U.S. patent application number 10/760709 was filed with the patent office on 2004-10-07 for novel human calcium dependent proteases, polynucleotides encoding the same, and uses thereof.
Invention is credited to Donoho, Gregory, Friedrich, Glenn, Nehls, Michael C., Sands, Arthur T., Turner, C. Alexander JR., Zambrowicz, Brian.
Application Number | 20040198968 10/760709 |
Document ID | / |
Family ID | 26849208 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040198968 |
Kind Code |
A1 |
Donoho, Gregory ; et
al. |
October 7, 2004 |
Novel human calcium dependent proteases, polynucleotides encoding
the same, and uses thereof
Abstract
Novel human polunucleotide and polypeptide sequences are
disclosed that can be used in therapeuutic, diagnostic, and
pharmacogenomic applications.
Inventors: |
Donoho, Gregory; (Portage,
MI) ; Turner, C. Alexander JR.; (The Woodlands,
TX) ; Nehls, Michael C.; (Stockdorf, DE) ;
Friedrich, Glenn; (Houston, TX) ; Zambrowicz,
Brian; (The Woodlands, TX) ; Sands, Arthur T.;
(The Woodlands, TX) |
Correspondence
Address: |
LEXICON GENETICS INCORPORATED
8800 TECHNOLOGY FOREST PLACE
THE WOODLANDS
TX
77381-1160
US
|
Family ID: |
26849208 |
Appl. No.: |
10/760709 |
Filed: |
January 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10760709 |
Jan 20, 2004 |
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10202619 |
Jul 23, 2002 |
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6716614 |
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10202619 |
Jul 23, 2002 |
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09653839 |
Sep 1, 2000 |
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6433153 |
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60152057 |
Sep 2, 1999 |
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Current U.S.
Class: |
536/23.2 ;
435/226; 435/320.1; 435/325; 435/6.16; 435/69.1; 530/388.26 |
Current CPC
Class: |
C12N 9/6472
20130101 |
Class at
Publication: |
536/023.2 ;
435/006; 435/069.1; 435/226; 435/320.1; 435/325; 530/388.26 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/64; C07K 016/40 |
Claims
1.-3. (Cancelled)
4. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO:2.
5. An antibody having immunospecificity for the polypeptide
sequence of SEQ ID NO:2.
6. A process for identifying a compound useful for the treatment of
low white blood cell count in a patient, comprising contacting a
NHP preparation with said compound for a time sufficient to allow
the compound to interact with or bind to the NHP preparation, and
determining whether said compound inhibits the activity of the NHP
preparation.
7. The process of claim 6, wherein said NHP preparation comprises
an isolated NIP protein, polypeptide or peptide.
8. The process of claim 6, wherein said NHP preparation comprises a
recombinant cell that expresses a NHP protein, polypeptide, or
peptide.
9. The process of claim 6, wherein said NHP preparation comprises a
transgenic nonhuman animal that expresses a human NHP protein,
polypeptide or peptide.
10. The process of claim 6, wherein said NHP preparation comprises
a human NHP protein, polypeptide, or peptide.
11. A process for treating low white blood cell count, in a mammal
in need of such treatment, comprising administering a
therapeutically effective amount of a compound that inhibits NHP
activity to said mammal.
12. The process of claim 11, wherein said mammal is a human.
13. A method for selectively inhibiting NHP activity in a human
patient, comprising administering a compound that selectivity of a
NHP protein, polypeptide or peptide in said patient, wherein the
activity of the compound does not result in significant toxic side
effects in said patient.
14. The method of claim 13, wherein the inhibition of the NHP
protein, polypeptide or peptide increases white blood cell count in
said patient.
Description
[0001] The present application is a continuation-in-part of
co-pending U.S. application Ser. No. 09/653,839, filed on Sep. 1,
2000, which claims the benefit of U.S. Provisional Application
Serial No. 60/152,057, filed Sep. 2, 1999, both of which are herein
incorporated by reference in their entirety.
1.0 INTRODUCTION
[0002] The present invention relates to the discovery,
identification, and characterization of novel human polynucleotides
encoding proteins that share sequence similarity with human calcium
dependent proteases, specifically calpains. 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 overexpress the
disclosed polynucleotides, antagonists and agonists of the
proteins, and other compounds that modulate the expression or
activity of the proteins encoded by the disclosed polynucleotides,
which can be used for diagnosis, drug screening, clinical trial
monitoring, the treatment of diseases and disorders, such as a
reduced white blood cell count, and cosmetic or nutriceutical
applications.
2.0 BACKGROUND OF THE INVENTION
[0003] Proteases are enzymes that mediate the proteolytic cleavage
of polypeptide sequences. In particular, calcium-dependent
proteases, such as calpains, have been found in virtually every
vertebrate cell that has been examined for their presence. The
calpain system has at least three well-characterized protein
members that are activated in response to changes in calcium
concentration. These proteins include at least two calpains that
are activated at different concentrations of calcium, and a
calpastatin that specifically inhibits the two calpains. Various
tissue/species specific cDNAs have been described that are
homologous to the calpains. Given the near ubiquitous expression of
calpains, they have been implicated in a wide variety of cellular
functions including, but not limited to, cell proliferation and
differentiation, signal transduction, processes involving
interactions between the cell membrane and cytoskeleton secretion,
platelet aggregation, cytokinesis, and disease. Accordingly,
calpains represent a key target for the regulation of a variety of
biological pathways.
[0004] Reduced white blood cell count, or neutropenia, is a major
complication that occurs during many forms of chemotherapy,
particularly those regimens involving myelosuppressive anti-cancer
drugs, and as a result of certain infectious diseases. Although
treatments for neutropenia currently exist in the art, they are not
ideal for use in all circumstances, and are actually
contraindicated in certain patients. Therefore, new treatments for
neutropenia would represent a significant advance in the art.
3.0 SUMMARY OF THE INVENTION
[0005] 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 animal
calcium-activated proteases, or calpains. As such, the novel genes
represent a new class of protease proteins with a range of
homologues and orthologs that transcend phyla and a broad range of
species.
[0006] The novel human nucleic acid sequences described herein,
encode proteins/open reading frames (ORFs) of 739, 723, 702, and
686 amino acids in length (see SEQ ID NOS:2, 4, 6, and 8
respectively).
[0007] 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 NHPs,
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 open reading frame or
regulatory sequence replacement constructs) or to enhance the
expression of the described NHPs (e.g., expression constructs that
place the described polynucleotide under the control of a strong
promoter system), and transgenic animals that express a NHP
sequence, or "knock-outs" (which can be conditional) that do not
express a functional NHP. Knock-out mice can be produced in several
ways, one of which involves the use of mouse embryonic stem cell
("ES cell") lines that contain gene trap mutations in a murine
homolog of at least one of the described NHPs. When the unique NHP
sequences described in SEQ ID NOS:1-9 are "knocked-out" they
provide a method of identifying phenotypic expression of the
particular gene, as well as a method of assigning function to
previously unknown genes. In addition, animals in which the unique
NHP sequences described in SEQ ID NOS:1-9 are "knocked-out" provide
an unique source in which to elicit antibodies to homologous and
orthologous proteins, which would have been previously viewed by
the immune system as "self" and therefore would have failed to
elicit significant antibody responses.
[0008] To these ends, gene trapped knockout ES cells have been
generated in murine homologs of the described NHPs.
Characterization of mice in which both copies of a NHP have been
disrupted (homozygotes) has allowed the identification of a novel
role for this enzyme, and a model for the study of certain
disorders. In particular, NHP knockout mice (that are homozygous
for the mutated gene) display, intra alia, increased white blood
cell counts. This suggests that these mice can be used as models
for the study of the treatment of a variety of human conditions,
including, but not limited to, neutropenia, as exemplified by
neutropenia associated with the administration of myelosuppressive
anti-cancer drugs.
[0009] In addition, the invention includes animals containing at
least a single disrupted NHP allele (e.g., "knock-out" mice) that
do not express normal levels of a NHP, humanized "knock-in" animals
where the endogenous murine NHP gene has been replaced by one or
more polynucleotides encoding at least a first human NHP protein,
or animals harboring one or more NHP transgene (e.g., mice
overexpressing a NHP). These animals may either transiently,
inducibly, or constitutively express a NHP.
[0010] Additionally, the unique NHP sequences described in SEQ ID
NOS:1-9 are useful for the identification of protein coding
sequences, and mapping an unique gene to a particular chromosome.
These sequences identify biologically verified exon splice
junctions, as opposed to splice junctions that may have been
bioinformatically predicted from genomic sequence alone. The
sequences of the present invention are also useful as additional
DNA markers for restriction fragment length polymorphism (RFLP)
analysis, and in forensic biology, particularly given the presence
of nucleotide polymorphisms within the described sequences.
[0011] 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 products, 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, such
as reduced white blood cell count.
[0012] The present invention also provides novel methods and
compositions that can be used to facilitate drug discovery, drug
development, and/or as treatments of conditions such as reduced
white blood cell count, and the complications resulting therefrom.
The present invention is based on the identification and novel
functional characterization of the NHPs described herein.
[0013] The invention encompasses diagnostic assays that make use of
the NHP polynucleotide sequences, or portions thereof, host cells
expressing such nucleotides, and the expression products of such
nucleotides, nucleotides that encode mammalian versions of the
NHPs, including human NHPs, nucleotides that encode NHP mutants and
the corresponding mutant NHP expression products, nucleotides that
encode portions of a NHP that correspond to one or more of the NHP
functional domains and the polypeptide products specified by such
nucleotide sequences, and nucleotides that encode fusion proteins
containing a NHP or one or more of its domains fused to another
polypeptide.
[0014] The present invention also features assays for the
identification of compounds that modulate NHP activity in the body.
Such compounds can be used as agents to affect NHP-mediated
processes, for example, as therapeutic agents for the treatment of
low white blood cell count. The present invention also contemplates
methods of using mammalian NHP protein(s), and particularly
recombinantly expressed human NHP protein(s), in cell-free and/or
cell-based assays for identifying compounds (modulators) that bind
to and/or antagonize or otherwise modulate (i.e., increase or
decrease) NHP activity. Compounds developed using such assays are
then typically used in in vivo assays to determine the effect of
such compounds on NHP-mediated processes, and to discern or verify
the observed phenotypic effects. Such phenotypic effects include,
but are not limited to, increased white blood cell count or
reduction in one or more of the complications associated with
reduced white blood cell count. The invention thus additionally
contemplates compounds that bind to and/or activate or inhibit the
activity of a NHP, as well as pharmaceutical compositions
comprising such compounds, and the use of such compounds to treat
NHP-related disorders.
[0015] In addition to small molecule agonists and antagonists of
the NHPs, the invention also contemplates the use of large
molecules to effect the levels or bioavailability of a NHP in vivo,
including, but not limited to, mutant NHP proteins that compete
with native NHPs, anti-NHP antibodies, anti-idiotypic antibodies
that bind anti-NHP antibodies or NHP binding partners, nucleotide
sequences that can be used to inhibit NHP expression (e.g.,
antisense, ribozyme and/or triplex molecules, and coding sequence
or regulatory sequence replacement constructs) or to enhance NHP
expression (e.g., expression constructs that place a NHP sequence
under the control of a strong promoter or expression system).
[0016] In addition, the invention encompasses methods and
compositions for the diagnostic evaluation, typing and prognosis of
NHP-mediated disorders, including, inter alia, low white blood cell
count, and for the identification of subjects having a
predisposition to such conditions.
[0017] For example, in another embodiment of the present invention,
NHP nucleic acid molecules of the invention can be used as
diagnostic hybridization probes or as primers for diagnostic PCR
analysis for the identification of NHP gene mutations, allelic
variations, and/or regulatory defects in a NHP gene. NHP sequences
may be used in hybridization or amplification assays of biological
samples to detect abnormalities involving NHP gene structure,
including point mutations, insertions, deletions and/or chromosomal
rearrangements. Such diagnostic assays include, but are not limited
to, Southern analyses, single stranded conformational polymorphism
analyses (SSCP), restriction fragment length polymorphisms (RFLP),
coding single nucleotide polymorphisms (cSNP) and PCR analyses.
These assays can be combined with "gene chip" technology and used
to screen pre-existing genetic databases of patients suffering from
various NHP-mediated disorders. The sequences of the present
invention are also useful as additional DNA markers for forensic
biology. The present invention further provides for diagnostic kits
for practicing such methods.
4.0 DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES
[0018] The Sequence Listing provides the sequences of 4
calpain-like ORFs that encode the described NHP amino acid
sequences.
5.0 DETAILED DESCRIPTION OF THE INVENTION
[0019] The NHPs, described for the first time herein, are novel
proteins that are expressed in, inter alia, human cell lines, and
human prostate and testis cells. The described sequences were
compiled from gene trapped cDNAs and clones isolated from a human
testis cDNA library (Edge Biosystems, Gaithersburg, Md.).
[0020] The present invention encompasses the nucleotides presented
in the Sequence Listing, host cells expressing such nucleotides,
the sexpression products of such nucleotides, and: (a) nucleotides
that encode mammalian homologs of the described polynucleotides,
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; (d) nucleotides that
encode chimeric fusion proteins containing all or a portion of a
coding region of a NHP, or one of its domains (e.g., a receptor
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.
[0021] The present invention also includes murine NHPs, mutated
muririe embryonic stem cell clones, and animals derived from these
embryonic stem cells. Characterization of mice in which NHP gene
function has been disrupted (knock-outs) indicates that the NHPs
play a role in conditions including, but not limited to, reduced
white blood cell count, one or more of the complications arising
from reduced white blood cell count, and other disorders, as
detailed herein.
[0022] The invention encompasses the use of NHP nucleotides, NHP
proteins and peptides, as well as antibodies to NHPs (that can, for
example, act as NHP agonists or antagonists), antagonists
(peptides, small organic molecules, fusion proteins, etc.) that
inhibit NHP activity or expression, or agonists that activate NHP
activity orincrease its expression, in the identification,
diagnosis, prognosis, and/or treatment of NHP-mediated disorders.
The diagnosis of a NHP abnormality in a patient, or an abnormality
in a NHP regulatory pathway, can also facilitate the development of
treatments or therapeutic regimens. In addition, NHP nucleotides
and NHP proteins can be used to identify compounds effective in the
treatment of, among other things, NHP-mediated disorders,
including, but not limited to, reduced white blood cell count. In
addition, the present invention encompasses methods and
compositions for the diagnostic evaluation, typing and prognosis of
NHP-mediated disorders including, but not limited to, reduced white
blood cell count.
[0023] An additional embodiment of the present invention relates to
methods of using NHP polynucleotides and/or NHP gene products
(proteins, polypeptides and/or peptides) for the identification of
compounds that modulate, i.e., act as agonists or antagonists, of
NHP gene expression and/or NHP gene product activity. Such
compounds can be used as agents to manipulate NHP-mediated
disorders and, in particular, as therapeutic agents for the
treatment of NHP-mediated disorders. Such methods and compositions
are typically capable of modulating the level of NHP gene
expression and/or the level of NHP gene product activity. The basis
for these aspects of the present invention is the novel discovery
that the elimination of both NHP alleles results in, among other
effects, increased white blood cell counts, as shown herein
below.
[0024] The invention described in the subsections below thus
encompasses NHP polypeptides or peptides corresponding to one or
more of the functional domains of a NHP, mutated, truncated or
deleted NHPs, NHP fusion proteins (e.g., a NHP or one or more
functional domains of a NHP fused to an unrelated protein or
peptide, such as albumin or an immunoglobulin constant region,
i.e., IgFc), nucleotide sequences encoding such products, and host
cell expression systems that can produce such NHP products.
[0025] The invention also encompasses antibodies and anti-idiotypic
antibodies, or fragments thereof (including Fab and F(ab').sub.2
fragments), antagonists and agonists of a NHP, as well as compounds
or nucleotide constructs that inhibit expression of a NHP gene
(transcription factor inhibitors, antisense and ribozyme molecules,
and/or coding sequence or regulatory sequence replacement
constructs), or promote expression or overexpression 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.).
[0026] The NHP proteins, polypeptides or peptides, NHP fusion
proteins, NHP nucleotide sequences, antibodies, antagonists and/or
agonists can be useful for the detection of mutant NHPs or
inappropriately expressed NHPs, which can be used, for example, to
diagnose NHP-mediated disorders. 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 also 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 a NHP in the
body. The use of engineered host cells and/or animals can offer an
advantage in that such systems allow not only for the
identification of compounds that bind to the endogenous
receptor/ligand of a NHP, but can also identify compounds that
trigger NHP-mediated activities or pathways.
[0027] Where, as in the present instance, some of the described NHP
peptides or polypeptides are thought to be cytoplasmic or nuclear
proteins, expression systems can be engineered that produce soluble
derivatives of a NHP (such as those corresponding to NHP
extracellular and/or intracellular domains, or truncated NHP
polypeptides lacking one or more hydrophobic domains) and/or NHP
fusion protein products (especially NHP-Ig fusion proteins, i.e.,
fusions of one or more NHP domain(s) to an IgFc). These expression
products, as well as NHP. antibodies, anti-idiotypic antibodies
(including Fab fragments), and NHP 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 a NHP could activate or
effectively antagonize the endogenous NHP or a protein interactive
therewith. 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 (and, consequently, modulating
white blood cell counts higher or lower). Thus, the invention also
encompasses pharmaceutical formulations and methods for treating
biological disorders.
[0028] Various aspects of the invention are described in greater
detail in the subsections below.
5.1 The NHP Nucleotide Sequences
[0029] The cDNA sequences (SEQ ID NOS:1, 3, 5, 7, and 9) and the
corresponding deduced amino acid sequences (SEQ ID NOS:2, 4, 6, and
8) of the described NHPs are presented in the Sequence Listing. The
NHP genes were obtained from a human testis cDNA library using
probes and/or primers generated from human gene trapped sequence
tags. Expression analysis has provided evidence that the described
NHPs can be expressed, for example, in human testis, prostate, and
gene trapped human cells. In addition to human calpain genes, the
described NHPS share significant similarity to a variety of
proteases from mice, pigs, chickens, and rats.
[0030] The described open reading frames can also contain several
polymorphisms, including: an A to G transition corresponding to,
for example, base 1474 of SEQ ID NOS:1 or 3, which can result in
either a K or an E being present at the corresponding amino acid
position of SEQ ID NOS:2 or 4; a C to T transition corresponding
to, for example, base 1669 of SEQ ID NOS:1 or 3, which can result
in a Q or a stop codon that truncates the ORF at the corresponding
amino acid position of SEQ ID NOS:2 or 4; and a T to A transversion
corresponding to, for example, base, 1673 of SEQ ID NOS:1 or 3,
which can result in a L or a H at the corresponding amino acid
position of SEQ ID NOS:2 or 4.
[0031] As discussed above, the present invention includes 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 et al., eds.,
1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing Associates, Inc., and John Wiley & Sons, Inc., N.Y.,
at p. 2.10.3) and encodes a functionally equivalent expression
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 encode a functionally equivalent NHP product.
Functional equivalents of a NHP include, but are not limited to,
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.
[0032] 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 to
corresponding regions of, for example, SEQ ID NO:1 (as measured by
BLAST sequence comparison analysis using, for example, the
University of Wisconsin GCG sequence analysis package (SEQUENCHER
3.0, Gene Codes Corp., Ann Arbor, Mich.) using default
parameters).
[0033] The invention also includes nucleic acid molecules,
preferably DNA molecules, that hybridize to, and are therefore the
complements of, the described NHP-encoding polynucleotides. Such
hybridization conditions can be highly stringent or less highly
stringent, as described herein. 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 bases long, 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.
[0034] 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 NHP oligonucleotide sequence(s) first
disclosed in SEQ ID NOS:1-9. 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, 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 relative to one
or more additional sequence(s), or one or more restriction sites,
present in the disclosed sequence.
[0035] These nucleic acid molecules may encode or act as NHP
antisense molecules, useful, for example, in NHP gene regulation
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 one or more of the biological
functions associated with a NHP, as described herein. Further, such
sequences can be used as part of ribozyme and/or triple helix
sequences that are also useful for NHP gene regulation.
[0036] Inhibitory antisense or double stranded oligonucleotides can
additionally comprise at least one modified base moiety that is
selected from the group including, but not limited to,
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 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.
[0037] The antisense oligonucleotides can also comprise at least
one modified sugar moiety selected from the group including, but
not limited to, arabinose, 2-fluoroarabinose, xylulose, and
hexose.
[0038] In yet another embodiment, the antisense oligonucleotides
will comprise at least one modified phosphate backbone selected
from the group including, but not limited to a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0039] In yet another embodiment, the antisense oligonucleotides
are .alpha.-anomeric oligonucleotides. 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 can also be 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 NHP.
