U.S. patent application number 16/564209 was filed with the patent office on 2020-03-05 for anti-nuclear antibody detection and diagnostics for systemic and non-systemic autoimmune disorders.
The applicant listed for this patent is IMMCO DIAGNOSTICS, INC.. Invention is credited to KISHORE MALYAVANTHAM, LAKSHMANAN SURESH.
Application Number | 20200072845 16/564209 |
Document ID | / |
Family ID | 54196278 |
Filed Date | 2020-03-05 |
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United States Patent
Application |
20200072845 |
Kind Code |
A1 |
SURESH; LAKSHMANAN ; et
al. |
March 5, 2020 |
ANTI-NUCLEAR ANTIBODY DETECTION AND DIAGNOSTICS FOR SYSTEMIC AND
NON-SYSTEMIC AUTOIMMUNE DISORDERS
Abstract
Provided are compositions that contain mammalian cells for use
in detecting antibodies. The mammalian cells are modified such that
they do not contain LEDGF protein. The mammalian cells are
immobilized on a solid substrate. The compositions can also contain
mammalian cells that contain the LEDGF protein. Methods for using
the cell compositions in diagnostic approaches are included, as are
kits for performing diagnostic tests.
Inventors: |
SURESH; LAKSHMANAN;
(WILLIAMSVILLE, NY) ; MALYAVANTHAM; KISHORE;
(WILLIAMSVILLE, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IMMCO DIAGNOSTICS, INC. |
BUFFALO |
NY |
US |
|
|
Family ID: |
54196278 |
Appl. No.: |
16/564209 |
Filed: |
September 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15124175 |
Sep 7, 2016 |
10451633 |
|
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PCT/US15/22120 |
Mar 24, 2015 |
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16564209 |
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61969771 |
Mar 24, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/564 20130101;
C12N 15/85 20130101; G01N 33/5005 20130101; G01N 33/6854
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; G01N 33/50 20060101 G01N033/50; G01N 33/564 20060101
G01N033/564; C12N 15/85 20060101 C12N015/85 |
Claims
1-9. (canceled)
10. A method for determining whether a biological sample comprises
antibodies that bind to LEDGF protein (anti-LEDGF Abs), the method
comprising: i) exposing the biological sample to mammalian cells
that are modified such that they do not comprise LEDGF protein
(LEDGF- cells), ii) exposing the biological sample to mammalian
cells that comprise the LEDGF protein (LEDGF+ cells), and iii)
comparing the amount of anti-LEDGF Abs bound to the LEDGF- cells to
the amount of anti-LEDGF Abs bound to the LEDGF+ cells, wherein
determining a greater amount of anti-LEDGF Abs bound to the LEDGF+
cells relative to the amount of anti-LEDGF Abs bound to the LEDGF-
cells is indicative that the biological sample comprised the
anti-LEDGF Abs, and wherein the same or less anti-LEDGF Abs bound
to the LEDGF+ cells relative to the amount of anti-LEDGF Abs bound
to the LEDGF- cells is indicative that the biological sample did
not comprise the anti-LEDGF Abs.
11. The method of claim 10 wherein the LEDGF- cells are killed and
permeabilized and are immobilized on a solid substrate.
12. The method of claim 11, wherein the LEDGF+ cells are killed and
permeabilized, and are immobilized on the same solid substrate as
the LEDGF- cells, or are immobilized on a distinct solid
substrate.
13. The method of claim 12, wherein the determining the amount of
anti-LEDGF Abs is performed using an indirect immunofluorescence
(IIF) assay.
14. The method of claim 10, wherein a chromosome in the LEDGF-
cells comprises a Cas9 DNA coding sequence, or a DNA sequence
encoding a clustered regularly interspaced short palindromic
repeats (CRISPR) guide RNA targeted to a DNA sequence encoding the
LEDGF protein, or a combination thereof.
15. The method of claim 12, wherein the LEDGF- and LEGDF+ cells are
the same mammalian cell type.
16-20. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application No. 61/969,771, filed on Mar. 24, 2014, the disclosure
of which is incorporated herein by reference.
FIELD
[0002] The present invention relates generally to autoimmune
disease and more particularly to compositions and methods for use
in detecting anti-nuclear antibodies
BACKGROUND
[0003] Autoimmune diseases are typically challenging to diagnose,
and in general individuals with one autoimmune disease are at
higher risk for developing others, such as systemic autoimmune
rheumatic diseases (SARDs). The presence of anti-nuclear
autoantibodies (ANAs) is considered to be a hallmark of SARDS, and
this association has been known for some time. The American College
of Rheumatology (ACR) recommends testing for ANAs by indirect
immunofluorescence (IIF) assay using HEp-2 cells, as described in
their position statement in 2009. This statement explained that
HEp-2 cells are able to express 100-150 relevant auto-antigens for
use in ANA antibody detection. Thus, immobilized and preserved
monolayers of HEp2 cells are the most commonly used substrates in
IIF detection of ANAs.
