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Title: Pollen allergy-related
gene 513
United States Patent: 6,986,990
Issued: January 17, 2006
Inventors:
Nagasu; Takeshi (Ibaraki, JP); Sugita; Yuji
(Kanagawa, JP); Fujishima; Tomoko (Tokyo, JP); Oshida; Tadahiro (Kanagawa,
JP); Obayashi; Masaya (Tokyo, JP); Gunji; Shigemichi (Tokyo, JP);
Obayashi; Izumi (Kanagawa, JP); Imai; Yukiho (Kanagawa, JP); Yoshida; Nei
(Kanagawa, JP); Ogawa; Kaoru (Kanagawa, JP); Matsui; Keiko (Kanagawa, JP)
Assignee: Genox Research, Inc. (JP)
Appl. No.: 019832
Filed: April 26, 2000
PCT Filed: April 26, 2000
PCT NO: PCT/JP00/02733
371 Date: March 28, 2002
102(e) Date: March 28, 2002
PCT PUB.NO.: WO00/65049
PCT PUB. Date: November 2, 2000
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Executive MBA in Pharmaceutical Management, U. Colorado
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Abstract
T cells from multiple subjects who showed
different cedar pollen specific IgE titers were prepared before and after
cedar pollen dispersal, and differential display analysis was performed to
screen for pollen antigen responsive genes. As a result, a novel gene was
successfully isolated whose expression is significantly higher in subjects
with high cedar pollen specific IgE titer. The inventors have found that
it is possible to use this gene to test for an allergic disease and to
screen for candidate compounds for therapeutic drugs for an allergic
disease.
Description of the Invention
TECHNICAL FIELD
The present invention relates to a gene
involved in allergic disease, in particular, pollen allergy, and a method
for testing for an allergic disease and a method for screening for a
candidate compound for a therapeutic drug for an allergy using the
expression of the gene as an index.
BACKGROUND ART
Allergic diseases including pollen
allergy are considered to be multifactorial diseases. These diseases
involve the expression of many different genes each of which is influenced
by various environmental factors. Thus, it is very difficult to identify a
specific gene which causes a specific disease.
Additionally, allergic diseases are thought to involve the expression of
mutated or defective genes, or overexpression or reduction of the specific
gene expression. To elucidate the role of gene expression in diseases, it
is necessary to understand how a gene is involved in triggering disease
onset and how expression of the gene is altered by external stimulants
such as drugs.
Recent developments in gene expression analysis techniques have made it
possible to analyze and compare the gene expression of many clinical
samples. Among these methods, the differential display (DD) method is
significantly useful. The differential display method was originally
developed by Liang and Pardee in 1992 (Science, 1992, 257:967-971). By
using this method, one can screen several tens or more different samples
at one time and it is possible to detect altered gene expression among the
samples. By revealing alterations in gene expression due to mutation as
well as time- and environment-dependent gene expression, it is expected to
provide important information about disease-related genes. Such genes
include those of which expression is influenced by environmental factors.
Among allergic diseases, pollen allergy has recently become more common.
It is considered that some genes whose expression is affected by pollen,
one of the environmental factors, are involved in cause of pollen allergy.
Based on this situation, it was hoped that genes involved in pollen
allergy could be isolated.
DISCLOSURE OF THE
INVENTION
An objective of the present invention is
to provide a gene involved in allergic disease, in particular, pollen
allergy. Another objective of the invention is to provide a method for
testing for an allergic disease and a method for screening for a candidate
compound for a therapeutic drug for an allergy using the expression of the
gene as an index.
According to the previously established techniques of the "Fluorescent DD
method" (T. Ito et al. FEBS Letter 351:231-236, 1994), the inventors
developed a new DD system that can analyze T-cell RNA samples prepared
from multiple human blood samples. The inventors isolated T cells from
blood samples of multiple subjects including pollen allergy patients
before and after exposure to pollen dispersal and conducted screening for
a gene showing differential expression between subjects showing different
cedar pollen-specific IgE titers, or before and after pollen dispersal.