[0040] Further, NHP homologs and orthologs can be isolated from
nucleic acids from additional mammalian species, for example, by
performing PCR using two degenerate or "wobble" oligonucleotide
primer pools designed on the basis of amino acid sequences within
the NHP sequences disclosed herein. The template for the reaction
may be genomic DNA, or total RNA, mRNA, and/or cDNA obtained by
reverse transcription of mRNA prepared from, for example, human or
non-human cell lines, cell types, or tissues known to express, or
suspected of expressing, an allele of a NHP gene.
[0041] The PCR product can be sequenced directly, or subcloned and
sequenced, to ensure that the amplified sequences represent NHP
coding sequences. 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.
[0042] 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 to express, or suspected of expressing, a NHP). 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,
Molecular Cloning, A Laboratory. Manual, Cold Spring Harbor Press,
N.Y. (and periodic updates thereof).
[0043] NHP sequences can also be used to isolate mutant alleles of
a NHP. Such mutant alleles can be isolated from individuals either
known to have, or suspected of having, a genotype that contributes
to increased white blood cell counts. Mutant alleles and/or
peptides, polypeptides or proteins may then be utilized in the
therapeutic and diagnostic programs described herein. Additionally,
such sequences of any of the genes corresponding to NHPs can be
used to detect gene regulatory (e.g., promoter or
promoter/enhancer) defects that can affect, for example, white
blood cell counts.
[0044] A cDNA encoding a mutant NHP gene or sequence 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 to express or
suspected of expressing a mutant NHP gene 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 NHP 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.
[0045] Alternatively, a genomic library can be constructed using
DNA obtained from an individual suspected of carrying, or known to
carry, a mutant NHP allele (e.g., a person manifesting a
NHP-associated phenotype such as, for example, increased white
blood cell counts), or a cDNA library can be constructed using RNA
from a tissue known to express, or suspected of expressing, 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.
[0046] Additionally, an expression library can be constructed
utilizing cDNA synthesized from, for example, RNA isolated from a
tissue known to express, or suspected of expressing, a mutant NHP
allele in an individual suspected of carrying, or known to carry,
such a mutant allele. In this manner, gene products made by the
putatively mutant tissue may 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 and Lane, eds.,
1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press,
Cold Spring Harbor, N.Y., incorporated herein by reference in its
entirety).
[0047] 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.
[0048] The invention also encompasses nucleotide sequences that
encode mutant isoforms of any of the NHP amino acid sequences,
peptide fragments thereof, truncated versions thereof, and/or
fusion proteins, including any of the above fused to another
unrelated polypeptide. Examples of such polypeptides can include,
but are not limited to, an epitope tag that aids in purification or
detection of the resulting fusion protein, or an enzyme,
fluorescent protein, or luminescent protein that can be used as a
marker.
[0049] The present invention additionally encompasses: (a) RNA or
DNA vectors that contain any portion of a NHP and/or its
complement, as well as any of the peptides or proteins encoded
thereby; (b) DNA vectors that contain a cDNA that substantially
spans the entire open reading frame corresponding to any of the NHP
sequences and/or their complements; (c) DNA expression vectors that
contain any of the foregoing sequences, or a portion thereof,
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 contain a cDNA that spans
the entire open reading frame, or any portion thereof,
corresponding to any of the NHP sequences, operatively associated
with a regulatory element, which may be exogenously controlled
(such as in gene activation), either in vivo and/or in vitro, which
directs the expression of NHP coding sequences in the host
cell.
[0050] 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 baculovirus polyhedron
promoter, the cytomegalovirus (hCMV) immediate early gene promoter,
regulatable, viral elements, (particularly retroviral LTR
promoters), the early or late promoters of SV40 and 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.
[0051] The present invention also encompasses nucleotide constructs
encoding NHP products that can be used to genetically engineer host
cells to express such NHP products in vivo. These genetically
engineered cells function as "bioreactors" in the body, delivering
a continuous supply of a NHP, NHP peptides or polypeptides, soluble
NHPs, or NHP fusion proteins. Nucleotide constructs encoding
functional versions of a NHP, mutant versions of a NHP, as well as
antisense and ribozyme molecules, can be used in "gene therapy"
approaches for the modulation of NHP expression and/or activity in
the treatment of NHP-mediated disorders. Thus, the invention also
encompasses pharmaceutical formulations and methods for treating
NHP-mediated disorders such as reduced white blood cell count.
[0052] An additional application of the described novel human
polynucleotide sequences is their use in the molecular
mutagenesis/evolution of proteins that are at least partially
encoded by the described novel sequences using, for example,
polynucleotide shuffling or related methodologies. Such approaches
are described in U.S. Pat. Nos. 5,830,721 and 5,837,458, which are
herein incorporated by reference in their entirety.
[0053] 5.1.1 Cells that Contain NHP Disrupted Alleles
[0054] Another aspect of the current invention includes cells that
contain a disrupted NHP gene. There are a variety of techniques
that can be used to disrupt genes in cells, and especially ES
cells. Examples of such methods are described in co-pending U.S.
patent application Ser. No. 08/728,963, and U.S. Pat. Nos.
5,789,215, 5,487,992, 5,627,059, 5,631,153, 6,087,555, 6,136,566,
6,139,833, and 6,207,371, all of which are herein incorporated by
reference in their entirety.
[0055] 5.1.2 Identification of Cells that Express a NHP
[0056] Host cells that contain NHP coding sequence and/or express a
biologically active NHP gene product, or fragment thereof, can be
identified by at least four general approaches: (a) DNA-DNA or
DNA-RNA hybridization; (b) the presence or absence of "marker" gene
functions; (c) assessing the level of NHP transcription as measured
by the expression of NHP mRNA transcripts in the host cell; and (d)
detection of NHP gene product as measured by immunoassay, enzymatic
assay, chemical assay, or one or more of the biological activities
of NHPs. These identification methods are described in greater
detail below. Prior to screening for gene expression, the host
cells can first be treated in an effort to increase the level of
expression of sequences encoding NHP polynucleotides, especially in
cell lines that produce low amounts of NHP mRNAs and/or NHP
peptides and proteins.
[0057] In approach (a) above, the presence of a NHP coding sequence
can be detected by DNA-DNA or DNA-RNA hybridization using probes
comprising nucleotide sequences that are homologous or
complementary to the NHP coding sequences, as described herein, or
portions or derivatives thereof.
[0058] In approach (b), the recombinant expression vector/host
system can be identified and selected based upon the presence or
absence of certain "marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics, resistance to methotrexate,
transformation phenotype, occlusion body formation in baculovirus,
etc.). For example, if a NHP polynucleotide sequence that encodes a
NHP peptide or protein is inserted within a marker gene sequence of
a vector, recombinants containing a NHP coding sequence can be
identified by the absence of marker gene function. Alternatively, a
marker gene can be placed in tandem with a NHP sequence, under the
control of the same or a different promoter used to control the
expression of the NHP coding sequence. Expression of the marker
gene product in response to induction or selection indicates the
presence of the NHP coding sequence.
[0059] In approach (c), transcriptional activity of a coding region
of a NHP can be assessed by hybridization assays. For example, RNA
can be isolated and analyzed by Northern blot using a probe derived
from a NHP, or any portion thereof. Alternatively, total nucleic
acids of the host cell may be extracted and assayed for
hybridization to such probes. Additionally, RT-PCR (using NHP
specific oligos) may be used to detect low levels of gene
expression in a sample, or in RNA isolated from a spectrum of
different tissues, or in cDNA libraries derived from different
tissues, to determine which tissues express a given NHP.
[0060] In approach (d), the expression of the peptides and proteins
of the current invention can be assessed immunologically, for
example by Western blots, immunoassays such as
radioimmuno-precipitation, radioimmunoassays, enzyme-linked
immunosorbent assays, and the like. This can be achieved by using
an antibody, or a binding partner, specific to a NHP peptide or
protein. Additionally, expression can be assessed by monitoring one
or more of the biological activities of a NHP. The NHPs have, among
others activities, activity as a protease, and is therefore
involved in protein degradation. Thus assays described herein, as
well as those commonly known to those of skill in the art to
examine proteases, can be used to access NHP biological
activity.
[0061] 5.1.3. The Use NHP Polynucleotide Sequences to Diagnose
NHP-mediated Disorders
[0062] The NHP polynucleotide sequences, as described herein, can
be used in hybridization based assays to identify and diagnose
NHP-mediated disorders that result from mutant NHP sequences, or to
quantify levels of NHP expression, thus identifying individuals
that are at risk for developing NHP-mediated disorders. These
assays could be in the form of fluorescence or enzyme based in situ
hybridization, PCR, or in a preferred embodiment, hybridization
probes used to assess gene expression patterns using a microarray
or high-throughput "chip" format.
[0063] The present invention includes assays that utilize, among
others, NHP sequences (and vectors comprising the same), a open
reading frame (ORF) encoding a naturally occurring protein having
NHP activity and that hybridizes to a complement of a NHP DNA
sequence under highly stringent conditions, as described herein,
and encodes a functionally equivalent gene product, as described
herein. The present assays also contemplate the use of any
nucleotide sequences that hybridize to the complement of a
nucleotide sequence that encodes a NHP under moderately stringent
conditions, as described herein, yet still encodes a functionally
equivalent NHP product, as described herein.
[0064] The invention also includes the use of 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 herein. In instances where the
nucleic acid molecules are "DNA oligos", such molecules are
generally about 16 to about 100 bases long, or about 20 to about 80
bases long, or about 34 to about 45 bases long, or any variation or
combination of sizes represented therein that incorporate a
contiguous region of NHP sequence. Such oligonucleotides can be
used, for example, in conjunction with the polymerase chain
reaction (PCR) to screen libraries, isolate clones, and prepare
cloning and sequencing templates, etc.
[0065] For oligonucleotide probes, highly stringent conditions can
typically 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). 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
(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. USA 85:7448-7451), etc.
[0066] 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, supra, and Ausubel, et al., 1989, supra
(and periodic updates of both).
[0067] Alternatively, NHP oligonucleotides and/or amino acids can
be used as hybridization probes for screening libraries, or
assessing gene expression patterns (particularly using a microarray
or high-throughput "chip" format). Such assays would be applicable
to the screening of large databases containing, for example,
sequences obtained from patients suspected of having a NHP defect.
This methodology would therefore link functional information with
large amounts of genetic information.
[0068] Additionally, a series of 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:1-9 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:1-9, 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.
[0069] Addressable arrays comprising sequences first disclosed in
SEQ ID NOS:1-9 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 usually 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:1-9.
[0070] For example, a series of NHP oligonucleotide sequences, or
the complements thereof, can be used in chip format to represent
all or a portion of the described NHP 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 (3'-to-5') orientation.
[0071] Microarray-based analysis allows the discovery of broad
patterns of genetic activity, providing new understanding of gene
functions, and generating novel and unexepected insight into
transcriptional processes and biological mechanisms. The use of
addressable arrays comprising sequences first disclosed in SEQ ID
NOS:1-9 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.
[0072] Probes consisting of sequences first disclosed in SEQ ID
NOS:1-9 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
intended target of the drug. These unique sequences therefore also
have utility in defining and monitoring both drug action and
toxicity.
[0073] As an example of utility, the sequences first disclosed in
SEQ ID NOS:1-9 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:1-9 in silico, and by comparing previously collected genetic
databases and the disclosed sequences using computer software known
to those in the art. Thus the sequences first disclosed in SEQ ID
NOS:1-9 can be used to identify mutations associated with a
particular disease, and also in diagnostic or prognostic
assays.
[0074] In addition to the NHP nucleotide sequences described
herein, additional full length NHP cDNA or gene sequences present
in the same or similar species (such as, for example, additional
splice variants, polymorphisms, pseudogenes, etc.), and/or homologs
or orthologs of the NHP gene present in other species, can be
identified and readily isolated by standard molecular biological
techniques using the NHP sequences presented herein. The
identification of homologs of a NHP in related species can be
useful, for example, in developing alternative animal model systems
for the purpose of drug discovery.
[0075] Labeled NHP nucleotide probes can also be used to screen a
genomic library derived from an organism of interest, again, using
appropriately stringent conditions. In particular, 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 and clinical protocols for treating
NHP-related disorders in human patients. 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 prognostics and/or diagnostics.
[0076] For example, the present sequences can be used in
restriction fragment length polymorphism (RFLP) analysis to
identify specific individuals. In this technique, an individuals
genomic DNA is digested with one or more restriction enzymes, and
probed on a Southern blot to yield unique bands for identification
(as generally described in U.S. Pat. No. 5,272,057, incorporated
herein by reference). In addition, the sequences of the present
invention can be used to provide polynucleotide reagents, e.g., PCR
primers, targeted to specific loci in the human genome, which can
enhance the reliability of DNA-based forensic identifications by,
for example, providing another "identification marker" (i.e.,
another DNA sequence that is unique to a particular individual).
Actual base sequence information can be used for identification as
an accurate alternative to patterns formed by restriction enzyme
generated fragments.
5.2 NHP Polypeptides
[0077] NHPs, NHP polypeptides, NHP 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, and as reagents in assays for screening
for compounds that can be used 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 augment
the efficacy of therapeutic agents.
[0078] The Sequence Listing discloses the amino acid sequences
encoded by the described NHP polynucleotide sequences. The NHPs
have initiator methionines in DNA sequence contexts consistent with
a translation initiation site. The sequence data presented herein
indicate that alternatively spliced forms of the NHPs exist (which
may or may not be tissue specific).
[0079] The NHP amino acid sequences of the invention include the
nucleotide and 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 herein 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.
[0080] 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 signal transduction pathway, or a
change in cellular metabolism (e.g., proteolytic activity, ion
flux, tyrosine phosphorylation, 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 herein, but that
result in a silent change, thus producing a functionally equivalent
expression 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.
[0081] While random mutations can be made to NHP DNA (using random
mutagenesis techniques well-known in the art), and the resulting
NHP mutants tested for activity, site-directed mutations of a NHP
coding sequence can be engineered (using site-directed mutagenesis
techniques well-known to those skilled in the art) to generate NHP
mutants with increased or decreased function.
[0082] For example, the novel amino acid sequence of peptides,
polypeptides and proteins encoded by a NHP can be aligned with
homologs from different species. Mutant peptides, polypeptides and
proteins can be engineered so that regions of interspecies identity
are maintained, whereas the variable residues are altered, e.g., by
deletion or insertion of an amino acid residue(s) or by
substitution of one or more different amino acid residues. For
example, alterations in variable residues may be designed to
produce a mutant form of a NHP peptide, polypeptide or protein that
is more stable but retains function. Other alterations may be
designed to alter function, such as those designed to enhance
binding or enzymatic activity of a NHP product. One of skill in the
art could easily test such mutant or deleted forms of a NHP
peptide, polypeptide or protein for the effect of such alterations
on function using the teachings presented herein.
[0083] Other mutations to the coding sequences described herein can
be made to generate peptides, polypeptides and proteins that are
better suited for expression, scale up, etc., in the host cells
chosen. For example, the triplet code for each amino acid can be
modified to conform more closely to the preferential codon usage of
the translational machinery of the particular host cell, or, for
example, to yield a messenger RNA molecule with a longer half-life.
Those skilled in the art would readily know what modifications of
the nucleotide sequence would be desirable to conform the
nucleotide sequence to preferential codon usage or to make the
messenger RNA more stable. Such information would be obtainable,
for example, through use of computer programs, through review of
available research data on codon usage and messenger RNA stability,
and through other means known to those of skill in the art.
[0084] Additionally contemplated are oligopeptides that are modeled
on an amino acid sequence first described in the Sequence Listing.
Such NHP oligopeptides are generally between about 10 to about 100
amino acids long, or between about 16 to about 80 amino acids long,
or between about 20 to about 35 amino acids long, or any variation
or combination of sizes represented therein that incorporate a
contiguous region of sequence first disclosed in the Sequence
Listing. Such NHP oligopeptides can be of any length disclosed
within the above ranges and can initiate at any amino acid position
represented in the Sequence Listing.
[0085] The invention also contemplates "substantially isolated" or
"substantially pure" proteins or polypeptides. By a "substantially
isolated" or "substantially pure" protein or polypeptide is meant a
protein or polypeptide that has been separated from at least some
of those components that naturally accompany it. Typically, the
protein or polypeptide is substantially isolated or pure when it is
at least 60%, by weight, free from the proteins and other
naturally-occurring organic molecules with which it is naturally
associated in vivo. Preferably, the purity of the preparation is at
least 75%, more preferably at least 90%, and most preferably at
least 99%, by weight. A substantially isolated or pure protein or
polypeptide may be obtained, for example, by extraction from a
natural source, by expression of a recombinant nucleic acid
encoding the protein or polypeptide, or by chemically synthesizing
the protein or polypeptide.
[0086] Purity can be measured by any appropriate method, e.g.,
column chromatography such as immunoaffinity chromatography using
an antibody specific for the protein or polypeptide, polyacrylamide
gel electrophoresis, or HPLC analysis. A protein or polypeptide is
substantially free of naturally associated components when it is
separated from at least some of those contaminants that accompany
it in its natural state. Thus, a polypeptide that is chemically
synthesized or produced in a cellular system-different from the
cell from which it naturally originates will be, by definition,
substantially free from its naturally associated components.
Accordingly, substantially isolated or pure proteins or
polypeptides include eukaryotic proteins synthesized in E. coli,
other prokaryotes, or any other organism in which they do not
naturally occur.
[0087] 5.2.1 NHP Fusion Proteins
[0088] Peptides corresponding to one or more portions of a NHP,
truncated or deleted NHPs, as well as fusion proteins in which a
full length NHP, a NHP Peptide or truncated NHP is fused to an
unrelated protein are also within the scope of the invention, and
can be designed on the basis of NHP nucleotide and/or amino acid
sequences disclosed herein. Such fusion proteins include, but are
not limited to: IgFc fusions, which stabilize NHP proteins or
peptides and prolong half-life in vivo; fusions to any amino acid
sequence that allows the fusion protein to be anchored to the cell
membrane; or fusions to an enzyme, fluorescent protein, or
luminescent protein that provides a marker function.
[0089] Also encompassed by the present invention are fusion
proteins that direct a NHP to a target organ and/or facilitate
transport across the membrane into the cytosol. Conjugation of NHPs
to antibody molecules, or their Fab or F(ab').sub.2 fragments,
could be used to target cells bearing a particular epitope.
Attaching an appropriate signal sequence to a NHP would also
transport a NHP to a desired location within the cell.
Alternatively targeting of a NHP or its nucleic acid sequence might
be achieved using liposome or lipid complex based delivery systems.
Such technologies are described in "Liposomes: A Practical
Approach", New, R.R.C., ed., Oxford University Press, N.Y., and in
U.S. Pat. Nos. 4,594,595, 5,459,127, 5,948,767 and 6,110,490 and
their respective disclosures, which are herein incorporated by
reference in their entirety. Additionally embodied are novel
protein constructs engineered in such a way that they facilitate
transport of NHPs to a target site or desired organ, where they
cross the cell membrane and/or the nucleus where the NHPs can exert
their functional activity. This goal may be achieved by coupling of
a NHP to a cytokine or other ligand that provides targeting
specificity, and/or to a protein transducing domain (see generally
U.S. Provisional Patent Application Ser. Nos. 60/111,701 and
60/056,713, both of which are herein incorporated by reference, for
examples of such transducing sequences), to facilitate passage
across cellular membranes, and can optionally be engineered to
include nuclear localization signals.
[0090] Alternatively, any fusion protein can be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. Another exemplary system 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 sequence of interest is
subcloned into a vaccinia recombination plasmid such that the
sequence'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.
[0091] The novel gene products/peptide sequences encoded by NHPs
are also useful as epitope tags for antigenic or other tagging of
proteins and polypeptides that have been engineered to incorporate
or comprise at least a portion of a NHP peptide sequence.
[0092] 5.2.2 NHP Expression Systems
[0093] While NHP polypeptides and peptides can be chemically
synthesized (e.g., see Creighton, 1983, Proteins: Structures and
Molecular Principles, W. H. Freeman & Co., N.Y.), large
polypeptides derived from NHPs, and full length NHPs themselves,
may advantageously be produced by recombinant DNA technology using
techniques well-known in the art for expressing nucleic acids
containing NHP gene sequences and/or coding sequences. Such methods
can be used to construct expression vectors containing NHP
nucleotide sequences and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination (see, for example, the techniques
described in Sambrook et al., 1989, supra, and Ausubel et al.,
1989, supra). Alternatively, RNA and/or DNA encoding NHP nucleotide
sequences may be chemically synthesized using, for example,
synthesizers (see, for example, the techniques described in
"Oligonucleotide Synthesisi", 1984, Gait, ed., IRL Press, Oxford,
which is incorporated by reference herein in its entirety).
