexual, sleep, weight gain, etc.), there are recent
concerns relating to the undesirable effects of antidepressants on
metabolic syndromes, such as diabetes and hypercholesteremia.
[0005] Clearly, there is a need for methods of obtaining accurate
and objective information about the physiological and/or genetic
status of depressed or potentially suicidal patients, particularly
as the patient's physiological and/or genetic status relates to the
likely response of the patient to a particular treatment
regimen.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention provides novel assays for the
diagnosis and detection of various mental illnesses. The assays,
which include assays for detecting the level of expression of
various genes associated with mental disorders, allow the
practitioner to obtain a more accurate diagnosis of mental illness
in a subject, and allow the practitioner to distinguish between
various mental illnesses and associated pathologies. The invention
also provides compositions useful for practicing the methods of
invention, and for developing further diagnostic methodologies, as
well as new therapeutics, to aid in the treatment of mental
illness.
[0007] In one embodiment, the present invention provides methods of
correlating the expression of FGFR2 splice variants with MDD.
Because of the relationship between MDD, BP and psychotic disorders
such as schizoaffective disorders or psychotic depression, the
splice variants described herein are unique to MDD and can be used
for differential diagnosis, treatment and prevention of MDD.
[0008] In another embodiment, the present invention provides
methods for altering the behavioral profiles of rats by injecting
the rats with FGF2, an NCAM peptide mimetic, and a peptide
inhibitor of FGF receptors. Both FGF2 and the NCAM peptide mimetic
have antidepressant-like effects in the forced swim test when
injected intracerebroventricularly. The description and examples
presented herein show that the presence of a peptide inhibitor
reverses the effect. In one embodiment of the invention, ligands
that activate FGF receptors are used for their antidepressant
effects in the therapeutic treatment of individuals with MDD.
[0009] In another embodiment, the invention provides a set of genes
associated with suicide in the amygdala and methods for correlating
expression of one or more of those genes with suicidal tendencies
(Table 1A and Table 1B). In a related embodiment, the invention
provides a set of genes associated with suicide, co-morbidity with
substance abuse in MDD patients (Table 1C) and methods of detecting
one or more of those genes, correlating those genes with suicide
risk in appropriate patients, and methods of treating individuals
identified as suicidal or likely to become suicidal.
[0010] In another embodiment, the invention also provides methods
of correlating the differential expression of particular
lithium-responsive genes (Table 2A and Table 2B) with bipolar
affective disorder.
[0011] In yet other embodiment, the invention provides methods for
increasing the memory and learning abilities of adult animals by
treating early postnatal animals with FGF2. In yet another
embodiment, the invention provides methods for treating memory and
learning disabilities in animals deficient in active FGF2 by
treating early postnatal animals with FGF2.
[0012] In another embodiment, the invention provides a method for
facilitating the diagnosis of a mood disorder in a subject,
comprising the steps of: (i) measuring the level of expression of a
gene, wherein the gene is selected from the group consisting of the
genes listed in FIG. 5, FIG. 6, FIG. 7, FIG. 8; Table 2B, Table 3,
and/or Table 4; (ii) determining whether the gene is dysregulated
relative to a control, wherein dysregulation of the gene indicates
an increased likelihood that the subject suffers from a mood
disorder; and (iii) recording or reporting any finding with respect
to the increased likelihood, i.e., reporting whether there is or is
not an increased likelihood that the subject suffers from a mood
disorder.
[0013] In a related embodiment, the mood disorder in question is
bipolar disorder, and the gene whose dysregulation is analyzed is
selected from the group consisting of the genes listed in FIG. 5,
FIG. 7, FIG. 8, Table 2B, and/or Table 4. In another related
embodiment, the mood disorder is major depression disorder and the
gene is selected from the group consisting of the genes listed in
FIG. 6, FIG. 8, Table 1 and/or Table 3.
[0014] In yet another related embodiment of the method for
facilitating the diagnosis of a mood disorder in a subject, the
gene dysregulation, which is detected and measured, occurs in the
subject's brain. In yet another related embodiment, the brain
tissue in which the dysregulation occurs is selected from the group
consisting of the locus coeruleus, the dorsal raphe, the anterior
cingulate cortex, the dorsolateral prefrontal cortex, the
hippocampus, and the amygdala. In yet another related embodiment,
the gene dyrsegulation is detected in a cell in which the observed
gene expression reflects the gene expression observed in the brain,
e.g., a lymphocyte cell.
[0015] In yet another related embodiment of the method for
facilitating the diagnosis of a mood disorder in a subject, the
dysregulation of gene expression is assayed by detecting messenger
RNA transcribed from the gene or genes of interest. In yet another
related embodiment, gene expression is assayed by selectively
detecting, directly or indirectly, the protein product of the gene
or gene of interest. In yet another related embodiment, detecting
messenger RNA transcribed from the gene of interest comprises the
steps of (i) contacting said mRNA with a reagent which selectively
associates with said messenger RNA; and (ii) detecting the level of
said reagent which selectively associates with said mRNA.
[0016] In another related embodiment of the method for facilitating
the diagnosis of mood disorder in a subject, the measured level of
expression of the gene of interest is higher than a level
associated with humans without a mood disorder. In yet another
related embodiment, the level of expression of the gene is lower
than a level associated with humans without a mood disorder, i.e.,
the gene is downregulated in subjects with a mood disorder.
[0017] In another related embodiment of the method for facilitating
the diagnosis of mood disorder in a subject, the level of
expression of the gene is detected using a microarray assay, and
wherein said gene is one of at least two genes on the
microarray.
[0018] In other embodiment of the method for facilitating the
diagnosis of mood disorder in a subject, the gene is selected from
Table 1 and the mood disorder is suicidal. In a related embodiment,
the subject was previously diagnosed with a mood disorder
associated with an increased likelihood of suicidal activity. In
yet another related embodiment, the subject was previously
diagnosed with a mood disorder selected from the group consisting
of major depression, bipolar disorder, and schizophrenia. In yet
another related embodiment, the method further comprises
prescribing a treatment for the subject which reduces the
likelihood of a suicide attempt by the subject.
[0019] In other embodiment of the method for facilitating the
diagnosis of mood disorder in a subject, the subject has symptoms
of both bipolar disorder and major depressive disorder, and the
gene of interest is differently expressed in bipolar subjects
versus major depression disorder subjects, thereby facilitating a
diagnosis of bipolar disorder or major depressive disorder in said
subject.
[0020] In other embodiment of the method for facilitating the
diagnosis of mood disorder in a subject, the gene of interest is
dysregulated in substance-abusing MDD subjects versus MDD subjects
who are not substance abusers, and the gene of interest is selected
from the dysregulated genes listed in Table 1C, thereby
facilitating a diagnosis of MDD versus a diagnosis of MDD in
addition to substance abuse.
[0021] In another embodiment, the invention provides a method of
identifying a compound for treatment or prevention of a mood
disorder, the method comprising the steps of: (i) contacting the
compound with a polypeptide or polynucleotide corresponding to a
dysregulated gene selected from the group of dysregulated genes
listed in FIG. 5, FIG. 6, FIG. 7, FIG. 8; Table 2B, Table 3, and
Table 4; and (ii) determining the functional effect of the compound
upon the polypeptide or polynucleotide (e.g., inhibition or
enhancement of activity), thereby identifying a compound for
treatment or prevention of a mood disorder. In a related
embodiment, the contacting step is performed in vitro. In yet
another related embodiment, the polypeptide is expressed in a cell
and the cell is contacted with the compound. In yet another related
embodiment, the mood disorder is selected from the group consisting
of bipolar disorder, major depression disorder, suicidal, and
substance abuse comorbidity. In yet another related embodiment, the
method further comprising administering the identified or candidate
compound to an animal and determining the effect on the animal,
e.g., determining the effect on the animal's mental health and
behavioral phenotype.
[0022] In another embodiment, the invention also provides a method
of treating a subject who is prone to suicide, comprising the step
of administering to the subject a therapeutically effective amount
of a polypeptide, the polypeptide encoded by a polynucleotide
corresponding to a gene listed in Table 1 or Table 2.
[0023] In another embodiment, the invention also provides a method
of treating symptoms of anxiety in a subject (e.g., an animal such
as a mouse, cat, dog, horse or human), comprising the step of
administering a sufficient amount of FGF2 peptide to the subject
after the subject has been diagnosed with anxiety or an illness
associated with anxiety. In a related embodiment, the subject is a
human. In another related embodiment, the sufficient amount of FGF2
is a dose administered at least twice weekly over a period at least
one week in length. In yet another related embodiment, the illness
being treated is Major Depression or Major Depressive Disorder.
[0024] In another embodiment, the invention provides a method for
diagnosing a human suffering from chronic stress comprising a)
obtaining a nucleic acid sample from the subject; and b)
determining the exon IIIb:IIIc splice variant ratio of the
expressed gene selected from the group consisting of FGFR2 and
FGFR3, wherein a ratio less than approximately 10 is associated
with an increased likelihood that said human is suffering from
chronic stress. In a related embodiment, the gene is FGFR2. In
another related embodiment, the gene is FGFR3. In another related
embodiment, the method further comprises administering a
pharmacological treatment to a human diagnosed with chronic stress
using the method.
[0025] In another embodiment, the invention provides a method for
identifying a compound which alters the exon IIIb:IIIc splice
variant ratio of an expressed gene selected from the group
consisting of FGFR2 and FGFR3 in a living animal, comprising a)
identifying at least one animal suffering from chronic stress; b)
measuring the exon IIIb:IIIc splice variant ratio in said at least
one animal; c) administering a test compound to said at least one
animal; d) measuring the splice variant ratio a second time after
the administration of said test compound; recording the identity of
the test compound If said measurement shows that the splice variant
ratio is increased.
[0026] In another embodiment, the invention provides a method for
treating a subject suffering from a glutamatergic imbalance
comprising administering to the subject a sufficient amount of a
compound which targets a molecule selected from the group
consisting of: glial transporters, glutamine synthetase, AMPA,
kainate, GRM1 and GRM7.
[0027] In yet another embodiment, the invention provides a method
for increasing neurite growth in a subject suffering from MDD,
comprising administering to the subject a sufficient amount of a
compound which targets FGFR3, TrkB receptor, or a growth hormone
receptor, or which mimics the actions of FGF2.
[0028] In another embodiment, the invention provides a method for
detecting global glial alterations in a subject suffering from MDD,
comprising the steps of determining the level of gene expression in
the LC region of a subject, wherein at least one gene whose level
is examined is a glial marker gene selected from the group of glial
marker genes in Table 3.
[0029] In yet another embodiment, the invention provides a method
for distinguishing between BP and MDD in a human subject,
comprising a) measuring the level of expression of at least one
MDD- or BP-specific gene in DR tissue of said subject, wherein said
MDD- or BP-specific gene is selected from the MDD- or BP-specific
dysregulated genes listed in Table 4; b) and identifying an
increased likelihood that said subject suffers from BP versus MDD,
wherein downregulation of an MDD-specific gene in Table 4
correlates with an increased likelihood of MDD in said subject, and
wherein downregulation of a BP-specific gene in Table 4 correlates
with an increased likelihood of BP in said subject. In a related
embodiment, the method further comprises recording or reporting the
risk of developing BP or MDD. In a related embodiment, the risk is
reported to a physician or to the subject.
[0030] In yet another embodiment, the invention provides a method
for identifying a human subject with an increased risk of BP or
MDD, comprising: (i) measuring the level of expression of a
dysregulated gene selected from the genes listed in Table 3; and
(ii) correlating said measurement with an increased risk of BP or
MDD in said subject. In a related embodiment, the method further
comprises recording or reporting the risk of developing BP or MDD.
In a related embodiment, the risk is reported to a physician or to
the subject.
[0031] In yet another embodiment, the invention provides a method
for facilitating the diagnosis of major depression disorder in a
subject, comprising the steps of: (i) measuring the ratio of
expression of FGFR2 exon 5 to FGFR2 exon 11; (ii) determining
whether said ratio is lower than a control, wherein a lower ratio
indicates an increased likelihood that said subject suffers from
major depression disorder; and (iii) recording or reporting any
finding with respect to said increased likelihood. In a related
embodiment, the expression ratio is the ration in said subject's
dorsolatereral prefrontal cortex.
[0032] In another embodiment, the invention provides a method for
facilitating the diagnosis of major depression disorder in a
subject, comprising the steps of: (i) measuring the expression of
FGFR2 exon 9; (ii) determining whether said expression is lower
than a control, wherein a lower level of expression indicates an
increased likelihood that said subject suffers from major
depression disorder; and (iii) recording or reporting any finding
with respect to said increased likelihood. In a related embodiment,
the expression is in said subject's dorsolatereral prefrontal
cortex.
[0033] In another embodiment, the invention provides a method for
improving memory in an animal, comprising administering FGF2 to
said animal. In a related embodiment, the animal is a rodent, cat,
dog, horse, primate or human. In yet another related embodiment,
administration occurs within 48 hours of the birth of said animal.
In another related embodiment, the FGF2 is administered
subcutaneously. In yet another related embodiment, the FGF2 is
administered at 20 ng/g body weight. In yet another related
embodiment, the invention provides a non-human animal, e.g., a
rodent, which has been treated with FGF2. In a related embodiment,
the animal is an adult animal, previously treated with FGF2, with
an improved memory relative to an adult animal which was not
previously treated (e.g., treated shortly after birth). In other
related embodiments, the expression of NCAM in the FGF2-treated
animal is decreased relative to that observed in similar untreated
animals. In other related embodiments, the expression of at least
one gene selected from the group consisting of GAP-43, Rgs4, trkB,
CCK, SST and Vgf is increased in the FGF2-treated animals relative
to an untreated animal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows FGFR2 variant differences in Mood Disorders.
FGFR2 soluble receptor splice variants may represent a smaller
percentage of the total receptors in MDD than in controls.
[0035] FIG. 2 shows the effect of acute injection of FGF2 on mouse
depression and anxiety, as measured by mobility tests (top) and the
elevated plus maze (EPM) test (bottom), respectively. "Open,"
"center," and "closed" refer to time spent in the open, center and
closed parts of the EPM, respectively.
[0036] FIG. 3 shows the effects of injections of NCAM peptide on
mouse depression and anxiety, as measured by the climbing and
forced swim test (top) and the elevated plus maze (EPM) test
(bottom), respectively. "Open," "center," and "closed" refer to
time spent in the open, center and closed parts of the EPM,
respetively.
[0037] FIG. 4 shows the effects of injections of a peptide
inhibitor on mouse depression and anxiety, as measured by the
climbing and forced swim test (top) and the elevated plus maze
(EPM) test (bottom), respectively. "Open," "center," and "closed"
refer to time spent in the open, center and closed parts of the
EPM, respetively.
[0038] FIG. 5 shows a table listing genes in the cAMP signalling
pathway whose expression is significantly dysregulated in the
anterior cingulate cortex (AnCg) from patients with bipolor
disorder (BPD).
[0039] FIG. 6 shows a table listing genes in cAMP signalling
pathways whose expression is significantly dysregulated in the
anterior cingulate cortex (AnCg) of patients with major depression
disorder (MDD).
[0040] FIG. 7 shows a table listing genes in the
phosphatidylinositol signalling pathway whose expression is
significantly dysregulated in the anterior cingulate cortex (AnCg)
of patients with bipolar disorder (BPD).
[0041] FIG. 8 shows a table listing genes in the
phosphatidylinositol signalling pathway whose expression is
significantly dysregulated in the anterior cingulate cortex (AnCg)
of patients with major depression disorder (MDD).
[0042] FIG. 9 shows two charts which illustrate the effect of
chronic FGF-2 administration (5 ng/g, 3 weeks) on anxiety in
rodents with different, as measured by the time the rodents spend
in the light compartment of the test system. LR, animals with
intrinsic high anxiety; HR, animals with intrinsic low anxiety;
HRFGF-2, low anxiety animals administered FGF-2; LRFGF-2, high
anxiety animals administered FGF-2.
[0043] FIG. 10 shows the inverse relationship between FGF-2 gene
expression and anxiety behavior using the "open arms" test. CA-2,
hippocampus region CA-2.
[0044] FIG. 11, top, shows a schematic of the basic structure of
FGFR2 and FGFR3 aligned with the exons amplified and described in
the Example. Emphasis is placed on the IIIb/IIIc splice variants in
the C-terminus of the third Ig-like domain of both receptors (R2
and R3). Exon sequences for FGFR2 and FGFR3 are in no way identical
(see FIG. 11, bottom), but exon nomenclature was synchronized to
match each exon number to corresponding regions on both R2 and R3
protein structures. The truncated and cleaved isoforms of the FGF
receptors are excluded from the schematic. FIG. 11, bottom, shows
sequences of forward and reverse FGFR2 and FGFR3 primers designed
for real time RT-PCR quantitative analysis. Primers were optimized
and designed for maximum efficiency with differential detection for
IIIb/IIIc splice variants for both FGFR2 and FGFR3.
[0045] FIG. 12 shows two charts which illustrate the chronic
stress-induced decrease in the exon IIIc:IIIb splice variant
expression ratio in both FGFR2 (top panel) and FGFR3 (bottom
panel). V (vehicle); NS (non-stress); Chronic stress (S); FGF-2
(F).
DEFINITIONS
[0046] A "mental disorder" or "mental illness" or "mental disease"
or "psychiatric or neuropsychiatric disease or illness or disorder"
refers to mood disorders (e.g., major depression, mania, and
bipolar disorders), psychotic disorders (e.g., schizophrenia,
schizoaffective disorder, schizophreniform disorder, delusional
disorder, brief psychotic disorder, and shared psychotic disorder),
personality disorders, anxiety disorders (e.g.,
obsessive-compulsive disorder) as well as other mental disorders
such as substance-related disorders, childhood disorders, dementia,
autistic disorder, adjustment disorder, delirium, multi-infarct
dementia, and Tourette's disorder as described in Diagnostic and
Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV).
Typically, such disorders have a complex genetic and/or a
biochemical component.
[0047] A "mood disorder" refers to disruption of feeling tone or
emotional state experienced by an individual for an extensive
period of time. Mood disorders include major depression disorder
(i.e., unipolar disorder), mania, dysphoria, bipolar disorder,
dysthymia, cyclothymia and many others. See, e.g., Diagnostic and
Statistical Manual of Mental Disorders, Fourth Edition, (DSM
IV).
[0048] "Major depression disorder," "major depressive disorder," or
"unipolar disorder" refers to a mood disorder involving any of the
following symptoms: persistent sad, anxious, or "empty" mood;
feelings of hopelessness or pessimism; feelings of guilt,
worthlessness, or helplessness; loss of interest or pleasure in
hobbies and activities that were once enjoyed, including sex;
decreased energy, fatigue, being "slowed down"; difficulty
concentrating, remembering, or making decisions; insomnia,
early-morning awakening, or oversleeping; appetite and/or weight
loss or overeating and weight gain; thoughts of death or suicide or
suicide attempts; restlessness or irritability; or persistent
physical symptoms that do not respond to treatment, such as
headaches, digestive disorders, and chronic pain. Various subtypes
of depression are described in, e.g., DSM IV.
[0049] "Bipolar disorder" is a mood disorder characterized by
alternating periods of extreme moods. A person with bipolar
disorder experiences cycling of moods that usually swing from being
overly elated or irritable (mania) to sad and hopeless (depression)
and then back again, with periods of normal mood in between.
Diagnosis of bipolar disorder is described in, e.g., DSM IV.
Bipolar disorders include bipolar disorder I (mania with or without
major depression) and bipolar disorder II (hypomania with major
depression), see, e.g., DSM IV.
[0050] "A psychotic disorder" refers to a condition that affects
the mind, resulting in at least some loss of contact with reality.
Symptoms of a psychotic disorder include, e.g., hallucinations,
changed behavior that is not based on reality, delusions and the
like. See, e.g., DSM IV. Schizophrenia, schizoaffective disorder,
schizophreniform disorder, delusional disorder, brief psychotic
disorder, substance-induced psychotic disorder, and shared
psychotic disorder are examples of psychotic disorders.
