U.S. patent application number 10/436356 was filed with the patent office on 2003-12-11 for novel human gaba receptor proteins and polynucleotides encoding the same.
Invention is credited to Friedrich, Glenn, Mathur, Brian, Sands, Arthur T., Turner, C. Alexander JR., Zambrowicz, Brian.
Application Number | 20030229217 10/436356 |
Document ID | / |
Family ID | 22645440 |
Filed Date | 2003-12-11 |
United States Patent
Application |
20030229217 |
Kind Code |
A1 |
Turner, C. Alexander JR. ;
et al. |
December 11, 2003 |
Novel human GABA receptor proteins and polynucleotides encoding the
same
Abstract
Novel human polynucleotide and polypeptide sequences are
disclosed that can be used in therapeutic, diagnostic, and
pharmacogenomic applications.
Inventors: |
Turner, C. Alexander JR.;
(The Woodlands, TX) ; Mathur, Brian; (The
Woodlands, TX) ; Friedrich, Glenn; (Houston, TX)
; Zambrowicz, Brian; (The Woodlands, TX) ; Sands,
Arthur T.; (The Woodlands, TX) |
Correspondence
Address: |
Lance K. Ishimoto
LEXICON GENETICS INCORPORATED
8800 Technology Forest Place
The Woodlands
TX
77381
US
|
Family ID: |
22645440 |
Appl. No.: |
10/436356 |
Filed: |
May 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10436356 |
May 12, 2003 |
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09765069 |
Jan 18, 2001 |
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6586582 |
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60176692 |
Jan 18, 2000 |
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Current U.S.
Class: |
536/23.5 |
Current CPC
Class: |
C07K 14/705 20130101;
C07K 14/70571 20130101; A61P 9/12 20180101; A01K 2217/05 20130101;
A61K 38/00 20130101; A61K 48/00 20130101; A61P 15/00 20180101; A61P
3/04 20180101; A61P 27/02 20180101; A61P 25/24 20180101; A61P 43/00
20180101 |
Class at
Publication: |
536/23.5 |
International
Class: |
C07H 021/04 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising at least 24
contiguous bases of nucleotide sequence first disclosed in SEQ ID
NO: 1.
2. An isolated nucleic acid molecule comprising a nucleotide
sequence that: (a) encodes the amino acid sequence shown in SEQ ID
NO: 2; and (b) hybridizes under stringent conditions to the
nucleotide sequence of SEQ ID NO: 1 or the complement thereof.
3. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes the amino acid sequence shown in SEQ ID NO:
2.
4. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes the amino acid sequence shown in SEQ ID NO:
8.
Description
[0001] The present application claims the benefit of U.S.
Provisional Application No. 60/176,692 which was filed on Jan. 18,
2000 and is herein incorporated by reference in its entirety.
1. INTRODUCTION
[0002] The present invention relates to the discovery,
identification, and characterization of novel human polynucleotides
encoding proteins that share sequence similarity with animal
gamma-amino butyric acid (GABA) receptor subunits. The invention
encompasses the described polynucleotides, host cell expression
systems, the encoded proteins, fusion proteins, polypeptides and
peptides, antibodies to the encoded proteins and peptides, and
genetically engineered animals that either lack or over express the
disclosed sequences, antagonists and agonists of the proteins, and
other compounds that modulate the expression or activity of the
proteins encoded by the disclosed sequences that can be used for
diagnosis, drug screening, clinical trial monitoring and the
treatment of diseases and disorders.
2. BACKGROUND OF THE INVENTION
[0003] Membrane proteins play important roles as, inter alia, cell
surface markers, receptors, and mediators of signal transduction.
GABA receptors bind a potent inhibitory neurotransmitter and this
interaction serves as a target for a variety of pharmaceutical
agents such as benzodiazepines, barbiturates and alcohol.
3. SUMMARY OF THE INVENTION
[0004] The present invention relates to the discovery,
identification, and characterization of nucleotides that encode
novel human proteins, and the corresponding amino acid sequences of
these proteins. The novel human proteins (NHPs) described for the
first time herein share structural similarity with membrane
receptors such as, but not limited to human and other mammalian
GABA receptors.
[0005] The novel human nucleic acid sequences described herein,
encode alternative proteins/open reading frames (ORFs) of 467, 392,
180, 420, 345, and 133 amino acids in length (see SEQ ID NOS: 2, 4,
6, 8, 10, and 12 respectively).
