Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR
DIAGNOSIS OF RHEUMATOID ARTHRITIS
1 Introduction The present invention relates to the
identification of proteins and protein isoforms that are
associated with Rheumatoid Arthritis and to their use
for screening, diagnosis, prognosis, therapy and drug
development.
2 Background Of The Invention Rheumatoid Arthritis (RA)
is a multisystem disorder in which immunological
abnormalities characteristically result in symmetrical
joint inflammation, articular erosions and extra-
articular complications. It is the most common and
disabling autoimmune arthritis, and genetic
susceptibility is well defined. It affects about l-3~
of the population. Prevalence increases with age, but
peaks between 30 and 55 years.
Rheumatoid factors, i.e., antibodies directed against
the Fc fragment of immunoglobulin G (IgG?, are present in 75~
of patients with RA. Rheumatoid factors may be IgM, IgG or
IgA. Naturally occurring rheumatoid factors are thought to
play a role in the clearance of foreign antigen from the
body. In RA, rheumatoid factors are produced by synovial
plasma cells and have the ability to form immune complexes,
to activate complement, and to participate in the
inflammatory response, suggesting that they are involved in
the pathogenesis of RA. Coexistence of rheumatoid factors
and reduced IG galactosylation in RA is predictive of severe
disease.
Synovial tissue is the main focus of inflammation in RA
and there is a chronic synovial infiltrate of CD4+ T
lymphocytes, activated B-lymphocytes, mononuclear cells and
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polymorphonuclear leukocytes. HLA class II expression is
increased and is found on nearly all cell types, indicating
that they are in an active state. Chronic disease is
characterized by erosion of cartilage and bone, and synovial
hypertrophy.
Diagnosis of RA has been difficult. A disease pattern
may not be evident from the history, examination and
investigations in early disease. There is no diagnostic test
for RA, but the American College of Rheumatology has defined
criteria for its classification (Medicine, ed. Axford, J.,
Blackwell Science, 1996, pp. 3.18-3.22). These criteria are
merely observational landmarks which, in combination, can be
deemed to indicate RA.
75% of adults with RA are IgM rheumatoid factor
positive. This may be absent early in the disease. The
level of rheumatoid factor may be used prognostically at
diagnosis, but fluctuations are unhelpful in monitoring the
disease. Antinuclear antibodies are present in 30% of
patients.
TgM rheumatoid factor is usually measured using
agglutination tests. These include the latex test in which
particles coated with IgG are agglutinated, and the sheep-
cell agglutination test (SCAT or Rose-Waaler test), in which
sheep red cells are agglutinated. At least 70% of RA
patients diagnosed by other criteria have a positive
rheumatoid factor latex agglutination test. Rheumatoid
factor tests are also positive in other rheumatic diseases,
viral infections, chronic inflammatory diseases, neoplasm or
chemotherapy and, significantly, 4% of healthy individuals.
Drug treatment of RA is aimed at: (1) alleviating pain
(analgesics); (2) modifying the inflammatory events
themselves once they have been triggered (anti-inflammatory
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drugs); and (3) modifying the immunological events leading to
inflammation (disease-modifying drugs).
3 Summary Of The Invention The present invention provides
methods and compositions for screening, diagnosis and
prognosis of RA, for monitoring the effectiveness of RA
treatment, for therapy of RA and for drug development.
1
A first aspect of the invention provides methods for
diagnosis of RA that comprise analyzing a sample (e. g.,
plasma, serum, or synovial fluid) by two-dimensional
electrophoresis to detect the level of at least one
Rheumatoid Arthritis-Diagnostic Feature (RADF), e.g. an RADF
selected from the group of RADFs disclosed herein. These
methods are also suitable for screening, prognosis,
monitoring the results of therapy, and drug development.
A second aspect of the invention provides methods for
diagnosis of RA that comprise detecting in a sample (e. g.,
plasma, serum, or synovial fluid) the level of at least one
Rheumatoid Arthritis-Diagnostic Protein Isoform (RPI), e.g.
an RPI selected from the group of RPIs disclosed herein.
These methods are also suitable for screening, prognosis,
monitoring the results of therapy, and drug development.
A third aspect of the invention provides monoclonal and
polyclonal antibodies capable of immunospecific binding to an
RPI, e.g. an RPT disclosed herein.
A fourth aspect of the invention provides a preparation
comprising an isolated RPI, i.e., an RPI free from proteins
or protein isoforms having a significantly different
isoelectric point or a significantly different apparent
molecular weight from the RPI.
A fifth aspect of the invention provides methods for
treatment or prevention of RA that comprise administering a
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compound which is able to decrease the level of at least one
RADF -- or the level or activity of at least one RPI -- that
is present at an increased level in the synovial fluid of RA
patients as compared with the serum of RA patients.
Preferably, the administered compound is an antibody, an
anti-sense oligonucleotide, a ribozyme, or an oligonucleotide
capable of forming a triple helix.
A sixth aspect of the invention provides methods for
treatment or prevention of RA that comprise administering a
compound which is able to increase the level of at least one
RADF -- or the level or activity of at least one RPI -- that
is present at a decreased level in the synovial fluid of RA
patients as compared with the serum of RA patients.
Preferably, the administered compound is an RPI, a nucleic
acid encoding an RPI, (e.g. a nucleic acid that is part of an
expression vectory, or a cell that is able to express and
secrete one or more RPIs.
4 Brief Description Of The Figures Figure 1 is an image
obtained from 2-dimensional electrophoresis of normal
human serum, which has been annotated to identify 14
landmark features, designated PL1 to PL16.
Detailed Description Of The Invention The invention
described in detail below encompasses methods and
compositions for screening, diagnosis and prognosis of
RA in a subject, methods for monitoring the results of
RA therapy, methods for drug development, and methods
for treating RA. Preferably, the subject is a mammal,
more preferably a human, and most preferably a human
adult.
For clarity of disclosure, and not by way of limitation,
the invention will be described with respect to the analysis
of serum and synovial fluid samples. However, as one skilled
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in the art will appreciate, the assays and techniques
described herein can be applied to other types of patient
samples, including a body fluid (e. g., plasma, urine,
cerebrospinal fluid, joint aspirate), a tissue sample, or
homogenate thereof.
5.1 Rheumatoid Arthritis-Diagnostic Features (RADFs)
In one aspect of the invention, two-
dimensional electrophoresis is used to analyze
serum from a subject in order to measure the
abundance of one or more Rheumatoid Arthritis-
Diagnostic Features (RADFs) for screening or
diagnosis of RA, to determine the prognosis of an
RA patient, to monitor the effectiveness of RA
therapy or for drug development. As used herein,
"two-dimensional electrophoresis" (2D-
electrophoresis) means a technique comprising
isoelectric focusing, followed by denaturing
electrophoresis; this generates a two-dimensional
gel (2D-gel) containing a plurality of separated
proteins. Preferably, the step of denaturing
electrophoresis uses polyacrylamide electrophoresis
in the presence of sodium dodecyl sulfate (SDS-
PAGE). Especially preferred are the highly
accurate and automatable methods and apparatus
("the Preferred Technology") described in U.S.
Application No. 08/980,574, which is incorporated
herein by reference in its entirety. Briefly, the
Preferred Technology provides efficient, computer-
assisted methods and apparatus for identifying,
selecting and characterizing biomolecules in a
biological sample. A two-dimensional array is
generated by separating biomolecules on a two-
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dimensional gel according to their electrophoretic
mobility and isoelectric point. A computer-
generated digital profile of the array is
generated, representing the identity, apparent
molecular weight, isoelectric point, and relative
abundance of a plurality of biomolecules detected
in the two-dimensional array, thereby permitting
computer-mediated comparison of profiles from
multiple biological samples, as well as computer
aided excision of separated proteins of interest.
As used herein, the term "Rheumatoid Arthritis-
Diagnostic Feature" (RADF) refers to a feature (e. g., a spot
in a 2D gel), detectable by 2D electrophoresis of a
biological sample, that is differentially present in one
sample compared to another, relevant sample, e.g., (1) in
serum from a subject with RA compared with serum from a
subject without RA or (2) in synovial fluid taken from a
subject with RA compared with serum taken from a subject with
RA.
As used herein, a feature (or a protein isoform) is
"differentially present" in a first sample with respect to a
second sample when a method for detecting the feature or
isoform (e. g., 2D electrophoresis or an immunoassay) reveals
that the feature (or protein isoform) is present at a
different relative abundance in a first sample as compared
with a second sample. If the measured feature in the first
sample is at a higher relative abundance than in the second
sample, the feature or isoform is "increased" in the first
sample with respect to the second; conversely, if the
measured feature in the first sample is at a lower relative
abundance than in the second sample, the feature or isoform
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is "decreased" in the first sample with respect to the
second.
Preferably, the relative abundance of a feature in two
samples is determined in two steps. First, the signal
obtained upon detecting the feature in a sample is normalized
by reference to a suitable background parameter, e.g., to the
total protein in the sample being analyzed (e. g., total
protein loaded onto a gel), to an invariant feature, ,i..e., a
feature whose abundance is known to be similar in the samples
being compared, e.g., one or more Expression Reference
Features (ERFs), such as the ERFs disclosed below, or to the
total signal detected from all proteins in the sample.
Secondly, the normalized signal for the feature in one
sample or sample set is compared with the normalized signal
for the same feature in another sample or sample set in order
to identify features that are "differentially present" in the
first sample (or sample set) with respect to the second.
The RADFs disclosed herein have been identified by one
of two sample comparisons. The first group of RADFs are
those features that (a) are differentially present in the
svnovial fluid of subjects with RA as compared with the serum
of subjects with RA, and (b) are not differentially present
in the synovial fluid of subjects without RA as compared with
the serum of subjects without RA. Subjects without RA can
include normal subjects with no known disease or condition,
or subjects with joint diseases or conditions other than RA,
including gout, osteoarthritis, or synovitis (e. g., traumatic
synovitis). The second group of RADFs are those features
that are differentially present in serum from a subject with
RA compared with serum from a subject without RA.
Four groups of RADFs have been identified through the
methods and apparatus of the Preferred Technology. The first
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group consists of RADFs that are increased in synovial fluid
verses serum in subjects with RA, but are not increased in
synovial fluid versus serum in subjects without RA. These
RADFs can be described by apparent molecular weight (MW) and
isolectric point (pI), as follows:
Table I. RADFs Increased in RA Synovial Fluid vs. R.A Serum
Name Fold pI MW
increase (kd)
RADF-1 118.3 6.76 74,447
RADF-2 94.7 7.42 25,049
RADF-3 62.5 4.92 54,948
RADF-4 26.0 5.1 53,241
RA17F-5 20.1 6.94 27,221
RADF-6 11.0 5.14 137,225
RADF-7 9.9 5.86 26,217
RADF-8 8.2 5.79 58,161
RADF-9 5.0 6.17 57,613
RADF-10 3.7 5.43 39,842
RADF-11 3.4 5.98 52,631
RADF-12 3.3 7.06 72,543
The second group consists of R.ADFs that are decreased in
synovial fluid of versus serum in subjects with R.A, but are
not decreased the the synovial fluid versus serum in subjects
without RA. These RADFs can be described by apparent
molecular weight (MW) and isolectric point (pI), as follows:
Table II. RADFs Decreased in RA Synovial Fluid vs. R.A Serum
Name Fold pI MW
decrease (kd)
RADF-13 37.5 4.92 53,578
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RADF-14 24.6 6.2 76,789
R.A.DF-15 12 . 6 4.56 63, 737
R.ADF-16 11.9 5.36 24,124
RADF-17 10.1 5.96 158,868
R.ADF-18 9.0 9.52 14,953
RADF-19 5.3 5.1 131,608
RADF-20 3.7 5.12 60,216
RADF-21 2.9 ~ 4.95 32,321
R.ADF-22 3.0 6.95 27,812
The third group consists of RADFs that are increased in
the serum of subjects with R.A as compared with the serum of
subjects without RA. These RADFs can be described by
apparent molecular weight (MW) and isoelectric point (pI) as
follows:
Table III. RADFs Increased In RA Serum vs. Non-RA Serum
Name Fold pI MW
increase (kd)
RADF-13 2.7 4.92 53,578
RADF-16 4.3 5.36 24,124 I
RADF-22 2.7 6.95 27,812
RADF-23 10.1 9.00 47,978
RADF-24 5.5 5.31 74,447
~ADF-25 3.5 5.34 40,271
R.ADF-26 3.4 4.81 40,997
R.ADF-27 3.2 6.97 56,354 I
RADF-28 3.1 5.41 16,807
RADF-29 2.9 5.02 36,372
R.ADF-30 2.8 4.52 17,629
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RADF-31 2.6 [ 5.26 ~ 16,687
The fourth group consists of RADFs that are decreased in
the serum of subjects with RA as compared with the serum of
subjects without RA. These RADFs can be described by
apparent molecular weight (MW) and isoelectric point (pI) as
follows:
Table IV. R.ADFs Decreased In RA Serum vs. Non-RA Serum
Name Fold pI MW
decrease (kd)
RADF-32 36.5 6.61 70,511
RA.DF-33 4.3 6.29 76,112
R.ADF-34 3.7 5.65 37,966
RADF-35 3.3 5.93 34,471
RADF-36 3.1 6.09 57,613
RADF-37 3.0 5.41 183,864
'~ RADF-382.9 5.04 81,696
RADF-39 2.8 6.25 53,917
RADF-40 2.6 6.37 82,423
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For any given RADF in the first and second groups, the
ratio obtained upon comparing the signal measured in synovial
fluid from subjects with RA relative to the signal measured
in serum from subjects with RA will depend upon the
particular analytical protocol and detection technique that
is used. Accordingly, the present invention contemplates
that for each RADF in the first and second groups, a
laboratory will establish reference ranges for the ratio of
each R.ADF in synovial fluid versus serum in subjects with and
without RA, according to the analytical protocol and
detection technique in use, as is conventional in the
diagnostic art. Preferably, at least one control pair of
synovial fluid and serum samples from a subject known to have
R.A, or at least one control pair of synovial fluid and serum
samples from a subject known not to have RA (and more
preferably at least one of each such control pairs) is
included with each batch of test samples analyzed.
Similarly, for any given RADF in the third and fourth
groups, the ratio obtained upon comparing the signal measured
in serum from subjects with RA relative to the signal
measured in serum from subjects without RA will depend upon
the particular analytical protocol and detection technique
that is used. Accordingly, the present invention
contemplates that each laboratory will establish reference
ranges for the ratio of each R.ADF in the third and fourth
groups in subjects with and without RA according to the
analytical protocol and detection technique in use, as is
conventional in the diagnostic art. Preferably, at least one
positive control serum sample from a subject known to have
R.A, or a negative control serum sample from a subject known
to be free of RA (and more preferably at least one positive
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and at least one negative control sample) is included with
each batch of test samples analyzed.
In a preferred embodiment, the signal associated with an
RADF is normalized with reference to one or more Expression
Reference Features (ERFs) detected in the same 2D gel. As
will be apparent to one of ordinary skill in the art, such
ERFs may readily be determined by comparing different samples
using the Preferred Technology. Suitable ERFs for this
purpose include (but are not limited to) those described in
the following table:
TABLE V
ERF-# MW pI
ERF-1 38719 5.34
ERF-2 13915 5.26
ERF-3 73465 5.14
ERF-4 40924 4.87
ERF-5 104893 7.22
ERF-6 37294 7.62
ERF-7 24124 5.48
ERF-8 53409 4.56
ERF-9 36372 5.02
ERF-10 26930 5.52
By way of example, Table VI shows the levels of the
RADFs identified in Tables I and II normalized to ERF-1 and
ERF-3; one of skill in the art will realize that the levels
of RADFs can be normalized relative to any desired ERF. The
values shown in Table VI are the ratios of each RADF relative
to the ERF in question, where a positive ratio indicates
enhanced levels of the RADF relative to the ERF, and a
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negative ratio indicates diminished levels of the RADF
relative to the ERF. As will be evident to one of skill in
the art, the RADF/ERF ratios can serve as a diagnostic
parameters in their own right.
TABLE VI Ratio of RADFs to ERFs in RA Synovial Fluid
RADF# Normalized to ERF- Normalized to.ERF-3
1
RADF-1 -22.2 -26.0
RADF-2 -96.6 -124.6
RADF-3 -59.4 -71.3
RADF-13 38.1 31.8
RADF-4 -23.0 -27.6
RADF-14 25.2 21.9
RADF-5 -6.4 -7.3
RADF-15 13.2 11.1
RADF-16 12.3 9.9
RADF-6 -10.3 -12.2
RADF-17 10.2 8.4
RADF-7 -2.3 -2.8
RADF-18 9.2 7.6
RADF-8 -7.3 -9.2
RADF-19 5.5 4.6
RADF-9 -4.8 -5.6
RADF-20 3.8 3.3
RADF-10 -3.7 -4.4
RADF-11 -3.5 -4.7
RADF-12 -2.5 -3.1
RADF-22 2.7 2.3
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In similar fashion Table VII shows the levels of RADFs
identified in Tables III and IV normalized to ERF-1 and ERF-
3. One of skill in the art will realize that the levels of
RADFs can be expressed relative to any desired ERF, and that
the RADF/ERF ratios can be used as diagnostic parameters in
their own right.
TABLE VII Ratio of RADFs to ERFs in RA Serum
RADF# Normalized to ERF- Normalized to ERF-3
1
RADF-32 -13.4 -9.5
RADF-23 5.6 7.3
RADF-24 4.4 5.3
RADF-16 3.5 4.0
RADF-33 -1.4 -1.3
RADF-34 -2.9 -2.5
RADF-25 2.9 3.2
RADF-26 2.6 2.8
RADF-35 -4.0 -3.7
RADF-27 2.4 2.6
RADF-36 -3.0 -2.6
RADF-28 -2.2 2.8
RADF-37 -4.2 -3.5
As the skilled artisan will appreciate, the measured MW
and pI of a given feature or protein .isoform will vary to
some extent depending on the precise protocol used for each
step of the 2D electrophoresis and for landmark matching. As
used herein, the terms "MW" and "pI" are defined,
respectively, to mean the apparent molecular weight and the
isoelectric point of a feature or protein isoform as measured
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in exact accordance with the experimental protocol set forth
in Section 6 below ("the Reference Protocol"). When the
Reference Protocol is followed and when samples are run in
duplicate or a higher number of replicates, variation in the
measured mean pI of an RADF or RPI is typically less than +1~
and variation in the measured mean MW of an RADF or RPI is
typically less than +5~. Where the skilled artisan wishes to
deviate from the Reference Protocol, calibration experiments
should be performed to compare the MW and pI for each RADF or
protein isoform as detected (a) by the Reference Protocol and
(b) by the deviant protocol.
RADFs can be used for detection, prognosis, diagnosis,
or monitoring of RA or for drug development. In one
embodiment of the invention, synovial fluid and serum from a
subject are analyzed by 2D electrophoresis for quantitative
detection of one or more RADFs selected from the group
consisting of RADF-1 to RADF-12, wherein an increased
abundance of an RADF in synovial fluid relative to serum
indicates the presence of RA.
In another embodiment of the invention, synovial fluid
and serum from a subject are analyzed by 2D electrophoresis
for quantitative detection of one or more RADFs selected from
the group consisting of R.ADF-13 to RADF-22 wherein a
decreased abundance of an RADF in synovial fluid relative to
serum indicates the presence of RA.
In another embodiment of the invention, synovial fluid
from a subject is analyzed by 2D electrophoresis for
quantitative detection of one or more RADFs selected from the
group consisting of RADF-1 to RADF-22 wherein the ratio of
the one or more RADFs relative to an Expression Reference
Feature (ERF) indicates the presence of RA; ERF-1 and ERF-3
are preferred ERFs for this purpose.
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In yet another embodiment of the invention, serum from a
subject with RA is analyzed by 2D electrophoresis for
quantitative detection of one or more RADFs selected from the
group consisting of RADF-13, RADF-16, and RADF-22 to RADF-31,
wherein an increased abundance of an RADF in serum from the
subject relative to serum from a subject or subjects without
RA (e. g. a control sample or a previously determined
reference range) indicates the presence of RA.
In another embodiment of the invention, synovial fluid
from a subject is analyzed by 2D electrophoresis for
quantitative detection of one or more RADFs selected from the
group consisting of RADF-32 to RADF-40, wherein a decreased
abundance of an RADF in serum from the subject relative to
serum from a subject or subjects without RA (e. g. a control
sample or a previously determined reference range) indicates
the presence of RA.
In another embodiment of the invention, serum from a
subject is analyzed by 2D electrophoresis for quantitative
detection of one or more RADFs selected from the group
consisting of RADF-13, RADF-16, and RADF-22 to RADF-40,
wherein the ratio of the one or more RADFs relative to an ERF
indicates the presence of RA; ERF-1 and ERF-3 are preferred
ERFs for this purpose.
5.2 Rheumatoid Arthritis-Diagnostic Protein
Isoforms (RPIs)
1.1
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In another aspect of the invention, serum from a subject
is analyzed for quantitative detection of one or more
Rheumatoid Arthritis-Diagnostic Protein Isoforms (RPIs) for
screening or diagnosis of RA, to determine the prognosis of
an RA patient, or to monitor the effectiveness of RA therapy
or for drug development. As used herein, the term
"Rheumatoid Arthritis-Diagnostic Protein Isoform" includes
(1) a protein isoform that is differentially present in serum
from subjects with RA compared with serum from subjects
without RA, and (2) a protein isoform that (a) is
differentially present in synovial fluid from subjects with
RA compared with serum from subjects with RA, and (b) is not
differentially present in the synovial fluid compared with
serum from subjects without RA. As is well known in the art,
the protein product of a single gene may be expressed as
variants (isoforms) that differ as a result of differential
post-translational modification (e. g., glycosylation,
phosphorylation or acylation), so that proteins of identical
amino acid sequence can differ in their pI, MW, or both,
and/or that differ in their amino acid composition (e.g., as
a result of alternative splicing or limited proteolysis). It
follows that differential presence of a protein isoform does
not require differential expression of the gene encoding the
protein in question.
Four groups of RPIs have been identified by partial
amino acid sequencing of RADFs, using the methods and
apparatus of the Preferred Technology. The first group
consists of RPIs that are increased in synovial fluid versus
serum in subjects with RA, but are not increased in synovial
fluid versus serum in subjects without RA. The MWs, pIs and
partial amino acid sequences of these RPIs are presented in
Table VIII, as follows:
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Table VIII. RPI~s Increased In Synovial Fluid
RPI RADF Homologous ProteinPartial AminC pI MW (kd)
Acid Sequences
RPI-1 RADF-1 Transferrin DQYELLCLDNTR 6.76 74,447
EGYYGYTGAFR
DYELLCLDGTR
CQSFR
INHCR
SCHTGLGR
SCHTAVGR
APNHAVVTR
ASYLDCIR
WCALSHHER
DSGFQMNQLR
SASDLTWDNLK
WCAVSEHEATK
KDSGFQMNQLR
MYLGYEYVTAIR
MYLGYEYVTAIR
CSTSSLLEACTFR
FDEFFSEGCAPGSK
KPVEEYANCHLAR
EDPQTFYYAVAWK
DCHLAQVPSHTWAR
ELDIWELLNQAQEHFGK
SAGWNIPIGLLYCDLPEPR
RPI-2 RADF-2 IgG x light chainSGTASWCLLNNFYPR7.42 25,049
FSGSGSGTDFTLTISR
EIVLTQSPATLSLSPGER
RPI-3 RADF-3 Alpha-1-antitrypsinWERPFEVK 4.92 54,948
SVLGQLGITK
LSITGTYDLK
SPLFMGK
FLENEDR
QINDYVEK
KQINDYV$x
LGMFNIQHCK
GKWERPFEVK
TDTSHHDQDHPTFNK
LQHLENELTHDIITK
VFSNGADLSGVTEEAPLK
LYHSEAFTVNFGDTEEAK
RPI-4 RADF-4 Vitamin D bindingYTFELSR 5.1 53,241
protein FEDCCQEK
HLSLLTTLSNR
VCSQYAAYGEK
RTHLPEVFLSK
RPI-5 RADF-6 Ceruloplasmin EYTDASFTNR 5.14 137,225
precursor DDEEFIESNK
QSEDSTFYLGER
ALYLQYTDETFR
LISVDTEHSNIYLQNGPDR
QYTDSTFR
MYYSAVDPTK
GAYPLSIEPIGVR
NNEGTWSPNYNPQSR
RPI-6 RADF-8 Ig alpha-1&2 ChainWLQGSQELPR 5.79 58,161
c region YLTWASR
QEPSQGTTTFAVTSILR
RPI-7 RADF-9 Ig alpha-1&2 chainWLQGSQELPR 6.17 57,613
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c region
RPI-8 RADF-10 Haptoglobin precursorVGYVSGWGR 5.43 39,842
YVMLPVADQDQCIR
RPI-9 RADF-11 Heta-2-glycoproteinVCPFAGILENGAVR 5.98 52,631
1
precursor ATVVYQGER
(Apo-lipoproteinEHSSLAFWK
H)
TCPKPDDLPFSTWPLK
RPI-10RADF-11 NOVEL VAA(I/L)EHFGR 5.98 52,631
RPI-11RADF-12 Transferrin DYELLCLDGTR 7.06 72,543
The second group consists of RPIs that are decreased in
synovial fluid verses serum serum in subjects with RA, but
are not decreased in synovial fluid verses serum in subjects
without RA. The MWs, pIs and partial amino acid sequences of
these RPIs are presented in Table IX, as follows:
Table IX.