[0051] "Schizophrenia" refers to a psychotic disorder involving a
withdrawal from reality by an individual. Symptoms comprise for at
least a part of a month two or more of the following symptoms:
delusions (only one symptom is required if a delusion is bizarre,
such as being abducted in a space ship from the sun);
hallucinations (only one symptom is required if hallucinations are
of at least two voices talking to one another or of a voice that
keeps up a running commentary on the patient's thoughts or
actions); disorganized speech (e.g., frequent derailment or
incoherence); grossly disorganized or catatonic behavior; or
negative symptoms, i.e., affective flattening, alogia, or
avolition. Schizophrenia encompasses disorders such as, e.g.,
schizoaffective disorders. Diagnosis of schizophrenia is described
in, e.g., DSM IV. Types of schizophrenia include, e.g., paranoid,
disorganized, catatonic, undifferentiated, and residual.
[0052] An "antidepressant" refers to an agents typically used to
treat clinical depression. Antidepressants includes compounds of
different classes including, for example, specific serotonin
reuptake inhibitors (e.g., fluoxetine), tricyclic antidepressants
(e.g., desipramine), and dopamine reuptake inhibitors (e.g,
bupropion). Typically, antidepressants of different classes exert
their therapeutic effects via different biochemical pathways. Often
these biochemical pathways overlap or intersect. Additional
diseases or disorders often treated with antidepressants include,
chronic pain, anxiety disorders, and hot flashes.
[0053] An "agonist" refers to an agent that binds to a polypeptide
or polynucleotide of the invention, stimulates, increases,
activates, facilitates, enhances activation, sensitizes or up
regulates the activity or expression of a polypeptide or
polynucleotide of the invention.
[0054] An "antagonist" refers to an agent that inhibits expression
of a polypeptide or polynucleotide of the invention or binds to,
partially or totally blocks stimulation, decreases, prevents,
delays activation, inactivates, desensitizes, or down regulates the
activity of a polypeptide or polynucleotide of the invention.
[0055] "Inhibitors," "activators," and "modulators" of expression
or of activity are used to refer to inhibitory, activating, or
modulating molecules, respectively, identified using in vitro and
in vivo assays for expression or activity, e.g., ligands, agonists,
antagonists, and their homologs and mimetics. The term "modulator"
includes inhibitors and activators. Inhibitors are agents that,
e.g., inhibit expression of a polypeptide or polynucleotide of the
invention or bind to, partially or totally block stimulation or
enzymatic activity, decrease, prevent, delay activation,
inactivate, desensitize, or down regulate the activity of a
polypeptide or polynucleotide of the invention, e.g., antagonists.
Activators are agents that, e.g., induce or activate the expression
of a polypeptide or polynucleotide of the invention or bind to,
stimulate, increase, open, activate, facilitate, enhance activation
or enzymatic activity, sensitize or up regulate the activity of a
polypeptide or polynucleotide of the invention, e.g., agonists.
Modulators include naturally occurring and synthetic ligands,
antagonists, agonists, small chemical molecules and the like.
Assays to identify inhibitors and activators include, e.g.,
applying putative modulator compounds to cells, in the presence or
absence of a polypeptide or polynucleotide of the invention and
then determining the functional effects on a polypeptide or
polynucleotide of the invention activity. Samples or assays
comprising a polypeptide or polynucleotide of the invention that
are treated with a potential activator, inhibitor, or modulator are
compared to control samples without the inhibitor, activator, or
modulator to examine the extent of effect. Control samples
(untreated with modulators) are assigned a relative activity value
of 100%. Inhibition is achieved when the activity value of a
polypeptide or polynucleotide of the invention relative to the
control is about 80%, optionally 50% or 25-1%. Activation is
achieved when the activity value of a polypeptide or polynucleotide
of the invention relative to the control is 110%, optionally 150%,
optionally 200-500%, or 1000-3000% higher.
[0056] The term "test compound" or "drug candidate" or "modulator"
or grammatical equivalents as used herein describes any molecule,
either naturally occurring or synthetic, e.g., protein,
oligopeptide (e.g., from about 5 to about 25 amino acids in length,
preferably from about 10 to 20 or 12 to 18 amino acids in length,
preferably 12, 15, or 18 amino acids in length), small organic
molecule, polysaccharide, lipid, fatty acid, polynucleotide, RNAi,
oligonucleotide, etc. The test compound can be in the form of a
library of test compounds, such as a combinatorial or randomized
library that provides a sufficient range of diversity. Test
compounds are optionally linked to a fusion partner, e.g.,
targeting compounds, rescue compounds, dimerization compounds,
stabilizing compounds, addressable compounds, and other functional
moieties. Conventionally, new chemical entities with useful
properties are generated by identifying a test compound (called a
"lead compound") with some desirable property or activity, e.g.,
inhibiting activity, creating variants of the lead compound, and
evaluating the property and activity of those variant compounds.
Often, high throughput screening (HTS) methods are employed for
such an analysis.
[0057] A "small organic molecule" refers to an organic molecule,
either naturally occurring or synthetic, that has a molecular
weight of more than about 50 Daltons and less than about 2500
Daltons, preferably less than about 2000 Daltons, preferably
between about 100 to about 1000 Daltons, more preferably between
about 200 to about 500 Daltons.
[0058] An "siRNA" or "RNAi" refers to a nucleic acid that forms a
double stranded RNA, which double stranded RNA has the ability to
reduce or inhibit expression of a gene or target gene when the
siRNA expressed in the same cell as the gene or target gene.
"siRNA" or "RNAi" thus refers to the double stranded RNA formed by
the complementary strands. The complementary portions of the siRNA
that hybridize to form the double stranded molecule typically have
substantial or complete identity. In one embodiment, an siRNA
refers to a nucleic acid that has substantial or complete identity
to a target gene and forms a double stranded siRNA. Typically, the
siRNA is at least about 15-50 nucleotides in length (e.g., each
complementary sequence of the double stranded siRNA is 15-50
nucleotides in length, and the double stranded siRNA is about 15-50
base pairs in length, preferable about preferably about 20-30 base
nucleotides, preferably about 20-25 or about 24-29 nucleotides in
length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides in length.
[0059] The term "Table #" when used herein includes all sub-tables
of the Table referred to (e.g., "Table 1" refers to Table 1A, 1B,
and Table 1C) unless otherwise indicated.
[0060] "Determining the functional effect" refers to assaying for a
compound that increases or decreases a parameter that is indirectly
or directly under the influence of a polynucleotide or polypeptide
of the invention (such as a polynucleotide of FIG. 1, FIGS. 5-8, or
Tables 1-4, or a polypeptide encoded by a gene of FIG. 1, FIGS.
5-8, or Tables 1-4), e.g., measuring physical and chemical or
phenotypic effects. Such functional effects can be measured by any
means known to those skilled in the art, e.g., changes in
spectroscopic (e.g., fluorescence, absorbance, refractive index),
hydrodynamic (e.g., shape), chromatographic, or solubility
properties for the protein; measuring inducible markers or
transcriptional activation of the protein; measuring binding
activity or binding assays, e.g. binding to antibodies; measuring
changes in ligand binding affinity; measurement of calcium influx;
measurement of the accumulation of an enzymatic product of a
polypeptide of the invention or depletion of an substrate;
measurement of changes in protein levels of a polypeptide of the
invention; measurement of RNA stability; G-protein binding; GPCR
phosphorylation or dephosphorylation; signal transduction, e.g.,
receptor-ligand interactions, second messenger concentrations
(e.g., cAMP, IP3, or intracellular Ca.sup.2+); identification of
downstream or reporter gene expression (CAT, luciferase,
.beta.-gal, GFP and the like), e.g., via chemiluminescence,
fluorescence, calorimetric reactions, antibody binding, inducible
markers, and ligand binding assays.
[0061] Samples or assays comprising a nucleic acid or protein
disclosed herein that are treated with a potential activator,
inhibitor, or modulator are compared to control samples without the
inhibitor, activator, or modulator to examine the extent of
inhibition. Control samples (untreated with inhibitors) are
assigned a relative protein activity value of 100%. Inhibition is
achieved when the activity value relative to the control is about
80%, preferably 50%, more preferably 25-0%. Activation is achieved
when the activity value relative to the control (untreated with
activators) is 110%, more preferably 150%, more preferably 200-500%
(i.e., two to five fold higher relative to the control), more
preferably 1000-3000% higher.
[0062] "Biological sample" includes sections of tissues such as
biopsy and autopsy samples, and frozen sections taken for
histologic purposes. Such samples include blood, sputum, tissue,
lysed cells, brain biopsy, cultured cells, e.g., primary cultures,
explants, and transformed cells, stool, urine, etc. A biological
sample is typically obtained from a eukaryotic organism, most
preferably a mammal such as a primate, e.g., chimpanzee or human;
cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a
bird; reptile; or fish.
[0063] "Antibody" refers to a polypeptide substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof which specifically bind and recognize an analyte (antigen).
The recognized immunoglobulin genes include the kappa, lambda,
alpha, gamma, delta, epsilon and mu constant region genes, as well
as the myriad immunoglobulin variable region genes. Light chains
are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0064] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0065] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially an Fab with part of
the hinge region (see, Paul (Ed.) Fundamental Immunology, Third
Edition, Raven Press, NY (1993)). While various antibody fragments
are defined in terms of the digestion of an intact antibody, one of
skill will appreciate that such fragments may be synthesized de
novo either chemically or by utilizing recombinant DNA methodology.
Thus, the term antibody, as used herein, also includes antibody
fragments either produced by the modification of whole antibodies
or those synthesized de novo using recombinant DNA methodologies
(e.g., single chain Fv).
[0066] The terms "peptidomimetic" and "mimetic" refer to a
synthetic chemical compound that has substantially the same
structural and functional characteristics of the polynucleotides,
polypeptides, antagonists or agonists of the invention. Peptide
analogs are commonly used in the pharmaceutical industry as
non-peptide drugs with properties analogous to those of the
template peptide. These types of non-peptide compound are termed
"peptide mimetics" or "peptidomimetics" (Fauchere, Adv. Drug Res.
15:29 (1986); Veber and Freidinger TINS p. 392 (1985); and Evans et
al., J. Med. Chem. 30:1229 (1987), which are incorporated herein by
reference). Peptide mimetics that are structurally similar to
therapeutically useful peptides may be used to produce an
equivalent or enhanced therapeutic or prophylactic effect.
Generally, peptidomimetics are structurally similar to a paradigm
polypeptide (i.e., a polypeptide that has a biological or
pharmacological activity), such as a CCX CKR, but have one or more
peptide linkages optionally replaced by a linkage selected from the
group consisting of, e.g., --CH.sub.2NH--, --CH.sub.2S--,
--CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--. The mimetic
can be either entirely composed of synthetic, non-natural analogues
of amino acids, or, is a chimeric molecule of partly natural
peptide amino acids and partly non-natural analogs of amino acids.
The mimetic can also incorporate any amount of natural amino acid
conservative substitutions as long as such substitutions also do
not substantially alter the mimetic's structure and/or activity.
For example, a mimetic composition is within the scope of the
invention if it is capable of carrying out the binding or enzymatic
activities of a polypeptide or polynucleotide of the invention or
inhibiting or increasing the enzymatic activity or expression of a
polypeptide or polynucleotide of the invention.
[0067] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0068] The term "isolated," when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is essentially
free of other cellular components with which it is associated in
the natural state. It is preferably in a homogeneous state although
it can be in either a dry or aqueous solution. Purity and
homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high
performance liquid chromatography. A protein that is the
predominant species present in a preparation is substantially
purified. In particular, an isolated gene is separated from open
reading frames that flank the gene and encode a protein other than
the gene of interest. The term "purified" denotes that a nucleic
acid or protein gives rise to essentially one band in an
electrophoretic gel. Particularly, it means that the nucleic acid
or protein is at least 85% pure, more preferably at least 95% pure,
and most preferably at least 99% pure.
[0069] The term "nucleic acid" or "polynucleotide" refers to
deoxyribonucleotides or ribonucleotides and polymers thereof in
either single- or double-stranded form. Unless specifically
limited, the term encompasses nucleic acids containing known
analogues of natural nucleotides that have similar binding
properties as the reference nucleic acid and are metabolized in a
manner similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions), alleles, orthologs, SNPs, and
complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell.
Probes 8:91-98 (1994)). The term nucleic acid is used
interchangeably with gene, cDNA, and mRNA encoded by a gene.
[0070] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers. As used herein, the terms encompass amino acid
chains of any length, including full-length proteins (i.e.,
antigens), wherein the amino acid residues are linked by covalent
peptide bonds.
[0071] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. "Amino acid mimetics" refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0072] Amino acids may be referred to herein by either the commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes.
[0073] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, "conservatively modified variants" refers to those
nucleic acids that encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein that encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid that encodes a polypeptide is implicit in each described
sequence.
[0074] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0075] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
[0076] 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
[0077] (see, e.g., Creighton, Proteins (1984)).
[0078] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide sequence in the comparison window
may comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
[0079] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%,
90%, or 95% identity over a specified region), when compared and
aligned for maximum correspondence over a comparison window, or
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
Such sequences are then said to be "substantially identical." This
definition also refers to the complement of a test sequence.
Optionally, the identity exists over a region that is at least
about 50 nucleotides in length, or more preferably over a region
that is 100 to 500 or 1000 or more nucleotides in length.
[0080] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0081] A "comparison window," as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0082] An example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad.
Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0083] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0084] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0085] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0086] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, optionally 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C. Such washes can be performed for 5, 15, 30,
60, 120, or more minutes. Nucleic acids that hybridize to the genes
listed in FIG. 1, FIGS. 5-8, or Tables 1-4 are encompassed by the
invention. Also encompassed by the invention are arrays comprising
nucleotides for detecting the expression of two or more of the
genes listed in FIG. 1, FIGS. 5-8, or Tables 1-4.
[0087] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides that they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. Such washes can be performed for 5,
15, 30, 60, 120, or more minutes. A positive hybridization is at
least twice background. Those of ordinary skill will readily
recognize that alternative hybridization and wash conditions can be
utilized to provide conditions of similar stringency.
[0088] For PCR, a temperature of about 36.degree. C. is typical for
low stringency amplification, although annealing temperatures may
vary between about 32.degree. C. and 48.degree. C. depending on
primer length. For high stringency PCR amplification, a temperature
of about 62.degree. C. is typical, although high stringency
annealing temperatures can range from about 50.degree. C. to about
65.degree. C., depending on the primer length and specificity.
Typical cycle conditions for both high and low stringency
amplifications include a denaturation phase of 90.degree.
C.-95.degree. C. for 30 sec-2 min., an annealing phase lasting 30
sec.-2 min., and an extension phase of about 72.degree. C. for 1-2
min. Protocols and guidelines for low and high stringency
amplification reactions are provided, e.g., in Innis et al., PCR
Protocols, A Guide to Methods and Applications (1990).
[0089] The phrase "a nucleic acid sequence encoding" refers to a
nucleic acid that contains sequence information for a structural
RNA such as rRNA, a tRNA, or the primary amino acid sequence of a
specific protein or peptide, or a binding site for a trans-acting
regulatory agent. This phrase specifically encompasses degenerate
codons (i.e., different codons which encode a single amino acid) of
the native sequence or sequences which may be introduced to conform
with codon preference in a specific host cell.
[0090] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(nonrecombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under-expressed or not expressed at
all.
[0091] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0092] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a host cell. The expression vector can
be part of a plasmid, virus, or nucleic acid fragment. Typically,
the expression vector includes a nucleic acid to be transcribed
operably linked to a promoter.
[0093] The phrase "specifically (or selectively) binds to an
antibody" or "specifically (or selectively) immunoreactive with",
when referring to a protein or peptide, refers to a binding
reaction which is determinative of the presence of the protein in
the presence of a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein and do not bind
in a significant amount to other proteins present in the sample.
Specific binding to an antibody under such conditions may require
an antibody that is selected for its specificity for a particular
protein. For example, antibodies raised against a protein having an
amino acid sequence encoded by any of the polynucleotides of the
invention can be selected to obtain antibodies specifically
immunoreactive with that protein and not with other proteins,
except for polymorphic variants. A variety of immunoassay formats
may be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays,
Western blots, or immunohistochemistry are routinely used to select
monoclonal antibodies specifically immunoreactive with a protein.
See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, NY (1988) for a description of immunoassay
formats and conditions that can be used to determine specific
immunoreactivity. Typically, a specific or selective reaction will
be at least twice the background signal or noise and more typically
more than 10 to 100 times background.
[0094] One who is "predisposed for a mental disorder" as used
herein means a person who has an inclination or a higher likelihood
of developing a mental disorder when compared to an average person
in the general population.
DETAILED DESCRIPTION OF THE INVENTION
[0095] Evidence based on analysis of only a restricted number of
molecules suggests altered and unique gene disregulation that may
be involved in the pathophysiology of bipolar disorder (BPD) and
major depressive disorder (MDD) as well as in the mechanism of drug
treatment for these disorders. The recent development of microarray
technology allows a comprehensive view of the mRNA expression
profiles of specific genes, systems and signaling pathways.
I. Introduction
[0096] To understand the genetic basis of mental disorders, studies
have been conducted to investigate the expression patterns of genes
that are differentially expressed specifically in central nervous
system of subjects with mood disorders. In several studies, the
differential and unique expression of known and novel genes was
determined by way of interrogating total RNA samples purified from
postmortem brains of BP and MDD patients with Affymetrix Gene
Chips.RTM. (containing high-density oligonucleotide probe set
arrays). The fundamental principle is that by identifying genes and
pathways that are differentially expressed in BP and/or MDD
(relative to healthy control subjects), via global expression
profiling of the transcriptomes as above, one can identify genes
that cause, effect, or are associated with the disease, or that
interact with drugs used to treat the disease, for use in
diagnostic and therapeutic applications.
[0097] The Examples provided herein describe the microarray gene
expression profiling of the dorsolateral prefrontal, anterior
cingulate, hippocampus, Nucleus Accumbens, Amigdala and cerebellar
cortices of BPD and MDD patients. In particular, preferred
embodiments of the invention disclosed herein relate to the mRNA
expression levels of genes related to the FGF and GPCRs pathways.
Other preferred embodiments focus on the detection of splice
variants of FGFR2, which are also dysregulated. In still other
embodiments, the invention provides genes which are disregulated by
lithium for the specific treatment of BP disorders.
[0098] The invention also provides nucleic acid sequences and
protein sequences which are useful for deciphering the mode of
action of currently used mood stabilizers such as lithium. The
sequences provided are also useful for drug discovery, e.g.,
discovering new leads to identifying more efficacious therapeutic
targets in the form of a central molecule/pathway through which an
entire system or network of pathways can be modulated to remedy the
perturbed cellular process underlying MDD, BP, or a principal
endophenotype of these disorders. For instance, manipulating GSK3B
can affect one or more of the inositol triphosphate, NF-kB family,
mitochondrial apoptosis, and ubiquitin-proteasome pathways.
Improved knowledge of target-specificity of drugs could help to
minimize side effects associated with numerous mood stabilizers
currently in use. The invention provides combinations of biomarkers
and relevant genes which can be used in methods for diagnosing MDD,
BP and related disorders, as well as for developing additional
tools for that purpose, and for monitoring drug efficacy.
[0099] The present invention provides methods for exploiting the
altered expression (either higher or lower expression as indicated
herein) or unique differential expression of the genes of FIG. 1,
FIGS. 5-8, or Tables 1-4 which is observed in selected brain
regions of patients diagnosed with mood disorders (e.g., bipolar
disorder and major depression disorder) in comparison with normal
individuals. This invention thus provides methods for diagnosis of
mental disorders such as mood disorders (e.g., bipolar disorder,
major depression, and the like) and other mental disorders having a
genetic component by detecting the level of a transcript or
translation product of the genes listed in FIG. 1, FIGS. 5-8, or
Tables 1-4, as well as their corresponding biochemical
pathways.