[0006] The invention also encompasses agonists and antagonists of
the described NHPs, including small molecules, large molecules,
mutant NHPs, or portions thereof that compete with native NHP,
peptides, and antibodies, as well as nucleotide sequences that can
be used to inhibit the expression of the described NHPs (e.g.,
antisense and ribozyme molecules, and gene or regulatory sequence
replacement constructs) or to enhance the expression of the
described NHP sequences (e.g., expression constructs that place the
described sequence under the control of a strong promoter system),
and transgenic animals that express a NHP transgene, or
"knock-outs" (which can be conditional) that do not express a
functional NHP. A gene trapped murine ES cell line has been
produced that knocks-out a murine ortholog of the described
NHPs.
[0007] Further, the present invention also relates to processes for
identifying compounds that modulate, i.e., act as agonists or
antagonists, of NHP expression and/or NHP activity that utilize
purified preparations of the described NHPs and/or NHP product, or
cells expressing the same. Such compounds can be used as
therapeutic agents for the treatment of any of a wide variety of
symptoms associated with biological disorders or imbalances.
4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
[0008] The Sequence Listing provides the sequences of the described
NHP ORFs that encode the described NHP amino acid sequences.
5. DETAILED DESCRIPTION OF THE INVENTION
[0009] The NHPs, described for the first time herein, are novel
proteins that are expressed in, inter alia, human cell lines, human
testis, brain, adrenal gland cells, and gene trapped human
cells.
[0010] The present invention encompasses the nucleotides presented
in the Sequence Listing, host cells expressing such nucleotides,
the expression products of such nucleotides, and: (a) nucleotides
that encode mammalian homologs of the described sequences,
including the specifically described NHPs, and the NHP products;
(b) nucleotides that encode one or more portions of the NHPs that
correspond to functional domains, and the polypeptide products
specified by such nucleotide sequences, including but not limited
to the novel regions of any active domain(s); (c) isolated
nucleotides that encode mutant versions, engineered or naturally
occurring, of the described NHPs in which all or a part of at least
one domain is deleted or altered, and the polypeptide products
specified by such nucleotide sequences, including but not limited
to soluble proteins and peptides in which all or a portion of the
signal sequence is deleted; (d) nucleotides that encode chimeric
fusion proteins containing all or a portion of a coding region of
an NHP, or one of its domains (e.g., a receptor or ligand binding
domain, accessory protein/self-association domain, etc.) fused to
another peptide or polypeptide; or (e) therapeutic or diagnostic
derivatives of the described polynucleotides such as
oligonucleotides, antisense polynucleotides, ribozymes, dsRNA, or
gene therapy constructs comprising a sequence first disclosed in
the Sequence Listing.
[0011] As discussed above, the present invention includes: (a) the
human DNA sequences presented in the Sequence Listing (and vectors
comprising the same) and additionally contemplates any nucleotide
sequence encoding a contiguous NHP open reading frame (ORF) that
hybridizes to a complement of a DNA sequence presented in the
Sequence Listing under highly stringent conditions, e.g.,
hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% sodium
dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel F. M. et al.,
eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing Associates, Inc., and John Wiley & sons, Inc., New
York, at p. 2.10.3) and encodes a functionally equivalent gene
product. Additionally contemplated are any nucleotide sequences
that hybridize to the complement of a DNA sequence that encodes and
expresses an amino acid sequence presented in the Sequence Listing
under moderately stringent conditions, e.g., washing in
0.2.times.SSC/0.1% SDS at 42.degree. C. (Ausubel et al., 1989,
supra), yet still encodes a functionally equivalent NHP product.
Functional equivalents of a NHP include naturally occurring NHPs
present in other species and mutant NHPs whether naturally
occurring or engineered (by site directed mutagenesis, gene
shuffling, directed evolution as described in, for example, U.S.
Pat. No. 5,837,458). The invention also includes degenerate nucleic
acid variants of the disclosed NHP polynucleotide sequences.
[0012] Additionally contemplated are polynucleotides encoding NHP
ORFs, or their functional equivalents, encoded by polynucleotide
sequences that are about 99, 95, 90, or about 85 percent similar or
identical to corresponding regions of the nucleotide sequences of
the Sequence Listing (as measured by BLAST sequence comparison
analysis using, for example, the GCG sequence analysis package
(Madison, Wis.) using standard default settings).