[0094] A variety of host-expression vector systems can be used to
express the NHP nucleotide sequences of the invention. Where the
NHP peptide or polypeptide is a soluble derivative of, for example,
a membrane protein (e.g., NHP peptides derived from an
extracellular domain (ECD) of a NHP, or truncated or deleted NHPs
in which a transmembrane (TM) and/or cytoplasmic domain (CD) have
been deleted, etc.) the peptide or polypeptide can be recovered
from the culture, i.e., from the host cell in cases where the NHP
peptide or polypeptide is not secreted, or from the culture media
in cases where the NHP peptide or polypeptide is secreted by the
cells. However, such expression systems also encompass engineered
host cells that express a NHP, or functional equivalent, in situ,
i.e., anchored in the cell membrane. 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 a NHP,
but to assess biological activity, e.g., in certain drug screening
assays.
[0095] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express the NHP sequences described herein may be
engineered. Rather than using expression vectors that 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
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 that 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.
[0096] 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 and Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980,Cell 22:817) genes,
which 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, Proc. 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 and 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).
[0097] The expression systems that may 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., Saccharomyces,
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 nucleotide 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 NHP nucleotide sequences and 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).
[0098] 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 a 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 and Inouye, 1985, Nucleic Acids Res.
13:3101-3109; Van Heeke and Schuster, 1989, J. Biol. Chem.
264:5503-5509); and the like. pGEX vectors may 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
expression product can be released from the GST moiety.
[0099] In an exemplary insect system, Autographa californica
nuclear polyhedrosis virus (AcNPV) is used as a vector to
phosphoribosyltransfera- se (Szybalska and Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes,
which 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, Proc. 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 and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo,
which confers resistance to the aminoglycoside G-418
(Colbere-Garapin et al., 1981, J. Mol. Biol. 150:1); and hygro,
which confers resistance to hygromycin (Santerre et al., 1984, Gene
30:147).
[0100] The expression systems that may 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., Saccharomyces,
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 nucleotide 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 NHP nucleotide sequences and 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). 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 Bitter et al., 1987, Methods
in Enzymol. 153:516-544).
[0101] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review, see Ausubel et al.,
1989, supra, Ch. 13; Grant et al., 1987, Methods in Enzymol.
153:516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash.,
D.C., Ch. 3; Bitter, 1987, Methods in Enzymol. 152:673-684; and
Strathern et al., eds., "The Molecular Biology of the Yeast
Saccharomyces", 1982, Cold Spring Harbor Press, Vols. I and II.
[0102] In cases where plant expression vectors are used, expression
of a NHP coding sequence may be driven by any of a number of
promoters. For example, viral promoters such as the 35S RNA-and 19S
RNA promoters of CaMV (Brisson et al., 1984, Nature 310:511-514),
or the coat protein promoter of TMV. (Takamatsu et al., 1987, EMBO
J. 6:307-311) may be used; alternatively, plant promoters such as
the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.
3:1671-1680; Broglie et al., 1984, Science 224:838-843); or heat
shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et
al., 1986, Mol. Cell. Biol. 6:559-565) may be used. These
constructs can be introduced into plant cells using Ti plasmids, Ri
plasmids, plant virus vectors, direct DNA transformation,
microinjection, electroporation, etc. For reviews of such
techniques, see, for example, Weissbach and Weissbach, 1988,
Methods for Plant Molecular Biology, Academic Press, NY, Section
VIII, pp. 421-463; and Grierson and Corey, 1988, Plant Molecular
Biology, 2d Ed., Blackie, London, Ch. 7-9.
[0103] 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 or desired modification
and processing of the NHP-protein, polypeptide or peptide
expressed. To this end, eukaryotic host cells that possess the
cellular machinery for proper processing of the primary transcript,
glycosylation, and phosphorylation of the NHP 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 U937 cells, and
in particular human cell lines.
[0104] 5.2.3 NHP Transgenic Animals
[0105] The present invention provides for transgenic animals that
carry a NHP transgene in all their cells, as well as animals that
carry a NHP transgene in some, but not all their cells, i.e.,
mosaic animals or somatic cell transgenic animals. Animals of any
species, including, but not limited to, mice, rats, rabbits, guinea
pigs, pigs, micro-pigs, goats, and non-human primates, e.g.,
baboons, monkeys, and chimpanzees, can be used to generate
transgenic animals carrying NHP polynucleotides. NHP transgenes may
be integrated as a single transgene or in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene may
also be selectively introduced into and activated in a particular
cell type by following, for example, the teaching of Lasko et al.,
1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. The regulatory
sequences required for such a cell-type specific activation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art.
[0106] When it is desired that a NHP transgene be integrated into
the chromosomal site of the endogenous copy of the NHP gene, gene
targeting is preferred. Briefly, when such a technique is to be
utilized, vectors containing some nucleotide sequences homologous
to the endogenous NHP gene are designed for the purpose of
integrating, via homologous recombination with chromosomal
sequences, into and disrupting the function of the nucleotide
sequence of the endogenous NHP gene (i.e., "knockout" animals). In
this way, the expression of the endogenous NHP gene may also be
eliminated by inserting nonfunctional sequences into the endogenous
NHP gene. The transgene may also be selectively introduced into a
particular cell type, thus inactivating the endogenous NHP gene in
only that cell type, by following, for example, the teaching of Gu
et al., 1994, Science 265:103-106. The regulatory sequences
required for such a cell-type specific inactivation will depend
upon the particular cell type of interest.
[0107] Any technique known in the art may be used to introduce a
NHP transgene into animals to produce the founder lines of
transgenic animals. Such techniques include, but are not limited
to, pronuclear microinjection (U.S. Pat. No. 4,873,191,
incorporated herein by reference); retrovirus-mediated gene
transfer into germ lines (Van der Putten et al., 1985, Proc. Natl.
Acad. Sci. USA 82:6148-6152); gene targeting in embryonic stem
cells.(Thompson et al., 1989, Cell 56:313-321); electroporation of
embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); sperm-mediated
gene transfer (Lavitrano et al., 1989, Cell 57:717-723); and
positive-negative selection as described in U.S. Pat. No.
5,464,764, herein incorporated by reference. For a review of such
techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol.
115:171-229, which is incorporated by reference herein in its
entirety.
[0108] Once transgenic animals have been generated, the expression
of the recombinant NHP gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to assay
whether integration of the NHP transgene has taken place. The level
of mRNA expression of the NHP transgene in the tissues of the
transgenic animals may also be assessed using techniques that
include, but are not limited to, Northern blot analysis of cell
type samples lines or host systems can be chosen to ensure the
correct or desired modification and processing of the NHP protein,
polypeptide or peptide expressed. To this end, eukaryotic host
cells that possess the cellular machinery for proper processing of
the primary transcript, glycosylation, and phosphorylation of the
NHP 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 U937 cells, and in particular human cell lines.
[0109] 5.2.3 NHP Transgenic Animals
[0110] The present invention provides for transgenic animals that
carry a NHP transgene in all their cells, as well as animals that
carry a NHP transgene in some, but not all their cells, i.e.,
mosaic animals or somatic cell transgenic animals. Animals of any
species, including, but not limited to, mice, rats, rabbits, guinea
pigs, pigs, micro-pigs, goats, and non-human primates, e.g.,
baboons, monkeys, and chimpanzees, can be used to generate
transgenic animals carrying NHP polynucleotides. NHP transgenes may
be integrated as a single transgene or in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene may
also be selectively introduced into and activated in a particular
cell type by following, for example, the teaching of Lakso et al.,
1992, Proc. Natl. Acad. Sci. USA 89:6232-6236. The regulatory
sequences required for such a cell-type specific activation will
depend upon the particular cell type of interest, and will be
apparent to those of skill in the art.
[0111] When it is desired that a NHP transgene be integrated into
the chromosomal site of the endogenous copy of the NHP gene, gene
targeting is preferred. Briefly, when such a technique is to be
utilized, vectors containing some nucleotide sequences homologous
to the endogenous NHP gene are designed for the purpose of
integrating, via homologous recombination with chromosomal
sequences, into and disrupting the function of the nucleotide
and/or activity of a NHP expression product. Additionally, such
antibodies can be used in conjunction with gene therapy to, for
example, evaluate normal and/or engineered NHP-expressing cells
prior to their introduction into a patient. Such antibodies may
additionally be used in methods for the inhibition of abnormal NHP
activity. Thus, such antibodies may be utilized as a part of
treatment methods.
[0112] For the production of antibodies, various host animals may
be immunized by injection with a NHP, a NHP peptide (e.g., one
corresponding to a functional domain of a NHP), a truncated NHP
polypeptide (a NHP in which one or more domains have been deleted),
functional equivalents of a NHP, or mutated variants of a 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, chitosan, 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, diphtheria toxoid, ovalbumin, cholera toxin, or
fragments thereof. Polyclonal antibodies are heterogeneous
populations of antibody molecules derived from the sera of the
immunized animals.
[0113] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, may be obtained by any
technique that 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, and IgD, and any subclass thereof. The hybridomas
producing the mAbs 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.
[0114] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. USA 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. 6,114,598, 6,075,181
and 5,877,397 and their respective disclosures, which are herein
incorporated by reference in their entirety. Also encompassed by
the present invention is the use 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.
[0115] 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 341:544-546) can be
adapted to produce single chain antibodies against NHP expression
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.
[0116] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, such fragments include,
but are not limited to: F(ab').sub.2 fragments, which can be
produced by pepsin digestion of an antibody molecule; and Fab
fragments, which can be generated by reducing the disulfide bridges
of 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.
[0117] 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 and
Bona, 1993, FASEB J. 7:437-444; and Nissinoff, 1991, J. Immunol.
147:2429-2438). For example, antibodies that 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 fragments of such anti-idiotypes
can be used in therapeutic regimens involving a NHP signaling
pathway.
[0118] Additionally given the high degree of relatedness of
mammalian NHPs, the presently described knock-out mice (having
never seen a NHP, and thus never been tolerized to a NHP) have an
unique utility, as they can be advantageously applied to the
generation of antibodies against the disclosed mammalian NHPs
(i.e., a NHP will be immunogenic in NHP knock-out animals).
5.4 Diagnosis of NHP-mediated Disorders
[0119] A variety of methods can be employed for the diagnostic and
prognostic evaluation of NHP-mediated disorders. These methods can
also be used to identify subjects having a predisposition to such
disorders. Such methods may, for example, utilize reagents such as
NHP nucleotide sequences, NHP proteins or peptides, and/or anti-NHP
antibodies. Specifically, such reagents may be used, for example,
for: (1) the detection of the presence of NHP gene mutations, or
the detection of either over- or under-expression of a NHP mRNA
relative to the non-NHP disorder state; (2) the detection of either
an over- or an under-abundance of a NHP gene product 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;
Cote 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, incuding IgG, IgM,
IgE, IgA, and IgD, and any subclass thereof. The hybridomas
producing the mAbs 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.
[0120] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. USA 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. 6,114,598, 6,075,181
and 5,877,397 and their respective disclosures, which are herein
incorporated by reference in their entirety. Also encompassed by
the present invention is the use 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.
[0121] 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 341:544-546) can be
adapted to produce single chain antibodies against NHP expression
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.
[0122] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, such fragments include,
but are not limited to: F(ab').sub.2 fragments, which can be
produced by pepsin digestion of an antibody molecule; and Fab
fragments, which can be generated by reducing the disulfide bridges
of 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.
[0123] 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 and
Bona, 1993, FASEB J. 7:437-444; and Nisonoff, 1991, J. Immunol.
147:2429-2438). For example, antibodies that 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 fragments of such anti-idiotypes
can be used in therapeutic regimens involving a NHP signaling
pathway.
[0124] Additionally given the high degree of relatedness of
mammalian NHPs, the presently described knock-out mice (having
never seen a NHP, and thus never been tolerized to a NHP) have an
unique utility, as they can be advantageously applied to the
generation of antibodies against the disclosed mammalian NHPs
(i.e., a NHP will be immunogenic in NHP knock-out animals).
5.4 Diagosis of NHP-Mediated Disorders
[0125] A variety of methods can be employed for the diagnostic and
prognostic evaluation of NHP-mediated disorders. These methods Such
techniques include, for example, the use of restriction fragment
length polymorphisms (RFLPs), which involve sequence variations in
one or more of the recognition sites for any particular restriction
enzyme used.
[0126] Additionally, improved methods for analyzing DNA
polymorphisms that can be utilized for the identification of NHP
gene mutations have been described that capitalize on the presence
of variable numbers of short, tandemly repeated DNA sequences
between certain restriction enzyme sites. For example, Weber (U.S.
Pat. No. 5,075,217, which is incorporated herein by reference in
its entirety) describes a DNA marker based on length polymorphisms
in blocks of (dC-dA).sub.n-(dG-dT).sub.n short tandem repeats. The
average separation of (dC-dA).sub.n-(dG-dT).sub.n blocks is
estimated to be 30,000-60,000 bp. Markers that are so closely
spaced exhibit a high frequency of co-inheritance, and are
extremely useful in the identification of genetic mutations, such
as, for example, mutations within a NHP gene, and the diagnosis of
diseases and disorders related to NHP mutations.
[0127] Also, Caskey et al. (U.S. Pat. No. 5,364,759, which is
incorporated herein by reference in its entirety) describe a DNA
profiling assay for detecting short tri- and tetra-nucleotide
repeat sequences. The process includes extracting the DNA of
interest, amplifying the extracted DNA, and labeling the repeat
sequences to form a genotypic map of the individual's DNA.
[0128] The level of NHP gene expression can also be assayed by
detecting and measuring NHP transcription. For example, RNA from a
cell type or tissue known to express, or suspected of expressing, a
NHP-gene may be isolated and tested utiizing hybridization or PCR
techniques such as those described herein. The isolated cells can
be derived from cell culture or from a patient sample. The analysis
of cells taken from culture may be a necessary step in the
assessment of cells to be used as part of a cell-based gene therapy
technique or, alternatively, to test the effect of compounds on the
expression of a NHP gene. Such analyses may reveal both
quantitative and qualitative aspects of the expression pattern of a
NHP gene, including activation or inactivation of NHP gene
expression.
[0129] In one embodiment of such a detection scheme, cDNAs are
synthesized from the RNAs of interest (e.g., by reverse
transcription of the RNA molecule into cDNA). A sequence within the
cDNA is then used as the template for a nucleic acid amplification
reaction, such as a PCR amplification reaction, or the like. For
detection of the amplified product, the nucleic acid amplification
may be performed using radioactively or non-radioactively labeled
nucleotides. Alternatively, enough amplified product may be made
such that the product may be visualized by utilizing standard
ethidium bromide staining or any other suitable nucleic acid
staining method.
[0130] Additionally, it is possible to perform such NHP gene
expression assays in situ, i.e., directly upon tissue sections
(fixed and/or frozen) of patient tissue obtained from biopsies or
resections, such that no nucleic acid purification is necessary.
Nucleic acid reagents may be used as probes and/or primers for such
in situ procedures (see, for example, Nuovo, 1992, "PCR In Situ
Hybridization: Protocols And Applications", Raven Press, N.Y.).
Alternatively, if a sufficient quantity of the appropriate cells
can be obtained, standard Northern analysis can be performed to
determine the level of mRNA expression of a NHP gene.
[0131] Additionally, NHP oligonucleotide or polynucleotide
sequences can be used as hybridization probes in conjunction with a
solid support matrix/substrate (e.g., resins, beads, membranes,
plastics, polymers, metal or metallized substrates, gene chips, and
crystalline or polycrystalline substrates, etc.).
[0132] 5.4.2 Detection of NHP Gene Products
[0133] Antibodies directed against wild-type or mutant NHP gene
products, or conserved variants or peptide fragments thereof, which
are discussed above, may also be used in diagnostic and prognostic
assays, as described herein. Such diagnostic methods may be used to
detect abnormalities in the level of NHP gene expression, or
abnormalities in the structure and/or temporal, tissue, cellular,
or subcellular location of a NHP, and may be performed in vivo or
in vitro, such as, for example, on biopsy tissue.
[0134] For example, antibodies directed to epitopes of a NHP can be
used in vivo to detect the pattern and level of expression of a NHP
in the body. Such antibodies can be labeled, e.g., with a
radio-opaque or other appropriate compound, and injected into a
subject, in order to visualize binding to a NHP expressed in the
body, using methods such as X-rays, CAT-scans, or MRI. Labeled
antibody fragments, e.g., a Fab or single chain antibody comprising
the smallest portion of the antigen binding region, may be
preferred for this purpose, to promote crossing the blood-brain
barrier and permit labeling of a NHP expressed in the brain.
Additionally, any NHP fusion protein or NHP conjugated protein
whose presence can be detected can be administered. For example NHP
fusion or conjugated proteins labeled with a radio-opaque or other
appropriate compound can be administered and visualized in vivo, as
discussed above for labeled antibodies. Further, NHP fusion
proteins, such as alkaline phosphatase-NHP or NHP-alkaline
phosphatase fusion proteins, can be utilized for in vitro
diagnostic procedures.
[0135] Alternatively, immunoassays or fusion protein detection
assays can be utilized on biopsy and autopsy samples in vitro to
permit assessment of the expression pattern of a NHP. Such assays
can include the use of antibodies directed to epitopes of any of
the domains of a NHP. The use of each or all of these labeled
antibodies will yield useful information regarding translation and
intracellular transport of a NHP, and can identify alterations in
processing.
[0136] The tissue or cell type to be analyzed will generally
include those that are known to express, or suspected of
expressing, a NHP gene. The protein isolation methods employed
herein may, for example, be such as those previously described
(Harlow and Lane, 1988, supra). The isolated cells can be derived
from cell culture or from a patient. The analysis of cells taken
from culture may be a necessary step in the assessment of cells
that could be used as part of a cell-based gene therapy technique
or, alternatively, to test the effect of compounds on the
expression of a NHP gene.
[0137] For example, antibodies, or fragments of antibodies, useful
in the present invention may be used to quantitatively or
qualitatively detect the presence of NHP gene products, or
conserved variants or peptide fragments thereof. This can be
accomplished, for example, by immunofluorescence techniques
employing a fluorescently labeled antibody coupled with light
microscopic, flow cytometric, or fluorimetric detection.
[0138] The antibodies (or fragments thereof) or NHP fusion or
conjugated proteins useful in the present invention may,
additionally, be employed histologically, as in immunofluorescence,
immunoelectron microscopy or non-immuno assays, for in situ
detection of NHP gene products or conserved variants or peptide
fragments thereof. In situ detection may be accomplished by
removing a histological specimen from a patient, and applying
thereto a labeled antibody or fusion protein of the present
invention. The antibody (or fragment) or fusion protein is
preferably applied by overlaying the labeled antibody (or fragment)
onto a biological sample. Through the use of such a procedure, it
is possible to determine not only the presence of a NHP gene
product, or conserved variants or peptide fragments, but also its
distribution in the examined tissue. Using the present invention,
those of ordinary skill will readily perceive that any of a wide
variety of histological methods (such as staining procedures) can
be modified in order to achieve such in situ detection.
[0139] Immunoassays and non-immunoassays for NHP gene products, or
conserved variants or peptide fragments thereof, will typically
comprise incubating a sample, such as a biological fluid, a tissue
extract, freshly harvested cells, or lysates of cells that have
been incubated in cell culture, in the presence of a detectably
labeled antibody capable of identifying NHP gene products, or
conserved variants or peptide fragments thereof, and detecting the
bound antibody by any of a number of techniques well-known in the
art. The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support that is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers, followed by treatment
with the detectably labeled NHP antibody or NHP fusion protein. The
solid phase support may then be washed with the buffer a second
time to remove unbound antibody or fusion protein. The amount of
bound label on solid support may then be detected by conventional
means.
[0140] The terms "solid phase support or carrier" are intended to
include any support capable of binding an antigen or an antibody.
Well-known supports or carriers include, but are not limited to,
glass, polystyrene, polypropylene, polyethylene, dextran, nylon,
amylases, natural and modified celluloses, polyacrylamides,
gabbros, and magnetite. The nature of the carrier can be either
soluble to some extent or insoluble for the purposes of the present
invention. The support material may have virtually any possible
structural configuration so long as the coupled molecule is capable
of binding to an antigen or antibody. Thus, the support
configuration may be spherical, as in a bead, or cylindrical, as in
the inside surface of a test tube, or the external surface of a
rod. Alternatively, the surface may be flat, such as a sheet, test
strip, etc. Preferred supports include polystyrene beads. Those
skilled in the art will know many other suitable carriers for
binding antibody or antigen, or will be able to ascertain the same
by use of routine experimentation.
