U.S. patent application number 12/032012 was filed with the patent office on 2009-01-15 for ly6h gene.
This patent application is currently assigned to OTSUKA PHARMACEUTICAL CO.,LTD.. Invention is credited to Masato HORIE, Yutaka Ohbuchi, Keiichi Okutomi, Mikio Suzuki, Yoshihiro Taniguchi.
Application Number | 20090017479 12/032012 |
Document ID | / |
Family ID | 17391112 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090017479 |
Kind Code |
A1 |
HORIE; Masato ; et
al. |
January 15, 2009 |
LY6H GENE
Abstract
The invention provides a brain-specific gene useful in treating
Alzheimer's disease, for instance, which comprises a nucleotide
sequence cording for the amino acid sequence shown in SEQ ID NO:1
and fragments thereof; an expression vector comprising the gene; a
host cell comprising the expression vector; an expression product
of the gene; an antibody against the product; a therapeutic and
prophylactic composition for neurodegenerative disease; and the
like.
Inventors: |
HORIE; Masato; (Naruto-shi,
JP) ; Okutomi; Keiichi; (Tokushima-shi, JP) ;
Taniguchi; Yoshihiro; (Tokushima-shi, JP) ; Suzuki;
Mikio; (Tokushima-shi, JP) ; Ohbuchi; Yutaka;
(Tokushima-shi, JP) |
Correspondence
Address: |
SUGHRUE, MION, ZINN, MACPEAK & SEAS, PLLC
2100 Pennsylvania Avenue, N.W.
Washington
DC
20037-3202
US
|
Assignee: |
OTSUKA PHARMACEUTICAL
CO.,LTD.
|
Family ID: |
17391112 |
Appl. No.: |
12/032012 |
Filed: |
February 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10743018 |
Dec 23, 2003 |
7432363 |
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12032012 |
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09787360 |
Mar 16, 2001 |
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PCT/JP99/05039 |
Sep 16, 1999 |
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10743018 |
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Current U.S.
Class: |
435/7.92 ;
435/7.1; 530/387.9 |
Current CPC
Class: |
A61P 25/16 20180101;
A61K 48/00 20130101; C07K 14/705 20130101; A61P 25/28 20180101;
A61K 38/00 20130101; A61P 9/10 20180101 |
Class at
Publication: |
435/7.92 ;
530/387.9; 435/7.1 |
International
Class: |
G01N 33/533 20060101
G01N033/533; C07K 16/18 20060101 C07K016/18; G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 17, 1998 |
JP |
1998-263550 |
Claims
1-19. (canceled)
20. An antibody which binds specifically to a protein comprising
the amino acid sequence of SEQ ID NO: 1.
21. The antibody according to claim 20, wherein the protein is an
expression product of an expression vector comprising: a
polynucleotide consisting of the nucleotide sequence of SEQ ID NO:
2 or a polynucleotide consisting of the nucleotide sequence of SEQ
ID NO: 3.
22. The antibody according to claim 21, wherein the expression
vector comprises the polynucleotide consisting of the nucleotide
sequence of SEQ ID NO: 2.
23. The antibody according to claim 21, wherein the expression
vector comprises the polynucleotide consisting of the nucleotide
sequence of SEQ ID NO: 3.
24. A method for detecting a wild-type or mutant LY6H in a sample,
comprising subjecting said sample to a technique selected from the
group consisting of immunoprecipitation, immunostaining, western
blotting, ELISA, radioimmunoassay (RIA), immuno-radiometric assay
(IRMA) and immuno-enzymometric assay (IEMA), and wherein said
technique is performed with the antibody of claim 20 or 21.
25. The method of claim 24, wherein said sample is a biological
sample obtained from a human body.
Description
TECHNICAL FIELD
[0001] The invention relates to a gene expressed with high
specificity at a high level in the brain, more particularly a gene
coding for a novel protein belonging to the Ly6 Family (cf. the
literature cited below) which has been utilized in the purification
of blood stem cells, studies on the differentiation of blood cells,
activation of immune cells, inhibition of production of active
immune cells, treatment of tumors and the like. The invention
further relates to a novel protein encoded by said gene and to its
specific antibody. In addition, the invention relates to a
therapeutic and prophylactic composition for neurodegenerative
disease such as Alzheimer's disease.
BACKGROUND ART
[0002] Proteins of the Ly6 family have a low-molecular weight
GPI-anchored structure and have been identified as a class of cell
surface glycoproteins forming a gene cluster on mouse chromosome 15
[Proc. Natl. Acad. Sci., USA., 84, 1638-1643 (1987)].
[0003] The Ly6 family is specifically expressed at high levels in
bone marrow cells and lymphoid cells and, therefore, has been
utilized as a marker for T-cell differentiation and hematopoietic
stem cells [Immunol. Cell Biol., 73, 277-296 (1995)]. While much
remains to be known about its functions in vivo, the finding that
its expression is highly modulated in the lymphocytic system
suggests that these proteins are playing important roles in the
immune system, particularly in the differentiation and function of
T cells. It is reported that Ly6c, for instance, mediates the
homing of CD8.sup.+ T cells to the lymph node through
integrin-dependent adhesion [Proc. Natl. Acad. Sci., USA., 94,
6898-6903 (1997)].
[0004] Furthermore, many GPI-anchored proteins are known to
interact with protein kinases [Science, 254, 1016-1019 (1991)]. For
example, the interaction of Ly6 with p56lck and p59fyn suggests the
likelihood of its involvement in the signal transduction of T cells
[Eur. J. Immunol., 23, 825-831 (1993)]. It is also reported that T
cells derived from Ly6a-defective mice have been enhanced in the
ability to proliferate in response to antigenic stimulation [J.
Exp. Med., 186, 705-717 (1997)]. The possibility of its regulating
not only the activation of T cells but also that of B cells has
also been suggested [J. Immunol., 144, 2197-2204 (1990)].
[0005] Furthermore, several GPI-anchored proteins are known to have
been expressed and be functioning in both the lymphocytic system
and the nervous system [Nature, 379, 826-829 (1996); Curr. Biol.,
7, 705-708 (1997)]. In the Ly6 family, Ly6a.2 and Ly6E are
reportedly present and functioning in both systems [Proc. Natl.
Acad. Sci., USA., 85, 2255-2259 (1996); J. Immunol, 157, 969-973
(1996)].
[0006] Elucidation of the physiological roles played by such
proteins of the Ly6 family and the genes coding for the proteins
and the resulting information are considered to be of use in the
field of fundamental scientific research as well as in the
pharmaceutical field in connection with the purification of blood
stem cells, studies on the differentiation of blood cells,
activation of immune cells, inhibition of activation of immune
cells, therapy of tumors, and the like.
[0007] Recently, in patients with Alzheimer's disease, an excessive
cerebral temporal lobe atrophy as compared with age-associated
brain atrophy has been reported [Jobst, K. A., et al., Lancet, 343,
829-830 (1994)], suggesting that some gene or genes having a
bearing on the cerebral temporal lobe are somehow associated with
the onset and progression of Alzheimer's disease. It is logical to
assume that should such a gene be identified or characterized,
there might be provided information useful for the therapy and
prophylaxis of Alzheimer's disease.
[0008] Therefore, an object of the present invention is to provide
the above information needed by those concerned, particularly a
novel human protein belonging to the Ly6 family and a gene coding
for the protein.
[0009] A further object of the invention is to provide a
pharmaceutical composition for the therapy and prophylaxis of
various neurodegenerative diseases, represented by Alzheimer's
disease.
[0010] The present inventor explored into the genes derived from
various human tissues and succeeded in isolating and characterizing
a novel brain-specific gene meeting the above objects. The inventor
further found that the level of expression of this newly isolated
gene is markedly depressed in the temporal lobe, inclusive of the
hippocampus and entorhinal cortex, of a patient with Alzheimer's
disease, that this is a causative factor in the onset and
progression of Alzheimer's disease and in dementia and other
disturbances and that this gene and its expression product can be
exploited with advantage in the therapy and prophylaxis of
Alzheimer's disease. The present invention has been accomplished on
the basis of the above findings.
DISCLOSURE OF INVENTION
[0011] The present invention provides a gene comprising a
nucleotide sequence coding for the following protein (a) or
(b).
(a) a protein having the amino acid sequence shown in SEQ ID NO:1
(b) a protein having an amino acid sequence derived from the amino
acid sequence shown in SEQ ID NO:1 by deletion, substitution or
addition of one or a plurality of amino acids and having at least
one physiological activity selected from the group consisting of
neuronal survival-supporting activity, nerve elongating activity,
nerve regenerating activity, neuroglia-activating activity, and
mnemonic (brain memory-forming) activity.
[0012] The invention also provides the above gene wherein the
nucleotide sequence is shown in SEQ ID NO:2, in particular, which
is a human gene.
[0013] Furthermore, the invention provides a gene comprising the
following polynucleotides (a) or (b), particularly the
corresponding human gene.
(a) a polynucleotide containing the nucleotide sequence shown in
SEQ ID NO:3 (b) a polynucleotide which hybridizes under stringent
condition with a DNA having the nucleotide sequence shown in SEQ ID
NO:3.
[0014] The invention further provides a gene expression vector
harboring said gene; a host cell harboring said gene expression
vector; an expression product which is expressed by said host cell;
a protein encoded by the gene of the invention; and an antibody
which binds said expression product or said protein.
[0015] The invention further provides a therapeutic and
prophylactic composition for neurodegenerative disease, which
comprises said protein or an equivalent thereof or said expression
product as an active ingredient in combination with a
pharmaceutical carrier. More particularly, the invention provides
the therapeutic and prophylactic composition for neurodegenerative
disease, wherein said active ingredient is a protein having the
amino acid sequence shown in SEQ ID NO:1 or an equivalent thereof
or a gene product obtainable by expression of the whole or part of
a gene comprising a nucleotide sequence shown in SEQ ID NO:2 and
having at least one physiological action selected from the group
consisting of neuronal survival-supporting action, nerve elongating
action, nerve-regenerating action, neuroglia-activating action, and
brain memory-forming (mnemonic, encoding) action.
[0016] Especially, the invention provides the therapeutic and
prophylactic composition for Alzheimer's disease, Alzheimer type
dementia, brain ischemia and Parkinson's disease.
[0017] In addition, the invention provides a sense strand
oligonucleotide comprising at least 20 consecutive constituent
nucleotides of the nucleotide sequence shown in SEQ ID NO:2; a gene
therapy composition comprising said sense strand oligonucleotide as
an active ingredient in combination with a pharmaceutical carrier;
and a gene-specific probe comprising an oligonucleotide sequence of
at least 10 consecutive constituent nucleotides of the nucleotide
sequence shown in SEQ ID NO:2.
[0018] Furthermore, the invention provides a method of screening
for candidate compounds either capable of binding to said protein,
equivalent thereof or expression product or influencing its
activity which comprises using said protein, equivalent or
expression product; a kit for said screening; and said compounds so
screened.
[0019] Representation of amino acids, peptides, nucleotide
sequences, nucleotides, etc. by abbreviations in the specification
is in conformity with the rules recommended by IUPAC-IUB [IUPAC-IUB
Communication on Biological Nomenclature, Eur. J. Biochem., 138, 9
(1984)], "Guideline for drafting patent specifications relative to
nucleotide sequences and/or amino acid sequences" (edited by the
Patent Office of Japan) and the conventions relating to the use of
codes or symbols in the art.
[0020] A specific example of the gene of the invention is the gene
deduced from the DNA sequence of the PCR product designated "LY6H"
as described in the example which appears later herein. Its
nucleotide sequence is as shown in SEQ ID NO:3.
[0021] The LY6H gene is a cDNA containing a 420-codon open reading
frame (ORF) coding for a novel brain-specific protein (LY6H
protein) having a 140-residue amino acid sequence as shown in SEQ
ID NO:1, and having a full-length sequence of 854 nucleotides.
[0022] The LY6H protein which is the expression product of the gene
of the invention was found to have high homology to mouse Ly6
family proteins [Immunol. Cell Biol., 73, 277-296 (1995)] by a
GenBank/EMBL database search using FASTA Program (Person, W. R. et
al., Proc. Natl. Acad. Sci., USA., 85, 2444-2448 (1988)).
Furthermore, high gene-to-gene homology was recognized. Therefore,
the gene of the invention is considered to be a novel human Ly6
gene.
[0023] The LY6 gene of the invention was identified to be a gene
which is specifically expressed in the brain by the sequencing of
more than 28000 cDNA clones selected at random from a fetal human
brain cDNA library. By RH chromosome mapping [Hum. Mol. Genet., 5,
339-346 (1996)], the locus of the gene on the chromosome was found
to be 8q24. 3.
[0024] Thus, the gene and expression product of the invention, thus
provided, contribute to detection of the expression of the gene in
various tissues, production of human LY6H protein by genetic
engineering techniques, and construction of an antibody thereto,
hence enabling the purification of hematopoietic stem cells, study
of blood cell differentiation, activation or suppression of immune
cells, therapy of tumors, and the like.
[0025] In addition, the expression product (polypeptide) of the
invention, thus provided, enables provision of a drug for
prophylaxis and therapy of neurodegenerative diseases such as
Alzheimer's disease, Alzheimer type dementia, Parkinson's disease
and ischemic brain. Furthermore, the sense strand of the gene
according to the invention can be utilized as a pharmaceutical
composition for gene therapy, with which the onset and progression
of the above-mentioned neurodegenerative diseases can be inhibited
or arrested.
[0026] The invention further provides a method of screening for
compounds either binding or influencing the activity of the
expression product (polypeptide) of the invention and a relevant
kit for screening, hence compounds so screened as well. For
identification of such compounds screened, an antibody binding to
the expression product of the gene of the invention can be
utilized.
[0027] In the specification, the term "gene" is used to mean a
double-stranded DNA and its constituent single-stranded DNA,
whether sense or antisense, without regard to its length.
Therefore, unless otherwise indicated, the gene (DNA) of the
invention includes a double-stranded DNA containing a human genomic
DNA, a single-stranded DNA (sense strand) inclusive of the cDNA, a
single-stranded DNA (antisense strand) having a sequence
complementary to said sense strand, and fragments of said DNAs.
[0028] The gene (DNA) of the invention may contain a leader
sequence, a coding region, exons and introns. The polynucleotide
includes both RNA and DNA. The DNA includes cDNA, genomic DNA and
synthetic DNA. The polypeptide includes its fragments, homologs,
derivatives and mutants. The mutants include alleles which occur
naturally, mutants not existing naturally, mutants having amino
acid sequences mutated by deletion, substitution, addition and/or
insertion, and mutants having functionally equivalent modified
amino acid sequences.
[0029] Such modifications (e.g. mutations) of amino acid sequences
may for example occur from spontaneous mutation or
posttranslational modification but may be artificially induced by
utilizing a native gene (for example, specific genes of the
invention).
[0030] The homology of such mutants to the unmutated polypeptide
may be at least 70%, preferably 80%, more preferably 95%, still
more preferably 97%. The above polypeptide and its mutants and
homologs have a structural feature conserved in common and may have
the biological activities of the expression product of the gene of
the invention, such as neuronal survival-supporting action,
neuronal growth-stimulating action, nerve generating action, and
neuroglia-stimulating action. The homology of polypeptides can be
analyzed by searching through a database such as SWISSPLOTS
Database using a sequence analysis software such as FASTA [Clustal,
V., Methods Mol. Biol., 25, 307-318 (1994)].
[0031] The gene coding for such a mutant is silent or conserved for
amino acid substitution. Thus, the amino acid residues encoded by
the nucleotide sequence are not altered.
[0032] The conservatively substitutable amino acid residues, i.e.
the amino acid residues substitutable with other amino acid
residues without losing the activities of the polypeptide having
such original amino acid residues, and the corresponding original
amino acid residues are as follows.
TABLE-US-00001 Original amino Conservatively substituting acid
residue amino acid residue Ala Ser Arg Lys Asn Gln, His Asp Glu Cys
Ser Gln Asn Glu Asp Gly Pro His Asn or Gln Ile Leu or Val Leu Ile
or Val Lys Arg, Aln or Glu Met Leu or Ile Phe Met, Leu or Tyr Ser
Thr Thr Ser Trp Tyr Tyr Trp or Phe Val Ile or Leu
[0033] In addition, Cys may be substituted for a different kind of
amino acid residue, e.g. Ser, Ala or Val.