[0013] The invention also includes nucleic acid molecules,
preferably DNA molecules, that hybridize to, and are therefore the
complements of, the described NHP nucleotide sequences. Such
hybridization conditions may be highly stringent or less highly
stringent, as described above. In instances where the nucleic acid
molecules are deoxyoligonucleotides ("DNA oligos"), such molecules
are generally about 16 to about 100 bases long, or about 20 to
about 80, or about 34 to about 45 bases long, or any variation or
combination of sizes represented therein that incorporate a
contiguous region of sequence first disclosed in the Sequence
Listing. Such oligonucleotides can be used in conjunction with the
polymerase chain reaction (PCR) to screen libraries, isolate
clones, and prepare cloning and sequencing templates, etc.
[0014] Alternatively, such NHP oligonucleotides can be used as
hybridization probes for screening libraries, and assessing gene
expression patterns (particularly using a micro array or
high-throughput "chip" format). Additionally, a series of the
described NHP oligonucleotide sequences, or the complements
thereof, can be used to represent all or a portion of the described
NHP sequences. An oligonucleotide or polynucleotide sequence first
disclosed in at least a portion of one or more of the sequences of
SEQ ID NOS: 1-12 can be used as a hybridization probe in
conjunction with a solid support matrix/substrate (resins, beads,
membranes, plastics, polymers, metal or metallized substrates,
crystalline or polycrystalline substrates, etc.). Of particular
note are spatially addressable arrays (i.e., gene chips, microtiter
plates, etc.) of oligonucleotides and polynucleotides, or
corresponding oligopeptides and polypeptides, wherein at least one
of the biopolymers present on the spatially addressable array
comprises an oligonucleotide or polynucleotide sequence first
disclosed in at least one of the sequences of SEQ ID NOS: 1-12, or
an amino acid sequence encoded thereby. Methods for attaching
biopolymers to, or synthesizing biopolymers on, solid support
matrices, and conducting binding studies thereon are disclosed in,
inter alia, U.S. Pat. Nos. 5,700,637, 5,556,752, 5,744,305,
4,631,211, 5,445,934, 5,252,743, 4,713,326, 5,424,186, and
4,689,405 the disclosures of which are herein incorporated by
reference in their entirety.
[0015] Addressable arrays comprising sequences first disclosed in
SEQ ID NOS: 1-12 can be used to identify and characterize the
temporal and tissue specific expression of a gene. These
addressable arrays incorporate oligonucleotide sequences of
sufficient length to confer the required specificity, yet be within
the limitations of the production technology. The length of these
probes is within a range of between about 8 to about 2000
nucleotides. Preferably the probes consist of 60 nucleotides and
more preferably 25 nucleotides from the sequences first disclosed
in SEQ ID NOS: 1-12.
[0016] For example, a series of the described oligonucleotide
sequences, or the complements thereof, can be used in chip format
to represent all or a portion of the described sequences. The
oligonucleotides, typically between about 16 to about 40 (or any
whole number within the stated range) nucleotides in length can
partially overlap each other and/or the sequence may be represented
using oligonucleotides that do not overlap. Accordingly, the
described polynucleotide sequences shall typically comprise at
least about two or three distinct oligonucleotide sequences of at
least about 8 nucleotides in length that are each first disclosed
in the described Sequence Listing. Such oligonucleotide sequences
can begin at any nucleotide present within a sequence in the
Sequence Listing and proceed in either a sense (5'-to-3')
orientation vis-a-vis the described sequence or in an antisense
orientation.
[0017] Microarray-based analysis allows the discovery of broad
patterns of genetic activity, providing new understanding of gene
functions and generating novel and unexpected insight into
transcriptional processes and biological mechanisms. The use of
addressable arrays comprising sequences first disclosed in SEQ ID
NOS: 1-12 provides detailed information about transcriptional
changes involved in a specific pathway, potentially leading to the
identification of novel components or gene functions that manifest
themselves as novel phenotypes.
[0018] Probes consisting of sequences first disclosed in SEQ ID
NOS: 1-12 can also be used in the identification, selection and
validation of novel molecular targets for drug discovery. The use
of these unique sequences permits the direct confirmation of drug
targets and recognition of drug dependent changes in gene
expression that are modulated through pathways distinct from the
drugs intended target. These unique sequences therefore also have
utility in defining and monitoring both drug action and
toxicity.
[0019] As an example of utility, the sequences first disclosed in
SEQ ID NOS: 1-12 can be utilized in microarrays or other assay
formats, to screen collections of genetic material from patients
who have a particular medical condition. These investigations can
also be carried out using the sequences first disclosed in SEQ ID
NOS: 1-12 in silico and by comparing previously collected genetic
databases and the disclosed sequences using computer software known
to those in the art.
[0020] Thus the sequences first disclosed in SEQ ID NOS: 1-12 can
be used to identify mutations associated with a particular disease
and also as a diagnostic or prognostic assay.