[0100] The invention further provides methods of identifying a
compound useful for the treatment of such disorders by selecting
compounds, e.g., FGF2, NCAM and peptide inhibitors of the FGF
system, that modulate the functional effect of the translation
products or the expression of the transcripts described herein. The
invention also provides for methods of treating patients with such
mental disorders, e.g., by administering the compounds of the
invention or by gene therapy.
[0101] The invention also provides much-needed tools for
researching mental illness and the underlying molecular causes of
mental illness. These tools include animal models which have been
engineered to exhibit phenotypes which are useful for elucidating
the molecular basis for mental abnormalities and for identifying
treatments for mental abnormalities. For example, the invention
provides in one embodiment a mouse with improved memory and
learning ability.
[0102] The genes and the polypeptides that they encode, which are
associated with mood disorders such as bipolar disease and major
depression, are useful for facilitating the design and development
of various molecular diagnostic tools such as GeneChips.TM.
containing probe sets specific for all or selected mental
disorders, including but not limited to mood disorders, and as an
ante- and/or post-natal diagnostic tool for screening newborns in
concert with genetic counseling. Other diagnostic applications
include evaluation of disease susceptibility, prognosis, and
monitoring of disease or treatment process, as well as providing
individualized medicine via predictive drug profiling systems,
e.g., by correlating specific genomic motifs with the clinical
response of a patient to individual drugs. In addition, the present
invention is useful for multiplex SNP and haplotype profiling,
including but not limited to the identification of therapeutic,
diagnostic, and pharmacogenetic targets at the gene, mRNA, protein,
and pathway level. Profiling of splice variants and deletions is
also useful for diagnostic and therapeutic applications.
[0103] The genes and the polypeptides that they encode, described
herein, are also useful as drug targets for the development of
therapeutic drugs for the treatment or prevention of mental
disorders, including but not limited to mood disorders.
[0104] Antidepressants belong to different classes, e.g.,
desipramine, bupropion, and fluoxetine are in general equally
effective for the treatment of clinical depression, but act by
different mechanisms. The similar effectiveness of the drugs for
treatment of mood disorders suggests that they act through a
presently unidentified common pathway. Animal models of depression,
including treatment of animals with known therapeutics such as
SSRIs, can be used to examine the mode of action of the genes of
the invention. Lithium is drug of choice for treating BP.
[0105] The genes and the polypeptides that they encode, described
herein, as also useful as drug targets for the development of
therapeutic drugs for the treatment or prevention of mental
disorders, including but not limited to mood disorders. Mental
disorders have a high co-morbidity with other neurological
disorders, such as Parkinson's disease or Alzheimer's. Therefore,
the present invention can be used for diagnosis and treatment of
patients with multiple disease states that include a mental
disorder such as a mood disorder. These mood disorders include BP,
MDD, and other disorders such as psychotic-depression, depression
and anxiety features, melancholic depression, chronic depression,
BPI and BPII.
II. General Recombinant Nucleic Acid Methods for Use with the
Invention
[0106] In numerous embodiments of the present invention,
polynucleotides of the invention will be isolated and cloned using
recombinant methods. Such polynucleotides include, e.g., those
listed in FIG. 1, FIGS. 5-8, or Tables 1-4, which can be used for,
e.g., protein expression or during the generation of variants,
derivatives, expression cassettes, to monitor gene expression, for
the isolation or detection of sequences of the invention in
different species, for diagnostic purposes in a patient, e.g., to
detect mutations or to detect expression levels of nucleic acids or
polypeptides of the invention. In some embodiments, the sequences
of the invention are operably linked to a heterologous promoter. In
one embodiment, the nucleic acids of the invention are from any
mammal, including, in particular, e.g., a human, a mouse, a rat, a
primate, etc.
A. General Recombinant Nucleic Acids Methods
[0107] This invention relies on routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of
use in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994)).
[0108] For nucleic acids, sizes are given in either kilobases (kb)
or base pairs (bp). These are estimates derived from agarose or
acrylamide gel electrophoresis, from sequenced nucleic acids, or
from published DNA sequences. For proteins, sizes are given in
kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are
estimated from gel electrophoresis, from sequenced proteins, from
derived amino acid sequences, or from published protein
sequences.
[0109] Oligonucleotides that are not commercially available can be
chemically synthesized according to the solid phase phosphoramidite
triester method first described by Beaucage & Caruthers,
Tetrahedron Letts. 22:1859-1862 (1981), using an automated
synthesizer, as described in Van Devanter et. al., Nucleic Acids
Res. 12:6159-6168 (1984). Purification of oligonucleotides is by
either native acrylamide gel electrophoresis or by anion-exchange
HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149
(1983).
[0110] The sequence of the cloned genes and synthetic
oligonucleotides can be verified after cloning using, e.g., the
chain termination method for sequencing double-stranded templates
of Wallace et al., Gene 16:21-26 (1981).
B. Cloning Methods for the Isolation of Nucleotide Sequences
Encoding Desired Proteins
[0111] In general, the nucleic acids encoding the subject proteins
are cloned from DNA sequence libraries that are made to encode cDNA
or genomic DNA. The particular sequences can be located by
hybridizing with an oligonucleotide probe, the sequence of which
can be derived from the sequences of the genes listed in FIG. 1,
FIGS. 5-8, or Tables 1-4, which provide a reference for PCR primers
and defines suitable regions for isolating specific probes.
Alternatively, where the sequence is cloned into an expression
library, the expressed recombinant protein can be detected
immunologically with antisera or purified antibodies made against a
polypeptide comprising an amino acid sequence encoded by a gene
listed in FIG. 1, FIGS. 5-8, or Tables 1-4.
[0112] Methods for making and screening genomic and cDNA libraries
are well known to those of skill in the art (see, e.g., Gubler and
Hoffman Gene 25:263-269 (1983); Benton and Davis Science,
196:180-182 (1977); and Sambrook, supra). Brain cells are an
example of suitable cells to isolate RNA and cDNA sequences of the
invention.
[0113] Briefly, to make the cDNA library, one should choose a
source that is rich in mRNA. The mRNA can then be made into cDNA,
ligated into a recombinant vector, and transfected into a
recombinant host for propagation, screening and cloning. For a
genomic library, the DNA is extracted from a suitable tissue and
either mechanically sheared or enzymatically digested to yield
fragments of preferably about 5-100 kb. The fragments are then
separated by gradient centrifugation from undesired sizes and are
constructed in bacteriophage lambda vectors. These vectors and
phage are packaged in vitro, and the recombinant phages are
analyzed by plaque hybridization. Colony hybridization is carried
out as generally described in Grunstein et al., Proc. Natl. Acad.
Sci. USA., 72:3961-3965 (1975).
[0114] An alternative method combines the use of synthetic
oligonucleotide primers with polymerase extension on an mRNA or DNA
template. Suitable primers can be designed from specific sequences
of the invention. This polymerase chain reaction (PCR) method
amplifies the nucleic acids encoding the protein of interest
directly from mRNA, cDNA, genomic libraries or cDNA libraries.
Restriction endonuclease sites can be incorporated into the
primers. Polymerase chain reaction or other in vitro amplification
methods may also be useful, for example, to clone nucleic acids
encoding specific proteins and express said proteins, to synthesize
nucleic acids that will be used as probes for detecting the
presence of mRNA encoding a polypeptide of the invention in
physiological samples, for nucleic acid sequencing, or for other
purposes (see, U.S. Pat. Nos. 4,683,195 and 4,683,202). Genes
amplified by a PCR reaction can be purified from agarose gels and
cloned into an appropriate vector.
[0115] Appropriate primers and probes for identifying
polynucleotides of the invention from mammalian tissues can be
derived from the sequences provided herein. For a general overview
of PCR, see, Innis et al. PCR Protocols: A Guide to Methods and
Applications, Academic Press, San Diego (1990).
[0116] Synthetic oligonucleotides can be used to construct genes.
This is done using a series of overlapping oligonucleotides,
usually 40-120 bp in length, representing both the sense and
anti-sense strands of the gene. These DNA fragments are then
annealed, ligated and cloned.
[0117] A gene encoding a polypeptide of the invention can be cloned
using intermediate vectors before transformation into mammalian
cells for expression. These intermediate vectors are typically
prokaryote vectors or shuttle vectors. The proteins can be
expressed in either prokaryotes, using standard methods well known
to those of skill in the art, or eukaryotes as described infra.
III. Purification of Proteins of the Invention
[0118] Either naturally occurring or recombinant polypeptides of
the invention can be purified for use in functional assays.
Naturally occurring polypeptides, e.g., polypeptides encoded by
genes listed in FIG. 1, FIGS. 5-8, or Tables 1-4, can be purified,
for example, from mouse or human tissue such as brain or any other
source of an ortholog. Recombinant polypeptides can be purified
from any suitable expression system.
[0119] The polypeptides of the invention may be purified to
substantial purity by standard techniques, including selective
precipitation with such substances as ammonium sulfate; column
chromatography, immunopurification methods, and others (see, e.g.,
Scopes, Protein Purification Principles and Practice (1982); U.S.
Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et al.,
supra).
[0120] A number of procedures can be employed when recombinant
polypeptides are purified. For example, proteins having established
molecular adhesion properties can be reversible fused to
polypeptides of the invention. With the appropriate ligand, the
polypeptides can be selectively adsorbed to a purification column
and then freed from the column in a relatively pure form. The fused
protein is then removed by enzymatic activity. Finally the
polypeptide can be purified using immunoaffinity columns.
A. Purification of Proteins from Recombinant Bacteria
[0121] When recombinant proteins are expressed by the transformed
bacteria in large amounts, typically after promoter induction,
although expression can be constitutive, the proteins may form
insoluble aggregates. There are several protocols that are suitable
for purification of protein inclusion bodies. For example,
purification of aggregate proteins (hereinafter referred to as
inclusion bodies) typically involves the extraction, separation
and/or purification of inclusion bodies by disruption of bacterial
cells typically, but not limited to, by incubation in a buffer of
about 100-150 .mu.g/ml lysozyme and 0.1% Nonidet P40, a non-ionic
detergent. The cell suspension can be ground using a Polytron
grinder (Brinkman Instruments, Westbury, N.Y.). Alternatively, the
cells can be sonicated on ice. Alternate methods of lysing bacteria
are described in Ausubel et al. and Sambrook et al., both supra,
and will be apparent to those of skill in the art.
[0122] The cell suspension is generally centrifuged and the pellet
containing the inclusion bodies resuspended in buffer which does
not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl
(pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic
detergent. It may be necessary to repeat the wash step to remove as
much cellular debris as possible. The remaining pellet of inclusion
bodies may be resuspended in an appropriate buffer (e.g., 20 mM
sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers
will be apparent to those of skill in the art.
[0123] Following the washing step, the inclusion bodies are
solubilized by the addition of a solvent that is both a strong
hydrogen acceptor and a strong hydrogen donor (or a combination of
solvents each having one of these properties). The proteins that
formed the inclusion bodies may then be renatured by dilution or
dialysis with a compatible buffer. Suitable solvents include, but
are not limited to, urea (from about 4 M to about 8 M), formamide
(at least about 80%, volume/volume basis), and guanidine
hydrochloride (from about 4 M to about 8 M). Some solvents that are
capable of solubilizing aggregate-forming proteins, such as SDS
(sodium dodecyl sulfate) and 70% formic acid, are inappropriate for
use in this procedure due to the possibility of irreversible
denaturation of the proteins, accompanied by a lack of
immunogenicity and/or activity. Although guanidine hydrochloride
and similar agents are denaturants, this denaturation is not
irreversible and renaturation may occur upon removal (by dialysis,
for example) or dilution of the denaturant, allowing re-formation
of the immunologically and/or biologically active protein of
interest. After solubilization, the protein can be separated from
other bacterial proteins by standard separation techniques.
[0124] Alternatively, it is possible to purify proteins from
bacteria periplasm. Where the protein is exported into the
periplasm of the bacteria, the periplasmic fraction of the bacteria
can be isolated by cold osmotic shock in addition to other methods
known to those of skill in the art (see, Ausubel et al., supra). To
isolate recombinant proteins from the periplasm, the bacterial
cells are centrifuged to form a pellet. The pellet is resuspended
in a buffer containing 20% sucrose. To lyse the cells, the bacteria
are centrifuged and the pellet is resuspended in ice-cold 5 mM
MgSO.sub.4 and kept in an ice bath for approximately 10 minutes.
The cell suspension is centrifuged and the supernatant decanted and
saved. The recombinant proteins present in the supernatant can be
separated from the host proteins by standard separation techniques
well known to those of skill in the art.
B. Standard Protein Separation Techniques for Purifying
Proteins
1. Solubility Fractionation
[0125] Often as an initial step, and if the protein mixture is
complex, an initial salt fractionation can separate many of the
unwanted host cell proteins (or proteins derived from the cell
culture media) from the recombinant protein of interest. The
preferred salt is ammonium sulfate. Ammonium sulfate precipitates
proteins by effectively reducing the amount of water in the protein
mixture. Proteins then precipitate on the basis of their
solubility. The more hydrophobic a protein is, the more likely it
is to precipitate at lower ammonium sulfate concentrations. A
typical protocol is to add saturated ammonium sulfate to a protein
solution so that the resultant ammonium sulfate concentration is
between 20-30%. This will precipitate the most hydrophobic
proteins. The precipitate is discarded (unless the protein of
interest is hydrophobic) and ammonium sulfate is added to the
supernatant to a concentration known to precipitate the protein of
interest. The precipitate is then solubilized in buffer and the
excess salt removed if necessary, through either dialysis or
diafiltration. Other methods that rely on solubility of proteins,
such as cold ethanol precipitation, are well known to those of
skill in the art and can be used to fractionate complex protein
mixtures.
2. Size Differential Filtration
[0126] Based on a calculated molecular weight, a protein of greater
and lesser size can be isolated using ultrafiltration through
membranes of different pore sizes (for example, Alicon or Millipore
membranes). As a first step, the protein mixture is ultrafiltered
through a membrane with a pore size that has a lower molecular
weight cut-off than the molecular weight of the protein of
interest. The retentate of the ultrafiltration is then
ultrafiltered against a membrane with a molecular cut off greater
than the molecular weight of the protein of interest. The
recombinant protein will pass through the membrane into the
filtrate. The filtrate can then be chromatographed as described
below.
3. Column Chromatography
[0127] The proteins of interest can also be separated from other
proteins on the basis of their size, net surface charge,
hydrophobicity and affinity for ligands. In addition, antibodies
raised against proteins can be conjugated to column matrices and
the proteins immunopurified. All of these methods are well known in
the art.
[0128] It will be apparent to one of skill that chromatographic
techniques can be performed at any scale and using equipment from
many different manufacturers (e.g., Pharmacia Biotech).
IV. Detection of Gene Expression
[0129] Those of skill in the art will recognize that detection of
expression of polynucleotides of the invention has many uses. For
example, as discussed herein, detection of the level of
polypeptides or polynucleotides of the invention in a patient is
useful for diagnosing mood disorders or psychotic disorders or a
predisposition for a mood disorder or psychotic disorders.
Moreover, detection of gene expression is useful to identify
modulators of expression of the polypeptides or polynucleotides of
the invention.
[0130] A variety of methods of specific DNA and RNA measurement
using nucleic acid hybridization techniques are known to those of
skill in the art (see, Sambrook, supra). Some methods involve an
electrophoretic separation (e.g., Southern blot for detecting DNA,
and Northern blot for detecting RNA), but measurement of DNA and
RNA can also be carried out in the absence of electrophoretic
separation (e.g., by dot blot). Southern blot of genomic DNA (e.g.,
from a human) can be used for screening for restriction fragment
length polymorphism (RFLP) to detect the presence of a genetic
disorder affecting a polypeptide of the invention.
[0131] The selection of a nucleic acid hybridization format is not
critical. A variety of nucleic acid hybridization formats are known
to those skilled in the art. For example, common formats include
sandwich assays and competition or displacement assays.
Hybridization techniques are generally described in Hames and
Higgins Nucleic Acid Hybridization, A Practical Approach, IRL Press
(1985); Gall and Pardue, Proc. Natl. Acad. Sci. U.S.A., 63:378-383
(1969); and John et al. Nature, 223:582-587 (1969).
[0132] Detection of a hybridization complex may require the binding
of a signal-generating complex to a duplex of target and probe
polynucleotides or nucleic acids. Typically, such binding occurs
through ligand and anti-ligand interactions as between a
ligand-conjugated probe and an anti-ligand conjugated with a
signal. The binding of the signal generation complex is also
readily amenable to accelerations by exposure to ultrasonic
energy.
[0133] The label may also allow indirect detection of the
hybridization complex. For example, where the label is a hapten or
antigen, the sample can be detected by using antibodies. In these
systems, a signal is generated by attaching fluorescent or enzyme
molecules to the antibodies or in some cases, by attachment to a
radioactive label (see, e.g., Tijssen, "Practice and Theory of
Enzyme Immunoassays," Laboratory Techniques in Biochemistry and
Molecular Biology, Burdon and van Knippenberg Eds., Elsevier
(1985), pp. 9-20).
[0134] The probes are typically labeled either directly, as with
isotopes, chromophores, lumiphores, chromogens, or indirectly, such
as with biotin, to which a streptavidin complex may later bind.
Thus, the detectable labels used in the assays of the present
invention can be primary labels (where the label comprises an
element that is detected directly or that produces a directly
detectable element) or secondary labels (where the detected label
binds to a primary label, e.g., as is common in immunological
labeling). Typically, labeled signal nucleic acids are used to
detect hybridization. Complementary nucleic acids or signal nucleic
acids may be labeled by any one of several methods typically used
to detect the presence of hybridized polynucleotides. The most
common method of detection is the use of autoradiography with
.sup.3H, .sup.125, .sup.35S, .sup.14C, or .sup.32P-labeled probes
or the like.
[0135] Other labels include, e.g., ligands that bind to labeled
antibodies, fluorophores, chemiluminescent agents, enzymes, and
antibodies which can serve as specific binding pair members for a
labeled ligand. An introduction to labels, labeling procedures and
detection of labels is found in Polak and Van Noorden Introduction
to Immuocytochemistry, 2nd ed., Springer Verlag, NY (1997); and in
Haugland Handbook of Fluorescent Probes and Research Chenmicals, a
combined handbook and catalogue Published by Molecular Probes, Inc.
(1996).
[0136] In general, a detector which monitors a particular probe or
probe combination is used to detect the detection reagent label.
Typical detectors include spectrophotometers, phototubes and
photodiodes, microscopes, scintillation counters, cameras, film and
the like, as well as combinations thereof. Examples of suitable
detectors are widely available from a variety of commercial sources
known to persons of skill in the art. Commonly, an optical image of
a substrate comprising bound labeling moieties is digitized for
subsequent computer analysis.
[0137] Most typically, the amount of RNA is measured by quantifying
the amount of label fixed to the solid support by binding of the
detection reagent. Typically, the presence of a modulator during
incubation will increase or decrease the amount of label fixed to
the solid support relative to a control incubation which does not
comprise the modulator, or as compared to a baseline established
for a particular reaction type. Means of detecting and quantifying
labels are well known to those of skill in the art.
[0138] In preferred embodiments, the target nucleic acid or the
probe is immobilized on a solid support. Solid supports suitable
for use in the assays of the invention are known to those of skill
in the art. As used herein, a solid support is a matrix of material
in a substantially fixed arrangement.
[0139] A variety of automated solid-phase assay techniques are also
appropriate. For instance, very large scale immobilized polymer
arrays (VLSIPS.TM.), available from Affymetrix, Inc. (Santa Clara,
Calif.) can be used to detect changes in expression levels of a
plurality of genes involved in the same regulatory pathways
simultaneously. See, Tijssen, supra., Fodor et al. (1991) Science,
251: 767-777; Sheldon et al. (1993) Clinical Chemistry 39(4):
718-719, and Kozal et al. (1996) Nature Medicine 2(7): 753-759.
[0140] Detection can be accomplished, for example, by using a
labeled detection moiety that binds specifically to duplex nucleic
acids (e.g., an antibody that is specific for RNA-DNA duplexes).