[0034] The gene and expression product according to the invention
provide information and means of great use for the elucidation,
expatiation, diagnosis, prophylaxis and therapy of
neurodegenerative diseases such as Alzheimer's disease, brain
ischemia and Parkinson's disease. The gene of the invention can
also be used with advantage for the development of new drugs
capable of inducing expression of the gene for use in the treatment
of said neurode-generative diseases. In addition, the detection of
expression of the gene of the invention and of the resulting
expression product in individuals or tissues, and the detection of
mutation (deletion or point mutation) of the gene or abnormal
expression thereof can be utilized with advantage for the
elucidation and diagnosis of said neurodegenerative diseases.
[0035] The gene of the invention includes but is not limited to the
gene having the nucleotide sequence shown in SEQ ID NO:2 which
codes for a protein having the amino acid sequence shown in SEQ ID
NO:1, for example a gene (LY6H gene) having the nucleotide sequence
shown in SEQ ID NO:3. For example, the gene of the invention may be
a gene coding for an amino acid sequence derived from the
above-defined amino acid sequence by a given modification, a gene
coding for an amino acid sequence having a given degree of homology
to the above-defined amino acid sequence, or a gene having a
nucleotide sequence having a given degree of homology to any of the
above genes.
[0036] The above-mentioned given degree of homology to a defined
amino acid sequence or nucleotide sequence means a homology of at
least not less than 70%, preferably not less than 90%, more
preferably not less than 95%, still more preferably not less than
97%. The present invention encompasses homologs (gene homologs and
protein homologs) having such homology.
[0037] The gene of the invention includes "a gene coding for a
polypeptide having an amino acid sequence (modified amino acid
sequence) derived from the amino acid sequence shown in SEQ ID NO:1
by deletion, substitution or addition of one or a plurality of
amino acids". The extent and position or positions of "deletion,
substitution or addition" are not particularly restricted insofar
as the resulting polypeptide having a modified amino acid sequence
is equivalent, in biological function, to the polypeptide (LY6H
protein) having the amino acid sequence shown in SEQ ID NO:1. The
biological "function" mentioned above includes physiological
functions such as neuronal survival-supporting action, nerve
elongating action, nerve regenerating action, neuroglia-activating
action, and mnemonic action, and the "equivalent" is a polypeptide
having such functions. Therefore, the protein having such a
modified amino acid sequence includes a protein (equivalent) having
a fragment (a consecutive-residue fraction) of the amino acid
sequence shown in SEQ ID NO:1 and having physiological activities
similar to those mentioned above for the full-length of said amino
acid sequence. Furthermore, the gene coding for a polypeptide
having the above-modified amino acid sequence may be a gene with
which the gene of the invention encoding a polypeptide having the
pre-modification amino acid sequence can be detected. The term
plurality as used in connection with said modification usually
means not less than 2 but up to several, although the range is not
restrictive.
[0038] The homolog of LY6H gene (and the homolog of the expression
product of the gene) according to the invention means any of a
series of related genes (and their expression products) which are
homologous in sequence and recognized as one gene family from their
structural characteristics, common gene expression pattern, and
similarities in said biological function. This, of course, includes
alleles of the genes of the invention.
[0039] The modification (mutation) of an amino acid sequence may
occur naturally, for example by spontaneous mutation and
posttranslational modification but may be induced artificially on
the basis of the native gene (for example the specific gene of the
invention). The invention covers any and all modified genes having
the above characteristics without regard to the cause or means of
modification or mutation.
[0040] The artificial means for said modification (mutation) of the
amino acid sequence includes genetic engineering techniques such as
site-specific mutagenesis [Methods in Enzymology, 154, 350, 367-382
(1987); ibid., 100, 468 (1983); Nucleic Acids Res., 12, 9441
(1984); "Zoku Seikagaku Jikken Koza (Experiments in Biochemistry,
Second Series) 1": Idenshi Kenkyuho (Methods in Gene Research) II,
the Biochemical Society of Japan (ed.), p 105 (1986), etc.],
methods of chemical synthesis such as the phosphotriester method
and phosphoamidite method [J. Am. Chem. Soc., 89, 4801 (1967);
ibid., 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron
Lett., 22, 1859 (1981); ibid., 24, 245 (1983)], and combinations of
such methods.
[0041] More particularly, the DNA can be synthesized by a chemical
method such as the phosphoamidite method or the phosphotriester
method, and this synthesis can be effected on a commercially
available automated oligo-nucleotide synthesizer. The
double-stranded fragment can be obtained from the chemically
synthesized single-strand fragment by synthesizing a complementary
strand and annealing them under suitable conditions or adding the
complementary strand using a suitable primer sequence and a DNA
polymerase.
[0042] A specific example of the gene according to the invention is
the gene having the nucleotide sequence shown in SEQ ID NO:3. The
coding region (the sequence shown in SEQ ID NO:2) of this
nucleotide sequence is an example of combination of codons
specifying the respective amino acid residues of the amino acid
sequence shown under SEQ ID NO:1. The gene of the invention is not
limited to the gene having said defined nucleotide sequence but
includes any gene having a nucleotide sequence obtainable by
selecting any arbitrary combination of codons for each amino acid
residue. Selection of codons can be made in the routine manner,
with reference to the codon usage in the host to be employed
[Nucleic Acids Res., 9, 43 (1981)].
[0043] Furthermore, the gene of the invention includes one having a
nucleotide sequence showing a certain level of homology to the
nucleotide sequence shown in SEQ ID NO:3. Inferred by said level of
homology are polynucleotides and complementary polynucleotides
having at least 70% homology, preferably at least 90% homology,
more preferably at least 95% homology, to the nucleotide sequence
shown in SEQ ID NO:3. The gene having such a level of homology may
for example be characterized as a polynucleotide which hybridizes
with a DNA having the nucleotide sequence shown in SEQ ID NO:3
under stringent conditions. More particularly, the gene having a
nucleotide sequence which hybridizes with the DNA having the
nucleotide sequence shown in SEQ ID NO:3 under the condition of
6.times.SSC at 65.degree. C. overnight or 50% formamide-4.times.SSC
at 37.degree. C. overnight is subsumed in the concept of the gene
having said level of homology. Here, SSC stands for standard saline
citrate (1.times.SSC=0.15 M NaCl, 0.015 M sodium citrate).
[0044] The gene of the invention can be easily produced and
isolated by the general genetic engineering technology based on the
sequence information on any specific example of the gene of the
invention as disclosed in this specification [e.g. Molecular
Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); Zoku Seikagaku
Jikken Koza (Experiments in Biochemistry, Second Series): "Idenshi
Kenkyuho (Methods in Gene Research) I, II, III, the Biochemical
Society of Japan (ed.), (1986)].
[0045] More particularly, this can be done by preparing a cDNA
library from a suitable source, in which the gene of the invention
can be expressed, by a routine procedure and selecting a desired
clone from this library using a suitable probe or antibody specific
to the gene of the invention [Proc. Natl. Acad. Sci., USA., 78,
6613 (1981); Science, 222, 778 (1983)].
[0046] The source of cDNA, which can be used in the above procedure
includes various cells and tissues expressing the gene of the
invention, as well as cultured cells derived therefrom,
particularly brain tissues. Isolation of the total RNA from such a
source, isolation and purification of mRNA, and acquisition and
cloning of cDNA can also be carried out in the conventional manner.
Moreover, cDNA libraries are commercially available and the present
invention can be carried into practice using such cDNA libraries,
for example those cDNA libraries available from CLONTECH Lab.
Inc.
[0047] The method of screening for the gene of the invention from a
cDNA library is not particularly restricted but the conventional
procedure can be employed. Examples of the screening methods
include an immuno-screening method using a specific antibody to the
protein produced by a cDNA to select the corresponding cDNA clone,
a method using a probe selectively binding to the objective DNA
sequence, such as a plaque hybridization method, and a colony
hybridization method, and a combination of such methods.
[0048] As the probe for the above method, the DNA chemically
synthesized according to the nucleotide sequence information on the
gene of the invention can be generally employed. The gene of the
invention which has already been obtained or a fragment thereof can
also be used as the probe with advantage. The sense primer and
antisense primer established according to the nucleotide sequence
information on the gene of the invention can be used as screening
probes.
[0049] The nucleotide sequence for use as the probe may be a
partial nucleotide sequence corresponding to SEQ ID NO:2 and
comprising at least 10 consecutive nucleotides, preferably 20
consecutive nucleotides, more preferably 30 consecutive
nucleotides, most preferably 50 consecutive nucleotides. Moreover,
the positive clone having the oligonucleotide sequence shown in SEQ
ID NO:2 as such can be used as the probe.
[0050] In obtaining the gene of the invention, the DNA/RNA
amplification by PCR [Science, 230, 1350 (1985)] can be used with
advantage. Particularly when a full-length cDNA can hardly be
obtained from a library, the RACE method [Rapid amplification of
cDNA ends; Jikken Igaku (Experimental Medicine), 12(6), 35 (1994)],
especially 5'-RACE method [M. A. Frohman, et al., Proc. Natl. Acad.
Sci., USA., 8, 8998 (1988)], can be used with advantage.
[0051] The primers for use in such PCR methods can be judiciously
established with reference to the sequence information on the gene
of the invention as disclosed herein and can be synthesized by the
routine procedure. The isolation and purification of the amplified
DNA/RNA fragment can be carried out in the routine manner as
mentioned above, for example by the gel electrophoresis method.
[0052] Sequencing of the gene of the invention as obtained in the
above manner or various DNA fragments can be made in accordance
with the dideoxy method [Proc. Natl. Acad. Sci., USA., 74, 5463
(1977)] or the Maxam and Gilbert method [Methods in Enzymology, 65,
499 (1980)] or more expediently by using a commercial sequencing
kit.
[0053] For example, with the gene of the invention thus obtained,
the expression or non-expression of the gene of the invention in an
individual or a given tissue can be specifically detected by
utilizing a portion or the whole of the nucleotide sequence of the
gene of the invention.
[0054] The above detection can be made by the conventional
procedures, such as RNA amplification by RT-PCR [reverse
transcribed-polymerase chain reaction; E. S. Kawasaki, et al.,
Amplification of RNA. In PCR Protocol. A Guide to Methods and
Applications, Academic Press, Inc., SanDiego, 21-27 (1991)],
Northern blot analysis [Molecular Cloning, Cold Spring Harbor Lab.
(1989)], determination on cellular level by in situ RT-PCR [Nucl.
Acids Res., 21, 3159-3166 (1993)] or in situ hybridization, NASBA
[nucleic acid sequence-based amplification, Nature, 350, 91-92
(1991)], and the like conventional techniques. The preferred is the
RT-PCR detection method.
[0055] The primer which is to be used when the PCR method is chosen
for the above purpose is not particularly restricted insofar as it
is characteristic of the gene of the invention and capable of
selective amplification of the particular gene only and can be
judiciously established based on the sequence information on the
gene of the invention. Usually, one having a partial sequence of
the gene of the invention, which is about 10-35 nucleotides long,
preferably about 15-30 nucleotides long can be used as the
primer.
[0056] The gene of the invention, thus, includes the DNA fragment
which can be used as a specific primer and/or specific probe for
the detection of the LY6H gene of the invention.
[0057] The DNA fragment mentioned above can be defined as a
polynucleotide which hybridizes with the polynucleotide having the
nucleotide sequence shown in SEQ ID NO:2 under stringent
conditions. The stringent conditions mentioned above may be the
ordinary conditions for primers or probes and, as such, are not
particularly restricted. For example, the above-mentioned
conditions of 6.times.SSC, 65.degree. C., overnight or the
condition of 50% formamide-4.times.SSC, 37.degree. C., overnight
can be mentioned.
[0058] By applying the gene of the invention to the standard
genetic engineering technology, the expression product
(polypeptide) of the gene or a protein containing it can be easily
produced in large quantities and with good reproducibility.
[0059] Therefore, the invention further provides a polypeptide
having the amino acid sequence encoded by the gene of the invention
(the expression product of the invention), a vector harboring the
gene of the invention for the production of the polypeptide, a host
cell transfected with the vector, and a method of producing the
polypeptide of the invention which comprises growing the host
cell.
[0060] The polypeptide (LY6H protein) having the amino acid
sequence shown under SEQ ID NO:1 is a specific embodiment of the
polypeptide of the invention. The polypeptide of the invention is
not limited to this LY6H protein but includes its homolog. The
homolog may be a polypeptide having an amino acid sequence derived
from the amino acid sequence shown in SEQ ID NO:1 by the deletion,
substitution or addition of one or more amino acids and retaining
the same function as the LY6H protein. A specific example of the
homolog is the expression product of a homolog of said LY6H gene
(the LY6H equivalent gene inclusive of the allele).
[0061] Furthermore, the homolog of the LY6H protein of the
invention includes proteins having the same activity or function as
the polypeptide having the amino acid sequence shown in SEQ ID NO:1
as derived from any of mammals such as equine, sheep, bovine,
canine, monkey, cat, bear, etc. and rodents such as rat, mouse and
rabbit.
[0062] The polypeptide of the invention can be produced by the
conventional recombinant DNA technology [e.g. Science, 224, 1431
(1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Natl.
Acad. Sci., USA., 80, 5990 (1983)] based on the gene sequence
information provided by the present invention.
[0063] More particularly, the production of said polypeptide is
carried out by the procedure comprising constructing a recombinant
DNA (expression vector) which permits expression of the gene coding
for the desired protein in a host cell, transforming the host cell
with the vector, growing the resulting transformant, and harvesting
the polypeptide from the culture broth.
[0064] The host cell mentioned above may be whichever of a
prokaryotic cell and an eukaryotic cell. As the prokaryotic host,
Escherichia coli, Bacillus subtilis and other common bacteria can
be mentioned and preferably cells of Escherichia coli, particularly
cells of Escherichia coli K12, can be employed. The eukaryotic host
cell includes cells of vertebrates and yeasts and the former
include the monkey cell line COS [Cell, 23: 175 (1981)], Chinese
hamster ovarian cells, and the dihydrofolate reductase-defective
cells thereof [Proc. Natl. Acad. Sci., USA., 77: 4216 (1980)]. As
the latter, yeast cells of the genus Saccharomyces can be used with
advantage, but these are not exclusive choices.
[0065] When prokaryotic cells are used as host cells, an expression
plasmid construct prepared by using a vector which is replicatable
in the particular host cell and adding a promoter and SD (Shine and
Dalgarno) sequence upstream of the gene of the invention so that
the gene may be expressed therein as well as an initiation codon
(e.g. ATG) necessary for initiation of protein synthesis can be
used with advantage. As the vector mentioned above, it is usual to
employ plasmids derived from Escherichia coli, such as pBR322,
pBR325, pUC12, pUC13, etc. However, these are not exclusive choices
but various known vectors can be utilized. Examples of the
commercial vectors for use in expression systems using E. coli
include pGEX-4T (Amersham Pharmacia Biotech), pMAL-C2, pMA1-P2 (New
England Biolabs), pET21, pET21/lacq (Invitrogen) and pBAD/H is
(Invitrogen).
[0066] As the expression vector for use when cells of a vertebrate
are used as host cells, the vector having a promoter upstream of
the gene of the invention to be expressed, RNA splice sites,
polyadenylation site and a transcription termination sequence is
usually employed, and this vector may further have a replication
origin where necessary. A specific example of the expression vector
is pSV2dhfr harboring an early promoter of SV40 [Mol. Cell. Biol.,
1: 854 (1981)]. Aside from the above, various known vectors
available commercially can be employed. Examples of the commercial
vectors which are used in expression systems using animal cells
include vectors for animal cells, such as pEGFP-N, pEGFP-C
(CLONTECH), pIND (Invitrogen), pcDNA3.1/H is (Invitrogen), etc.,
and vectors for insect cells, such as pFastBac HT (Gibci BRL),
pAcGHLT (PharMingen), pAc5/V5-His, pMT/V5-His and pMT/Bip/V5-his
(all Invitrogen).
[0067] pAM82 having a promoter for the acid phosphatase gene [Proc.
Natl. Acad. Sci., USA., 80: 1 (1983)] is a specific example of the
expression vector for use when yeast cells are used as host cells.
The commercial expression vectors for yeast cells include pPICZ
(invitrogen) and pPICZ.alpha. (Invitrogen).
[0068] The promoter is not particularly restricted, either. When a
strain of the genus Escherichia is used as the host, tryptophan
(trp) promoter, lpp promoter, lac promoter, recA promoter, PL/PR
promoter, etc. can be utilized with advantage. When the host is a
strain of the genus Bacillus, SP01 promoter, SP02 promoter, penP
promoter, etc. are preferably used. When a yeast is used as the
host, pH05 promoter, PGK promoter, GAP promoter, ADH promoter, etc.
can be utilized with advantage. The preferred promoter for use when
host cells are animal cells include SV40-derived promoters,
retrovirus promoters, metallothionein promoter, heat shock
promoter, cytomegalovirus promoter, and SR.alpha. promoter.