[0021] Although the presently described sequences have been
specifically described using nucleotide sequence, it should be
appreciated that each of the sequences can uniquely be described
using any of a wide variety of additional structural attributes, or
combinations thereof. For example, a given sequence can be
described by the net composition of the nucleotides present within
a given region of the sequence in conjunction with the presence of
one or more specific oligonucleotide sequence(s) first disclosed in
the SEQ ID NOS: 1-12. Alternatively, a restriction map specifying
the relative positions of restriction endonuclease digestion sites,
or various palindromic or other specific oligonucleotide sequences
can be used to structurally describe a given sequence. Such
restriction maps, which are typically generated by widely available
computer programs (e.g., the University of Wisconsin GCG sequence
analysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor,
Mich., etc.), can optionally be used in conjunction with one or
more discrete nucleotide sequence(s) present in the sequence that
can be described by the relative position of the sequence relatve
to one or more additional sequence(s) or one or more restriction
sites present in the disclosed sequence.
[0022] For oligonucleotide probes, highly stringent conditions may
refer, e.g., to washing in 6.times.SSC/0.05% sodium pyrophosphate
at 37.degree. C. (for 14-base oligos), 48.degree. C. (for 17-base
oligos), 55.degree. C. (for 20-base oligos), and 60.degree. C. (for
23-base oligos). These nucleic acid molecules may encode or act as
NHP gene antisense molecules, useful, for example, in NHP gene
regulation (for and/or as antisense primers in amplification
reactions of NHP nucleic acid sequences). With respect to NHP gene
regulation, such techniques can be used to regulate biological
functions. Further, such sequences may be used as part of ribozyme
and/or triple helix sequences that are also useful for NHP gene
regulation.
[0023] Inhibitory antisense or double stranded oligonucleotides can
additionally comprise at least one modified base moiety which is
selected from the group including but not limited to
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluraci- l, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0024] The antisense oligonucleotide can also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0025] In yet another embodiment, the antisense oligonucleotide
will comprise at least one modified phosphate backbone selected
from the group consisting of a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0026] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., 1987, Nucl.
Acids Res. 15:6625-6641). The oligonucleotide is a
2'-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987,
FEBS Lett. 215:327-330). Alternatively, double stranded RNA can be
used to disrupt the expression and function of a targeted NHP.
[0027] Oligonucleotides of the invention can be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides can be synthesized by the method of Stein et al.
(1988, Nucl. Acids Res. 16:3209), and methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451), etc.
[0028] Low stringency conditions are well known to those of skill
in the art, and will vary predictably depending on the specific
organisms from which the library and the labeled sequences are
derived. For guidance regarding such conditions see, for example,
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual (and
periodic updates thereof), Cold Springs Harbor Press, N.Y.; and
Ausubel et al., 1989, Current Protocols in Molecular Biology, Green
Publishing Associates and Wiley Interscience, N.Y.
[0029] Alternatively, suitably labeled NHP nucleotide probes can be
used to screen a human genomic library using appropriately
stringent conditions or by PCR. The identification and
characterization of human genomic clones is helpful for identifying
polymorphisms (including, but not limited to, nucleotide repeats,
microsatellite alleles, single nucleotide polymorphisms, or coding
single nucleotide polymorphisms), determining the genomic structure
of a given locus/allele, and designing diagnostic tests. For
example, sequences derived from regions adjacent to the intron/exon
boundaries of the human gene can be used to design primers for use
in amplification assays to detect mutations within the exons,
introns, splice sites (e.g., splice acceptor and/or donor sites),
etc., that can be used in diagnostics and pharmacogenomics.
[0030] Further, a NHP gene homolog can be isolated from nucleic
acid obtained from an organism of interest by performing PCR using
two degenerate or "wobble" oligonucleotide primer pools designed on
the basis of amino acid sequences within the NHP products disclosed
herein. The template for the reaction may be total RNA, mRNA,
and/or cDNA obtained by reverse transcription of mRNA prepared from
human or non-human cell lines or tissue known or suspected to
express an allele of a NHP gene.
[0031] The PCR product can be subcloned and sequenced to ensure
that the amplified sequences represent the sequence of the desired
NHP gene. The PCR fragment can then be used to isolate a full
length cDNA clone by a variety of methods. For example, the
amplified fragment can be labeled and used to screen a cDNA
library, such as a bacteriophage cDNA library. Alternatively, the
labeled fragment can be used to isolate genomic clones via the
screening of a genomic library.