The inventors thus isolated a novel gene (gene 513).
The inventors divided the subjects into those with a high IgE titer
against cedar pollen (cedar pollen allergy diathesis group) and others
(normal group) and measured the expression level of the isolated gene 513
between the two groups. The results showed that the gene expression level
was significantly higher in the cedar pollen allergy diathesis group
compared to the normal group. Based on these observations, the inventors
found that the gene expression level can be used as an index to test for
an allergic disease and to screen for candidate compounds for a
therapeutic drug for allergic diseases.
Therefore, the present invention relates to a gene showing high expression
in individuals having the allergy diathesis and a method for testing for
an allergic disease and a method for screening for a candidate compound
for a therapeutic drug for allergic diseases using the gene expression as
an index. More specifically, the present invention relates to:
(1) a nucleic acid molecule comprising
the nucleotide sequence of SEQ ID NO: 1;
(2) a nucleic acid molecule comprising
the coding region of the nucleotide sequence of SEQ ID NO: 1;
(3) a DNA that specifically hybridizes
to the nucleic acid molecule of (1) or (2) and that is at least 15
nucleotides long;
(4) a method for detecting the nucleic
acid molecule of (1) wherein said method uses the DNA of (3);
(5) a method for testing for an allergic
disease, said method comprising the steps of:
- (a) preparing T cells from a subject,
- (b) preparing an RNA sample from said
T cells,
- (c) conducting hybridization with said
RNA sample using the DNA of (3) as probe, wherein said DNA is labeled,
and
- (d) measuring the amount of RNA that
is derived from said subject and that hybridizes with said DNA and
comparing said amount with a control (normal group);
(6) a method for testing for an allergic
disease, said method comprising the steps of:
- (a) preparing T cells from a subject,
- (b) preparing an RNA sample from said
T cells,
- (c) synthesizing cDNA by conducting
reverse transcription reaction with said RNA sample,
- (d) conducting polymerase chain
reaction (PCR) using said cDNA as template and the DNA of (3) as primer,
and
- (e) comparing the amount of a DNA
amplified by said PCR with a control (normal group);
(7) the method of (6), wherein said PCR
is carried out by a PCR amplification monitoring method;
(8) the method of any one of (5) to (7),
wherein said T cells are prepared from peripheral blood of said subject;
(9) the method of any one of (5) to (8),
wherein said allergic disease is a cedar pollen allergy;
(10) a method for screening for a
candidate compound for a therapeutic drug for an allergic disease, said
method comprising the steps of:
- (a) administering a test compound to a
pollen allergy model animal and stimulating with pollen antigen,
- (b) preparing T cells from said model
animal,
- (c) preparing an RNA sample from said
T cells,
- (d) conducting hybridization with said
RNA sample using the DNA of (3) as probe, wherein said DNA is labeled,
- (e) measuring the amount of RNA that
is derived from said T cells and that hybridizes with said DNA, and
- (f) selecting a compound that reduces
the amount of said RNA measured in (e), compared to a control (a case
where said test compound is not administered);
(11) a method for screening for a
candidate compound for a therapeutic drug for an allergic disease, said
method comprising the steps of:
- (a) administering a test compound to a
pollen allergy model animal and stimulating with pollen antigen,
- (b) preparing T cells from said model
animal,
- (c) preparing an RNA sample from said
T cells,
- (d) synthesizing cDNA by conducting
reverse transcription reaction with said RNA sample,
- (e) conducting polymerase chain
reaction (PCR) using said cDNA as template and the DNA of (3) as primer,
and
- (f) selecting a compound that reduces
the amount of said DNA amplified in (e), compared to a control (a case
where said test compound is not administered);
(12) a method for screening for a
candidate compound for a therapeutic drug for an allergic disease, said
method comprising the steps of:
- (a) administering a test compound to a
pollen allergy model animal,
- (b) preparing lymphocytes from said
model animal,
- (c) stimulating said lymphocytes with
pollen antigen,
- (d) separating T cells from said
lymphocytes stimulated with said antigen,
- (e) preparing an RNA sample from said
T cells,