One preferred example uses an antibody that recognizes DNA-RNA
heteroduplexes in which the antibody is linked to an enzyme
(typically by recombinant or covalent chemical bonding). The
antibody is detected when the enzyme reacts with its substrate,
producing a detectable product. Coutlee et al. (1989) Analytical
Biochemistry 181:153-162; Bogulavski (1986) et al. J. Immunol.
Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res.
141:397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) Proc.
Nat'l Acad. Sci. USA 65:993-1000; Ballard (1982) Mol. Intmunol.
19:793-799; Pisetsky and Caster (1982) Mol. Immunol. 19:645-650;
Viscidi et al. (1988) J. Clin. Microbial. 41:199-209; and Kiney et
al. (1989) J. Clin. Microbiol. 27:6-12 describe antibodies to RNA
duplexes, including homo and heteroduplexes. Kits comprising
antibodies specific for DNA:RNA hybrids are available, e.g., from
Digene Diagnostics, Inc. (Beltsville, Md.).
[0141] In addition to available antibodies, one of skill in the art
can easily make antibodies specific for nucleic acid duplexes using
existing techniques, or modify those antibodies that are
commercially or publicly available. In addition to the art
referenced above, general methods for producing polyclonal and
monoclonal antibodies are known to those of skill in the art (see,
e.g., Paul (3rd ed.) Fundamental Immunology Raven Press, Ltd., NY
(1993); Coligan Current Protocols in Immunology Wiley/Greene, NY
(1991); Harlow and Lane Antibodies: A Laboratory Manual Cold Spring
Harbor Press, NY (1988); Stites et al. (eds.) Basic and Clinical
Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif.,
and references cited therein; Goding Monoclonal Antibodies:
Principles and Practice (2d ed.) Academic Press, New York, N.Y.,
(1986); and Kohler and Milstein Nature 256: 495-497 (1975)). Other
suitable techniques for antibody preparation include selection of
libraries of recombinant antibodies in phage or similar vectors
(see, Huse et al. Science 246:1275-1281 (1989); and Ward et al.
Nature 341:544-546 (1989)). Specific monoclonal and polyclonal
antibodies and antisera will usually bind with a K.sub.D of at
least about 0.1 .mu.M, preferably at least about 0.01 .mu.M or
better, and most typically and preferably, 0.001 .mu.M or
better.
[0142] The nucleic acids used in this invention can be either
positive or negative probes. Positive probes bind to their targets
and the presence of duplex formation is evidence of the presence of
the target. Negative probes fail to bind to the suspect target and
the absence of duplex formation is evidence of the presence of the
target. For example, the use of a wild type specific nucleic acid
probe or PCR primers may serve as a negative probe in an assay
sample where only the nucleotide sequence of interest is
present.
[0143] The sensitivity of the hybridization assays may be enhanced
through use of a nucleic acid amplification system that multiplies
the target nucleic acid being detected. Examples of such systems
include the polymerase chain reaction (PCR) system, in particular
RT-PCR or real time PCR, and the ligase chain reaction (LCR)
system. Other methods recently described in the art are the nucleic
acid sequence based amplification (NASBA, Cangene, Mississauga,
Ontario) and Q Beta Replicase systems. These systems can be used to
directly identify mutants where the PCR or LCR primers are designed
to be extended or ligated only when a selected sequence is present.
Alternatively, the selected sequences can be generally amplified
using, for example, nonspecific PCR primers and the amplified
target region later probed for a specific sequence indicative of a
mutation.
[0144] An alternative means for determining the level of expression
of the nucleic acids of the present invention is in situ
hybridization. In situ hybridization assays are well known and are
generally described in Angerer et al., Methods Enzymol. 152:649-660
(1987). In an in situ hybridization assay, cells, preferentially
human cells from the cerebellum or the hippocampus, are fixed to a
solid support, typically a glass slide. If DNA is to be probed, the
cells are denatured with heat or alkali. The cells are then
contacted with a hybridization solution at a moderate temperature
to permit annealing of specific probes that are labeled. The probes
are preferably labeled with radioisotopes or fluorescent
reporters.
V. Immunological Detection of the Polypeptides of the Invention
[0145] In addition to the detection of polynucleotide expression
using nucleic acid hybridization technology, one can also use
immunoassays to detect polypeptides of the invention. Immunoassays
can be used to qualitatively or quantitatively analyze
polypeptides. A general overview of the applicable technology can
be found in Harlow & Lane, Antibodies: A Laboratory Manual
(1988).
A. Antibodies to Target Polypeptides or Other Immunogens
[0146] Methods for producing polyclonal and monoclonal antibodies
that react specifically with a protein of interest or other
immunogen are known to those of skill in the art (see, e.g.,
Coligan, supra; and Harlow and Lane, supra; Stites et al., supra
and references cited therein; Goding, supra; and Kohler and
Milstein Nature, 256:495-497 (1975)). Such techniques include
antibody preparation by selection of antibodies from libraries of
recombinant antibodies in phage or similar vectors (see, Huse et
al., supra; and Ward et al., supra). For example, in order to
produce antisera for use in an immunoassay, the protein of interest
or an antigenic fragment thereof, is isolated as described herein.
For example, a recombinant protein is produced in a transformed
cell line. An inbred strain of mice or rabbits is immunized with
the protein using a standard adjuvant, such as Freund's adjuvant,
and a standard immunization protocol. Alternatively, a synthetic
peptide derived from the sequences disclosed herein and conjugated
to a carrier protein can be used as an immunogen.
[0147] Polyclonal sera are collected and titered against the
immunogen in an immunoassay, for example, a solid phase immunoassay
with the immunogen immobilized on a solid support. Polyclonal
antisera with a titer of 10.sup.4 or greater are selected and
tested for their cross-reactivity against unrelated proteins or
even other homologous proteins from other organisms, using a
competitive binding immunoassay. Specific monoclonal and polyclonal
antibodies and antisera will usually bind with a K.sub.D of at
least about 0.1 mM, more usually at least about 1 .mu.M, preferably
at least about 0.1 .mu.M or better, and most preferably, 0.01 .mu.M
or better.
[0148] A number of proteins of the invention comprising immunogens
may be used to produce antibodies specifically or selectively
reactive with the proteins of interest. Recombinant protein is the
preferred immunogen for the production of monoclonal or polyclonal
antibodies. Naturally occurring protein, such as one comprising an
amino acid sequence encoded by a gene listed in Tables 1-4 and FIG.
1 may also be used either in pure or impure form. Synthetic
peptides made using the protein sequences described herein may also
be used as an immunogen for the production of antibodies to the
protein. Recombinant protein can be expressed in eukaryotic or
prokaryotic cells and purified as generally described supra. The
product is then injected into an animal capable of producing
antibodies. Either monoclonal or polyclonal antibodies may be
generated for subsequent use in immunoassays to measure the
protein.
[0149] Methods of production of polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably a
purified protein, is mixed with an adjuvant and animals are
immunized. The animal's immune response to the immunogen
preparation is monitored by taking test bleeds and determining the
titer of reactivity to the polypeptide of interest. When
appropriately high titers of antibody to the immunogen are
obtained, blood is collected from the animal and antisera are
prepared. Further fractionation of the antisera to enrich for
antibodies reactive to the protein can be done if desired (see,
Harlow and Lane, supra).
[0150] Monoclonal antibodies may be obtained using various
techniques familiar to those of skill in the art. Typically, spleen
cells from an animal immunized with a desired antigen are
immortalized, commonly by fusion with a myeloma cell (see, Kohler
and Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative
methods of immortalization include, e.g., transformation with
Epstein Barr Virus, oncogenes, or retroviruses, or other methods
well known in the art. Colonies arising from single immortalized
cells are screened for production of antibodies of the desired
specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells may be enhanced by
various techniques, including injection into the peritoneal cavity
of a vertebrate host. Alternatively, one may isolate DNA sequences
which encode a monoclonal antibody or a binding fragment thereof by
screening a DNA library from human B cells according to the general
protocol outlined by Huse et al., supra.
[0151] Once target protein specific antibodies are available, the
protein can be measured by a variety of immunoassay methods with
qualitative and quantitative results available to the clinician.
For a review of immunological and immunoassay procedures in general
see, Stites, supra. Moreover, the immunoassays of the present
invention can be performed in any of several configurations, which
are reviewed extensively in Maggio Enzyme Immunoassay, CRC Press,
Boca Raton, Fla. (1980); Tijssen, supra; and Harlow and Lane,
supra.
[0152] Immunoassays to measure target proteins in a human sample
may use a polyclonal antiserum that was raised to the protein
(e.g., one has an amino acid sequence encoded by a gene listed FIG.
1, FIGS. 5-8, or Tables 1-4) or a fragment thereof. This antiserum
is selected to have low cross-reactivity against different proteins
and any such cross-reactivity is removed by immunoabsorption prior
to use in the immunoassay.
B. Immunological Binding Assays
[0153] In a preferred embodiment, a protein of interest is detected
and/or quantified using any of a number of well-known immunological
binding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;
4,517,288; and 4,837,168). For a review of the general
immunoassays, see also Asai Methods in Cell Biology Volume 37:
Antibodies in Cell Biology, Academic Press, Inc. NY (1993); Stites,
supra. Immunological binding assays (or immunoassays) typically
utilize a "capture agent" to specifically bind to and often
immobilize the analyte (in this case a polypeptide of the present
invention or antigenic subsequences thereof). The capture agent is
a moiety that specifically binds to the analyte. In a preferred
embodiment, the capture agent is an antibody that specifically
binds, for example, a polypeptide of the invention. The antibody
may be produced by any of a number of means well known to those of
skill in the art and as described above.
[0154] Immunoassays also often utilize a labeling agent to
specifically bind to and label the binding complex formed by the
capture agent and the analyte. The labeling agent may itself be one
of the moieties comprising the antibody/analyte complex.
Alternatively, the labeling agent may be a third moiety, such as
another antibody, that specifically binds to the antibody/protein
complex.
[0155] In a preferred embodiment, the labeling agent is a second
antibody bearing a label. Alternatively, the second antibody may
lack a label, but it may, in turn, be bound by a labeled third
antibody specific to antibodies of the species from which the
second antibody is derived. The second antibody can be modified
with a detectable moiety, such as biotin, to which a third labeled
molecule can specifically bind, such as enzyme-labeled
streptavidin.
[0156] Other proteins capable of specifically binding
immunoglobulin constant regions, such as protein A or protein G,
can also be used as the label agents. These proteins are normal
constituents of the cell walls of streptococcal bacteria. They
exhibit a strong non-immunogenic reactivity with immunoglobulin
constant regions from a variety of species (see, generally,
Kronval, et al. J. Immunol., 111:1401-1406 (1973); and Akerstrom,
et al. J. Immunol., 135:2589-2542 (1985)).
[0157] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. The incubation time will depend
upon the assay format, analyte, volume of solution, concentrations,
and the like. Usually, the assays will be carried out at ambient
temperature, although they can be conducted over a range of
temperatures, such as 10.degree. C. to 40.degree. C.
1. Non-Competitive Assay Formats
[0158] Immunoassays for detecting proteins of interest from tissue
samples may be either competitive or noncompetitive. Noncompetitive
immunoassays are assays in which the amount of captured analyte (in
this case the protein) is directly measured. In one preferred
"sandwich" assay, for example, the capture agent (e.g., antibodies
specific for a polypeptide encoded by a gene listed in FIG. 1,
FIGS. 5-8, or Tables 1-4) can be bound directly to a solid
substrate where it is immobilized. These immobilized antibodies
then capture the polypeptide present in the test sample. The
polypeptide thus immobilized is then bound by a labeling agent,
such as a second antibody bearing a label. Alternatively, the
second antibody may lack a label, but it may, in turn, be bound by
a labeled third antibody specific to antibodies of the species from
which the second antibody is derived. The second can be modified
with a detectable moiety, such as biotin, to which a third labeled
molecule can specifically bind, such as enzyme-labeled
streptavidin.
2. Competitive Assay Formats
[0159] In competitive assays, the amount of analyte (such as a
polypeptide encoded by a gene listed in FIG. 1, FIGS. 5-8, or
Tables 1-4) present in the sample is measured indirectly by
measuring the amount of an added (exogenous) analyte displaced (or
competed away) from a capture agent (e.g., an antibody specific for
the analyte) by the analyte present in the sample. In one
competitive assay, a known amount of, in this case, the protein of
interest is added to the sample and the sample is then contacted
with a capture agent, in this case an antibody that specifically
binds to a polypeptide of the invention. The amount of immunogen
bound to the antibody is inversely proportional to the
concentration of immunogen present in the sample. In a particularly
preferred embodiment, the antibody is immobilized on a solid
substrate. For example, the amount of the polypeptide bound to the
antibody may be determined either by measuring the amount of
subject protein present in a protein/antibody complex or,
alternatively, by measuring the amount of remaining uncomplexed
protein. The amount of protein may be detected by providing a
labeled protein molecule.
[0160] Immunoassays in the competitive binding format can be used
for cross-reactivity determinations. For example, a protein of
interest can be immobilized on a solid support. Proteins are added
to the assay which compete with the binding of the antisera to the
immobilized antigen. The ability of the above proteins to compete
with the binding of the antisera to the immobilized protein is
compared to that of the protein of interest. The percent
cross-reactivity for the above proteins is calculated, using
standard calculations. Those antisera with less than 10%
cross-reactivity with each of the proteins listed above are
selected and pooled. The cross-reacting antibodies are optionally
removed from the pooled antisera by immunoabsorption with the
considered proteins, e.g., distantly related homologs.
[0161] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein, thought to be perhaps a protein of the present
invention, to the immunogen protein. In order to make this
comparison, the two proteins are each assayed at a wide range of
concentrations and the amount of each protein required to inhibit
50% of the binding of the antisera to the immobilized protein is
determined. If the amount of the second protein required is less
than 10 times the amount of the protein partially encoded by a
sequence herein that is required, then the second protein is said
to specifically bind to an antibody generated to an immunogen
consisting of the target protein.
3. Other Assay Formats
[0162] In a particularly preferred embodiment, western blot
(immunoblot) analysis is used to detect and quantify the presence
of a polypeptide of the invention in the sample. The technique
generally comprises separating sample proteins by gel
electrophoresis on the basis of molecular weight, transferring the
separated proteins to a suitable solid support (such as, e.g., a
nitrocellulose filter, a nylon filter, or a derivatized nylon
filter) and incubating the sample with the antibodies that
specifically bind the protein of interest. For example, the
antibodies specifically bind to a polypeptide of interest on the
solid support. These antibodies may be directly labeled or
alternatively may be subsequently detected using labeled antibodies
(e.g., labeled sheep anti-mouse antibodies) that specifically bind
to the antibodies against the protein of interest.
[0163] Other assay formats include liposome immunoassays (LIA),
which use liposomes designed to bind specific molecules (e.g.,
antibodies) and release encapsulated reagents or markers. The
released chemicals are then detected according to standard
techniques (see, Monroe et al. (1986) Amer. Clin. Prod. Rev.
5:34-41).
4. Labels
[0164] The particular label or detectable group used in the assay
is not a critical aspect of the invention, as long as it does not
significantly interfere with the specific binding of the antibody
used in the assay. The detectable group can be any material having
a detectable physical or chemical property. Such detectable labels
have been well developed in the field of immunoassays and, in
general, most labels useful in such methods can be applied to the
present invention. Thus, a label is any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g., Dynabeads.TM.), fluorescent
dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and
the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S,
.sup.14C, or .sup.32P), enzymes (e.g., horse radish peroxidase,
alkaline phosphatase and others commonly used in an ELISA), and
calorimetric labels such as colloidal gold or colored glass or
plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
[0165] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. As indicated above, a wide variety of labels may be used,
with the choice of label depending on the sensitivity required, the
ease of conjugation with the compound, stability requirements,
available instrumentation, and disposal provisions.
[0166] Non-radioactive labels are often attached by indirect means.
The molecules can also be conjugated directly to signal generating
compounds, e.g., by conjugation with an enzyme or fluorescent
compound. A variety of enzymes and fluorescent compounds can be
used with the methods of the present invention and are well-known
to those of skill in the art (for a review of various labeling or
signal producing systems which may be used, see, e.g., U.S. Pat.
No. 4,391,904).
[0167] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is a
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence. The fluorescence may be detected visually,
by means of photographic film, by the use of electronic detectors
such as charge-coupled devices (CCDs) or photomultipliers and the
like. Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the resulting
reaction product. Finally simple calorimetric labels may be
detected directly by observing the color associated with the label.
Thus, in various dipstick assays, conjugated gold often appears
pink, while various conjugated beads appear the color of the
bead.
[0168] Some assay formats do not require the use of labeled
components. For instance, agglutination assays can be used to
detect the presence of the target antibodies. In this case,
antigen-coated particles are agglutinated by samples comprising the
target antibodies. In this format, none of the components need to
be labeled and the presence of the target antibody is detected by
simple visual inspection.
[0169] In some embodiments, BP or MDD in a patient may be diagnosed
or otherwise evaluated by visualizing expression in situ of one or
more of the appropriately dysregulated gene sequences identified
herein, e.g., FIG. 1, FIGS. 5-8, or Tables 1-4. Those skilled in
the art of visualizing the presence or expression of molecules
including nucleic acids, polypeptides and other biochemicals in the
brains of living patients will appreciate that the gene expression
information described herein may be utilized in the context of a
variety of visualization methods. Such methods include, but are not
limited to, single-photon emission-computed tomography (SPECT) and
positron-emitting tomography (PET) methods. See, e.g., Vassaux and
Groot-wassink, "In Vivo Noninvasive Imaging for Gene Therapy," J.
Biomedicine and Biotechnology, 2: 92-101 (2003).
[0170] PET and SPECT imaging shows the chemical functioning of
organs and tissues, while other imaging techniques--such as X-ray,
CT and MRI--show structure. The use of PET and SPECT imaging is
useful for qualifying and monitoring the development of brain
diseases, including bipolar disorder, major depression disorder,
schizophrenia and associated disorders. In some instances, the use
of PET or SPECT imaging allows diseases to be detected years
earlier than the onset of symptoms. The use of small molecules for
labelling and visualizing the presence or expression of
polypeptides and nucleotides has had success, for example, in
visualizing proteins in the brains of Alzheimer's patients, as
described by, e.g., Herholz K et al., Mol Imaging Biol.,
6(4):239-69 (2004); Nordberg A, Lancet Neurol., 3(9):519-27 (2004);
Neuropsychol Rev., Zakzanis K K et al., 13(1): 1-18 (2003); Kung M
P et al, Brain Res., 1025(1-2):98-105 (2004); and Herholz K, Ann
Nucl Med., 17(2):79-89 (2003).
[0171] The dysregulated genes disclosed in FIG. 1, FIGS. 5-8, or
Tables 1-4, or their encoded peptides (if any), or fragments
thereof, can be used in the context of PET and SPECT imaging
applications. After modification with appropriate tracer residues
for PET or SPECT applications, molecules which interact or bind
with the transcripts in Tables FIG. 1, FIGS. 5-8, or Tables 1-4 or
with any polypeptides encoded by those transcripts may be used to
visualize the patterns of gene expression and facilitate diagnosis
of schizophrenia, MDD, BP, and related disorders as described
herein. Similarly, if the encoded polypeptides encode enzymes,
labeled molecules which interact with the products of catalysis by
the enzyme may be used for the in vivo imaging and diagnostic
application described herein.
[0172] Antisense technology is particularly suitable for detecting
the transcripts identified in FIG. 1, FIGS. 5-8, or Tables 1-4. For
example, the use of antisense peptide nucleic acid (PNA) labeled
with an appropriate radionuclide, such as .sup.111In, and
conjugated to a brain drug-targeting system to enable transport
across biologic membrane barriers, has been demonstrated to allow
imaging of endogenous gene expression in brain cancer. See Suzuki
et al., Journal of Nuclear Medicine, 10: 1766-1775 (2004). Suzuki
et al. utilize a delivery system comprising monoclonal antibodies
that target transferring receptors at the blood-brain barrier and
facilitate transport of the PNA across that barrier. Modified
embodiments of this technique may be used to target upregulated
genes associated with schizophrenia, BP or MDD, such as the
upregulated genes which appear in FIG. 1, FIGS. 5-8, or Tables 1-4,
in methods of treating schizophrenic, BP or MDD patients.