[0032] PCR technology can also be used to isolate full length cDNA
sequences. For example, RNA can be isolated, following standard
procedures, from an appropriate cellular or tissue source (i.e.,
one known, or suspected, to express a NHP sequence). A reverse
transcription (RT) reaction can be performed on the RNA using an
oligonucleotide primer specific for the most 5' end of the
amplified fragment for the priming of first strand synthesis. The
resulting RNA/DNA hybrid may then be "tailed" using a standard
terminal transferase reaction, the hybrid may be digested with
RNase H, and second strand synthesis may then be primed with a
complementary primer. Thus, cDNA sequences upstream of the
amplified fragment can be isolated. For a review of cloning
strategies that can be used, see e.g., Sambrook et al., 1989,
supra.
[0033] A cDNA encoding a mutant NHP gene can be isolated, for
example, by using PCR. In this case, the first cDNA strand may be
synthesized by hybridizing an oligo-dT oligonucleotide to mRNA
isolated from tissue known or suspected to be expressed in an
individual putatively carrying a mutant NHP allele, and by
extending the new strand with reverse transcriptase. The second
strand of the cDNA is then synthesized using an oligonucleotide
that hybridizes specifically to the 5' end of the normal gene.
Using these two primers, the product is then amplified via PCR,
optionally cloned into a suitable vector, and subjected to DNA
sequence analysis through methods well known to those of skill in
the art. By comparing the DNA sequence of the mutant NHP allele to
that of a corresponding normal NHP allele, the mutation(s)
responsible for the loss or alteration of function of the mutant
NHP gene product can be ascertained.
[0034] Alternatively, a genomic library can be constructed using
DNA obtained from an individual suspected of or known to carry a
mutant NHP allele (e.g., a person manifesting a NHP-associated
phenotype such as, for example, obesity, vision disorders, high
blood pressure, depression, infertility, etc.), or a cDNA library
can be constructed using RNA from a tissue known, or suspected, to
express a mutant NHP allele. A normal NHP gene, or any suitable
fragment thereof, can then be labeled and used as a probe to
identify the corresponding mutant NHP allele in such libraries.
Clones containing mutant NHP gene sequences can then be purified
and subjected to sequence analysis according to methods well known
to those skilled in the art.
[0035] Additionally, an expression library can be constructed
utilizing cDNA synthesized from, for example, RNA isolated from a
tissue known, or suspected, to express a mutant NHP allele in an
individual suspected of or known to carry such a mutant allele. In
this manner, gene products made by the putatively mutant tissue can
be expressed and screened using standard antibody screening
techniques in conjunction with antibodies raised against a normal
NHP product, as described below. (For screening techniques, see,
for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A
Laboratory Manual", Cold Spring Harbor Press, Cold Spring
Harbor.)
[0036] Additionally, screening can be accomplished by screening
with labeled NHP fusion proteins, such as, for example, alkaline
phosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In
cases where a NHP mutation results in an expressed gene product
with altered function (e.g., as a result of a missense or a
frameshift mutation), polyclonal antibodies to a NHP are likely to
cross-react with a corresponding mutant NHP gene product. Library
clones detected via their reaction with such labeled antibodies can
be purified and subjected to sequence analysis according to methods
well known in the art.
[0037] The invention also encompasses (a) DNA vectors that contain
any of the foregoing NHP coding sequences and/or their complements
(i.e., antisense); (b) DNA expression vectors that contain any of
the foregoing NHP coding sequences operatively associated with a
regulatory element that directs the expression of the coding
sequences (for example, baculo virus as described in U.S. Pat. No.
5,869,336 herein incorporated by reference); (c) genetically
engineered host cells that contain any of the foregoing NHP coding
sequences operatively associated with a regulatory element that
directs the expression of the coding sequences in the host cell;
and (d) genetically engineered host cells that express an
endogenous NHP gene under the control of an exogenously introduced
regulatory element (i.e., gene activation). As used herein,
regulatory elements include, but are not limited to, inducible and
non-inducible promoters, enhancers, operators and other elements
known to those skilled in the art that drive and regulate
expression. Such regulatory elements include but are not limited to
the cytomegalovirus (hCMV) immediate early gene, regulatable, viral
elements (particularly retroviral LTR promoters), the early or late
promoters of SV40 adenovirus, the lac system, the trp system, the
TAC system, the TRC system, the major operator and promoter regions
of phage lambda, the control regions of fd coat protein, the
promoter for 3-phosphoglycerate kinase (PGK), the promoters of acid
phosphatase, and the promoters of the yeast .alpha.-mating
factors.