[0069] As the expression vector for the gene of the invention, the
conventional fusion protein expression vector can be used with
advantage. pGEX (Promega) for the expression of
glutathione-S-transferase (GST)-fused proteins is a specific
example of the vector.
[0070] The polynucleotide sequence wherein the coding sequence for
a mature polypeptide assists in the expression and secretion of a
polypeptide from host cells includes the secretory sequence, the
leader sequence and the marker sequence (hexahistidine tag,
histidin tag) used in the purification of a fusion mature
polypeptide in the case of bacterial cells, and the hemaglutinin
(HA) tag in the case of mammalian cells.
[0071] The method of introducing the recombinant DNA (expression
vector) into the host cell and the associated transforming method
are not particularly restricted but various standardized methods
can be utilized.
[0072] The transformant obtained can be cultured in the routine
manner, whereby the objective protein encoded by the deliberately
designed gene according to the invention is expressed and produced
(accumulated/secreted) intracellularly, extracellularly or on the
cell membrane.
[0073] The culture medium to be used can be judiciously selected
from among various routine media according to the kind of adopted
host cell and the culture is also performed under conditions
favoring growth of the host cell.
[0074] The resulting recombinant protein (LY6H protein) according
to the invention can be optionally isolated and purified by various
separation techniques taking advantage of its physical and/or
chemical properties, for instance ["Seikagaku Data Book
(Biochemical Data Book) II", 1175-1259, First Edition, 1st
impression, Jun. 23, 1980, Tokyo Kagaku Dojin K. K.; Biochemistry,
25(25), 8274 (1986); Eur. J. Biochem., 163, 313 (1987), etc.].
[0075] Examples of such techniques are the conventional
reconstitution method, treatment with a protein precipitating agent
(salting-out method), centrifugation, osmotic shock method, sonic
disruption, ultrafiltration, various types of chromatography such
as molecular sieve chromatography (gel filtration), adsorption
chromatography, ion exchange chromatography, affinity
chromatography and high performance liquid chromatography (HPLC),
dialysis, and combinations of these techniques. The particularly
preferred technique includes affinity chromatography using a column
to which a specific antibody to the protein of the invention has
been coupled.
[0076] In designing the objective gene encoding the polypeptide of
the invention, the nucleotide sequence of Y6H gene as shown in SEQ
ID NO:2 can be utilized with advantage. If desired, this gene can
be used after the codons specifying the respective amino acid
residues have been judiciously altered. Furthermore, when any amino
acid residue or partial sequence of the amino acid sequence encoded
by the LY6H gene is to be modified by substitution, deletion or
addition, such modifications can be made by the various methods
described above, for example by site-specific mutagenesis.
[0077] The polypeptide of the invention can be produced by the
standard protocol for chemical synthesis according to the amino
acid sequence shown in SEQ ID NO:1. The method includes the
conventional liquid-phase method and solid-phase method for peptide
synthesis.
[0078] More particularly, the method for peptide synthesis includes
the so-called stepwise elongation method in which the constituent
amino acids are coupled one by one for chain extension and the
fragment condensation method which comprises synthesizing fragments
each consisting of several amino acids beforehand and coupling the
fragments together. The synthesis of the protein of the invention
can be carried out by whichever of the above two methods.
[0079] The method of condensation for use in the above peptide
synthesis may also be a conventional one, including the azide
process, mixed acid anhydride process, DCC process, active ester
process, redox process, DPPA (diphenylphosphoryl azide) process,
DCC+additive (1-hydroxybenzotriazole, N-hydroxysuccinamide,
N-hydroxy-5-norbornene-2,3-dicarboximide or the like) process and
Woodward's reagent process.
[0080] The solvent to be used in these processes can also be
judiciously selected from among the common solvents well known in
the art for use in such peptide-forming condensation reactions.
Examples of the solvents include dimethylformamide (DMF), dimethyl
sulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran
(THF), ethyl acetate, etc., and mixtures thereof.
[0081] In conducting the peptide synthesizing reactions, the
carboxyl group of any amino acid or fragment peptide that should
not take part in the reaction can be protected in advance,
generally by esterification in the form of a lower alkyl ester such
as methyl ester, ethyl ester, tert-butyl ester, etc. or an aralkyl
ester such as benzyl ester, p-methoxybenzyl ester, p-nitrobenzyl
ester, etc.
[0082] Referring to any amino acid having a functional group in its
side chain., the hydroxyl group of a tyrosine residue, for
instance, may be protected in advance with an acetyl, benzyl,
benzyloxycarbonyl, tertiary butyl or other group, although such
protection is not necessarily indispensable. Furthermore, the
guanidino group of an arginine residue can be protected with a
suitable protective group such as nitro, tosyl,
p-methoxybenzene-sulfonyl, methylene-2-sulfonyl, benzyloxycarbonyl,
isobornyloxycarbonyl, adamantyloxycarboxyl or the like.
[0083] The reactions for eliminating such protective groups from
the protected amino acids, peptides or the end product protein of
the invention can also be carried out in the routine manner, for
example by catalytic reduction or a method using liquid
ammonia/sodium, hydrogen fluoride, hydrogen bromide, hydrogen
chloride, trifluoroacetic acid, acetic acid, formic acid,
methane-sulfonic acid or other reagent.
[0084] The polypeptide of the invention, thus produced, can be
purified as needed by the various techniques mentioned above, such
as ion exchange resin chromatography, partition chromatography, gel
chromatography, countercurrent distribution and the like methods in
routine us in the field of peptide chemistry.
[0085] The polypeptide of the invention can be used with advantage
as an immunogen for preparation of its specific antibody. By
utilizing this immunogen, the antiserum (polyclonal antibody) and
the monoclonal antibody can be provided.
[0086] The technology of producing antibodies is well known to
those skilled in the art and the known procedures can be employed
in the present invention [e.g. Zoku Seikagaku Jikken Koza
(Experiments in Biochemistry, second series) "Men-eki Seikagaku
Kenkyuho (Methods in Immunobiochemistry)", edited by the
Biochemical Society of Japan (1986)].
[0087] For example, as the immune animal for harvesting the desired
antiserum therefrom, the ordinary animals such as rabbit, guinea
pig, rat, mouse, chicken, etc. can be arbitrarily selected and the
immunization with said immunogen and the collection of blood can
also be carried out by the conventional procedures.
[0088] Preparation of a Monoclonal Antibody can Also be carried out
by the conventional technique which comprises constructing a
hybridoma between the plasma cell (immune cell) of an animal
immunized with said immunogen and a plasmacytoma cell, selecting
clones producing the desired antibody, and cultivating the clones.
The immune animal is generally selected in consideration of its
compatibility with the plasmacytoma cell to be used for cell fusion
and usually the mouse or the rat is used with advantage. The
immunization procedure may be the same as used for the preparation
of said antiserum and, if desired, the immunization can be made
using a conventional adjuvant in combination.
[0089] The plasmacytoma cell for use in said hybridization is not
particularly restricted, either, but includes various myeloma cells
such as p3 (p3/x63-Ag8) [Nature, 256: 495-497 (1975)], p3-U1
[Current Topics in Microbiology and Immunology, 81: 1-7 (1978)],
NS-1 [Eur. J. Immunol., 6: 511-519 (1976)], MPC-11 [Cell, 8:
405-415 (1976)], SP2/0 [Nature, 276: 269-271 (1978)], etc., R210
[Nature, 277: 131-133 (1979)] and others in rats, and cells derived
therefrom.
[0090] The hybridization between said immune cell and said
plasmacytoma cell can be effected by the known technology in the
presence of a conventional hybridization promoter such as
polyethylene glycol (PEG) or Sendai virus (HVJ) and the separation
of the objective hybridoma can also be carried out in the known
manner [Meth. in Enzymol., 73: 3 (1981); Zoku Seikagaku Jikken Koza
(ditto)].
[0091] The search for the objective antibody-producing cell clone
and the monoclonal antibody preparation can also be carried out in
the routine manner. For example, the search for the
antibody-producing hybridoma can be made by any of the various
techniques in routine use for the detection of antibodies, such as
ELISA [Meth. in Enzymol., 70: 419-439 (1980)], plaque method, spot
method, agglutination reaction method, Ouchterlony method,
radioimmunoassay, and the like, using the protein of the invention
as an antigen.
[0092] Harvesting of the antibody of the invention from the
resulting hybridoma can be achieved by cultivating the hybridoma in
the routine manner and recovering the antibody as a culture
supernatant or administering the hybridoma to a compatible mammal
and recovering the antibody in the form of ascites. The former
method is suitable for production of the antibody of high purity,
while the latter method is suitable for high-production of the
antibody. The antibody thus produced can be further purified by the
conventional means such as salting-out, gel filtration, affinity
chromatography and the like.
[0093] The antibody thus obtained is characterized by its binding
affinity for the LY6H protein of the invention and can be used with
advantage for the purification of LY6H protein and determination or
differentiation of the protein by immunological techniques.
Furthermore, since a decreased expression of the gene of the
invention has been confirmed in the temporal lobe of the brain of a
patient with Alzheimer's disease which is a neurodegenerative
disease, this antibody can be utilized in the screening for
agonists or antagonists of LY6H protein.
[0094] The present invention provides the novel antibody described
above, too.
[0095] The polypeptide of the invention is useful in the field of
medicine as pharmaceutical products containing it as an active
ingredient. Therefore, the invention provides a pharmaceutical
composition comprising the polypeptide of the invention as an
active ingredient.
[0096] The usefulness of the polypeptide of the invention in or as
said pharmaceutical composition is ascribable to the neuronal
survival-supporting action, nerve elongating action, nerve
regenerating action, neuroglia-activating action and mnemonic
action inherent in this brain-specific polypeptide. Examples of the
methods for confirming these actions include the following methods
for each action.
1) Neuronal Survival-Supporting Action
[0097] The following method can be used for quantitating the
neuronal survival-supporting action of the polypeptide of the
invention. For example, the hippocampus is aseptically isolated
from the whole brain of a fetal SD rat and treated with an enzyme,
and seeded in a poly-L-lysine (Sigma)-precoated 96-well plate
containing 10% fetal calf serum-DMEM at a final concentration of
2.times.10.sup.5 cells/cm.sup.2.
[0098] The cells are grown for 24 hours., at the end of which time
the culture medium is changed to 1% N2 Supplement
(Gibco)-containing DMEM. Then, the active ingredient polypeptide of
the invention is added (the invention group). As control, the
polypeptide of the invention which has been heat-treated in a
boiling water bath for 5 minutes is added (the boiled protein
group).
[0099] The cells (culture) in each group as prepared in the above
manner are cultured for 72 hours. Then, by performing an MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay using Promega Cell Titer 96-Well Assay System, the neuronal
survival-supporting effect of the polypeptide of the invention on
hippocampal neurons can be evaluated.
[0100] Similarly, by isolating the ventral midbrain aseptically
from the same whole brain of a fetal SD rat as above and carrying
out an MTT assay in the same manner as above, the neuronal
survival-supporting effect of the polypeptide of the invention on
the midbrain neurons can be investigated.
2) Dopaminergic Neuron Survival-Supporting Action
[0101] As a method of evaluating the neuronal survival-supporting
action of the polypeptide of the invention, the following method of
quantitating the dopaminergic neuron survival-supporting activity
can be mentioned. Thus, the cells (culture) in each group as
prepared above under 1) are cultured for 72 hours and, then, fixed
with 4% paraformaldehyde-PBS by 15-minute standing at room
temperature. Then, using 1% Triton X 100/PBS, the culture is passed
through a membrane.
[0102] To prevent non-specific binding of the antibody, the cells
are incubated in 10% goat serum-PBS for 1 hour and, then, using an
anti-tyrosine hydroxylase polyclonal antibody (Chemicon, diluted
1000-fold in PBS), further incubated at 4.degree. C. for 16 hours.
After removal of the antibody fluid, the cells are washed with PBS
and, after addition of peroxidase-labeled dextran polymer-coupled
goat anti-rabbit immunoglobulin (Dako), are incubated at room
temperature for 1 hour.
[0103] Detection of the tyrosine-hydroxylase-positive cells can be
made by the color reaction using diaminobenzidine as the substrate.
In this manner, the dopaminergic neuronal survival-supporting
activity of the polypeptide of the invention can be assayed using
the number of tyrosine hydroxylase-positive cells as the
indicator.
3) Nerve Elongating Action
[0104] The determination of the nerve elongating action (axonal
elongation-promoting action) of the polypeptide of the invention
can be carried out using PC12 cells [ATCC Accession Number CRL1721;
Science, 229, 393-395 (1985)] as follows. Thus, PC12 cells
subcultured in modified Dalbecco's MEM (D-MEM) containing 5% of
heat-inactivated (56.degree. C., 30 min) horse serum and 10% fetal
calf serum (FCS) are transplanted in a collagen-coated plastic
petri dish, 35 mm in diameter, at a concentration of
6.times.10.sup.4 cells/3 ml. On day 2 after transplantation, the
medium was replaced with D-MEM containing a varying concentration
of the polypeptide of the invention as well as nerve growth factor
(NGF; Wako Pure Chemical Ind.) and FCS and the cultivation is
continued in each case. On day 3, morphological changes of the
cells are examined with a phase-contrast microscope. By assessing
whether the formation of neurites or the promotion of neurite
outgrowth is observed in comparison with control, the axonal
elongation-promoting potential of the polypeptide of the invention
can be evaluated.
4) Neuroglia-Activating Action
[0105] The neuroglia-activating action can be evaluated, for
example by determining the effect of the polypeptide of the
invention on the activation of neuroglia by FGF in accordance with
the method of Kniss et al. or the method of Bogler et al. [Kniss,
D. A., and Burry, R. W., Brain Res., 439, 281-288 (1988); Bogler,
O., et al., Proc. Natl. Acad. Sci., USA., 87(16), 6368-6372
(1990)].
5) Mnemonic Action
[0106] The mnemonic action can be evaluated, for example, in
accordance with the water-maze protocol of Morris [Morris. R. G.
M., J. Neurosci. Meth., 11, 47-60 (1984)].
[0107] Another evaluation method comprises administering the LY6H
protein or an agonist or antagonist of the LY6H protein as selected
by a screening to an animal model of Alzheimer's disease such as a
mutant .beta.-amyloid precursor protein gene or mutant presenilin 1
gene transgenic mouse [e.g. Nature, 373, 523-527 (1995); Nature
Med., 5, 560-564 (1999)] and evaluating the degree of progression
of the disease or the degree of nerve degeneration in comparison
with a non-treated control group.
[0108] Moreover, in order to have the gene expressed in the human
temporal lobe (gene therapy), an adenovirus vector [Straus, E. S.,
Plenum Press New York, 451-496 (1984); Setoguchi, Y., et al.,
Blood, 84, 2953-2964 (1994)], for instance, is used. Thus, a
possible procedure comprises cloning the gene of the invention in
an adenovirus vector, culturing it in the stem cell, administering
it directly into the temporal lobe or intravenously through a
peripheral blood vessel and checking to see whether Alzheimer type
dementia or Alzheimer's disease has been improved or its
progression inhibited.
[0109] The polypeptide as the active ingredient of the
pharmaceutical composition of the invention includes its
pharmaceutically acceptable salt. Such salt includes nontoxic salts
with alkali metals, alkaline earth metals or ammonium, such as
salts with sodium, potassium, lithium, calcium, magnesium, barium
and ammonium. These salts can be prepared by the conventional
methods in the art. Furthermore, said salt includes nontoxic acid
addition salts which can be prepared by reacting the active
ingredient polypeptide of the invention with suitable organic or
inorganic acids. The representative nontoxic acid addition salt
includes the hydrochloride, hydrobromide, sulfate, bisulfate,
acetate, oxalate, valerate, oleate, laurate, borate, benzoate,
lactate, phosphate, p-toluenesulfonate (tosylate), citrate,
maleate, fumarate, succinate, tartrate, sulfonate, glycolate,
maleate, ascorbate, benzenesulfonate, naphthalenesulfonate, and the
like.
[0110] The pharmaceutical composition of the invention includes a
composition comprising a pharmacologically effective amount of the
polypeptide of the invention and a suitable nontoxic pharmaceutical
carrier or diluent.
[0111] The pharmaceutical carrier which can be used for said
pharmaceutical composition (pharmaceutical preparation) includes
diluents or excipients which are conventionally utilized according
to dosage forms, such as fillers, volume builders, binders,
humectants, disintegrators, surfactants, and lubricants. These can
be judiciously selected and used according to the unit dosage form
of the composition.