- (f) conducting hybridization with said
RNA sample using the DNA of (3) as probe, wherein said DNA is labeled,
- (g) measuring the amount of RNA that
is derived from said T cells and that hybridizes with said DNA, and
- (h) selecting a compound that reduces
the amount of said RNA measured in (g), compared to a control (a case
where said test compound is not administered);
(13) a method for screening for a
candidate compound for a therapeutic drug for an allergic disease, said
method comprising the steps of:
- (a) administering a test compound to a
pollen allergy model animal,
- (b) preparing lymphocytes from said
model animal,
- (c) stimulating said lymphocytes with
pollen antigen;
- (d) separating T cells from said
lymphocytes stimulated with said antigen,
- (e) preparing an RNA sample from said
T cells,
- (f) synthesizing cDNA by conducting
reverse transcription reaction with said RNA sample,
- (g) conducting polymerase chain
reaction (PCR) using said cDNA as template and the DNA of (3) as primer,
and
- (h) selecting a compound that reduces
the amount of said DNA amplified in (g), compared to a control (a case
where said test compound is not administered);
(14) a method for screening for a
candidate compound for a therapeutic drug for an allergic disease, said
method comprising the steps of:
- (a) preparing lymphocytes from a
pollen allergy model animal or from a human having a pollen allergy,
- (b) stimulating said lymphocytes with
pollen antigen in the presence of a test compound,
- (c) separating T cells from said
lymphocytes stimulated with said antigen,
- (d) preparing an RNA sample from said
T cells,
- (e) conducting hybridization with said
RNA sample using the DNA of (3) as probe, wherein said DNA is labeled,
- (f) measuring the amount of RNA that
is derived from said T cells and that hybridizes with said DNA, and
- (g) selecting a compound that reduces
the amount of said RNA measured in (f), compared to a control (a case
where said test compound is not administered);
(15) a method for screening for a
candidate compound for a therapeutic drug for an allergic disease, said
method comprising the steps of:
- (a) preparing lymphocytes from a
pollen allergy model animal or from a human having a pollen allergy,
- (b) stimulating said lymphocytes with
pollen antigen in the presence of a test compound,
- (c) separating T cells from said
lymphocytes stimulated with said antigen,
- (d) preparing an RNA sample from said
T cells,
- (e) synthesizing cDNA by conducting
reverse transcription reaction with said RNA sample,
- (f) conducting polymerase chain
reaction (PCR) using said cDNA as template and the DNA of (3) as primer,
and
- (g) selecting a compound that reduces
the amount of said DNA amplified in (f) compared to a control (a case
where said test compound is not administered);
(16) a method for screening for a
candidate compound for a therapeutic drug for an allergic disease, said
method comprising the steps of:
- (a) stimulating a T-cell line with a
lymphocyte-stimulating substance in the presence of a test compound,
- (b) preparing an RNA sample from said
stimulated T-cell line,
- (c) conducting hybridization with said
RNA sample using the DNA of (3) as probe, wherein said DNA is labeled,
- (d) measuring the amount of RNA that
is derived from said T-cell line and that hybridizes with said DNA, and
- (e) selecting a compound that reduces
the amount of said RNA measured in (d), compared to a control (a case
where said test compound is not administered);
(17) a method for screening for a
candidate compound for a therapeutic drug for an allergic disease, said
method comprising the steps of:
- (a) stimulating a T-cell line with a
lymphocyte-stimulating substance in the presence of a test compound,
- (b) preparing an RNA sample from said
stimulated T-cell line,
- (c) synthesizing cDNA by conducting
reverse transcription reaction with said RNA sample,
- (d) conducting polymerase chain
reaction (PCR) using said cDNA as template and the DNA of (3) as primer,
and
- (e) selecting a compound that reduces
the amount of said DNA amplified in (d), compared to a control (a case
where said test compound is not administered);
(18) the method of (10) or (11), wherein
said T cells are prepared from peripheral blood of said pollen allergy
model animal;
(19) the method of any one of (12) to
(15), wherein said lymphocytes are prepared from peripheral blood; and
(20) the method of any one of (10) to
(19), wherein said allergic disease is a cedar pollen allergy.