RPI RADF Homologous ProteinPartial Amino pI MW
Acid Se ences (kd)
RPI-12RADF-13Alpha-1-antitrypsinDTEEEDFHVDQVTTVK4.9253,578
VFSNGADLSGVTEEAPLK
LSITGTYDLK
LGMFNIQHCK
SVLGQLGITK
FLENEDR
FLENEDRR
WERPFEVK
GKWERPFEVK
KLYHSEAFTVNFGDTEEAK
LYHSEAFTVNFGDTEEAKK
RPI-13RADF-14Transferrin DSGFQMNQLR 6.2 76,789
precursor WCALSHHER
SASDLTWDNLK
EGYYGYTGAFR
SCHTGLGR
SCHTAVGR
APNHAVVTR
DCHLAQVPSHTWAR
RPI-14RADF-14IgM chain LICQATGFSPR 6.2 76,789
VSVFVPPR
DGFFGNPR
QIQVSWLR
QVGSGVTTDQVQAEAK
FTCTVTHTDLPSPLK
GVALHRPDVYLLPPAR
EQLNLR
GFPSVLR
VQHPNGNK
NVPLPVIAELPPK
DVMQGTDEHWCK
YVTSAPMPEPQAPGR
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RPI RADF Homologous ProteinPartial Amino pI MW
Acid Sequences (kd)
ESDWLSQSMFTCR
RPI-15RADF-15Alpha-1- DSLEFR 4.56 63,737
antichymotrypainADLSGITGAR
precursor EQLSLLDR
ITLLSALVETR
WEMPFDPQDTHQSR
LYGSEAFATDFQDSAAAK
AVLDVFEEGTEASAATAVK
RPI-16RADF-16Apolipoprotein LSPLGEEMR 5.36 24,124
A-1
precursor VQPYLDDFQK
DLATVYVDVLK
AHVDALR
AELQEGAR
WQEEMELYR
VSFLSALEEYTK
RPI-17RADF-16NOVEL DSGAD(I/L)S 5.36 24,124
RPI-18RADF-17Complement factorQMSKYPSGER 5.96 158,868
H
precursor MDGASNVTCINSR
CGKDGWSAQPTCIK
GNTAKCTSTGWIPAPR
IDVHLVPDR
EFDHNSNIR
RPYFPVAVGK
SLGNVIMVCR
LYSTCEGGFR
HGGLXHENMR
EIMSNYNIALR
TDCLSLPSFENAIPMGEK
RPI-19RADF-17Copper transportingDRSASHLDHK 5.96 158,868
ATPase ASINSLLSDKR
QIEAMGFPAFVK
VFAEVLPSHKVAK
CYIQVTGMTCASCVANIER
RPI-20RADF-17NOVEL NV(I/L)DAPHAR 5.96 158,868
RPI-21RADF-18Hemoglobin alphaVGAHAGEYGAEALER9.52 14,953
chain MFLSFPTTK
TYFPHFDLSHGSAQVK
RPI-22RADF-19Ceruloplasmin GAYPLSIEPIGVR 5.1 131,608
precursor ALYLQYTDETFR
GSLHANGR
YTVNQCR
QXTDSTFR
DNEDFQESNR
QSEDSTFYLGER
DLYSGLIGPLIVCR
VDKDNEDFQESNR
NNEGTYYSPNYNPQSR
RPI-23RADF-20Ig alpha-1&2 QEPSQGTTTFAWSILR5.12 60,216
chain c
region YLTWASR
SAVQGPPER
WLQGSQELPR
DASGATFTWTPSSGK
RPI-24RADF-22Complement C3C HQQTVTIPPK 6.95 27,812
precursor SSLSVPYVIVPLK
LPYSWR
DSITTWEILAVSMSDK
SEFPESWLWNVEDLK
TLDPER
WPEGIR
NEQVEIR
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RPI RADF Homologous ProteinPartial Amino pI MW
Acid Sequences (kd)
AVLYNYR
RHQQTVTIPPK
AAVYHHFISDGVR
VELLHNPAFCSLATTK
SNLDEDIIAEENIVSR
The third group comprises RPIs that are increased in the
serum of subjects with RA as compared with the serum of
subjects without RA. The MWs, pIs and partial amino acid
sequences of these RPIs are presented in Table X, as follows:
Table X.
RPI RADF Homologous Partial Amino pI MW
Protein Acid Sequence (kd)
RPI-25RADF-23Ig y chain EPQVYTLPPSR 9 47,978
C
region DTLMISR
GPSVFPLAPSSK
STSGGTAALGCLVK
FNWYVDGVEVHNAK
TPBVTCVVVDVSHEDPEVK
RPI-26RADF-24Hemopexin NFPSPVDAAFR 5.31 74,447
GGYTLVSGYPK
GECQAEGVLFFQGDR
RPI-16RADF-16ApolipoproteinLSPLGEEMR 5.36 24,124
A-1 precursor VQPYLDDFQK
DLATVYVDVLK
AHVDALR
AELQEGAR
WQEEMELYR
VSFLSALEEYTK
RPI-17RADF-16NOVEL DSGAD(I/L)S 5.36 24,124
RPI-27RADF-25Haptoglobin VGYVSGWGR 5.34 40,271
precursor GSPPWQAK
YVMLPVADQDQCIR
VTSIQDWVQK
SCAVAEYGVYVK
RPI-28RADF-26Complement ENEGFTVTAEGK 4.81 40,997
C3
precursor VYAYYNLEESCTR
NTMILEICTR
VSHSEDDCLAFK
RPI-29RADF-26Haptoglobin VGYVSGWGR 4.81 40,997
precursor
RPI-30RADF-26Zn alpha2 glyco-QDSQLQK 4.81 40,997
protein IDVHWTR
SQPMGLWR
WEAEPVYVQR
AREDIFMETLK
AYLEEECPATLR
RPI-31RADF-27Ig alpha-1&2 WLQGSQELPR 6.97 56,354
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RPI RADF Homologous Partial Amino pI MW
Protein Acid Sequence (kd)
chain c region
RPI-32 RADF-27Complement QVPAHAR 6.97 56,359
factor
B (HH Fragment)ISVIRPSK
VASYGVKPR
DISEVVTPR
GDSGGPLIVHK
YGLVTYATYPK
LPPTTTCQQQK
FLCTGGVSPYADPNTCR
RPI-12 RADF-13Alpha-1-anti-DTEEEDFHVDQVTTVK 4.92 53,578
trypsin VFSNGADLSGVTEEAPLK
LSITGTYDLK
LGMFNIQHCK
SVLGQLGITK
FLENEDR
FLENEDRR
WSRPFEVK
GKWERPFEVK
KLYHSEAFTVNFGDTEEAK
RPI-24 RADF-22Complement HQQTVTIPPK 6.95 27,812
C3C
precursor SSLSVPYVIVPLK
LPYSVVR
DSITTWEILAVSMSDK
SEFPESWLWNVEDLK
TLDPER
WPEGIR
NEQVEIR
AVLYNYR
RHQQTVTIPPK
AAVYHHFISDGVR
VELLHNPAFCSLATTK
SNLDEDIIAEENIVSR
The fourth group comprises RPIs that are decreased in
the serum of subjects with RA as compared with the serum of
subjects without RA. The MWs, pIs and partial amino acid
sequences of these RPIs are presented in Table XI, as
follows:
Table XI.
RPI RADF Homologous ProteinPartial Amino pI MW
Acid Sequence (kd)
RPI-33 RADF-32Transferrin DSGFQMNQLR 6.61 70,511
RPI-34 RADF-33Transferrin DSGFQMNQLR 6.29 76,112
CDEWSVNSVGK
RPI-35 RADF-34C-terminal trypticCTSTGWIPAPR 5.65 37,966
fragment ComplementSCDNPYIPNGDYSPLR
factor H EYHFGQAVR
SLGNVIMVCR
TGDEITYQCR
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RPI RADF Homologous ProteinPartial Amino pI MW
Acid Sequence (kd)
KGEWVALNPLR
GSWVALNPLRK
RPI-36RADF-35Haptoglobin relatedVGWSGWGQSDNFK 5.93 34,471
protein precursorNYAEVGR
WLHPNYHQVDIGLIK
RPI-37RADF-36Ig alpha-1&2 WLQGSQELPR 6.09 57,613
chain c
region QEPSQGTTTFAVTSILR
YLTWASR
SAVQGPPER
RPI-38RADF-37Complement factorEIMENYNIALR 5.41 183,864
H
precursor IDVHLVPDR
EFDHNSNIR
RPYFPVAVGK
SLGNVIMVCR
SCDIPVFMNAR
TDCLSLPSFENAIPMGEK
In one embodiment of the invention, synovial fluid and
serum from a subject are analyzed for quantitative detection
of one or more RPIs selected from the group consisting of
RPI-1 to RPI-11 wherein an increased abundance of one or more
such RPIs in synovial fluid relative to serum indicates the
presence of RA. In a further embodiment of the invention,
synovial fluid and serum from a subject are analyzed for
quantitative detection of one or more RPIs selected from the
group consisting of RPI-12 to RPI-24 wherein a decreased
abundance of one or more such RPIs in synovial fluid relative
to serum indicates the presence of RA.
In another embodiment of the invention, serum from a
subject is analyzed for quantitative detection of one or more
RPIs selected from the group consisting of RPI-12, RPI-16,
RPI-17 and RPI-24 to RPI-32, wherein an increased abundance
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of one or more such RPIs in serum from the subject indicates
the presence of RA. In another embodiment of the invention,
serum from a subject is analyzed for quantitative detection
of one or more RPIs selected from the group consisting of
RPI-33 to RPI-38, wherein a decreased abundance of one or
more such RPIs in serum from the subject indicates the
presence of RA.
Preferably, the abundance of an RPI is normalized to an
Expression Reference Protein Isoform (ERPI). ERPIs have been
identified by partial amino acid sequencing of the ERFs
described above, using the methods and apparatus of the
Preferred Technology. The partial amino acid sequences of
these ERPIs, and the known proteins to which they are
homologous are presented in table XIb.
TABLE XIb
ERPI-# ERF-# Homologous Protein Partial Amino
Acid Sequences
ERPI-1 ERF-4 Zn-a-2-glycoprotein EDIFMETLK
precursor
ERPI-2 ERF-6 Immunoglobulin heavy EEQYNSTYR
chain y (intermediate
segment)
ERPI-3 ERF-8 a-2-HS-glycoprotein LDGKFSVVYAK
precursor
As shown above, the RPIs described herein include
previously unknown proteins, as well as isoforms of known
proteins where the isoforms were not previously known to be
associated with RA. For each RPI, the present invention
additionally provides a preparation comprising the isolated
RPI or fragments thereof, and antibodies that bind to said
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RPI or to said fragments, or to said RPI and said fragments.
As used herein, an "isolated" RPI is an RPI free of proteins
or protein isoforms having a significantly different pI or MW
from those of the RPI, as determined by 2D electrophoresis.
As used herein, a "significantly different" pI or MW is one
that permits the contaminating protein isoform to be resolved
from the RPI on 2D electrophoresis, performed according to
the Reference Protocol.
In one embodiment, an isolated protein is provided, said
protein comprising a peptide with the amino acid sequence
identified in Table VIII, IX, X or XI for an RPI, said
protein having a pI and MW within 10% (preferably within 5%,
more preferably within 1%) of the values identified in Tables
VIII, IX, X and XI for that RPI.
The RPIs of the invention can be assayed by any method
known to those skilled in the art. In one embodiment, the
RPIs are separated on a 2-D gel by virtue of their MWs and
pIs and visualized by staining the gel.
Alternatively, RPIs can be detected in assays, such as
immunoassays, for detection, prognosis, diagnosis, or
monitoring of RA or for drug development. In one embodiment,
an immunoassay is performed by contacting a sample derived
from a subject to be tested with an anti-RPI antibody under
conditions such that immunospecific binding can occur, and
detecting or measuring the amount of any immunospecific
binding by the antibody. Preferably, the anti-RPI antibody
preferentially binds to the RPI rather than to other isoforms
of the same protein. In a preferred embodiment, the anti-RPI
antibody binds to the RPI with at least 2-fold greater
affinity, more preferably at least 5-fold greater affinity,
still more preferably at least 10-fold greater affinity,. than
to said other isoforms of the same protein.
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In another embodiment, an immunoassay is performed
by contacting a sample derived, for example, from a
subject to be tested, with a plurality of anti-RPI anti
bodies under conditions such that immunospecific bin
ding can occur, and simultaneouslydetecting or
measuring the amount of any immunospecific binding
by the plurality of antibodies to a plurality of RPIs
Preferably, each anti-RPI antibody binds to a
different RPI, and is optionally fixed to a solid supp
ort. For example, antibodies can be fixed in a two di
mensional array arrangment, wherein each position of
the array is occupied by antibodies that
specifically bind a single RPI, and wherein the
array has antibodies specific for one or more RPIs.
Preferably, each anti-RPI antibody preferentially
binds to an RPI rather than to other isoforms of the s
arne protein. In a preferred embodiment, the anti-RP
I antibodies bind to the RPIs with at least 2-fold grea
ter affinity, more preferably at least 5-fold
greater affinity, still more preferably at least 10-fol
d greater affinity, than to said other isoforms of the
same proteins.
Tn one embodiment, binding of antibody in tissue
sections can be used to detect aberrant RPI localization or
aberrant (e.g., high, low, absent) levels of an RPI. In a
specific embodiment, antibody to an RPI can be used to assay
in a patient tissue or serum sample for the presence of the
RPI where an aberrant level of RPI is indicative of RA. As
used herein, an "aberrant level" means an increased or
decreased level relative to that present, or relative to a
standard level representing that present, in an analogous
sample from a portion of the body or from a subject not
having RA.
The immunoassays which can be used include without
limitation competitive and non-competitive assay systems
using techniques such as western blots, radioimmunoassays,
ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions,
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immunodiffusion assays, agglutination assays, complement-
fixation assays, immunoradiometric assays, fluorescent
immunoassays, protein A immunoassays, to name but a few.
If desired, an RPI can be detected by means of a two-
step sandwich assay. Where an RPI represents a particular
glycoform of a protein; the first step can employ an anti-RPI
antibody (which can optionally be immobilized on a solid
phase) to capture the RPI; in the second step, a directly or
indirectly labelled lectin is used to detect the captured
RPI. Any lectin can be used for this purpose that
preferentially binds to the RPI rather than (a) to other
glycoforms that have the same core protein as the RPI or (b)
to other isoforms that share the antigenic determinant
recognized by the antibody. In a preferred embodiment, the
chosen lectin binds to the RPI with at least 2-fold greater
affinity, more preferably at least 5-fold greater affinity,
still more preferably at least 10-fold greater affinity, than
to said other isoforms that have the same core protein as the
RPI. A lectin that is suitable for detecting a given RPI can
readily be identified by methods well known in the art, for
instance upon testing one or more lectins enumerated in Table
I on pages 158-159 of Sumar et al., Lectins as Indicators of
Disease-Associated Glycoforms, In: Gabius H-J & Gabius S
(eds.), 1993, Lectins and Glycobiology, at pp. 158-174 (which
is incorporated herein by reference in its entirety).
If desired, a gene encoding an RPI, a related gene and
related nucleic acid sequences and subsequences, including
complementary sequences, can also be used in hybridization
assays. A nucleotide encoding an RPI, or subsequences
thereof comprising about at least 8 nucleotides, or the
complement of the foregoing, can be used as hybridization
probes. Hybridization assays can be used far detection,
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prognosis, diagnosis, or monitoring of conditions, disorders,
or disease states, associated with aberrant changes in RPI
gene expression, in particular RA or recrudescence of RA
following therapy. In particular, such a hybridization assay
is carried out by a method comprising contacting a sample
containing nucleic acid with a nucleic acid probe capable of
hybridizing to a DNA or RNA encoding an RPI, under conditions
such that hybridization can occur, and detecting or measuring
any resulting hybridization.
The invention also provides diagnostic kits, comprising
in one or more containers an anti-RPI antibody. In addition,
such a kit may optionally comprise one or more of the
following: (1) instructions for using the anti-RPI antibody
for diagnosis, prognosis, therapeutic monitoring, drug
development or any combination of these applications; (2) a
notice in the form prescribed by a governmental agency
regulating the manufacture, use or sale of pharmaceuticals or
biological products, which notice reflects approval by the
agency of manufacture, use or sale for human administration;
(3) a labeled binding partner to the antibody; and (4) a
solid phase (such as a reagent strip) upon which the anti-RPI
antibody is immobilized. If no labeled binding partner to
the antibody is provided, the anti-RPI antibody itself can be
labeled with a detectable marker, e.g., a chemiluminescent,
enzymatic, fluorescent, or radioactive moiety.
The invention also provides a kit comprising in one or
more containers a nucleic acid probe capable of hybridizing
to RNA encoding a distinct RPI. In a specific embodiment, a
kit can comprise in one or more containers a pair of primers
(e. g., each in the size range of 6-30 nucleotides, more
preferrably 10-20 nucleotides) that are capable of priming
amplification, -- such as by polymerase chain reaction (see
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e.g., Innis et al., 1990, PCR Protocols, Academic Press,
Inc., San Diego, CA), lipase chain reaction (see EP 320,308)
use of Q(3 replicase, cyclic probe reaction, or other methods
known in the art -- under appropriate reaction conditions of
at least a portion of a nucleic acid encoding an RPI.
Kits are also provided which allow for the detection of
a plurality of RPIs or a plurality of nucleic acids each
encoding an RPI. A kit can optionally further comprise a
predetermined amount of an isolated RPI protein or a nucleic
acid encoding an RPI, e.g., for use as a standard or control.
5.3 Use in Clinical Studies The diagnostic methods and
compositions of the present invention can assist in
monitoring a clinical study, e.g., for testing
drugs for therapy of RA. In one embodiment,
candidate molecules are tested for their ability to
restore R.ADF or RPI levels in a patient suffering
from RA towards levels found in subjects not
suffering from RA or, in a treated patient to
maintain RADF or RFI levels at or near non-RA or
serum values. The levels of one or more RADFs or
RPIs can be assayed.
In another embodiment, the methods and compositions of
the present invention are used to identify individuals with
RA when screening candidates for a clinical study; such
individuals can then be included in or excluded from the
study or can be placed in a separate cohort for treatment or
analysis.
5.4 Purification of RPIs In particular aspects, the
invention provides isolated RPIs, preferably human
RPIs, and fragments and.derivatives thereof which
comprise an antigenic determinant (i.e., can be
recognized by an antibody) or which are otherwise
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functionally active, as well as nucleic acid
sequences encoding the foregoing. "Functionally
active" RPI as used herein refers to that material
displaying one or more known functional activities
associated with a full-length (wild-type) RPI,
e.g., binding to an RPI substrate or RPI binding
partner, antigenicity (binding to an anti-target
antibody), immunogenicity, etc.
In specific embodiments, the invention provides
fragments of an RPI comprising at least 6 amino acids, 10
amino acids, 50 amino acids, or at least 75 amino acids.
Fragments, or proteins comprising fragments, lacking some or
all of the regions of an RPI are also provided. Nucleic
acids encoding the foregoing are provided.
Once a recombinant nucleic acid which expresses the RPI
gene sequence is identified, the gene product can be
analyzed. This is achieved by assays based on the physical
or functional properties of the product, including
radioactive labelling of the product followed by analysis by
gel electrophoresis, immunoassay, etc.
Once the RPI is identified, it can be isolated and
purified by standard methods including chromatography (e. g.,
ion exchange, affinity, and sizing column chromatography),
centrifugation, differential solubility, or by any other
standard technique for the purification of proteins.
Alternatively, once an RPI produced by a recombinant
nucleic acid is identified, the entire amino acid sequence of
the RPI can be deduced from the nucleotide sequence of the
chimeric gene contained in the recombinant. As a result, the
protein can be synthesized by standard chemical methods known
in the art (e. g., see Hunkapille, et al., M., 1984, Nature
x:105-111) .
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In another alternative embodiment, native RPIs can be
purified from natural sources, by standard methods such as
those described above (e. g., immunoaffinity purification).
In a preferred embodiment, RPIs are isolated by the
Preferred Technology described in U.S. Application No.
08/980,574, which is incorporated herein by reference. For
preparative-scale runs, a narrow-range "zoom gel" having a pH
range of 2 pH units or less is preferred for the isoelectric
step, according to the method described in Westermeier, 1993,
Electrophoresis in Practice (VCH, Weinheim, Germany), pp.
197-209 (which is incorporated herein by reference in its
entirety); this modification permits a larger quantity of a
target protein to be loaded onto the gel, and thereby
increases the quantity of isolated RPI that can be recovered
from the gel. When used in this way for preparative-scale
runs, the Preferred Technology typically provides up to 100
ng, and can provide up to 1000 ng, of an isolated RPI in a
single run. Those of skill in the art will appreciate that a
zoom gel can be used in any separation strategy which employs
gel isoelectric focusing.
In a specific embodiment of the present invention, such
RPIs, whether produced by recombinant DNA techniques or by
chemical synthetic methods or by purification of native
proteins, include (but are not limited to) those containing'
all or part of the amino acid sequence of the RPI, as well as
fragments and other derivatives, and analogs thereof,
including proteins homologous thereto.
5.5 Production of Antibodies to RPIs According to the
invention, an RPI, its fragments or other
derivatives, or analogs thereof, may be used as an
immunogen to generate antibodies which
immunospecifically bind such an immunogen. Such
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proteins, fragments, derivatives, or analogs can be
isolated by any convenient means, including the
methods described in the preceding section of this
application. The antibodies generated include but
are not limited to polyclonal, monoclonal,
chimeric, single chain, Fab fragments, and an Fab
expression library. In a specific embodiment,
antibodies to a human RPI are produced. In another
embodiment, antibodies to a domain of an RPI are
produced. In a specific embodiment, hydrophilic
fragments of an RPI are used as immunogens for
antibody production.
1.1
Various procedures known in the art may be used for the
production of polyclonal antibodies to an RPI or derivative
or analog. In a particular embodiment, rabbit polyclonal
antibodies to an epitope of an RPI, or a subsequence thereof,
can be obtained. For the production of antibody, various
host animals can be immunized by injection with the native
RPI, or a synthetic version, or derivative (e. g., fragment)
thereof, including but not limited to rabbits, mice, rats,
horses, goats etc. Various adjuvants may be used to increase
the immunological response, depending on the host species,
and including but not limited to complete or incomplete
Freund's adjuvant, mineral gels such as aluminum hydroxide,
surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human
adjuvants such as BCG (bacille Calmette-Guerin) and
Corynebacterium parvum.
For preparation of monoclonal antibodies directed toward
an RPI sequence or analog thereof, any technique which
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provides for the production of antibody molecules by
continuous cell lines in culture may be used. For example,
the hybridoma technique originally developed by Kohler and
Milstein (1975, Nature 256:495-497), as well as the trioma
technique, the human B-cell hybridoma technique (Kozbor et
al., 1983, Immunology Today 4_:72), and the EBV-hybridoma
technique to produce human monoclonal antibodies (Cole et
al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan
R. Liss, Inc., pp. 77-96). (Each of the foregoing references
is incorporated herein by reference.) In an additional
embodiment of the invention, monoclonal antibodies can be
produced in germ-free animals as described in PCT/US90/02545,
which is incorporated herein by reference. According to the
invention, human antibodies may be used and can be obtained
by using human hybridomas (Cote et al., 1983, Prvc. Natl.
Acad. Sci. USA 80:2026-2030) or by transforming human B cells
with EBV virus in vitro (Cole et al., 1985, in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In
fact, according to the invention, 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 specific for an RPI together with genes from a human
antibody molecule of appropriate biological activity can be
used; such antibodies are within the scope of this invention.
(Each of the foregoing references is incorporated herein by
reference.)
According to the invention, techniques described for the
production of single chain antibodies (U. S. Patent 4,946,778,
incorporated herein by reference) can be adapted to produce
RPI-specific single-chain antibodies. An additional
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embodiment of the invention utilizes the techniques described
for the construction of Fab expression libraries (Ruse et
al., 1989, Science 246:1275-1281) to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity for RPIs, derivatives, or analogs.
Antibody fragments which contain the idiotype of the
molecule can be generated by known techniques. For example,
such fragments include but are not limited to: the F(ab')2
fragment which can be produced by pepsin digestion of the
antibody molecule; the Fab' fragments which can be generated
by reducing the disulfide bridges of the F(ab')2 fragment, the
Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent, and Fv fragments.
In the production of antibodies, screening for the
desired antibody can be accomplished by techniques known in
the art, e.g., ELISA (enzyme-linked immunosorbent assay).
For example, to select antibodies which recognize a specific
domain of an RPI, one may assay generated hybridomas for a
product which binds to an RPI fragment containing such
domain. For selection of an antibody that specifically binds
a first RPI homolog but which does not specifically bind a
different RPI homolog, one can select on the basis of
positive binding to the first RPI homolog and a lack of
binding to the second RPI homolog. Similarly, for selection
of an antibody that specifically binds an RPI but which does
not specifically bind a different isoform of the same protein
(e.g., a different glycoform having the same core peptide as
the RPI), one can select on the basis of positive binding to
the RPI and a lack of binding to the different isoform (e. g.,
glycoform) .
In addition, techniques developed for the production
of "chimeric antibodies" (Morrison, et al., 1984, Proc
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Natl. Acad.Sci., 81, 6851-6855; Neuberger, et al.,
1984, Nature 312, 604-608; Takeda, et al.,
1985, Nature, 314, 452-454) by splicing the genes
from a mouse antibody molecule of appropriate
antigen specificity together with genes from a human
antibody molecule of appropriate biological activity c
an be used. A chimeris antibody is a molecule in whi
ch different portions are derived from different anim
al species, such as those having a variable
region derived from a murine mAb and a human
immunoglobulin constant region. (See, e.g., Cabilly
et al. , U. S . Patent No. 4, 8 16,567; and Boss et al. , U
.S. Patent No. 4,816397, which are incorporated
herein by reference in their entirety.)
In addition, techniques have been developed for
the production of humanized antibodies. (See, e.g.,
Queen, U.S Patent No. 5,585,089 and Winter, U.S. Pa
tent No. 5,225,539, which are incorporated
herein by reference in their entirety.) An
immunoglobulin light or heavy chain variable region
consists of a"framework" region interrupted by
three hypervariable regions, referred to as
complementarity determining regions (CDRs). The
extent of the framework region and CDRs have been
precisely defined (see, "Sequences of Proteins of
Immunological Interest", Kabat, E. et al., U.S.Depart
ment of Health and Human Services (1983)). Briefly,
humanized antibodies are antibody molecules from no
n-human species having one or more CDRs from the n
on-human species and a framework region from a
human immunoglobulin molecule.