[0112] The particularly preferred pharmaceutical composition of the
invention can be prepared using various additives which can be
formulated in ordinary protein preparations, such as the
stabilizer, biocide, buffer, isotonizing agent, chelating agent, pH
control agent and surfactant.
[0113] The stabilizer includes human serum albumin, an L-amino
acid, a sugar, and a cellulose derivative, for instance, can be
mentioned. These may be used singly or in combination with a
surfactant or the like where necessary. The use in combination with
a surfactant may lead to a more effective stabilization of the
active ingredient in particular.
[0114] The L-amino acid is not particularly restricted but may for
example be any of glycine, cysteine and glutamic acid.
[0115] The sugar is not particularly restricted but includes
monosaccharides such as glucose, mannose, galactose, fructose,
etc.; sugar alcohols such as mannitol, inositol, xylitol, etc.,
disaccharides such as sucrose, maltose, lactose, etc.;
polysaccharides such as dextran, hydroxypropylstarch, chondroitin
sulfate, hyaluronic acid, etc.; and their derivatives.
[0116] The surfactant is not particularly restricted, either, but
both ionic and nonionic surfactants can be employed. Examples of
the surfactant are polyoxyethylene glycol sorbitan alkyl esters,
polyoxyethylene alkyl ethers, sorbitan monoacyl esters and fatty
acid glycerides.
[0117] The cellulose derivative that can be used is not
particularly restricted, either, but includes methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, and carboxymethylcellulose
sodium.
[0118] The level of addition of any of said sugar and other
additives can be judiciously selected with reference to the amount
in common use. Generally, the sugar is used in a proportion of not
less than about 0.0001 mg, preferably within the range of about
0.01 to about 10 mg, per .mu.g of the active ingredient. The
surfactant is used generally in a proportion of not less than about
0.00001 mg, preferably within the range of about 0.0001 to about
0.01 mg, per .mu.g of the active ingredient. Human serum albumin,
an example of the stabilizer, can be used in a proportion of not
less than about 0.0001 mg, preferably within the range of about
0.001 to about 0.1 mg, per .mu.g of the active ingredient. The
amount of the amino acid, another example of the stabilizer, can be
selected from the range of about 0.001 to about 10 mg per .mu.g of
the active ingredient. The level of addition of the cellulose
derivative is not less than about 0.00001 mg and is preferably
selected from the range of about 0.001 to about 0.1 mg.
[0119] The amount of the active ingredient in the pharmaceutical
composition of the invention can be liberally selected from a broad
range but is generally selected from the range of about 0.00001 to
about 70 weight %, preferably about 0.0001 to about 5 weight %.
[0120] The pharmaceutical composition of the invention may be
further supplemented with a buffer, an isotonizing agent, and a
chelating agent. The buffer includes boric acid, phosphoric acid,
acetic acid, citric acid, .epsilon.-aminocaproic acid, glutamic
acid, and the corresponding salts (the alkali metal or alkaline
earth metal salts thereof, such as sodium salts, potassium salts,
calcium salts and magnesium salts). The isotonizing agent includes
sodium chloride, potassium chloride, sugars and glycerol. The
chelating agent includes sodium edetate, and citric acid. The level
of addition of any of these additives may be within the
conventional range.
[0121] The pharmaceutical preparation of the invention can be
provided in the form of a solution, and in a lyophilized form which
can be stored. Such lyophilized preparations can be
extemporaneously dissolved in, for example, a buffer inclusive of
water, saline or the like at a suitable concentration.
[0122] As regards the unit dosage form of the pharmaceutical
composition of the invention, various forms can be selected
according to the therapeutic objective. The representative form
includes solid dosage forms such as tablets, pills, powders, neat
powders, granules, capsules, etc. and liquid dosage forms such as
solutions, suspensions, emulsions, syrups, elixirs and so on. These
dosage forms are generally classified, by route of administration,
into oral preparations, parenteral preparations, nasal
preparations, vaginal suppositories, rectal suppositories,
sublingual tablets, ointments, and others. Each of such dosage
forms can be formulated and molded or otherwise prepared by the
established pharmaceutical procedure.
[0123] For example, tablets can be manufactured using, as said
pharmaceutical carrier, any of various excipients such as lactose,
sucrose, sodium chloride, glucose, urea, starch, calcium carbonate,
kaolin, crystalline cellulose, silicic acid, potassium phosphate,
etc.; binders such as water, ethanol, propanol, simple syrup,
glucose solution, starch solution, gelatin solution,
carboxymethylcellulose, hydroxypropylcellulose, methylcellulose,
polyvinylpyrrolidone, etc.; disintegrators such as
carboxymethylcellulose sodium, carboxymethylcellulose calcium,
low-substitution-degree hydroxypropylcellulose, dried starch,
sodium alginate, agar powder, laminaran powder, sodium hydrogen
carbonate, calcium carbonate, etc.; surfactants such as
polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate,
monoglycerol stearate, etc.; disintegration inhibitors such as
sucrose, stearin, cacao butter, hydrogenated oil, etc.; absorption
promoters such as quaternary ammonium bases, sodium lauryl sulfate,
etc.; humectants such as glycerol, starch, etc.; adsorbents such as
starch, lactose, kaolin, bentonite, colloidal silica, etc.; and
lubricants such as purified talc, salts of stearic acid, boric acid
powder, polyethylene glycol and so on.
[0124] Where necessary, such tablets can be coated with
conventional coatings to provide sugar-coated tablets,
gelatin-coated tablets, enteric-coated tablets and film-coated
tablets. Double-layer or multi-layer tablets may also be
employed.
[0125] The pharmaceutical carrier which can be used for the
production of pills includes various excipients such as glucose,
lactose, starch, cacao butter, hydrogenated vegetable oil, kaoline,
talc, etc.; binders such as gum arabic powder, tragacanth powder,
gelatin, ethanol, etc.; and disintegrators such as laminaran and
agar.
[0126] The capsules can be generally manufactured in the
conventional manner by blending the active ingredient of the
invention with the pharmaceutical carrier or carriers and filling
the resulting composition into hard gelatin capsule shells, soft
capsule shells or the like.
[0127] The liquid preparation for oral administration includes
pharmaceutically acceptable solutions, emulsions, suspensions,
syrups, elixirs, etc. as formulated with routine inert diluents,
such as water, and these dosage forms may contain a wetting agent,
an emulsifier, a suspending agent and/or other auxiliary additives.
These can be manufactured by the established pharmaceutical
procedures.
[0128] The liquid preparation for parenteral administration,
inclusive of sterile aqueous and non-aqueous solutions, emulsions
and suspension, can be prepared using such diluents as water, ethyl
alcohol, propylene glycol, polyethylene glycol, ethoxylated
isostearyl alcohol, polyoxylated isostearyl alcohol,
polyoxyethylene sorbitan fatty acid esters, and vegetable oils such
as olive oil. In addition, injectable organic esters, such as ethyl
oleate, may be formulated. Furthermore, any of the conventional
solubilizers, buffers, wetting agents, emulsifiers, suspending
agents, preservatives, dispersants, etc. can also be added.
[0129] The above various pharmaceutical dosage forms are sterilized
in the routine manner. This sterilization can be achieved by
filtration through a bacterial filter, formulation of a biocide,
irradiation, or a heat treatment. Furthermore, these may be
provided in the form of sterile solid compositions which can be
extemporaneously dissolved in sterile water or a suitable
sterilizable medium.
[0130] For the manufacture of dosage forms for rectal or vaginal
administration, such pharmaceutical carriers as polyethylene
glycol, cacao butter, higher alcohols, higher alcohol esters,
gelatin, semisynthetic glycerides, etc. can be employed.
[0131] Ointments such as pastes, creams and gels can be prepared
using a diluent such as white petrolatum, paraffin, glycerol,
cellulose derivatives, propylene glycol, polyethylene glycol,
silicone, bentonite, and vegetable oils such as olive oil.
[0132] Compositions for transnasal or sublingual administration can
be prepared in the routine manner using a well-known standard
excipient.
[0133] Where necessary, those pharmaceutical preparations of the
invention may be supplemented with coloring agents, preservatives,
perfumes, flavoring agents, sweeteners, and other drugs.
[0134] The method for administration of such pharmaceutical
preparations is not particularly restricted but can be selected
according to the specific dosage form, patient's age, sex and other
factors, severity of illness, and other variables. For example,
tablets, pills, solutions, suspensions, emulsions, granules and
capsules are administered orally, while parenteral products are
administered intravenously, either alone or in admixture with the
conventional glucose, amino acid or other infusion, or, where
necessary, administered alone intramuscularly, intradermally,
subcutaneously or intraperitoneally. The rectal suppositories are
administered into the rectum; the vaginal suppositories are
administered into the vagina; the nasal preparations are
administered into the nostrils, the sublingual preparations are
administered buccally, and the ointments are administered topically
for transdermal drug delivery.
[0135] The dosage for any of the above pharmaceutical preparations
is not particularly restricted but can be judiciously selected from
a broad range according to the expected therapeutic effect,
administration method, duration of treatment, patient background
such as age and sex, and other factors. Generally, the recommended
usual dosage of the active ingredient is about 0.01 .mu.g-10
mg/day, preferably about 0.1 .mu.g-1 mg/day, per kg of the
patient's body weight. The above dose may be administered once a
day or in 2 or more divided doses.
[0136] Furthermore, as pointed out in the working example to be
presented later herein, the expression of the gene of the invention
has been abolished or decreased in the temporal lobe of patients
with Alzheimer's disease. Therefore, by constructing an arbitrary
expression vector harboring the whole or part of the gene of the
invention and introducing the expression vector into the temporal
lobe tissue for forced expression of the gene in the tissue,
neurodegenerative changes inclusive of an excessive atrophy of
neurons in the temporal lobe may be inhibited and, hence, the
progression of Alzheimer's disease may be arrested. Therefore, the
present invention further provides a pharmaceutical composition for
gene therapy (gene therapeutic agent) which is possessed of such a
neurodegeneration-inhibitory action.
[0137] The present invention further provides the above expression
vector or vector for gene therapy, cells transfected with the gene
of the invention through the introduction of said vector, and a
pharmaceutical composition for gene therapy which comprises any of
the above as the active component.
[0138] The gene therapy using said gene therapeutic agent is
performed by administering at least one member selected from the
group consisting of the vector for introduction and expression of
the gene of the invention and cells transfected with the gene of
the invention through the introduction of said vector into the
brain neurons or temporal lobe tissue of a patient with
neurodegenerative disease. By such a procedure, neurodegenerative
changes in such tissue can be inhibited and symptoms of Alzheimer's
disease, Alzheimer type dementia, Parkinson's disease, brain
ischemia, etc. can be alleviated.
[0139] The gene therapy is now described in further detail. In the
following execution of a gene therapy, the routine chemical,
molecular biological, microbiological, recombinant DNA, genetic,
and immunological techniques can be employed unless otherwise
specified. These techniques are described in, inter alia, Maniatis,
T., et al., Molecular Cloning: A laboratory manual (Cold Spring
Harbor Laboratory), Cold Spring Harbor, N.Y. (1982)), Sambrook, J.,
et al., Molecular Cloning: A laboratory manual, 2nd Ed. (Cold
Spring harbor Laboratory), Cold Spring harbor, New York (1981)),
Ausbel, F. M., et al., Current Protocols in Molecular Biology, John
Wiley and Sons, New York (1992)), Glover, D., DNA Cloning, I and II
(Oxford Press (1985)), Anand, Techniques for the Analysis of
Complex Genomes (Academic Press (1992), Guthrie, G., et al., Guide
to Yeast Genetics and Molecular Biology (Academic Press (1991)),
and Fink, et al., Hum. Gene Ther., 3, 11-19 (1992).
[0140] The gene therapy can be carried out using a gene therapy
vector harboring the whole or part of the gene of the invention or
cells transfected with the gene of the invention through the
introduction of said vector. This gene therapy may for example be a
method of supplying the LY6H gene or its function to cells in which
said gene has not been expressed. By such gene therapy,
neurodegeneration around the receptor cell/target cell is
inhibited.
[0141] The gene of the invention or a fragment of the gene can be
introduced into cells by means of a vector adapted to maintain the
gene extrachromosomally. In such cases, the particular gene can be
caused to be expressed by the cells from an extrachromosomal
position. Moreover, when the LY6H gene is to be expressed by
introducing a fragment of the gene into the temporal lobe site of
the brain nervous system where no expression of the gene is found,
the particular fragment of the gene may be a fragment encoding a
part of LY6H protein which is necessary for the survival or
non-tumorigenic growth of cells.
[0142] The gene transfer vector may be any of various known vectors
in which the gene of the invention has been subcloned as will be
described later herein.
[0143] The introduction of the gene transfer vector into the target
cell can be easily effected by the established technology of
introducing DNA into various cells which is already known to those
skilled in the art, such as electroporation, calcium phosphate
transfection (coprecipitation), virus transduction and other
techniques. The cells transfected with the gene of the invention
can be utilized as a drug for neurodegenerative disorders of the
brain, inclusive of an inhibitor of premature atrophy of the brain
nervous system, or as models for therapeutic research.
[0144] As mentioned above, the gene or gene fragment of the
invention as introduced by the gene therapy according to the
invention increases the expression of the corresponding gene
product in the brain nerve or surrounding tissue to thereby inhibit
atrophy of the brain nerve in the tissue expressing the gene. Such
gene therapy can be applied with advantage to the brain neuronal
tissue where the expression of LY6H gene or the LY6H protein has
been abolished as well as to the brain neuronal tissue where the
level of expression of said gene has been depressed.
[0145] The gene therapy according to the present invention is
performed as follows. First, a screening is carried out for
candidate patients for the gene therapy by recording a computer
tomogram (CT) with the scanning position fixed to the temporal lobe
of the patient with Alzheimer type dementia or Alzheimer's disease
to check for atrophy of the temporal lobe or progression of the
atrophy.
[0146] Then, to achieve expression of the gene of the invention,
the intracellular LY6HmRNA is created in the target cell and its
translation is promoted to accelerate expression of the LY6H gene.
For this purpose, preferably a sense oligonucleotide corresponding
to the mRNA of the gene is produced and supplied to the target
cell. By providing the cell with the activity to promote expression
of the LY6H gene by the above gene therapy, the neurodegenerative
change in the brain receptor cell/target cell can be inhibited.
[0147] According to the above gene therapy using said sense
oligonucleotide, the objective inhibition of neurodegenerative
change of the brain and consequent alleviation or arrest of
progression of neurodegenerative symptoms may be successfully
attained by subcloning the LY6H gene into a retrovirus, adenovirus
or AAV-derived vector and infecting the target brain nerve cells
with the vector to thereby cause expression of the sense
oligonucleotide.
[0148] When a sense oligonucleotide of the gene of the invention is
introduced into the cerebral neuron or tissue to increase the
expression of LY6H protein, the sense oligonucleotide need not be
the full-length nucleotide of the LY6H gene but may be the
modification product insofar as it retains a function substantially
identical to the function of the parent gene and promotes
expression of the LY6H gene or a fragment gene comprising a partial
sequence retaining said function.
[0149] Vectors which can be used for introducing an objective gene
for both DNA recombination and extrachromosomal gene maintenance
are already known in the art and any of such known vectors can be
used in the practice of the invention. For example, a virus or
plasmid vector which includes a copy of LY6H gene sense
oligonucleotide ligated to the expression control element and is
capable of expressing the sense-oligonucleotide product in the
target cell can be used as such vectors. Any of the expression
vectors mentioned above can be usually employed but the preferred
are vectors constructed by using any of the vectors disclosed in
U.S. Pat. No. 5,252,479 and WO 93/07282 (specifically pWP-7A,
pWP-19, pWU-1, pWP-8A, pWP-21 and/or pRSVL) or the pRC/CMV
(Invitrogen) as the source vector. The still more preferred are the
various virus vectors described later herein.
[0150] As the promoter for use in the vector for gene therapy,
those promoters, which are intrinsic to the affected tissues to be
treated in various diseases, are preferably employed. Examples of
the promoters are albumin, .alpha.-fetoprotein,
.alpha.1-antitrypsin, transferrin, and transthyretin for the liver,
and carbonic anhydrase I and carcinoembryonic antigen for the
colon. When the affected tissues are the uterus and placenta,
estrogen, aromatase, cytochrome P450, cholesterol
side-chain-cleaving enzyme P450, and 17.alpha.-hydroxylase P450 can
be exemplified.