[0004] ANA detection using IIF assay can reveal a multitude of
patterns such as homogeneous, fine granular, coarse granular,
nucleolar, centromere, nuclear dots, pleomorphic, mitochondrial and
a variety of cytoskeletal patterns. Patients can have one or more
patterns in combination with varying intensity of reactivity for
each pattern. These patterns are a result of specific autoantibody
binding to nuclear and cytoplasmic antigens which include but are
not necessarily limited to dsDNA, nucleosomes, histones, SS-A
Ro52/Ro60, SS-B/La, Ku, Mi-2, RNPs (Ribonucleoproteins: U1SnRNP 68,
U1SnRNP A, U1SnRNP C, U2SnRNPs etc,), Scl-70, PM-Scl, Fibrillarin,
Th/To, CENP-B, CENP-A, Sp100, PCNA, Ribo-P, Jo1, AMA-M2, Actin,
Vimentin, and others.
[0005] Other methodologies have been utilized for screening and
confirmation of ANAs. However, due to a variety of reasons, which
include but are not limited to prevalence of false negative and
false positive results, lack of standardization of test algorithms
(i.e., reflex testing), and an inability to detect the diverse
arrays of ANAs prevalent in individuals with SARDS, use of HEp-2
cells as the substrate for ANA testing remains the gold standard.
Unfortunately, use of HEp-2 cells also involves complex test
interpretation, false results and specialized skills, in part
because it has been reported that up to 20% of apparently healthy
subjects give a positive ANA IIF test result due to the presence of
autoantibodies that recognize the so-called "dense fine speckles
70" (DFS70) antigen, which is also referred to herein and in the
art as lens epithelium-derived growth factor (LEDGF). PSIP1/LEDGF
is also known as AA408851, AU015605, Dfs70, Ledgf, Ledgfa, Ledgfb,
mLEDGF, PC4 and SFRS1-interacting protein (PSIP1), Psip2 (isoform),
p52, p75, PAIP encoded by the PSIP1 gene. Moreover, the DFS IIF
pattern has been reported in up to 20% of ANA positive healthy
subjects, but often not in ANA positive sera obtain from SARD
patients (Mahler and Fritzler 2012). Since the main objective of
the ANA HEp-2 test is to function as a tool for diagnosing and
classifying SARD, as well as potentially other autoimmune diseases,
the anti-DFS70 antibodies and the DFS pattern they produce reduce
the usefulness of the ANA test, such as by increasing false results
and otherwise complicating test interpretation. This has important
ramifications for a variety of approaches that rely on accurate
detection of ANA and treatment decisions for patients who are
tested for ANA antibodies. Thus, there is an ongoing and unmet need
for improved compositions and methods for detecting ANA. The
present disclosure meets these and other needs.
SUMMARY
[0006] The present disclosure comprises in various embodiments
compositions and methods for use in detecting ANA autoantibodies,
and/or for determining whether a biological sample obtained or
derived from an individual comprises antibodies that recognize the
LEDGF protein. The disclosure includes compositions and methods
that can be used for diagnosing and/or aiding in the diagnosis of
autoimmune disorders that are positively correlated with the
presence of ANA autoantibodies. Kits/products comprising reagents
for use in detection of ANA autoantibodies, such as antibodies to
LEDGF protein, are also provided
[0007] In one aspect the present disclosure comprises modified
mammalian cells for use in detecting antibodies. The mammalian
cells are modified such that they do not express or comprise LEDGF
protein (LEDGF- cells). In embodiments, the disclosure includes
mixtures of the mammalian LEDGF- cells, and mammalian cells that do
express and comprise LEDGF protein (LEDGF+ cells). In embodiments,
the mammalian cells are immobilized on a solid substrate, such as a
glass, plastic, or other polymer-based substrate. In embodiments,
the solid substrate comprises a microscope slide, a diagnostic
slide, a microtitre plate, or beads formed of glass or a
polymer.
[0008] In embodiments, the cells are killed and permeabilized.
Those skilled in the art will recognize that permeabilized cells
are those cells which have been exposed to organic reagents
(commonly referred to in the art as `fixatives`) which can include
but are not limited to organic solvents, such as acetone, alcohol,
and aldehyde containing solutions, such as formaldehyde,
paraformaldehyde, and the like. Cells exposed to such reagents are
commonly referred to as "fixed" and the process of treating them
with such reagents is referred to as "fixing" the cells. Fixing the
mammalian cells such that they are permeabilized is lethal, and
thus the fixed/permeabilized cells are also considered to be killed
cells.
[0009] The LEDGF- cells are modified using any suitable techniques,
reagents and the like such that LEDGF protein is not expressed, or
its expression is reduced. In embodiments, mRNA encoding the LEDGF
protein is degraded using any of a variety of RNAi-mediated
approaches. In another embodiment, the gene encoding the LEDGF
protein, which is described more fully below, is disrupted by any
suitable technique including but not limited to the use of a
clustered regularly interspaced short palindromic repeats (CRISPR)
system comprising a CRISPR-associated (Cas) nuclease and a CRISPR
guide RNA (gRNA). In embodiments, the modification to the cells
comprises integration of a polynucleotide sequence encoding the Cas
enzyme and/or the gRNA into at least one chromosome of the cells,
such as the LEDGF- cells.