[0038] The present invention also encompasses antibodies and
anti-idiotypic antibodies (including Fab fragments), antagonists
and agonists of the NHP, as well as compounds or nucleotide
constructs that inhibit expression of a NHP gene (transcription
factor inhibitors, antisense and ribozyme molecules, or gene or
regulatory sequence replacement constructs), or promote the
expression of a NHP (e.g., expression constructs in which NHP
coding sequences are operatively associated with expression control
elements such as promoters, promoter/enhancers, etc.).
[0039] The NHPs or NHP peptides, NHP fusion proteins, NHP
nucleotide sequences, antibodies, antagonists and agonists can be
useful for the detection of mutant NHPs or inappropriately
expressed NHPs for the diagnosis of disease. The NHP proteins or
peptides, NHP fusion proteins, NHP nucleotide sequences, host cell
expression systems, antibodies, antagonists, agonists and
genetically engineered cells and animals can be used for screening
for drugs (or high throughput screening of combinatorial libraries)
effective in the treatment of the symptomatic or phenotypic
manifestations of perturbing the normal function of NHP in the
body. The use of engineered host cells and/or animals may offer an
advantage in that such systems allow not only for the
identification of compounds that bind to the endogenous receptor
for an NHP, but can also identify compounds that trigger
NHP-mediated activities or pathways.
[0040] Finally, the NHP products can be used as therapeutics. For
example, soluble derivatives such as NHP peptides/domains
corresponding to the NHPs, NHP fusion protein products (especially
NHP-Ig fusion proteins, i.e., fusions of a NHP, or a domain of a
NHP, to an IgFc), NHP antibodies and anti-idiotypic antibodies
(including Fab fragments), antagonists or agonists (including
compounds that modulate or act on downstream targets in a
NHP-mediated pathway) can be used to directly treat diseases or
disorders. For instance, the administration of an effective amount
of soluble NHP, or a NHP-IgFc fusion protein or an anti-idiotypic
antibody (or its Fab) that mimics the NHP could activate or
effectively antagonize the endogenous NHP receptor. Nucleotide
constructs encoding such NHP products can be used to genetically
engineer host cells to express such products in vivo; these
genetically engineered cells function as "bioreactors" in the body
delivering a continuous supply of a NHP, a NHP peptide, or a NHP
fusion protein to the body. Nucleotide constructs encoding
functional NHPs, mutant NHPs, as well as antisense and ribozyme
molecules can also be used in "gene therapy" approaches for the
modulation of NHP expression. Thus, the invention also encompasses
pharmaceutical formulations and methods for treating biological
disorders.
[0041] Various aspects of the invention are described in greater
detail in the subsections below.
5.1 THE NHP SEQUENCES
[0042] The cDNA sequences and the corresponding deduced amino acid
sequences of the described NHPs are presented in the Sequence
Listing. The NHP nucleotides were obtained from clustered human
gene trapped sequences, ESTs, and cDNA isolated from a human testis
cell library. The described sequences share structural similarity
with GABA receptor proteins, and particularly rho 3 subunits of the
GABA receptor.
5.2 NHPS AND NHP POLYPEPTIDES
[0043] NHPs, polypeptides, peptide fragments, mutated, truncated,
or deleted forms of the NHPs, and/or NHP fusion proteins can be
prepared for a variety of uses. These uses include but are not
limited to the generation of antibodies, as reagents in diagnostic
assays, for the identification of other cellular gene products
related to a NHP, as reagents in assays for screening for compounds
that can be as pharmaceutical reagents useful in the therapeutic
treatment of mental, biological, or medical disorders and diseases.
Given the similarity information and expression data, the described
NHPs can be targeted (by drugs, oligos, antibodies, etc,) in order
to treat disease, or to therapeutically augment the efficacy of
therapeutic agents.
[0044] The Sequence Listing discloses the amino acid sequences
encoded by the described NHP sequences. The NHPs typically display
initiator methionines in DNA sequence contexts consistent with a
translation initiation site, and a signal sequence characteristic
of membrane or secreted proteins.
[0045] The NHP amino acid sequences of the invention include the
amino acid sequences presented in the Sequence Listing as well as
analogues and derivatives thereof. Further, corresponding NHP
homologues from other species are encompassed by the invention. In
fact, any NHP protein encoded by the NHP nucleotide sequences
described above are within the scope of the invention, as are any
novel polynucleotide sequences encoding all or any novel portion of
an amino acid sequence presented in the Sequence Listing. The
degenerate nature of the genetic code is well known, and,
accordingly, each amino acid presented in the Sequence Listing, is
generically representative of the well known nucleic acid "triplet"
codon, or in many cases codons, that can encode the amino acid. As
such, as contemplated herein, the amino acid sequences presented in
the Sequence Listing, when taken together with the genetic code
(see, for example, Table 4-1 at page 109 of "Molecular Cell
Biology", 1986, J. Darnell et al. eds., Scientific American Books,
New York, N.Y., herein incorporated by reference) are generically
representative of all the various permutations and combinations of
nucleic acid sequences that can encode such amino acid
sequences.