In the present invention allergic disease is a general term for diseases
that involve allergic reactions. More specifically, for a disease to be
considered allergic, the allergen there must be identified, there exists a
strong correlation between exposure to the allergen and the onset of the
pathological change, and the pathological change has been proven to have
an immunological mechanism. An immunological mechanism means that the T
cells show an immune response to allergen stimulation. Representative
allergic diseases are bronchial asthma, allergic rhinitis, atopic
dermatitis, pollen allergy, insect allergy, etc. Allergic diathesis is a
genetic factor which is inherited from allergic parents to children.
Familial allergic diseases are also called atopic diseases, and their
causative factor which can be inherited is atopic diathesis.
In the present invention, "nucleic acid molecule" includes DNA and RNA.
Also in the present invention, "testing for an allergic disease" includes
not only testing a patient who has been affected with the disease but also
testing a subject who has not affected with the disease for whether the
subject has an allergic diathesis or not.
The present invention relates to a novel gene 513 that correlates with IgE
production response against cedar pollen in individuals. The nucleotide
sequence of gene 513 cDNA which was found by the inventors is shown in SEQ
ID NO: 1.
The nucleotide sequence of gene 513 cDNA which was isolated by the
inventors is a partial sequence of 513 cDNA, but one skilled in the art
can usually isolate the full length cDNA of gene 513, based on the
sequence information of 513 cDNA described in SEQ ID NO: 1. Examples of
methods for isolating the full length cDNA include a method for screening
a T-cell cDNA library and such by hybridization using a gene 513-derived
sequence as a probe and a method for obtaining the full length of the cDNA
by screening libraries with an amplification product to be obtained having
a primer-specific size as an index using gene 513-derived sequences as
primers and using the T-cell cDNA library and such as a template. The
examples further include a method for extending the sequence of gene 513
by the RACE method (Frohman, M. A. et al.: Proc. Natl. Acad. Sci. USA, 85:
8992, 1988), in which mRNAs of T cells and such are converted into single
stranded cDNAs by using a gene 513-derived sequence as a primer, an
oligomer is attached to the end of the cDNA thus obtained, and then PCR is
carried out.
Herein, "nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO: 1" includes the full length gene 513 cDNA which can be isolated
from information of the 513 cDNA nucleotide sequence according to SEQ ID
NO: 1.
Gene 513 expression was significantly higher in the atopic diathesis group
(IgE titer against cedar pollen is 3.5 AU/ml or more) than in the non-atopic
diathesis group. Therefore, using gene 513 expression (including
transcription to mRNA and translation to protein) as an index, it may be
possible to test for an allergic disease and to screen for a candidate
compound for a therapeutic drug for allergic diseases.
As a disease that can be a target for the testing and treatment in the
present invention, cedar pollen allergy is particularly preferable.
Expression of gene 513 can be detected in testing for an allergic disease
in the present invention by using a hybridization technique with nucleic
acids which can hybridize with gene 513 as probes or by using a gene
amplification technique with DNAs which can hybridize with the gene of the
present invention as primers.
The probe or primer used for testing for a disease in the present
invention means nucleic acid molecules which specifically hybridize to
gene 513 and are at least 15 nucleotide long. Herein "specifically
hybridize" means that under normal hybridization conditions, preferably
stringent conditions, there is no significant cross hybridization with DNA
and/or RNA encoding other genes. Stringent condition means, for example,
carrying out hybridization using a probe and transfer membrane at 68° C.
in Express Hybridization Solution (CLONTECH) and a final wash of 0.1×SSC,
0.05% SDS at 50° C.
These nucleic acid molecules could be either synthetic or natural ones.