Alternatively, techniques described for the
production of single chain antibodies (U.S. Patent 4,
946,778; Bird, 1988, Science 242, 423-426; Huston, a
t al., 1988, Proc. Natl. Acad. Sci. USA 85, 5879-588
3; and Ward, et al., 1989, Nature 334, 544- 546) can
be used. Single chain antibodies are formed by linki
ng the heavy and light chain fragments of the
Fv region via an amino acid bridge, resulting in a
single chain polypeptide.
Antibody fragments that recognize specific
epitopes may begenerated by known techniques. For
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example, such fragments include but are not limited t
o: the F(ab')2 fragments, which can be produced by p
epsin digestion of the antibody molecule and the
Fab fragments, which can be generated by reducing t
he disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries may be constr
ucted (Huse, et al., 1989, Science, 246, 1275-1281) t
o allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
LABELING OF ANTIBODIES AND USES THEREOF
Described herein are methods for detectably
labeling molecules capable of specifically
recognizing one or more RPI epitopes or epitopes of c
onserved variants or peptide fragmentsof an RPI. Th
a labeling and detection methods employed herein
may, for example, be such as those described in Harl
ow and Lane (Harlow, E. and Lane, D., 1988, "Antibo
dies: A Laboratory Manual", Cold Spring Harbor Lab
oratory Press, Cold Spring Harbor, New York),
which is incorporated herein by reference in its
entirety.
One of the ways in which the RPI-specific
antibody or peptide mimetic can be labeled is by linki
ng the same to an enzyme, such labeled molecules can
be used in an enzyme immunoassay such as
ELISA (enzyme linked immunosorbent assay).
The enzyme which is bound to the antibody will
react with an appropriate substrate, preferably a chr
omogenic substrate, in such a manner as to produce a
chemical moiety which can be detected, for example,
by spectrophotometric, fluorimetric or by visual mea
ns. Enzymes which can be used to detectably
label the antibodies, derivatives and analogs thereof,
and peptides include, but are not limited to, malate
dehydrogenase, staphylococcal nuclease, delta-5-
steroid isomerase, yeast alcoholdehydrogenase, alpha
-glycerophosphate, dehydrogenase, triosephosphate is
omerase, horseradish peroxidase, alkaline phosphatas
e, asparaginase, glucose oxidase, beta-galactosidase,
ribonuclease, urease, catalase, glucose-6-
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phosphate-dehydrogenase, glucoamylase and acetylchol
inesterase. The detection can be accomplished by col
orimetric methods whichemploy a chromogenic substr
ate for the enzyme. Detection may also be
accomplished by visual comparison of the extent of
enzymatic reaction of a substrate in comparison withs
imilarly prepared standards.
For use in detection methods, the molecules are
preferably labeled with a radioisotope, including but n
of limited to: 125I, 131I, or 99mTc. Such peptides a
nd antibodies can be detected in in vivo assays
using a radioimmunoassay (RIA) or radioprobe. The
radioactive isotope can be detected by such means ast
he use of agamma counter or a scintillation counter o
r by autoradiography.It is also possible to label the
antibodies, derivatives and analogs thereof, and pepti
des with a fluorescent compound. When
the fluorescently labeled peptide is exposed to light
of theproper wave length, its presence can then be
detected due to fluorescence. Among the most commo
nly used fluorescent labeling compounds are fluoresce
in isothiocyanate, rhodamine, phycoerythrin, phycocy
anin, allophycocyanin, o-phthaldehyde and fluorescam
ine.
It is also possible to label the antibodies,
derivatives and analogs thereof, and peptides with bi
otin. The biotin labeled peptide can be exposed to an
avidin-conjugated detectable marker, such as a fluor
escent label conjugated to avidin. Because
avidin binds with high affinity to biotin, the avidin-c
onjugated detectable marker becomes associated
with the biotin labeled peptide, thereby allowing for
the detection of the peptide.
The antibodies, derivatives and analogs thereof,
andpeptides can also be detectably labeled using
fluorescenceemitting metals such as 152Eu, or others
of the lanthanide series. These metals can be attache
d to the antibodies, derivatives andanalogs
thereof, and peptides using such metal chelating
groupsas diethylenetriaminepentacetic acid (DTPA)
or ethylenediaminetetraacetic acid (EDTA).
The antibodies, .derivatives and analogs thereof,
and peptides also can be detectably labeled by
coupling to achemiluminescent compound. The presen
ce of the chemiluminescent- tagged peptides are
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then determined by detecting the presence of luminesc
ence that arises during the course of a chemical
reaction. Examples of particularly useful chemilumi
nescent labeling compounds are luminol, isoluminol, t
heromatic acridinium ester, imidazole, acridinium sal
t and oxalate ester.
Likewise, a bioluminescent compound may be
used to label theantibodies, derivatives and analogs t
hereof, and peptides of the present invention. Biolu
minescence is a type of chemiluminescence found in b
iological systems in, which acatalytic protein increas
es the efficiency of the chemiluminescent reaction. T
he presence of a bioluminescent protein is
determined by detecting the presence ofluminescence
Important bioluminescent compounds for purposes o
f labeling areluciferin, luciferase and aequorin.
The labeled antibodies that are determined to
specifically bind an RPI can be used to detect the
presence of the RPI in a variety of biological samples
including a body fluid (e.g.,plasma, urine, cerebros
pinal fluid, joint aspirate), a tissue
sample, or homogenate thereof. Such labeled
antibodies may also be used to visualize the
localization of RPIs within or on individual cells.
The labeled antibodies that are determined to sp
ecifically
bind an RPI can be administered to a patient at diagn
ostically
effective doses to detect the presence of an RPI. A
diagnostically effective dose refers to that amount o
f the
molecule sufficient to target a diagnostic to a cell c
ontaining
an RPI on its surface such that the cell can be detect
ed using
methods commonly available in the art.
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Antibodies specific to a domain of an RPI are also
provided.
The foregoing antibodies can be used in methods known in
the art relating to the localization and activity of the RPIs
of the invention, e.g., for imaging these proteins, measuring
levels thereof in appropriate physiological samples, in
diagnostic methods, etc.
5.6 Isolation Of DNA Encoding An RPI Specific
embodiments for the cloning of an RPI gene, are
presented below by way of example and not of
limitation.
The nucleotide sequences of the present invention,
including DNA and RNA, and comprising a sequence encoding the
RPI or a fragment or analog thereof, may be synthesized using
methods known in the art, such as using conventional chemical
approaches or polymerase chain reaction (PCR) amplification
of overlapping oligonucleotides. The sequences also provide
for the identification and cloning of the RPI gene from any
species, for instance for screening cDNA libraries, genomic
libraries or expression libraries.
The nucleotide sequences comprising a sequence encoding
an RPI of the present invention are useful for their ability
to selectively form duplex molecules with complementary
stretches of other protein genes. Depending on the
application, a variety of hybridization conditions may be
employed to achieve varying sequence identities.
For a high degree of selectivity, relatively stringent
conditions are used to form the duplexes, such as low salt or
high temperature conditions. As used herein, "highly
stringent conditions" means hybridization to filter-bound DNA
in 0.5 M NaHP04, 7~ sodium dodecyl sulfate (SDS), 1 mM EDTA at
65°C, and washing in O.lxSSC/0.1~ SDS at 68°C (Ausubel F.M. et
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al., eds., 1989, Current Protocols in Molecular Biology, Vol.
I, Green Publishing Associates, Inc., and John Wiley & sons,
Inc., New York, at p. 2.10.3; incorporated herein by
reference in its entirety.) For some applications, less
stringent hybridization conditions are required. As used
herein "moderately stringent conditions" means washing in
0.2xSSC/0.1% SDS at 42°C (Ausubel et al., 1989, supra).
Hybridization conditions can also be rendered more stringent
by the addition of increasing amounts of formamide, to
destabilize the hybrid duplex. Thus, particular
hybridization conditions can be readily manipulated, and will
generally be chosen depending on the desired results. For
example, convenient hybridization temperatures in the
presence of 50% formamide are: 42°C for a probe which is 95
to 100% homologous to the RPI gene fragment, 37°C for 90 to
95% homology and 32°C for 70 to 90% homology.
In the preparation of genomic libraries, DNA fragments
are generated, some of which will encode a part or the whole
of an RPI. The DNA may be cleaved at specific sites using
various restriction enzymes. Alternatively, one may use
DNase in the presence of manganese to fragment the DNA, or
the DNA can be physically sheared, as for example, by
sonication. The DNA fragments can then be separated
according to size by standard techniques, including but not
limited to, agarose and polyacrylamide gel electrophoresis,
column chromatography and sucrose gradient centrifugation.
The DNA fragments can then be inserted into suitable vectors,
including but not limited to plasmids, cosmids,
bacteriophages lambda or T4, and yeast artificial chromosome
(YAC). (See, for example, Sambrook et al., 1989, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory Press,' Cold Spring Harbor, New York; Glover, D.M.
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(ed.), 1985, DNA Cloning: A Practical Approach, MRL Press,
Ltd., Oxford, U.K. Vol. I, II; Ausubel F.M. et al., eds.,
1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing Associates, Inc., and John Wiley & sons, Inc., New
Yark). The genomic library may be screened by nucleic acid
hybridization to labeled probe (Benton and Davis, 1977,
Science 196:180; Grunstein and Hogness, 1975, Proc. Natl.
Acad. Sci. USA 72:3961) .
The genomic libraries may be screened with labeled
degenerate oligonucleotide probes corresponding to the amino
acid sequence of any peptide of the RPI using optimal
approaches well known in the art. Any probe used preferably
is 10 nucleotides or longer, more preferably 15 nucleotides
or longer.
As shown in Tables VIII to XI above, some RPIs disclosed
herein correspond to previously identified proteins encoded
by genes whose sequences are publicly known. To screen such
a gene, any probe may be used that is complementary to the
gene or its complement; preferably the probe is l0
nucleotides or longer, more preferably 15 nucleotides or
longer. The Entrez database held by the National Center for
Biotechnology Information (NCBI) -- which is accessible at
http://www.ncbi.nlm.nih.gov/ -- provides gene sequences for
these RPIs under the following accession numbers, and each
sequence is incorporated herein by reference:
Table XII. Gene sequences of RPI-related proteins
RADF # RPI Accession numbers
RADF-1 RPI-1 T40090, T40068
RADF-3 RPI-3 AA551927, AA260531, W97741, N99366,
T70526, T40177, T40060, T40034, T39910,
~ T39894
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RADF-4 RPI-4 T41010, T40102, T40058, T39954
RADF-6 RPI-5 AA269874
RADF-8 RPI-6 220858, AA503766, H51308, H03365
RADF-9 RPI-7 220858, AA503766, H51308, H03365
RADF-10 RPI-8 221022, 219947
RADF-11 RPI-9 T41063, T41020, T41005, T40881, T40190,
T40139, T40125, T40114, T40096, T39908.
RADF-12 RPI-11 T40090, T40068
RADF-13 RPI-12 AA551927, AA260531, W97741, N99366,
T70526, T40177, T40060, T40034, T39910,
T39894
RADF-14 RPI-13 T40090, T40068
RADF-15 RPI-15 T40940, T40002
RADF-16 RPI-16 T73244, T71043, T71032, T40181, T40116
RADF-18 RPI-21 N99641, N99445, N99528, 220485, 220465
RADF-19 RPI-22 AA269874
.RADF-20 RPI-23 220858, AA503766, H51308, H03365
RADF-22 RPI-24 T1952, H73939, 220894, T40182, T40167,
T40158
RADF-23 RPI-25 AA614684, AA523377, AA715907, AA580429,
AA630254, AA617854, AA580356
RADF-24 RPI-26 AA268201, T64416, T62149, T40186
RADF-25 RPI-27 221017, 220888, 219984, 219971, T41056,
T40108
RADF-26 RPI-28 T1952, H73939, 220894, T40182, T40167,
T40158
RADF-26 RPI-29 221017, 220888, 219984, 219971, T41056,
T40108
RADF-26 RPI-30 T64707
RADF-27 RPI-31 220858, AA503766, H51308, H03365
RADF-32 RPI-33 T40090, T40068
RADF-33 RPI-34 T40090, T40068
RADF-35 RPI-36 221022, 219947
~ ~
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RADF-36 ~ RPI-37 ~ Z20858, AA503766, H51308, H03365
For each of RPI-10, RPI-17, and RPI-20, a degenerate set
of probes is provided, as follows:
(a) Probes for RPI-10
5'- A C G A C C T T T A T G T T C T A T A G C A G C A A C -3'
G T G G G C C G G G G
T T A A T T T
C C G C C C
(b) Probes for RPI-17
5'- A C G A G C A T G A G G A G C A T C T A T A A G A T T -3'
G T G G G G G C G G G
T T T T A A T
C C C C G C
(c) Probes for RPI-20
5' - A G A T A T A T C A G C A C C A G A A T C -3'
G C T C G G G G G C T G
T A A T T T
C G C C C
Clones in libraries with insert DNA encoding the RPI or
fragments thereof will hybridize to one or more of the
degenerate oligonucleotide probes (or their complement).
Hybridization of such oligonucleotide probes to genomic
libraries are carried out using methods known in the art.
For example, hybridization with one of the above-mentioned
degenerate sets of aligonucleotide probes, or their
complement (or with any member of such a set, or its
complement) can be performed under highly stringent or
moderately stringent conditions as defined above, or can be
carried out in 2X SSC, 1.0~ SDS at 50° C and washed using the
same conditions.
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In yet another aspect, clones of nucleotide sequences
encoding a part or the entire RPI or RPI-derived polypeptides
may also be obtained by screening expression libraries. For
example, DNA from the relevant source is isolated and random
fragments are prepared and ligated into an expression vector
(e. g., a bacteriophage, plasmid, phagemid or cosmid) such
that the inserted sequence in the vector is capable of being
expressed by the host cell into which the vector is then
introduced. various screening assays can then be used to
select for the expressed RPI or RPI-derived polypeptides. In
one embodiment, the various anti-RPI antibodies of the
invention can be used to identify the desired clones using
methods known in the art. See, for example, Harlow and Lane,
1988, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, Appendix IV.
Clones or plaques from the library are brought into contact
with the antibodies to identify those clones that bind.
In an embodiment, colonies or plaques containing DNA
that encodes an RPI or RPI-derived polypeptide can be
detected using DYNA Beads according to Olsvick et al., 29th
ICAAC, Houston, Tex. 1989, incorporated herein by reference.
Anti-RPI antibodies are crosslinked to tosylated DYNA Beads
M280, and these antibody-containing beads would then be used
to adsorb to colonies or plaques expressing RPI or RPI-
derived polypeptide. Colonies or plaques expressing an RPI
or RPI-derived polypeptide are identified as any of those
that bind the beads.
Alternatively, the anti-RPI antibodies can be
nonspecifically immobilized to a suitable support, such as
silica or CeliteTM resin. This material would then be used to
adsorb to bacterial colonies expressing the RPI protein or
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RPI-derived polypeptide as described in the preceding
paragraph.
In another aspect, PCR amplification may be used to
produce substantially pure DNA encoding a part of or the
whole of an RPI from genomic DNA. Oligonucleotide primers,
degenerate or otherwise, corresponding to known RPI sequences
can be used as primers.
PCR can be carried out, e.g., by use of a Perkin-Elmer
Cetus thermal cycler and Taq polymerase (Gene AmpT~). One can
choose to synthesize several different degenerate primers,
for use in the PCR reactions. It is also possible to vary
the stringency of hybridization conditions used in priming
the PCR reactions, to allow for greater or lesser degrees of
nucleotide sequence similarity between the degenerate primers
and the corresponding sequences in the DNA. After successful
amplification of a segment of the sequence encoding an RPI,
that segment may be molecularly cloned and sequenced, and
utilized as a probe to isolate a complete genomic clone.
This, in turn, will permit the determination of the gene's
complete nucleotide sequence, the analysis of its expression,
and the production of its protein product for functional
analysis, as described infra.
The RPI gene can also be identified by mRNA selection by
nucleic acid hybridization followed by in vitro translation.
In this procedure, fragments are used to isolate
complementary mRNAs by hybridization. Such DNA fragments may
represent available, purified RPI DNA of another species
(e.g., mouse, human). Immunoprecipitation analysis or
functional assays (e. g., aggregation ability in vitro;
binding to receptor) of the in vitro translation products of
the isolated products of the isolated mRNAs identifies the
mRNA and, therefore, the complementary DNA fragments that
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contain the desired sequences. In addition, specific mRNAs
may be selected by adsorption of polysomes isolated from
cells to immobilized antibodies specifically directed against
an RPI. A radiolabelled RPI cDNA can be synthesized using
the selected mRNA (from the adsorbed polysomes) as a
template. The radiolabelled mRNA or cDNA may then be used as
a probe to identify the RPI DNA fragments from among other
genomic DNA fragments.
Alternatives to isolating RPI genomic DNA include, but
are not limited to, chemically synthesizing the gene sequence
itself from a known sequence or making cDNA to the mRNA which
encodes the RPI. For example, RNA for cDNA cloning of the
RPI gene can be isolated from cells which express the RPI.
Other methods are possible and within the scope of the
invention.
Any eukaryotic cell potentially can serve as the nucleic
acid source for the molecular cloning of the RPI gene. The
nucleic acid sequences encoding the RPI can be isolated from
vertebrate, mammalian, human, porcine, bovine, feline, avian,
equine, canine, as well as additional primate sources,
insects, plants, etc. The DNA may be obtained by standard
procedures known in the art from cloned DNA (e.g., a DNA
"library"), by chemical synthesis, by cDNA cloning, or by the
cloning of genomic DNA, or fragments thereof, purified from
the desired cell. (See, for example, Sambrook et al., 1989,
Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York;
Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach,
MRL Press, Ltd., Oxford, U.K. Vol. I, II.) Clones derived
from genomic DNA may contain regulatory and intron DNA
regions in addition to coding regions; clones derived from
cDNA will contain only exon sequences. Whatever the source,
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the RPI gene should be molecularly cloned into a suitable
vector for propagation.
The identified and isolated gene or cDNA can then be
inserted into an appropriate cloning vector. A large number
of vector-host systems known in the art may be used.
Possible vectors include, but are not limited to, plasmids or
modified viruses, but the vector system must be compatible
with the host cell used. Such vectors include, but are not
limited to, bacteriophages such as lambda derivatives, or
plasmids such as PBR322 or pUC plasmid derivatives or the
Bluescript vector (Stratagene). The insertion into a cloning
vector can, for example, be accomplished by ligating the DNA
fragment into a cloning vector which has complementary
cohesive termini. However, if the complementary restriction
sites used to fragment the DNA are not present in the cloning
vector, the ends of the DNA molecules may be enzymatically
modified. Alternatively, any site desired may be produced by
ligating nucleotide sequences (linkers) onto the DNA termini;
these ligated linkers may comprise specific chemically
synthesized oligonucleotides encoding restriction
endonuclease recognition sequences. In an alternative
method, the cleaved vector and RPI gene may be modified. by
homopolymeric tailing. Recombinant molecules can be
introduced into host cells via transformation, transfection,
infection, electroporation, etc., so that many copies of the
gene sequence are generated.
In specific embodiments, transformation of host cells
with recombinant DNA molecules that incorporate the isolated
RPI gene, cDNA, or synthesized DNA sequence enables
generation of multiple copies of the gene. Thus, the gene
may be obtained in large quantities by growing transformants,
isolating the recombinant DNA molecules from the
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transformants and, when necessary, retrieving the inserted
gene from the isolated recombinant DNA.
The RPI sequences provided by the instant invention
include those nucleotide sequences encoding substantially the
same amino acid sequences as found in native RPIs, and those
encoded amino acid sequences with functionally equivalent
amino acids, as well as those encoding other target
derivatives or analogs.
5.7 Expression of DNA Encodincr an RPI The nucleotide
sequence encoding an RPI or a functionally active
analog or fragment or other derivative thereof can
be inserted into an appropriate expression vector,
i.e., a vector which contains the necessary
elements for the transcription and translation of
the inserted protein-coding sequence. The
necessary transcriptional and translational signals
can also be supplied by the native RPI gene or its
flanking regions. A variety of host-vector systems
may be utilized to express the protein-coding
sequence. These include but are not limited to
mammalian cell systems infected with virus (e. g.,
vaccinia virus, adenovirus, etc.); insect cell
systems infected with virus (e. g., baculovirus);
microorganisms such as yeast containing yeast
vectors, or bacteria transformed with
bacteriophage, DNA, plasmid DNA, or cosmid DNA.
The expression elements of vectors vary in their
strengths and specificities. Depending on the
host-vector system utilized, any one of a number of
suitable transcription and translation elements may
be used. In specific embodiments, the human RPI
gene is expressed, or a sequence encoding a
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functionally active portion of the human RPI. In
yet another embodiment, a fragment of target
comprising a domain of the RPI is expressed.
1.1
1.1
Any of the methods previously described for the
insertion of DNA fragments into a vector may be used to
construct expression vectors containing a chimeric gene
consisting of appropriate transcriptional and translational
control signals and the protein coding sequences. These
methods may include in vitro recombinant DNA and synthetic
techniques and in vivo recombinants (genetic recombination).
Expression of nucleic acid sequence encoding an RPI or
peptide fragment may be regulated by a second nucleic acid
sequence so that the RPI or peptide is expressed in a host
transformed with the recombinant DNA molecule. For example,
expression of an RPI may be controlled by any promoter or
enhancer element known in the art. Promoters which may be
used to control RPI gene expression include, but are not
limited to, the SV40 early promoter region (Bernoist and
Chambon, 1981, Nature 290:304-310), the promoter contained in
the 3' long terminal repeat of Rous sarcoma virus (Yamamoto,
et al., 1980, Cell 22:787-797), the herpes thymidine kinase
promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA
78:1441-1445), the regulatory sequences of the
metallothionein gene (Brinster et al., 1982, Nature 296:39-
42); prokaryotic expression vectors such as the p-lactamase
promoter (Villa-Kamaroff et al., 1978, Proc. Nat.. Acad. Sci.
USA 75:3727-3731), or the tac promoter (DeBoer et al., 1983,
Proc. Natl. Acad. Sci. USA 80:21-25); see also "Useful
proteins from recombinant bacteria" in Scientific American,
1980, 242:74-94; plant expression vectors comprising the
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nopaline synthetase promoter region (Herrera-Estrella et al.,
Nature 303:209-213) or the cauliflower mosaic virus 35S RNA
promoter (Gardner et al., 1981, Nucl. Acids Res. 9:2871), and
the promoter of the photosynthetic enzyme ribulose
biphosphate carboxylase {Herrera-Estrella et al., 1984,
Nature 310:115-120); promoter elements from yeast or other
fungi such as the Gal 4 promoter, the ADC (alcohol
dehydrogenase) promoter, PGK (phosphoglycerol kinase)
promoter, alkaline phosphatase promoter, and the following
animal transcriptional control regions, which exhibit tissue
specificity and have been utilized in transgenic animals:
elastase I gene control region which is active in pancreatic
acinar cells {Swift et al., 1984, Cell 38:639-646; Ornitz et
al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409;
MacDonald, 1987, Hepatology 7:425-515); insulin gene control
region which is active in pancreatic beta cells (Hanahan,
1985, Nature 315:115-122), immunoglobulin gene control region
which is active in lymphoid cells (Grosschedl et al,, 1984,
Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;
Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse
mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al.,
1986, Cell 45:485-495), albumin gene control region which is
active in liver (Pinkert et al., 1987, Genes and Devel.
1:268-276), alpha-fetoprotein gene control region which is
active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.
5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-
antitrypsin gene control region which is active in the liver
(Kelsey et al., 1987, Genes and Devel. 1_:161-171), beta-
globin gene control region which is active in myeloid cells
(Mogram et al., 1985, Nature 315:338-340; Kollias et al.,
1986, CeI1 46:89-94; myelin basic protein gene control region
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which is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-712); myosin light chain-
2 gene control region which is active in skeletal muscle
(Sani, 1985, Nature 314:283-286), and gonadotropic releasing
hormone gene control region which is active in the
hypothalamus (Mason et al., 1986, Science 234:1372-1378).
In a specific embodiment, a vector is used that
comprises a promoter operably linked to an RPI-encoding
nucleic acid, one or more origins of replication, and,
optionally, one or more selectable markers (e.g., an
antibiotic resistance gene).
In a specific embodiment, an expression construct is
made by subcloning an RPI coding sequence into the EcoRI
restriction site of each of the three pGEX vectors
(Glutathione S-Transferase expression vectors; Smith and
Johnson, 1988, Gene 7:31-40). This allows for the expression
of the RPI product from the subclone in the correct reading
f rame .
Expression vectors containing RPI gene inserts can be
identified by three general approaches: (a) nucleic acid
hybridization, (b) presence or absence of "marker" gene
functions, and (c) expression of inserted sequences. In the
first approach, the presence of an RPI gene inserted in an
expression vector can be detected by nucleic acid
hybridization using probes comprising sequences that are
homologous to an inserted RPI gene. In the second approach,
the recombinant 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, transformation phenotype,
occlusion body formation in baculovirus, ete.) caused by the
insertion of an RPI gene in the vector. For example; if the
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RPI gene is inserted within the marker gene sequence of the
vector, recombinants containing the RPI gene insert can be
identified by the absence of the marker gene function. In
the third approach, recombinant expression vectors can be
identified by assaying the RPI gene product expressed by the
recombinant. Such assays can be based, for example, on the
physical or functional properties of the RPI in in vitro
assay systems, e.g., binding with anti-RPI antibody.
Once a particular recombinant DNA molecule is identified
and isolated, several methods known in the art may be used to
propagate it. Once a suitable host system and growth
conditions are established, recombinant expression vectors
can be propagated and prepared in quantity. As previously
explained, the expression vectors which can be used include,
but are not limited to, the following vectors or their
derivatives: human or animal viruses such as vaccinia virus
or adenovirus; insect viruses such as baculovirus; yeast
vectors; bacteriophage vectors (e. g., lambda), and plasmid
and cosmid DNA vectors, to name but a few.