[0151] For the prostate, prostate-specific antigens, gp91-fox gene,
and prostate-specific kallikrein can be exemplified. For the
breast, erb-B2, erb-B3, .beta.-casein, .beta.-lactoglobin, and whey
protein can be exemplified. For the lung, surfactant protein C, and
uroglobulin can be exemplified. For the skin, K-14-keratin, human
keratin 1 or 6, and leucline can be exemplified. For the brain,
glial fibrillary acidic protein, mature astrocyte-specific protein,
myelin, tyrosine hydroxylase pancreatic villin, glucagon, and
Langerhans islet amyloid polypeptide can be exemplified. For the
thyroid, thyroglobulin, and calcitonin can be exemplified. For the
bone, .alpha.1 collagen, osteocalcin, and bone sialoglycoprotein
can be exemplified. For the kidney, renin, liver/bone/kidney
alkaline phosphatase, and erythropoietin can be exemplified, and
for the pancreas, amylase, and PAP1 can be exemplified.
[0152] Furthermore, in the production of a vector for introduction
of a sense oligonucleotide, the sense oligonucleotide to be
introduced (one having a full-length or partial sequence
corresponding to the sequence of the gene of the invention) can be
easily prepared and acquired by the standard genetic engineering
techniques based on the nucleotide sequence information on the gene
of the invention as described hereinbefore.
[0153] The transfer of such a vector for introduction of a sense
oligonucleotide into cells can be carried out by various techniques
already known in the art, such as electroporation, calcium
phosphate transfection (coprecipitation), virus transduction and
the like. The cells transfected with said sense oligonucleotide, as
such and in an isolated form, have a brain
neurodegeneration-inhibitory action so that they can be used as a
drug, or a therapeutic research model, for the inhibition or arrest
of progression of neurodegenerative lesions as well.
[0154] In gene therapy, the above vector for introduction of a
sense oligonucleotide can be injected either topically into the
temporal lobe or surrounding region of the patient or systemically.
Furthermore, it may be cultured together with stem cells and, then,
administered by local or systemic injection. By such
administration, the vector can be introduced into the nerve cells
of the patient's brain. In the event the transduced gene is not
permanently taken up in the chromosome of each target cell, the
administration may be repeated periodically.
[0155] The method for gene therapy according to the invention
includes both the in vivo technique which comprises administering a
construct for introduction of said sense oligonucleotide (a sense
oligonucleotide transfer vector) directly into the body and the ex
vivo technique which comprises transferring the gene into cultured
stem cells and, after culturing, transplanting or otherwise
introducing the cells into the patient's body. A gene therapy
comprising introducing said sense oligonucleotide directly into the
cell is also feasible.
[0156] The target cells into which the sense oligonucleotide of the
gene of the invention is to be introduced can be judiciously
selected according to the object of gene therapy (treatment). For
example, the target cells include brain neurons and brain nerve
tissues as well as lymphocytes, fibroblasts, hepatocytes and
hemopoietic cells.
[0157] The method of introducing the sense oligonucleotide in the
above gene therapy includes a viral introduction technique and a
non-viral introduction technique.
[0158] As to the viral introduction technique, in consideration of
the fact that the sense oligonucleotide to be transferred is a
foreign substance which is expressed especially in the normal brain
cells, the method using a retrovirus vector, for instance, can be
exemplified. Other virus vectors which can be used include the
adenovirus vector, HIV (human immunodeficiency virus) vector,
adeno-associated virus (AAV) vector, herpes virus vector, herpes
simplex virus (HSV) vector, and Epstein-Barr virus (EBV)
vector.
[0159] The method of constructing a virus vector for transfer of a
sense oligonucleotide and the method for transfer of the sense
oligonucleotide to the target cell or target tissue are now
specifically described.
[0160] The retrovirus vector system consists of a virus vector and
a helper cell (packaging cell). The helper cell means a cell which
has expressed genes encoding the structural protein gag (structural
protein within the virus particle), pol (reverse transcriptase),
env (coat protein), etc. of a retrovirus but which has not formed
virus particles. On the other hand, the virus vector has the
packaging signal and LTR (long terminal repeats) but lacks
structural genes, such as gag, pol, env, etc., which are necessary
for virus replication. The packaging signal is a sequence which
functions as a tag in the assembly of a virus particle. Selective
genes (neo, hyg) and the object sense oligonucleotide ligated in
the cloning site are inserted in lieu of the virus genes. In order
that a high titer of virus particles may be obtained, it is
important to use an insert as short as possible, provide a broad
packaging signal including a part of the gag gene, and use care not
to leave ATG of the gag gene.
[0161] As the vector DNA harboring the object sense oligonucleotide
is transferred to the helper cell, the vector genomic RNA is
packaged by the virus structural protein formed by the helper cell,
whereby virus particles are formed and secreted. The virus particle
as a recombinant virus infects the target cell and, as a result,
the DNA sequence reverse-transcribed from the virus genomic RNA is
integrated into the cell nucleus so that the sense gene inserted in
the vector is expressed.
[0162] It may be employed a technique using a fibronectin fragment
containing the cell adhesion domain, heparin-binding site and
conjugating segment [Hanenberg, H., et al., Exp. Hemat., 23, 747
(1995)], for enhancing the efficiency of transfer of the object
gene.
[0163] An example of the retrovirus vector for use in the above
retrovirus vector system is the retrovirus derived from mouse
leukemia virus [McLachlin, J. R., et al., Proc. Natl. Acad. Res.
Molec. Biol., 38, 91-135 (1990)].
[0164] The method using an adenovirus vector is now described in
detail. The adenovirus vector can be constructed in accordance with
the methods described in Berkner, K. L., Curr. Topics Microbiol.
Immunol., 158, 39-66 (1992), Setoguchi, Y., et al., Blood, 84,
2946-2953 (1994), Kanegae, H. et al. [Jikken Igaku (Experimental
Medicine), 12, 28-34 (1994)] and Ketner, G. et al., Proc. Natl.
Acad. Sci., USA., 91, 6186-6190 (1994).
[0165] For example, to construct a non-proliferative adenovirus
vector, the early region E1 and/or E3 of the adenovirus is excised
in the first place. Then, a plasmid vector containing the desired
foreign gene expression unit (which consists of the sense
oligonucleotide to be transferred, the promoter for transcription
of said sense oligonucleotide, Poly A for insuring the stability of
the transcribed gene) and a part of the adenovirus genomic DNA and
a plasmid containing the adenovirus genome are used to cotransfect
the 293 cell, for instance. As a homologous recombination is thus
caused to take place between them for substitution of the gene
expression unit for E1, a nonproliferative adenovirus vector is
obtained as a vector harboring the object sense oligonucleotide. A
3'-end adenovirus vector with a terminal protein added can also be
constructed by ligating the adenovirus genomic DNA in a cosmid
vector. Furthermore, the YAC vector may also be utilized for the
construction of an adenovirus vector.
[0166] Production of an adeno-associated virus (AAV) vector is now
described briefly. AAV was discovered as a small virus
contaminating adenovirus culture systems. As to this virus, the
existence of the genus Parvovirus capable of autonomous
proliferation within the host cell without requiring a helper virus
for virus replication and the genus Dependovirus which requires a
helper virus has been identified. This AAV has a broad host range
and is one of the common viruses infecting various kinds of cells.
The virus genome is a linear single-stranded DNA consisting of 4680
nucleotides, with the 145 nucleotides at both ends having a
characteristic sequence known as ITR (inverted terminal repeat).
This ITR region functions as the replication origin and plays the
role of a primer. This ITR is also essential to packaging for virus
particles and integration of AAV into the chromosome DNA of the
host cell. In regard of the virus protein, the left-half of the
genome codes for the nonstructural protein, that is the regulatory
protein Rep which controls replication and transcription.
[0167] Construction of the recombinant AAV can be carried out by
utilizing the property of AAV to become integrated into the
chromosome DNA, whereby the desired gene transfer vector can be
prepared. This method may be described in detail as follows. First,
a plasmid (AAV vector plasmid) retaining the ITRs at 5'- and
3'-ends of a wild-type AVV and harboring the sense oligonucleotide
to be transferred as interposed therebetween is constructed. The
virus protein necessary for virus replication and formation of
virus particles is supplied from a separate helper plasmid. It is
necessary to insure that no common nucleotide sequence will exist
between the two plasmids so that a wild-type virus will not appear
on DNA recombination. Thereafter, the two plasmids are transferred
into the 293 cell by transfection, for example, and, further, the
cells are infected with an adenovirus as the helper virus (when the
293 cell is used, this adenovirus may be a non-proliferative one),
whereby the desired non-proliferative recombinant AAV is produced.
Since this recombinant AAV is present in the nucleus, the cells are
subjected to freeze-thawing and recovered and the contaminant
adenovirus is inactivated by heating at 56.degree. C. Then, where
necessary, the recombinant AAV is separated and concentrated by
ultracentrifugation using cesium chloride. In this manner, the
desired recombinant AAV for gene transfer can be obtained.
[0168] Production of an EBV vector can be carried out by the method
of Shimidzu et al. [Shimidzu, N., SAIBO KOUGAKU (Cell Technology,
14(3), 280-287 (1995)].
[0169] Production of the EBV vector for transfer of the sense
oligonucleotide according to the invention is now described
briefly. EB virus (Epstein-Barr virus) is a virus of the family
Herpesviridae, which was first isolated by Epstein and coworkers
from cultured cells derived from Burkitt lymphoma [Kieff, E. and
Liebowitz, D.: Virology, 2nd ed. Raven Press, New York, 1990, pp.
1889-1920]. This EBV has cell-transforming activity and in order to
use it as a vector for gene transfer, it is necessary to prepare a
virus defected of this transforming activity. This can be done as
follows.
[0170] Thus, first of all, the EBV genome in the vicinity of the
target DNA in which the desired foreign gene is to be inserted is
cloned. Then, a DNA fragment of the foreign gene and a
drug-resistant gene are inserted to construct a vector for
preparation of a recombinant virus. Then, the vector for
preparation of a recombinant virus as excised with a suitable
restriction enzyme is transfected to EBV-positive Akata cells. The
recombinant virus formed by homologous recombination is recovered,
together with the wild type Akata EBV, through stimulation of virus
production by anti-surface immunoglobulin treatment. The
recombinant virus is infected to EBV-negative Akata cells and, in
the presence of a drug, resistant clones are selected, whereby
Akata cells infected exclusively with the recombinant virus free of
wild type EBV can be obtained. Further, by inducing viral activity
in the recombinant virus-infected Akata cells, the objective
recombinant virus vector can be produced in quantities.
[0171] The method of introducing the object gene into the target
cell or target tissue in the gene therapy of the invention includes
the following representative two methods.
[0172] The first method comprises harvesting the target cells from
a patient to be treated, growing the cells ex vivo, for example
under addition of interleukin-2 (IL-2) or the like, to transfer the
objective sense oligonucleotide harbored in the retrovirus vector,
and retransplanting the resulting cells (ex vivo method). This
method is suitable for the therapy of genetic diseases caused by
defective genes and cancer, for instance.
[0173] The second method is a method for direct gene transfer which
comprises injecting the object sense oligonucleotide directly into
the patient's body or the target site such as the cerebral tissue
(direct method).
[0174] More particularly, the first method can be carried out in
the following manner, for instance. Thus, the mononuclear cells,
such as stem cells, harvested from the patient are fractionally
separated from monocytes using a blood sorter and cultured in the
presence of IL-2 in a suitable medium such as AIM-V medium for
about 72 hours, followed by addition of the vector harboring the
sense oligonucleotide to be introduced. For enhancing the
efficiency of transfer of the sense oligonucleotide, the cells may
be grown in the presence of protamine at 32.degree. C. for 1 hour,
centrifuged at 2500 ppm, and then cultured under 10% carbon dioxide
gas at 37.degree. C. for 24 hours. After this procedure is repeated
a few times, the cells are further cultured in the presence of IL-2
in, for example, AIM-V medium for 48 hours and then washed with
saline. The viable cells are counted and the efficiency of
introduction of the sense oligonucleotide is evaluated by said in
situ PCR or, when the object is enzymatic activity, assaying the
degree of the enzymatic activity.
[0175] The safety checks such as culture of bacteria and fungi in
cultured cells, check for the presence or absence of mycoplasma
infection, search for endotoxin, etc. are carried out to confirm
safety. Thereafter, the cultured cells transformed with the
predicted effective dose of the sense oligonucleotide are returned
to the patient by intravenous drip injection. The above procedure
is repeated at intervals of several weeks or a few months to
consummate the gene therapy.
[0176] The dosage of the virus vector is judiciously selected
according to the target cell. The usually preferred dose may for
example be 1.times.10.sup.3 cfu-1.times.10.sup.8 cfu in terms of
virus titer per 1.times.10.sup.8 target cells.
[0177] It can be adopted an alternative version of the above first
method that comprises co-cultivating the virus-producer cells
having the retrovirus vector harboring the object sense
oligonucleotide and the patient's cells to thereby introduce the
sense oligonucleotide into the target cells.
[0178] In carrying out the second method (direct method) for gene
therapy, it is particularly preferable to perform a preliminary
experiment ex vivo to check whether the objective sense
oligonucleotide can be actually introduced by the gene transfer
method by carrying out PCR of the vector gene cDNA or in situ PCR
or check whether the desired therapeutic effect, for example
elevation of a specific activity or the growth or inhibition of
growth of the target cell can be actually achieved by introduction
of the objective sense oligonucleotide. Moreover, when a virus
vector is used, it is, of course, of great importance to confirm
the safety of introduction of the sense oligonucleotide in gene
therapy by performing a PCR search for proliferative retrovirus and
the like, determining the reverse transcriptase activity, or
monitoring the coat protein (env) gene by the RCR technique.
[0179] The gene therapy of the invention in Alzheimer's disease,
Alzheimer type dementia or Parkinson's disease may for example be a
therapy of neurodegenerative disease which comprises harvesting
stem cells or brain nerve cells from the patient, establishing a
cultured cell line by enzymatic treatment or the like, introducing
the object sense oligonucleotide into the target brain nerve cells
utilizing AAV or the like, carrying out a screening with G418
cells, measuring the amount of expression of IL-12 or the like in
vivo, giving a radiation treatment, and inoculating the cells into
the patient's brain tissue or the temporal lobe site.
[0180] The present invention further provides a pharmaceutical
composition or preparation (a gene therapeutic agent) comprising a
sense oligonucleotide transfer vector of the invention or a cell
line transformed with the sense oligonucleotide as an active
ingredient in a pharmacologically effective amount in combination
with a suitable nontoxic pharmaceutical carrier or diluent.
[0181] The pharmaceutical carrier that can be utilized in the
pharmaceutical composition (pharmaceutical preparation) of the
invention includes those diluents or excipients, e.g. fillers,
volume builders, binders, humectants, disintegrators, surfactants,
lubricants, etc., which are usually employed depending on the mode
of use of such a composition, and these can be selectively used
according to the contemplated unit dosage form of the
preparation.
[0182] The unit dosage form of the pharmaceutical preparation of
the invention may be the same as mentioned for the polypeptide
preparation of the invention, and a suitable one can be judiciously
selected according to the therapeutic objective.
[0183] The therapeutic and prophylactic method for
neurodegenerative disease according to the invention is now
described in detail.
[0184] The present invention provides a method for therapy of
neurodegenerative diseases, such as Alzheimer's disease, Alzheimer
type dementia, brain ischemia, Parkinson's disease, and like
diseases in which either an excess or a shortage of the LY6H
polypeptide is involved. When LY6H activity is excessive, several
approaches can be taken. The first method comprises administering
an inhibitor compound (antagonist) in an effective amount to
inhibit the function of LY6H polypeptide by blocking its binding to
a ligand, substrate, receptor, enzyme or the like or inhibiting a
secondary signal in combination with a pharmaceutically acceptable
carrier to thereby improve an abnormal state. An alternative method
comprises administering a soluble-form LY6H polypeptide capable of
binding to a ligand, substrate, enzyme, receptor or the like in
competition with the endogenous LY6H. A typical example of such
competitive substance includes a fragment of LY6H polypeptide. In
another method, a soluble-form LY6H polypeptide capable of binding
to a ligand in competition with endogenous LY6H can be
administered. A typical example of such competitive substance
includes a fragment of LY6H polypeptide.
[0185] In a still another method, expression of the gene coding for
endogenous LY6H polypeptide can be inhibited by applying a gene
expression inhibition technique to the LY6H gene product. The known
technique of this kind includes the use of an internally generated
or separately administered antisense sequence [e.g.
oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988), O'Connor, J. Neurochem 56: 560
(1991)]. As an alternative method, an oligonucleotide capable of
forming a triple helix with the gene can be supplied [e.g. Lee et
al., Nucleic Acids Res., 6: 3073 (1979); Cooney et al., Science,
241: 456 (1988); Dervan et al., Science, 251: 1360 (1991)]. These
oligomers can be administered as such or related oligomers may be
caused to be expressed in vivo.