[0141] The binding activity of a given lot of NHP antibody or NHP
fusion protein may be determined according to well-known methods.
Those skilled in the art will be able to determine operative and
optimal assay conditions for each determination by employing
routine experimentation.
[0142] With respect to antibodies, one of the ways in which a NHP
antibody can be detectably labeled is by linking the same to an
enzyme for use in an enzyme immunoassay (EIA; see, for example,
Gosling, ed., 2000, "Immunoassays: A Practical Approach", Oxford
University Press, Inc., N.Y.). The enzyme that is bound to the
antibody will react with an appropriate substrate, preferably a
chromogenic substrate, in such a manner as to produce a chemical
moiety that can be detected, for example, by spectrophotometric,
fluorimetric or visual means. Enzymes that can be used to
detectably label the antibody include, but are not limited to,
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods that employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0143] Additionally, detection may also be accomplished using any
of a variety of other immunoassays. For example, by radioactively
labeling the antibodies or antibody fragments, it is possible to
detect NHPs through the use of a radioimmunoassay (RIA). The
radioactive isotope can be detected by such means as the use of a
gamma or scintillation counter, or by autoradiography.
[0144] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wavelength, its presence can then be detected
due to fluorescence. Exemplary fluorescent labeling compounds
include, but are not limited to, fluorescein isothiocyanate,
rhodamine, phycoerythrin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine. The antibody can also be
detectably labeled using fluorescence emitting metals such as
.sup.152Eu, or others of the lanthanide series. These metals can be
attached to the antibody using such metal chelating groups as
diethylenetriaminepentacetic acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA). The antibody also can be
detectably labeled by coupling it to a chemiluminescent compound.
The presence of the chemiluminescent-tagged antibody is then
determined by detecting the presence of luminescence that arises
during the course of a chemical reaction. Examples of particularly
useful chemiluminescent labeling compounds include, but are not
limited to, luminol, isoluminol, theromatic acridinium ester,
imidazole, acridinium salt and oxalate ester.
[0145] Likewise, a bioluminescent compound may be used to label the
NHP antibodies of the present invention. Bioluminescence is a type
of chemiluminescence found in biological systems, in which a
catalytic protein increases the efficiency of the chemiluminescent
reaction. The presence of a bioluminescent protein is determined by
detecting the presence of luminescence. Exemplary bioluminescent
compounds for purposes of labeling include, but are not limited to,
luciferin, luciferase and aequorin (green fluorescent protein; as
described in U.S. Pat. Nos. 5,491,084, 5,625,048, 5,777,079,
5,795,737, 5,804,387, 5,874,304, 5,968,750, 5,976,796, 6,020,192,
6,027,881, 6,054,321, 6,096,865, 6,146,826, 6,172,188 and
6,265,548, each of which is hereby incorporated by: reference).
5.5 Screening Assays for Compounds that Modulate NHP Expression or
Activity
[0146] The following assays are designed to identify compounds that
interact with (e.g., bind to) NHPs, compounds that interact with
(e.g., bind to) intracellular proteins that interact with NHPs,
compounds that interact with (e.g., bind to) both intracellular and
extracellular proteins or receptors that regulate NHP activity and
expression, compounds that interfere with the interaction of NHPs
or proteins or compounds involved in NHP-mediated activity, and
compounds that modulate the activity of a NHP gene (i.e., modulate
the level of NHP gene expression) or modulate the level of NHPs.
Assays may additionally be utilized that identify compounds that
bind to NHP gene regulatory sequences (e.g., promoter sequences)
and that may modulate NHP gene expression.
[0147] The compounds that can be screened in accordance with the
present invention include, but are not limited to, peptides,
antibodies and fragments thereof, and other organic compounds
(e.g., peptidomimetics, small organic compounds) that bind to a NHP
and either mimic or increase the activity of a NHP (i.e., agonists)
or inhibit the activity of a NHP (i.e., antagonists); as well as
peptides, antibodies or fragments thereof, and other organic
compounds that mimic or increase NHP activity or inhibit the
activity of a NHP.
[0148] Such compounds may include, but are not limited to, peptides
such as, for example, soluble peptides, including, but not limited
to, members of random peptide libraries (see, e.g., Lam et al.,
1991, Nature 354:82-84; Houghten et al., 1991, Nature 354:84-86),
and combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids, phosphopeptides (including, but
not limited to, members of random or partially degenerate, directed
phosphopeptide libraries; see, e.g., Songyang et al., 1993, Cell
72:767-778), antibodies (including, but not limited to, polyclonal,
monoclonal, humanized, anti-idiotypic, chimeric or single chain
antibodies, and Fab, F(ab').sub.2 and Fab expression library
fragments, and epitope-binding fragments thereof), and small
organic or inorganic molecules.
[0149] Other compounds that can be screened in accordance with the
invention include, but are not limited to, small organic molecules
that are able to gain entry into an appropriate cell and affect the
expression of a NHP gene, or some other gene involved in a NHP
pathway (e.g., by interacting with the regulatory region or
transcription factors involved in gene expression); or such
compounds that affect the activity of NHPs or the activity of some
other intracellular factor involved in a NHP pathway.
[0150] Computer modeling and searching technologies permit
identification of compounds, or the improvement of already
identified compounds, that can modulate NHP expression or activity.
Having identified such a compound or composition, the active sites
or regions are identified. The active site can be identified using
methods known in the art including, for example, from the amino
acid sequences of peptides, from the nucleotide sequences of
nucleic acids, or from study of complexes of the relevant compound
or composition with its natural ligand. In the latter case,
chemical or X-ray crystallographic methods can be used to find the
active site by finding where on the factor the complexed ligand is
found. Next, the three dimensional geometric structure of the
active site is determined. This can be done by known methods,
including X-ray crystallography, which can determine a complete
molecular structure. On the other hand, solid or liquid phase NMR
can be used to determine certain intra-molecular distances. Any
other experimental method of structure determination can be used to
obtain partial or complete geometric structures. The geometric
structures may be measured with a complexed ligand, natural or
artificial, which may increase the accuracy of the active site
structure determined.
[0151] If an incomplete or insufficiently accurate structure is
determined, the methods of computer based numerical modeling can be
used to complete the structure or improve its accuracy. Any
recognized modeling method can be used, including parameterized
models specific to particular biopolymers such as proteins or
nucleic acids, molecular dynamics models based on computing
molecular motions, statistical mechanics models based on thermal
ensembles, or combined models. For most types of models, standard
molecular force fields, representing the forces between constituent
atoms and groups, are necessary, and can be selected from force
fields known in physical chemistry. The incomplete or less accurate
experimental structures can serve as constraints on the complete
and more accurate structures computed by these modeling
methods.
[0152] Finally, having determined the structure of the active site,
either experimentally, by modeling, or a combination thereof,
candidate modulating compounds can be identified by searching
databases containing compounds along with information on their
molecular structure. Such a search seeks compounds having
structures that match the determined active site structure and that
interact with the groups defining the active site. Such a search
can be manual, but is preferably computer assisted. The compounds
found from such a search are potential NHP modulating
compounds.
[0153] Alternatively, these methods can be used to identify
improved modulating compounds from an already known modulating
compound or ligand. The composition of the known compound can be
modified and the structural effects of modification can be
determined using the experimental and computer modeling methods
described above applied to the new composition. The altered
structure is then compared to the active site structure of the
compound to determine if an improved fit or interaction results. In
this manner systematic variations in composition, such as by
varying side groups, can be quickly evaluated to obtain modified
modulating compounds or ligands of improved specificity or
activity.
[0154] Further experimental and computer modeling methods useful to
identify modulating compounds based upon identification of the
active sites of NHPs, and related transduction and transcription
factors, will be apparent to those of skill in the art.
[0155] Examples of molecular modeling systems are the CHARMM and
QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMM
performs the energy minimization and molecular dynamics functions.
QUANTA performs the construction, graphic modeling and analysis of
molecular structure. QUANTA allows interactive construction,
modification, visualization, and analysis of the behavior of
molecules with each other.
[0156] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen et al., 1988,
Acta Pharmaceutical Fennica 97:159-166; Ripka, New scientist 54-57
(Jun. 16, 1988); McKinaly and Rossmann, 1989, Ann. Rev. Pharmacol.
Toxiciol. 29:111-122; Perry and Davies, OSAR: Quantitative
Structure-Activity Relationships in Drug Design, pp. 189-193 (Alan
R. Liss, Inc. 1989); Lewis and Dean, 1989, Proc. R. Soc. Lond.
236:125-140 and 141-162; and, with respect to a model receptor for
nucleic acid components, Askew et al., 1989, J. Am. Chem. Soc.
111:1082-1090. Other computer programs that screen and graphically
depict chemicals are available from companies such as BioDesign,
Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario,
Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these
are primarily designed for application to drugs specific to
particular proteins, they can be adapted to the design of drugs
specific to regions of DNA or RNA, once that region is
identified.
[0157] Although described above with reference to design and
generation of compounds that could alter binding, one could also
screen libraries of known compounds, including natural products or
synthetic chemicals, and biologically active materials, including
proteins, for compounds that are inhibitors or activators of
NHPs.
[0158] Compounds identified via assays such as those described
herein may be useful, for example, in further elaborating the
biological function of a NHP gene product, and for ameliorating
NHP-related disorders.
[0159] 5.5.1 In Vitro Screening Assays for Compounds that Bind to a
NHP
[0160] In vitro systems may be designed to identify compounds
capable of interacting with (e.g., binding to) NHPs. The compounds
thus identified (such as NHP modulators, natural NHP substrates,
etc.) can be useful, for example, in modulating the activity of
wild-type and/or mutant NHP gene products; in elaborating the
biological function of NHPs; in screens for identifying compounds
that disrupt normal NHP interactions; or in themselves directly
disrupt such interactions.
[0161] The principle of the assays used to identify compounds that
bind to a NHP involves preparing a reaction mixture of a NHP and
the test compound under conditions and for a time sufficient to
allow the two components to interact and bind, thus forming a
complex that can be removed and/or detected in the reaction
mixture. The NHP species used can vary depending upon the goal of
the screening assay. For example, where agonists of the natural
ligand/substrate are sought, full length NHPs, or a soluble
truncated NHP polypeptide that affords advantages in the assay
system (e.g., labeling, isolation of the resulting complex, etc.)
can be utilized.
[0162] The screening assays can be conducted in a variety of ways.
For example, one method to conduct such an assay would involve
anchoring a NHP protein, polypeptide, peptide, or fusion protein,
or the test substance, onto a solid phase and detecting NHP/test
compound complexes anchored on the solid phase at the end of the
reaction. In one embodiment of such a method, the NHP reactant may
be anchored onto a solid surface, and the test compound, which is
not anchored, may be labeled, either directly or indirectly.
Examples of some of the technologies available to immobilize the
molecules are discussed in Cass, ed., "Immobilized Biomolecules In
Analysis: A Practical Approach", Oxford University Press, N.Y.
[0163] In practice, microtiter plates may conveniently be utilized
as the solid phase. The anchored component may be immobilized by
non-covalent or covalent attachments. Non-covalent attachment may
be accomplished by simply coating the solid surface with a solution
of the protein and drying. Alternatively, an immobilized antibody,
preferably a monoclonal antibody, specific for the protein to be
immobilized may be used to anchor the protein to the solid surface.
The surfaces may be prepared in advance and stored. molecular
structure. Such a search seeks compounds having structures that
match the determined active site structure and that interact with
the groups defining the active site. Such a search can be manual,
but is preferably computer assisted. The compounds found from such
a search are potential NHP modulating compounds.
[0164] Alternatively, these methods can be used to identify
improved modulating compounds from an already known modulating
compound or ligand. The composition of the known compound can be
modified and the structural effects of modification can be
determined using the experimental and computer modeling methods
described above applied to the new composition. The altered
structure is then compared to the active site structure of the
compound to determine if an improved fit or interaction results. In
this manner systematic variations in composition, such as by
varying side groups, can be quickly evaluated to obtain modified
modulating compounds or ligands of improved specificity or
activity.
[0165] Further experimental and computer modeling methods useful to
identify modulating compounds based upon identification of the
active sites of NHPs, and related transduction and transcription
factors, will be apparent to those of skill in the art.
[0166] Examples of molecular modeling systems are the CHARMM and
QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMM
performs the energy minimization and molecular dynamics functions.
QUANTA performs the construction, graphic modeling and analysis of
molecular structure. QUANTA allows interactive construction,
modification, visualization, and analysis of the behavior of
molecules with each other.
[0167] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen et al., 1988,
Acta Pharmaceutical Fennica 97:159-166; Ripka, New Scientist 54-57
(Jun. 16, 1988); McKinlay and Rossmann, 1989, Ann. Rev. Pharmacol.
Toxiciol. 29:111-122; Perry and Davies, OSAR: employed for
identifying proteins that interact with NHPS. Among the traditional
methods that may be employed are co-immunoprecipitation,
crosslinking and co-purification through gradients or
chromatographic columns of cell lysates, or proteins obtained from
cell lysates, and a NHP to identify proteins in the lysate that
interact with NHPs. For these assays, the NHP component used can be
a full length NHP, a peptide or polypeptide corresponding to one or
more domains of a NHP, or a fusion protein containing one or more
domains of a NHP. Once isolated, such an intracellular protein can
be identified and can, in turn, be used in conjunction with
standard techniques to identify proteins with which it interacts.
For example, at least a portion of the amino acid sequence of an
intracellular protein that interacts with a NHP can be ascertained
using techniques well-known to those of skill in the art, such as
via the Edman degradation technique (see, e.g., Creighton, 1983,
supra, pp.34-49). The amino acid sequence obtained may be used as a
guide for the generation of oligonucleotide mixtures that can be
used to screen for gene sequences encoding such intracellular
proteins. Screening may be accomplished, for example, by standard
hybridization or PCR techniques. Techniques for the generation of
oligonucleotide mixtures and screening are well-known (see, e.g.,
Ausubel, supra., and Innis et al., eds. "PCR Protocols: A Guide to
Methods and Applications", 1990, Academic Press, Inc., N.Y.).
[0168] Additionally, methods may be employed that result in the
simultaneous identification of genes that encode proteins that are
capable of interacting with NHPs. These methods include, for
example, probing expression libraries, in a manner similar to the
well-known technique of antibody probing of lambda gt11 libraries,
using a labeled NHP protein, polypeptide, peptide or fusion
protein, e.g., a NHP polypeptide or NHP domain fused to a marker
(e.g., an enzyme, fluor, luminescent protein, or dye), or an Ig-Fc
domain.
[0169] One method that detects protein interactions in vivo, the
two-hybrid system, is described in detail for illustration only and
not by way of limitation. One version of this system utilizes yeast
cells (Chien et al., 1991, Proc. Natl. Acad. Sci. USA,
88:9578-9582), while another uses mammalian cells (Luo et al.,
1997, Biotechniques 22:350-352). Both the yeast and mammalian
two-hybrid systems are commercially available from Clontech (Palo
Alto, Calif.), and are further described in U.S. Pat. Nos.
5,283,173, 5,468,614, and 5,667,973, which are herein incorporated
by reference in their entirety.
[0170] Briefly, utilizing such a system, plasmids are constructed
that encode two hybrid proteins: one plasmid consists of
nucleotides encoding the DNA-binding domain of a transcription
activator protein fused to a NHP nucleotide sequence encoding a NHP
protein, polypeptide, peptide or fusion protein, and the other
plasmid consists of nucleotides encoding an activation domain of a
transcription activator protein fused to a cDNA encoding an unknown
protein to be tested for interaction with a NHP, which has been
recombined into this plasmid as part of a cDNA library. The
DNA-binding domain fusion plasmid and the cDNA library are
transformed into a strain of the yeast Saccharomyces cerevisiae or
a mammalian cell (such as Saos-2, CHO, CV1, Jurkat or HeLa) that
contains a reporter gene (e.g., HBS, lacZ, CAT, or a gene encoding
an essential amino acid synthetase) whose regulatory region
contains the binding site of the transcription activator. Either
hybrid protein alone cannot activate transcription of the reporter
gene: the DNA-binding domain hybrid cannot because it does not
provide activation function; and the activation domain hybrid
cannot because it cannot localize to the binding site of the
activator. Interaction of the two hybrid proteins reconstitutes the
functional activator protein and results in expression of the
reporter gene, which is detected by an assay for the reporter gene
product.
[0171] The two-hybrid system or related methodology may be used to
screen activation domain libraries for proteins that interact with
the "bait" gene product. By way of example, and not by way of
limitation, a NHP may be used as the bait gene product. Total
genomic or cDNA sequences are fused to DNA encoding an activation
domain. This library and a plasmid encoding a hybrid of a bait NHP
gene product fused to the DNA-binding domain are co-transformed
into a reporter strain, and the resulting transformants are
screened for those that express the reporter gene. For example, and
not by way of limitation, a bait NHP sequence, such as an open
reading frame of a NHP (or a domain of a NHP) can be cloned into a
vector such that it is translationally fused to DNA encoding the
DNA-binding domain of the GAL4 protein. These colonies are purified
and the library plasmids responsible for reporter gene expression
are isolated. DNA sequencing is then used to identify the proteins
encoded by the library plasmids.
[0172] A cDNA library of the cell line from which proteins that
interact with a bait NHP gene product are to be detected can be
made using methods routinely practiced in the art. According to one
particular system, for example, the cDNA fragments can be inserted
into a vector such that they are translationally fused to the
transcriptional activation domain of GAL4. This library can be
co-transformed along with the bait NHP gene-GAL4 fusion plasmid
into a yeast strain that cannot grow without added histidine, and
that contains a HIS3 gene driven by a promoter that contains GAL4
activation sequence. A cDNA encoded protein, fused to GAL4
transcriptional activation domain, which interacts with the bait
NHP gene product will reconstitute an active GAL4 protein and
thereby drive expression of the HIS3 gene. Colonies that express
HIS3 can be detected by their growth on petri dishes containing
semi-solid agar based media lacking histidine. The cDNA can then be
purified from these strains, and used to produce and isolate the
bait NHP gene-interacting protein using techniques routinely
practiced in the art.
[0173] 5.5.3 Assays for Compounds that Interfere with NHP
Activity
[0174] The macromolecules that interact with NHPs are referred to,
for purposes of this discussion, as "binding partners". These
binding partners are likely to be involved in a NHP pathway, and
therefore, may have a role in NHP-mediated disorders. Therefore, it
is desirable to identify compounds that interfere with or disrupt
the interaction of such binding partners with NHPs, and that may be
useful in regulating the activity of NHPs and controlling
NHP-mediated disorders.
[0175] The basic principle of the assay systems used to identify
compounds that interfere with the interaction between a NHP and its
binding partner or partners involves preparing a reaction mixture
containing a NHP protein, polypeptide, peptide or fusion protein,
and the binding partner under conditions and for a time sufficient
to allow the components to interact and bind, thus forming a
complex. In order to test a compound for inhibitory activity, the
reaction mixture is prepared in the presence and absence of the
test compound. The test compound may be initially included in the
reaction mixture, or may be added at a time subsequent to the
addition of the NHP moiety and its binding partner(s). Control
reaction mixtures are incubated without the test compound or with a
placebo. The formation of complexes between the NHP moiety and the
binding partner is then detected. The formation of a complex in the
control reaction, but not in the reaction mixture containing the
test compound, indicates that the compound interferes with the
interaction of the NHP and the interactive binding partner.
Additionally, complex formation within reaction mixtures containing
the test compound and a normal NHP protein may also be compared to
complex formation within reaction mixtures containing the test
compound and a mutant NHP. This comparison may be important in
those cases wherein it is desirable to identify compounds that
disrupt interactions of mutant but not normal NHPs.
[0176] Assays for compounds that interfere with the interaction of
NHPs and binding partner(s) can be conducted in a heterogeneous or
homogeneous format. Heterogeneous assays involve anchoring either
the NHP moiety product or the binding partner onto a solid phase
and detecting complexes anchored on the solid phase at the end of
the reaction. In homogeneous assays, the entire reaction is carried
out in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds that interfere
with the interaction by competition can be identified by conducting
the reaction in the presence of the test substance, i.e., by adding
the test substance to the reaction mixture prior to or
simultaneously with the NHP moiety and interactive binding partner.
Alternatively, test compounds that disrupt preformed complexes,
e.g., compounds with higher binding constants that displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are described briefly below.