In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or
modifies and processes the gene product in the specific
fashion desired. Expression from certain promoters can be
elevated in the presence of certain inducers; thus,
expression of the genetically engineered RPI may be
controlled. Furthermore, different host cells have
characteristic and specific mechanisms for the translational
and post-translational processing and modification (e. g.,
glycosylation, phosphorylation of proteins). Appropriate
cell lines or host systems can be chosen to ensure the
desired modification and processing of the foreign protein
expressed. For example, expression in a bacterial system can
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be used to produce an unglycosylated core protein product.
Expression in yeast will produce a glycosylated product.
Expression in mammalian cells can be used to ensure "native"
glycosylation of a heterologous protein. Furthermore,
different vector/host expression systems may effect
processing reactions to different extents.
For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell
lines which stably express the differentially expressed or
pathway gene protein may be engineered. Rather than using
expression vectors which contain viral origins of
replication, host cells can be transformed with DNA
controlled by appropriate expression control elements (e. g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker.
Following the introduction of the foreign DNA, engineered
cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers
resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form
foci which in turn can be cloned and expanded into cell
lines. This method may advantageously be used to engineer
cell lines which express the differentially expressed or
pathway gene protein. Such engineered cell lines may be
particularly useful in screening and evaluation of compounds
that affect the endogenous activity of the differentially
expressed or pathway gene protein.
A number of selection systems may be used, including but
not limited to the herpes simplex virus thymidine kinase
(Wigler, et al., 1977, Cell 11:223}, hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
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Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817)
genes can be employed in tk~, hgprt- or aprt- cells,
respectively. Also, antimetabolite resistance can be used as
the basis of selection for dhfr, which confers resistance to
methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA
77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072);
neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1); and
hygro, which confers resistance to hygromycin (Santerre et
al., 1984, Gene 30:147) genes.
In other specific embodiments, the RPI, fragment,
analog, or derivative may be expressed as a fusion, or
chimeric protein product (comprising the protein, fragment,
analog, or derivative joined via a peptide bond to a
heterologous protein sequence (of a different protein)).
Such a chimeric product can be made by ligating the
appropriate nucleic acid sequences encoding the desired amino
acid sequences to each other by methods known in the art, in
the proper coding frame, and expressing the chimeric product
by methods commonly known in the art. Alternatively, such a
chimeric product may be made by protein synthetic techniques,
e.g., by use of a peptide synthesizer.
Both cDNA and genomic sequences can be cloned and
expressed.
5.8 Therapeutic Use Of RPIs The invention provides for
treatment or prevention of various diseases and
disorders by administration of a therapeutic
compound. Such compounds include but are not
limited to: RPIs and analogs and derivatives
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(including fragments) thereof (e. g., as described
herein); antibodies thereto (as described herein);
nucleic acids encoding RPIs, analogs, or
derivatives (e. g., as described herein); RPI gene
antisense nucleic acids, and RPI gene agonists and
antagonists. As is described herein, an important
feature of the present invention is the
identification of RPT genes involved in rheumatoid
arthritis. Arthritis can be treated or prevented
by administration of a therapeutic compound that
promotes function or expression of RPIs which are
decreased in synovial fluid verses serum of RA
patients. Arthritis can also be treated or
prevented by administration of a therapeutic
compound that reduces function or expression of
RPIs which are increased in synovial fluid verses
serum of RA patients.
Generally, administration of products of a species
origin or species reactivity (in the case of antibodies) that
is the same species as that of the patient is preferred.
Thus, in a preferred embodiment, a human RPI, derivative, or
analog, or nucleic acid, or an antibody to a human RPT, is
administered to a human patient for therapy or prophylaxis.
5.8.1 Treatment And Prevention
Of Rheumatoid Arthritis
Rheumatoid arthritis is treated or prevented by
administration of a compound that promotes (i.e., increases
or supplies) the level or function of one or more RPIs -- or
the level of one or more RADFs -- that are decreased in
synovial fluid verses serum of subjects with RA. Examples of
such a compound include but are not limited to RPIs,
derivatives, or fragments that are functionally active,
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particularly that are active as demonstrated in in vitro
assays or in animal models, and nucleic acids encoding an RPI
or functionally active derivative or fragment thereof (e. g.,
for use in gene therapy). Other compounds that can be used,
e.g., RPI agonists, can be identified using in vitro assays.
Rheumatoid arthritis is also treated or prevented by
administration of a compound that inhibits (i.e., decreases)
the level or function of one or more RPIs -- or the level of
one or more RADFs -- that exhibit increased abundance in
synovial fluid verses serum of subjects with RA. Examples of
such a compound include but are not limited to RPI anti-sense
oligonucleotides, ribozymes, or antibodies directed against
RPIs. Other compounds that can be used, e.g., RPI
antagonists, can be identified using in vitro assays.
In specific embodiments, compounds that promote the
level or function of one or more RPIs, or the level of one or
more RADFs are administered therapeutically (including
prophylactically when an absent or decreased (relative to
normal or desired) RPI level or function, or RADF level has
been identified in synovial fluid of RA patients as compared
with serum in RA patients. In further embodiments, compounds
that inhibit RPI level or function, or RADF level are
administered therapeutically (including prophylactically when
an increased (relative to normal or desired) RPI level or
function, or RADF level has been identified in synovial fluid
of RA patients as compared with serum in RA patients. The
change in RPI function or level, or RADF level due to the
administration of such compounds can be readily detected,
e.g., by obtaining a patient tissue sample (e. g., from biopsy
tissue) and assaying it in vitro for RNA or protein levels,
or activity of the expressed RPI RNA or protein. The
Preferred Technology can also be used to detect levels of the
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RPI or RADF before and after the administration of the
compound. Many methods standard in the art can be thus
employed, including but not limited to kinase assays,
immunoassays to detect and/or visualize the RPI (e. g.,
Western blot, immunoprecipitation followed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis,
immunocytochemistry, etc.) and/or hybridization assays to
detect RPI expression by detecting and/or visualizing mRNA
encoding the RPI (e. g., Northern assays, dot blots, in situ
hybridization, etc.), etc.
The compounds of the invention include but are not
limited to any compound, e.g., a small organic molecule,
protein, peptide, antibody, nucleic acid, etc. that restores
the RA RPI or RADF profile towards normal with the proviso
that such compound is not a non-steroidal anti-inflammatory
agent (NSAID) (e. g. prednisone, ibuprofen, fenoprofen,
ketoprofen, flurbiprofen, indomethacin, sulindac, aspirin,
salicylsalicylic acid, diflunisal, naproxen, piroxicam,
tenoxicam, phenylbutazone, oxyphenbutazone), a gold salt, D-
penicillamine, an antimalarial such as hydroxychloroquine or
sulphasalazine, azathioprine, cyclophosphamide, chlorambucil,
methotrexate, a corticosteroid, anti-CD4 monoclonal antibody,
or anti-CDw52 antibody.
5.8.2 Gene Theranv In a specific embodiment;
nucleic acids comprising a sequence encoding
an RPI or functional derivative thereof, are
administered to promote RPI function, by way
of gene therapy. Gene therapy refers to
therapy performed by the administration to a
subject of an expressed or expressible nucleic
acid. In this embodiment of the invention,
the nucleic acid produces its encoded protein
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that mediates a therapeutic effect by
promoting RPI function.
Any of the methods for gene therapy available in the art
can be used according to the present invention. Exemplary
methods are described below.
For general reviews of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and
Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev.
Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science
260:926-932; and Morgan and~Anderson, 1993, Ann. Rev.
Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215).
Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al.
(eds.), 1993, Current Protocols in Molecular Biology, John
Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY.
In a preferred aspect, the compound comprises a nucleic
acid encoding an RPI, said nucleic acid being part of an
expression vector that expresses an RPI or fragment or
chimeric protein thereof in a suitable host. In particular,
such a nucleic acid has a promoter operably linked to the RPI
coding region, said promoter being inducible or constitutive,
and, optionally, tissue-specific. In another particular
embodiment, a nucleic acid molecule is used in which the RPI
coding sequences and any other desired sequences are flanked
by regions that promote homologous recombination at a desired
site in the genome, thus providing for intrachromosomal
expression of the RPI nucleic acid (Koller and Smithies,
1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et
al., 1989, Nature 342:435-438).
Delivery of the nucleic acid into a patient may be
either direct, in which case the patient is directly exposed
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to the nucleic acid or nucleic acid-carrying vector, or
indirect, in which case, cells are first transformed with the
nucleic acid in vitro, then transplanted into the patient.
These two approaches are known, respectively, as in vivo or
ex vivo gene therapy.
In a specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing it as part of
an appropriate nucleic acid expression vector and
administering it so that it becomes intracellular, e.g., by
infection using a defective or attenuated retroviral or other
viral vector (see U.S. Patent No. 4,980,286), or by direct
injection of naked DNA, or by use of microparticle
bombardment (e. g., a gene gun; Biolistic, Dupont), or coating
with lipids or cell-surface receptors or transfecting agents,
encapsulation in liposomes, microparticles, or microcapsules,
or by administering it in linkage to a peptide which is known
to enter the nucleus, by administering it in linkage to a
ligand subject to receptor-mediated endocytosis (see, e.g.,
Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be
used to target cell types specifically expressing the
receptors), etc. In another embodiment, a nucleic acid-
ligand complex can be formed in which the ligand comprises a
fusogenic viral peptide to disrupt endosomes, allowing the
nucleic acid to avoid lysosomal degradation. In yet another
embodiment, the nucleic acid can be targeted in vivo for cell
specific uptake and expression, by targeting a specific
receptor (see, e.g., PCT Publications WO 92/06180 dated April
16, 1992 {Wu et al.); WO 92/22635 dated December 23, 1992
(Wilson et al.); W092/20316 dated November 26, 1992 (Findeis
et al.); W093/14188 dated July 22, 1993 (Clarke et al.), WO
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93/20221 dated October 14, 1993 (Young)). Alternatively, the
nucleic acid can be introduced intracellularly and
incorporated within host cell DNA for expression, by
homologous recombination (Koller and Smithies, 1989, Proc.
Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989,
Nature 342:435-438).
In a specific embodiment, a viral vector that contains a
nucleic acid encoding an RPI is used. For example, a
retroviral vector can be used (see Miller et al., 1993, Meth.
Enzymol. 217:581-599). These retroviral vectors have been
modified to delete retroviral sequences that are not
necessary for packaging of the viral genome and integration
into host cell DNA. The nucleic acid encoding the RPI to be
used in gene therapy is cloned into the vector, which
facilitates delivery of the gene into a patient. Mare detail
about retroviral vectors can be found in Boesen et al., 1994,
Biotherapy 6:291-302, which describes the use of a_ retroviral
vector to deliver the mdrl gene to hematopoietic stem cells
in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., 1994,
J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood
83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy
4:129-141; and Grossman and Wilson, 1993, Curr. Op.in. in
Genetics and Devel. 3:110-114.
Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive
vehicles for delivering genes to respiratory epithelia.
Adenoviruses naturally infect respiratory epithelia where
they cause a mild disease. Other targets for adenovirus-
based delivery systems are liver, the central nervous system,
endothelial cells, and muscle. Adenoviruses have the
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advantage of being capable of infecting non-dividing cells.
Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based
gene therapy. Bout et al., 1994, Human Gene Therapy 5_:3-10
demonstrated the use of adenovirus vectors to transfer genes
to the respiratory epithelia of rhesus monkeys. Other
instances of the use of adenoviruses in gene therapy can be
found in Rosenfeld et al., 1991, Science 252:431-434;
Rosenfeld et al., 1992, CeI3 68:143-155; Mastrangeli et al.,
1993, J. Clin. Invest. 91:225-234; PCT Publication
W094/12649; and Wang, et al., 1995, Gene Therapy 2:775-783.
Adeno-associated virus (AAV) has also been proposed for
use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp.
Biol. Med. 204:289-300; U.S. Patent No. 5,436,146).
Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as
electroporation, lipofection, calcium phosphate mediated
transfection, or viral infection. Usually, the method of
transfer includes the transfer of a selectable marker to the
cells. The cells are then placed under selection to isolate
those cells that have taken up and are expressing the
transferred gene. Those cells are then delivered to a
patient.
In this embodiment, the nucleic acid is introduced into
a cell prior to administration in vivo of the resulting
recombinant cell. Such introduction can be carried out by
any method known in the art, including but not limited to
transfection, electroporation, microinjection, infection with
a viral or bacteriophage vector containing the nucleic acid
sequences, cell fusion, chromosome-mediated gene transfer,
microcell-mediated gene transfer, spheroplast fusion, etc.
Numerous techniques are known in the art for the introduction
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of foreign genes into cells (see, e.g., Loeffler and Behr,
1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993, Meth.
Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92)
and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The
technique should provide for the stable transfer of the
nucleic acid to the cell, so that the nucleic acid is
expressible by the cell and preferably heritable and
expressible by its cell progeny.
The resulting recombinant cells can be delivered to a
patient by various methods known in the art. In a preferred
embodiment, epithelial cells are injected, e.g.,
subcutaneously. In another embodiment, recombinant skin
cells may be applied as a skin graft onto the patient.
Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously.
The amount of cells envisioned for use depends on the
desired effect, patient state, etc., and can be determined by
one skilled in the art.
Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available
cell type, and include but are not limited to epithelial
cells, endothelial cells, keratinocytes, fibroblasts, muscle
cells, hepatocytes; blood cells such as T lymphocytes,
B lymphocytes, monocytes, macrophages, neutrophils,
eosinophils, megakaryocytes, granulocytes; various stem or
progenitor cells, in particular hematopoietic stem or
progenitor cells, e.g., as obtained from bone marrow,
umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene
therapy is autologous to the patient.
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In an embodiment in which recombinant cells axe used in
gene therapy, a nucleic acid encoding an RPI is introduced
into the cells such that it is expressible by the cells or
their progeny, and the recombinant cells are then
administered in vivo for therapeutic effect. In a specific
embodiment, stem or progenitor cells are used. Any stem
and/or progenitor cells which can be isolated and maintained
in vitro can potentially be used in accordance with this
embodiment of the present invention (see e.g. PCT Publication
WO 94/08598, dated April 28, 1994; Stemple and Anderson,
1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio.
21A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc.
61 : 771 ) .
In a specific embodiment, the nucleic acid to be
introduced for purposes of gene therapy comprises an
inducible promoter operably linked to the coding region, such
that expression of the nucleic acid is controllable by
controlling the presence or absence of the appropriate
inducer of transcription.
5.8.3 Inhibition Of RPIs To
Treat RheumatcZ~.d Arthritis
l.l.l
In one embodiment of the invention, RA is treated or
prevented by administration of a compound that antagonizes
(inhibits) the RPI levels and/or function of RPIs which are
elevated in synovial fluid compared with serum of patients
suffering from RA. Compounds that can be used include but
are not limited to anti-RPI antibodies (and fragments and
derivatives thereof containing the binding region thereof),
RPI antisense or ribozyme nucleic acids, and nucleic acids
encoding dysfunctional RPIs that are used to "knockout"
endogenous RPI function by homologous recombination (see,
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e.g., Capecchi, 1989, Science 24,:1288-1292). In a specific
embodiment of the invention, a nucleic acid containing a
portion of an RPI gene in which nucleotides encoding an RPI
flank (are both 5' and 3' to) a different gene sequence, is
used, as an RPI antagonist, to promote RPI inactivation by
homologous recombination (see also Koller and Smithies, 1989,
Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al.,
1989, Nature 342:435-438). Other compounds that inhibit RPI
function can be identified by use of known convenient in
vitro assays, e.g., based on their ability to inhibit binding
of RPIs to another protein, or inhibit any known RPI
function, as preferably assayed in vitro or in cell culture,
although genetic assays may also be employed. The Preferred
Technology can also be useful for detecting levels of the RPI
before and after the administration of the compound.
Preferably, suitable in vitro or in vivo assays, are utilized
to determine the effect of a specific compound and whether
its administration is indicated for treatment of the affected
tissue.
In specific embodiments, compounds that inhibit RPI
function are administered therapeutically (including
prophylactically) when an increased level of an RPI or RPI
function (e. g. greater than the normal level or desired
level) is detected in synovial fluid compared with serum of a
patient suffering from RA. The increased levels in RPI or
function can be readily detected, e.g., by quantifying
protein and/or RNA, by obtaining patient synovial fluid and
serum samples and assaying them in vitro for RNA or protein
levels, structure and/or activity of the expressed RNA
encoding the RPI, or the RPI itself. Many methods standard
in the art can be thus employed, including but not limited to
kinase assays, immunoassays to detect and/or visualize RPI
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(e. g., Western blot, immunoprecipitation followed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis,
immunocytochemistry, etc.) and/or hybridization assays to
detect RPI expression by detecting and/or visualizing
respectively mRNA encoding the RPI (e. g., Northern assays,
dot blots, in situ hybridization, etc.), etc.
5.8.4 Antisense Recrulation of RPIs
1.1.1
In a specific embodiment, RPI function is inhibited by
use of RPI antisense nucleic acids. The present invention
provides the therapeutic or prophylactic use of nucleic acids
comprising at least six nucleotides that are antisense to a
gene or cDNA encoding an RPI or a portion thereof. As used
herein, an RPI "antisense" nucleic acid refers to a nucleic
acid capable of hybridizing to a portion of an RNA encoding
an RPI (preferably mRNA) by virtue of some sequence
camplementarity. The antisense nucleic acid may be
complementary to a coding and/or noncoding region of an mRNA
encoding an RPI. Such antisense nucleic acids have utility
as compounds that inhibit RPI function, and can be used in
the treatment or prevention of rheumatoid arthritis.
The antisense nucleic acids of the invention can be
oligonucleotides that are double-stranded or single-stranded,
RNA or DNA or a modification or derivative thereof, which can
be directly administered to a cell, or which can be produced
intracellularly by transcription of exogenous, introduced
sequences.
The invention further provides pharmaceutical
compositions comprising an effective amount of the RPI
antisense nucleic acids of the invention in a
pharmaceutically acceptable carrier, as described infra.
In another embodiment, the invention is directed to
methods for inhibiting the expression of an RPI nucleic acid
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sequence in a prokaryotic or eukaryotic cell comprising
providing the cell with an effective amount of a composition
comprising an RPI antisense nucleic acid of the invention.
RPI antisense nucleic acids and their uses are described
in detail below.
RPI Antisense Nucleic Acids The RPT antisense nucleic
acids are of at least six nucleotides and are preferably
oligonucleotides (ranging from 6 to about 50
oligonucleotides). In specific aspects, the oligonucleotide
is at least 10 nucleotides, at least 15 nucleotides, at least
100 nucleotides, or at least 200 nucleotides. The
oligonucleotides can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the
base moiety, sugar moiety, or phosphate backbone. The
oligonucleotide may include other appending groups such as
peptides, or agents facilitating transport across the cell
membrane (see, e.g., Letsinger et al., 1989, Proc. Natl.
Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc.
Natl. Acad. Sci. 84:648-652; PCT Publication No. WO 88/09810,
published December 15, 1988) or blood-brain barrier {see,
e.g., PCT Publication No. WO 89/10134, published April 25,
1988), hybridization-triggered cleavage agents (see, e.g.,
Krol et al., 1988, BioTechniques 6:958-976) or intercalating
agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549).
In a preferred aspect of the invention, an RPI antisense
oligonucleotide is provided, preferably of single-stranded
DNA. The oligonucleotide may be modified at any position on
its structure with substituents generally known in the art.
The RPI antisense oligonucleotide may comprise at least
one modified base moiety which is selected from the group
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including but not limited to 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 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-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine, 2-thiocytosine, S-methyl-2-thiouracil,
2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-
5-oxyacetic acid methylester, uracil-S-oxyacetic acid (v),
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)
uracil, (acp3)w, and 2,6-diaminopurine.
In another embodiment, the oligonucleotide comprises at
least one modified sugar moiety, e.g. a sugar moiety selected
from the group consisting of arabinose, 2-fluoroarabinose,
xylulose, and hexose.
In yet another embodiment, the oligonucleotide comprises
at least one modified phosphate backbone selected from the
group consisting of a phosphorothioate, a phosphorodithioate,
a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the oligonucleotide is an
a-anomeric oligonucleotide. An a-anomeric oligonucleotide
forms specific double-stranded hybrids with complementary RNA
in which, contrary to the usual (3-units, the strands run
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parallel to each other (Gautier et al., 1987, Nucl. Acids
Res. 15:6625-6641).
The oligonucleotide may be conjugated to another
molecule, e.g., a peptide, hybridization triggered cross-
linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
Oligonucleotides of the invention may 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 may be synthesized by the
method of Stein et al. (1988, Nucl. Acids Res. 16:3209),
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.
In a specific embodiment, the RPI antisense nucleic acid
of the invention is produced intracellularly by transcription
from an exogenous sequence. Fox example, a vector can be
introduced in vivo such that it is taken up by a cell, within
which cell the vector or a portion thereof is transcribed,
producing an antisense nucleic acid (RNA) of the invention.
Such a vector would contain a sequence encoding the RPI
antisense nucleic acid. Such a vector can remain episomal or
become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such
vectors can be constructed by recombinant DNA technology
methods standard in the art. Vectors can be plasmid, viral,
or others known in the art, used for replication and
expression in mammalian cells. Expression of the sequence
encoding the RPI antisense RNA can be by any promoter known
in the art to act in mammalian, preferably human, cells.
Such promoters can be inducible or constitutive. Such
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promoters include but are not limited to: the SV40 early
promoter region (Bernoist and Chambon, 1981, Nature 290:304-
310), the promoter contained in the 3' long terminal repeat of
Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797),
the herpes thymidine kinase promoter (Wagner et al., 1981,
Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory
sequences of the metallothionein gene (Brinster et al., 1982,
Nature 296:39-42), etc.
The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA
transcript of an RPI gene, preferably a human RPI gene.
However, absolute complementarity, although preferred, is not
required. A sequence "complementary to at least a portion of
an RNA," as referred to herein, means a sequence having
sufficient complementarity to be able to hybridize with the
RNA, forming a stable duplex; in the case of double-stranded
RPI antisense nucleic acids, a single strand of the duplex
DNA may thus be tested, or triplex formation may be assayed.
The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid.
Generally, the longer the hybridizing nucleic acid, the more
base mismatches with an RNA encoding an RPI it may contain
and still form a stable duplex (or triplex, as the case may
be). One skilled in the art can ascertain a tolerable degree
of mismatch by use of standard procedures to determine the
melting point of the hybridized complex.
5.8.5 Therapeutic Use Of RPI
Antisense Nucleic Acids
The RPI antisense nucleic acids can be used to treat (or
prevent) rheumatoid arthritis when the RPI of interest is
overexpressed in the synovial fluid patients suffering from
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rheumatoid arthritis. In a preferred embodiment, a single-
stranded DNA antisense RPI oligonucleotide is used.
Cell types which express or overexpress RNA encoding an
RPI can be identified by various methods known in the art.
Such cell types include but are not limited to leukocytes
(e. g., neutrophils, macrophages, monocytes) and resident
cells (e.g., synoviocytes). Such methods include, but are
not limited to, hybridization with an RPI-specific nucleic
acid (e. g., by Northern hybridization, dot blot
hybridization, in situ hybridization), observing the ability
of RNA from the cell type to be translated in vitro into an
RPI, immunoassay, etc. In a preferred aspect, primary tissue
from a patient can be assayed for RPI expression prior to
treatment, e.g., by immunocytochemistry or in situ
hybridization.
Pharmaceutical compositions of the invention, comprising
an effective amount of an RPI antisense nucleic acid in a
pharmaceutically acceptable carrier, can be administered to a
patient having rheumatoid arthritis.
The amount of RPI antisense nucleic acid which will be
effective in the treatment of RA can be determined by
standard clinical techniques. Where possible, it is
desirable to determine the antisense cytotoxicity of the
tumor type to be treated in vitro, and then in useful animal
model systems prior to testing and use in humans.
In a specific embodiment, pharmaceutical compositions
comprising RPI antisense nucleic acids are administered via
liposomes, microparticles, or microcapsules. In various
embodiments of the invention, it may be useful to use such
compositions to achieve sustained release of the RPI
antisense nucleic acids. In a specific embodiment, it may be
desirable to utilize liposomes targeted via antibodies to
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specific identifiable tumor antigens (Leonetti et al., 1990,
Proc. Natl. Acad. Sci. USA 87:2448-2451; Renneisen et al.,
1990, J. Biol. Chem. 265:16337-16342).
5.8.6 Inhibitory Ribozyme And
Triple Helix Approaches
Tn another embodiment, symptoms of RA may be ameliorated
by decreasing the level of RPI gene expression and/or RPI
gene product activity by using RPI gene sequences in
conjunction with well-known gene "knock-out," ribozyme and/or
triple helix methods to decrease the level of RPI gene
expression. Among the compounds that may exhibit the ability
to modulate the activity, expression or synthesis of the RPI
gene, including the ability to ameliorate the symptoms of a
RA, are ribozyme, and triple helix molecules. Such molecules
may be designed to reduce or inhibit either unimpaired, or if
appropriate, mutant target gene activity. Techniques for the
production and use of such molecules are well known to those
of skill in the art.
Ribozyme molecules designed to catalytically cleave RPI
gene mRNA transcripts can be used to prevent translation of
target gene mRNA and, therefore, expression of RPI gene
product. (See, e.g., PCT International Publication
W090/11364, published October 4, 1990; Sarver et al., 1990,
Science x:1222-1225).
Ribozymes are enzymatic RNA molecules capable of
catalyzing the specific cleavage of RNA. (For a review, see
Rossi, 1994, Current Biology 4, 469-471). The mechanism of
ribozyme action involves sequence specific hybridization of
the ribozyme molecule to complementary target RNA, followed
by an endonucleolytic cleavage event. The composition of
ribozyme molecules must include one or more sequences
complementary to the target gene mRNA, and must include the
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well known catalytic sequence responsible for mRNA cleavage.
For this sequence, see, e.g., U.S. Patent No. 5,093,246,
which is incorporated herein by reference in its entirety.
While ribozymes that cleave mRNA at site specific
recognition sequences can be used to destroy mRNAs encoding
ari RPI, the use of hammerhead ribozymes is preferred.