[0186] For the therapy of abnormal symptoms related to an
under-expression of LY6H and its activity, several methods can be
utilized. The first method comprises administering a compound
capable of activating LY6H (agonist) in a therapeutically effective
amount together with a pharmaceutically acceptable carrier to a
subject to thereby improve the abnormal symptoms. In another
method, the endogenous production of LY6H by related cells in the
subject can be actuated by gene therapy. For example, the
polynucleotide of the present invention may be manipulated so as to
be expressed with a defective retrovirus vector as mentioned
hereinbefore. Then, this retrovirus expression construct is
isolated and introduced into packaging cells transduced with a
retrovirus plasmid vector harboring the RNA encoding the
polypeptide of the invention so that the packaging cells will form
infective virus particles containing the object gene. These
producer cells are administered to the subject for in vivo
manipulation of the cells so that the polypeptide may be expressed
in vivo. For an overview of gene therapy, reference may be made to
Human Molecular Genetics, T. Strachan and A. P. Read, BIOS
Scientific Publishers Ltd. (1996), Chapter 20--Gene Therapy and
Other Molecular Genetic-based Therapeutic Approaches, inclusive of
the specific references cited therein. An alternative method
comprises administering a therapeutic dose of LY6H polypeptide in
combination with a suitable pharmaceutical carrier.
[0187] The cells may for example be formulated in
phosphate-buffered saline (pH 7.4), Ringer's solution or an
intracellular composition injection or in such a dosage form as can
be administered in combination with a substance conducive to an
enhanced gene transfer efficiency, such as protamine.
[0188] The method of administering the above pharmaceutical
preparation is not particularly restricted but a suitable regimen
can be established according to the particular dosage form, the
patient's age, sex and other factors, the severity of illness, and
the like.
[0189] The amount of the active ingredient to be incorporated in
the pharmaceutical preparation and the dosage are not particularly
restricted but each can be liberally selected from a broad range
according to the expected therapeutic benefit, method of
administration, duration of treatment, patient background inclusive
of age and sex, and other variables.
[0190] Generally, the dosage of the retrovirus vector harboring the
sense oligonucleotide as a pharmaceutical preparation may for
example be about 1.times.10.sup.3 pfu through 1.times.10.sup.15 pfu
in terms of retrovirus titer per kilogram body weight per day.
[0191] In the case of cells carrying the sense oligonucleotide for
introduction, the dosage can be properly selected from the range of
about 1.times.10.sup.4 cells/body through 1.times.10.sup.15
cells/body.
[0192] The above preparation can be administered once a day or in a
few divided doses a day, or even intermittently at intervals of 1
or several weeks. Preferably, a substance conducive to an enhanced
gene transfer efficiency, such as protamine, or a preparation
containing the same can be administered in combination.
[0193] When the gene therapy according to the invention is applied
to the therapy of a neurodegenerative disease, it can be performed
in a suitable combination with other gene therapies (conjunctive
gene therapy) or in combination with a pharmacotherapy utilizing an
acetylcholinesterase inhibitor or the like and/or a rehabilitation
therapy. The gene therapy of the invention can be performed with
reference to the NIH guidelines, inclusive of its safety aspect
[Recombinant DNA Advisory Committee, Human Gene Therapy, 4, 365-389
(1993)].
[0194] Furthermore, in accordance with the invention, for the
purpose of detecting the presence of LY6H gene, it is possible to
prepare a biological sample such as blood or serum, optionally
extract the nucleic acid, and analyzing it for LY6H gene.
[0195] The method of detecting the gene may comprise preparing a
DNA fragment of the gene of the invention and design it so that it
may be used in the screening for LY6H gene and/or its
amplification. More specifically, it is possible to construct a DNA
fragment having the properties of a probe for plaque hybridization,
colony hybridization, Southern blotting, Northern blotting, etc. or
a probe for the preparation of a full-length or partial DNA of the
gene of the invention as amplified by a polymerase chain reaction
(PCR) which amplifies a nucleotide sequence with a polymerase. For
this purpose, a primer having the same sequence as LY6H gene is
first prepared. Then, this primer is reacted, as a probe for
screening, with a biological sample (nucleic acid sample) to check
for the presence of the particular LY6H gene sequence. The nucleic
acid sample may be prepared by any of various techniques
facilitating detection of the target sequence, such as
denaturation, restriction enzyme digestion, electrophoresis or dot
blotting.
[0196] As the method for said screening, the use of a PCR technique
is particularly preferred from sensitivity points of view, and this
technique is not particularly restricted inasmuch as a fragment of
the gene of the invention is used as a primer. Thus, It can be
utilized that any of the hitherto-known techniques [Science, 230,
1350-1354 (1985)] and the modified versions of PCR which have been
developed of late or will be developed in the future [Sakaki,
Yoshiyuki et al. (ed.), Jikken Igaku (Experimental Medicine),
Supplement 8(9) (1990), Y dosha; Protein, Nucleic Acid, Enzyme:
Special Supplement, Kyoritsu Shuppan, 35(7) (1990)].
[0197] The DNA fragment for use as the primer is a chemically
synthesized oligo-DNA, and such oligo-DNA can be synthesized using
an automated DNA synthesizer or the like, for example Pharmacia LKB
Gene Assembler Plus (Pharmacia). The preferred length of the primer
(sense primer or antisense primer) to be synthesized may for
example be about 10-30 nucleotides. The probe for us in said
screening is usually a labeled probe but may be an unlabeled one,
or the detection may be made according to specific binding to a
directly or indirectly labeled ligand. The suitable label and the
method of labeling the probe or ligand belong to the prior art.
Thus, the prior art label includes radioisotopes, biotin,
fluorescent groups, chemiluminescent groups, enzymes, antibodies,
etc., which can be taken up through known procedures such as nick
translation, random priming and kinase treatment.
[0198] The PCR technique to be used for detection may for example
be RT-PCR but various modifications of the technique which are in
routine use in the art can be utilized.
[0199] Furthermore, the above assay method can be expediently
carried out by utilizing an reagent kit for detecting an LY6H gene
in samples.
[0200] Therefore, the present invention provides an LY6H gene
detection reagent kit comprising a DNA fragment of the gene of the
invention.
[0201] This reagent kit comprises at least a DNA fragment which
hybridizes with a part or the whole of the nucleotide sequence
shown in SEQ ID NO:2 or its complementary nucleotide sequence as an
essential component and may optionally contain other components
such as a labeling agent and PCR reagents (for example, Taq DNA
polymerase, deoxynucleotide triphosphates, primers, etc.).
[0202] The labeling agent may be a radioisotope or a chemical
modifier such as a fluorescent substance but the DNA fragment as
such may have been conjugated with such a labeling agent. This
reagent kit may further contain a suitable reaction solvent or
diluent, standard antibody, buffer, wash solution, reaction stopper
solution, etc. which make an assay easier to perform.
[0203] The present invention in a further aspect provides a method
for diagnosis of neurodegenerative diseases which comprises using
the above assay method and a diagnostic agent or diagnostic reagent
kit for use in practicing said method.
[0204] By the direct or indirect sequencing of the LY6H genes
obtained from test samples by utilizing the above method, it is
possible to find new LY6H gene-related genes having high homology
to the wild-type LY6H gene.
[0205] Therefore, the present invention further provides a method
of screening for human LY6H gene-related genes in samples which
comprises performing said assay and sequencing of the LY6H genes
contained in test samples.
[0206] The wild-type LY6H and/or mutant LY6H can be determined by
utilizing the protein encoded by the human LY6H gene of the
invention (a polypeptide having the amino acid sequence shown in
SEQ ID NO:1), a polypeptide having an amino acid sequence derived
from the sequence shown in SEQ ID NO:1 by the deletion,
substitution or addition of 1 or a plurality of amino acids, a
fragment of either of them, or an antibody to any of such
proteins.
[0207] Therefore, the invention provides a method of determining an
anti-wild-type LY6H and/or mutant LY6H antibody or a method of
determining the antigen. By this method, the degree of impairment
of the brain nerve can be detected from a change in wild-type LY6H
(polypeptide). Such changes can be detected by the sequencing of
LY6H by the well-established technology described hereinabove, more
preferably by detecting differences in the LY6H polypeptide or the
presence or absence of LY6H polypeptide by the use of said antibody
(polyclonal or monoclonal antibody).
[0208] The following is a specific example of determination of said
wild-type and/or mutant LY6H. The anti-LY6H antibody can be used to
immunoprecipitate LY6H polypeptide from a solution containing a
biological sample obtained from a human body, such as blood or
serum or can be reacted with the LY6H polypeptide on polyacrylamide
gel of Western blot or immunoblot. The LY6H polypeptide in a
paraffin section or frozen tissue specimen can be detected by an
immunohistochemical technique using the anti-LY6H antibody. The
antibody production and purification technology are well known in
the art and suitable techniques can be selectively employed.
[0209] The preferred technology relevant to the detection of a
wild-type LY6H or a mutant thereof includes enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA),
immunoradiometric assay (IRMA) and immuno-enzymometric assay (IEMA)
with a sandwich technique using a monoclonal antibody and/or a
polyclonal antibody.
[0210] The invention further provides an LY6H ligand or an LY6H
receptor existing in a cell membrane fraction or on a cell surface
and having binding affinity for LY6H polypeptide. The LY6H receptor
can be obtained by conjugating a labeled LY6H polypeptide in a
biological sample containing a cell membrane fraction, extracting,
isolating and purifying the conjugation product and identifying the
amino acid sequence of the isolated product. The procedure for
preparation and the method of sequencing this LY6H receptor
polypeptide is obvious to one skilled in the art.
[0211] Furthermore, by applying the LY6H receptor or a fragment
thereof to a screening for various drugs, the invention enables
selecting out various compounds (which react with the LY6H
receptor, inclusive of low molecular compounds, high molecular
compounds, proteins, protein fragments, antigens, antibodies,
etc.). Preferably, the LY6H receptor as a whole is used. The LY6H
receptor polypeptide or fragment thereof for use in such screening
may have been immobilized on a solid matrix or be a free substance
in a solution to be transported to the cell surface.
[0212] An example of the above pharmacoscreening is a screening
system in which prokaryotic or eukaryotic host cells transformed
stably with a recombinant DNA coding for an LY6H polypeptide, or a
fragment thereof, are used in, preferably, a competitive binding
assay. As an alternative, said host cells, whether in the free form
or as immobilized, are used in the standard binding assay. More
particularly, the above pharmacoscreening may comprise reacting the
LY6H receptor polypeptide, or a fragment thereof, with the LY6H
polypeptide, or a fragment thereof, in the presence of a candidate
drug, to cause formation of a complex and detecting the degree of
inhibition of the complex formation by the above candidate
drug.
[0213] Thus, in accordance with the invention, there can be
provided a method for pharmacoscreening which comprises contacting
a candidate drug with the LY6H receptor polypeptide, or a fragment
thereof and, then, detecting the presence of the resulting complex
or the presence of a complex of the LY6H receptor polypeptide, or a
fragment thereof, with a ligand by a per se known technique.
Furthermore, by assaying LY6H receptor activity, it is possible to
evaluate whether a candidate drug is capable of antagonizing the
LY6H receptor and accordingly may modify the above-defined LY6H
activity, i.e. may be able to modulate growth of neurons, or
modulate protein-protein conjugation or complex-forming activity.
In such a competitive binding assay, the LY6H receptor polypeptide,
or a fragment thereof, is labeled. When the free LY6H receptor
polypeptide or fragment thereof is separated from the
protein-protein complex and the labeling amount of the free
(non-complex-forming) substance is measured, the measured value
serves as a yardstick of the binding of the test factor to the LY6H
receptor. The measured value serves also as a measure of inhibition
of the binding of the LY6H receptor to the LY6H polypeptide. By
analyzing a small peptide (pseudopeptide) of the LY6H polypeptide
in this manner, the candidate drug can be assayed as a substance
having LY6H receptor antagonizing activity.
[0214] Another protocol for pharmacoscreening in accordance with
the invention is that of screening for a compound having an
adequate binding affinity for the LY6H receptor polypeptide.
Briefly, this procedure comprises synthesizing a large number of
different test peptide compounds on a solid support such as the
surface of a plastic pin or other material, reacting the test
peptide compounds with the LY6H receptor polypeptide and, after
washing, detecting the binding reaction products of LY6H receptor
polypeptide by a known method [e.g. PCT patent publication No. WO
84-03564]. The purified LY6H receptor can be directly coated on the
plate to be used in said pharmacoscreening procedure. The antibody
may be captured with a non-neutralizing antibody against the
polypeptide and the LY6H receptor polypeptide be immobilized on a
solid phase.
[0215] The invention is further directed to the use of a
competitive pharmacoscreening assay. For the binding to the LY6H
receptor polypeptide, or a fragment thereof, a neutralizing
antibody capable of specific binding to the LY6H receptor
polypeptide is caused to compete with the candidate compound. By
such a competitive reaction with the neutralizing antibody, the
presence of any peptide having one or more antigenic determinants
of the LY6H receptor polypeptide can be detected.
[0216] As a further method for drug screening, the LY6H polypeptide
of the invention or the LY6H gene product of the invention can be
used in the screening for compounds which activate (agonists) or
inhibit (antagonists or inhibitors) the activity of the LY6H
polypeptide or LY6H gene product.
[0217] By using the LY6H polypeptide or LY6H gene product of the
invention, agonists or antagonists can be identified from cells,
cell-free preparations, chemical libraries and naturally-occurring
compositions. These agonists or antagonists may be natural or
modified substrates, ligands, enzymes or receptors of the LY6H
polypeptide of the invention or structural or functional copies of
the polypeptide of the invention [Coligan et al., Current Protocols
in Immunology, 1(2), Chapter 5 (1991)].
[0218] In situ hybridization studies revealed the expression of
LY6H gene of the invention in various tissues of the human normal
brain, at particularly high levels in the hippocampus and
entorhinal cortex which are usually severely impaired in Alzheimer
patients, and its expression level has been found to be
considerably depressed in the temporal lobe inclusive of the
hippocampus and entorhinal cortex of patients with Alzheimer's
disease. It is, therefore, very likely that this gene is associated
with the onset and progression of said disease.
[0219] Therefore, an agonist or antagonist of this LY6H protein or
an LY6H gene product is expected to find application as a
therapeutic or prophylactic drug for neurodegenerative diseases
such as Alzheimer's disease, Alzheimer type dementia, brain
ischemia and Parkinson's disease.
[0220] Compounds obtainable by the screening for candidate drugs
for said LY6H gene-related diseases have the functions of the
protein of the invention (the expression product of the gene of the
invention), such as neuronal survival-supporting action, nerve
elongating action, nerve regenerating action, neuroglia-activating
action, etc. in the central and other nerve systems and brain
mnemonic (memory-forming) action, among other physiological
actions, and, therefore, can be used as a therapeutic or
prophylactic drug for various neurodegenerative diseases such as
Alzheimer's disease, Alzheimer type dementia, brain ischemia and
Parkinson's disease. Thus, the proteins of the invention (inclusive
of the gene expression products, partial peptides thereof, and
salts thereof) are of use as reagents for the screening for
compounds which promote the functions of the protein of the
invention.
[0221] The invention provides a method of screening for compounds
which promote the functions of the protein of the invention
(hereinafter each referred to sometimes as a functional enhancer of
the protein of the invention). More particularly, the invention
provides (a) a method of screening for a functional enhancer of the
protein of the invention which comprises contacting (1) the protein
of the invention with nerve cells or a nerve tissue on one hand and
(2) the protein and a test compound with said nerve cells or tissue
on the other hand and comparing the results and (b) a method of
screening for a functional enhancer of the protein of the invention
which comprises administering (1) the protein of the invention to a
vertebrate on one hand and (2) the protein of the invention and a
test compound to the vertebrate on the other hand and comparing the
results.
[0222] More particularly, in the above screening method (a), a
physiological activity in the central or other nervous systems,
such as neuronal survival-supporting activity, nerve elongating
activity, nerve regenerating activity or neuroglia-activating
activity, is measured under the above conditions (1) and (2) and
the results are compared. In the screening method (b), the mnemonic
(memory-forming) activity in the brain, for instance, is measured
under said two conditions (1) and (2) and the results are
compared.