[0177] In a heterogeneous assay system, either the NHP moiety or
the interactive binding partner is anchored onto a solid surface,
while the non-anchored species is labeled, either directly or
indirectly. In practice, microtiter plates are conveniently
utilized. The anchored species may be immobilized by non-covalent
or covalent attachments. Non-covalent attachment may be
accomplished simply by coating the solid surface with a solution of
a NHP gene product or binding partner and drying. Alternatively, an
immobilized antibody specific for the species to be anchored may be
used to anchor the species to the solid surface. The surfaces may
be prepared in advance and stored.
[0178] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing), and any
complexes formed will remain immobilized on the solid surface. The
detection of complexes anchored on the solid surface can be
accomplished in a number of ways. Where the non-immobilized species
is pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the non-immobilized
species is not pre-labeled, an indirect label can be used to detect
complexes anchored on the surface, e.g., using a labeled antibody
specific for the initially non-immobilized species (the antibody,
in turn, may be directly labeled or indirectly labeled with a
labeled anti-Ig antibody). Depending upon the order of addition of
reaction components, test compounds that inhibit complex formation
or that disrupt preformed complexes can be detected.
[0179] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected, e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds that inhibit complex
or that disrupt preformed complexes can be identified.
[0180] In an alternate embodiment of the invention, a homogeneous
assay can be used. In this approach, a preformed complex of a NHP
moiety and the interactive binding partner is prepared in which
either the NHP or its binding partner is labeled, but the signal
generated by the label is quenched due to formation of the complex
(see, e.g., U.S. Pat. No. 4,109,496, incorporated herein by
reference, which utilizes this approach for immunoassays). The
addition of a test substance that competes with and displaces one
of the species from the preformed complex will result in the
generation of a signal above background. In this way, test
substances that disrupt NHP/intracellular binding partner
interactions can be identified.
[0181] In a particular embodiment, a NHP fusion protein can be
prepared for immobilization. For example, a NHP, or a peptide
fragment thereof, e.g., corresponding to one or more particular
domain(s), can be fused to glutathione-S-transferase (GST) using a
fusion vector, such as pGEX-5X-1, in such a manner that the GST
binding activity is maintained in the resulting fusion protein. An
interactive binding partner, identified as described herein, can be
purified and used to raise polyclonal and monoclonal antibodies,
using methods routinely practiced in the art. Such antibodies can
be labeled with a radioactive isotope, .sup.125I for example, by
methods routinely practiced in the art. In a heterogeneous assay,
such GST-NHP fusion proteins can be anchored to glutathione-agarose
beads. The interactive binding partner can then be added in the
presence or absence of the test compound in a manner that allows
interaction and binding to occur. At the end of the reaction
period, unbound material can be washed away, and a labeled
monoclonal antibody that binds the binding partner can be added to
the system and allowed to bind to complexed binding partner. The
interaction between the NHP and the interactive binding partner can
be detected by measuring the amount of radioactivity that remains
associated with the glutathione-agarose beads. A successful
inhibition of the interaction by the test compound will result in a
decrease in measured radioactivity.
[0182] Alternatively, a GST-NHP fusion protein and an interactive
binding partner can be mixed together in liquid in the absence of
the solid glutathione-agarose beads. The test compound can be added
either during or after the species are allowed to interact. This
mixture can then be added to the glutathione-agarose beads and
unbound material is washed away. Again the extent of inhibition of
the NHP/binding partner interaction can be detected by adding a
labeled antibody against the binding partner and measuring the
radioactivity associated with the beads.
[0183] In another embodiment of the invention, where the binding
partner is a protein, these same techniques can be employed using
peptide fragments that correspond to one or more of the binding
domains of a NHP and/or the interactive binding partner, in place
of one or both of the full length proteins. Any number of methods
routinely practiced in the art can be used to identify and isolate
the binding domains or regions. These methods include, but are not
limited to, mutagenesis of the gene encoding one of the proteins,
and screening for disruption of binding in a co-immunoprecipitation
assay. Compensating mutation(s) in the sequence encoding the second
species in the complex can then be selected. Sequence analysis of
the sequences encoding the respective proteins will reveal the
mutation(s) that correspond to the region of the protein involved
in interactive binding. Alternatively, one protein can be anchored
to a solid surface using methods described above, and allowed to
interact with and bind to its labeled binding partner, which has
been treated with a proteolytic enzyme, such as trypsin. After
washing, a short, labeled peptide comprising the binding domain may
remain associated with the solid material, which can be isolated
and identified by amino acid sequencing. Also, once a sequence
encoding the binding partner is obtained, short polynucleotide
segments can be engineered to express peptide fragments of the
protein, which can then be tested for binding activity, and
purified or synthesized.
[0184] For example, and not by way of limitation, a NHP protein,
polypeptide or peptide can be anchored to a solid material, as
described above, by making a GST-NHP fusion protein and allowing it
to bind to glutathione agarose beads. The interactive binding
partner can be labeled with a radioactive isotope, such as
.sup.35S, and cleaved with a proteolytic enzyme, such as trypsin.
Cleavage products can then be added to the anchored GST-NHP fusion
protein and allowed to bind. After washing away unbound peptides,
labeled bound material, representing the intracellular binding
partner binding domain, can be eluted, purified, and analyzed to
determine the amino acid sequence by well-known methods. Peptides
so identified can be produced synthetically or fused to appropriate
facilitative proteins using recombinant DNA technology.
[0185] 5.5.4 The Use of Compounds to Treat NHP-Mediated
Disorders
[0186] The invention also encompasses the use of agonists and
antagonists of a NHP (including small molecules and large
molecules), mutant versions of a NHP or portions thereof that
compete with native NHPs, peptides, and antibodies, as well as
nucleotide sequences that can be used to inhibit the expression of
a NHP (e.g., antisense and ribozyme molecules, and gene or
regulatory sequence replacement constructs) or to enhance the
expression of NHP polynucleotides (e.g., expression constructs that
place the described polynucleotide under the control of a strong
promoter system) in the treatment of NHP-mediated disorders.
Compounds including, but not limited to, those identified via assay
techniques such as those described above, can be tested for the
ability to ameliorate symptoms associated with NHP-mediated
disorders.
[0187] The assays described above can identify compounds that
affect NHP activity, or compounds that affect NHP gene activity (by
affecting NHP gene expression, including molecules, e.g., proteins
or small organic molecules, that affect or interfere with splicing
events so that expression of a full length or a truncated form of a
NHP can be modulated). However, it should be noted that the assays
described can also be used to identify compounds that indirectly
modulate NHPs. The identification and use of compounds that affect
a NHP-independent step in a NHP pathway are also within the scope
of the invention. Compounds that indirectly affect NHP activity can
also be used as part of a therapeutic method for the treatment of
NHP-mediated disorders.
[0188] The invention additionally encompasses cell-based and animal
model-based assays for the identification of compounds exhibiting
an ability to ameliorate the symptoms of NHP-mediated disorders.
Cell-based systems used to identify compounds that may act to
ameliorate NHP-mediated disorder symptoms can include, for example,
recombinant or non-recombinant cells, such as cell lines that
express a NHP sequence. Host cells (e.g., COS cells, CHO cells,
fibroblasts) genetically engineered to express a functional NHP can
also be used. The presence of a functional NHP can be determined,
for example, by a chemical or a phenotypic change, the induction of
another host cell gene, a change in ion flux (e.g., Ca.sup.++), or
tyrosine phosphorylation of host cell proteins, etc.
[0189] In utilizing such cell systems, cells may be exposed to a
compound suspected of exhibiting an ability to ameliorate the
symptoms of NHP-mediated disorders, at a sufficient concentration
and for a time sufficient to elicit such an amelioration of the
symptoms of NHP-mediated disorders in the exposed cells. After
exposure, the cells can be assayed to measure alterations in NHP
expression, e.g., by assaying cell lysates for NHP mRNA transcripts
(e.g., by Northern analysis or RT-PCR), or by assaying for the
level of a NHP protein expressed in the cell (e.g., by SDS-PAGE and
Western blot or immunoprecipitation); compounds that regulate or
modulate NHP expression are good candidates as therapeutics.
Alternatively, the cells can be examined to determine whether one
or more NHP disorder-like cellular phenotype has been altered to
resemble a more normal or more wild-type, non-NHP disorder
phenotype, or a phenotype more likely to produce a lower incidence
or severity of disorder symptoms. Still further, the expression
and/or activity of components of the signal transduction pathway(s)
of which a NHP is a part, or the activity of a NHP signal
transduction pathway itself, can be assayed.
[0190] In addition, animal-based NHP-mediated disorder systems may
be used to identify compounds capable of treating or ameliorating
symptoms associated with NHP-mediated disorders. These animals may
be transgenic, knockout, or knock-in (preferably humanized
knock-ins where, for example, the endogenous animal NHP gene has
been replaced by a human NHP sequence) animals, as described
herein. Such animal models may be used as test substrates for the
identification of drugs, pharmaceuticals, therapies and
interventions that may be effective in treating such disorders. For
example, animal models can be exposed to a compound suspected of
exhibiting an ability to ameliorate symptoms of NHP-mediated
disorders, at a sufficient concentration and for a time sufficient
to elicit such an amelioration of NHP disorder associated symptoms
in the exposed animals. The response of the animals to the exposure
may be monitored by assessing the reversal of symptoms associated
with NHP-mediated disorders. With regard to intervention, any
treatments that reverse, halt or slow the progression of any aspect
of symptoms associated with NHP disorders should be considered as
candidates for therapeutic intervention in treatment of human NHP
disorders. Dosages of test agents may be determined by deriving
toxicity and dose-response curves.
5.6 Pharmaceutical Preparations and Methods of Administration
[0191] Compounds that are determined to affect expression of the
sequences of the current invention, or the interaction of the
peptides and proteins of the present invention with any of their
binding partners, can be administered to a patient at
therapeutically effective doses to treat or ameliorate low white
blood cell counts. A therapeutically effective dose refers to that
amount of the compound sufficient to result in any delay in onset,
amelioration or retardation of disease symptoms.
[0192] 5.6.1 Effective Dose
[0193] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index, and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
that exhibit large therapeutic indices are preferred. Compounds
that exhibit toxic side effects may be used in certain embodiments,
however care should usually be taken to design delivery systems
that target such compounds preferentially to the site of affected
tissue, in order to minimize potential damage to uninfected cells
and, thereby, reduce side effects.
[0194] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosages for use in
humans. The dosages of such compounds lie preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the methods of
the invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0195] When the therapeutic treatment of disease is contemplated,
the appropriate dosage may also be determined using animal studies
to determine the maximal tolerable dose, or MTD, of a bioactive
agent per kilogram weight of the test subject. In general, at least
one animal species tested is mammalian. Those skilled in the art
regularly extrapolate doses for efficacy and avoiding toxicity to
other species, including human. Before human studies of efficacy
are undertaken, Phase I clinical studies in normal subjects help
establish safe doses.
[0196] Additionally, the bioactive agent may be complexed with a
variety of well established compounds or structures that, for
instance, enhance the stability of the bioactive agent, or
otherwise enhance its pharmacological properties (e.g., increase in
vivo half-life, reduce toxicity, etc.).
[0197] The therapeutic agents will be administered by any number of
methods known to those of ordinary skill in the art including, but
not limited to, inhalation; subcutaneous (sub-q); intravenous
(I.V.); intraperitoneal (I.P.); intramuscular (I.M.), or
intrathecal injection; or topically applied (transderm, ointments,
creams, salves, eye drops, and the like).
[0198] 5.6.2 Formulations and Use
[0199] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manners using
one or more physiologically acceptable carriers or excipients.
[0200] Thus, the compounds and their physiologically acceptable
salts and solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose),
or oral, buccal, parenteral or rectal administration.
[0201] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring agents, coloring agents and
sweetening agents as appropriate.
[0202] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner.
[0203] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges of, e.g., gelatin for use in an inhaler or
insufflator may be formulated containing a powder mix of the
compound and a suitable powder base such as lactose or starch.
[0204] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0205] The compounds may also be formulated as compositions for
rectal administration such as suppositories or retention enemas,
e.g., containing conventional suppository bases such as cocoa
butter or other glycerides.
[0206] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil), ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt. The compositions may, if desired, be presented in a pack or
dispenser device, which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
[0207] The examples below are provided to illustrate the subject
invention. These examples are provided by way of illustration only,
and are not included for the purpose of limiting the invention in
any way whatsoever.
6.0 EXAMPLES
6.1 NHP Gene Disrupted Mice
[0208] Gene trapping is a method of nonspecific insertional
mutagenesis that uses a fragment of DNA coding for a reporter or
selectable marker gene as a mutagen. Gene trap vectors have been
designed to integrate into introns or exons in a manner that allows
the cellular splicing machinery to splice vector encoded exons to
cellular mRNAs. Commonly, gene trap vectors contain selectable
marker sequences that are preceded by strong splice acceptor
sequences and are not preceded by a promoter. Thus, when such
vectors integrate into a gene, the cellular splicing machinery
splices exons from the trapped gene onto the 5' end of the
selectable marker sequence. Typically, such selectable marker genes
can only be expressed if the vector encoding the gene has
integrated into an intron. The resulting gene trap events are
subsequently identified by selecting for cells that can survive
selective culture.
[0209] Embryonic stem cells (Lex-1 cells derived from murine strain
129SvEv.sup.Brd) were mutated by a process involving the insertion
of at least a portion of a genetically engineered vector sequence
into the NHP gene. The mutated embryonic stem cells were then
microinjected into blastocysts, which were subsequently introduced
into pseudopregnant female hosts and carried to term using
established methods, such as those described in, for example,
Zambrowicz et al., eds., "Mouse Mutagenesis", 1998, Lexicon Press,
The Woodlands, Tex., and periodic updates thereof, herein
incorporated by reference. In this case, the virus inserted in the
forward orientation, close to the nucleotides coding for amino acid
135, and disrupted the NHP gene. The resulting chimeric animals
were subsequently bred to produce offspring capable of germline
transmission of an allele containing the engineered mutation in the
NHP gene.
[0210] Techniques useful to disrupt a gene in a cell, and
especially an ES cell, that may already have a disrupted gene are
disclosed in U.S. Pat. Nos. 6,136,566, 6,139,833 and 6,207,371, and
U.S. patent application Ser. No. 08/728,963, each of which are
hereby incorporated herein by reference in their entirety.
[0211] 6.1.1 The effect of NHP Disruption on Mouse Physiology
[0212] The genetic distribution of wild-type (11), heterozygous
(28) and homozygous (19) animals appears to be normal. Thus, for
the remaining studies, mice homozygous for the disruption of the
NHP gene were studied in conjunction with heterozygous and
wild-type litter mates. During this analysis, the mice were
subjected to a medical work-up using an integrated suite of medical
diagnostic procedures designed to assess the function of the major
mammalian organ systems in the subject. By studying numerous mice
in which the NHP gene had been disrupted, in conjunction with
wild-type litter mates, more reliable and repeatable data was
obtained. Disruption of the NHP gene resulted in, among other
effects, an unexpected increase in white blood cell counts, as
described in greater detail below. The disruption of the NHP gene
was confirmed by RT-PCR.
[0213] Additional studies of the expression patterns of human and
murine NHPs showed that the NHPs can be detected in certain mouse
and human tissues by RT-PCR. NHP transcripts were detected in mouse
tissue derived from mouse brain, thymus, and heart. NHP transcripts
were detected in human tissue derived from human prostate and
testis.
[0214] 6.1.2 Size, Percent Body Fat, and Bone Mineral Density of
NHP Knockouts
[0215] Body composition and percent body fat was measured by dual
energy X-ray absorptiometry (DEXA) using the Piximus small animal
densitometer (Lunar Corporation, Madison, Wis.). Individual mice
were sedated with Avertin (1.25% solution, 2.5 mg/10 gm body weight
delivered by intraperitoneal injection), immobilized on a
positioning tray and then placed on the Piximus imaging window. All
scans were performed using the total body mode (0.18.times.0.18
mm), and the analysis was performed on the total body region of
interest. The entire body, except the head, of each mouse was
exposed for 5 minutes to a cone shaped beam of both high and low
energy x-rays. A high-resolution digital picture was taken of the
image of the x-rays hitting a luminescent panel. Lunar PIXImus
software (version 1.45) was used to calculate the ratio of
attenuation of the high and low energies to separate bone from soft
tissue compartments and, within the soft tissue compartment, to
separate lean tissue mass from fat mass and thus determine the bone
mineral density (BMD), volumetric bone mineral density (vBMD),
total bone mineral content (BMC), fat composition (% fat), lean
body mass (LBM), the ratio of BMC/LBM, and total tissue mass (TTM)
in the regions of interest (total body for all tests, and
additionally, vertebrae and both femurs for BMD). Previous studies
have determined that this technique precisely measures fat and lean
tissue mass, and that there is a close relationship between fat and
lean tissue mass estimated by this technique with those measured
using chemical carcass analysis (Nagy and Clair, 2000, Obesity
Research 8:392-398).
[0216] Body composition and percent body fat was measured in eight
(8) homozygous (6 males, 2 females), four (4) heterozygous (2 males
and 2 females), and four (4) wild-type (2 males and 2 females)
mice. The mean percent total body fat was notably decreased in both
the male and female (-/-) mice compared to the (+/-) and (+/+)
animals. However, the values for the (-/-) animals fell within
historical normal ranges. There was no difference between groups in
any of the other parameters measured (TTM, LBM, vBMD, total body
BMD, femur BMD, vertebrae BMD, total body BMC, and the BMC/LBM
ratio).
[0217] Mouse body weight was determined to the nearest 0.1 gm using
an Ohaus Scout scale. Body length was determined from nose to the
base of tail and is reported in cm. Body weight and body length
data were obtained for mice at eight (8) weeks of age. The body
weight of eight (8) homozygous (6 males, 2 females), four (4)
heterozygous (2 males and 2 females), and four (4) wild-type (2
males and 2 females) mice was determined and analyzed. Decreased
body weight was noted for the two female (-/-) mice compared to the
female (+/+) and (+/-) mice.
[0218] Body length data was determined and analyzed for eight (8)
homozygous (6 males, 2 females), four (4) heterozygous (2 males and
2 females), and four (4) wild-type (2 males and 2 females) mice.
There was no significant difference in body length between
groups.
[0219] 6.1.3 The Effect of NHP Disruption on Hematology and Blood
Chemistry
[0220] Whole blood was collected by retro-orbital bleed and placed
in a capillary blood collection tube that contained EDTA. The blood
was analyzed using the Cell-Dyn 3500R analyzer (Abbott
Diagnostics). The analyzer employs dual technologies to provide the
basis for a five-part white blood cell (WBC) differential
identification. Multi-Angle Polarized Scatter Separation (MAPSS)
provides the primary white blood cell count and differential
information, while impedance provides additional information in the
presence of fragile lymphocytes and hypotonically resistant red
blood cells. Approximately 135 microliters of whole blood is
aspirated into the analyzer using a peristaltic pump. Four
independent measurement techniques are used by the Cell-Dyn 3500R
System to obtain the hematologic parameters. The WBC Optical Count
(WOC) and the WBC differential data are measured in the optical
flow channel, resulting in the identification of the WBC
subpopulations (neutrophils, lymphocytes, monocytes, eosinophils,
and basophils) for the five part WBC differential. The WBC
Impedance Count (WIC) is measured in one electrical impedance
channel. The RBC and platelet data are measured in a second
electrical impedance channel. The hemoglobin is measured in the
spectrophotometric channel. The sample was aspirated, diluted,
mixed, and the measurements for each parameter were obtained during
each instrument cycle. The final hematological analysis parameters
obtained are white blood cell count, neutrophils, lymphocytes,
monocytes, eosinophils, basophils, red blood cells, hemoglobin,
hematocrit, platlets, red cell distribution width, mean corpuscular
volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin
concentration and mean platelet volume.
[0221] Blood samples were obtained from a total of sixteen (16)
mice; eight (8) homozygous (6 males, 2 females), four (4)
heterozygous (2 males and 2 females), and four (4) wild-type (2
males and 2 females) mice. Analysis and comparison of the blood
samples revealed that disruption of the NHP gene results in an
upward trend in mean white blood cell (WBC) counts, with subsequent
increases in absolute neutrophil (NEUTRO), lymphocyte (LYM),
monocyte (MONO), eosinophil (EOS) and basophil (BASO) counts in the
(-/-) and (+/-) mice. The WBC values for the +/+ mice were
5.9.+-.2.3, for the +/- mice were 9.7.+-.0.7, and for the -/- mice
were 9.6.+-.2.8. The NEUTRO values for the +/+ mice were
0.91.+-.0.29, for the +/- mice were 1.40.+-.0.39, and for the -/-
mice were 1.26.+-.0.34. The LYM values for the +/+ mice were
4.61.+-.2.03, for the +/- mice were 7.70.+-.0.53, and for the -/-
mice were 7.83.+-.2.58. The MONO values for the +/+ mice were
0.30.+-.0.13, for the +/- mice were 0.53.+-.0.19, and for the -/-
mice were 0.42.+-.0.18. The EOS values for the +/+ mice were
0.033.+-.0.030, for the +/- mice were 0.063.+-.0.050, and for the
-/- mice were 0.050.+-.0.059. The BASO values for the +/+ mice were
0.008.+-.0.008, for the +/- mice were 0.016.+-.0.009, and for the
-/- mice were 0.018.+-.0.010. However, the relative distribution of
these cells remained normal.