Hammerhead ribozyrnes cleave mRNAs at locations dictated by
flanking regions that form complementary base pairs with the
target mRNA. The sole requirement is that the target mRNA
have the following sequence of two bases: 5'-UG-3'. The
construction and production of hammerhead ribozymes is well
known in the art and is described more fully in Myers, 1995,
Molecular Biology and Biotechno.~ogy: A Comprehensive Desk
Reference, VCH Publishers, New York, (see especially Figure
4, page 833) and in Haseloff and Gerlach, 1988, Nature, 334,
585-591, which is incorporated herein by reference in its
entirety.
Preferably the ribozyme is engineered so that the
cleavage recognition site is located near the 5' end of the
mRNA encoding the RPI, i.e., to increase efficiency and
minimize the intracellular accumulation of non-functional
mRNA transcripts.
The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as
the one that occurs naturally in Tetrahymena thermophila
(known as the IVS, or L-19 IVS RNA) and that has been
extensively described by Thomas Cech and collaborators (Zaug,
et a.l., 1984, Science, 224, 574-578; Zaug and Cech, 1986,
Science, 231, 470-475; Zaug, et al., 1986, Nature, 324, 429-
433; published International patent application No. WO
88/04300 by University Patents Inc.; Been and Cech, 1986,
Cell, 47, 207-216). The Cech-type ribozymes have an eight
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base pair active site which hybridizes to.a target RNA
sequence whereafter cleavage of the target RNA takes place.
The invention encompasses those Cech-type ribozymes which
target eight base-pair active site sequences that are present
in the gene encoding the RPI.
As in the antisense approach, the ribozymes can be
composed of modified oligonucleotides (e. g., for improved
stability, targeting, etc.) and should be delivered to cells
that express the RPI in vivo. A preferred method of delivery
involves using a DNA construct "encoding" the ribozyme under
the control of a strong constitutive pol III or pol II
promoter, so that transfected cells will produce sufficient
quantities of the ribozyme to destroy endogenous mRNA
encoding the RPI and inhibit translation. Because ribozymes,
unlike antisense molecules, are catalytic, a lower
intracellular concentration is required for efficiency.
Endogenous RPI expression can also be reduced by
inactivating or "knocking out" the RPI gene or its promoter
using targeted homologous recombination (e. g., see Smithies,
et al., 1985, Nature 317:230-234; Thomas and Capecchi, 1987,
Cell x:503-512; Thompson et al., 1989, CeII 5:313-321; each
of which is incorporated by reference herein in its
entirety). For example, a mutant, non-functional RPI gene
(or a completely unrelated DNA sequence) flanked by DNA
homologous to the endogenous RPI gene (either the coding
regions or regulatory regions of the gene encoding the RPI)
can be used, with or without a selectable marker and/or a
negative selectable marker, to transfect cells that express
the target gene in vivo. Insertion of the DNA construct, via
targeted homologous recombination, results in inactivation of
the target gene. Such approaches are particularly suited in
the agricultural field where modifications to ES (embryonic
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stem) cells can be used to generate animal offspring with an
inactive target gene (e.g., see Thomas and Capecchi, 1987 and
Thompson, 1989, supra). However this approach can be adapted
for use in humans provided the recombinant DNA constructs are
directly administered or targeted to the required site in
vivo using appropriate viral vectors.
Alternatively, endogenous RPI gene expression can be
reduced by targeting deoxyribonucleotide sequences
complementary to the regulatory region of the RPI gene (i.e.,
the RPI gene promoter and/or enhancers) to form triple
helical structures that prevent transcription of the RPI gene
in target cells in the body. (See generally, Helene, 1991,
Anticancer Drug Des., 6(6}, 569-584; Helene, et al., 1992,
Ann. N.Y. Acad. Sci., 660, 27-36; and Maher, 1992, Bioassays
14(12), 807-815).
Nucleic acid molecules to be used in triplex helix
formation for the inhibition of transcription should be
single stranded and composed of deoxynucleotides. The base
composition of these oligonucleotides must be designed to
promote triple helix formation via Hoogsteen base pairing
rules, which generally require sizeable stretches of either
purines or pyrimidines to be present on one strand of a
duplex. ,Nucleotide sequences may be pyrimidine-based, which
will result in TAT and CGC' triplets across the three
associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarity to a
purine-rich region of a single strand of the duplex in a
parallel orientation to that strand. In addition, nucleic
acid molecules may be chosen that are purine-rich, for
example, contain a stretch of G residues. These molecules
will form a triple helix with a DNA duplex that is rich in GC
pairs, in which the majority of the purine residues are
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located on a single strand of the targeted duplex, resulting
in GGC triplets across the three strands in the triplex.
Alternatively, the potential sequences that can be
targeted for triple helix formation may be increased by
creating a so called "switchback" nucleic acid molecule.
Switchback molecules are synthesized in an alternating 5'-3',
3'-5' manner, such that they base pair with first one strand
of a duplex and then the other, eliminating the necessity for
a sizeable stretch of either purines or pyrimidines to be
present on one strand of a duplex.
In instances wherein the antisense, ribozyme, and/or
triple helix molecules described herein are utilized to
inhibit mutant gene expression, it is possible that the
technique may so efficiently reduce or inhibit the
transcription (triple helix) and/or translation (antisense,
ribozyme) of mRNA produced by normal RPI gene alleles that
the possibility may arise wherein the concentration of normal
RPI gene product present may be lower than is necessary for a
normal phenotype. In such cases, to ensure that
substantially normal levels of RPI gene activity are
maintained, therefore, nucleic acid molecules that encode and
express RPI gene polypeptides exhibiting normal RPI gene
activity may, be introduced into cells via gene therapy
methods that do not contain sequences susceptible to whatever
antisense, ribozyme, or triple helix treatments are being
utilized. Alternatively, in instances whereby the RPI gene
encodes an extracellular protein, it may be preferable to co-
administer normal RPI gene protein in order to maintain the
requisite level of target gene activity.
Anti-sense RNA and DNA, ribozyme, and triple helix
molecules of the invention may be prepared by any method
known in the art for the synthesis of DNA and RNA molecules,
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as discussed above. These include techniques for chemically
synthesizing oligodeoxyribonucleotides and
oligoribonucleotides well known in the art such as for
example solid phase phosphoramidite chemical synthesis.
Alternatively, RNA molecules may be generated by in vitro and
in vivo transcription of DNA sequences encoding the antisense
RNA molecule. Such DNA sequences may be incorporated into a
wide variety of vectors that incorporate suitable RNA
polymerase promoters such as the T7 or SP6 polymerase
promoters. Alternatively, antisense cDNA constructs that
synthesize antisense RNA constitutively or inducibly,
depending on the promoter used, can be introduced stably into
cell lines.
5.9 Demonstration Of Therapeutic Or Prophylactic
Utilitv
The compounds of the invention are preferably tested in
vitro, and then in vivo for the desired therapeutic or
prophylactic activity, prior to use in humans. For example,
in vitro assays which can be used to determine whether
administration of a specific compound is indicated, include
in vitro cell culture assays in which a patient tissue sample
is grown in culture, and exposed to or otherwise administered
a compound, and the effect of such compound upon the tissue
sample is observed.
Test compounds can be tested for their ability to
restore RADF or RPI levels in patients suffering from RA
towards levels found in subjects not suffering from RA or to
produce similar changes in experimental animal models of RA
(e.g., adjuvant arthritis in rats). Compounds able to
restore said levels can be used as lead compounds for further
drug discovery, or used therapeutically. RADF and RPI
expression can be assayed by the Preferred Technology,
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immunoassays, gel electrophoresis followed by visualization,
or any other method known to those skilled in the art. Such
assays can be used to screen candidate drugs or in clinical
monitoring or drug development, where abundance of an RADF or
RPI can serve as a surrogate marker for clinical disease.
In various specific embodiments, in vitro assays can be
carried out with representative cells of cell types involved
in a patient's disorder, to determine if a compound has a
desired effect upon such cell types.
Compounds for use in therapy can be tested in suitable
animal model systems prior to testing in humans, including
but not limited to rats, mice, chicken, cows, monkeys,
rabbits, etc. For in vivo testing, prior to administration
to humans, any animal model system known in the art may be
used.
5.10 Therapeutic/Prophylactic
Administration And Compositions
The invention provides methods of treatment (and
prophylaxis) by administration to a subject of an effective
amount of a compound of the invention. In a preferred
aspect, the compound is substantially purified (e. g.
substantially free from substances that limit its effect or
produce undesired side-effects). The subject is preferably
an animal, including but not limited to animals such as cows;
pigs, horses, chickens, cats, dogs, etc., and is preferably a
mammal, and most preferably human. In a specific embodiment,
a non-human mammal is the subj ect .
Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid are
described above; additional appropriate formulations and
routes of administration can be selected from among those
described hereinbelow.
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Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation
in liposomes, microparticles, microcapsules, recombinant
cells capable of expressing the compound, receptor-mediated
endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem.
262:4429-4432), construction of a nucleic acid as part of a
retroviral or other vector, etc. Methods of introduction
include but are not limited to intradermal, intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal,
epidural, and oral routes. The compounds may be administered
by any convenient route, for example by infusion or bolus
injection, by absorption through epithelial or mucocutaneous
linings (e. g., oral mucosa, rectal and intestinal mucosa,
etc.) and may be administered together with other
biologically active agents. Administration can be systemic
or local. In addition, it may be desirable to introduce the
pharmaceutical compositions of the invention into the central
nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter,
for example, attached to a reservoir, such as an Omrnaya
reservoir. Pulmonary administration can also be employed,
e.g., by use of an inhaler or nebulizer, and formulation with
an aerosolizing agent.
In a specific embodiment, it may be desirable to
administer the pharmaceutical compositions of the invention
locally to the area in need of treatment; this may be
achieved by, for example, and not by way of limitation, local
infusion during surgery, topical application, e.g., in
conjunction with a wound dressing after surgery, by
injection, by means of a catheter, by means of a suppository,
or by means of an implant, said implant being of a porous,
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non-porous, or gelatinous material, including membranes, such
as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or
former site) of a malignant tumor or neoplastic or pre-
neoplastic tissue.
In another embodiment, the compound can be delivered in
a vesicle, in particular a liposome (see Langer, Science
249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-365 {1989);
Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
In yet another embodiment, the compound can be delivered
in a controlled release system. In one embodiment, a pump
may be used (see Langer, supra; Sefton, CRC Crit. Ref.
Biomed. Eng. 14;201 (1987); Buchwald et al., Surgery 88:507
(1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In
another embodiment, polymeric materials can be used (see
Medical Applications of Controlled Release, Langer and Wise
(eds.), CRC Pres., Boca Raton, Florida (1974); Controlled
Drug Bioavailability, Drug Product Design and Performance,
Smolen and Ball (eds.), Wiley, New York (1984); Ranger and
Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983);
see also Levy et al., Science 28:190 (1985); During et al.,
Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)). In yet another embodiment, a controlled
release system can be placed in proximity of the therapeutic
target, i.e., the brain, thus requiring only a fraction of
the systemic dose (see, e.g., Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)).
Other controlled release systems are discussed in the
review by Langer (Science 249:1527-1533 {1990)).
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In a specific embodiment where the compound of the
invention is a nucleic acid encoding a protein, the nucleic
acid can be administered in vivo to promote expression of its
encoded protein, by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that
it becomes intracellular, e.g., by use of a retroviral vector
(see U.S. Patent No. 4,980,286), or by direct injection, or
by use of microparticle bombardment (e. g., a gene gun;
Biolistic, Dupont), or coating with lipids or cell-surface
receptors ar transfecting agents, or by administering it in
linkage to a homeobox-like peptide which is known to enter
the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.
Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid
can be introduced intracellularly and incorporated within
host cell DNA for expression, by homologous recombination.
The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically
acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory
agency of the Federal or a state government or listed in the
U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals, and more particularly in humans. The
term "carrier" refers to a diluent, adjuvant, excipient, or
vehicle with which the therapeutic is administered. Such
pharmaceutical carriers can be sterile liquids, such as water
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral
oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered
intravenously. Saline solutions and aqueous dextrose and
glycerol solutions can also be employed as liquid carriers,
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particularly for injectable solutions. Suitable
pharmaceutical excipients include starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol,
water, ethanol and the like. The composition, if desired,
can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. These compositions can take
the form of solutions, suspensions, emulsion, tablets, pills,
capsules, powders, sustained-release formulations and the
like. The composition can be formulated as a suppository,
with traditional binders and carriers such as triglycerides.
Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate,
etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E.W.
Martin. Such compositions will contain a therapeutically
effective amount of the compound, preferably in purified
form, together with a suitable amount of carrier so as to
provide the form for proper administration to the patient.
The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated
in accordance with routine procedures as a pharmaceutical
composition adapted for intravenous administration to human
beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous
buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocaine
to ease pain at the site of the injection. Generally, the
ingredients are supplied either separately or mixed together
in unit dosage form, for example, as a dry lyophilized powder
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or water free concentrate in a hermetically sealed container
such as an ampoule or sachette indicating the quantity of
active agent. Where the composition is to be administered by
infusion, it can be dispensed with an infusion bottle
containing sterile pharmaceutical grade water or saline.
Where the composition is administered by injection, an
ampoule of sterile water for injection or saline can be
provided so that the ingredients may be mixed prior to
administration.
The compounds of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts
include those formed with free amino groups such as those
derived from hydrochloric, phosphoric, acetic, oxalic,
tartaric acids, etc., and those formed with free carboxyl
groups such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine,
etc.
The amount of the compound of the invention which will
be effective in the treatment of RA can be determined by
standard clinical techniques. In addition, in vitro assays
may optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the formulation
will also depend on the route of administration, and the
seriousness of the disease or disorder, and should be decided
according to the judgment of the practitioner and each
patient s circumstances. However, suitable dosage ranges for
intravenous administration are generally about 20-500
micrograms of active compound per kilogram body weight.
Suitable dosage ranges for intranasal administration are
generally about 0.01 pg/kg body weight to 1 mg/kg body
weight. Effective doses may be extrapolated from dose-
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response curves derived from .in vitro or animal model test
systems.
Suppositories generally contain active ingredient in the
range of 0.5~ to 10~ by weight; oral formulations preferably
contain 10~s to 95~ active ingredient.
The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of
the ingredients of the pharmaceutical compositions of the
invention. Optionally associated with such containers) can
be a notice in the form prescribed by a governmental agency
regulating the manufacture, use or sale of pharmaceuticals or
biological products, which notice reflects approval by the
agency of manufacture, use or sale for human administration.
6 EXAMPLE: PROTEINS FROM SERUM AND SYNOVIAL
FLUID OF PATIENTS WITH RA
Using the following Reference Protocol, proteins in
serum and synovial fluid from patients with rheumatoid
arthritis (RA) or from patients without RA (i.e., patients
with gout, osteoarthritis, or traumatic synovitis) were
separated by isoelectric focusing followed by SDS-PAGE and
compared. Each sample was run in duplicate.
6.1 Sample preparation
1.1
A protein assay was carried out on the sample as
received (Pierce BCA Cat # 23225). A volume of serum
corresponding to 300~.g of total protein was aliquoted and an
equal volume of 10~ (w/v) SDS (Fluka 71729), 2.3~ (w/v)
dithiothreitol (BDH 443852A) was added. The sample was
heated at 95°C for 5 mins, and then allowed to cool to 20°C .
1251 of the following buffer was then added to the sample:
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8M urea (BDH 452043w )
4~ CHAPS (Sigma C3023)
65mM dithiotheitol (DTT)
2~ (v/v) Resolytes 3.5-10 (BDH 44338 2x)
This mixture was vortexed, and centrifuged at 13000 rpm for 5
mins at 15°C, and the supernatant was analyzed by isoelectric
focusing.
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6.2 Isoelectric Focusincr
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Use in Clinical Studies The diagnostic methods and
compositions of the present invention can assist in
monitoring a clinical study, e.g., for testing drugs for
therapy of RA. In one embodiment, candidate molecules are
tested for their ability to restore RADF or RPI levels in a
patient suffering from RA towards levels found in subjects
not suffering from RA or, in a treated patient to maintain
RADF or RFI levels at or near non-RA or serum values. The
levels of one or more RADFs or RPIs can be assayed. In another
embodiment, the methods and compositions of the present
invention are used to identify individuals with RA when
screening candidates for a clinical study; such individuals
can then be included in or excluded from the study or can be
placed in a separate cohort for treatment or
analysis.Purification of RPIs In particular aspects, the
invention provides isolated RPIs, preferably human RPIs, and
fragments and derivatives thereof which comprise an antigenic
determinant (i.e., can be recognized by an antibody) or which
are otherwise functionally-active, as well as nucleic acid
sequences encoding the foregoing. Functionallyactive RPI as
used herein refers to that material displaying one or more
known functional activities associated with a full-length
(wild-type) RPI, e.g., binding to an RPI substrate or RPI
binding partner, antigenicity (binding to an anti-target
antibody), immunogenicity, etc. In specific embodiments,
the invention provides fragments of an RPI comprising at
least 6 amino acids, 10 amino acids, 50 amino acids, or at
least 75 amino acids. Fragments, or proteins comprising
fragments, lacking some or all of the regions of an RPI are
also provided. Nucleic acids encoding the foregoing are
provided.Once a recombinant nucleic acid which expresses the
RPI gene sequence is identified, the gene product can be
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analyzed. This is achieved by assays based on the physical
or functional properties of the product, including
radioactive labelling of the product followed by analysis by
gel electrophoresis, immunoassay, etc.Once the RPI is
identified, it can be isolated and purified by standard
methods including chromatography (e. g., ion exchange,
affinity, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique
for the purification of proteins.Alternatively, once an RPI
produced by a recombinant nucleic acid is identified, the
entire amino acid sequence of the RPI can be deduced from the
nucleotide sequence of the chimeric gene contained in the
recombinant. As a result, the protein can be synthesized by
standard chemical methods known in the art (e.g., see
Hunkapille, et al., M., 1984, Nature 310105-111). In another
alternative embodiment, native RPIs can be purified from
natural sources, by standard methods such as those described
above (e. g., immunoaffinity purification). In a preferred
embodiment, RPIs are isolated by the Preferred Technology
described in U.S. Application No. 08/980,574, which is
incorporated herein by reference. For preparative-scale
runs, a narrow-range zoomgel having a pH range of 2 pH units
or less is preferred for the isoelectric step, according to
the method described in Westermeier, 1993, Electrophoresis in
Practice (vCH, Weinheim, Germany}, pp. 197-209 (which is
incorporated herein by reference in its entirety); this
modification permits a larger quantity of a target protein to
be loaded onto the gel, and thereby increases the quantity of
isolated RPI that can be recovered from the gel. When used
in this way for preparative-scale runs, the Preferred
Technology typically provides up to 100 ng, and can provide
up to 1000 ng, of an isolated RPI in a single run. Those of
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skill in the art will appreciate that a zoom gel can be used
in any separation strategy which employs gel isoelectric
focusing. In a specific embodiment of the present invention,
such RPIs, whether produced by recombinant DNA techniques or
by chemical synthetic methods or by purification of native
proteins, include (but are not limited to) those containing
all or part of the amino acid sequence of the RPI, as well as
fragments and other derivatives, and analogs thereof,
including proteins homologous thereto.Production of
Antibodies to RPIs According to the invention, an RPI, its
fragments or other derivatives, or analogs thereof, may be
used as an immunogen to generate antibodies which
immunospecifically bind such an immunogen. Such proteins,
fragments, derivatives, or analogs can be isolated by any
convenient means, including the methods described in the
preceding section of this application. The antibodies
generated include but are not limited to polyclonal,
monoclonal, chimeric, single chain, Fab fragments, and an Fab
expression library. In a specific embodiment, antibodies to
a human RPI are produced. In another embodiment, antibodies
to a domain of an RPI are produced. In a specific
embodiment, hydrophilic fragments of an RPI are used as
immunogens for antibody production. Various procedures known
in the art may be used for the production of polyclonal
antibodies to an RPI or derivative or analog. In a
particular embodiment, rabbit polyclonal antibodies to an
epitope of an RPI, or a subsequence thereof, can be obtained.
For the production of antibody, various host animals can be
immunized by injection with the native RPI, or a synthetic
version, or derivative (e. g., fragment) thereof, including
but not limited to rabbits, mice, rats, horses, goats etc.
Various adjuvants may be used to increase the immunological
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response, depending on the host species, and including but
not limited to complete or incomplete Freund~s adjuvant,
mineral gels such as aluminum hydroxide, surface active
substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human
adjuvants such as BCG (bacille Calmette-Guerin) and
Corynebacterium parvum.For preparation of monoclonal
antibodies directed toward an RPI sequence or analog thereof,
any technique which provides for the production of antibody
molecules by continuous cell lines in culture may be used.
For example, the hybridoma technique originally developed by
Kohler and Milstein (1975, Nature 256495-497), as well as the
trioma technique, the human B-cell hybridoma technique
(Kozbor et al., 1983, Immunology Today 472), and the EBV-
hybridoma technique to produce human monoclonal antibodies
(Cole et al., 1985, in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96). (Each of the
foregoing references is incorporated herein by reference.)
In an additional embodiment of the invention, monoclonal
antibodies can be produced in germ-free animals as described
in PCT/US90/02545, which is incorporated herein by reference.
According to the invention, human antibodies may be used and
can be obtained by using human hybridomas (Cote et al., 1983',
Proc. Natl. Acad. Sci. USA 8-002026-2030) or by transforming
human B cells with EBV virus in vitro (Cole et al., 1985, in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp.
77-96). In fact, according to the invention, techniques
developed for the production of chimericantibodies (Morrison
et al., 1984, Proc. Natl. Acad. Sc.i. USA 816851-6855;
Neuberger et al., 1984, Nature 312604-608; Takeda et al.,
1985, Nature 314452-454) by splicing the genes from a mouse
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antibody molecule specific for an RPI together with genes
from a human antibody molecule of appropriate biological
activity can be used; such antibodies are within the scope of
this invention. (Each of the foregoing references is
incorporated herein by reference.)According to the invention,
techniques described for the production of single chain
antibodies (U.S. Patent 4,946,778, incorporated herein by
reference) can be adapted to produce RPI-specific single-
chain antibodies. An additional embodiment of the invention
utilizes the techniques described for the construction of Fab
expression libraries (Huse et al., 1989, Science 2461275-
1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity for RPIs,
derivatives, or analogs.Antibody fragments which contain the
idiotype of the molecule can be generated by known
techniques. For example, such fragments include but are not
limited to the F(ab')2 fragment which can be produced by
pepsin digestion of the antibody molecule; the Fab'.fragments
which can be generated by reducing the disulfide bridges of
the F(ab')z fragment, the Fab fragments which can be generated
by treating the antibody molecule with papain and a reducing
agent, and Fv fragments. In the production of antibodies,
screening for the desired antibody can be accomplished by
techniques known in the art, e.g., ELISA (enzyme-linked
immunosorbent assay). For example, to select antibodies
which recognize a specific domain of an RPI, one may assay
generated hybridomas for a product which binds to an RPI
fragment containing such domain. For selection of an
antibody that specifically binds a first RPI homolog but
which does not specifically bind a different RPI homolog, one
can select on the basis of positive binding to the first RPI
homolog and a lack of binding to the second RPI homolog.
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Similarly, for selection of an antibody that specifically
binds an RPI but which does not specifically bind a different
isoform of the same protein (e. g., a different glycoform
having the same core peptide as the RPI), one can select on
the basis of positive binding to the RPI and a lack of
binding to the different isoform (e. g., glycoform).Antibadies
specific to a domain of an RPI are also provided. The
foregoing antibodies can be used in methods known in the art
relating to the localization and activity of the RPIs of the
invention, e.g., for imaging these proteins, measuring levels
thereof in appropriate physiological samples, in diagnostic
methods, etc.Isolation Of DNA Encoding An RPI Specific
embodiments for the cloning of an RPI gene, are presented
below by way of example and not of limitation. The nucleotide
sequences of the present invention, including DNA and RNA,
and comprising a sequence encoding the RPI or a fragment or
analog thereof, may be synthesized using methods known in the
art, such as using conventional chemical approaches or
polymerase chain reaction (PCR) amplification of overlapping
oligonucleotides. The sequences~also provide for the
identification and cloning of the RPI gene from any species,
for instance for screening cDNA libraries, genomic libraries
or expression libraries.The nucleotide sequences comprising a
sequence encoding an RPI of the present invention are useful
for their ability to selectively form duplex molecules with
complementary stretches of other protein genes. Depending on
the application, a variety of hybridization conditions may be
employed to achieve varying sequence identities. For a high
degree of selectivity, relatively stringent conditions are
used to form the duplexes, such as low salt or high
temperature conditions. As used herein,
highlystringentconditions means hybridization to filter-bound
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DNA in 0.5 M NaHP04, 7~ sodium dodecyl sulfate (SDS), 1 mM
EDTA at 65°C, and washing in O.lxSSC/0.1~ SDS at 68°C
(Ausubel
F.M. et al., eds., 1989, Current Protocols in Molecular
Biology, Vol. I, Green Publishing Associates, Inc., and John
Wiley & sons, Inc., New York, at p. 2.10.3; incorporated
herein by reference in its entirety.) For some applications,
less stringent hybridization conditions are required. As
used herein moderatelystringentconditions means washing in
0.2xSSC/0.1~ SDS at 42°C (Ausubel et al., 1989, supra).
Hybridization conditions can also be rendered more stringent
by the addition of increasing amounts of formamide, to
destabilize the hybrid duplex. Thus, particular
hybridization conditions can be readily manipulated, and will
generally be chosen depending on the desired results. For
example, convenient hybridization temperatures in the
presence of 50~ formamide are 42°C for a probe which is 95
to 100 homologous to the RPI gene fragment, 37°C for 90 to
95~ homology and 32°C for 70 to 90~ homology.In the
preparation of genomic libraries, DNA fragments are
generated, some of which will encode a part or the whole of
an RPI. The DNA may be cleaved at specific sites using
various restriction enzymes. Alternatively, one may use
DNase in the presence of manganese to fragment the DNA, or
the DNA can be physically sheared, as for example, by
sonication. The DNA fragments can then be separated
according to size by standard techniques, including but not
limited to, agarose and polyacrylamide gel electrophoresis,
column chromatography and sucrose gradient centrifugation.