[0046] The invention also encompasses proteins that are
functionally equivalent to the NHPs encoded by the presently
described nucleotide sequences as judged by any of a number of
criteria, including, but not limited to, the ability to bind and
cleave a substrate of a NHP, or the ability to effect an identical
or complementary downstream pathway, or a change in cellular
metabolism (e.g., proteolytic activity, ion flux, tyrosine
phosphorylation, transport, etc.). Such functionally equivalent NHP
proteins include, but are not limited to, additions or
substitutions of amino acid residues within the amino acid sequence
encoded by the NHP nucleotide sequences described above, but which
result in a silent change, thus producing a functionally equivalent
gene product. Amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved. For example, nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine; polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; positively charged (basic) amino acids include arginine,
lysine, and histidine; and negatively charged (acidic) amino acids
include aspartic acid and glutamic acid.
[0047] A variety of host-expression vector systems can be used to
express the NHP nucleotide sequences of the invention. Where, as in
the present instance, the NHP peptide or polypeptide is thought to
be membrane protein, the hydrophobic regions of the protein can be
excised and the resulting soluble peptide or polypeptide can be
recovered from the culture media. Such expression systems also
encompass engineered host cells that express a NHP, or functional
equivalent, in situ. Purification or enrichment of a NHP from such
expression systems can be accomplished using appropriate detergents
and lipid micelles and methods well known to those skilled in the
art. However, such engineered host cells themselves may be used in
situations where it is important not only to retain the structural
and functional characteristics of the NHP, but to assess biological
activity, e.g., in drug screening assays.
[0048] The expression systems that can be used for purposes of the
invention include but are not limited to microorganisms such as
bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing NHP nucleotide sequences; yeast (e.g., Saccharomiyces,
Pichia) transformed with recombinant yeast expression vectors
containing NHP nucleotide sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
containing NHP sequences; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing NHP nucleotide sequences; or mammalian cell systems
(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression
constructs containing promoters derived from the genome of
mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses (e.g., the adenovirus late promoter; the vaccinia virus
7.5K promoter).
[0049] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the NHP
product being expressed. For example, when a large quantity of such
a protein is to be produced for the generation of pharmaceutical
compositions of or containing NHP, or for raising antibodies to a
NHP, vectors that direct the expression of high levels of fusion
protein products that are readily purified may be desirable. Such
vectors include, but are not limited, to the E. coli expression
vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which a NHP
coding sequence may be ligated individually into the vector in
frame with the lacZ coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids
Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. pGEX vectors (Pharmacia or American
Type Culture Collection) can also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione. The
PGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0050] In an insect system, Autographa callifornica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. A NHP coding
sequence may be cloned individually into non-essential regions (for
example the polyhedrin gene) of the virus and placed under control
of an AcNPV promoter (for example the polyhedrin promoter).
Successful insertion of NHP coding sequence will result in
inactivation of the polyhedrin gene and production of non-occluded
recombinant virus (i.e., virus lacking the proteinaceous coat coded
for by the polyhedrin gene). These recombinant viruses are then
used to infect Spodoptera frugiperda cells in which the inserted
sequence is expressed (e.g., see Smith et al., 1983, J. Virol.
46:584; Smith, U.S. Pat. No. 4,215,051).
[0051] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the NHP nucleotide sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing a NHP
product in infected hosts (e.g., See Logan & Shenk, 1984, Proc.
Natl. Acad. Sci. USA 81:3655-3659). Specific initiation signals may
also be required for efficient translation of inserted NHP
nucleotide sequences. These signals include the ATG initiation
codon and adjacent sequences. In cases where an entire NHP gene or
cDNA, including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only a portion of a NHP coding sequence is inserted,
exogenous translational control signals, including, perhaps, the
ATG initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (See Bittner et al., 1987, Methods in Enzymol.
153:516-544).
[0052] In addition, a host cell strain may be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include, but are not limited to, CHO, VERO, BHK, HeLa,
COS, MDCK, 293, 3T3, WI38, and in particular, human cell lines.