[0222] Approximately 200 microliters of whole blood was collected
from the retro-orbital plexus. The blood was placed in a 2.5 ml
micro-collection tube and centrifuged to obtain the serum. The
sample was analyzed for the following analytes: albumin, alkaline
phosphatase, alanine aminotransferase (ALT), total bilirubin, blood
urea nitrogen (BUN), calcium, glucose, phosphorus, cholesterol,
triglycerides, creatinine and uric acid using a Cobas Integra 400
(Roche Diagnostics). The Cobas Integra 400 is a random and
continuous access, sample selective analyzer. The analyzer uses
four measuring principles: absorbance photometry, turbidimetry,
fluorescence polarimetry and ion-selective electrode potentiometry
to assay the analytes described above.
[0223] A total of sixteen (16) mice were analyzed; eight (8)
homozygous (6 males, 2 females), four (4) heterozygous (2 males and
2 females), and four (4) wild-type (2 males and 2 females) mice.
There were no significant differences in any of the above analytes
between the three groups.
[0224] The mononuclear cell profile is derived by staining a single
sample of lysed peripheral blood from each mouse with a panel of
six lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE,
CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled
antibodies stain-mutually exclusive cell types. The samples are
analyzed using a Becton Dickinson FACSCalibur flow cytometer with
CellQuest software. All leukocytes are identified by CD45 staining,
and granulocytes are excluded by scatter. T cells are identified by
expression of TCR b-chain, and are further divided into CD4+CD8-
(mature helper) and CD4-CD8+ (mature cytotoxic/suppressor). NK
cells and B cells are identified from the TCRb- (non-T) population
by staining with pan-NK and CD19 antibody, respectively. Monocytes
are defined as CD45+ mononuclear cells which are negative for all
T, B, and NK markers.
[0225] A total of six (6) mice were analyzed: two (2) wild-type
(+/+); two (2) heterozygotes (+/-); and two (2) homozygotes. There
were no significant differences in the percentage of CD4+CD8- or
CD4-CD8+ cells, the CD4+/CD8+ ratio, or the percentage of B cells
or monocytes between the two groups.
[0226] Additionally, quantitative insulin assays were performed on
of eight (8) homozygous mice (6 males and 2 females) and four (4)
wild-type mice (2 males and 2 females) using a Cobra II Series
Auto-Gamma Counting System to determine the insulin levels. There
was no significant difference in the insulin levels between the two
groups of mice.
[0227] 6.1.4 Urinalysis
[0228] Approximately 100 microliters of urine was collected by
placing the mouse in a clean cage lined with aluminum foil and
carefully watching the mouse for urination. Immediately following
urination, the sample was collected into a micro-collection tube.
The specimen was analyzed using a calibrated Chemstrip 101 Urine
Analyzer (Ames Diagnostics) urinalysis test strip. The urine was
placed on the test pad and was read as indicated by the
manufacturer according to the package insert. This urinalysis
determines urine osmolality, the presence of leukocytes, nitrite,
protein, glucose, ketones, urobilinogen, bilirubin and blood.
[0229] Urine samples were obtained from fifteen (15) mice; eight
(8) homozygous (6 males, 2 females), four (4) heterozygous (2 males
and 2 females), and three (4) wild-type (2 males and 1 female)
mice. Analysis and comparison of the urine samples revealed
abnormal protein levels (30 mg/dl) in 5 of the 8 homozygous mice
(compared to only 1 of the 4 heterozygous and 1 of the 3 wild-type
mice), and abnormal ketone levels in 6 of the 8 homozygous mice
(compared to only 1 of the 4 heterozygous and 1 of the 3 wild-type
mice). No differences between the groups in the other analytes were
detected.
[0230] 6.1.5 Opthalmology
[0231] Slit Lamp Analysis: The slit lamp is a biomicroscope that
allows examination of the anatomy of the anterior eye segment, as
well as the localization of some abnormalities. It is a rapid and
convenient method for preliminary eye examination prior to fundus
photography. Mouse eye analysis began with examination utilizing a
slit lamp (Nikon, Tokyo, Japan) in combination with a 60 or 90
diopter (D) condensing lens. In preparation for examination, mouse
pupils were dilated by adding a drop of 1% cyclopentolate and 1%
atropine (Alcon Laboratory Inc., Fort Worth, Tex.) to each eye.
[0232] Fundus Photography: Fundus photography is a noninvasive
method of examining the eye that is adaptable to high throughput
analysis. The appearance of the ocular fundus is representative of
overall health. Variation in the appearance of the ocular fundus
can be indicative of different diseases, including, but not limited
to, diabetes, obesity, cardiovascular disorders, angiogenesis,
oxidant related disorders and cancer. Selected animals were
subjected to fundus photography using a Kowa Genesis small animal
fundus digital camera (Tokyo, Japan) to photograph mouse fundi. The
instrument was used with a condensing lens, Volk 60D or 90D
(Mentor, Ohio, USA), mounted between the camera and the object to
be viewed (mouse eye). In order to avoid complications of
anesthesia, such as clouding of the ocular media, photographs were
obtained on conscious mice, whose vibrissae were trimmed with fine
scissors to prevent them from obscuring the photograph.
[0233] Retinal Angiography: Fluor escein angiography is an
established technique used to examine the circulation of the
retina. In particular it enables the progression of diabetic
retinopathy to be monitored, and provides valuable information on
the presence or absence of vascular lesions such as edema (leakage)
and ischemia (occlusion of the capillaries). The retinal
angiography procedure was similar to the procedure used for fundus
photography, except that the standard light was replaced with blue
light in combination with a barrier filter. To facilitate imaging,
mice were injected intraperitoneally with 25% sodium fluorescein
(Akorn Inc., Decator, Ill.) at a dose of 0.01 ml per 5-6 gm body
weight. For viewing, the eyepiece was fitted with the
manufacturer-supplied barrier filter. The digital imaging system
used consists of a camera, a computer, and Komit+software (Kowa,
Tokyo, Japan) especially designed for ophthalmological
applications, which facilitates image data acquisition, analysis
and storage.
[0234] The artery to vein ratio (A/V) is the ratio of the artery
diameter to the vein diameter (measured before the bifurcation of
the vessels). The A/V ratio is measured and calculated according to
fundus images. Many diseases will influence the ratio, i.e.,
diabetes, cardiovascular disorders, papilledema and optic
atrophy.
[0235] Ophthalmological analysis was performed on sixteen (16)
mice; eight (8) homozygous (6 males, 2 females), four (4)
heterozygous (2 males and 2 females), and four (4) wild-type (2
males and 2 females). The analysis revealed no significant
ophthalmological differences between mice with or without
functional NHP alleles, with the exception of an increased retinal
arteriolar light reflex (ALR) noted in 4/8 (-/-) mice compared to
the (+/+) mice, indicating abnormal thickening of the arteriolar
wall. Additionally, 1/8 (-/-) mice had a subcapsular cataract.
[0236] 6.1.6 Neurological and Behavioral Analysis
[0237] Functional Observational Battery (FOB): A subset of tests
from the Irwin neurological screen (Irwin, 1968,
Psychopharmacologia 13:222-257) were used to evaluate the gross
neurological function of the mice. This battery of simple
neurological tests took 10 minutes and was useful for detecting
severe neurological impairments.
[0238] Mice were first examined for their overall physical
characteristics, such as presence of whiskers, bald patches,
piloerection, exopthalmus, palpebral closure, kyphosis, lordosis,
and tail abnormalities. The mice were then placed into an empty
cage for one minute. Any abnormal spontaneous behaviors such as
wild-running, excessive grooming, freezing behavior, hunched body
posture when walking, etc., were recorded. Mice were next placed
into an empty cage to assess motor reflexes. The cage was quickly
moved side to side and up and down. The normal postural reflex is
to extend all four legs in order to maintain an upright, balanced
position. The righting reflex was measured by turning the mice on
their back and determining how long it took the mice to return to
an upright position. Normal mice will immediately right themselves.
If a mouse did not right itself within 60 seconds, the mouse was
returned to its normal upright position.
[0239] The eye blink reflex, ear twitch reflex, and flank reflex
were measured by lightly touching the eye, tip of the ear, and
torso once each with a thin clear piece of plastic. The
whisker-orienting response was measured by lightly touching the
whiskers with a thin clear piece of plastic while the animal was
allowed to move freely. The whiskers are typically moving. When
touched the whiskers of normal mice will stop moving and in many
cases the mouse will turn its head to the side where the whiskers
were touched. To determine a mouse's visual response to light, the
mouse was examined in a dimly lit room. Pupil constriction and
dilation reflexes were measured by quickly directing a penlight at
the mouse's eye and observing pupil constriction and subsequently
pupil dilatation once the light source was removed.
[0240] The visual reaching response was measured by suspending a
mouse by its tail and moving it down towards the edge of a cage. A
mouse that can see the cage will reach towards it when the cage is
moved in the direction of the mouse.
[0241] The tail suspension response was determined by holding the
mouse approximately six inches in the air by the tail for 20
seconds and recording normal behaviors such as grabbing of the
hindlimbs with the forelimbs and turning up on its sides. If
present, abnormal behaviors such as hindlimb and forelimb clutch
were also recorded.
[0242] The cateleptic response was measured by using a small rod at
a fixed vertical position. The mouse was positioned such that its
forelimbs were resting on the rod. Normal mice in this situation
will quickly remove their forelimbs from the rod. A 60 second time
limit was allowed, after which a non-responsive mouse was returned
to its normal posture.
[0243] The olfactory response was tested by using an odor such as
vanilla extract as an olfactory cue. A small amount of vanilla was
placed on cotton swab and held behind and to the side of a mouse.
If the mouse turns and orients itself to the position of the
vanilla extract-containing cotton swab, the result is interpreted
as an indication that the mouse can smell this olfactory cue.
[0244] Mouse body temperature was determined by gently inserting a
small probe into the rectum and recording the temperature with a
digital read-out (Physitemp, Clifton). This process took less than
5 sec and the mice appeared calm and unstressed throughout the
procedure.
[0245] This entire battery of simple neurological tests took about
10 minutes and provided for the detection of severe neurological
impairment. At the completion of these tests the mice were returned
to their home cage.
[0246] Hot Plate Assay for Nociception: Mice were removed from
their home cage and placed on a 55.0.degree. C. (+/-0.2.degree. C.)
hot plate, and the latency to the first hind limb response was
recorded. A Plexiglas enclosure was placed around the subject to
keep them from walking off of the plate. The hind paw response is a
foot shake, paw lick, or jump. The maximum time allowed for a hind
limb response to occur was 30 seconds, after which the mouse was
removed if a hind limb response had not occurred.
[0247] Open Field Assay for Anxiety Related Responses and
Locomotor/Exploratory Activity: Anxiety-related, locomotor, and
exploratory responses were measured in a clear Plexiglas (40
cm.times.40 cm.times.30 cm) open-field arena. A mouse was placed in
the center of the arena and allowed to explore for 20 minutes.
Overhead high-level lighting provides additional room lighting to
enhance anxiety-related behaviors. Activity in the open field was
quantified by a computer-controlled Vetsamax optical animal
activity system (Accuscan Instruments, Columbus, Ohio) containing
16 photoreceptor beams on each side of the arena, thereby dividing
the arena into 256 equally-sized squares. An additional set of
photobeams was placed above this set to record vertical activity,
and a set was placed below to record nose poke activity, thus
giving three levels of recordable activity. Total distance traveled
(locomotor activity), number of rearing and nose poke events
(exploratory activity), and center distance (i.e., the distance
traveled in the center of the arena) were recorded. The center
distance was divided by the total distance traveled to obtain a
center distance:total distance ratio. The center distance:total
distance ratio can be used as an index of anxiety-related
responses. Data was collected in four-minute intervals over the 20
minute test session.
[0248] Rotarod Assay for Motor Coordination: Motor coordination and
balance were tested using an accelerating rotarod (Accuscan
Instruments, Columbus, Ohio). The test was performed by placing a
mouse on a rotating drum (measuring 3 cm in diameter) and recording
the time each animal was able to stay on the rotating rod. Some
mice hold on to the rotating rod as they begin to fall and ride
completely around the rod. The speed of the rod accelerates from 0
to 40 rpm over the length of the 5 minute test. Mice were given
four consecutive trials with a maximum time of 300 seconds (5
min).
[0249] Prepulse Inhibition of the Acoustic Startle Response:
Prepulse inhibition of the acoustic startle response was measured
using the SR-Lab System (San Diego Instruments, San Diego, Calif.).
A test session began by placing a mouse in the Plexiglas cylinder
where it was left undisturbed for 3 min. A test session consists of
three trial types. One trial type was a 40 ms, 120 decibel (dB)
sound burst alone that is termed the startle stimulus. There were
four different acoustic prepulse plus startle stimulus trial types.
The prepulse sound is presented 100 ms before the startle stimulus.
The 20 ms prepulse sounds are at 74, 78, 82, and 90 dB. Finally,
there were trials where no stimulus is presented to measure
baseline movement in the cylinders. Six blocks of the six trial
types were presented in pseudorandom order, such that each trial
type was presented once within a block of seven trials. The average
intertrial interval was 15 sec with a range of 10 to 20 seconds.
The startle response is recorded for 65 ms (measuring the response
every 1 ms) starting at the onset of the startle stimulus. The
background noise level in each chamber is approximately 70 dB. The
maximum startle amplitude recorded during the 65 ms sampling window
(Vmax) was used.
[0250] The formula used to calculate % prepulse inhibition of a
startle response is:
[0251] 100-[(startle on acoustic prepulse+startle stimulus
trials/startle response alone trials) .times.100].
[0252] Sixteen (16) mice were analyzed; eight (8) homozygous (6
males, 2 females), four (4) heterozygous (2 males and 2 females),
and four (4) wild-type (2 males and 2 females) mice. There were no
notable differences in any of the parameters measured between the
groups.
[0253] 6.1.7 Radiology
[0254] One (1) male wild type (+/+) mouse and four (4) homozygous
(-/-) mice (2 males and 2 females) were subject to examination
using a mouse-size computer aided tomography (CT) scanning unit,
the MicroCAT.TM. (ImTek, Inc., Knoxville, Tenn.). The mice were
injected with a CT contrast agent, omnipaque 300 (Nycomed Amersham,
300 mg of iodine per ml, 0.25 ml per animal, or 2.50-3.75 g
iodine/kg body weight), intraperitoneally. After resting in the
cage for approximately 10 minutes, the mice were sedated by
intraperitoneal injection of Avertin (1.25% 2,2,2,-tribromoethanol,
20 ml/kg body weight). The CT-scan was done with the anesthetized
animal lying prone on the test bed. Three dimensional images were
reconstructed by the Feldkamp algorithm in a cluster of
workstations using ImTek 3DRECON software.
[0255] Significant depletion of fat depots in the abdominal and
subcutaneous regions was noted for the 2 female (-/-) mice. There
were no additional radiologic findings of significance that
differentiated the mice of any genotypic group. The following
observations were made for all groups of mice. There were no
abnormalities observed in the skull, spine, tail or individual
bones. The head, brain and neck appeared normal. The cervical lymph
nodes were not enlarged. The lung fields were clear. The hearts
were of normal size. The mediastinum and vessels revealed no
abnormalities. The liver, spleen and kidneys were normal in size,
shape and position. The rate of excretion of contrast media from
the kidneys was within normal limits, indicating normal kidney
function. The lymph nodes, and other abdominal organs, such as the
adrenals, ovaries and prostate were normal. No lesions were
observed in the soft tissues (skin, muscle or fat).
[0256] 6.1.8 Blood Pressure and Heart Rate Determination
[0257] In an additional study, blood pressure was determined in
eight (8) homozygous (6 males, 2 females) and four (4) wild-type (2
males and 2 females) mice. Mice were subjected to a conscious
systolic blood pressure protocol similar to that previously
described (Krege et al., 1995, Hypertension 25:1111-1115). Briefly,
mice were placed on a heated platform (37.degree. C.) with their
tails placed through a cuff and in a sensor to detect the systolic
blood pressure. The blood pressure was measured 20 times a day for
4 consecutive days--the first ten measurements are discarded to
allow the animals to acclimate, and then the next ten measurements
are recorded. There was no significant difference in the average
systolic blood pressure between the wild-type mice and the
homozygous mice.
[0258] Additionally, the heart rate was measured in eight (8)
homozygous (6 males, 2 females) and four (4) wild-type (2 males and
2 females) mice. There was no significant difference in the heart
rate between the wild-type mice and the homozygous mice.
[0259] 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 incorported by reference in their
entirety.