The DNA fragments can then be inserted into suitable vectors,
including but not limited to plasmids, cosmids,
bacteriophages lambda or T4, and yeast artificial chromosome
(YAC). (See, for example, Sambrook et al., 1989, Molecular
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Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York; Glover, D.M.
(ed.), 1985, DNA Cloning A Practical Approach, MRL Press,
Ltd., Oxford, U.K. Vol. I, II; Ausubel F.M. et al., eds.,
1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing Associates, Inc., and John Wiley & sons, Inc., New
York). The genomic library may be screened by nucleic acid
hybridization to labeled probe (Benton and Davis, 1977,
Science 196180; Grunstein and Hogness, 1975, Proc. Natl.
Acad. Sci. USA 723961). The genomic libraries may be
screened with labeled degenerate oligonucleotide probes
corresponding to the amino acid sequence of any peptide of
the RPI using optimal approaches well known in the art. Any
probe used preferably is 10 nucleotides or longer, more
preferably 15 nucleotides or longer. As shown in Tables VIII
to XI above, some RPIs disclosed herein correspond to
previously identified proteins encoded by genes whose
sequences are publicly known. To screen such a gene, any
probe may be used that is complementary to the gene or its
complement; preferably the probe is 10 nucleotides or longer,
more preferably 15 nucleotides or longer. The Entrez
database held by the National Center for Biotechnology
Information (NCBI) -- which is accessible at
http//www.ncbi.nlm.nih.gov/ -- provides gene sequences for
these RPIs under the following accession numbers, and each
sequence is incorporated herein by referenceTable XII. Gene
sequences of RPI-related proteinsRADF #RPIAccession
numbersRADF-1 RPI-1 T40090, T40068RADF-3 RPI-3 AA551927,
AA260531, W97741, N99366, T70526, T40177, T40060, T40034,
T39910, T39894RADF-4 RPI-4 T41010, T40102, T40058,
T39954RADF-6 RPI-5 AA269874RADF-8 RPI-6 220858,
AA503766, H51308, H03365RADF-9 RPI-7 220858, AA503766,
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H51308, H03365RADF-10 RPI-8 221022, Z19947RADF-11 RPI-9
T41063, T41020, T41005, T40881, T40190, T40139, T40125,
T40114, T40096, T39908RADF-12 RPI-11T40090, T40068RADF-13
RPI-I2AA551927, AA260531, W97741, N99366, T70526,
T40177, T40060, T40034, T39910, T39894RADF-14 RPI-
13T40090, T40068RADF-15 RPI-15T40940, T40002RADF-16 RPI-
16T73244, T71043, T71032, T40181, T40116RADF-18 RPI-
21N99641, N99445, N99528, 220485, Z20465RADF-19 RPI-
22AA269874RADF-20 RPI-23220858, AA503766, H51308,
H03365RADF-22 RPI-24T1952, H73939, 220894, T40182, T40167,
T40158RADF-23 RPI-25AA614684, AA523377, AA715907, AA580429,
AA630254, AA617854, AA580356RADF-24 RPI-26AA268201,
T64416, T62149, T40186RADF-25 RPI-27221017, 220888, 219984,
219971, T41056, T40108RADF-26 RPI-28T1952, H73939, 220894,
T40182, T40167, T40158RADF-26 RPI-29221017, 220888, 219984,
219971, T41056, T40108RADF-26 RPI-30T64707RADF-27 RPI-
31Z20858, AA503766, H51308, H03365RADF-32 RPI-33T40090,
T40068RADF-33 RPI-34T40090, T40068RADF-35 RPI-36221022,
Z19947RADF-36 RPI-37220858, AA503766, H51308, H03365For each
of RPI-10, RPI-17, and RPI-20, a degenerate set of probes is
provided, as follows(a) Probes for RPI-105'- A C G A C C T T
T A T G T T C T A T A G C A G C A A C -3' G T G G
G C C G G G G T T
A A T T T C C G
C C C(b) Probes for RPI-175'- A C G A G C A T G
A G G A G C A T C T A T A A G A T T -3' G T G G
G G G C G G G T T T
T A A T C C C
C G C (c) Probes for RPI-205'-
A G A T A T A T C A G C A C C A G A A T C -3' G C T C G
G G G G C T G T A A T
T T C G C C C
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Clones in libraries with insert DNA encoding the RPI
or fragments thereof will hybridize to one or more of the
degenerate oligonucleotide probes (or their complement).
Hybridization of such oligonucleotide probes to genomic
libraries are carried out using methods known in the art.
For example, hybridization with one of the above-mentioned
degenerate sets of oligonucleotide probes, or their
complement (or with any member of such a set, or its
complement) can be performed under highly stringent or
moderately stringent conditions as defined above, or can be
carried out in 2X SSC, I.O~ SDS at 50° C and washed using the
same conditions. In yet another aspect, clones of nucleotide
sequences encoding a part or the entire RPI or RPI-derived
polypeptides may also be obtained by screening expression
libraries. For example, DNA from the relevant source is
isolated and random fragments are prepared and ligated into
an expression vector (e. g., a bacteriophage, plasmid,
phagemid or cosmid) such that the inserted sequence in the
vector is capable of being expressed by the host cell into
which the vector is then introduced. Various screening
assays can then be used to select for the expressed RPI or
RPI-derived polypeptides. In one embodiment, the various
anti-RPI antibodies of the invention can be used to identify
the desired clones using methods known in the art. See, for
example, Harlow and Lane, 1988, Antibodies A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, Appendix IV. Clones or plaques from the library
are brought into contact with the antibodies to identify
those clones that bind. In an embodiment, colonies or plaques
containing DNA that encodes an RPI or RPI-derived polypeptide
can be detected using DYNA Beads according to Olsvick et al.,
29th ICAAC, Houston, Tex. 1989, incorporated herein by
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reference. Anti-RPI antibodies are crosslinked to tosylated
DYNA Beads M280, and these antibody-containing beads would
then be used to adsorb to colonies or plaques expressing RPI
or RPI-derived polypeptide. Colonies or plaques expressing
an RPI or RPI-derived polypeptide are identified as any of
those that bind the beads. Alternatively, the anti-RPI
antibodies can be nonspecifically immobilized to a suitable
support, such as silica or CeliteTM resin. This material
would then be used to adsorb to bacterial colonies expressing
the RPI protein or RPI-derived polypeptide as described in
the preceding paragraph. In another aspect, PCR amplification
may be used to produce substantially pure DNA encoding a part
of or the whole of an RPI from genomic DNA. Oligonucleotide
primers, degenerate or otherwise, corresponding to known RPI
sequences can be used as primers.PCR can be carried out,
e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq
polymerise (Gene AmpT~). One can choose to synthesize several
different degenerate primers, for use in the PCR reactions.
It is also possible to vary the stringency of hybridization
conditions used in priming the PCR reactions, to allow for
greater or lesser degrees of nucleotide sequence similarity
between the degenerate primers and the corresponding
sequences in the DNA. After successful amplification of a
segment of the sequence encoding an RPI, that segment may be
molecularly cloned and sequenced, and utilized as a probe to
isolate a complete genomic clone. This, in turn, will permit
the determination of the gene's complete nucleotide sequence,
the analysis of its expression, and the production of its
protein product for functional analysis, as described
infra. The RPI gene can also be identified by mRNA selection
by nucleic acid hybridization followed by in vitro
translation. In this procedure, fragments are used to
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isolate complementary mRNAs by hybridization. Such DNA
fragments may represent available, purified RPI DNA of
another species (e. g., mouse, human). Immunoprecipitation
analysis or functional assays (e.g., aggregation ability in
vitro; binding to receptor) of the in vitro translation
products of the isolated products of the isolated mRNAs
identifies the mRNA and, therefore, the complementary DNA
fragments that contain the desired sequences. In addition,
specific mRNAs may be selected by adsorption of polysomes
isolated from cells to immobilized antibodies specifically
directed against an RPI. A radiolabelled RPI cDNA can be
synthesized using the selected mRNA (from the adsorbed
polysomes) as a template. The radiolabelled mRNA or cDNA may
then be used as a probe to identify the RPI DNA fragments
from among other genomic DNA fragments.Alternatives to
isolating RPI genomic DNA include, but are not limited to,
chemically synthesizing the gene sequence itself from a known
sequence or making cDNA to the mRNA which encodes the RPI.
For example, RNA for cDNA cloning of the RPI gene can be
isolated from cells which express the RPI. Other methods are
possible and within the scope of the invention. Any
eukaryotic cell potentially can serve as the nucleic acid
source for the molecular cloning of the RPI gene. The
nucleic acid sequences encoding the RPI can be isolated from
vertebrate, mammalian, human, porcine, bovine, feline, avian,
equine, canine, as well as additional primate sources,
insects, plants, etc. The DNA may be obtained by standard
procedures known in the art from cloned DNA (e.g., a DNA
library), by chemical synthesis, by cDNA cloning, or by the
cloning of genomic DNA, or fragments thereof, purified from
the desired cell. (See, for example, Sambrook et al., 1989,
Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring
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Harbor Laboratory Press, Cold Spring Harbor, New York;
Glover, D.M. (ed.), 1985, DNA Cloning A Practical Approach,
MRL Press, Ltd., Oxford, U.K. Vol. I, II.) Clones derived
from genomic DNA may contain regulatory and intron DNA
regions in addition to coding regions; clones derived from
cDNA will contain only exon sequences. Whatever the source,
the RPI gene should be molecularly cloned into a suitable
vector for propagation.The identified and isolated gene or
cDNA can then be inserted into an appropriate cloning vector.
A large number of vector-host systems known in the art may
be used. Possible vectors include, but are not limited to,
plasmids or modified viruses, but the vector system must be
compatible with the host cell used. Such vectors include,
but are not limited to, bacteriophages such as lambda
derivatives, or plasmids such as PBR322 or pUC plasmid
derivatives or the Bluescript vector (Stratagene). The
insertion into a cloning vector can, for example, be
accomplished by ligating the DNA fragment into a cloning
vector which has complementary cohesive termini. However, if
the complementary restriction sites used to fragment the DNA
are not present in the cloning vector, the ends of the DNA
molecules may be enzymatically modified. Alternatively, any
site desired may be produced by ligating nucleotide sequences
(linkers) onto the DNA termini; these ligated linkers may
comprise specific chemically synthesized oligonucleotides
encoding restriction endonuclease recognition sequences. In
an alternative method, the cleaved vector and RPI gene may be
modified by homopolymeric tailing. Recombinant molecules can
be introduced into host cells via transformation,
transfection, infection, electroporation, etc., so that many
copies of the gene sequence are generated. In specific
embodiments, transformation of host cells with recombinant
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DNA molecules that incorporate the isolated RPI gene, cDNA,
or synthesized DNA sequence enables generation of multiple
copies of the gene. Thus, the gene may be obtained in large
quantities by growing transformants, isolating the
recombinant DNA molecules from the transformants and, when
necessary, retrieving the inserted gene from the isolated
recombinant DNA. The RPI sequences provided by the instant
invention include those nucleotide sequences encoding
substantially the same amino acid sequences as found in
native RPIs, and those encoded amino acid sequences with
functionally equivalent amino acids, as well as those
encoding other target derivatives or analogs.Expression of
DNA Encodin4 an RPI The nucleotide sequence encoding an
RPI or a functionally active analog or fragment or other
derivative thereof can be inserted into an appropriate
expression vector, i.e., a vector which contains the
necessary elements for the transcription and translation of
the inserted protein-coding sequence. The necessary
transcriptional and translational signals can also be
supplied by the native RPI gene or its flanking regions. A
variety of host-vector systems may be utilized to express the
protein-coding sequence. These include but are not limited
to mammalian cell systems infected with virus (e. g., vaccinia
virus, adenovirus, etc.); insect cell systems infected with
virus (e. g., baculovirus); microorganisms such as yeast
containing yeast vectors, or bacteria transformed with
bacteriophage, DNA, plasmid DNA, or cosmid DNA. The
expression elements of vectors vary in their strengths and
specificities. Depending on the host-vector system utilized,
any one of a number of suitable transcription and translation
elements may be used. In specific embodiments, the human RPI
gene is expressed, or a sequence encoding a functionally
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active portion of the human RPI. In yet another embodiment,
a fragment of target comprising a domain of the RPI is
expressed. Any of the methods previously described for
the insertion of DNA fragments into a vector may be used to
construct expression vectors containing a chimeric gene
consisting of appropriate transcriptional and translational
control signals and the protein coding sequences. These
methods may include in vitro recombinant DNA and synthetic
techniques and in vivo recombinants (genetic recombination).
Expression of nucleic acid sequence encoding an RPI or
peptide fragment may be regulated by a second nucleic acid
sequence so that the RPI or peptide is expressed in a host
transformed with the recombinant DNA molecule. For example,
expression of an RPI may be controlled by any promoter or
enhancer element known in the art. Promoters which may be
used to control RPI gene expression include, but are not
limited to, the SV40 early promoter region (Bernoist and
Chambon, 1981, Nature 290304-310), the promoter contained in
the 3' long terminal repeat of Rous sarcoma virus (Yamamoto,
et al., 1980, Cell 22787-797), the herpes thymidine kinase
promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA
781441-1445), the regulatory sequences of the metallothionein
gene (Brinster et al., 1982, Nature 2939-42); prokaryotic
expression vectors such as the (3-lactamase promoter (Villa-
Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 753727-
3731), or the tac promoter (DeBoer et al., 1983, Proc. Natl.
Acad. Sci. USA X21-25); see also
Usefulproteinsfromrecombinantbacteria in Scientific American,
1980, 24274-94; plant expression vectors comprising the
nopaline synthetase promoter region (Herrera-Estrella et al.,
Nature 303209-213) or the cauliflower mosaic virus 35S RNA
promoter (Gardner et al., 1981, Nucl. Acids Res. 92871), and
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the promoter of the photosynthetic enzyme ribulose
biphosphate carboxylase (Herrera-Estrella et al., 1984,
Nature 310115-120); promoter elements from yeast or other
fungi such as the Gal 4 promoter, the ADC (alcohol
dehydrogenase) promoter, PGK (phosphoglycerol kinase)
promoter, alkaline phosphatase promoter, and the following
animal transcriptional control regions, which exhibit tissue
specificity and have been utilized in transgenic animals
elastase I gene control region which is active in pancreatic
acinar cells (Swift et al., 1984, Cell 38639-646; Ornitz et
al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50399-409;
MacDonald, 1987, Hepatology 7425-515); insulin gene control
region which is active in pancreatic beta cells (Hanahan,
1985, Nature 315115-122), immunoglobulin gene control region
which is active in lymphoid cells (Grosschedl et al., 1984,
Cell 38647-658; Adames et al., 1985, Nature 318533-538;
Alexander et al., 1987, Mol. Cell. Biol. 71436-1444), mouse
mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al.,
1986, Cell 45485-495), albumin gene control region which is
active in liver (Pinkert et al., 1987, Genes and Devel. _1268-
276), alpha-fetoprotein gene control region which is active
in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 51639-1648;
Hammer et al., 1987, Science 2353-58; alpha 1-antitrypsin
gene control region which is active in the'liver (Kelsey et
al., 1987, Genes and Devel. 1161-171), beta-globin gene
control region which is active in myeloid cells (Mogram et
al., 1985, Nature 315338-340; Kollias et al., 1986, Cell
4_C89-94; myelin basic protein gene control region which is
active in oligodendrocyte cells in the brain (Readhead et
al., 1987, Cell 48703-712); myosin light chain-2 gene control
region which is active in skeletal muscle (Sani, 1985, Nature
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314283-286), and gonadotropic releasing hormone gene control
region which is active in the hypothalamus (Mason et al.,
1986, Science 2341372-1378).In a specific embodiment, a
vector is used that comprises a promoter operably linked to
an RPI-encoding nucleic acid, one or more origins of
replication, and, optionally, one or more selectable markers
(e. g., an antibiotic resistance gene). In a specific
embodiment, an expression construct is made by subcloning an
RPI coding sequence into the EcoRI restriction site of each
of the three pGEX vectors (Glutathione S-Transferase
expression vectors; Smith and Johnson, 1988, Gene 731-40).
This allows for the expression of the RPI product from the
subclone in the correct reading frame. Expression vectors
containing RPI gene inserts can be identified by three
general approaches (a) nucleic acid hybridization, (b)
presence or absence of marker gene functions, and (c)
expression of inserted sequences. In the first approach, the
presence of an RPI gene inserted in an expression vector can
be detected by nucleic acid hybridization using probes
comprising sequences that are homologous to an inserted RPI
gene. In the second approach, the recombinant 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, transformation
phenotype, occlusion body formation in baculovirus, etc.)
caused by the insertion of an RPI gene in the vector. For
example, if the RPI gene is inserted within the marker gene
sequence of the vector, recombinants containing the RPI gene
insert can be identified by the absence of the marker gene
function. In the third approach, recombinant expression
vectors can be identified by assaying the RPI gene product
expressed by the recombinant. Such assays can be based, for
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example, on the physical or functional properties of the RPI
in in vitro assay systems, e.g., binding with anti-RPI
antibody.Once a particular recombinant DNA molecule is
identified and isolated, several methods known in the art may
be used to propagate it. Once a suitable host system and
growth conditions are established, recombinant expression
vectors can be propagated and prepared in quantity. As
previously explained, the expression vectors which can be
used include, but are not limited to, the following vectors
or their derivatives human or animal viruses such as
vaccinia virus or adenovirus; insect viruses such as
baculovirus; yeast vectors; bacteriophage vectors (e. g.,
lambda), and plasmid and cosmid DNA vectors, to name but a
few. In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or
modifies and processes the gene product in the specific
fashion desired. Expression from certain promoters can be
elevated in the presence of certain inducers; thus,
expression of the genetically engineered RPI may be
controlled. Furthermore, different host cells have
characteristic and specific mechanisms for the translational
and post-translational processing and modification (e. g.,
glycosylation, phosphorylation of proteins). Appropriate
cell lines or host systems can be chosen to ensure the
desired modification and processing of the foreign protein
expressed. For example, expression in a bacterial system can
be used to produce an unglycosylated core protein product.
Expression in yeast will produce a glycosylated product.
Expression in mammalian cells can be used to ensure native
glycosylation of a heterologous protein. Furthermore,
different vector/host expression systems may effect
processing reactions to different extents. For long-term,
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high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which
stably express the differentially expressed or pathway gene
protein may be engineered. Rather than using expression
vectors which contain viral origins of replication, host
cells can be transformed with DNA controlled by appropriate
expression control elements (e. g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites,
etc.), and. a selectable marker. Following the introduction
of the foreign DNA, engineered cells may be allowed to grow
for 1-2 days in an enriched media, and then are switched to a
selective media. The selectable marker in the recombinant
plasmid confers resistance to the selection and allows cells
to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded
into cell lines. This method may advantageously be used to
engineer cell lines which express the differentially
expressed or pathway gene protein. Such engineered cell
lines may be particularly useful in screening and evaluation
of compounds that affect the endogenous activity of the
differentially expressed or pathway gene protein.A number of
selection systems may be used, including but not limited to
the herpes simplex virus thymidine kinase (Wigler, et al.,
1977, Cell 11223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 482026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22817)
genes can be employed in tk~, hgprt- or aprt- cells,
respectively. Also, antimetabolite resistance can be used as
the basis of selection for dhfr, which confers resistance to
methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA
773567; O~Hare et al., 1981, Proc. Natl. Acad. Sci. USA
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781527); gpt, which confers resistance to mycophenolic acid
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 782072);
neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin et al., 1981, J. MoI. Biol. 1501); and
hygro, which confers resistance to hygromycin (Santerre et
al., 1984, Gene 30147) genes. In other specific embodiments,
the RPI, fragment, analog, or derivative may be expressed as
a fusion, or chimeric protein product (comprising the
protein, fragment, analog, or derivative joined via a peptide
bond to a heterologous protein sequence (of a different
protein)). Such a chimeric product can be made by ligating
the appropriate nucleic acid sequences encoding the desired
amino acid sequences to each other by methods known in the
art, in the proper coding frame, and expressing the chimeric
product by methods commonly known in the art. Alternatively,
such a chimeric product may be made by protein synthetic
techniques, e.g., by use of a peptide synthesizer. Both cDNA
and genomic sequences can be cloned and expressed. Therapeutic
Use Of RPIs The invention provides for treatment or
prevention of various diseases and disorders by
administration of a therapeutic compound. Such compounds
include but are not limited to RPIs and analogs and
derivatives (including fragments) thereof (e. g., as described
herein); antibodies thereto (as described herein); nucleic
acids encoding RPIs, analogs, or derivatives (e.g., as
described herein); RPI gene antisense nucleic acids, and RPI
gene agonists and antagonists. As is described herein, an
important feature of the present invention is the
identification of RPI genes involved in rheumatoid arthritis.
Arthritis can be treated or prevented by administration of a
therapeutic compound that promotes function or expression of
RPIs which are decreased in synovial fluid verses serum of RA
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patients. Arthritis can also be treated or prevented by
administration of a therapeutic compound that reduces
function or expression of RPIs which are increased in
synovial fluid verses serum of RA patients. Generally,
administration of products of a species origin or species
reactivity (in the case of antibodies) that is the same
species as that of the patient is preferred. Thus, in a
preferred embodiment, a human RPI, derivative, or analog, or
nucleic acid, or an antibody to a human RPI, is administered
to a human patient for therapy or prophylaxis.Treatment And
Prevention Of Rheumatoid ArthritisRheumatoid
arthritis is treated or prevented by administration of a
compound that promotes (i.e., increases or supplies) the
level or function of one or more RPIs -- or the level of one
or more RADFs -- that are decreased in synovial fluid verses
serum of subjects with RA. Examples of such a compound
include but are not limited to RPIs, derivatives, or
fragments that are functionally active, particularly that are
active as demonstrated in in vitro assays or in animal
models, and nucleic acids encoding an RPT or functionally
active derivative or fragment thereof (e. g., for use in gene
therapy). Other compounds that can be used, e.g., RPI
agonists, can be identified using in vitro assays. Rheumatoid
arthritis is also treated or prevented by administration of a
compound that inhibits (i.e., decreases) the level or
function of one or more RPIs -- or the level of one or more
RADFs -- that exhibit increased abundance in synovial fluid
verses serum of subjects with RA. Examples of such a
compound include but are not limited to RPI anti-sense
oligonucleotides, ribozymes, or antibodies directed against
RPIs. Other compounds that can be used, e.g., RPI
antagonists, can be identified using in vitro assays. In
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specific embodiments, compounds that promote the level or
function of one or more RPIs, or the level of one or more
RADFs are administered therapeutically (including
prophylactically when an absent or decreased (relative to
normal or desired) RPI level or function, or RADF level has
been identified in synovial fluid of RA patients as compared
with serum in RA patients. In further embodiments, compounds
that inhibit RPI level or function, or RADF level are
administered therapeutically (including prophylactically when
an increased (relative to normal or desired) RPI level or
function, or RADF level has been identified in synovial fluid
of RA patients as compared with serum in RA patients. The
change in RPI function or level, or RADF level due to the
administration of such compounds can be readily detected,
e.g., by obtaining a patient tissue sample (e. g., from biopsy
tissue) and assaying it in vitro for RNA or protein levels,
or activity of the expressed RPI RNA or protein. The
Preferred Technology can also be used to detect levels of the
RPI or RADF before and after the administration of the
compound. Many methods standard in the art can be thus
employed, including but not limited to kinase assays,
immunoassays to detect and/or visualize the RPI (e. g.,
Western blot, immunoprecipitation followed by sodium dodecyl
sulfate polyacrylamide gel electrophoresis,
immunocytochemistry, etc.) and/or hybridization assays to
detect RPI expression by detecting and/or visualizing mRNA
encoding the RPI (e. g., Northern assays, dot blots, in situ
hybridization, etc.), etc.The compounds of the invention
include but are not limited to any compound, e.g., a small
organic molecule, protein, peptide, antibody, nucleic acid,
etc. that restores the RA RPI or RADF profile towards normal
with the proviso that such compound is not a non-steroidal
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anti-inflammatory agent (NSAID) (e. g. prednisone, ibuprofen,
fenoprofen, ketoprofen, flurbiprofen, indomethacin, sulindac,
aspirin, salicylsalicylic acid; diflunisal, naproxen,
piroxicam, tenoxicam, phenylbutazone, oxyphenbutazone), a
gold salt, D-penicillamine, an antimalarial such as
hydroxychloroquine or sulphasalazine, azathioprine,
cyclophosphamide, chlorambucil, methotrexate, a
corticosteroid, anti-CD4 monoclonal antibody, or anti-CDw52
antibody. Gene Therabv In a specific embodiment, nucleic
acids comprising a sequence encoding an RPI or functional
derivative thereof, are administered to promote RPI function,
by way of gene therapy. Gene therapy refers to therapy
performed by the administration to a subject of an expressed
or expressible nucleic acid. In this embodiment of the
invention, the nucleic acid produces its encoded protein that
mediates a therapeutic effect by promoting RPI function. Any
of the methods for gene therapy available in the art can be
used according to the present invention. Exemplary methods
are described below.For general reviews of the methods of
gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy
12488-505; Wu and Wu, 1991, Biotherapy 387-95; Tolstoshev,
1993, Ann. Rev. Pharmacol. Toxicol. 32573-596; Mulligan,
1993, Science 260926-932; and Morgan and Anderson, 1993, Ann.
Rev. Biochem. 62191-217; May, 1993, TIBTECH 11(5)155-215).
Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al.