[0173] In other embodiments, the dysregulated genes listed in FIG.
1, FIGS. 5-8, or Tables 1-4 may be used in the context of prenatal
and neonatal diagnostic methods. For example, fetal or neonatal
samples can be obtained and the expression levels of appropriate
transcripts (e.g., the transcripts in FIG. 1, FIGS. 5-8, or Tables
1-4) may be measured and correlated with the presence or increased
likelihood of a mental disorder, e.g., MDD. Similarly, the presence
of one or more of the SNPs identified in FIG. 1 may be used to
infer or corroborate dysregulated expression of a gene and the
likelihood of a mood disorder in prenatal, neonatal, children and
adult patients.
[0174] In other embodiments, the brain labeling and imaging
techniques described herein or variants thereof may be used in
conjunction with any of the dysregulated gene sequences in FIG. 1,
FIGS. 5-8, or Tables 1-4 in a forensic analysis, i.e., to determine
whether a deceased individual suffered from schizophrenia, BP, or
MDD.
VI. Screening for Modulators of Polypeptides and Polynucleotides of
the Invention
[0175] Modulators of polypeptides or polynucleotides of the
invention, i.e. agonists or antagonists of their activity or
modulators of polypeptide or polynucleotide expression, are useful
for treating a number of human diseases, including mood disorders
or psychotic disorders. Administration of agonists, antagonists or
other agents that modulate expression of the polynucleotides or
polypeptides of the invention can be used to treat patients with
mood disorders or psychotic disorders.
A. Screening Methods
[0176] A number of different screening protocols can be utilized to
identify agents that modulate the level of expression or activity
of polypeptides and polynucleotides of the invention in cells,
particularly mammalian cells, and especially human cells. In
general terms, the screening methods involve screening a plurality
of agents to identify an agent that modulates the polypeptide
activity by binding to a polypeptide of the invention, modulating
inhibitor binding to the polypeptide or activating expression of
the polypeptide or polynucleotide, for example.
1. Binding Assays
[0177] Preliminary screens can be conducted by screening for agents
capable of binding to a polypeptide of the invention, as at least
some of the agents so identified are likely modulators of
polypeptide activity. The binding assays usually involve contacting
a polypeptide of the invention with one or more test agents and
allowing sufficient time for the protein and test agents to form a
binding complex. Any binding complexes formed can be detected using
any of a number of established analytical techniques. Protein
binding assays include, but are not limited to, methods that
measure co-precipitation, co-migration on non-denaturing
SDS-polyacrylaimde gels, and co-migration on Western blots (see,
e.g., Bennet and Yamamura, (1985) "Neurotransmitter, Hormone or
Drug Receptor Binding Methods," in Neurotransmitter Receptor
Binding (Yamamura, H. I., et al., eds.), pp. 61-89. The protein
utilized in such assays can be naturally expressed, cloned or
synthesized.
[0178] Binding assays are also useful, e.g., for identifying
endogenous proteins that interact with a polypeptide of the
invention. For example, antibodies, receptors or other molecules
that bind a polypeptide of the invention can be identified in
binding assays.
2. Expression Assays
[0179] Certain screening methods involve screening for a compound
that up or down-regulates the expression of a polypeptide or
polynucleotide of the invention. Such methods generally involve
conducting cell-based assays in which test compounds are contacted
with one or more cells expressing a polypeptide or polynucleotide
of the invention and then detecting an increase or decrease in
expression (either transcript, translation product, or catalytic
product). Some assays are performed with peripheral cells, or other
cells, that express an endogenous polypeptide or polynucleotide of
the invention.
[0180] Polypeptide or polynucleotide expression can be detected in
a number of different ways. As described infra, the expression
level of a polynucleotide of the invention in a cell can be
determined by probing the mRNA expressed in a cell with a probe
that specifically hybridizes with a transcript (or complementary
nucleic acid derived therefrom) of a polynucleotide of the
invention. Probing can be conducted by lysing the cells and
conducting Northern blots or without lysing the cells using in
situ-hybridization techniques. Alternatively, a polypeptide of the
invention can be detected using immunological methods in which a
cell lysate is probed with antibodies that specifically bind to a
polypeptide of the invention.
[0181] Other cell-based assays are reporter assays conducted with
cells that do not express a polypeptide or polynucleotide of the
invention. Certain of these assays are conducted with a
heterologous nucleic acid construct that includes a promoter of a
polynucleotide of the invention that is operably linked to a
reporter gene that encodes a detectable product. A number of
different reporter genes can be utilized. Some reporters are
inherently detectable. An example of such a reporter is green
fluorescent protein that emits fluorescence that can be detected
with a fluorescence detector. Other reporters generate a detectable
product. Often such reporters are enzymes. Exemplary enzyme
reporters include, but are not limited to, .beta.-glucuronidase,
chloramphenicol acetyl transferase (CAT); Alton and Vapnek (1979)
Nature 282:864-869), luciferase, .beta.-galactosidase, green
fluorescent protein (GFP) and alkaline phosphatase (Toh, et al.
(1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J. Mol.
Appl. Gen. 2:101).
[0182] In these assays, cells harboring the reporter construct are
contacted with a test compound. A test compound that either
activates the promoter by binding to it or triggers a cascade that
produces a molecule that activates the promoter causes expression
of the detectable reporter. Certain other reporter assays are
conducted with cells that harbor a heterologous construct that
includes a transcriptional control element that activates
expression of a polynucleotide of the invention and a reporter
operably linked thereto. Here, too, an agent that binds to the
transcriptional control element to activate expression of the
reporter or that triggers the formation of an agent that binds to
the transcriptional control element to activate reporter
expression, can be identified by the generation of signal
associated with reporter expression.
[0183] The level of expression or activity can be compared to a
baseline value. As indicated above, the baseline value can be a
value for a control sample or a statistical value that is
representative of expression levels for a control population (e.g.,
healthy individuals not having or at risk for mood disorders or
psychotic disorders). Expression levels can also be determined for
cells that do not express a polynucleotide of the invention as a
negative control. Such cells generally are otherwise substantially
genetically the same as the test cells.
[0184] A variety of different types of cells can be utilized in the
reporter assays. Cells that express an endogenous polypeptide or
polynucleotide of the invention include, e.g., brain cells,
including cells from the cerebellum, anterior cingulate cortex,
dorsolateral prefrontal cortex, amygdala, hippocampus, or nucleus
accumbens. Cells that do not endogenously express polynucleotides
of the invention can be prokaryotic, but are preferably eukaryotic.
The eukaryotic cells can be any of the cells typically utilized in
generating cells that harbor recombinant nucleic acid constructs.
Exemplary eukaryotic cells include, but are not limited to, yeast,
and various higher eukaryotic cells such as the COS, CHO and HeLa
cell lines.
[0185] Various controls can be conducted to ensure that an observed
activity is authentic including running parallel reactions with
cells that lack the reporter construct or by not contacting a cell
harboring the reporter construct with test compound. Compounds can
also be further validated as described below.
3. Catalytic Activity
[0186] Catalytic activity of polypeptides of the invention can be
determined by measuring the production of enzymatic products or by
measuring the consumption of substrates. Activity refers to either
the rate of catalysis or the ability to the polypeptide to bind
(K.sub.m) the substrate or release the catalytic product
(K.sub.d).
[0187] Analysis of the activity of polypeptides of the invention
are performed according to general biochemical analyses. Such
assays include cell-based assays as well as in vitro assays
involving purified or partially purified polypeptides or crude cell
lysates. The assays generally involve providing a known quantity of
substrate and quantifying product as a function of time.
4. Validation
[0188] Agents that are initially identified by any of the foregoing
screening methods can be further tested to validate the apparent
activity. Preferably such studies are conducted with suitable
animal models. The basic format of such methods involves
administering a lead compound identified during an initial screen
to an animal that serves as a model for humans and then determining
if expression or activity of a polynucleotide or polypeptide of the
invention is in fact upregulated. The animal models utilized in
validation studies generally are mammals of any kind. Specific
examples of suitable animals include, but are not limited to,
primates, mice, and rats. As described herein, models using
admininstration of known therapeutics can be useful.
5. Animal Models
[0189] Animal models of mental disorders also find use in screening
for modulators. In one embodiment, invertebrate models such as
Drosophila models can be used, screening for modulators of
Drosophila orthologs of the human genes disclosed herein. In
another embodiment, transgenic animal technology including gene
knockout technology, for example as a result of homologous
recombination with an appropriate gene targeting vector, or gene
overexpression, will result in the absence, decreased or increased
expression of a polynucleotide or polypeptide of the invention. The
same technology can also be applied to make knockout cells. When
desired, tissue-specific expression or knockout of a polynucleotide
or polypeptide of the invention may be necessary. Transgenic
animals generated by such methods find use as animal models of
mental illness and are useful in screening for modulators of mental
illness.
[0190] Knockout cells and transgenic mice can be made by insertion
of a marker gene or other heterologous gene into an endogenous gene
site in the mouse genome via homologous recombination. Such mice
can also be made by substituting an endogenous polynucleotide of
the invention with a mutated version of the polynucleotide, or by
mutating an endogenous polynucleotide, e.g., by exposure to
carcinogens.
[0191] For development of appropriate stem cells, a DNA construct
is introduced into the nuclei of embryonic stem cells. Cells
containing the newly engineered genetic lesion are injected into a
host mouse embryo, which is re-implanted into a recipient female.
Some of these embryos develop into chimeric mice that possess germ
cells partially derived from the mutant cell line. Therefore, by
breeding the chimeric mice it is possible to obtain a new line of
mice containing the introduced genetic lesion (see, e.g., Capecchi
et al., Science 244:1288 (1989)). Chimeric targeted mice can be
derived according to Hogan et al., Manipulating the Mouse Embryo: A
Laboratory Manual, Cold Spring Harbor Laboratory (1988) and
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,
Robertson, ed., IRL Press, Washington, D.C., (1987).
B. Modulators of Polypeptides or Polynucleotides of the
Invention
[0192] The agents tested as modulators of the polypeptides or
polynucleotides of the invention can be any small chemical
compound, or a biological entity, such as a protein, sugar, nucleic
acid or lipid. Alternatively, modulators can be genetically altered
versions of a polypeptide or polynucleotides, e.g., recombinant or
altered versions of FGF2, NCAM, or a peptide inhibitor of the FGF
system. Typically, test compounds will be small chemical molecules
and peptides. Essentially any chemical compound can be used as a
potential modulator or ligand in the assays of the invention,
although most often compounds that can be dissolved in aqueous or
organic (especially DMSO-based) solutions are used. The assays are
designed to screen large chemical libraries by automating the assay
steps and providing compounds from any convenient source to assays,
which are typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays). It will be appreciated that
there are many suppliers of chemical compounds, including Sigma
(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St.
Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs,
Switzerland) and the like. Modulators also include agents designed
to reduce the level of mRNA of the invention (e.g. antisense
molecules, ribozymes, DNAzymes and the like) or the level of
translation from an mRNA.
[0193] In one preferred embodiment, high throughput screening
methods involve providing a combinatorial chemical or peptide
library containing a large number of potential therapeutic
compounds (potential modulator or ligand compounds). Such
"combinatorial chemical libraries" or "ligand libraries" are then
screened in one or more assays, as described herein, to identify
those library members (particular chemical species or subclasses)
that display a desired characteristic activity. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics.
[0194] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0195] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication WO 93/20242),
random bio-oligomers (e.g., PCT Publication No. WO 92/00091),
benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.
Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides
(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal
peptidomimetics with glucose scaffolding (Hirschmann et al., J.
Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses
of small compound libraries (Chen et al., J. Amer. Chem. Soc.
116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303
(1993)), and/or peptidyl phosphonates (Campbell et al., J. Org.
Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger
and Sambrook, all supra), peptide nucleic acid libraries (see,
e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g.,
Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and
PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,
Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small
organic molecule libraries (see, e.g., benzodiazepines, Baum
C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines,
5,288,514, and the like).
[0196] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied
Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J.; Tripos; Inc., St. Louis, Mo.; 3D Pharmaceuticals, Exton, Pa.;
Martek Biosciences, Columbia, Md., etc.).
C. Solid State and Soluble High Throughput Assays
[0197] In the high throughput assays of the invention, it is
possible to screen up to several thousand different modulators or
ligands in a single day. In particular, each well of a microtiter
plate can be used to run a separate assay against a selected
potential modulator, or, if concentration or incubation time
effects are to be observed, every 5-10 wells can test a single
modulator. Thus, a single standard microtiter plate can assay about
100 (e.g., 96) modulators. If 1536 well plates are used, then a
single plate can easily assay from about 100 to about 1500
different compounds. It is possible to assay several different
plates per day; assay screens for up to about 6,000-20,000
different compounds are possible using the integrated systems of
the invention. More recently, microfluidic approaches to reagent
manipulation have been developed.
[0198] The molecule of interest can be bound to the solid state
component, directly or indirectly, via covalent or non-covalent
linkage, e.g., via a tag. The tag can be any of a variety of
components. In general, a molecule that binds the tag (a tag
binder) is fixed to a solid support, and the tagged molecule of
interest is attached to the solid support by interaction of the tag
and the tag binder.
[0199] A number of tags and tag binders can be used, based upon
known molecular interactions well described in the literature. For
example, where a tag has a natural binder, for example, biotin,
protein A, or protein G, it can be used in conjunction with
appropriate tag binders (avidin, streptavidin, neutravidin, the Fc
region of an inmiunoglobulin, etc.). Antibodies to molecules with
natural binders such as biotin are also widely available and
appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue
SIGMA, St. Louis Mo.).
[0200] Similarly, any haptenic or antigenic compound can be used in
combination with an appropriate antibody to form a tag/tag binder
pair. Thousands of specific antibodies are commercially available
and many additional antibodies are described in the literature. For
example, in one common configuration, the tag is a first antibody
and the tag binder is a second antibody which recognizes the first
antibody. In addition to antibody-antigen interactions,
receptor-ligand interactions are also appropriate as tag and
tag-binder pairs, such as agonists and antagonists of cell membrane
receptors (e.g., cell receptor-ligand interactions such as
transferrin, c-kit, viral receptor ligands, cytokine receptors,
chemokine receptors, interleukin receptors, immunoglobulin
receptors and antibodies, the cadherin family, the integrin family,
the selectin family, and the like; see, e.g., Pigott & Power,
The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and
venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),
intracellular receptors (e.g., which mediate the effects of various
small ligands, including steroids, thyroid hormone, retinoids and
vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both
linear and cyclic polymer configurations), oligosaccharides,
proteins, phospholipids and antibodies can all interact with
various cell receptors.
[0201] Synthetic polymers, such as polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, and polyacetates
can also form an appropriate tag or tag binder. Many other tag/tag
binder pairs are also useful in assay systems described herein, as
would be apparent to one of skill upon review of this
disclosure.
[0202] Common linkers such as peptides, polyethers, and the like
can also serve as tags, and include polypeptide sequences, such as
poly-Gly sequences of between about 5 and 200 amino acids. Such
flexible linkers are known to those of skill in the art. For
example, poly(ethelyne glycol) linkers are available from
Shearwater Polymers, Inc., Huntsville, Ala. These linkers
optionally have amide linkages, sulfhydryl linkages, or
heterofunctional linkages.
[0203] Tag binders are fixed to solid substrates using any of a
variety of methods currently available. Solid substrates are
commonly derivatized or functionalized by exposing all or a portion
of the substrate to a chemical reagent which fixes a chemical group
to the surface which is reactive with a portion of the tag binder.
For example, groups which are suitable for attachment to a longer
chain portion would include amines, hydroxyl, thiol, and carboxyl
groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to
functionalize a variety of surfaces, such as glass surfaces. The
construction of such solid phase biopolymer arrays is well
described in the literature (see, e.g., Merrifield, J. Am. Chem.
Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,
e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)
(describing synthesis of solid phase components on pins); Frank and
Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of
various peptide sequences on cellulose disks); Fodor et al.,
Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry
39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759
(1996) (all describing arrays of biopolymers fixed to solid
substrates). Non-chemical approaches for fixing tag binders to
substrates include other common methods, such as heat,
cross-linking by UV radiation, and the like.
[0204] The invention provides in vitro assays for identifying, in a
high throughput format, compounds that can modulate the expression
or activity of the polynucleotides or polypeptides of the
invention. In a preferred embodiment, the methods of the invention
include such a control reaction. For each of the assay formats
described, "no modulator" control reactions that do not include a
modulator provide a background level of binding activity.
[0205] In some assays it will be desirable to have positive
controls to ensure that the components of the assays are working
properly. At least two types of positive controls are appropriate.
First, a known activator of a polynucleotide or polypeptide of the
invention can be incubated with one sample of the assay, and the
resulting increase in signal resulting from an increased expression
level or activity of polynucleotide or polypeptide determined
according to the methods herein. Second, a known inhibitor of a
polynucleotide or polypeptide of the invention can be added, and
the resulting decrease in signal for the expression or activity can
be similarly detected.
D. Computer-Based Assays
[0206] Yet another assay for compounds that modulate the activity
of a polypeptide or polynucleotide of the invention involves
computer assisted drug design, in which a computer system is used
to generate a three-dimensional structure of the polypeptide or
polynucleotide based on the structural information encoded by its
amino acid or nucleotide sequence. The input sequence interacts
directly and actively with a pre-established algorithm in a
computer program to yield secondary, tertiary, and quaternary
structural models of the molecule. Similar analyses can be
performed on potential receptors or binding partners of the
polypeptides or polynucleotides of the invention. The models of the
protein or nucleotide structure are then examined to identify
regions of the structure that have the ability to bind, e.g., a
polypeptide or polynucleotide of the invention. These regions are
then used to identify polypeptides that bind to a polypeptide or
polynucleotide of the invention.
[0207] The three-dimensional structural model of a protein is
generated by entering protein amino acid sequences of at least 10
amino acid residues or corresponding nucleic acid sequences
encoding a potential receptor into the computer system. The amino
acid sequences encoded by the nucleic acid sequences provided
herein represent the primary sequences or subsequences of the
proteins, which encode the structural information of the proteins.
At least 10 residues of an amino acid sequence (or a nucleotide
sequence encoding 10 amino acids) are entered into the computer
system from computer keyboards, computer readable substrates that
include, but are not limited to, electronic storage media (e.g.,
magnetic diskettes, tapes, cartridges, and chips), optical media
(e.g., CD ROM), information distributed by internet sites, and by
RAM. The three-dimensional structural model of the protein is then
generated by the interaction of the amino acid sequence and the
computer system, using software known to those of skill in the
art.
[0208] The amino acid sequence represents a primary structure that
encodes the information necessary to form the secondary, tertiary,
and quaternary structure of the protein of interest. The software
looks at certain parameters encoded by the primary sequence to
generate the structural model. These parameters are referred to as
"energy terms," and primarily include electrostatic potentials,
hydrophobic potentials, solvent accessible surfaces, and hydrogen
bonding. Secondary energy terms include van der Waals potentials.
Biological molecules form the structures that minimize the energy
terms in a cumulative fashion. The computer program is therefore
using these terms encoded by the primary structure or amino acid
sequence to create the secondary structural model.
[0209] The tertiary structure of the protein encoded by the
secondary structure is then formed on the basis of the energy terms
of the secondary structure. The user at this point can enter
additional variables such as whether the protein is membrane bound
or soluble, its location in the body, and its cellular location,
e.g., cytoplasmic, surface, or nuclear. These variables along with
the energy terms of the secondary structure are used to form the
model of the tertiary structure. In modeling the tertiary
structure, the computer program matches hydrophobic faces of
secondary structure with like, and hydrophilic faces of secondary
structure with like.
[0210] Once the structure has been generated, potential ligand
binding regions are identified by the computer system.