Sequence CWU 1
1
9 1 2220 DNA homo sapiens 1 atgctgtact ccccagggcc gagtcttccg
gagtcagcag agagcctgga tggatcacag 60 gaggataagc ctcggggctc
atgtgcggag cccactttta ctgatacggg aatggtggct 120 cacataaaca
acagccggct caaggccaag ggcgtgggcc agcacgacaa cgcccagaac 180
tttggtaacc agagctttga ggagctgcga gcagcctgtc taagaaaggg ggagctcttc
240 gaggacccct tattccctgc tgaacccagc tcactgggct tcaaggacct
gggccccaac 300 tccaaaaatg tgcagaacat ctcctggcag cggcccaagg
atatcataaa caaccctcta 360 ttcatcatgg atgggatttc tccaacagac
atctgccagg ggatcctcgg ggactgctgg 420 ctgctggctg ccatcggctc
ccttaccacc tgccccaaac tgctataccg cgtggtgccc 480 agaggacaga
gcttcaagaa aaactatgct ggcatcttcc attttcagat ttggcagttt 540
ggacagtggg tgaacgtggt ggtagatgac cggctgccca caaagaatga caagctggtg
600 tttgtgcact caaccgaacg cagtgagttc tggagtgccc tgctggagaa
ggcgtatgcc 660 aagctgagtg ggtcctatga agcattgtca gggggcagta
ccatggaggg ccttgaggac 720 ttcacaggag gcgtggccca gagcttccaa
ctccagaggc cccctcagaa cctgctcagg 780 ctccttagga aggccgtgga
gcgatcctcc ctcatgggtt gctccattga agtcaccagt 840 gatagtgaac
tggaatccat gactgacaag atgctggtga gagggcacgc ttactctgtg 900
actggccttc aggatgtcca ctacagaggc aaaatggaaa cactgattcg ggtccggaat
960 ccctggggcc ggattgagtg gaatggagct tggagtgaca gtgccaggga
gtgggaagag 1020 gtggcctcag acatccagat gcagctgctg cacaagacgg
aggacgggga gttctggatg 1080 tcctaccaag atttcctgaa caacttcacg
ctcctggaga tctgcaacct cacgcctgat 1140 acactctctg gggactacaa
gagctactgg cacaccacct tctacgaggg cagctggcgc 1200 agaggcagct
ccgcaggggg ctgcaggaac caccctggca cgttctggac caacccccag 1260
tttaagatct ctcttcctga gggggatgac ccagaggatg acgcagaggg caatgttgtg
1320 gtctgcacct gcctggtggc cctaatgcag aagaactggc ggcatgcacg
gcagcaggga 1380 gcccagctgc agaccattgg ctttgtcctc tacgcggtcc
caaaagagtt tcagaacatt 1440 caggatgtcc acttgaagaa ggaattcttc
acgaagtatc aggaccacgg cttctcagag 1500 atcttcacca actcacggga
ggtgagcagc caactccggc tgcctccggg ggaatatatc 1560 attattccct
ccacctttga gccacacaga gatgctgact tcctgcttcg ggtcttcacc 1620
gagaagcaca gcgagtcatg ggaattggat gaagtcaact atgctgagca actccaagag
1680 gaaaaggtct ctgaggatga catggaccag gacttcctac atttgtttaa
gatagtggca 1740 ggagagggca aggagatagg ggtgtatgag ctccagaggc
tgctcaacag gatggccatc 1800 aaattcaaaa gcttcaagac caagggcttt
ggcctggatg cttgccgctg catgatcaac 1860 ctcatggata aagatggctc
tggcaagctg gggcttctag agttcaagat cctgtggaaa 1920 aaactcaaga
aatggatgga catcttcaga gagtgtgacc aggaccattc aggcaccttg 1980
aactcctatg agatgcgcct ggttattgag aaagcaggca tcaagctgaa caacaaggta
2040 atgcaggtcc tggtggccag gtatgcagat gatgacctga tcatagactt
tgacagcttc 2100 atcagctgtt tcctgaggct aaagaccatg ttcacattct
ttctaaccat ggaccccaag 2160 aatactggcc atatttgctt gagcctggaa
cagtggctgc agatgaccat gtggggatag 2220 2 739 PRT homo sapiens 2 Met
Leu Tyr Ser Pro Gly Pro Ser Leu Pro Glu Ser Ala Glu Ser Leu 1 5 10
15 Asp Gly Ser Gln Glu Asp Lys Pro Arg Gly Ser Cys Ala Glu Pro Thr
20 25 30 Phe Thr Asp Thr Gly Met Val Ala His Ile Asn Asn Ser Arg
Leu Lys 35 40 45 Ala Lys Gly Val Gly Gln His Asp Asn Ala Gln Asn
Phe Gly Asn Gln 50 55 60 Ser Phe Glu Glu Leu Arg Ala Ala Cys Leu
Arg Lys Gly Glu Leu Phe 65 70 75 80 Glu Asp Pro Leu Phe Pro Ala Glu
Pro Ser Ser Leu Gly Phe Lys Asp 85 90 95 Leu Gly Pro Asn Ser Lys
Asn Val Gln Asn Ile Ser Trp Gln Arg Pro 100 105 110 Lys Asp Ile Ile
Asn Asn Pro Leu Phe Ile Met Asp Gly Ile Ser Pro 115 120 125 Thr Asp
Ile Cys Gln Gly Ile Leu Gly Asp Cys Trp Leu Leu Ala Ala 130 135 140
Ile Gly Ser Leu Thr Thr Cys Pro Lys Leu Leu Tyr Arg Val Val Pro 145
150 155 160 Arg Gly Gln Ser Phe Lys Lys Asn Tyr Ala Gly Ile Phe His
Phe Gln 165 170 175 Ile Trp Gln Phe Gly Gln Trp Val Asn Val Val Val
Asp Asp Arg Leu 180 185 190 Pro Thr Lys Asn Asp Lys Leu Val Phe Val
His Ser Thr Glu Arg Ser 195 200 205 Glu Phe Trp Ser Ala Leu Leu Glu
Lys Ala Tyr Ala Lys Leu Ser Gly 210 215 220 Ser Tyr Glu Ala Leu Ser
Gly Gly Ser Thr Met Glu Gly Leu Glu Asp 225 230 235 240 Phe Thr Gly
Gly Val Ala Gln Ser Phe Gln Leu Gln Arg Pro Pro Gln 245 250 255 Asn
Leu Leu Arg Leu Leu Arg Lys Ala Val Glu Arg Ser Ser Leu Met 260 265
270 Gly Cys Ser Ile Glu Val Thr Ser Asp Ser Glu Leu Glu Ser Met Thr
275 280 285 Asp Lys Met Leu Val Arg Gly His Ala Tyr Ser Val Thr Gly
Leu Gln 290 295 300 Asp Val His Tyr Arg Gly Lys Met Glu Thr Leu Ile
Arg Val Arg Asn 305 310 315 320 Pro Trp Gly Arg Ile Glu Trp Asn Gly
Ala Trp Ser Asp Ser Ala Arg 325 330 335 Glu Trp Glu Glu Val Ala Ser
Asp Ile Gln Met Gln Leu Leu His Lys 340 345 350 Thr Glu Asp Gly Glu
Phe Trp Met Ser Tyr Gln Asp Phe Leu Asn Asn 355 360 365 Phe Thr Leu
Leu Glu Ile Cys Asn Leu Thr Pro Asp Thr Leu Ser Gly 370 375 380 Asp
Tyr Lys Ser Tyr Trp His Thr Thr Phe Tyr Glu Gly Ser Trp Arg 385 390
395 400 Arg Gly Ser Ser Ala Gly Gly Cys Arg Asn His Pro Gly Thr Phe
Trp 405 410 415 Thr Asn Pro Gln Phe Lys Ile Ser Leu Pro Glu Gly Asp
Asp Pro Glu 420 425 430 Asp Asp Ala Glu Gly Asn Val Val Val Cys Thr
Cys Leu Val Ala Leu 435 440 445 Met Gln Lys Asn Trp Arg His Ala Arg
Gln Gln Gly Ala Gln Leu Gln 450 455 460 Thr Ile Gly Phe Val Leu Tyr
Ala Val Pro Lys Glu Phe Gln Asn Ile 465 470 475 480 Gln Asp Val His
Leu Lys Lys Glu Phe Phe Thr Lys Tyr Gln Asp His 485 490 495 Gly Phe
Ser Glu Ile Phe Thr Asn Ser Arg Glu Val Ser Ser Gln Leu 500 505 510
Arg Leu Pro Pro Gly Glu Tyr Ile Ile Ile Pro Ser Thr Phe Glu Pro 515
520 525 His Arg Asp Ala Asp Phe Leu Leu Arg Val Phe Thr Glu Lys His
Ser 530 535 540 Glu Ser Trp Glu Leu Asp Glu Val Asn Tyr Ala Glu Gln
Leu Gln Glu 545 550 555 560 Glu Lys Val Ser Glu Asp Asp Met Asp Gln
Asp Phe Leu His Leu Phe 565 570 575 Lys Ile Val Ala Gly Glu Gly Lys
Glu Ile Gly Val Tyr Glu Leu Gln 580 585 590 Arg Leu Leu Asn Arg Met
Ala Ile Lys Phe Lys Ser Phe Lys Thr Lys 595 600 605 Gly Phe Gly Leu
Asp Ala Cys Arg Cys Met Ile Asn Leu Met Asp Lys 610 615 620 Asp Gly
Ser Gly Lys Leu Gly Leu Leu Glu Phe Lys Ile Leu Trp Lys 625 630 635
640 Lys Leu Lys Lys Trp Met Asp Ile Phe Arg Glu Cys Asp Gln Asp His
645 650 655 Ser Gly Thr Leu Asn Ser Tyr Glu Met Arg Leu Val Ile Glu
Lys Ala 660 665 670 Gly Ile Lys Leu Asn Asn Lys Val Met Gln Val Leu
Val Ala Arg Tyr 675 680 685 Ala Asp Asp Asp Leu Ile Ile Asp Phe Asp
Ser Phe Ile Ser Cys Phe 690 695 700 Leu Arg Leu Lys Thr Met Phe Thr
Phe Phe Leu Thr Met Asp Pro Lys 705 710 715 720 Asn Thr Gly His Ile
Cys Leu Ser Leu Glu Gln Trp Leu Gln Met Thr 725 730 735 Met Trp Gly
3 2172 DNA homo sapiens 3 atgctgtact ccccagggcc gagtcttccg
gagtcagcag agagcctgga tggatcacag 60 gaggataagc ctcggggctc
atgtgcggag cccactttta ctgatacggg aatggtggct 120 cacataaaca
acagccggct caaggccaag ggcgtgggcc agcacgacaa cgcccagaac 180
tttggtaacc agagctttga ggagctgcga gcagcctgtc taagaaaggg ggagctcttc
240 gaggacccct tattccctgc tgaacccagc tcactgggct tcaaggacct
gggccccaac 300 tccaaaaatg tgcagaacat ctcctggcag cggcccaagg
atatcataaa caaccctcta 360 ttcatcatgg atgggatttc tccaacagac
atctgccagg ggatcctcgg ggactgctgg 420 ctgctggctg ccatcggctc
ccttaccacc tgccccaaac tgctataccg cgtggtgccc 480 agaggacaga
gcttcaagaa aaactatgct ggcatcttcc attttcagat ttggcagttt 540
ggacagtggg tgaacgtggt ggtagatgac cggctgccca caaagaatga caagctggtg
600 tttgtgcact caaccgaacg cagtgagttc tggagtgccc tgctggagaa
ggcgtatgcc 660 aagctgagtg ggtcctatga agcattgtca gggggcagta
ccatggaggg ccttgaggac 720 ttcacaggag gcgtggccca gagcttccaa
ctccagaggc cccctcagaa cctgctcagg 780 ctccttagga aggccgtgga
gcgatcctcc ctcatgggtt gctccattga agtcaccagt 840 gatagtgaac
tggaatccat gactgacaag atgctggtga gagggcacgc ttactctgtg 900
actggccttc aggatgtcca ctacagaggc aaaatggaaa cactgattcg ggtccggaat
960 ccctggggcc ggattgagtg gaatggagct tggagtgaca gtgccaggga
gtgggaagag 1020 gtggcctcag acatccagat gcagctgctg cacaagacgg
aggacgggga gttctggatg 1080 tcctaccaag atttcctgaa caacttcacg
ctcctggaga tctgcaacct cacgcctgat 1140 acactctctg gggactacaa
gagctactgg cacaccacct tctacgaggg cagctggcgc 1200 agaggcagct
ccgcaggggg ctgcaggaac caccctggca cgttctggac caacccccag 1260
tttaagatct ctcttcctga gggggatgac ccagaggatg acgcagaggg caatgttgtg
1320 gtctgcacct gcctggtggc cctaatgcag aagaactggc ggcatgcacg
gcagcaggga 1380 gcccagctgc agaccattgg ctttgtcctc tacgcggtcc
caaaagagtt tcagaacatt 1440 caggatgtcc acttgaagaa ggaattcttc
acgaagtatc aggaccacgg cttctcagag 1500 atcttcacca actcacggga
ggtgagcagc caactccggc tgcctccggg ggaatatatc 1560 attattccct
ccacctttga gccacacaga gatgctgact tcctgcttcg ggtcttcacc 1620
gagaagcaca gcgagtcatg ggaattggat gaagtcaact atgctgagca actccaagag
1680 gaaaaggtct ctgaggatga catggaccag gacttcctac atttgtttaa
gatagtggca 1740 ggagagggca aggagatagg ggtgtatgag ctccagaggc
tgctcaacag gatggccatc 1800 aaattcaaaa gcttcaagac caagggcttt
ggcctggatg cttgccgctg catgatcaac 1860 ctcatggata aagatggctc
tggcaagctg gggcttctag agttcaagat cctgtggaaa 1920 aaactcaaga
aatggatgga catcttcaga gagtgtgacc aggaccattc aggcaccttg 1980
aactcctatg agatgcgcct ggttattgag aaagcaggca tcaagctgaa caacaaggta
2040 atgcaggtcc tggtggccag gtatgcagat gatgacctga tcatagactt
tgacagcttc 2100 atcagctgtt tcctgaggct aaagaccatg ttcatggctg
cagatgacca tgtggggata 2160 gaggcgctgt ag 2172 4 723 PRT homo
sapiens 4 Met Leu Tyr Ser Pro Gly Pro Ser Leu Pro Glu Ser Ala Glu
Ser Leu 1 5 10 15 Asp Gly Ser Gln Glu Asp Lys Pro Arg Gly Ser Cys
Ala Glu Pro Thr 20 25 30 Phe Thr Asp Thr Gly Met Val Ala His Ile
Asn Asn Ser Arg Leu Lys 35 40 45 Ala Lys Gly Val Gly Gln His Asp
Asn Ala Gln Asn Phe Gly Asn Gln 50 55 60 Ser Phe Glu Glu Leu Arg
Ala Ala Cys Leu Arg Lys Gly Glu Leu Phe 65 70 75 80 Glu Asp Pro Leu
Phe Pro Ala Glu Pro Ser Ser Leu Gly Phe Lys Asp 85 90 95 Leu Gly
Pro Asn Ser Lys Asn Val Gln Asn Ile Ser Trp Gln Arg Pro 100 105 110
Lys Asp Ile Ile Asn Asn Pro Leu Phe Ile Met Asp Gly Ile Ser Pro 115
120 125 Thr Asp Ile Cys Gln Gly Ile Leu Gly Asp Cys Trp Leu Leu Ala
Ala 130 135 140 Ile Gly Ser Leu Thr Thr Cys Pro Lys Leu Leu Tyr Arg
Val Val Pro 145 150 155 160 Arg Gly Gln Ser Phe Lys Lys Asn Tyr Ala
Gly Ile Phe His Phe Gln 165 170 175 Ile Trp Gln Phe Gly Gln Trp Val
Asn Val Val Val Asp Asp Arg Leu 180 185 190 Pro Thr Lys Asn Asp Lys
Leu Val Phe Val His Ser Thr Glu Arg Ser 195 200 205 Glu Phe Trp Ser
Ala Leu Leu Glu Lys Ala Tyr Ala Lys Leu Ser Gly 210 215 220 Ser Tyr
Glu Ala Leu Ser Gly Gly Ser Thr Met Glu Gly Leu Glu Asp 225 230 235
240 Phe Thr Gly Gly Val Ala Gln Ser Phe Gln Leu Gln Arg Pro Pro Gln
245 250 255 Asn Leu Leu Arg Leu Leu Arg Lys Ala Val Glu Arg Ser Ser
Leu Met 260 265 270 Gly Cys Ser Ile Glu Val Thr Ser Asp Ser Glu Leu
Glu Ser Met Thr 275 280 285 Asp Lys Met Leu Val Arg Gly His Ala Tyr
Ser Val Thr Gly Leu Gln 290 295 300 Asp Val His Tyr Arg Gly Lys Met
Glu Thr Leu Ile Arg Val Arg Asn 305 310 315 320 Pro Trp Gly Arg Ile
Glu Trp Asn Gly Ala Trp Ser Asp Ser Ala Arg 325 330 335 Glu Trp Glu
Glu Val Ala Ser Asp Ile Gln Met Gln Leu Leu His Lys 340 345 350 Thr
Glu Asp Gly Glu Phe Trp Met Ser Tyr Gln Asp Phe Leu Asn Asn 355 360
365 Phe Thr Leu Leu Glu Ile Cys Asn Leu Thr Pro Asp Thr Leu Ser Gly
370 375 380 Asp Tyr Lys Ser Tyr Trp His Thr Thr Phe Tyr Glu Gly Ser
Trp Arg 385 390 395 400 Arg Gly Ser Ser Ala Gly Gly Cys Arg Asn His
Pro Gly Thr Phe Trp 405 410 415 Thr Asn Pro Gln Phe Lys Ile Ser Leu
Pro Glu Gly Asp Asp Pro Glu 420 425 430 Asp Asp Ala Glu Gly Asn Val
Val Val Cys Thr Cys Leu Val Ala Leu 435 440 445 Met Gln Lys Asn Trp
Arg His Ala Arg Gln Gln Gly Ala Gln Leu Gln 450 455 460 Thr Ile Gly
Phe Val Leu Tyr Ala Val Pro Lys Glu Phe Gln Asn Ile 465 470 475 480
Gln Asp Val His Leu Lys Lys Glu Phe Phe Thr Lys Tyr Gln Asp His 485
490 495 Gly Phe Ser Glu Ile Phe Thr Asn Ser Arg Glu Val Ser Ser Gln
Leu 500 505 510 Arg Leu Pro Pro Gly Glu Tyr Ile Ile Ile Pro Ser Thr
Phe Glu Pro 515 520 525 His Arg Asp Ala Asp Phe Leu Leu Arg Val Phe
Thr Glu Lys His Ser 530 535 540 Glu Ser Trp Glu Leu Asp Glu Val Asn
Tyr Ala Glu Gln Leu Gln Glu 545 550 555 560 Glu Lys Val Ser Glu Asp
Asp Met Asp Gln Asp Phe Leu His Leu Phe 565 570 575 Lys Ile Val Ala
Gly Glu Gly Lys Glu Ile Gly Val Tyr Glu Leu Gln 580 585 590 Arg Leu
Leu Asn Arg Met Ala Ile Lys Phe Lys Ser Phe Lys Thr Lys 595 600 605
Gly Phe Gly Leu Asp Ala Cys Arg Cys Met Ile Asn Leu Met Asp Lys 610
615 620 Asp Gly Ser Gly Lys Leu Gly Leu Leu Glu Phe Lys Ile Leu Trp
Lys 625 630 635 640 Lys Leu Lys Lys Trp Met Asp Ile Phe Arg Glu Cys
Asp Gln Asp His 645 650 655 Ser Gly Thr Leu Asn Ser Tyr Glu Met Arg
Leu Val Ile Glu Lys Ala 660 665 670 Gly Ile Lys Leu Asn Asn Lys Val
Met Gln Val Leu Val Ala Arg Tyr 675 680 685 Ala Asp Asp Asp Leu Ile
Ile Asp Phe Asp Ser Phe Ile Ser Cys Phe 690 695 700 Leu Arg Leu Lys
Thr Met Phe Met Ala Ala Asp Asp His Val Gly Ile 705 710 715 720 Glu
Ala Leu 5 2109 DNA homo sapiens 5 atggtggctc acataaacaa cagccggctc
aaggccaagg gcgtgggcca gcacgacaac 60 gcccagaact ttggtaacca
gagctttgag gagctgcgag cagcctgtct aagaaagggg 120 gagctcttcg
aggacccctt attccctgct gaacccagct cactgggctt caaggacctg 180
ggccccaact ccaaaaatgt gcagaacatc tcctggcagc ggcccaagga tatcataaac
240 aaccctctat tcatcatgga tgggatttct ccaacagaca tctgccaggg
gatcctcggg 300 gactgctggc tgctggctgc catcggctcc cttaccacct
gccccaaact gctataccgc 360 gtggtgccca gaggacagag cttcaagaaa
aactatgctg gcatcttcca ttttcagatt 420 tggcagtttg gacagtgggt
gaacgtggtg gtagatgacc ggctgcccac aaagaatgac 480 aagctggtgt
ttgtgcactc aaccgaacgc agtgagttct ggagtgccct gctggagaag 540
gcgtatgcca agctgagtgg gtcctatgaa gcattgtcag ggggcagtac catggagggc
600 cttgaggact tcacaggagg cgtggcccag agcttccaac tccagaggcc
ccctcagaac 660 ctgctcaggc tccttaggaa ggccgtggag cgatcctccc
tcatgggttg ctccattgaa 720 gtcaccagtg atagtgaact ggaatccatg
actgacaaga tgctggtgag agggcacgct 780 tactctgtga ctggccttca
ggatgtccac tacagaggca aaatggaaac actgattcgg 840 gtccggaatc
cctggggccg gattgagtgg aatggagctt ggagtgacag tgccagggag 900
tgggaagagg tggcctcaga catccagatg cagctgctgc acaagacgga ggacggggag
960 ttctggatgt cctaccaaga tttcctgaac aacttcacgc tcctggagat
ctgcaacctc 1020 acgcctgata cactctctgg ggactacaag agctactggc
acaccacctt ctacgagggc 1080 agctggcgca gaggcagctc cgcagggggc
tgcaggaacc accctggcac gttctggacc 1140 aacccccagt ttaagatctc