(eds.), 1993, Current Protocols in Molecular Biology, John
Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY.In a
preferred aspect, the compound comprises a nucleic acid
encoding an RPI, said nucleic acid being part of an
expression vector that expresses an RPI or fragment or
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chimeric protein thereof in a suitable host. In particular,
such a nucleic acid has a promoter operably linked to the RPI
coding region, said promoter being inducible or constitutive,
and, optionally, tissue-specific. In another particular
embodiment, a nucleic acid molecule is used in which the RPI
coding sequences and any other desired sequences are flanked
by regions that promote homologous recombination at a desired
site in the genome, thus providing for intrachromosomal
expression of the RPI nucleic acid (Koller and Smithies,
1989, Proc. Natl. Acad. Sci. USA 88932-8935; Zijlstra et
al., 1989, Nature 342435-438). Delivery of the nucleic acid
into a patient may be either direct, in which case the
patient is directly exposed to the nucleic acid or nucleic
acid-carrying vector, or indirect, in which case, cells are
first transformed with the nucleic acid in vitro, then
transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.In a
specific embodiment, the nucleic acid is directly
administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing it as part of
an appropriate nucleic acid expression vector and
administering it so that it becomes intracellular, e.g., by
infection using a defective or attenuated retroviral or other
viral vector (see U.S. Patent No. 4,980,286), or by direct
injection of naked DNA, or by use of microparticle
bombardment (e. g., a gene gun; Biolistic, Dupont), or coating
with lipids or cell-surface receptors or transfecting agents,
encapsulation in liposomes, microparticles, or microcapsules,
or by administering it in linkage to a peptide which is known
to enter the nucleus, by administering it in linkage to a
ligand subject to receptor-mediated endocytosis (see, e.g.,
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Wu and Wu, 1987, J. Biol. Chem. 2624429-4432) (which can be
used to target cell types specifically expressing the
receptors), etc. In another embodiment, a nucleic acid-
ligand complex can be farmed in which the ligand comprises a
fusogenic viral peptide to disrupt endosomes, allowing the
nucleic acid to avoid lysosomal degradation. In yet another
embodiment, the nucleic acid can be targeted in vivo for cell
specific uptake and expression, by targeting a specific
receptor (see, e.g., PCT Publications WO 92/06180 dated April
16, 1992 (Wu et al.); WO 92/22635 dated December 23, 1992
(Wilson et al.); W092/20316 dated November 26, 1992 (Findeis
et al.); W093/14188 dated July 22, 1993 (Clarke et al.), WO
93/20221 dated October 14, 1993 (Young)). Alternatively, the
nucleic acid can be introduced intracellularly and
incorporated within host cell DNA for expression, by
homologous recombination (Koller and Smithies, 1989, Proc.
Natl. Acad. Sci. USA 868932-8935; Zijlstra et al., 1989,
Nature 342435-438). In a specific embodiment, a viral vector
that contains a nucleic acid encoding an RPI is used. For
example, a retraviral vector can be used (see Miller et al.,
1993, Meth. Enzymol. 217581-599). These retroviral vectors
have been modified to delete retroviral sequences that are
not necessary for packaging of the viral genome and
integration into host cell DNA. The nucleic acid encoding
the RPI to be used in gene therapy is cloned into the vector,
which facilitates delivery of the gene into a patient. More
detail about retroviral vectors can be found in Boesen et
al., 1994, Biotherapy 6291-302, which describes the use of a
retroviral vector to deliver the mdrl gene to hematopoietic
stem cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are Clowes et al., 1994,
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J. Clin. Invest. 93644-651; Kiem et al., 1994, Blood 831467-
1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4129-
141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics
and Devel. 3110-114.Adenoviruses are other viral vectors that
can be used in gene therapy. Adenoviruses are especially
attractive vehicles for delivering genes to respiratory
epithelia. Adenoviruses naturally infect respiratory
epithelia where they cause a mild disease. Other targets for
adenovirus-based delivery systems are liver, the central
nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in
Genetics and Development 3499-503 present a review of
adenovirus-based gene therapy. Bout et al., 1994, Human Gene
Therapy 5_3-10 demonstrated the use of adenovirus vectors to
transfer genes to the respiratory epithelia of rhesus
monkeys. Other instances of the use of adenoviruses in gene
therapy can be found in Rosenfeld et al., 1991, Science
252431-434; Rosenfeld et al., 1992, Cell 68143-155;
Mastrangeli et al., 1993, J. Clin. Invest. 91225-234; PCT
Publication W094/12649; and Wang, et al., 1995, Gene Therapy
2775-783.Adeno-associated virus (AAV) has also been proposed
for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp.
Biol. Med. 204289-300; U.S. Patent No. 5,436,146).Another
approach to gene therapy involves transferring a gene to
cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or
viral infection. Usually, the method of transfer includes
the transfer of a selectable marker to the cells. The cells
are then placed under selection to isolate those cells that
have taken up and are expressing the transferred gene. Those
cells are then delivered to a patient. In this embodiment,
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the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell.
Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences,
cell fusion, chromosome-mediated gene transfer, microcell-
mediated gene transfer, spheroplast fusion, etc. Numerous
techniques are known in the art for the introduction of
foreign genes into cells (see, e.g., Loeffler and Behr, 1993,
Meth. Enzymol. 217599-618; Cohen et al., 1993, Meth. Enzymol.
217518-644; Cline, 1985, Pharmac. Ther. 2969-92) and may be
used in accordance with the present invention, provided that
the necessary developmental and physiological functions of
the recipient cells are not disrupted. The technique should
provide for the stable transfer of the nucleic acid to the
cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny. The
resulting recombinant cells can be delivered to a patient by
various methods known in the art. In a preferred embodiment,
epithelial cells are injected, e.g., subcutaneously. In
another embodiment, recombinant skin cells may be applied as
a skin graft onto the patient. Recombinant blood cells
(e. g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned
for use depends on the desired effect, patient state, etc.,
and can be determined by one skilled in the art. Cells into
which a nucleic acid can be introduced for purposes of gene
therapy encompass any desired, available cell type, and
include but are not limited to epithelial cells, endothelial
cells, keratinocytes, fibroblasts, muscle cells, hepatocytes;
blood cells such as T lymphocytes, B lymphocytes, monocytes,
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macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained
from bone marrow, umbilical cord blood, peripheral blood,
fetal liver, etc.In a preferred embodiment, the cell used for
gene therapy is autologous to the patient.In an embodiment in
which recombinant cells are used in gene therapy, a nucleic
acid encoding an RPI is introduced into the cells such that
it is expressible by the cells or their progeny, and the
recombinant cells are then administered in vivo for
therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells
which can be isolated and maintained in vitro can potentially
be used in accordance with this embodiment of the present
invention (see e.g. PCT Publication WO 94/08598, dated April
28, 1994; Stemple and Anderson, 1992, Cell 71973-985;
Rheinwald, 1980, Meth. Cell Bio. 21A229; and Pittelkow and
Scott, 1986, Mayo Clinic Proc. X1771). In a specific
embodiment, the nucleic acid to be introduced for purposes of
gene therapy comprises an inducible promoter operably linked
to the coding region, such that expression of the nucleic
acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.Tnhibition Of
RPIs To Treat Rheumatoid ArthritisIn one
embodiment of the invention, RA is treated or prevented by
administration of a compound that antagonizes (inhibits) the
RPI levels and/or function of RPIs which are elevated in
synovial fluid compared with serum of patients suffering from
RA. Compounds that can be used include but are not limited
to anti-RPI antibodies (and fragments and derivatives thereof
containing the binding region thereof), RPI antisense or
ribozyme nucleic acids, and nucleic acids encoding
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dysfunctional RPIs that are used to knockout endogenous RPI
function by homologous recombination (see, e.g., Capecchi,
1989, Science 21288-1292). In a specific embodiment of the
invention, a nucleic acid containing a portion of an RPI gene
in which nucleotides encoding an RPI flank (are both 5' and 3'
to) a different gene sequence, is used, as an RPI antagonist,
to promote RPI inactivation by homologous recombination (see
also Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
868932-8935; Zijlstra et al., 1989, Nature 342435-438).
Other compounds that inhibit RPI function can be identified
by use of known convenient in vitro assays, e.g., based on
their ability to inhibit binding of RPIs to another protein,
or inhibit any known RPI function, as preferably assayed in
vitro or in cell culture, although genetic assays may also be
employed. The Preferred Technology can also be useful for
detecting levels of the RPI before and after the
administration of the compound. Preferably, suitable in
vitro or in vivo assays, are utilized to determine the effect
of a specific compound and whether its administration is
indicated for treatment of the affected tissue. In specific
embodiments, compounds that inhibit RPI function are
administered therapeutically (including prophylactically)
when an increased level of an RPI or RPI function (e. g.
greater than the normal level or desired level) is detected
in synovial fluid compared with serum of a patient suffering
from RA. The increased levels in RPI or function can be
readily detected, e.g., by quantifying protein and/or RNA, by
obtaining patient synovial fluid and serum samples and
assaying them in vitro for RNA or protein levels, structure
and/or activity of the expressed RNA encoding the RPI, or the
RPI itself. Many methods standard in the art can be thus
employed, including but not limited to kinase assays,
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immunoassays to detect and/or visualize RPI (e. g., Western
blot, immunoprecipitation followed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis, immunocytochemistry,
etc.) and/or hybridization assays to detect RPI expression by
detecting and/or visualizing respectively mRNA encoding the
RPI (e. g., Northern assays, dot blots, in situ hybridization,
etc.), etc.Antisense Reaulation of RPIsIn a specific
embodiment, RPI function is inhibited by use of RPI antisense
nucleic acids. The present invention provides the
therapeutic or prophylactic use of nucleic acids comprising
at least six nucleotides that are antisense to a gene or cDNA
encoding an RPI or a portion thereof. As used herein, an RPI
antisense nucleic acid refers to a nucleic acid capable of
hybridizing to a portion of an RNA encoding an RPI
(preferably mRNA) by virtue of some sequence complementarity.
The antisense nucleic acid may be complementary to a coding
and/or noncoding region of an mRNA encoding an RPI. Such
antisense nucleic acids have utility as compounds that
inhibit RPI function, and can be used in the treatment or
prevention of rheumatoid arthritis. The antisense nucleic
acids of the invention can be oligonucleotides that are
double-stranded or single-stranded, RNA or DNA or a
modification or derivative thereof, which can be directly
administered to a cell, or which can be produced
intracellularly by transcription of exogenous, introduced
sequences. The invention further provides pharmaceutical
compositions comprising an effective amount of the RPI
antisense nucleic acids of the invention in a
pharmaceutically acceptable carrier, as described infra. In
another embodiment, the invention is directed to methods for
inhibiting the expression of an RPI nucleic acid sequence in
a prokaryotic or eukaryotic cell comprising providing the
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cell with an effective amount of a composition comprising an
RPI antisense nucleic acid of the invention.RPT antisense
nucleic acids and their uses are described in detail
below.RPI Antisense Nucleic Acids The RPI antisense nucleic
acids are of at least six nucleotides and are preferably
oligonucleotides (ranging from 6 to about 50
oligonucleotides). In specific aspects, the oligonucleotide
is at least 10 nucleotides, at least 15 nucleotides, at least
100 nucleotides, or at least 200 nucleotides. The
oligonucleotides can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the
base moiety, sugar moiety, or phosphate backbone. The
oligonucleotide may include other appending groups such as
peptides, or agents facilitating transport across the cell
membrane (see, e.g., Letsinger et al., 1989, Proc. Natl.
Acad. Sci. USA 866553-6556; Lemaitre et al., 1987, Proc.
Natl. Acad. Sci. 84648-652; PCT Publication No. WO 88/09810,
published December 15, 1988) or blood-brain barrier (see,
e.g., PCT Publication No. WO 89/10134, published April 25,
1988), hybridization-triggered cleavage agents (see, e.g.,
Krol et al., 1988, BioTechrliques 6958-976) or intercalating
agents (see, e.g., Zon, 1988, Pharm. Res. 5539-549).In a
preferred aspect of the invention, an RPI antisense
oligonucleotide is provided, preferably of single-stranded
DNA. The oligonucleotide may be modified at any position on
its structure with substituents generally known in the
art. The RPI antisense oligonucleotide may comprise at least
one modified base moiety which is selected from the group
including but not limited to 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
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5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, 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-isopentenyladenine,
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.In another embodiment,
the oligonucleotide comprises at least one modified sugar
moiety, e.g. a sugar moiety selected from the group
consisting of arabinose, 2-fluoroarabinose, xylulose, and
hexose.In yet another embodiment, the oligonucleotide
comprises at least one modified phosphate backbone selected
from the group consisting of a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an
alkyl phosphotriester, and a formacetal or analog thereof. In
yet another embodiment, the oligonucleotide is an a-anomeric
oligonucleotide. An a-anomeric oligonucleotide forms
specific double-stranded hybrids with complementary RNA in
which, contrary to the usual (3-units, the strands run
parallel to each other (Gautier et al., 1987, Nucl. Acids
Res. 156625-6641).The oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
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cleavage agent, etc.Oligonucleotides of the invention may 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 may be
synthesized by the method of Stein et al. (1988, Nucl. Acids
Res. 163209), methylphosphonate oligonucleotides can be
prepared by use of controlled pore glass polymer supports
(Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 857448-7451),
etc.In a specific embodiment, the RPI antisense nucleic acid
of the invention is produced intracellularly by transcription
from an exogenous sequence. For example, a vector can be
introduced in vivo such that it is taken up by a cell, within
which cell the vector or a portion thereof is transcribed,
producing an antisense nucleic acid (RNA) of the invention.
Such a vector would contain a sequence encoding the RPI
antisense nucleic acid. Such a vector can remain episomal or
become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such
vectors can be constructed by recombinant DNA technology
methods standard in the art. Vectors can be plasmid, viral,
or others known in the art, used for replication and
expression in mammalian cells. Expression of the sequence
encoding the RPI antisense RNA can be by any promoter known
in the art to act in mammalian, preferably human, cells.
Such promoters can be inducible or constitutive. Such
promoters include but are not limited to the SV40 early
promoter region (Bernoist and Chambon, 1981, Nature 290304-
310), the promoter contained in the 3' long terminal repeat of
Rous sarcoma virus (Yamamoto et al., 1980, Cell 22787-797),
the herpes thymidine kinase promoter (Wagner et al., 1981,
Proc. Natl. Acad. Sci. USA 781441-1445), the regulatory
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sequences of the metallothionein gene (Brinster et al., 1982,
Nature ,2"639-42), etc.The antisense nucleic acids of the
invention comprise a sequence complementary to at least a
portion of an RNA transcript of an RPI gene, preferably a
human RPI gene. However, absolute complementarity, although
preferred, is not required. A sequence
complementarytoatleastaportionofanRNA, as referred to herein,
means a sequence having sufficient complementarity to be able
to hybridize with the RNA, forming a stable duplex; in the
case of double-stranded RPI antisense nucleic acids, a single
strand of the duplex DNA may thus be tested, or triplex
formation may be assayed. The ability to hybridize will
depend on both the degree of complernentarity and the length
of the antisense nucleic acid. Generally, the longer the
hybridizing nucleic acid, the more base mismatches with an
RNA encoding an RPI it may contain and still form a stable
duplex (or triplex, as the case may be). One skilled in the
art can ascertain a tolerable degree of mismatch by use of
standard procedures to determine the melting point of the
hybridized complex.Therapeutic Use Of RPI
Antisense Nucleic AcidsThe RPI antisense nucleic acids
can be used to treat (or prevent) rheumatoid arthritis when
the RPI of interest is overexpressed in the synovial fluid
patients suffering from rheumatoid arthritis. In a preferred
embodiment, a single-stranded DNA antisense RPI
oligonucleotide is used.Cell types which express or
overexpress RNA encoding an RPI can be identified by various
methods known in the art. Such cell types include but are
not limited to leukocytes (e. g., neutrophils, macrophages,
monocytes} and resident cells (e. g., synoviocytes}. Such
methods include, but are not limited to, hybridization with
an RPI-specific nucleic acid (e. g., by Northern
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hybridization, dot blot hybridization, in situ
hybridization), observing the ability of RNA from the cell
type to be translated in vitro into an RPI, immunoassay, etc.
In a preferred aspect, primary tissue from a patient can be
assayed for RPI expression prior to treatment, e.g., by
immunocytochemistry or in situ hybridization. Pharmaceutical
compositions of the invention, comprising an effective amount
of an RPI antisense nucleic acid in a pharmaceutically
acceptable carrier, can be administered to a patient having
rheumatoid arthritis. The amount of RPI antisense nucleic acid
which will be effective in the treatment of RA can be
determined by standard clinical techniques. Where possible,
it is desirable to determine the antisense cytotoxicity of
the tumor type to be treated in vitro, and then in useful
animal model systems prior to testing and use in humans.In a
specific embodiment, pharmaceutical compositions comprising
RPI antisense nucleic acids are administered via liposomes,
microparticles, or microcapsules. In various embodiments of
the invention, it may be useful to use such compositions to
achieve sustained release of the RPI antisense nucleic acids.
In a specific embodiment, it may be desirable to utilize
liposomes targeted via antibodies to specific identifiable
tumor antigens (Leonetti et al., 1990, Proc. Natl. Acad. Sci.
USA 872448-2451; Renneisen et al., 1990, J. Biol. Chem.
26516337-16342). Inhibitory Ribozyme And Triple Helix
AnproachesIn another embodiment, symptoms of RA may be
ameliorated by decreasing the level of RPI gene expression
and/or RPI gene product activity by using RPI gene sequences
in conjunction with well-known gene knock-out, ribozyme
and/or triple helix methods to decrease the level of RPI gene
expression. Among the compounds that may exhibit the ability
to modulate the activity, expression or synthesis of the RPI
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gene, including the ability to ameliorate the symptoms of a
RA, are ribozyme, and triple helix molecules. Such molecules
may be designed to reduce or inhibit either unimpaired, or if
appropriate, mutant target gene activity. Techniques for the
production and use of such molecules are well known to those
of skill in the art.Ribozyme molecules designed to
catalytically cleave RPI gene mRNA transcripts can be used to
prevent translation of target gene mRNA and, therefore,
expression of RPI gene product. (See, e.g., PCT
International Publication W090/11364, published October 4,
1990; Sarver et al., 1990, Science 2471222-1225). Ribozymes
are enzymatic RNA molecules capable of catalyzing the
specific cleavage of RNA. (For a review, see Rossi, 1994,
Current Biology 4, 469-471). The mechanism of ribozyme
action involves sequence specific hybridization of the
ribozyme molecule to complementary target RNA, followed by an
endonucleolytic cleavage event. The composition of ribozyme
molecules must include one or more sequences complementary to
the target gene~mRNA, and must include the well known
catalytic sequence responsible for mRNA cleavage. For this
sequence, see, e.g., U.S. Patent No. 5,093,246, which is
incorporated herein by reference in its entirety. While
ribozymes that cleave mRNA at site specific recognition
sequences can be used to destroy mRNAs encoding an RPI, the
use of hammerhead ribozymes is preferred. Hammerhead
ribozymes cleave mRNAs at locations dictated by flanking
regions that form complementary base pairs with the target
mRNA. The sole requirement is that the target mRNA have the
following sequence of two bases 5'-UG-3'. The construction
and production of hammerhead ribozymes is well known in the
art and is described more fully in Myers, 1995, Molecular
Biology and Biotechnology A Comprehensive Desk Reference,
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VCH Publishers, New York, (see especially Figure 4, page 833)
and in Haseloff and Gerlach, 1988, Nature, 334, 585-591,
which is incorporated herein by reference in its
entirety.Preferably the ribozyme is engineered so that the
cleavage recognition site is located near the 5' end of the
mRNA encoding the RPI, i.e., to increase efficiency and
minimize the intracellular accumulation of non-functional
mRNA transcripts. The ribozymes of the present invention also
include RNA endoribonucleases (hereinafter Cech-
typeribozymes) such as the one that occurs naturally in
Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA)
and that has been extensively described by Thomas Cech and
collaborators (Zaug, et al., 1984, Science, 224, 574-578;
Zaug and Cech, 1986, Science, 231, 470-475; Zaug, et al.,
1986, Nature, 324, 429-433; published International patent
application No. WO 88/04300 by University Patents Inc.; Been
and Cech, 1986, Cell, 47, 207-216). The Cech-type ribozymes
have an eight base pair active site which hybridizes to a
target RNA sequence whereafter cleavage of the target RNA
takes place. The invention encompasses those Cech-type
ribozymes which target eight base-pair active site sequences
that are present in the gene encoding the RPI.As in the
antisense approach, the ribozymes can be composed of modified
oligonucleotides (e. g., for improved stability, targeting,
etc.) and should be delivered to cells that express the RPI
in vivo. A preferred method of delivery involves using a DNA
construct encoding the ribozyme under the control of a strong
constitutive pol III or pol II promoter, so that transfected
cells will produce sufficient quantities of the ribozyme to
destroy endogenous mRNA encoding the RPI and inhibit
translation. Because ribozymes, unlike antisense molecules,
are catalytic, a lower intracellular concentration is
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required for efficiency.Endogenous RPI expression can also be
reduced by inactivating or knockingout the RPI gene or its
promoter using targeted homologous recombination (e.g., see
Smithies, et al., 1985, Nature 317230-234; Thomas and
Capecchi, 1987, Cell 51503-512; Thompson et al., 1989, Cell
5313-321; each of which is incorporated by reference herein
in its entirety). For example, a mutant, non-functional RPI
gene (or a completely unrelated DNA sequence) flanked by DNA
homologous to the endogenous RPI gene (either the coding
regions or regulatory regions of the gene encoding the RPI)
can be used, with or without a selectable marker and/or a
negative selectable marker, to transfect cells that express
the target gene in vivo. Insertion of the DNA construct, via
targeted homologous recombination, results in inactivation of
the target gene. Such approaches are particularly suited in
the agricultural field where modifications to ES (embryonic
stem) cells can be used to generate animal offspring with an
inactive target gene (e.g., see Thomas and Capecchi, 1987 and
Thompson, 1989, supra). However this approach can be adapted
for use in humans provided the recombinant DNA constructs are
directly administered or targeted to the required site in
vivo using appropriate viral vectors. Alternatively,
endogenous RPI gene expression can be reduced by targeting
deoxyribonucleotide sequences complementary to the regulatory
region of the RPI gene (i.e., the RPI gene promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the RPI gene in target cells in the body.
(See generally, Helene, 1991, Anticancer Drug Des., 6(6).
569-584; Helene, et al., 1992, Ann. N.Y. Acad. Sci., 660, 27-
36; and Maher, 1992, Bioassays 14{12), 807-815). Nucleic acid
molecules to be used in triplex helix formation for the
inhibition of transcription should be single stranded and
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composed of deoxynucleotides. The base composition of these
oligonucleotides must be designed to promote triple helix
formation via Hoogsteen base pairing rules, which generally
require sizeable stretches of either purines or pyrimidines
to be present on .one strand of a duplex. Nucleotide
sequences may be pyrimidine-based, which will result in TAT
and CGC' triplets across the three associated strands of the
resulting triple helix. The pyrimidine-rich molecules provide
base complementarity to a purine-rich region of a single
strand of the duplex in a parallel orientation to that
strand. In addition, nucleic acid molecules may be chosen
that are purine-rich, for example, contain a stretch of G
residues. These molecules will form a triple helix with a
DNA duplex that is rich in GC pairs, in which the majority of
the purine residues are located on a single strand of the
targeted duplex, resulting in GGC triplets across the three
strands in the triplex. Alternatively, the potential sequences
that can be targeted for triple helix formation may be
increased by creating a so called switchback nucleic acid
molecule. Switchback molecules are synthesized in an
alternating 5'-3', 3'-5' manner, such that they base pair
with first one strand of a duplex and then the other,
eliminating the necessity for a sizeable stretch of either
purines or pyrimidines to be present on one strand of a
duplex. In instances wherein the antisense, ribozyme, and/or
triple helix molecules described herein are utilized to
inhibit mutant gene expression, it is possible that the
technique may so efficiently reduce or inhibit the
transcription (triple helix) and/or translation (antisense,
ribozyme) of mRNA produced by normal RPI gene alleles that
the possibility may arise wherein the concentration of normal
RPI gene product present may be lower than is necessary for a
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normal phenotype. In such cases, to ensure that
substantially normal levels of RPI gene activity are
maintained, therefore, nucleic acid molecules that encode and
express RPI gene polypeptides exhibiting normal RPI gene
activity may, be introduced into cells via gene therapy
methods that do not contain sequences susceptible to whatever
antisense, ribozyme, or triple helix treatments are being
utilized. Alternatively, in instances whereby the RPI gene
encodes an extracellular protein, it may be preferable to co-
administer normal RPI gene protein in order to maintain the
requisite level of target gene activity.Anti-sense RNA and
DNA, ribozyme, and triple helix molecules of the invention
may be prepared by any method known in the art for the
synthesis of DNA and RNA molecules, as discussed above.
These include techniques for chemically synthesizing
oligodeoxyribonucleotides and oligoribonucleotides well known
in the art such as for example solid phase phosphoramidite
chemical synthesis. Alternatively, RNA molecules may be
generated by in vitro and in vivo transcription of DNA
sequences encoding the antisense RNA molecule. Such DNA
sequences may be incorporated into a wide variety of vectors
that incorporate suitable RNA polymerase promoters such as
the T7 or SP6 polymerase promoters. Alternatively, antisense
cDNA constructs that synthesize antisense RNA constitutively
or inducibly, depending on the promoter used, can be
introduced stably into cell lines.Demonstration Of
Therapeutic Or Prophylactic i~TtilityThe compounds of
the invention are preferably tested in vitro, and then in
vivo for the desired therapeutic or prophylactic activity,
prior to use in humans. For example, in vitro assays which
can be used to determine whether administration of a specific
compound is indicated, include in vitro cell culture assays
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in which a patient tissue sample is grown in culture, and
exposed to or otherwise administered a compound, and the
effect of such compound upon the tissue sample is observed.