Three-dimensional structures for potential ligands are generated by
entering amino acid or nucleotide sequences or chemical formulas of
compounds, as described above. The three-dimensional structure of
the potential ligand is then compared to that of a polypeptide or
polynucleotide of the invention to identify binding sites of the
polypeptide or polynucleotide of the invention. Binding affinity
between the protein and ligands is determined using energy terms to
determine which ligands have an enhanced probability of binding to
the protein.
[0211] Computer systems are also used to screen for mutations,
polymorphic variants, alleles and interspecies homologs of genes
encoding a polypeptide or polynucleotide of the invention. Such
mutations can be associated with disease states or genetic traits
and can be used for diagnosis. As described above, GeneChip.TM. and
related technology can also be used to screen for mutations,
polymorphic variants, alleles and interspecies homologs. Once the
variants are identified, diagnostic assays can be used to identify
patients having such mutated genes. Identification of the mutated a
polypeptide or polynucleotide of the invention involves receiving
input of a first amino acid sequence of a polypeptide of the
invention (or of a first nucleic acid sequence encoding a
polypeptide of the invention), e.g., any amino acid sequence having
at least 60%, optionally at least 70% or 85%, identity with the
amino acid sequence of interest, or conservatively modified
versions thereof. The sequence is entered into the computer system
as described above. The first nucleic acid or amino acid sequence
is then compared to a second nucleic acid or amino acid sequence
that has substantial identity to the first sequence. The second
sequence is entered into the computer system in the manner
described above. Once the first and second sequences are compared,
nucleotide or amino acid differences between the sequences are
identified. Such sequences can represent allelic differences in
various polynucleotides of the invention, and mutations associated
with disease states and genetic traits.
VII. Compositions, Kits and Integrated Systems
[0212] The invention provides compositions, kits and integrated
systems for practicing the assays described herein using
polypeptides or polynucleotides of the invention, antibodies
specific for polypeptides or polynucleotides of the invention,
etc.
[0213] The invention provides assay compositions for use in solid
phase assays; such compositions can include, for example, one or
more polynucleotides or polypeptides of the invention immobilized
on a solid support, and a labeling reagent. For example, the kit
could include an array consisting of a set or subset of the
informative sequences listed in FIG. 1, FIGS. 5-8, or Tables 1-4.
In each case, the assay compositions can also include additional
reagents that are desirable for hybridization. Modulators of
expression or activity of polynucleotides or polypeptides of the
invention can also be included in the assay compositions.
[0214] The invention also provides kits for carrying out the
therapeutic and diagnostic assays of the invention. The kits
typically include a probe that comprises an antibody that
specifically binds to polypeptides or polynucleotides of the
invention, and a label for detecting the presence of the probe. The
kits may include several polynucleotide sequences encoding
polypeptides of the invention. Kits can include any of the
compositions noted above, and optionally further include additional
components such as instructions to practice a high-throughput
method of assaying for an effect on expression of the genes
encoding the polypeptides of the invention, or on activity of the
polypeptides of the invention, one or more containers or
compartments (e.g., to hold the probe, labels, or the like), a
control modulator of the expression or activity of polypeptides of
the invention, a robotic armature for mixing kit components or the
like.
[0215] The invention also provides integrated systems for
high-throughput screening of potential modulators for an effect on
the expression or activity of the polypeptides of the invention.
The systems typically include a robotic armature which transfers
fluid from a source to a destination, a controller which controls
the robotic armature, a label detector, a data storage unit which
records label detection, and an assay component such as a
microtiter dish comprising a well having a reaction mixture or a
substrate comprising a fixed nucleic acid or immobilization
moiety.
[0216] A number of robotic fluid transfer systems are available, or
can easily be made from existing components. For example, a Zymate
XP (Zymark Corporation; Hopkinton, Mass.) automated robot using a
Microlab 2200 (Hamilton; Reno, Nev.) pipetting station can be used
to transfer parallel samples to 96 well microtiter plates to set up
several parallel simultaneous STAT binding assays.
[0217] Optical images viewed (and, optionally, recorded) by a
camera or other recording device (e.g., a photodiode and data
storage device) are optionally further processed in any of the
embodiments herein, e.g., by digitizing the image and storing and
analyzing the image on a computer. A variety of commercially
available peripheral equipment and software is available for
digitizing, storing and analyzing a digitized video or digitized
optical image, e.g., using PC (Intel x86 or Pentium chip-compatible
DOS.RTM., OS2.RTM. WINDOWS.RTM., WINDOWS NT.RTM., WINDOWS95.RTM.,
WINDOWS98.RTM., or WINDOWS2000.RTM. based computers),
MACINTOSH.RTM., or UNIX.RTM. based (e.g., SUN.RTM. work station)
computers.
[0218] One conventional system carries light from the specimen
field to a cooled charge-coupled device (CCD) camera, in common use
in the art. A CCD camera includes an array of picture elements
(pixels). The light from the specimen is imaged on the CCD.
Particular pixels corresponding to regions of the specimen (e.g.,
individual hybridization sites on an array of biological polymers)
are sampled to obtain light intensity readings for each position.
Multiple pixels are processed in parallel to increase speed. The
apparatus and methods of the invention are easily used for viewing
any sample, e.g., by fluorescent or dark field microscopic
techniques.
VIII. Administration and Pharmaceutical Compositions
[0219] Modulators of the polynucleotides or polypeptides of the
invention (e.g., antagonists or agonists, such as FGF2, NCAM,
peptide inhibitors of the FGF system, or siRNA and/or antisense
inhibitors of genes which are overexpressed in subjects with mental
disorders) can be administered directly to a mammalian subject for
modulation of activity of those molecules in vivo. Administration
is by any of the routes normally used for introducing a modulator
compound into ultimate contact with the tissue to be treated and is
well known to those of skill in the art. Although more than one
route can be used to administer a particular composition, a
particular route can often provide a more immediate and more
effective reaction than another route.
[0220] Diseases that can be treated include the following, which
include the corresponding reference number from Morrison, DSM-IV
Made Easy, 1995: Schizophrenia, Catatonic, Subchronic, (295.21);
Schizophrenia, Catatonic, Chronic (295.22); Schizophrenia,
Catatonic, Subchronic with Acute Exacerbation (295.23);
Schizophrenia, Catatonic, Chronic with Acute Exacerbation (295.24);
Schizophrenia, Catatonic, in Remission (295.55); Schizophrenia,
Catatonic, Unspecified (295.20); Schizophrenia, Disorganized,
Subchronic (295.11); Schizophrenia, Disorganized, Chronic (295.12);
Schizophrenia, Disorganized, Subchronic with Acute Exacerbation
(295.13); Schizophrenia, Disorganized, Chronic with Acute
Exacerbation (295.14); Schizophrenia, Disorganized, in Remission
(295.15); Schizophrenia, Disorganized, Unspecified (295.10);
Schizophrenia, Paranoid, Subchronic (295.31); Schizophrenia,
Paranoid, Chronic (295.32); Schizophrenia, Paranoid, Subchronic
with Acute Exacerbation (295.33); Schizophrenia, Paranoid, Chronic
with Acute Exacerbation (295.34); Schizophrenia, Paranoid, in
Remission (295.35); Schizophrenia, Paranoid, Unspecified (295.30);
Schizophrenia, Undifferentiated, Subchronic (295.91);
Schizophrenia, Undifferentiated, Chronic (295.92); Schizophrenia,
Undifferentiated, Subchronic with Acute Exacerbation (295.93);
Schizophrenia, Undifferentiated, Chronic with Acute Exacerbation
(295.94); Schizophrenia, Undifferentiated, in Remission (295.95);
Schizophrenia, Undifferentiated, Unspecified (295.90);
Schizophrenia, Residual, Subchronic (295.61); Schizophrenia,
Residual, Chronic (295.62); Schizophrenia, Residual, Subchronic
with Acute Exacerbation (295.63); Schizophrenia, Residual, Chronic
with Acute Exacerbation (295.94); Schizophrenia, Residual, in
Remission (295.65); Schizophrenia, Residual, Unspecified (295.60);
Delusional (Paranoid) Disorder (297.10); Brief Reactive Psychosis
(298.80); Schizophreniform Disorder (295.40); Schizoaffective
Disorder (295.70); Induced Psychotic Disorder (297.30); Psychotic
Disorder NOS (Atypical Psychosis) (298.90); Personality Disorders,
Paranoid (301.00); Personality Disorders, Schizoid (301.20);
Personality Disorders, Schizotypal (301.22); Personality Disorders,
Antisocial (301.70); Personality Disorders, Borderline (301.83) and
bipolar disorders, maniac, hypomaniac, dysthymic or cyclothymic
disorders, substance-induced mood disorders, major depression,
psychosis, including paranoid psychosis, catatonic psychosis,
delusional psychosis, having schizoaffective disorder, and
substance-induced psychotic disorder.
[0221] In some embodiments, modulators of polynucleotides or
polypeptides of the invention can be combined with other drugs
useful for treating mental disorders including useful for treating
mood disorders, e.g., schizophrenia, bipolar disorders, or major
depression. In some preferred embodiments, pharmaceutical
compositions of the invention comprise a modulator of a polypeptide
of polynucleotide of the invention combined with at least one of
the compounds useful for treating schizophrenia, bipolar disorder,
or major depression, e.g., such as those described in U.S. Pat. No.
6,297,262; 6,284,760; 6,284,771; 6,232,326; 6,187,752; 6,117,890;
6,239,162 or 6,166,008. In other embodiments, modulators or
polypeptides of the invention, in particular FGF2, can be used to
treat and/or prevent learning disabilities, memory loss, or
disorders associated with learning disability and memory loss.
[0222] The pharmaceutical compositions of the invention may
comprise a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method
used to administer the composition. Accordingly, there is a wide
variety of suitable formulations of pharmaceutical compositions of
the present invention (see, e.g., Remington's Pharmaceutical
Sciences, 17.sup.th ed. 1985)).
[0223] The modulators (e.g., agonists or antagonists) of the
expression or activity of the a polypeptide or polynucleotide of
the invention, alone or in combination with other suitable
components, can be made into aerosol formulations (i.e., they can
be "nebulized") to be administered via inhalation or in
compositions useful for injection. Aerosol formulations can be
placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like.
[0224] Formulations suitable for administration include aqueous and
non-aqueous solutions, isotonic sterile solutions, which can
contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic, and aqueous and non-aqueous
sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. In
the practice of this invention, compositions can be administered,
for example, orally, nasally, topically, intravenously,
intraperitoneally, or intrathecally. The formulations of compounds
can be presented in unit-dose or multi-dose sealed containers, such
as ampoules and vials. Solutions and suspensions can be prepared
from sterile powders, granules, and tablets of the kind previously
described. The modulators can also be administered as part of a
prepared food or drug.
[0225] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
response in the subject over time. The optimal dose level for any
patient will depend on a variety of factors including the efficacy
of the specific modulator employed, the age, body weight, physical
activity, and diet of the patient, on a possible combination with
other drugs, and on the severity of the mental disorder. The size
of the dose also will be determined by the existence, nature, and
extent of any adverse side effects that accompany the
administration of a particular compound or vector in a particular
subject.
[0226] In determining the effective amount of the modulator to be
administered a physician may evaluate circulating plasma levels of
the modulator, modulator toxicity, and the production of
anti-modulator antibodies. In general, the dose equivalent of a
modulator is from about 1 ng/kg to 10 mg/kg for a typical
subject.
[0227] For administration, modulators of the present invention can
be administered at a rate determined by the LD-50 of the modulator,
and the side effects of the modulator at various concentrations, as
applied to the mass and overall health of the subject.
Administration can be accomplished via single or divided doses.
IX. Gene Therapy Applications
[0228] A variety of human diseases can be treated by therapeutic
approaches that involve stably introducing a gene into a human cell
such that the gene is transcribed and the gene product is produced
in the cell. Diseases amenable to treatment by this approach
include inherited diseases, including those in which the defect is
in a single or multiple genes. Gene therapy is also useful for
treatment of acquired diseases and other conditions. For
discussions on the application of gene therapy towards the
treatment of genetic as well as acquired diseases, see, Miller,
Nature 357:455-460 (1992); and Mulligan, Science 260:926-932
(1993).
[0229] In the context of the present invention, gene therapy can be
used for treating a variety of disorders and/or diseases in which
the polynucleotides and polypeptides of the invention has been
implicated. For example, compounds, including polynucleotides, can
be identified by the methods of the present invention as effective
in treating a mental disorder. Introduction by gene therapy of
these polynucleotides can then be used to treat, e.g., mental
disorders including mood disorders and psychotic disorders.
A. Vectors for Gene Delivery
[0230] For delivery to a cell or organism, the polynucleotides of
the invention can be incorporated into a vector. Examples of
vectors used for such purposes include expression plasmids capable
of directing the expression of the nucleic acids in the target
cell. In other instances, the vector is a viral vector system
wherein the nucleic acids are incorporated into a viral genome that
is capable of transfecting the target cell. In a preferred
embodiment, the polynucleotides can be operably linked to
expression and control sequences that can direct expression of the
gene in the desired target host cells. Thus, one can achieve
expression of the nucleic acid under appropriate conditions in the
target cell.
B. Gene Delivery Systems
[0231] Viral vector systems useful in the expression of the nucleic
acids include, for example, naturally occurring or recombinant
viral vector systems. Depending upon the particular application,
suitable viral vectors include replication competent, replication
deficient, and conditionally replicating viral vectors. For
example, viral vectors can be derived from the genome of human or
bovine adenoviruses, vaccinia virus, herpes virus, adeno-associated
virus, minute virus of mice (MVM), HIV, sindbis virus, and
retroviruses (including but not limited to Rous sarcoma virus), and
MoMLV. Typically, the genes of interest are inserted into such
vectors to allow packaging of the gene construct, typically with
accompanying viral DNA, followed by infection of a sensitive host
cell and expression of the gene of interest.
[0232] As used herein, "gene delivery system" refers to any means
for the delivery of a nucleic acid of the invention to a target
cell. In some embodiments of the invention, nucleic acids are
conjugated to a cell receptor ligand for facilitated uptake (e.g.,
invagination of coated pits and internalization of the endosome)
through an appropriate linking moiety, such as a DNA linking moiety
(Wu et al., J. Biol. Chem. 263:14621-14624 (1988); WO 92/06180).
For example, nucleic acids can be linked through a polylysine
moiety to asialo-oromucocid, which is a ligand for the
asialoglycoprotein receptor of hepatocytes.
[0233] Similarly, viral envelopes used for packaging gene
constructs that include the nucleic acids of the invention can be
modified by the addition of receptor ligands or antibodies specific
for a receptor to permit receptor-mediated endocytosis into
specific cells (see, e.g., WO 93/20221, WO 93/14188, and WO
94/06923). In some embodiments of the invention, the DNA constructs
of the invention are linked to viral proteins, such as adenovirus
particles, to facilitate endocytosis (Curiel et al., Proc. Natl.
Acad. Sci. U.S.A. 88:8850-8854 (1991)). In other embodiments,
molecular conjugates of the instant invention can include
microtubule inhibitors (WO/9406922), synthetic peptides mimicking
influenza virus hemagglutinin (Plank et al., J. Biol. Chem.
269:12918-12924 (1994)), and nuclear localization signals such as
SV40 T antigen (WO93/19768).
[0234] Retroviral vectors are also useful for introducing the
nucleic acids of the invention into target cells or organisms.
Retroviral vectors are produced by genetically manipulating
retroviruses. The viral genome of retroviruses is RNA. Upon
infection, this genomic RNA is reverse transcribed into a DNA copy
which is integrated into the chromosomal DNA of transduced cells
with a high degree of stability and efficiency. The integrated DNA
copy is referred to as a provirus and is inherited by daughter
cells as is any other gene. The wild type retroviral genome and the
proviral DNA have three genes: the gag, the pol and the env genes,
which are flanked by two long terminal repeat (LTR) sequences. The
gag gene encodes the internal structural (nucleocapsid) proteins;
the pol gene encodes the RNA directed DNA polymerase (reverse
transcriptase); and the env gene encodes viral envelope
glycoproteins. The 5' and 3' LTRs serve to promote transcription
and polyadenylation of virion RNAs. Adjacent to the 5' LTR are
sequences necessary for reverse transcription of the genome (the
tRNA primer binding site) and for efficient encapsulation of viral
RNA into particles (the Psi site) (see, Mulligan, In: Experimental
Manipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann
et al., Cell 33:153-159 (1983); Cone and Mulligan, Proceedings of
the National Academy of Sciences, U.S.A., 81:6349-6353 (1984)).
[0235] The design of retroviral vectors is well known to those of
ordinary skill in the art. In brief, if the sequences necessary for
encapsidation (or packaging of retroviral RNA into infectious
virions) are missing from the viral genome, the result is a
cis-acting defect which prevents encapsidation of genomic RNA.
However, the resulting mutant is still capable of directing the
synthesis of all virion proteins. Retroviral genomes from which
these sequences have been deleted, as well as cell lines containing
the mutant genome stably integrated into the chromosome are well
known in the art and are used to construct retroviral vectors.
Preparation of retroviral vectors and their uses are described in
many publications including, e.g., European Patent Application EPA
0 178 220; U.S. Pat. No. 4,405,712, Gilboa Biotechniques 4:504-512
(1986); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan
Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Eglitis et al.
Biotechniques 6:608-614 (1988); Miller et al. Biotechniques
7:981-990 (1989); Miller (1992) supra; Mulligan (1993), supra; and
WO 92/07943.
[0236] The retroviral vector particles are prepared by
recombinantly inserting the desired nucleotide sequence into a
retrovirus vector and packaging the vector with retroviral capsid
proteins by use of a packaging cell line. The resultant retroviral
vector particle is incapable of replication in the host cell but is
capable of integrating into the host cell genome as a proviral
sequence containing the desired nucleotide sequence. As a result,
the patient is capable of producing, for example, a polypeptide or
polynucleotide of the invention and thus restore the cells to a
normal phenotype.
[0237] Packaging cell lines that are used to prepare the retroviral
vector particles are typically recombinant mammalian tissue culture
cell lines that produce the necessary viral structural proteins
required for packaging, but which are incapable of producing
infectious virions. The defective retroviral vectors that are used,
on the other hand, lack these structural genes but encode the
remaining proteins necessary for packaging. To prepare a packaging
cell line, one can construct an infectious clone of a desired
retrovirus in which the packaging site has been deleted. Cells
comprising this construct will express all structural viral
proteins, but the introduced DNA will be incapable of being
packaged. Alternatively, packaging cell lines can be produced by
transforming a cell line with one or more expression plasmids
encoding the appropriate core and envelope proteins. In these
cells, the gag, pol, and env genes can be derived from the same or
different retroviruses.
[0238] A number of packaging cell lines suitable for the present
invention are also available in the prior art. Examples of these
cell lines include Crip, GPE86, PA317 and PG13 (see Miller et al.,
J. Virol. 65:2220-2224 (1991)). Examples of other packaging cell
lines are described in Cone and Mulligan Proceedings of the
National Academy of Sciences, USA, 81:6349-6353 (1984); Danos and
Mulligan Proceedings of the National Academy of Sciences, USA,
85:6460-6464 (1988); Eglitis et al. (1988), supra; and Miller
(1990), supra.
[0239] Packaging cell lines capable of producing retroviral vector
particles with chimeric envelope proteins may be used.
Alternatively, amphotropic or xenotropic envelope proteins, such as
those produced by PA317 and GPX packaging cell lines may be used to
package the retroviral vectors.
[0240] In some embodiments of the invention, an antisense
polynucleotide is administered which hybridizes to a gene encoding
a polypeptide of the invention. The antisense polypeptide can be
provided as an antisense oligonucleotide (see, e.g., Murayama et
al., Antisense Nucleic Acid Drug Dev. 7:109-114 (1997)). Genes
encoding an antisense nucleic acid can also be provided; such genes
can be introduced into cells by methods known to those of skill in
the art. For example, one can introduce an antisense nucleotide
sequence in a viral vector, such as, for example, in hepatitis B
virus (see, e.g., Ji et al., J. Viral Hepat. 4:167-173 (1997)), in
adeno-associated virus (see, e.g., Xiao et al., Brain Res.