Also, labeled DNA is usually used as the probe for hybridization. The
labeling methods may be nick translation using DNA polymerase I, end
labeling using polynucleotide kinase, fill-in end labeling using Klenow
fragment (Berger S L, Kimmel A R. (1987) Guide to Molecular Cloning
Techniques, Method in Enzymology, Academic Press; Hames B D, Higgins S J
(1985) Genes Probes: A Practical Approach. IRL Press; Sambrook J, Fritsch
E F, Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn.
Cold Spring Harbor Laboratory Press), transcription labeling using RNA
polymerase (Melton D A, Krieg, P A, Rebagkiati M R, Maniatis T, Zinn K,
Green M R. (1984) Nucleic Acid Res., 12, 7035-7056), non-isotopic DNA
labeling techniques by incorporating modified nucleotides (Kricka L J.
(1992) Nonisotopic DNA Probing Techniques. Academic Press), etc.
For testing for an allergic disease using hybridization techniques, for
example, Northern hybridization, dot blot hybridization, or DNA micro
array technique may be used.
As for gene amplification techniques, for example, RT-PCR may be used.
Using the PCR amplification monitoring method during the gene
amplification step in RT-PCR as described in Example 8, one can obtain a
more accurate quantification of gene 513 expression.
In the PCR amplification monitoring method, the target (DNA or reverse
transcribed product of RNA) is hybridized to probes that are dual-labeled
at both ends with different fluorescent dyes which, in combination, cancel
out their fluorescence each other. When the PCR proceeds and the 5′-3′
exonuclease activity of Taq polymerase degrades the probe, the two
fluorescent dyes become distant from each other and the fluorescence comes
to be detected. The fluorescence is detected in real time. By
simultaneously measuring for a standard sample in which the copy number of
the target is known, it is possible to determine the copy number of the
target in the subject sample with the cycle number where PCR amplification
is linear (Holland, P. M. et al., 1991, Proc. Natl. Acad. Sci. USA
88:7276-7280; Livak, K. J. et al., 1995, PCR Methods and Applications 4
(6):357-362; Heid, C. A. et al., Genome Research 6:986-994; Gibson, E. M.
U. et al., 1996, Genome Research 6:995-1001). For the PCR amplification
monitoring method, for example, ABI PRISM7700 (Perkin Elmer) may be used.
Testing for an allergic disease in the present invention can be also
carried out by detecting a protein encoded by gene 513. For such testing
methods, for, example, Western blotting analysis, immunoprecipitation, or
ELISA using antibody against the protein encoded by gene 513 may be used.
Antibodies against the protein encoded by the gene 513 of the present
invention, may be produced as polyclonal or monoclonal antibody by
techniques known to one skilled in the art (Milstein C, et al., 1983,
Nature 305 (5934): 537-40). Protein or its partial peptide used as an
antigen may be obtained, for example, by inserting gene 513 or its partial
sequence into an expression vector, by introducing it into an appropriate
host cell to prepare a transformant, by culturing the transformant to
express the recombinant protein, and by purifying the expressed
recombinant protein from the cultured transformant or the culture
supernatant.
In testing for an allergic disease in the present invention, if expression
of the gene of the invention is significantly higher in a subject, the
subject may be determined to have allergic diathesis with high IgE titer
against an allergen such as cedar pollen antigen. It is possible to use
the expression level of the gene of the invention together with allergen
specific antibody titer, symptoms, and such for testing for an allergic
disease.
Expression of gene 513 in T cells is increased in the pollen allergy
patient group, which shows high levels of pollen antigen specific IgE.
Allergic patients who show response toward antigens except cedar pollen
may also give high expression of gene 513 when the T-cell response is
accelerated by the antigens. In such a case, increased expression of gene
513 corresponds to an acceleration of the T-cell response, and thus by
monitoring the expression level of gene 513, it is possible to screen for
therapeutic drugs for an allergic disease.
Methods for screening for a candidate compound for the treatment of
allergic disease in this invention can be carried out in vivo or in vitro.
In the in vivo screening, for example, model animals such as mice may be
administered with a candidate agent and stimulated with pollen antigen.