[0223] The nerve cells (neurous and neuroglia) for use in the above
screening include neuroblastoma cells, glioma cells, and their
hybridoma cells (e.g. N18TG-2, IMR-32, GOTO (e.g. GOTO-P3), NB1,
C6BU-1, U251, KNS42, KNS81 and NG108-15 cells, and PC12 cells
having a potency of differentiation to nerve cells). The nerve
tissue which can be used includes the mouse neuroepithelial cell,
rat hippocampus primary culture cell, fetal mouse culture Prukinje
cell, and mouse dorsal root ganglia. The test compound includes
peptides, proteins, nonpeptide compounds, synthetic compounds,
fermentation products, cell extracts, plant extracts, animal tissue
extracts, and plasma. These compounds may be novel compounds or
known compounds.
[0224] In carrying out said screening method (a), the protein of
the invention (inclusive of a partial peptide thereof or a salt
thereof) is dissolved or suspended in a screening buffer to prepare
a sample of the protein of the invention. The buffer may be any
buffer solution that does not interfere with the contact between
the protein of the invention and the nerve cell or tissue (e.g.
phosphate buffer, Tris-HCl buffer, etc. at pH about 4-10,
preferably pH about 6-8). The duration of contact is usually about
1-10 days, preferably about 7-10 days. The contact temperature is
usually about 37.degree. C. The activities of the protein of the
invention in the central or other nervous systems, such as neuronal
survival-supporting activity, nerve elongating activity, nerve
regenerating activity, and neuroglia-activating activity, can be
determined by the routine methods such as visual assessment of
axonal elongation, measurement of intracellular Ca.sup.2+
concentration, and the like.
[0225] Any test compound promoting any of said physiological
activities, such as neuronal survival-supporting activity, nerve
elongating activity, nerve regenerating activity,
neuroglia-activating activity, by at least about 20%, preferably
not less than about 30%, more preferably not less than about 50%,
still more preferably not less than about 70%, under the
above-mentioned condition (2) as compared with the condition (1)
can be selected as a functional enhancer of the protein of the
invention.
[0226] In carrying out the above screening method (b), the protein
of the invention, alone or in combination with the test compound,
is administered to test animals by intravenous, subcutaneous or
intramuscular injection or orally. The dosage of the protein of the
invention for oral administration is generally about 0.1-100
mg/day, preferably about 1.0-50 mg/day, more preferably about
1.0-20 mg/day, per mammal (based on 50 kg body weight). The
parenteral dose should be selected according to the recipient and
the method of administration but it is preferable to administer
about 0.01-30 mg/day, preferably about 0.1-20 mg/day, more
preferably about 0.1-10 mg/day, per mammal (50 kg body weight) by
the intravenous route.
[0227] Test animals include such mammals as man, monkey,
chimpanzee, mouse, rat, rabbit, sheep, swine, bovine, horse, cat
and dog and fish (e.g. carp, salmon, herring, rainbow trout,
goldfish, etc.).
[0228] The mnemonic (memory-forming) activity of the protein of the
invention in the brain can be assayed in accordance with, for
example, a water maze test protocol [Morris, R. G. M., J. Neurosci.
Meth., 11, 47-60 (1984)]. Any test compound promoting the above
mnemonic effect by not less than about 20%, preferably not less
than 50%, more preferably not less than 70%, under said condition
(2) as compared with said condition (1) is of use as a functional
enhancer of the protein of the invention.
[0229] The screening kit as a further embodiment of the invention
contains the protein of the invention (inclusive of the expression
product of the gene, a partial peptide thereof, and any salt of
either of them) as an essential component. A kit consists of the
following components 1-4 is an example of the screening kit of the
invention.
Component 1: Hanks solution as assay buffer Component 2: Protein
standard (protein of the invention or a salt thereof) Component 3:
Nerve cells or a nerve tissue (a culture of said nerve cells or
nerve tissue in a 24-well plate, 10.sup.4 cell/well, as grown using
Eagle's MEM, Hanks solution under 5% CO.sub.2 at 37.degree. C.)
Component 4: An inverted microscope for observation
[0230] The screening with the above screening kit can be carried
out as follows.
[Method]
[0231] The number per field of vision of axonal elongation-positive
cells in the well containing the test compound is counted and
compared with the number of axonal elongation-positive cells in the
control (test compound-free) well and the difference is
statistically tested.
[0232] The compound or salt obtained by the screening method or
with the screening kit in accordance with the invention is a member
selected from the above-mentioned class consisting of peptides,
proteins, nonpeptide compounds, synthetic compounds, fermentation
products, cell extracts, plant extracts, animal tissue extracts,
etc. and is a compound capable of promoting the function of the
protein of the invention. The compound that promotes the functions
of the protein of the invention as such may show physiological
activities such as neuronal survival-supporting activity, nerve
elongating activity, nerve regenerating activity,
neuroglia-activating activity, etc. and thereby promote the
function of the protein of the invention or the like additively or
synergistically or, although not showing such physiological
activities by itself, may promote the function of the protein of
the invention. Examples of the salts of the compound include salts
with physiologically acceptable bases (e.g. alkali metals) or acids
(e.g. organic acids, inorganic acids). Particularly preferred are
physiologically acceptable acid addition salts, such as salts with
inorganic acids (e.g. hydrochloric acid, phosphoric acid,
hydrobromic acid, sulfuric acid) or organic acids (e.g. acetic
acid, formic acid, propionic acid, fumaric acid, maleic acid,
succinic acid, tartaric acid, citric acid, malic acid, oxalic acid,
benzoic acid, methanesulfonic acid, benzenesulfonic acid).
[0233] The compound or salt which promotes the function of the
protein of the invention is of value as a safe, low-toxicity
therapeutic-prophylactic drug for various neurodegenerative
diseases such as Alzheimer's disease, Alzheimer type dementia,
brain ischemia and Parkinson's disease.
[0234] The above screening procedure involves the use of cells
which express the LY6H polypeptide on the cell surface or respond
to the protein of the invention. Among such cells are cells derived
from mammalian animals, yeasts, Drosophilia and E. coli. The cells
which express the LY6H polypeptide (or the cell membrane having the
expressed polypeptide) or respond to the LY6H polypeptide is
contacted with the test compound to observe the stimulation or
inhibition of binding or functional response. Then, LY6H activity
of cells contacted with the candidate compound is compared with
that of similar cells not contacted.
[0235] The above assay can be carried out by detecting adhesion to
cells harboring the LY6H polypeptide using a label directly or
indirectly coupled to a candidate compound or in an assay system
utilizing a competition with a label-competitive substance. In this
manner, the binding of the candidate compound can be easily tested.
Furthermore, using a detection system suited to cells bearing the
LY6H polypeptide in such assays, it may be tested whether the
candidate compound will produce a signal ascribable to activation
of the LY6H polypeptide. The activation inhibitor is generally
assayed in the presence of a known agonist and the effect of the
candidate compound on the activation due to the agonist is
observed. The assay may comprise a simple procedure comprising
mixing the candidate compound with a solution containing the LY6H
polypeptide to form a mixture, determining the LY6H activity in the
mixture, and comparing the LY6H activity of the mixture with a
standard.
[0236] The low molecular compound (agonist or antagonist) which
binds to the LY6H protein can be obtained by a screening with
BIACORE 2000, for instance [Markgren, P. O., et al., Analytical
Biochemistry, 265, 340-350 (1998)].
[0237] In accordance with the invention, for the purpose of
developing a more active or stabilized LY6H polypeptide derivative
or a drug which enhances or blocks the function of the LY6H
polypeptide in vivo, it is possible to construct a biologically
active polypeptide or a structural analog thereof for interaction,
such as an LY6H agonist, LY6H antagonist, LY6H inhibitor or the
like. The structural analog mentioned above can be obtained, for
example, by determining the three-dimensional structure of a
complex of LY6H polypeptide with another protein by X-ray
crystallography, computer modeling or a combination of such
techniques. Information on the structure of a structural analog can
also be acquired by polypeptide modeling based on the structures of
homologous proteins.
[0238] To obtain said more active or stabilized LY6H polypeptide
derivative, analysis by alanine scan can be employed. This method
comprises substituting Ala for each amino acid residue to assess
the influence of substitution on peptide activity. Thus, as each
amino acid residue of a peptide is thus analyzed, the region of
importance to the activity or stability of the peptide is
determined. By this method, it is possible to design a more active
or stable LY6H polypeptide derivative.
[0239] It is also possible to isolate the target-specific antibody
selected by the functional assay and analyze its crystal structure.
As a rule, by this approach, the pharmacore providing a basis for
subsequent drug design is obtained. By producing an anti-ideotypic
antibody to the functional pharmacologically active antibody, it is
possible to identify and isolate a peptide from a chemically or
biologically generated peptide bank. Therefore, it is predictable
that the selected peptide may also serve as a pharmacore.
[0240] In this manner, it is possible to design and develop drugs
having improved or stabilized LY6H activity or acting as
inhibitors, agonists or antagonists of LY6H activity.
[0241] Evaluation of such a drug can be made by titrating its
effect on neuronal survival using primary culture hippocampal
neurons [Japan. J. Pharmacol, 53, 221-227 (1990)] or investigating
its effect on neurodegenerative lesions in Alzheimer model animals
such as mutant .beta.-amyloid precursor protein gene or mutant
presenilin 1 gene transgenic mice [Nature, 373, 523-527 (1995):
Nature Med., 5, 560-564 (1999)].
[0242] The compound thus obtained can be used not only as a drug
for Alzheimer's disease but also as a therapeutic drug for cerebral
infarction and other neurodegenerative diseases.
[0243] Furthermore, in accordance with the invention, by
constructing LY6H gene-bearing knockout mice (transgenic mice with
LY6H knockout backgrounds), it is possible to ascertain which site
or sites of the nucleotide sequence of the LY6H gene have
influences on said multiple LY6H activities in vivo, that is to say
what functions the expression products of LY6H gene and of a
modified LY6H gene have in vivo.
[0244] This method is a technique to intentionally modify the
genetic information of a living thing by utilizing homologous
recombinant genes, and includes a method using mouse embryonic stem
cells (ES cells) as an example [Capeccchi, M. R., Science, 244,
1288-1292 (1989)].
[0245] The method of constructing said mutant mice is by now a
routine technology for those skilled in the art, and mutant mice
can be easily constructed by applying a human wild-type Ly6H gene
or a mutant LY6H gene to a modified version of the above technology
[Noda, Testuo (ed.): Jikken Igaku (Experimental Medicine),
Supplement, 14(20) (1996), Yodosha]. Therefore, by utilizing this
technique, it is possible to design and develop drugs having
improved or stabilized LY6H activity or inhibitors, agonists, and
antagonists of LY6H activity.
BRIEF DESCRIPTION OF THE DRAWING
[0246] FIG. 1 is a diagrammatic representation of Northern blots
showing the pattern of expression of LY6H gene in various sites of
the brain of a patient with Alzheimer's disease.
BEST MODE FOR CARRYING OUT THE INVENTION
[0247] The following examples are intended to illustrate the
invention in further detail.
Example 1
(1) Cloning and DNA Sequencing of Human LY6H Gene
[0248] The mRNA extracted from the human fetal brain was purchased
from CLONTECH Laboratories and used as the starting material. From
this mRNA, a cDNA was synthesized and ligated into the vector
.lamda.ZAPII (Stratagene) to construct a cDNA library (Otsuka GEN
Research Institute, Otsuka Pharmaceutical Co.). Using the in vivo
excision method [in vivo excision: Short, J. M., et al., Nucleic
Acids Res., 16, 7583-7600 (1988)], colonies of Escherichia coli
bearing the human gene were formed on agar medium and randomly
picked up to register the human gene-bearing E. coli clones in a
96-well microplate. These clones were stored at -80.degree. C.
[0249] Then, each registered clone was cultured in 1.5 ml of LB
medium overnight and the DNA was extracted and purified using an
automatic plasmid extractor PI-100 (Kurabo). The contaminated E.
coli RNA was decomposed with RNase and removed. Finally, 30 .mu.l
of a DNA solution was prepared and using a 2 .mu.l portion, the
approximate DNA size and amount were checked by the minigel method.
A 7 .mu.l portion was used for a sequencing reaction and the
remaining 21 .mu.l was stored as plasmid DNA at 4.degree. C. By
this method, a cosmid which can also be used as a probe for FISH
(fluorescence in situ hybridization) described below can be
extracted by a minor modification of the program.
[0250] Then, a dideoxy terminator reaction of Sanger et al. using
T3, T7 or a synthetic oligonucleotide primer [Sanger, F., et al.,
Proc. Natl. Acad. Sci., USA., 74, 5463-5467 (1977)] or a cycle
sequencing reaction [Carothers, A. M., et al., Bio. Techniques, 7,
494-499 (1989)] which is the dideoxy terminator reaction plus PCR
was carried out. These are techniques for chain extension with
termination specific to 4 kinds of bases using a small amount
(about 0.1-0.5 .mu.g) of plasmid DNA as the template.
[0251] Using an FITC (fluorescein isothiocyanate)-labeled primer as
the sequence primer, about 25 cycles of reaction using Taq
polymerase were carried out. Of the fluorescence-labeled DNA
fragment, the sequence of about 400 nucleotides from the 5'-end of
the cDNA was determined with the automatic DNA sequencer ALF.TM.
DNA Sequencer (Pharmacia).
[0252] The 3'-nontranslated region is high in heterogeneity among
genes and suited for differentiation of individual genes.
Therefore, sequencing of the 3'-end region was also performed in
some cases.
[0253] The huge nucleotide sequence information generated with the
DNA sequencer was transmitted to the 64-bit computer DEC3400 for
computerized homology analysis. This homology analysis was carried
out by a database (GenBank, EMBL) search according to UWGCG's FASTA
Program [Pearson, W. R. and Lipman, D. J., Proc. Natl. Acad. Sci.,
USA., 85, 2444-2448 (1988)].
[0254] Fujiwara et al. describe in detail about the above method of
analysis for a human fetal brain cDNA library [Fujiwara, T., et
al., DNA Res., 2, 107-111 (1991)].
[0255] The ESTs (expressed sequence tags: partial DNA sequences of
the expressed gene fragment) randomly selected from the human fetal
brain cDNA library constructed as above were then sequenced.
[0256] The clone designated GEN-425D01 in the GenBank/EMBL sequence
search according to the FASTA Program was found to be highly
homologous to the gene coding for the mouse Ly6 family protein.
[0257] Using a double-stranded DNA inserted into a vector
(pBluescript vector; Stratagene) as a template and a synthetic
oligonucleotide as a primer, the nucleotide sequence of the cDNA
inclusive of the whole coding region of the above clone was
determined by Sanger's dideoxy chain termination method.
[0258] Sequencing with ABIPRISMTM377 automatic DNA sequencer
revealed that the cDNA sequence of the clone obtained above
contained a deduced amino acid coding region of 420 bases and the
amino acid sequence encoded thereby had 140 amino acid residues.
The nucleic acid sequence of the full-length cDNA clone was
composed of 854 nucleotides. The full sequence is shown in SEQ ID
NO:3; the nucleotide sequence of the open reading frame is shown in
SEQ ID NO:2; and the deduced amino acid sequence encoded by said
nucleotide sequence is shown in SEQ ID NO:1.
[0259] The amino acid sequence of the human LY6H protein was
compared with the sequences of other Ly6 family proteins, and the
nucleotide sequence conserved in the amino acid translation
initiation region [Kozak, M., J. Biol. Chem., 266, 19867-19870
(1991)] was compared with the 5'-region of the human LY6H gene. The
initiation codon thus determined was located in the position
99-101, which is the second ATG triplet, of the nucleotide sequence
shown in SEQ ID NO:3. Moreover, the polyadenylation signal (AATAAA)
was located in the position 832-837 of the same nucleotide
sequence.
(2) Northern Blot Analysis
[0260] To define the expression profile of LY6H in tissues, a
Northern blot analysis was performed using various human
tissues.
[0261] In the Northern blot analysis, Human MTN (Multiple-Tissue
Northern) Blot I and II (CLONTECH) were used.
[0262] The cDNA fragment was amplified by PCR using a primer set of
T3 and T7 promoter sequences.
[0263] The PCR amplification product of said GEN-425D01cDNA clone
was labeled with [.sup.32P]-dCTP (Random Primed DNA Labeling Kit,
Boehringer Mannheim GmbH) for use as a probe.
[0264] The blot containing the amplification product was
prehybridized (under conditions according to the product protocol)
and, then, subjected to hybridization according to the product
protocol.
[0265] The hybridization was performed at 65.degree. C. overnight
in a solution composed of 0.1 M NaCl/50 mM Tris-HCl (pH
7.5)/2.times.Denhardt's solution/10% dextran sulfate/1% SDS
solution (containing 100 .mu.g/ml denatured salmon sperm DNA).