756:76-83 (1997)), or in other systems including, but not limited,
to an HVJ (Sendai virus)-liposome gene delivery system (see, e.g.,
Kaneda et al., Ann. NY Acad. Sci. 811:299-308 (1997)), a "peptide
vector" (see, e.g., Vidal et al., CR Acad. Sci. III 32:279-287
(1997)), as a gene in an episomal or plasmid vector (see, e.g.,
Cooper et al., Proc. Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997),
Yew et al. Hum Gene Ther. 8:575-584 (1997)), as a gene in a
peptide-DNA aggregate (see, e.g., Niidome et al., J. Biol. Chem.
272:15307-15312 (1997)), as "naked DNA" (see, e.g., U.S. Pat. Nos.
5,580,859 and 5,589,466), in lipidic vector systems (see, e.g., Lee
et al., Crit. Rev Ther Drug Carrier Syst. 14:173-206 (1997)),
polymer coated liposomes (U.S. Pat. Nos. 5,213,804 and 5,013,556),
cationic liposomes (Epand et al., U.S. Pat. Nos. 5,283,185;
5,578,475; 5,279,833; and 5,334,761), gas filled microspheres (U.S.
Pat. No. 5,542,935), ligand-targeted encapsulated macromolecules
(U.S. Pat. Nos. 5,108,921; 5,521,291; 5,554,386; and
5,166,320).
[0241] Upregulated transcripts listed in the biomarker tables
herein which are correlated with mental disorders may be targeted
with one or more short interfering RNA (siRNA) sequences that
hybridize to specific sequences in the target, as described above.
Targeting of certain brain transcripts with siRNA in vivo has been
reported, for example, by Zhang et al., J. Gene. Med., 12:1039-45
(2003), who utilized monoclonal antibodies against the transferrin
receptor to facilitate passage of liposome-encapsulated siRNA
molecules through the blood brain barrier. Targeted siRNAs
represent useful therapeutic compounds for attenuating the
over-expressed transcripts that are associated with disease states,
e.g., MDD, BP, and other mental disorders.
[0242] In another embodiment, conditional expression systems, such
as those typified by the tet-regulated systems and the RU-486
system, can be used (see, e.g., Gossen & Bujard, PNAS 89:5547
(1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al.,
Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155
(1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)).
These systems impart small molecule control on the expression of
the target gene(s) of interest.
[0243] In another embodiment, stem cells engineered to express a
transcript of interest can implanted into the brain.
C. Pharmaceutical Formulations
[0244] When used for pharmaceutical purposes, the vectors used for
gene therapy are formulated in a suitable buffer, which can be any
pharmaceutically acceptable buffer, such as phosphate buffered
saline or sodium phosphate/sodium sulfate, Tris buffer, glycine
buffer, sterile water, and other buffers known to the ordinarily
skilled artisan such as those described by Good et al. Biochemistry
5:467 (1966).
[0245] The compositions can additionally include a stabilizer,
enhancer, or other pharmaceutically acceptable carriers or
vehicles. A pharmaceutically acceptable carrier can contain a
physiologically acceptable compound that acts, for example, to
stabilize the nucleic acids of the invention and any associated
vector. A physiologically acceptable compound can include, for
example, carbohydrates, such as glucose, sucrose or dextrans;
antioxidants, such as ascorbic acid or glutathione; chelating
agents; low molecular weight proteins or other stabilizers or
excipients. Other physiologically acceptable compounds include
wetting agents, emulsifying agents, dispersing agents, or
preservatives, which are particularly useful for preventing the
growth or action of microorganisms. Various preservatives are well
known and include, for example, phenol and ascorbic acid. Examples
of carriers, stabilizers, or adjuvants can be found in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Philadelphia,
Pa., 17th ed. (1985).
D. Administration of Formulations
[0246] The formulations of the invention can be delivered to any
tissue or organ using any delivery method known to the ordinarily
skilled artisan. In some embodiments of the invention, the nucleic
acids of the invention are formulated in mucosal, topical, and/or
buccal formulations, particularly mucoadhesive gel and topical gel
formulations. Exemplary permeation enhancing compositions, polymer
matrices, and mucoadhesive gel preparations for transdermal
delivery are disclosed in U.S. Pat. No. 5,346,701.
E. Methods of Treatment
[0247] The gene therapy formulations of the invention are typically
administered to a cell. The cell can be provided as part of a
tissue, such as an epithelial membrane, or as an isolated cell,
such as in tissue culture. The cell can be provided in vivo, ex
vivo, or in vitro.
[0248] The formulations can be introduced into the tissue of
interest in vivo or ex vivo by a variety of methods. In some
embodiments of the invention, the nucleic acids of the invention
are introduced into cells by such methods as microinjection,
calcium phosphate precipitation, liposome fusion, or biolistics. In
further embodiments, the nucleic acids are taken up directly by the
tissue of interest.
[0249] In some embodiments of the invention, the nucleic acids of
the invention are administered ex vivo to cells or tissues
explanted from a patient, then returned to the patient. Examples of
ex vivo administration of therapeutic gene constructs include Nolta
et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al.,
Seminars in Oncology 23 (1):46-65 (1996); Raper et al., Annals of
Surgery 223(2): 116-26 (1996); Dalesandro et al., J. Thorac. Cardi.
Surg., 11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad.
Sci. USA 93(1):402-6 (1996).
X. Diagnosis of Mood Disorders and Psychotic Disorders
[0250] The present invention also provides methods of diagnosing
mood disorders (such as major depression or bipolar disorder),
psychotic disorders (such as schizophrenia), or a predisposition of
at least some of the pathologies of such disorders. Diagnosis
involves determining the level of a polypeptide or polynucleotide
of the invention in a patient and then comparing the level to a
baseline or range. Typically, the baseline value is representative
of a polypeptide or polynucleotide of the invention in a healthy
person not suffering from a mood disorder or a psychotic disorder
or under the effects of medication or other drugs. Variation of
levels of a polypeptide or polynucleotide of the invention from the
baseline range (either up or down) indicates that the patient has a
mood disorder or a psychotic disorder or at risk of developing at
least some aspects of a mood disorder or a psychotic disorder. In
some embodiments, the level of a polypeptide or polynucleotide of
the invention are measured by taking a blood, urine or tissue
sample from a patient and measuring the amount of a polypeptide or
polynucleotide of the invention in the sample using any number of
detection methods, such as those discussed herein.
[0251] Antibodies can be used in assays to detect differential
protein expression in patient samples, e.g., ELISA assays,
immunoprecipitation assays, and immunohistochemical assays. PCR
assays can be used to detect expression levels of nucleic acids, as
well as to discriminate between variants in genomic structure or
transcription, such as the FGFR splice variants shown in FIG. 1 and
described in the Examples.
[0252] In the case where absence of gene expression is associated
with a disorder, the genomic structure of a gene can be evaluated
with known methods such as PCR to detect deletion or insertion
mutations associated with disease suspectibility. Conversely, the
presence of mRNA or protein corresponding to a particular gene
would indicate that an individual does not have the genetic
mutation associated with the lack of gene expression or the
associated disorder. Thus, diagnosis can be made by detecting the
presence or absence of mRNA or protein, or by examining the genomic
structure of the gene.
[0253] Single nucleotide polymorphism (SNP) analysis is also useful
for detecting differences between alleles of the polynucleotides
(e.g., genes) of the invention. SNPs linked to genes encoding
polypeptides of the invention are useful, for instance, for
diagnosis of diseases (e.g., mood disorders such as bipolar
disease, major depression, and schizophrenia disorders) whose
occurrence is linked to the gene sequences of the invention. For
example, if an individual carries at least one SNP linked to a
disease-associated allele of the gene sequences of the invention,
the individual is likely predisposed for one or more of those
diseases. If the individual is homozygous for a disease-linked SNP,
the individual is particularly predisposed for occurrence of that
disease. In some embodiments, the SNP associated with the gene
sequences of the invention is located within 300,000; 200,000;
100,000; 75,000; 50,000; or 10,000 base pairs from the gene
sequence.
[0254] Various real-time PCR methods can be used to detect SNPs,
including, e.g., Taqman or molecular beacon-based assays (e.g.,
U.S. Pat. Nos. 5,210,015; 5,487,972; Tyagi et al., Nature
Biotechnology 14:303 (1996); and PCT WO 95/13399 are useful to
monitor for the presence of absence of a SNP. Additional SNP
detection methods include, e.g., DNA sequencing, sequencing by
hybridization, dot blotting, oligonucleotide array (DNA Chip)
hybridization analysis, or are described in, e.g., U.S. Pat. No.
6,177,249; Landegren et al., Genome Research, 8:769-776 (1998);
Botstein et al., Am J Human Genetics 32:314-331 (1980); Meyers et
al., Methods in Enzymology 155:501-527 (1987); Keen et al., Trends
in Genetics 7:5 (1991); Myers et al., Science 230:1242-1246 (1985);
and Kwok et al., Genomics 23:138-144 (1994). PCR methods can also
be used to detect deletion/insertion polymorphisms, such as the
deletion polymorphism of the PSPHL gene associated with
suspectibility to BP.
[0255] In some embodiments, the level of the enzymatic product of a
polypeptide or polynucleotide of the invention is measured and
compared to a baseline value of a healthy person or persons.
Modulated levels of the product compared to the baseline indicates
that the patient has a mood disorder or a psychotic disorder or is
at risk of developing at least some aspects of a mood disorder or a
psychotic disorder. Patient samples, for example, can be blood,
urine or tissue samples. In some cases, one skilled in the art
could use expression of genes in readily obtainable cells, e.g.,
lymphocytes, as a proxy for evaluation expression of those genes in
one or more regions of the brain.
[0256] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended
claims.
Example 1
Differential Expression of Genes Associated with Suicide in Both BP
and MDD Subjects
[0257] Previous studies have investigated genes associated with
mood disorders and suicidal tendencies, using microarrays and PCRs
to analzye gene expression (Sibille et al>, 2004; Yanagi M, et
al., J Hum Genet., 50(4):210-6 (2005)). Neither investigation,
however, used a stringent analysis of suicide compared to mood
disorder and suicide compared to controls to detect genes that
might be most representative of suicide. This Example describes
microarray gene expression profiles in the amygdala, anterior
cingulate, and cerebellum in postmortem brains from BPD and MDD
patients that committed suicide, focusing on mRNA expression levels
of the molecules which regulate white-matter, oligodendrocyte,
myelin, and other pathways. The genes identified here may be used
as biomarkers for detecting and treating suicidal behavior.
[0258] Genes were discovered by selecting subjects run on U133P
chips with mRNA quality >1.4, pH>6.6, and AFS=0, Suicide
victims with a mood disorder (n=14) were compared to non-suicide
victims suffering from a mood disorder (n=9) and controls (n=27).
The age and pH were different between groups, and were entered as a
covariate in ANCOVA. Myelin and oligodendrocyte gene expression
were found to be dysregulated in suicidal mood disorder subjects
compared to non-suicidal mood disorder subjects or controls in the
amygdala. The complete list of identified genes which were
dysregulated in suicidal patients versus non-suicidal mood disorder
patients is presented in Table 1A. Table 1B lists genes which were
dysregulated in suicidal MDD patients versus non-suicidal MDD
patients, and genes which were dysregulated in suicidal MDD
patients versus control patients.
[0259] A similar study was performed using brains of MDD subjects
who were known to be drug abusers and comparing gene expression in
those subjects to gene expression in MDD subjects who were not
substance abusers, as well as to control subjects. Table 1C is a
list of genes which were shown to be dysregulated in
substance-abusing MDD patients versus MDD patients who were not
substance abusers. Table 1C also shows genes which were
dysregulated in substance-abusing MDD patients versus control
subjects.
[0260] In a related study, two cohorts were used to study and
compare gene expression in BP and MDD patients versus normal
patients. Cohort A consisted of 7 controls, 6 BPD patients, and 9
MDD patients. Cohort B included 7 controls and 5 MDD patients. The
subjects were selected to avoid possible confounding effects of
agonal events, tissue pH, RNA integrity, gender and age. The
results, summarized in FIGS. 5-8, show that changes were observed
in the expression levels of GPCRs and molecules regulating cAMP-
and phosphatidylinositol signaling pathways in the cerebral
cortices, especially in the anterior cingulate cortex, of mood
disorder patients. Expression levels of molecules acting as
negative regulators in cAMP signaling were increased in BPD, while
molecules activating cAMP signaling were not altered. Contrasted
with the changes in BPD, molecules suppressing cAMP signaling were
decreased in MDD. Expression levels of inositol
polyphosphate-1-phosphatase and phosphatidylinositol 3-kinases were
altered in BPD, while protein kinase C beta-1, inositol
triphosphate receptor-1, inositol polyphosphate-5-phosphatase were
increased in MDD. Two orphan GPCR genes, GPRC5B and GPR37,
consistently showed significant decreases in the three cortices in
MDD, and significant increases in anterior cingulate cortex of BPD.
Measuring differences in the expression of the genes identified in
FIGS. 5-8 is a useful tool for determining whether a subject is
suffering from a particular mental illness, particularly BP or
MDD.
Example 2
Identification of Lithium Responsive Genes which are Dysregulated
in BPD
[0261] This Example demonstrates that certain genes in non-human
primates (healthy rhesus macaque monkey) are differentially
expressed in response to treatment with the mood-stabilizing drug,
lithium (Li), the drug of choice for the treatment of BP. Gene
expression profiling was carried out on the anterior cingulate
cortex (AnCg), dorsolateral prefrontal cortex (DLPFC), hippocampus
(HC) and amygdala (AMY) of rhesus macaque monkeys, using the gene
expression detection methods described herein, and compared to the
human postmortem results described above. Table 2A shows the
lithium-responsive genes which had been previously identified in
the literature and which were confirmed by the present
investigation. Table 2B shows genes that are newly identified as
lithium-responsive in primates and which are also dysregulated in
human subjects with bipolar disorder.
Example 3
FGFR2 Variant Differences in Mood Disorders
[0262] The FGF receptor 2 (FGFR2) transcript is consistently found
to be decreased in several brain areas of depressed subjects (see,
e.g., U.S. patent application Ser. No. 10/701,263, filed Nov. 3,
2003, published as U.S. Pat. Publ. No. 20040152111-A1 on Aug. 5,
2004). The human FGFR2 gene contains 19 exons and produces as many
as 13 splice variants. These variants fall into three main
functional classes: first, variants that lack the transmembrane and
tyrosine kinase domain which are thought to be soluble receptors;
second, variants that contain the Ig IIIc type domain encoded by
exon 9; and third, variants that contain the Ig IIIb type domain
encoded by exon 8. The Ig III type domain confers ligand
specificity and thus these latter two variants have different
pharmacological profiles based on their use of the IIIc or IIIb
domain. This Example describes PCR-based measurements of exons
present in total RNA derived from human cortex (dorsolateral
prefrontal and anterior cingulated) and hippocampus.
[0263] Methods Post-mortem human brans were obtained and dissected
as previously described (Evans et al., PNAS 101(43):15506-11
(2004)). RNA for microarray analysis and semi-quantitative RT-PCR
was extracted from discrete brain regions using Trizol.
[0264] Microarray data was generated with a combination of
Affymetrix 133A anti 133plus 2.0 chips and was analyzed using a
custom probe mapping file based on a recent generation of the
RefSeq database
(http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF).
Each biological sample was run independently at two sites
(University of California-Irvine, University of run
California-Davis or the University of Michigan). Probe set signals
were calculated using RMA (Bolstad et al., Bioinformatics
19(2):185-93 (2003)) and statistical comparisons were made after
median centering the RMA data separately for each technical block
(across independent cohorts and sites). P-values were constructed
from t-tests between cases and controls. The final subject
composition included 13 major depressive subjects and 16 controls.
All were free of agonal factors, had brain pH measurements greater
than 6.8, and met other quality measures.
[0265] Semi-quantitative RT-PCR data were generated with exon
specific primers and the SVBR green method using the BioRad
iCycler. All primer pairs used in quantitative reactions were
tested for efficiency and determined to be at approximately 100%.
Cycle threshold (Ct) values were chosen within the linear range of
amplification and were normalized to total cDNA concentration as
determined by the PicoGreen assay (Molecular Probes). Contaminating
genomic DNA was eliminated with DNAse prior to cDNA synthesis with
a mixture of random hexamers and poly-T primers and was confirmed
eliminated by amplifying fragments across smaller introns (axon 3
to exon 4 and exon 7 to exon 10). No intron-containing amplicons
were detected.
[0266] Results The results of the above-mentioned study are
partially summarized in FIG. 1, which shows the differential
expression of exons 5 and 11 in depressed versus control subjects.
More specifically, the results show that the ratio of expression of
exon 5 to exon 11 is significantly lowered in MDD patients,
particularly in the DLPFC region. A similar analysis of the
expression of exon 9 (coding for the IIIc variant) showed that exon
9 expression in the AnCg and HC regions was decreased in MDD
subjects.
Example 4
Effect of Injection of FGF2, FGL Peptide (NCAM), and Peptide
Inhibitor of FGF Receptors on the Behavior of Rodents
[0267] A. Microinjection of FGF2
[0268] This set of experiments shows significant effects following
the microinjection of FGF2, using both the forced swim test (FST)
and the elevated plus maze (EPM) to evaluate depression in the
subject animals. In the FST, the FGF2-injected (n=12) animals
exhibited more swimming (t[23]=2.20, p<0.05) and less immobility
(t[23]=2.88, p<0.01) than controls (n=13). This is indicative of
less depression-like behavior in FGF2 animals. However, in the EPM,
the FGF2-injected animals spent significantly more time in the
closed arms (t[13]=3.18, p<0.01) and less time in the open arms
(t[13]=2.46, p<0.05). These results (FIG. 2) show that
anxiety-like behavior is increased after an acute injection of
FGF2.
[0269] B. Microinjection of NCAM (FGL Peptide)
[0270] This set of experiments shows a significant effect on animal
mood in the forced swim test after NCAM administration (amino acid
sequence=EVYVVAENQQGKSKA; see FIG. 3). Here, the NCAM-injected
animals (n=13) exhibited less immobility ([24]=2.13, p<0.05)
than controls (n=13). Again, this is an index of less
depression-like behavior. This is also in the same direction as the
FGF2 data, consistent with the fact that both FGF2 and NCAM
interact with the FGF receptor. Similarly, the NCAM-injected
animals spent more time (although not significantly more time) in
the closed arms and less time in the open arms, consistent with
increased anxiety-like behavior. NCAM-injected animals also spent
significantly less time in the center quadrant (t[18]=2.40,
p<0.05).
[0271] C. Microinjection of a Peptide Inhibitor of FGFR
[0272] This set of experiments shows a significant effect on animal
mood, using both the forced swim test and the elevated plus maze
test, after injection with an FGF system peptide inhibitor (amino
acid sequence=HFKDPKRLY). The results are shown in FIG. 4. In the
FST, the inhibitor-injected animals (n=7) exhibited significantly
less climbing (t[12]=2.06, p<0.05), less swimming (t[12]=1.92,
p<0.05) and more immobility (t[12]=3.58, p <0.005) than
controls (n=7). These results show that inhibition of the FGF
system can result in increased depression-like behavior. These
results confirm and advance the results of the previous data sets,
and are consistent with studies of the postmortem tissue of
individuals with major depression. The inhibitor-injected animals
also spent significantly more time in the center quadrant of the
EPM (T[7,7]=35.0, p=0.03). Although the same animals spent less
time in the closed arms and equal time in the open arms, indicative
of increased anxiety-like behavior, the lengths of time spent were
not significantly different. These observations are nevertheless
consistent with the conclusions drawn from the microinjection
studies above.
D. Administration of FGF2 Induces Long-Term Changes in Hippocampal
Gene Expression
[0273] Methods. Sprague-Dawley rats were injected with either
vehicle or FGF2 (20 ng/g, s.c.) the day after birth and sacrificed
after Morris water maze testing as adults. We assessed changes in
gene expression using both a candidate approach and a gene
discovery approach with laser-capture microdissection of the
dentate gyrus followed by microarray analyses.