tcttcctgag ggggatgacc cagaggatga cgcagagggc 1200 aatgttgtgg
tctgcacctg cctggtggcc ctaatgcaga agaactggcg gcatgcacgg 1260
cagcagggag cccagctgca gaccattggc tttgtcctct acgcggtccc aaaagagttt
1320 cagaacattc aggatgtcca cttgaagaag gaattcttca cgaagtatca
ggaccacggc 1380 ttctcagaga tcttcaccaa ctcacgggag gtgagcagcc
aactccggct gcctccgggg 1440 gaatatatca ttattccctc cacctttgag
ccacacagag atgctgactt cctgcttcgg 1500 gtcttcaccg agaagcacag
cgagtcatgg gaattggatg aagtcaacta
tgctgagcaa 1560 ctccaagagg aaaaggtctc tgaggatgac atggaccagg
acttcctaca tttgtttaag 1620 atagtggcag gagagggcaa ggagataggg
gtgtatgagc tccagaggct gctcaacagg 1680 atggccatca aattcaaaag
cttcaagacc aagggctttg gcctggatgc ttgccgctgc 1740 atgatcaacc
tcatggataa agatggctct ggcaagctgg ggcttctaga gttcaagatc 1800
ctgtggaaaa aactcaagaa atggatggac atcttcagag agtgtgacca ggaccattca
1860 ggcaccttga actcctatga gatgcgcctg gttattgaga aagcaggcat
caagctgaac 1920 aacaaggtaa tgcaggtcct ggtggccagg tatgcagatg
atgacctgat catagacttt 1980 gacagcttca tcagctgttt cctgaggcta
aagaccatgt tcacattctt tctaaccatg 2040 gaccccaaga atactggcca
tatttgcttg agcctggaac agtggctgca gatgaccatg 2100 tggggatag 2109 6
702 PRT homo sapiens 6 Met Val Ala His Ile Asn Asn Ser Arg Leu Lys
Ala Lys Gly Val Gly 1 5 10 15 Gln His Asp Asn Ala Gln Asn Phe Gly
Asn Gln Ser Phe Glu Glu Leu 20 25 30 Arg Ala Ala Cys Leu Arg Lys
Gly Glu Leu Phe Glu Asp Pro Leu Phe 35 40 45 Pro Ala Glu Pro Ser
Ser Leu Gly Phe Lys Asp Leu Gly Pro Asn Ser 50 55 60 Lys Asn Val
Gln Asn Ile Ser Trp Gln Arg Pro Lys Asp Ile Ile Asn 65 70 75 80 Asn
Pro Leu Phe Ile Met Asp Gly Ile Ser Pro Thr Asp Ile Cys Gln 85 90
95 Gly Ile Leu Gly Asp Cys Trp Leu Leu Ala Ala Ile Gly Ser Leu Thr
100 105 110 Thr Cys Pro Lys Leu Leu Tyr Arg Val Val Pro Arg Gly Gln
Ser Phe 115 120 125 Lys Lys Asn Tyr Ala Gly Ile Phe His Phe Gln Ile
Trp Gln Phe Gly 130 135 140 Gln Trp Val Asn Val Val Val Asp Asp Arg
Leu Pro Thr Lys Asn Asp 145 150 155 160 Lys Leu Val Phe Val His Ser
Thr Glu Arg Ser Glu Phe Trp Ser Ala 165 170 175 Leu Leu Glu Lys Ala
Tyr Ala Lys Leu Ser Gly Ser Tyr Glu Ala Leu 180 185 190 Ser Gly Gly
Ser Thr Met Glu Gly Leu Glu Asp Phe Thr Gly Gly Val 195 200 205 Ala
Gln Ser Phe Gln Leu Gln Arg Pro Pro Gln Asn Leu Leu Arg Leu 210 215
220 Leu Arg Lys Ala Val Glu Arg Ser Ser Leu Met Gly Cys Ser Ile Glu
225 230 235 240 Val Thr Ser Asp Ser Glu Leu Glu Ser Met Thr Asp Lys
Met Leu Val 245 250 255 Arg Gly His Ala Tyr Ser Val Thr Gly Leu Gln
Asp Val His Tyr Arg 260 265 270 Gly Lys Met Glu Thr Leu Ile Arg Val
Arg Asn Pro Trp Gly Arg Ile 275 280 285 Glu Trp Asn Gly Ala Trp Ser
Asp Ser Ala Arg Glu Trp Glu Glu Val 290 295 300 Ala Ser Asp Ile Gln
Met Gln Leu Leu His Lys Thr Glu Asp Gly Glu 305 310 315 320 Phe Trp
Met Ser Tyr Gln Asp Phe Leu Asn Asn Phe Thr Leu Leu Glu 325 330 335
Ile Cys Asn Leu Thr Pro Asp Thr Leu Ser Gly Asp Tyr Lys Ser Tyr 340
345 350 Trp His Thr Thr Phe Tyr Glu Gly Ser Trp Arg Arg Gly Ser Ser
Ala 355 360 365 Gly Gly Cys Arg Asn His Pro Gly Thr Phe Trp Thr Asn
Pro Gln Phe 370 375 380 Lys Ile Ser Leu Pro Glu Gly Asp Asp Pro Glu
Asp Asp Ala Glu Gly 385 390 395 400 Asn Val Val Val Cys Thr Cys Leu
Val Ala Leu Met Gln Lys Asn Trp 405 410 415 Arg His Ala Arg Gln Gln
Gly Ala Gln Leu Gln Thr Ile Gly Phe Val 420 425 430 Leu Tyr Ala Val
Pro Lys Glu Phe Gln Asn Ile Gln Asp Val His Leu 435 440 445 Lys Lys
Glu Phe Phe Thr Lys Tyr Gln Asp His Gly Phe Ser Glu Ile 450 455 460
Phe Thr Asn Ser Arg Glu Val Ser Ser Gln Leu Arg Leu Pro Pro Gly 465
470 475 480 Glu Tyr Ile Ile Ile Pro Ser Thr Phe Glu Pro His Arg Asp
Ala Asp 485 490 495 Phe Leu Leu Arg Val Phe Thr Glu Lys His Ser Glu
Ser Trp Glu Leu 500 505 510 Asp Glu Val Asn Tyr Ala Glu Gln Leu Gln
Glu Glu Lys Val Ser Glu 515 520 525 Asp Asp Met Asp Gln Asp Phe Leu
His Leu Phe Lys Ile Val Ala Gly 530 535 540 Glu Gly Lys Glu Ile Gly
Val Tyr Glu Leu Gln Arg Leu Leu Asn Arg 545 550 555 560 Met Ala Ile
Lys Phe Lys Ser Phe Lys Thr Lys Gly Phe Gly Leu Asp 565 570 575 Ala
Cys Arg Cys Met Ile Asn Leu Met Asp Lys Asp Gly Ser Gly Lys 580 585
590 Leu Gly Leu Leu Glu Phe Lys Ile Leu Trp Lys Lys Leu Lys Lys Trp
595 600 605 Met Asp Ile Phe Arg Glu Cys Asp Gln Asp His Ser Gly Thr
Leu Asn 610 615 620 Ser Tyr Glu Met Arg Leu Val Ile Glu Lys Ala Gly
Ile Lys Leu Asn 625 630 635 640 Asn Lys Val Met Gln Val Leu Val Ala
Arg Tyr Ala Asp Asp Asp Leu 645 650 655 Ile Ile Asp Phe Asp Ser Phe
Ile Ser Cys Phe Leu Arg Leu Lys Thr 660 665 670 Met Phe Thr Phe Phe
Leu Thr Met Asp Pro Lys Asn Thr Gly His Ile 675 680 685 Cys Leu Ser
Leu Glu Gln Trp Leu Gln Met Thr Met Trp Gly 690 695 700 7 2061 DNA
homo sapiens 7 atggtggctc acataaacaa cagccggctc aaggccaagg
gcgtgggcca gcacgacaac 60 gcccagaact ttggtaacca gagctttgag
gagctgcgag cagcctgtct aagaaagggg 120 gagctcttcg aggacccctt
attccctgct gaacccagct cactgggctt caaggacctg 180 ggccccaact
ccaaaaatgt gcagaacatc tcctggcagc ggcccaagga tatcataaac 240
aaccctctat tcatcatgga tgggatttct ccaacagaca tctgccaggg gatcctcggg
300 gactgctggc tgctggctgc catcggctcc cttaccacct gccccaaact
gctataccgc 360 gtggtgccca gaggacagag cttcaagaaa aactatgctg
gcatcttcca ttttcagatt 420 tggcagtttg gacagtgggt gaacgtggtg
gtagatgacc ggctgcccac aaagaatgac 480 aagctggtgt ttgtgcactc
aaccgaacgc agtgagttct ggagtgccct gctggagaag 540 gcgtatgcca
agctgagtgg gtcctatgaa gcattgtcag ggggcagtac catggagggc 600
cttgaggact tcacaggagg cgtggcccag agcttccaac tccagaggcc ccctcagaac
660 ctgctcaggc tccttaggaa ggccgtggag cgatcctccc tcatgggttg
ctccattgaa 720 gtcaccagtg atagtgaact ggaatccatg actgacaaga
tgctggtgag agggcacgct 780 tactctgtga ctggccttca ggatgtccac
tacagaggca aaatggaaac actgattcgg 840 gtccggaatc cctggggccg
gattgagtgg aatggagctt ggagtgacag tgccagggag 900 tgggaagagg
tggcctcaga catccagatg cagctgctgc acaagacgga ggacggggag 960
ttctggatgt cctaccaaga tttcctgaac aacttcacgc tcctggagat ctgcaacctc
1020 acgcctgata cactctctgg ggactacaag agctactggc acaccacctt
ctacgagggc 1080 agctggcgca gaggcagctc cgcagggggc tgcaggaacc
accctggcac gttctggacc 1140 aacccccagt ttaagatctc tcttcctgag
ggggatgacc cagaggatga cgcagagggc 1200 aatgttgtgg tctgcacctg
cctggtggcc ctaatgcaga agaactggcg gcatgcacgg 1260 cagcagggag
cccagctgca gaccattggc tttgtcctct acgcggtccc aaaagagttt 1320
cagaacattc aggatgtcca cttgaagaag gaattcttca cgaagtatca ggaccacggc
1380 ttctcagaga tcttcaccaa ctcacgggag gtgagcagcc aactccggct
gcctccgggg 1440 gaatatatca ttattccctc cacctttgag ccacacagag
atgctgactt cctgcttcgg 1500 gtcttcaccg agaagcacag cgagtcatgg
gaattggatg aagtcaacta tgctgagcaa 1560 ctccaagagg aaaaggtctc
tgaggatgac atggaccagg acttcctaca tttgtttaag 1620 atagtggcag
gagagggcaa ggagataggg gtgtatgagc tccagaggct gctcaacagg 1680
atggccatca aattcaaaag cttcaagacc aagggctttg gcctggatgc ttgccgctgc
1740 atgatcaacc tcatggataa agatggctct ggcaagctgg ggcttctaga
gttcaagatc 1800 ctgtggaaaa aactcaagaa atggatggac atcttcagag
agtgtgacca ggaccattca 1860 ggcaccttga actcctatga gatgcgcctg
gttattgaga aagcaggcat caagctgaac 1920 aacaaggtaa tgcaggtcct
ggtggccagg tatgcagatg atgacctgat catagacttt 1980 gacagcttca
tcagctgttt cctgaggcta aagaccatgt tcatggctgc agatgaccat 2040
gtggggatag aggcgctgta g 2061 8 686 PRT homo sapiens 8 Met Val Ala
His Ile Asn Asn Ser Arg Leu Lys Ala Lys Gly Val Gly 1 5 10 15 Gln
His Asp Asn Ala Gln Asn Phe Gly Asn Gln Ser Phe Glu Glu Leu 20 25
30 Arg Ala Ala Cys Leu Arg Lys Gly Glu Leu Phe Glu Asp Pro Leu Phe
35 40 45 Pro Ala Glu Pro Ser Ser Leu Gly Phe Lys Asp Leu Gly Pro
Asn Ser 50 55 60 Lys Asn Val Gln Asn Ile Ser Trp Gln Arg Pro Lys
Asp Ile Ile Asn 65 70 75 80 Asn Pro Leu Phe Ile Met Asp Gly Ile Ser
Pro Thr Asp Ile Cys Gln 85 90 95 Gly Ile Leu Gly Asp Cys Trp Leu
Leu Ala Ala Ile Gly Ser Leu Thr 100 105 110 Thr Cys Pro Lys Leu Leu
Tyr Arg Val Val Pro Arg Gly Gln Ser Phe 115 120 125 Lys Lys Asn Tyr
Ala Gly Ile Phe His Phe Gln Ile Trp Gln Phe Gly 130 135 140 Gln Trp
Val Asn Val Val Val Asp Asp Arg Leu Pro Thr Lys Asn Asp 145 150 155
160 Lys Leu Val Phe Val His Ser Thr Glu Arg Ser Glu Phe Trp Ser Ala
165 170 175 Leu Leu Glu Lys Ala Tyr Ala Lys Leu Ser Gly Ser Tyr Glu
Ala Leu 180 185 190 Ser Gly Gly Ser Thr Met Glu Gly Leu Glu Asp Phe
Thr Gly Gly Val 195 200 205 Ala Gln Ser Phe Gln Leu Gln Arg Pro Pro
Gln Asn Leu Leu Arg Leu 210 215 220 Leu Arg Lys Ala Val Glu Arg Ser
Ser Leu Met Gly Cys Ser Ile Glu 225 230 235 240 Val Thr Ser Asp Ser
Glu Leu Glu Ser Met Thr Asp Lys Met Leu Val 245 250 255 Arg Gly His
Ala Tyr Ser Val Thr Gly Leu Gln Asp Val His Tyr Arg 260 265 270 Gly
Lys Met Glu Thr Leu Ile Arg Val Arg Asn Pro Trp Gly Arg Ile 275 280
285 Glu Trp Asn Gly Ala Trp Ser Asp Ser Ala Arg Glu Trp Glu Glu Val
290 295 300 Ala Ser Asp Ile Gln Met Gln Leu Leu His Lys Thr Glu Asp
Gly Glu 305 310 315 320 Phe Trp Met Ser Tyr Gln Asp Phe Leu Asn Asn
Phe Thr Leu Leu Glu 325 330 335 Ile Cys Asn Leu Thr Pro Asp Thr Leu
Ser Gly Asp Tyr Lys Ser Tyr 340 345 350 Trp His Thr Thr Phe Tyr Glu
Gly Ser Trp Arg Arg Gly Ser Ser Ala 355 360 365 Gly Gly Cys Arg Asn
His Pro Gly Thr Phe Trp Thr Asn Pro Gln Phe 370 375 380 Lys Ile Ser
Leu Pro Glu Gly Asp Asp Pro Glu Asp Asp Ala Glu Gly 385 390 395 400
Asn Val Val Val Cys Thr Cys Leu Val Ala Leu Met Gln Lys Asn Trp 405
410 415 Arg His Ala Arg Gln Gln Gly Ala Gln Leu Gln Thr Ile Gly Phe
Val 420 425 430 Leu Tyr Ala Val Pro Lys Glu Phe Gln Asn Ile Gln Asp
Val His Leu 435 440 445 Lys Lys Glu Phe Phe Thr Lys Tyr Gln Asp His
Gly Phe Ser Glu Ile 450 455 460 Phe Thr Asn Ser Arg Glu Val Ser Ser
Gln Leu Arg Leu Pro Pro Gly 465 470 475 480 Glu Tyr Ile Ile Ile Pro
Ser Thr Phe Glu Pro His Arg Asp Ala Asp 485 490 495 Phe Leu Leu Arg
Val Phe Thr Glu Lys His Ser Glu Ser Trp Glu Leu 500 505 510 Asp Glu
Val Asn Tyr Ala Glu Gln Leu Gln Glu Glu Lys Val Ser Glu 515 520 525
Asp Asp Met Asp Gln Asp Phe Leu His Leu Phe Lys Ile Val Ala Gly 530
535 540 Glu Gly Lys Glu Ile Gly Val Tyr Glu Leu Gln Arg Leu Leu Asn
Arg 545 550 555 560 Met Ala Ile Lys Phe Lys Ser Phe Lys Thr Lys Gly
Phe Gly Leu Asp 565 570 575 Ala Cys Arg Cys Met Ile Asn Leu Met Asp
Lys Asp Gly Ser Gly Lys 580 585 590 Leu Gly Leu Leu Glu Phe Lys Ile
Leu Trp Lys Lys Leu Lys Lys Trp 595 600 605 Met Asp Ile Phe Arg Glu
Cys Asp Gln Asp His Ser Gly Thr Leu Asn 610 615 620 Ser Tyr Glu Met
Arg Leu Val Ile Glu Lys Ala Gly Ile Lys Leu Asn 625 630 635 640 Asn
Lys Val Met Gln Val Leu Val Ala Arg Tyr Ala Asp Asp Asp Leu 645 650
655 Ile Ile Asp Phe Asp Ser Phe Ile Ser Cys Phe Leu Arg Leu Lys Thr
660 665 670 Met Phe Met Ala Ala Asp Asp His Val Gly Ile Glu Ala Leu
675 680 685 9 2806 DNA homo sapiens 9 caagcaccga gctagccacc
agcatgctgt actccccagg gccgagtctt ccggagtcag 60 cagagagcct
ggatggatca caggaggata agcctcgggg ctcatgtgcg gagcccactt 120
ttactgatac gggaatggtg gctcacataa acaacagccg gctcaaggcc aagggcgtgg
180 gccagcacga caacgcccag aactttggta accagagctt tgaggagctg
cgagcagcct 240 gtctaagaaa gggggagctc ttcgaggacc ccttattccc
tgctgaaccc agctcactgg 300 gcttcaagga cctgggcccc aactccaaaa
atgtgcagaa catctcctgg cagcggccca 360 aggatatcat aaacaaccct
ctattcatca tggatgggat ttctccaaca gacatctgcc 420 aggggatcct
cggggactgc tggctgctgg ctgccatcgg ctcccttacc acctgcccca 480
aactgctata ccgcgtggtg cccagaggac agagcttcaa gaaaaactat gctggcatct
540 tccattttca gatttggcag tttggacagt gggtgaacgt ggtggtagat
gaccggctgc 600 ccacaaagaa tgacaagctg gtgtttgtgc actcaaccga
acgcagtgag ttctggagtg 660 ccctgctgga gaaggcgtat gccaagctga
gtgggtccta tgaagcattg tcagggggca 720 gtaccatgga gggccttgag
gacttcacag gaggcgtggc ccagagcttc caactccaga 780 ggccccctca
gaacctgctc aggctcctta ggaaggccgt ggagcgatcc tccctcatgg 840
gttgctccat tgaagtcacc agtgatagtg aactggaatc catgactgac aagatgctgg
900 tgagagggca cgcttactct gtgactggcc ttcaggatgt ccactacaga
ggcaaaatgg 960 aaacactgat tcgggtccgg aatccctggg gccggattga
gtggaatgga gcttggagtg 1020 acagtgccag ggagtgggaa gaggtggcct
cagacatcca gatgcagctg ctgcacaaga 1080 cggaggacgg ggagttctgg
atgtcctacc aagatttcct gaacaacttc acgctcctgg 1140 agatctgcaa
cctcacgcct gatacactct ctggggacta caagagctac tggcacacca 1200
ccttctacga gggcagctgg cgcagaggca gctccgcagg gggctgcagg aaccaccctg
1260 gcacgttctg gaccaacccc cagtttaaga tctctcttcc tgagggggat
gacccagagg 1320 atgacgcaga gggcaatgtt gtggtctgca cctgcctggt
ggccctaatg cagaagaact 1380 ggcggcatgc acggcagcag ggagcccagc
tgcagaccat tggctttgtc ctctacgcgg 1440 tcccaaaaga gtttcagaac
attcaggatg tccacttgaa gaaggaattc ttcacgaagt 1500 atcaggacca
cggcttctca gagatcttca ccaactcacg ggaggtgagc agccaactcc 1560
ggctgcctcc gggggaatat atcattattc cctccacctt tgagccacac agagatgctg
1620 acttcctgct tcgggtcttc accgagaagc acagcgagtc atgggaattg
gatgaagtca 1680 actatgctga gcaactccaa gaggaaaagg tctctgagga
tgacatggac caggacttcc 1740 tacatttgtt taagatagtg gcaggagagg
gcaaggagat aggggtgtat gagctccaga 1800 ggctgctcaa caggatggcc
atcaaattca aaagcttcaa gaccaagggc tttggcctgg 1860 atgcttgccg
ctgcatgatc aacctcatgg ataaagatgg ctctggcaag ctggggcttc 1920
tagagttcaa gatcctgtgg aaaaaactca agaaatggat ggacatcttc agagagtgtg
1980 accaggacca ttcaggcacc ttgaactcct atgagatgcg cctggttatt
gagaaagcag 2040 gcatcaagct gaacaacaag gtaatgcagg tcctggtggc
caggtatgca gatgatgacc 2100 tgatcataga ctttgacagc ttcatcagct
gtttcctgag gctaaagacc atgttcacat 2160 tctttctaac catggacccc
aagaatactg gccatatttg cttgagcctg gaacagtggc 2220 tgcagatgac
catgtgggga tagaggcgct gtaggagcct ggtcatctct accagcagca 2280
gcagcagcga ggttctagcc caggagggtg gggtgcttct tgtagccctc agctctccgg
2340 tctctgctga tgaaatgggc tccaggtggc agtgcccggg tcccaggtgc
cgtgtttact 2400 gcagcagtgg gacctccgtg cccactcccc cagctcagag
gctttctctt ttttccccaa 2460 cccggcttct gatggctggc tttcccccac
catcgctctc tcagagtata ttttactaaa 2520 gagtagttga tgcttcccca
gggtccccct ggctggggag gccaagaata gggaagggac 2580 ttgtagcccg
tttcttaccc tccatgcttg ctgtcctgct cacacctacc tgctgaccac 2640
ccatcctggc acagcctctg ttttcctccc catctgtgga tactattcta ataaatagca
2700 catgccattg gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2760 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa
2806
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