[0053] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the NHP sequences described above can be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the NHP product. Such engineered cell lines may
be particularly useful in screening and evaluation of compounds
that affect the endogenous activity of the NHP product.
[0054] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci.
USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072);
neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro,
which confers resistance to hygromycin (Santerre, et al., 1984,
Gene 30:147).
[0055] Alternatively, any fusion protein can be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl.
Acad. Sci. USA 88:8972-8976). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the
gene's open reading frame is translationally fused to an
amino-terminal tag consisting of six histidine residues. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+-nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
5.3 ANTIBODIES TO NHP PRODUCTS
[0056] Antibodies that specifically recognize one or more epitopes
of a NHP, or epitopes of conserved variants of a NHP, or peptide
fragments of a NHP are also encompassed by the invention. Such
antibodies include but are not limited to polyclonal antibodies,
monoclonal antibodies (mAbs), humanized or chimeric antibodies,
single chain antibodies, Fab fragments, F(ab').sub.2 fragments,
fragments produced by a Fab expression library, anti-idiotypic
(anti-Id) antibodies, and epitope-binding fragments of any of the
above.
[0057] The antibodies of the invention may be used, for example, in
the detection of NHP in a biological sample and may, therefore, be
utilized as part of a diagnostic or prognostic technique whereby
patients may be tested for abnormal amounts of NHP. Such antibodies
may also be utilized in conjunction with, for example, compound
screening schemes for the evaluation of the effect of test
compounds on expression and/or activity of a NHP gene product.
Additionally, such antibodies can be used in conjunction gene
therapy to, for example, evaluate the normal and/or engineered
NHP-expressing cells prior to their introduction into the patient.
Such antibodies may additionally be used as a method for the
inhibition of abnormal NHP activity. Thus, such antibodies may,
therefore, be utilized as part of treatment methods.
[0058] For the production of antibodies, various host animals may
be immunized by injection with the NHP, an NHP peptide (e.g., one
corresponding to a functional domain of an NHP), truncated NHP
polypeptides (NHP in which one or more domains have been deleted),
functional equivalents of the NHP or mutated variant of the NHP.
Such host animals may include but are not limited to pigs, rabbits,
mice, goats, and rats, to name but a few. Various adjuvants may be
used to increase the immunological response, depending on the host
species, including but not limited to Freund's adjuvant (complete
and incomplete), mineral salts such as aluminum hydroxide or
aluminum phosphate, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and Corynebacterium parvum. Alternatively, the
immune response could be enhanced by combination and or coupling
with molecules such as keyhole limpet hemocyanin, tetanus toxoid,
diptheria toxoid, ovalbumin, cholera toxin or fragments thereof.
Polyclonal antibodies are heterogeneous populations of antibody
molecules derived from the sera of the immunized animals.
[0059] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, can be obtained by any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique of Kohler and Milstein, (1975,
Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72;
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA. IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo makes this the, presently
preferred method of production.
[0060] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci., 81:6851-6855; Neuberger et al., .1984, Nature, 312:604-608;
Takeda et al., 1985, Nature, 314:452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable region derived from a
murine mAb and a human immunoglobulin constant region. Such
technologies are described in U.S. Pat. Nos. 6,075,181 and
5,877,397 and their respective disclosures which are herein
incorporated by reference in their entirety. Also favored is the
production of fully humanized monoclonal antibodies as described in
U.S. Pat. No. 6,150,584 and respective disclosures which are herein
incorporated by reference in their entirety.
[0061] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be
adapted to produce single chain antibodies against NHP gene
products. Single chain antibodies are formed by linking the heavy
and light chain fragments of the Fv region via an amino acid
bridge, resulting in a single chain polypeptide.
[0062] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, such fragments include,
but are not limited to: the F(ab').sub.2 fragments which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse et al., 1989, Science,
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
[0063] Antibodies to a NHP can, in turn, be utilized to generate
anti-idiotype antibodies that "mimic" a given NHP, using techniques
well known to those skilled in the art. (See, e.g., Greenspan &
Bona, 1993, FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.
147(8):2429-2438). For example antibodies which bind to a NHP
domain and competitively inhibit the binding of NHP to its cognate
receptor can be used to generate anti-idiotypes that "mimic" the
NHP and, therefore, bind and activate or neutralize a receptor.
Such anti-idiotypic antibodies or Fab fragments of such
anti-idiotypes can be used in therapeutic regimens involving a NHP
mediated pathway.
[0064] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing
description. Such modifications are intended to fall within the
scope of the appended claims. All cited publications, patents, and
patent applications are herein incorporated by reference in their
entirety.
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