[0274] Results. Rats injected with FGF2 performed significantly
better in learning and memory tests (e.g., 20 seconds on average to
find a hidden platform in Morris Water maze test versus 25 seconds
on average for vehicle-injected rats). Several genes associated
with neural plasticity were also found altered in the adult rats,
as shown by histochemical and RNA expression assays. For example,
expression of GAP-43, Rgs4, trkb, CCK, SST, and Vgf was increased,
while expression of NCAM was decreased.
Example 5
Anxiolytic Effect of Chronically Administered FGF2
[0275] Anxiety disorders have a high comorbidity with other
neuropsychiatric disorders including Major Depression (MD). This
Example shows that chronic FGF2 administration has an anxiolytic
effect in rats. Rats were placed into "high anxiety" (LR) or "low
anxiety" (HR) groups based on their behavior in a variety of motor
and behavioral tests. Both groups of animals were administered
either FGF2 (5 ng/g) or vehicle by intraperitoneal injection every
48 hours for 3 weeks. One day after the last FGF2 injection, all
animals were tested in the elevated plus-maze (EPM) and light-dark
(LD) anxiety test.
[0276] The apparatus for the EPM test is constructed of black
Plexiglass with four elevated open arms (70 cm from the floor, 45
cm long, and 12 cm wide). Illumination is provided by a 40-watt
desk lamp facing a wall and placed behind one of the closed arms.
The scientists put the animal inside the system. Animals that are
less anxious spend more time in the open arms whereas animals that
are more anxious spend less time in the open arms.
[0277] The LD test is conducted in a 30.times.60.times.30-cm
Plexiglas shuttle box with a translucent cover. Each box is divided
into two equal-sized compartments by a wall with a 12 cm-wide open
door. One compartment is painted white and brightly illuminated,
and the other is painted black with very dim light. The time each
rat spends in each compartment is monitored by rows of five
photocells located 2.5 cm above the grid floor of each compartment.
Animals that are less anxious spend more time in the light
compartment.
[0278] The results show that animals who received chronically
administered FGF2 are less anxious than animals who receive vehicle
(FIG. 9, top). The anxiety-reducing effects of FGF2 are clearly
more pronounced in rats who are inately more anxious (LR) prior to
the FGF2 regimen (FIG. 9, top).
[0279] To further understand the relationship between FGF2
expression and anxiety, FGF2 expression was measured in the CA-2
region of the hippocampus of rat brains taken from rats which
exhibited varying levels of anxiety (as measured by the EPM test).
The results (FIG. 10) show that FGF2 levels are inversely related
to anxiety, i.e., higher levels of inate FGF2 expression in rats
correlate with lower levels of anxiety.
[0280] Taken as a whole, the Example shows that chronic FGF2
administration is useful for alleviating symptoms of anxiety in
anxious animals and in subjects who are suffering from disorders
such as MDD which are associated with anxiety. The data also shows
that detection of FGF2 levels is useful for diagnosing anxiety or
characterizing disorders associated with anxiety, such as Major
Depression Disorder.
Example 6
Differential Regulation of FGFR Splice Variants Associated with
Chronic Stress
[0281] In the adult CNS, fibroblast growth factor receptor 2
(FGFR2) and fibroblast growth factor receptor 3 (FGFR3) are
differentially distributed. The mRNA of these receptors undergoes
alternative splicing in the exons coding for the carboxyl terminus
of the Ig-like domain III. This mutually exclusive mRNA splicing
produces two isoforms of FGFR2 and FGFR3 with significantly
different ligand binding profiles: one isoform expressing exon E1b
(FGFR2b/FGFR3b), and one isoform expressing exon IIIc
(FGFR2c/FGFR3c). Exon selection is strictly tissue-dependent during
development with exon IIIb expressed in epithelial lineages and
exon IIIc expressed in mesenchymal lineages. Cell cycle-dependent
IIIb to IIIc switches, however, have been induced in vitro by the
exogenous addition of FGF1 and FGF2. This Example shows that
chronic stress induces a decrease in the FGFR2 exon IIIc:IIIb
splice variant expression ratio.
[0282] Animals. Twenty-four male Sprague-Dawley rats weighing
220-250 g were ordered from Charles River (Wilmington, Mass.) and
remained undisturbed for one week to acclimatize to housing
conditions. The animals were housed in pairs on a 12 h light-dark
cycle (lights on 6:00 A.M.) with food and water available ad
libitum. All experiments were conducted in accordance with the NIH
Guide for the Care and Use of Animals and the University Committee
on the Use and Care of Animals.
[0283] FGF2 injection treatments and chronic unpredictable stress
(CUS) conditions. Half of the rats (n=12) were administered vehicle
(0.1M PBS with 100 .mu.g/mL bovine serum albumin), and the other
half (n=12) were administered human recombinant FGF2 dissolved in
vehicle in 5 ng/g dosages (Sigma, St. Louis, Mo.) every 48 hours
for three weeks. All treatments were injected intra-peritonealy.
During the same three week period as the FGF2 treatments, the
vehicle group was either handled (n=6) or exposed to CUS (n=6).
Likewise, the FGF2 injected group was either handled (n=6) or
exposed to CUS (n=6). The animals were exposed to the following
chronic unpredictable stressors (described in Isgor et al. (2004)):
ether (30 s), cold (2 h), noise (15 m), isolation (24 h), or
restraint (2 h). The stressors were randomized to avoid
habituation; sessions occurred once each day in either the morning
or afternoon. The 2.times.2 (condition by treatment) design divided
the subjects into nonstressed/vehicle (NS/V), nonstressed/FGF2
injection (NS/F), stressed/vehicle (S/V), and stressed/FGF2
injection (S/F) groups.
[0284] Forced swim testing. To test for possible FGF2 injection
effects on anti-depressant behavior for other studies, all animals
were subjected to forced swim testing according to Lucki (1997) 24
h after the termination of the FGF2 treatments and CUS
conditions.
[0285] Brains. The rats were sacrificed by the faculty by
decapitation three days after termination of the forced swim
testing. Brains were then removed and snap frozen in isopentane at
-80.degree. C.
[0286] Total RNA extraction. Gross dissections were performed on
the frontal cortex of all brains. Total RNA extraction was executed
following the reagent manufacturers' instructions. Tissues were
homogenized in TRIzol (Invitrogen, Carlsbad, Calif.; monophasic
phenol and guanidine isothiocyanate). Phase separation and RNA
precipitation were carried out with chloroform and 2-propanol
followed by centrifugation. RNA samples were purified using the
RNeasy Mini Kit (Qiagen, Valencia, Calif.), repeatedly washing
samples through spin columns. Pure RNA samples were reconstituted
with RNase-free water, followed by an integrity analysis for
28s/18s ribosomal RNA peaks with the 2100 Bioanalyzer and LabChip
(Agilent Technologies, Palo Alto, Calif.) system. Using the
Bioanalyzer's concentration readings, the samples were normalized
to 25 ng/.mu.L of total RNA per subject and stored at -80.degree.
C.
[0287] cDNA synthesis. cDNA was synthesized using the iScript cDNA
Synthesis Kit (Bio-Rad, Hercules, Calif.). All 24 RNA samples were
reversed transcribed with iScript Reverse Transcriptase (Bio-Rad)
and primed with oligo(dT) and random hexamer primers. Reaction
mixes were incubated in an iCycler PCR unit (Bio-Rad) according to
the manufacturer's standard 40 min protocol. The double-stranded
cDNA solutions were then analyzed for quality and concentration
using Invitrogen's Quant-iT PicoGreen dsDNA kit. 10-fold serial
dilutions were prepared with 2 .mu.g/1 mL (high range) and 50 ng/mL
(low range) stock DNA to generate standard curves. The
fluorescently labeled samples were analyzed with a 1420
Victor.sup.2 Multilabel Counter (EG&G Wallac, Wellesley, Mass.)
using the basic Fluorescein protocol. Total cDNA samples were
further normalized to fit the linear regression of the high range
standard curve.
[0288] Real time RT-PCR primer design. Sequences for rat FGFR2 and
FGFR3 exons were obtained from NCBI's Entrez Gene database
(www.ncbi.nlm.nih.gov) and the Ensembl-Rat gene database
(www.ensembl.org). Exon sequences were analyzed using NCBI's
nucleotide and protein BLAST and were matched with their
corresponding protein products within the receptor structures (FIG.
11). FGFR2 and FGFR3 exon sequences were analyzed for secondary
structure using the Mfold nucleic acid folding web server. Probable
hairpin regions were noted for exclusion in primer design.
Optimized primer sequences (FIG. 11) were generated using the
Primer3 web-based software. All primers were 18-22 base pairs,
targeted amplicons of 75-150 bps, and purchased from Invitrogen's
Custom Primer Synthesis service. The primer sequences (50 nmol/mL)
were validated and tested for efficiency with 5-fold serial
dilutions of pooled cDNA using iQ SYBR Green detection on an
iCycler iQ Real Time RT-PCR unit (Bio-Rad).
[0289] Real time RT-PCR quantification. Real time
reverse-transcriptase-PCR (RT-PCR) amplification reactions were
performed to quantify relative abundances of mRNA (reverse
transcribed to cDNA) of selected FGFR2 and R3 exons in each of the
four treatment by condition groups (n=6). iQ SYBR Green Supermix
detection was used on an iCycler iQ Real Time RT-PCR system
(Bio-Rad) according to the manufacturer's recommendations, with the
exception of preparing 19 .mu.L reactions instead of the instructed
50 .mu.L. Reference genes were omitted because total cDNA pools
were normalized. Reactions were run in duplicates, increasing each
group size to n=12. Two exons in juxtaposition were amplified per
plate for relative comparison (emphasis placed on exon IIIb and
exon IIIc). Reaction wells were arranged to equalize positional
representation amongst all groups. PCR protocol was as follows: hot
start at 95.degree. C. for 30 s, followed by 40 cycles of denature
at 95.degree. C. for 15 s, annealing at 60.degree. C. for 15 s, and
elongation at 72.degree. C. for 15 s. Florescence was quantified
after every cycle, and melt curve analysis was performed following
amplification to ensure single product reactions. Thorough
methodology is described by Kerman et al. (2006).
[0290] Data analysis Real time RT-PCR data was output as threshold
cycle (Ct) values, using Bio-Rad's iCycler iQ software's algorithm
to calculate the optimum fluorescence thresholds for reliable
detection (the mean florescence value of the first ten PCR cycles
plus 10 standard deviations). Essentially, lower Ct values indicate
higher amounts of initial target cDNA because fewer PCR cycles are
required to reach fluorescence thresholds. Ct values for all
reactions were then grouped and presented as means with standard
errors. Sample sizes were 9-12 per group due to outlier (.gtoreq.2
StDev from mean) exclusion. Mean fold changes were calculated using
a 2.sup..DELTA.Ct method (modification of technique described by
Livak and Schmittgen (2001)), comparing mean Ct values within one
variable (treatment effects within one condition or condition
effects within one treatment). This 2.sup..DELTA.Ct method assumes
equal primer efficiencies; however, for the purposes of quantifying
relative expression, it is acceptable if the primers are validated.
Exon IIIc to exon IIIb ratios were determined by calculating
individual 2.sup..DELTA.Ct values between corresponding reactions
of the two exons. Ratios were sorted similarly to the Ct value
groups and presented as mean ratios with standard errors.
Statistical significance tests for differences in multiple exon
mean Ct values and individual IIIc:IIIb ratios were performed using
two-factor ANOVA and Student's t-test for each comparison variable.
Significance level was set as p<0.05. All statistical analysis
was done in Microsoft Excel 2003.
[0291] Results. Chronic stress induced significant decreases in the
exon IIIc to exon IIIb (mutually exclusive expression) splice
variant ratio in the vehicle group (P<0.00004) and in the FGF2
injection group (P<0.005) (FIG. 12). While exon IIIc expression
remained relatively higher than IIIb in all groups, IIIb expression
increased significantly with stress while IIIc expression changed
only slightly. Thus, stress increases expression of the IIIb
variant relative to the IIIc variant for both FGFR2 and FGFR3. FGF2
injection did not alter the IIIc:IIIb ratio significantly in either
the NS or S group (P>0.1), nor did it significantly affect the
magnitude of IIIc:IIIb ratio changes caused by stress.
[0292] The results show that the affinity of FGFR2 or FGFR3 for
endogenous ligands such as FGF2 and FGF9 (which are differentially
expressed in MDD subjects) or for exogenous ligands (e.g.,
pharmacological peptides or other compounds) can be altered by
stress. The invention described herein provides methods of
detecting variations in FGFR2 and FGFR3 splicing and modifying
subject care accordingly. In another embodiment, the invention
provides methods for identifying and optimizing therapeutics for
treating depression and related ailments.
Example 7
Differential Regulation of Genes in the Locus Coeruleus and the
Dorsal Raphe in Subjects with Bipolar and Major Depression
Disorder
[0293] The Locus Coeruleus (LC) and the Dorsal Raphe (DR) are the
major sources for noradrenergic and serotonergic innervation of the
brain respectively. Dysregulation of these neurotransmitters has
been implicated in psychiatric disorders. This Example uses
postmortem brains of patients with MDD (N=12), BPD (N=6), and
healthy subjects (N=9) to contrast gene expression profiles in the
LC and DR regions of their brains. All subjects met inclusion
criteria of brain pH>6.6 and zero agonal factors. Total RNA
samples from laser capture microdissected LC and DR samples were
extracted, amplified, and probed with Affymetrix high density
oligonucleotide microarrays. Gene expression data were analyzed by
ANOVA of robust multichip average algorithm (p.ltoreq.0.1) and by
MAS5.0 "present" call algorithm (min.of 50% presences in one of the
3 health states). Genes meeting these criteria were analyzed using
Ingenuity Pathway Analysis (IPA). Compared to healthy individuals,
774 and 636 genes show altered expression in the LC and 627 and 656
genes show altered expression in the DR of MDD and BPD patients,
respectively.
[0294] LC gene expression patterns: The data is summarized in Table
3. Ingenuity Pathway Analysis revealed that 10 genes of the
glutamate receptor signaling pathway are significantly altered in
MDD (p<0.01) but not in BPD. Glutamate signaling gene expression
alterations are present in following synaptic compartments of the
locus coeruleus: glial cells, presynpatic neurons, and postsynaptic
neurons. This shows that glutamate signaling is altered in LC of
MDD patients. Glial transporters, glutamine synthetase, AMPA,
kainate, GRM1 and GRM7 are thus targets for treating glutamatergic
imbalance.
[0295] The expression of genes related to growth, i.e., fibroblast
growth factors, are also significantly altered in the LC of MDD
patients. Drugs that target FGFR3, TrkB receptor, growth hormone
receptor, or which mimic the actions of FGF-2, would increase
neurite outgrowth in the LC and reserve neuronal loss.
[0296] Genes that are almost exclusively expressed in glia are
significantly down-regulated. These genes are useful markers for
global glial alterations in MDD patients.
[0297] DR gene expression patterns. The data is summarized in Table
4. IPA analyses of the DR revealed alterations in the expression of
a number of genes in growth factor-related pathways in MDD.
Expression of most of the altered genes was downregulated.
Likewise, the expression of a number of genes in growth
factor-related pathways was downregulated in samples from the BP
cohort. However, these genes were distinct from those detected in
the MDD cohort. Several genes that were common to both disorders
were identified; their expression was altered in the same direction
in MDD and BPD subjects.
[0298] All publications, databases, Genbank sequences, patents, and
patent applications cited herein are hereby incorporated by
reference.
Sequence CWU 1
1
36121DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 2 amplicon real time reverse-transcriptase-PCR
amplification forward primer R2 F 1gccgtgatca gttggactaa g
21221DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 2 amplicon real time reverse-transcriptase-PCR
amplification forward primer R3 F 2agaggcttca agtgctaaac g
21321DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 2 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R2 R 3tgtggcacct tttatctgga g
21420DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 2 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R3 R 4gcacactaaa gtggcacagc
20521DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 5 amplicon real time reverse-transcriptase-PCR
amplification forward primer R2 F 5tatggaaagt gtggtcccat c
21620DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 5 amplicon real time reverse-transcriptase-PCR
amplification forward primer R3 F 6tggagcttgg tcatggaaag
20721DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 5 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R2 R 7acatcaaggt ggtaggtgtg g
21820DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 5 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R3 R 8ggatgctgcc aaacttgttc
20921DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 6 amplicon real time reverse-transcriptase-PCR
amplification forward primer R2 F 9ggaggggacg tagaatttgt c
211018DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 6 amplicon real time reverse-transcriptase-PCR
amplification forward primer R3 F 10ccaaccagac agccgttc
181121DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 6 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R2 R 11cttcaggacc ttgaggtagg g
211219DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 6 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R3 R 12cattcacctc cacgtgctt
191321DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon IIIb amplicon real time reverse-transcriptase-PCR
amplification forward primer R2 F 13ggggataaat agctccaatg c
211421DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon IIIb amplicon real time reverse-transcriptase-PCR
amplification forward primer R3 F 14cctggatcag tgagaatgtg g
211524DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon IIIb amplicon real time reverse-transcriptase-PCR
amplification reverse primer R2 R 15catatatatt ccccagcatc catc
241620DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon IIIb amplicon real time reverse-transcriptase-PCR
amplification reverse primer R3 R 16aaattggtgg ctcgacagag
201721DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon IIIc amplicon real time reverse-transcriptase-PCR
amplification forward primer R2 F 17acaccacgga caaagaaatt g
211820DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon IIIc amplicon real time reverse-transcriptase-PCR
amplification forward primer R3 F 18tgtccttgca caatgtcacc
201921DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon IIIc amplicon real time reverse-transcriptase-PCR
amplification reverse primer R2 R 19atagaattac ccgccaagca c
212020DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon IIIc amplicon real time reverse-transcriptase-PCR
amplification reverse primer R3 R 20acgcagagtg atgggaaaac
202122DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 8 amplicon real time reverse-transcriptase-PCR
amplification forward primer R2 F 21gatcacagct tccccagatt ac
222220DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 8 amplicon real time reverse-transcriptase-PCR
amplification forward primer R3 F 22ggaggagctg atggaagttg
202321DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 8 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R2 R 23tcttggtcgt ggtcttcatt c
212420DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 8 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R3 R 24ccaccaggat gaagaggaag
202522DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 11 amplicon real time reverse-transcriptase-PCR
amplification forward primer R2 F 25agagaaggac ctgtctgacc tg
222620DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 11 amplicon real time reverse-transcriptase-PCR
amplification forward primer R3 F 26atgccactga caaggacctg
202721DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 11 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R2 R 27cccaggaggt tgatgatgtt c
212820DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 11 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R3 R 28cccccaacag gttaatgatg
202921DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 15 amplicon real time reverse-transcriptase-PCR
amplification forward primer R2 F 29gtccttcggg gtgttaatgt g
213020DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 15 amplicon real time reverse-transcriptase-PCR
amplification forward primer R3 F 30tcctttggtg tcctcctctg
203121DNAArtificial Sequencefibroblast growth factor receptor 2
(FGFR2) exon 15 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R2 R 31agttcattgg tgcagttggt g
213218DNAArtificial Sequencefibroblast growth factor receptor 3
(FGFR3) exon 15 amplicon real time reverse-transcriptase-PCR
amplification reverse primer R3 R 32cagttggctg gcttgtcc
1833200PRTArtificial Sequencepoly-Gly flexible linker 33Gly Gly Gly
Gly Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa20 25 30Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa35 40
45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa50
55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa85 90 95Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa100 105 110Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa130 135 140Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa145 150 155 160Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa165 170 175Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa180 185
190Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa195 2003415PRTArtificial
SequenceNCAM (FGL peptide) 34Glu Val Tyr Val Val Ala Glu Asn Gln
Gln Gly Lys Ser Lys Ala1 5 10 15359PRTArtificial SequenceFGF system
peptide inhibitor 35His Phe Lys Asp Pro Lys Arg Leu Tyr1
5364PRTArtificial SequenceDDX50 DEAD box 36Asp Glu Ala Asp1
* * * * *
References