Then T cells are prepared from peripheral blood to measure the transcript
product level of gene 513. Alternatively, after administrating a candidate
agent to model animals such as mice, lymphocytes are separated from
peripheral blood and stimulated by cedar pollen antigen and such in vitro.
After the stimulation, T cells are prepared from the lymphocytes, and the
transcript product level of gene 513 is measured. As a result of this
measurement, a compound which can reduce transcription level of gene 513
may be selected. Here, the stimulation by pollen antigen is carried out to
trigger an antigen-specific allergic response in T cells and to judge the
treatment effect of the candidate compound on the response.
In an in vitro screening, for example, peripheral blood lymphocytes from a
pollen-allergic person, mouse, and such are collected and stimulated by
cedar pollen antigen in vitro. At the time of in vitro stimulation, a
candidate compound is added. Then T cells are prepared from the stimulated
peripheral blood lymphocytes, and the transcript product levels of gene
513 are measured. As a result of this measurement, a compound which can
reduce transcription level of gene 513 may be selected.
For screening for a candidate compound for a therapeutic drug for an
allergic disease in this invention, a T-cell line may be used. For
example, a T-cell line such as the Molt4 or Jurkat T-cell line may be
stimulated by a lymphocyte stimulating substance in vitro. As for the
lymphocyte stimulating substance, for example, calcium ionophore (A23187),
PMA, or phytohemagglutinin (PHA) may be used. At the time of in vitro
stimulation, the candidate agent is added. Then the transcript product
level of gene 513 is measured in the T-cell line. As a result of this
measurement, a compound which can reduce transcription level of gene 513
may be selected.
Like testing for an allergic disease in the invention, expression of gene
513 can be detected in screening for a candidate compound for a
therapeutic drug for an allergic disease by using a hybridization
technique with nucleic acids which can hybridize with gene 513 as probes
or by using a gene amplification technique with DNAs which can hybridize
with the gene of the present invention as primers.
As for hybridization techniques, for example, Northern hybridization, dot
blot hybridization, or DNA micro array technique may be used. As for gene
amplification techniques, for example, RT-PCR may be used. Using the PCR
amplification monitoring method as described in Example 8 during the gene
amplification step in RT-PCR, one can more accurately quantify the gene
513 expression.
Test compounds used in such screening include compound preparations
synthesized by existing chemical methods such as steroid derivatives,
compound preparations synthesized by combinatorial chemistry, extracts
from animal or plant tissues, mixtures of multiple compounds such as
microbial cultures and their purified preparations.
The compound isolated by the method for screening for a candidate compound
for a therapeutic drug for an allergic disease in this invention can be a
candidate agent for improving the condition of individuals having allergic
diathesis against allergens such as pollen antigen.
When a compound isolated by the screening method of the present invention
is used as a drug, it can be formulated by any known pharmaceutical
manufacturing methods. For example, the compound is administered to
patients with a pharmaceutically acceptable carrier or medium (e.g.
physiological saline, vegetable oil, suspending agent, surfactant,
stabilizer, etc.) The drug can be administered via various routes, for
example, percutaneously, intranasally, transbronchially, intramuscularly,
intravenously, or orally, depending on the properties of the compound.
Dosage may vary depending on some factors, such as age, weight, condition
of the patient and the administration method, but those skilled in the art
can properly determine the suitable dosage.
Claim 1 of 6 Claims
1. A method of testing for a
cedar pollen allergy in a human subject, said method comprising the steps
of:
(a) preparing T cells from the subject,
(b) preparing an RNA sample from said T cells,
(c) conducting hybridization with said RNA sample using a probe consisting
of the complement of a segment of SEQ ID NO:1, optionally wherein the probe
is linked to a label or vector nucleic acid, and
(d) measuring the amount of RNA that is derived from said subject and that
hybridizes with said probe, and comparing said amount with the amount of RNA
of a control group which has 3.5 AU/mL or less of cedar pollen specific IgE,
wherein if the amount of gene 513 RNA is significantly higher in a sample
from the subject than in the control group, then the subject is determined
to have a cedar pollen allergy.
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