Test compounds can be tested for their ability to restore
RADF or RPI levels in patients suffering from RA towards
levels found in subjects not suffering from RA or to produce
similar changes in experimental animal models of RA (e. g.,
adjuvant arthritis in rats). Compounds able to restore said
levels can be used as lead compounds for further drug
discovery, or used therapeutically. RADF and RPI expression
can be assayed by the Preferred Technology, immunoassays, gel
electrophoresis followed by visualization, or any other
method known to those skilled in the art. Such assays can be
used to screen candidate drugs or in clinical monitoring or
drug development, where abundance of an RADF or RPI can serve
as a surrogate marker for clinical disease. In various
specific embodiments, in vitro assays can be carried out with
representative cells of cell types involved in a patient's
disorder, to determine if a compound has a desired effect
upon such cell types.Compounds for use in therapy can be
tested in suitable animal model systems prior to testing in
humans, including but not limited to rats, mice, chicken,
cows, monkeys, rabbits, etc. For in vivo testing, prior to
administration to humans, any animal model system known in
the art may be used. Therapeutic/Prophylactic
Administratign And CompositionsThe invention provides
methods of treatment (and prophylaxis) by administration to a
subject of an effective amount of a compound of the
invention. In a preferred aspect, the compound is
substantially purified (e. g. substantially free from
substances that limit its effect or produce undesired side-
effects). The subject is preferably an animal, including but
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not limited to animals such as cows, pigs, horses, chickens,
cats, dogs, etc., and is preferably a mammal, and most
preferably human. In a specific embodiment, a non-human
mammal is the subject.Formulations and methods of
administration that can be employed when the compound
comprises a nucleic acid are described above; additional
appropriate formulations and routes of administration can be
selected from among those described hereinbelow.Various
delivery systems are known and can be used to administer a
compound of the invention, e.g., encapsulation in liposomes,
microparticles, microcapsules, recombinant cells capable of
expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, 1987, J. Biol. Chem. 2624429-4432),
construction of a nucleic acid as part of a retroviral or
other vector, etc. Methods of introduction include but are
not limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral
routes. The compounds may be administered by any convenient
route, for example by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e. g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be
administered together with other biologically active agents.
Administration can be systemic or local. In addition, it
may be desirable to introduce the pharmaceutical compositions
of the invention into the central nervous system by any
suitable route, including intraventricular and intrathecal
injection; intraventricular injection may be facilitated by
an intraventricular catheter, for example, attached to a
reservoir, such as an Ommaya reservoir. Pulmonary
administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.In a specific embodiment, it may be desirable to
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administer the pharmaceutical compositions of the invention
locally to the area in need of treatment; this may be
achieved by, for example, and not by way of limitation, local
infusion during surgery, topical application, e.g., in
conjunction with a wound dressing after surgery, by
injection, by means of a catheter, by means of a suppository,
or by means of an implant, said implant being of a porous,
non-porous, or gelatinous material, including membranes, such
as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or
former site) of a malignant tumor or neoplastic or pre-
neoplastic tissue.In another embodiment, the compound can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 2491527-1533 (1990); Treat et al., in Liposomes in
the Therapy of Infectious Disease and Cancer, Lopez-Berestein
and Fidler (eds.), Liss, New York, pp. 353-365 (1989);
Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)In
yet another embodiment, the compound can be delivered in a
controlled release system. In one embodiment, a pump may be
used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng.
14201 (1987); Buchwald et al., Surgery X8507 (1980); Saudek
et al., N. Engl. J. Med. 321574 (1989)). In another
embodiment, polymeric materials can be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Florida (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen
and Ball (eds.), Wiley, New York (1984); Ranger and Peppas,
J. Macromol. Sci. Rev. Macromol. Chem. X3,61 (1983); see also
Levy et al., Science 22$190 (1985); During et al., Ann.
Neurol. X351 (1989); Howard et al., J. Neurosurg. 71105
(1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target,
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i.e., the brain, thus requiring only a fraction of the
systemic dose (see, e.g., Goodson, in Medical Applications of
Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other
controlled release systems are discussed in the review by
Langer (Science X491527-1533 (1990)).In a specific embodiment
where the compound of the invention is a nucleic acid
encoding a protein, the nucleic acid can be administered in
vivo to promote expression of its encoded protein, by
constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S.
Patent No. 4,980,286), or by direct injection, or by use of
microparticle bombardment (e. g., a gene gun; Biolistic,
Dupont), or coating with lipids or cell-surface receptors or
transfecting agents, or by administering it in linkage to a
homeobox-like peptide which is known to enter the nucleus
(see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA
81864-1868), etc. Alternatively, a nucleic acid can be
introduced intracellularly and incorporated within host cell
DNA for expression, by homologous recombination. The present
invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a
compound, and a pharmaceutically acceptable carrier. In a
specific embodiment, the term pharmaceuticallyacceptable
means approved by a regulatory agency of the Federal or a
state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and
more particularly in humans. The term carrier refers to a
diluent, adjuvant, excipient, or vehicle with which the
therapeutic is administered. Such pharmaceutical carriers
can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin,
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such as peanut oil, soybean oil, mineral oil, sesame oil and
the like. Water is a preferred carrier when the
pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions
can also be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical excipients
include starch, glucose, lactose, sucrose, gelatin, malt,
rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk,
glycerol, propylene, glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents.
These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition
can be formulated as a suppository, with traditional binders
and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable pharmaceutical carriers are described in
RemingtonTsPharmaceuticalSciences by E.W. Martin. Such
compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for
proper administration to the patient. The formulation should
suit the mode of administration.In a preferred embodiment,
the composition is formulated in accordance with routine
procedures as a pharmaceutical composition adapted for
intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the
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composition may also include a solubilizing agent and a local
anesthetic such as lignocaine to ease pain at the site of the
injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent.
Where the composition is to be administered by infusion, it
can be dispensed with an infusion bottle containing sterile
pharmaceutical grade water or saline. Where the composition
is administered by injection, an ampoule of sterile water for
injection or saline can be provided so that the ingredients
may be mixed prior to administration.The compounds of the
invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with
free amino groups such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those
formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, etc.The amount of the compound of the
invention which will be effective in the treatment of RA can
be determined by standard clinical techniques. In addition,
in vitro assays may optionally be employed to help identify
optimal dosage ranges. The precise dose to be employed in
the formulation will also depend on the route of
administration, and the seriousness of the disease or
disorder, and should be decided according to the judgment of
the practitioner and each patient's circumstances. However,
suitable dosage ranges for intravenous administration are
generally about 20-500 micrograms of active compound per
kilogram body weight. Suitable dosage ranges for intranasal
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administration are generally about 0.01 pg/kg body weight to
1 mg/kg body weight. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal
model test systems. Suppositories generally contain active
ingredient in the range of 0.5% to 10% by weight; oral
formulations preferably contain 10% to 95% active
ingredient. The invention also provides a pharmaceutical pack
or kit comprising one or more containers filled with one or
more of the ingredients of the pharmaceutical compositions of
the invention. Optionally associated with such containers)
can be a notice in the form prescribed by a governmental
agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration. EXAMPLE PROTEINS FROM SERUM AND SYNOVIAL
FLUID OF PATIENTS WITH R.A Using the following Reference
Protocol, proteins in serum and synovial fluid from patients
with rheumatoid arthritis (RA) or from patients without RA
(i.e., patients with gout,.osteoarthritis, or traumatic
synovitis) were separated by isoelectric focusing followed by
SDS-PAGE and compared. Each sample was run in duplicate.
Sample preparationA protein assay was carried out on the
sample as received (Pierce BCA Cat # 23225). A volume of
serum corresponding to 300~,g of total protein was aliquoted
and an equal volume of 10% (w/v) SDS (Fluka 71729), 2.3%
(w/v) dithiothreitol (BDH 443852A) was added. The sample was
heated at 95°C for 5 mins, and then allowed to cool to 20°C .
125.1 of the following buffer was then added to the sampleBM
urea (BDH 452043w )4% CHAPS (Sigma C3023)65mM dithiotheitol
(DTT)2% (v/v) Resolytes 3.5-10 (BDH 44338 2x)This mixture was
vortexed, and centrifuged at 13000 rpm for 5 mins at 15°C,
and the supernatant was analyzed by isoelectric
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focusing.Isoelectric Focusincr Isoelectric focusing (IEF), was
performed using the Immobiline~ DryStrip Kit (Pharmacia
BioTech), following the procedure described in the
manufacturer's instructions, see Instructions for Immobiline°
DryStrip Kit, Pharmacia, # 18-1038-53, Edition AB
(incorporated herein by reference in its entirety).
Immobilized pH Gradient (IPG) strips (l8cm, pH 3-10 non-
linear strips; Pharmacia Cat. # 17-1235-O1) were rehydrated
overnight at 20°C in a solution of 8M urea, 2% (w/v) CHAPS,
lOmM DTT, 2% (v/v) Resolytes 3.5-10, as described in the
Immobiline DryStrip Users Manual. For IEF, 50,1 of
supernatant (prepared as above) Was loaded onto a strip, with
the cup-loading units being placed at the basic end of the
strip. The loaded gels were then covered with mineral oil
(Pharmacia 17-3335-01) and a voltage was immediately applied
to the strips according to the following profile, using a
Pharmacia EPS3500XL power supply (Cat 19-3500-Ol):
Initial voltage = 300V for 2 hrs
Linear Ramp from 300V to 3500V over 3hrs
Hold at 3500V for l9hrs
For all stages of the process, the current limit was set to
lOmA for 12 gels, and the wattage limit to 5W. The
temperature was held at 20°C throughout the run.
6.3 Gel Equilibration and SDS-PAGE Gel
Equilibration and SDS-PAGE
1.1
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After the final l9hr step, the strips were immediately
removed and immersed for 10 mins at 20°C in a first solution
of the following composition: 6M urea; 2% (w/v) DTT; 2%
(w/v) SDS; 30% (v/v) glycerol (Fluka 49767); 0:05M Tris/HC1,
pH 6.8 (Sigma Cat T-1503). The strips were removed from the
first solution and immersed for 10 minx at 20°C in a second
solution of the following composition: 6M urea; 2% (w/v)
iodoacetamide (Sigma I-6125); 2% (w/v) SDS; 30% (v/v)
glycerol; 0.05M Tris/HC1, pH 6.8. After removal from the
second solution, the strips were loaded onto supported gels
for SDS-PAGE according to Hochstrasser et al., 1988,
Analytical Biochemistry 173:412-423 (incorporated herein by
reference in its entirety), with modifications as specified
below.
6.4 Preparation of supported gels Preparation of
supported gels
The gels were cast between two glass plates of the
following dimensions: 23cm wide x 24cm long (back plate);
23cm wide x 24cm long with a 2cm deep notch in the central
l9cm (front plate). To promote covalent attachment of SDS-
PAGE gels, the back plate was treated with a 0.4% solution of
Y-methacryl-oxypropyltrimethoxysilane in ethanol
(BindSilaneTM; Pharmacia Cat. # 17-1330-Ol). The front plate
was treated with RepelSilaneT"' (Pharmacia Cat. # 17-1332-O1)
to reduce adhesion of the gel. Excess reagent was removed by
washing with water, and the plates were allowed to dry. At
this stage, both as identification for the gel, and as a
marker to identify the coated face of the plate, an adhesive
bar-code was attached to the back plate in a position such
that it would not come into contact with the gel matrix.
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The dried plates were assembled into a casting box with
a capacity of 13 gel sandwiches. The top and bottom plates
of each sandwich were spaced by means of lmm thick spacers,
2.5 cm wide. The sandwiches were interleaved with acetate
sheets to facilitate separation of the sandwiches after gel
polymerization. Casting was then carried out according to
Hochstrasser et al., op. cit.
A 9-16% linear polyacrylamide gradient was cast,
extending up to a point 2cm below the level of the notch in
the front plate, using the Angelique gradient casting system
(Large Scale Biology). Stock solutions were as follows.
Acrylamide (40% in water) was from Serva (Cat. # 10677). The
cross-linking agent was PDA (BioRad 161-0202), at a
concentration of 2.6% (w/w) of the total starting monomer
content. The gel buffer was 0.375M Tris/HC1, pH 8.8. The
polymerization catalyst was 0.05% (v/v) TEMED (BioRad 161-
0801), and the initiator was 0.1% (w/v) APS (BioRad 161-
0700). No SDS was included in the gel and no stacking gel
was used. The cast gels were allowed to polymerize at 20°C
overnight, and then stored at 4°C in sealed polyethylene bags
with 6m1 of gel buffer, and were used within 4 weeks.
6.5 SDS-PAGE SDS-PAGE
1.1
A solution of 0.5% (w/v) agarose (Fluky Cat 05075) was
prepared in running buffer (0.025M Tris, 0.198M glycine
(Fluky 50050), 1% (w/v) SDS, supplemented by a trace of
bromophenol blue). The agarose suspension was heated to 70°C
with stirring, until the agarose had dissolved. The top of
the supported 2nd D gel was filled with the agarose solution,
and the equilibrated strip was placed into the agarose, and
tapped gently with a palette knife until the gel was
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intimately in contact with the 2nd D gel. The gels were
placed in the 2nd D running tank, as described by Amess et
al., 1995, Electrophoresis 16:1255-1267 (incorporated herein
by reference in its entirety). The tank was filled with
running buffer (as above) until the level of the buffer was
just higher than the top of the region of the 2nd D gels which
contained polyacrylamide, so as to achieve efficient cooling
of the active gel area. Running buffer was added to the top
buffer compartments formed by the gels, and then voltage was
applied immediately to the gels using a Consort E-833 power
supply. For 1 hour, the gels were run at 20mA/gel. The
wattage limit was set to 150W for a tank containing 6 gels,
and the voltage limit was set to 600V. After 1 hour, the
gels were then run at 40mA/gel, with the same voltage and
wattage limits as before, until the bromophenol blue line was
0.5cm from the bottom of the gel. The temperature of the
buffer was held at 10°C throughout the run.
6.6 Staining Staining
Upon completion of the electrophoresis run, the gels
were immediately removed from the tank for fixation. The top
plate of the gel cassette was carefully removed, leaving the
gel bonded to the bottom plate. The bottom plate with~its
attached gel was then placed into a staining apparatus, which
can accommodate 12 gels. The gels were completely immersed
in fixative solution of 40~ (v/v) ethanol (BDH 28719), 10~
(v/v) acetic acid (BDH 100016X), 50~ (v/v) water (MilliQ-
Millipore), which was continuously circulated over the gels.
After an overnight incubation, the fixative was drained from
the tank, and the gels were primed by immersion in 7.5~ (v/v)
acetic acid, 0.05 (w/v) SDS, 92.5 (v/v) water for 30 mins.
The priming solution was then drained, and the gels were
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stained by complete immersion in a staining solution for 4
hours. A solution of fluorescent dye was prepared by
diluting Sypro Red (Molecular Probes, Inc., Eugene, Oregon)
according to the manufacturer's instructions; this diluted
solution was filtered under vacuum though a 0.4um filter.
6.7 Imaging of the gelcompletion of the
electrophoresis run, the gels were immediately
removed from the tank for fixation. The top plate
of the gel cassette was carefully removed, leaving
the gel bonded to the bottom plate. The bottom
plate with its attached gel was then placed into a
staining apparatus, which can accommodate 12 gels.
The gels were completely immersed in fixative
solution of 40% (v/v) ethanol (BDH 28719), 10%
(v/v) acetic acid (BDH 100016X), 50% (v/v) water
(MilliQ-Millipore), which was continuously
circulated over the gels. After an overnight
incubation, the fixative was drained from the tank,
and the gels were primed by immersion in 7.5% (v/v)
acetic acid, 0.05% (w/v) SDS, 92.5% (v/v) water for
30 minx. The priming solution was then drained,
and the gels were stained by complete immersion in
a staining solution for 4 hours. A solution of
fluorescent dye was prepared by diluting Sypro Red
(Molecular Probes, Inc., Eugene, Oregon) according
to the manufacturer's instructions; this diluted
solution was filtered under vacuum though a 0.4um
filter. Imaging of the ael
1.1
A computer-readable output was produced by imaging the
fluorescently stained gels with a Storm scanner (Molecular
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Dynamics, Sunnyvale, California) according to the
manufacturer's instructions, (see Storm User's Guide, 1995,
Version 4.0, Part No. 149-355, incorporated herein by
reference in its entirety) with modifications as described
below. The gels were removed from the stain, rinsed with
water briefly, and imaged on the Storm Scanner, in Red
Fluorescence mode with a PMT setting of 1000V, and a
resolution of 200 ~.m. Since the gel was rigidly bonded to a
glass plate, the gel was held in contact with the scanner bed
during imaging. To avoid interference patterns arising from
non-uniform contact between the gel and the scanner bed, a
film of water was introduced under the gel, taking care to
avoid air pockets. Moreover, the gel was placed in a frame
provided with two fluorescent buttons that were imaged
together with the gel to provide reference points (designated
Ml and M2) for determining the x,y coordinates of other
features detected in the gel. A matched frame was provided
on a robotic gel excisor in order to preserve accurate
alignment of the gel. After imaging, the gels were sealed in
polyethylene bags containing a small volume of staining
solution, and then stored at 4°C.
6.8 Digital Analysis of the Data
The data were processed as described in U.S. Application
No. 08/980,574, Sections 5.4 and 5.5 (incorporated herein by
reference), as set forth more particularly below.
6.8.1 Computer Analvais Of The Detector
Output Digital Analysis of the DataThe data
were processed as described in U.S.
Application No. 08/980,574, Sections 5.4 and
5.5 (incorporated herein by reference), as set
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forth more particularly below. Computer
Analysis Of The Detector Output
The output from the scanner was first processed using
the MELANIE~ II 2D PAGE analysis program (Release 2.2, 1997,
BioRad Laboratories, Hercules, California, Cat. # 170-7566)
to autodetect the registration points, M1 and M2; to autocrop
the images (i.e., to eliminate signals originating from areas
of the scanned image lying outside the boundaries of the gel,
e.g., the reference frame); to filter out artifacts due to
dust; to detect and quantify features; and to create image
files in GIF format. Features were detected using the
following parameters:
Smooths =2
Laplacian threshold 50
Partials threshold 1
Saturation = 100
Peakedness = 0
Minimum Perimeter = 10
6.9 Assignment of pI and MW Values Assignment
of pI and MW Values
Images were evaluated to reject images which had gross
abnormalities, or were of too low a loading or overall image
intensity, or were of too poor a resolution, or where
duplicates were too dissimilar. If one image of a duplicate
was rejected then the other image belonging to the duplicate
was also rejected regardless of image quality. Samples that
were rejected were scheduled for repeat analysis.
Landmark identification was used to determine the pI and
MW values of features detected in the images. This process
involves the identification of certain proteins which are
expected to be found in any given biological sample. As
these common proteins exhibit an identical isoelectric point
and molecular weight from sample to sample, they can be used
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as standards; this process also corrects for any possible gel
variation or distortion.
From the dataset of normal serum gels, a gel was
arbitrarily chosen as the Primary Master Gel. Landmark
features were then identified by comparing the features
detected in this Primary Master Gel with features previously
identified on 2D electrophoresis of normal human serum. (see
Bjellqvist et al., 1993, Electrophoresis 14:1357-1365;
incorporated herein by reference in its entirety).
Fourteen landmark features, designated PL1 to PL12 and
PL15 to PL16, were identified in the Primary Master Gel.
These landmark features are identified in Figure 1 and were
assigned the pI and/or MW values indicated in Table XII.
Table XII. Landmark Features used in this study
Name pI MW Name pI MW
(kd) (kd)
PL1 None 186,073 PL8 6.47 47,195
PL2 6.20 100,000 PL9 5.29 43,541
PL3 4.73 93,708 PL10 5.22 23,000
PL4 5.13 73,465 PL11 4.47 25,183
PL5 4.97 52,739 PL12 5.52 13,800
PL6 4.10 None PL15 7.80 36,962
PL7 4.80 40,997 PL16 8.58 None
As many of these landmarks as possible were identified
in each gel image in the dataset.
All features in the Master gel were then assigned a pI
value by linear interpolation/extrapolation (using the
MELANIE II software) to the two nearest landmarks that had
been assigned a pI value, and were assigned a MW value by
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linear interpolation/extrapolation (using the MELANIE II
software) to the MW of the two nearest landmarks that had
been assigned a MW value. These features were also labelled
with a unique number known as its Molecular Cluster Index (or
MCI ) .
Secondary Master gels were chosen for both the RA serum
and RA synovial gels. Features in these gels were paired
with common features in the Master gel, using the algorithm
supplied with the MELANIE II software, as described at
Section A, pp. 8-10 of the MELANIE II 2D PAGE (Release 2.2)
User Manual (The Melanie Group, Geneva, Switzerland).
Features that have been paired are linked to the
corresponding MCI, and hence to an associated pI and MW
value. Unpaired features present in these secondary master
gels were assigned pI and MW values by linear
interpolation/extrapolation (using the MELANIE II software)
with respect to the pI and MW of the landmarks. Additional
unique entries were then created in the MCI for these
features.
6.9.1 Construction of Profiles
construction of Profiles
All gels in the dataset were now matched to the Primary
and Secondary Master Gels, and paired features were linked to
the corresponding entries in the Molecular Cluster Index.
Duplicate gels were then aligned via the landmarks and a
matching process performed so as to pair identical spots on
the duplicate gels. This provided increased assurance that
subsequently measured isoelectric points and molecular
weights were accurate, as paired spots demonstrated the
reproducibility of the separation and also filtered out
artefacts.
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A measurement of the intensity of each protein spot was
taken and stored. Each protein spot was assigned an
identification code and matched to a spot on the Master gel.
The end result of this aspect of the analysis was the
generation, for each duplicate set of gels representing a
single serum or synovial fluid sample, of a digital profile
which contained, for each identified spot: 1) a unique
arbitrary identification code, 2) the x,y coordinates, 3) the
isoelectric point, 4) the molecular weight, 5) the signal
value, 6) the standard deviation for each of the preceding
measurements, and 7) a pointer to the MCI of the spot on the
master gel to which this spat was matched. By virtue of the
Laboratory Information Management System (LIMS), this profile
was traceable to the actual stored gel from which it was
generated, so that proteins identified by computer analysis
of gel profile databases could be retrieved. The LIMS also
permitted the profile to be traced back to the original
sample or patient.
6.9.2 Cross Matching Between Samples
1.1.1
Once the profile was generated, analysis was directed
toward the selection of interesting proteins. Each
significant feature in a profile was assigned an index, the
"Molecular Cluster Index" (MCI) that identifies the feature
in all gels and that serves as a pointer to parameters (1) to
(7) above of the feature. A molecular cluster table was
generated from the master gel for each sample type (e.g., R.A
serum and RA synovial fluid). Gels from all other samples of
the same type were matched with the relevant primary and
secondary master gels. The digital profiles for each sample
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were then annotated by adding, for each matched feature, the
MCI assigned to that feature in the master profile.
6.9.3 Differential Analysis of the
Profiles
Within each sample set (synovial fluid or serum), the
profiles were analyzed to identify and select those features
present in at least 50~ of the profiles. These selected
features were then assembled into a synovial fluid feature
set and a serum feature set. Matching features of each
feature set were then compared to identify those features
showing at least a 2-fold difference in mean intensity
between synovial fluid and serum. Then, the same features
were examined in the synovial and serum samples from subjects
without RA. Features which were differentially present in
the RA serum as compared to the RA synovial fluid samples but
not differentially present in the non-RA serum as compared to
the non-RA synovial fluid samples were identified as
Rheumatoid Arthritis-Diagnostic Features (RADFs).
6.10 Recovery and analysis of se~.ected proteinsRecovery
and analysis of selected ~,roteins
Proteins in RADFs were robotically excised and processed
to generate tryptic peptides; partial amino acid sequences of
these peptides were determined by mass spectroscopy, using de
novo sequencing.
6.11 Results Results
1.1
These initial experiments identified 12 features that
were increased and 10 features that were decreased in
synovial fluid verses serum from RA patients. Each RADF was
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differentially present only in synovial fluid verses serum
from subjects with RA but not in synovial fluid verses serum
from subjects without RA.
Partial amino acid sequences were determined for the
differentially present RPIs in these RADFs. Computer
analysis of public databases revealed that 21 of these
partially sequenced proteins were known in the art and that S
were not described in any public database examined. Table
VIII illustrates that several RPIs are isoforms of the same
protein. For example, RPI-1 and RPI-11 are isoforms of
transferrin. These isoforms are thought to arise from
differences in post-translational processing (e. g.,
glycosylation, phosphorylation, acylation or minimal
proteolysis) .
7 EXAMPLE: PROTEINS FROM SERUM OF
PATIENTS WITH AND WITHOUT RA
Proteins in serum from patients with rheumatoid
arthritis (RA) and from patients without RA were separated by
isoelectric focusing followed by SDS-PAGE and compared.
The analysis was performed as described in Example 6,
except that the comparison in Example 7 was between serum
from RA patients and serum from non-RA patients.
7.1 Results
These initial experiments identified 12 features that
were increased and 9 features that were decreased in serum
from RA patients as compared with serum from non-RA patients.
Details of these RADFs are provided in Tables III and IV.
Each RADF was differentially present in RA serum versus non-
RA serum.
Partial amino acid sequences were determined for the
differentially present RPIs in these RADFs. Computer
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analysis of public databases revealed that 27 of these
partially sequenced proteins were known in the art and that 4
were not described in any public database examined. Table XI
illustrates that several RPIs are isoforms of the same
protein. For example, RPI-44 and RPT-45 are isoforms of
transferrin. These isoforms are thought to arise from
differences in post-translational processing (e. g.,
glycosylation, phosphorylation, acylation or minimal
proteolysis).
Moreover, Tables VIII to XI demonstrate that RPI-2, RPI-
6, RPI-7, RPI-14, RPI-23, RPI-25, RPI-31, and RPI-37
represent immunoglobulin isoforms. RPI-2; in particular
represents an IgG light chain isoform. In other words, the
Preferred Technology has been used to identify a defined
subset of immunoglobulin isoforms that are specifically
associated with RA and that likely reflect an oligoclonal
humoral immune response associated with this disease. These
immunoglobulin isoforms (and fragments thereof, antibodies
thereto, etc.) are useful for diagnosis, prognosis,
therapeutic monitoring and drug development.
The present invention is not to be limited in scope by
the exemplified embodiments, which are intended as
illustrations of single aspects 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 and
accompanying drawings. Such modifications are intended to
fall within the scope of the appended claims.
All publications cited herein are incorporated by
reference in their entirety.
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