After wash twice with 2.times.SSC/0.1% SDS at room temperature, the
product was washed once with 0.1.times.SSC/0.1% SDS at 65.degree.
C. for 40 minutes. The filter was exposed against X-ray film
(Kodak) at -70.degree. C. for 18 hours.
[0266] The above test was performed using the following adult human
tissues: brain, pancreas, testis, small intestine, colon, thymus,
prostate, ovary, heart, placenta, lung, liver, skeletal muscle,
kidney, spleen, testis and peripheral blood leukocyte. As a result,
transcripts of about 1 kb showing homology to LY6H were observed in
the brain, pancreas, testis, small intestine, colon, thymus,
prostate and ovary, particularly high in the brain.
(3) Localization of the Gene on Chromosome by FISH Using Cosmid
Clones
[0267] FISH for chromosomal localization was carried out using 0.5
.mu.g of each cosmid DNA as a probe in accordance with the known
method [Takahashi, E. et al., Hum. Genet., 86, 14-16 (1990)]. It
was caught FISH signals by Provia 100 film (Fuji, ISO 100) or CCD
Camera System (Applied Imaging Cyto Vision).
[0268] As a result, the human LY6H gene was found to be located on
q24.3 of chromosome 8. Thus, GEN-425D01 was mapped on the
chromosome band 8q24.3.
[0269] The antibodies against proteins belonging to the Ly6 family
have been utilized in the purification of blood stem cells as a
target of gene therapy [van de Rijn, M., et al., Proc. Natl. Acad.
Sci., USA., 86, 4634-4638 (1989)], studies on the differentiation
of blood cells [van de Rijn. M., et al., Proc. Natl, Acad. Sci.,
USA., 86, 4634-4638 (1989); Classon, B. J. and Coverdale, L., Proc.
Natl. Acad. Sci., USA., 91, 5296-5300 (1994)], activation of immune
cells [Malek, T. R., et al., J. Exp. Med., 164, 709-722 (1986)],
inhibition of production of active immune cells [Haque, A., et al.,
Immunology, 69, 558-563 (1990)], and the like, and have also been
found to have antitumor effects [Lu, L., et al., J. Immunol., 142,
719-725 (1989)]. The human LY6H gene provided in the present
example enables detection of the expression of the gene in various
tissues, production of the human LY6H protein by genetic
engineering techniques, and construction of an antibody by
utilizing the gene, hence enabling said purification of blood stem
cells, research into the differentiation of blood cells, activation
of immune cells, inhibition of activation of immune cells, and
therapy of tumors.
[0270] Furthermore, the LY6H expressed at a high level in the brain
enables a research into the differentiation of nerve cells,
activation of neurons, and therapy of neural and mental
diseases.
[0271] Screening for compounds with the human LY6H protein as the
target is also made possible and the compounds thus obtained are as
useful as the anti-human LY6H protein antibody.
Example 2
(1) Northern Blot Analysis in the Brain Tissues of a Patient with
Alzheimer's Disease
[0272] Northern blot analysis was performed in accordance with
Example 1 (2).
[0273] To investigate the expression of the LY6H gene in the brain
tissues of patients with Alzheimer's disease, Northern blot
analysis was made using the brain tissues of an Alzheimer patient
and normal human brain tissues.
[0274] Northern blotting was performed using the human normal brain
blot II and human Alzheimer blot II (both Invitrogen) and the LY6H
gene expression in the various brain tissues, namely the frontal
lobe, temporal lobe, parietal lobe, occipital lobe, pons, thalamus
and corpus callosum, was compared between normal and Alzheimer
brains.
[0275] The results are shown in FIG. 1.
[0276] As was pointed out in Example 1, the LY6H gene is expressed
at a high level in the brain. The above analysis revealed that,
while the expression of the gene was confirmed in various tissues
of the human normal brain, the gene was expressed at particularly
high levels in the temporal lobe inclusive of the hippocampus and
entorhinal cortex which are known to be impaired severely in
patients with Alzheimer's disease while marked decreases were found
in the temporal lobe inclusive of the hippocampus and entorhinal
cortex in the patient with Alzheimer's disease, indicating that it
is very likely that the gene is involved in the onset and
progression of this disease.
[0277] Therefore, the LY6H gene sense strand, LY6H expression
product, and LY6H protein are expected to find application as
therapeutic drugs for Alzheimer's disease, Alzheimer type dementia,
brain ischemia and Parkinson's disease.
[0278] Furthermore, agonists and antagonists of LY6H protein are
also expected to be of use as therapeutic drugs for Alzheimer's
disease and other diseases.
Example 3
(1) Construction of an LY6H Expression Vector
[0279] The LY6H cDNA obtained by in vivo excision method is cleaved
with MvlI and XhoI to give an about 800-base fragment. This
fragment, containing the entire coding region of the LY6H gene
shown in SEQ ID NO:1, is ligated to the EcoRV/XhoI-cleaved
pAc5.1/V5-HisA (Invitrogen) to construct an expression vector
(pAC/LY6H expression vector).
(2) Expression and Purification of the Active Ingredient Protein of
the Invention
[0280] The pAC/LY6H expression vector DNA and pCoHYGRO vector
(Invitrogen) DNA are admixed in a ratio of 19:1 and introduced into
fruit fly (Schneider 2) cells by calcium phosphate transfection.
After the cells are cultured in 10% fetal calf serum-DES expression
medium (Invitrogen) at 23.degree. C. for 48 hours, 300 .mu.g/ml of
hygromycin (Hygromycini B, Boehringer Mannheim) is added to the
culture and the selection of drug-resistant cell clones is
performed for 2 weeks. A stable transformant is subjected to
stationary culture at a concentration of 5.times.10.sup.6 cells/ml
using 20 Falcon 5000 culture flasks (Becton Dickinson) containing
20 ml of 10% fetal calf serum-DES expression medium (Invitrogen)
and the cultured cells are harvested. After washing twice with
phosphate-buffered saline (PBS), the cells are suspended in PBS
containing 2% bovine serum albumin and 0.5 U/ml of
phosphatidyl-inositol-specific phospholipase C (PIPLC) and cultured
at 37.degree. C. for 1 hour. From the supernatant of the culture,
the objective protein can be purified by ion exchange column
chromatography or the like.
(3) Isolation and Culture of Hippocampal Neurons
[0281] The whole brain is aseptically isolated from fetal SD rats
on embryonic day 18 and the hippocampus is excised. The excised
tissue is cut to thin slices with a surgical knife and incubated
for enzymatic treatment in PBS containing 0.25% trypsin and 0.002%
DNase I at 37.degree. C. for 20 minutes. After the enzymatic
reaction is stopped by adding fetal calf serum, the
aspiration-ejection of the cell digest with a pipette having a
plastic tip is repeated 3 times to disperse the cells. The cell
dispersion is passed through a filter consisting of 2 stacked
sheets of lens paper to remove the undigested tissue and
centrifuged at 1000 rpm for 5 minutes. The cells are washed with
DMEM (Gibco) and seeded on a poly-L-lysine (Sigma)-coated 96-well
plate containing 10% FCS-DMEM at a final concentration of
2.times.10.sup.5 cells/cm.sup.2.
(4) Treatment with the Active Ingredient Protein of the
Invention
[0282] The above cells are cultured for 24 hours and after the
culture medium is changed to 1% N.sub.2 Supplement (Gibco)-DMEM,
the active ingredient protein of the invention as prepared under
(2) is added (the invention group).
[0283] For comparison, the active ingredient protein of the
invention is heat-treated in a boiling water bath for 5 minutes and
added (the boiled protein group).
(5) Evaluation of Hippocampal Neuronal Survival
[0284] The cells (culture) in each group as prepared under (4) are
cultured for 72 hours. Then, the hippocampal neuronal
survival-supporting effect of the active ingredient protein of the
invention can be evaluated by the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay. This MTT assay may be performed using Promega's "CellTiter
96" Assay System, for instance.
(6) Isolation and Culture of Midbrain Neurons
[0285] The whole brain is aseptically isolated from fetal SD rats
on embryonic day 14 and the ventral midbrain is excised. The tissue
is cut to thin slices with a surgical knife and incubated for
enzymatic treatment in phosphate-buffered saline (PBS) containing
0.25% trypsin and 0.002% DNase I at 37.degree. C. for 20 minutes.
After the enzymatic reaction is stopped by adding fetal calf serum,
the aspiration-ejection of the cell digest with a pipette having a
plastic tip is repeated 3 times to disperse the cells. The cell
dispersion is passed through a filter consisting of 2 stacked
sheets of lens paper to remove the undigested tissue and
centrifuged at 1000 rpm for 5 minutes. The cells are washed with
DMEM/F12 (Gibco) and seeded on a poly-L-lysine-coated 96-well plate
containing 10% FCS-DMEM/F12 at a final concentration of
3.times.10.sup.5 cell s/cm.sup.2.
(7) Treatment with the Active Ingredient Protein of the
Invention
[0286] The cells prepared under (6) are cultured for 24 hours and
after the culture medium is changed to 1% N.sub.2 Supplement
(Gibco)-DMEM/F12, the active ingredient protein of the invention as
prepared under (2) is added (the invention group).
[0287] For comparison, the active ingredient protein of the
invention is heat-treated in a boiling water bath for 5 minutes and
added (the boiled protein group).
(8) Evaluation of Midbrain Neuronal Survival Supporting Effect
[0288] The cells (culture) in each group as prepared under (7) are
cultured for 72 hours. Then, the midbrain neuronal
survival-supporting effect of the active ingredient protein of the
invention can be evaluated by the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay. This MTT assay may be performed using Promega's "CellTiter
96" Assay System, for instance.
(9) Evaluation of Dopaminergic Neuronal Survival-Supporting
Effect
[0289] The cells (culture) in each group as prepared under (7) are
cultured for 72 hours and, then, fixed by allowing them to stand in
4% paraformaldehyde-PBS at room temperature for 15 minutes.
Thereafter, using 1% Triton X100/PBS, it is passed through a
membrane.
[0290] To prevent nonspecific binding of the antibody, the cells
are incubated in 10% goat serum-PBS for 1 hour and, then, using an
anti-tyrosine hydroxylase polyclonal antibody (Chemicon; diluted
1000-fold with PBS), the cells are incubated at 4.degree. C. for 16
hours. After the antibody solution is removed, the cells are washed
with PBS and, with peroxidase-labeled dextran polymer-conjugated
goat anti-rabbit immunoglobulin (Dako) added, the cells are
incubated at room temperature for 1 hour.
[0291] The tyrosine hydroxylase-positive cells can be detected by
the color reaction using diaminobenzidine as the substrate. Using
the number of tyrosine hydroxylase-positive cells as a marker, the
dopaminergic neuronal survival-supporting effect can be
evaluated.
INDUSTRIAL APPLICABILITY
[0292] The present invention provides a novel brain-specific gene
and a protein encoded thereby and, by utilizing them, technologies
of value to the purification of blood stem cells, research into the
differentiation of blood cells, activation of immune cells,
inhibition of production of active immune cells, and therapy of
tumors can be provided. Also, the present invention provides novel
genes having physiologic activities such as brain neuronal
survival-supporting activity, nerve elongating activity, nerve
regenerating activity, neuroglia-activating activity and brain
memory-forming activity.
[0293] In view of the marked depression of its expression level in
the temporal lobe of the brain of patients with Alzheimer's
disease, the gene of the invention is considered to inhibit
neurodegenerative changes of the tissue, thus being of use as a
gene therapy drug. Moreover, the expression product of the gene of
the invention finds application as a prophylactic and therapeutic
drug for such neurodegenerative diseases.
Sequence CWU 1
1
31140PRTHomo sapinesmisc_featurehuman embryonic brain 1Met Leu Pro
Ala Ala Met Lys Gly Leu Gly Leu Ala Leu Leu Ala Val1 5 10 15Leu Leu
Cys Ser Ala Pro Ala His Gly Leu Trp Cys Gln Asp Cys Thr 20 25 30Leu
Thr Thr Asn Ser Ser His Cys Thr Pro Lys Gln Cys Gln Pro Ser 35 40
45Asp Thr Val Cys Ala Ser Val Arg Ile Thr Asp Pro Ser Ser Ser Arg
50 55 60Lys Asp His Ser Val Asn Lys Met Cys Ala Ser Ser Cys Asp Phe
Val65 70 75 80Lys Arg His Phe Phe Ser Asp Tyr Leu Met Gly Phe Ile
Asn Ser Gly 85 90 95Ile Leu Lys Val Asp Val Asp Cys Cys Glu Lys Asp
Leu Cys Asn Gly 100 105 110Ala Ala Gly Ala Gly His Ser Pro Trp Ala
Leu Ala Gly Gly Leu Leu 115 120 125Leu Ser Leu Gly Pro Ala Leu Leu
Trp Ala Gly Pro 130 135 1402420DNAHomo sapiensmisc_featurehuman
embryonic brain 2atgctgcctg cagccatgaa gggcctcggc ctggcgctgc
tggccgtcct gctgtgctcg 60gcgcccgctc atggcctgtg gtgccaggac tgcaccctga
ccaccaactc cagccattgc 120accccaaagc agtgccagcc gtccgacacg
gtgtgtgcca gtgtccgaat caccgatccc 180agcagcagca ggaaggatca
ctcggtgaac aagatgtgtg cctcctcctg tgacttcgtt 240aagcgacact
ttttctcaga ctatctgatg gggtttatta actctgggat cttaaaggtc
300gacgtggact gctgcgagaa ggatttgtgc aatggggcgg caggggcagg
gcacagcccc 360tgggccctgg ccggggggct cctgctcagc ctggggcctg
ccctcctctg ggctgggccc 4203854DNAHomo sapiensmisc_featurehuman
embryonic brain 3acgccgcccg agcccggagt gcggacaccc ccgggatgct
tgcgccccag aggacccgcg 60ccccaagccc ccgcgccgcc cccaggccca cccggagc
atg ctg cct gca gcc atg 116Met Leu Pro Ala Ala Met1 5aag ggc ctc
ggc ctg gcg ctg ctg gcc gtc ctg ctg tgc tcg gcg ccc 164Lys Gly Leu
Gly Leu Ala Leu Leu Ala Val Leu Leu Cys Ser Ala Pro 10 15 20gct cat
ggc ctg tgg tgc cag gac tgc acc ctg acc acc aac tcc agc 212Ala His
Gly Leu Trp Cys Gln Asp Cys Thr Leu Thr Thr Asn Ser Ser 25 30 35cat
tgc acc cca aag cag tgc cag ccg tcc gac acg gtg tgt gcc agt 260His
Cys Thr Pro Lys Gln Cys Gln Pro Ser Asp Thr Val Cys Ala Ser 40 45
50gtc cga atc acc gat ccc agc agc agc agg aag gat cac tcg gtg aac
308Val Arg Ile Thr Asp Pro Ser Ser Ser Arg Lys Asp His Ser Val
Asn55 60 65 70aag atg tgt gcc tcc tcc tgt gac ttc gtt aag cga cac
ttt ttc tca 356Lys Met Cys Ala Ser Ser Cys Asp Phe Val Lys Arg His
Phe Phe Ser 75 80 85gac tat ctg atg ggg ttt att aac tct ggg atc tta
aag gtc gac gtg 404Asp Tyr Leu Met Gly Phe Ile Asn Ser Gly Ile Leu
Lys Val Asp Val 90 95 100gac tgc tgc gag aag gat ttg tgc aat ggg
gcg gca ggg gca ggg cac 452Asp Cys Cys Glu Lys Asp Leu Cys Asn Gly
Ala Ala Gly Ala Gly His 105 110 115agc ccc tgg gcc ctg gcc ggg ggg
ctc ctg ctc agc ctg ggg cct gcc 500Ser Pro Trp Ala Leu Ala Gly Gly
Leu Leu Leu Ser Leu Gly Pro Ala 120 125 130ctc ctc tgg gct ggg ccc
tgatgtctcc tccttcccac ggggcttctg 548Leu Leu Trp Ala Gly Pro135
140agcttgctcc cctgagcctg tggctgccct ctccccagcc tggcgtggct
ggggctgggg 608gcagccttgg cccagctccg tggctgtggc ctgtggctct
cactcctccc ccgacgtgaa 668gcctccctgt ctctccgcca gctctgagtc
ccaggcagct ggacatctcc aggaaaccag 728gccatctggg caggaggcct
ggggatgagg gtgggggggg acccccaggt cccggagggg 788aagtgaagca
acagcccagc tggaagggcg tcttctgcgg agaaataaag tcacttttga 848gtcctg
854
* * * * *