[0010] In embodiments, the compositions and methods use modified
LEDGF+ and LEDGF- cells of the same type, i.e., they are both the
same type of cancer cell, or they are both of the same cell line,
or derived from the same cell line.
[0011] In embodiments the disclosure includes mixtures of LEDGF+
mammalian cells and modified LEDGF- mammalian cells that are useful
in diagnostic assays. The mixtures can be such that antibodies that
bind to antigens in the modified cells can be used to, for example,
establish a background amount of antibody binding that can be
compared to antibody binding using the LEDGF+ cells as a comparison
substrate. Thus, in certain aspects, a ratio of LEDGF- and LEDGF+
cells are provided. In embodiments, the ratio comprises a LEDGF-
cell amount to a LEDGF+ cell amount of 1:1, 1:2, 1:3, 1:4, 1:3,
1:6, 1:7, 1:8, 1:9, 1:10, as well as the reverse ratios.
[0012] In embodiments the disclosure includes modified cells,
wherein the modification is such that the cells do not express
LEDGF, cell cultures/cell lines derived from such cells, and their
progeny.
[0013] In embodiments the disclosure includes LEDF+ cells, wherein
the LEDGF protein in the LEGDF+ cells is present in a complex with
an antibody, and thus is suitable for use in a variety of
immuno-diagnostic tests. In an embodiment, the antibody is a first
antibody, such as a primary antibody. In embodiments, the primary
antibody that is bound the LEDGF protein the LEDGF+ cells is itself
present in a complex with a detectably labeled secondary
antibody.
[0014] In embodiments, the LEDGF+ and/or LEDGF- cells comprise one
or more nuclear antigens in present in a complex with an
antibody.
[0015] In another aspect the disclosure provides a method for
determining whether a biological sample comprises antibodies that
bind to LEDGF protein (anti-LEDGF Abs). The method generally
comprises the steps of: [0016] i) exposing the biological sample to
mammalian cells that are modified such that they do not comprise
LEDGF protein (LEDGF- cells), [0017] ii) exposing the biological
sample to mammalian cells that comprise the LEDGF protein (LEDGF+
cells), and [0018] iii) comparing the amount of anti-LEDGF Abs
bound to the LEDGF- cells to the amount of anti-LEDGF Abs bound to
the LEDGF+ cells, [0019] wherein determining a greater amount of
anti-LEDGF Abs bound to the LEDGF+ cells relative to the amount of
anti-LEDGF Abs bound to the LEDGF- cells is indicative that the
biological sample comprised the anti-LEDGF Abs, and [0020] wherein
the same or less anti-LEDGF Abs bound to the LEDGF+ cells relative
to the amount of anti-LEDGF Abs bound to the LEDGF- cells is
indicative that the biological sample did not comprise the
anti-LEDGF Abs.
[0021] In embodiments, the cells used in the method are killed and
permeabilized and are immobilized on a solid substrate. In
embodiments, the LEDGF- cells and the LEDGF+ cells are immobilized
on the same solid substrate; in embodiments they are immobilized on
distinct solid substrates. In embodiments, determining the amount
of anti-LEDGF Abs is performed using an indirect immunofluorescence
(IIF) assay.
[0022] In another aspect the disclosure comprises a kit comprising
modified LEDGF- cells and LEDGF+ cells, wherein the LEDGF- cells
and the LEDGF+ cells are immobilized on one or more solid
substrates, and wherein the LEDGF- cells and the LEDGF+ cells are
killed and permeabilized. In embodiments, the solid substrate(s)
and the cells are dried, and are provided in one or more suitable
containers.
[0023] In embodiments, the kit further comprises a composition
comprising primary antibodies that are capable of binding to
anti-nuclear autoantibodies (ANAs). The kit may further comprise
detectably labeled secondary antibodies that are capable of binding
to the primary antibodies. Any suitable detectable label can be
used and many are well known in the art. In embodiments, the
detectable label is a florescent label and is thus suitable for use
in, for example, an IIF assay.
BRIEF DESCRIPTION OF THE FIGURE
[0024] FIG. 1: Wild Type Hep2 expressing LEDGF and PISP1 disrupted
cell line using per Example Sequence 3 and not expressing LEDGF
were tested by IIF using confirmed human DFS70 positive anti-sera.
Top panel: two examples (A1 and B1) show brightly labeled cells
(WT) and cells with background fluorescence signal (PSIP KO).
Bottom panel: profile analysis (ImageJ version 1.421, National
Institute of Health, USA) plots the intensity of labeled nuclei
along the line (A2 is intensity plot for A1; B2 is intensity plot
for B1). High peaks correspond to WT cells and low peaks correspond
to PSIP-KO cells which do not express any detectable LEDGF
protein.
DETAILED DESCRIPTION
[0025] The present disclosure provides compositions and methods for
using the compositions in detecting ANA autoantibodies, and/or for
determining whether a biological sample obtained or derived from an
individual comprises antibodies that recognize the LEDGF protein,
and for diagnosing and/or aiding in the diagnosis of autoimmune
disorders that are positively correlated with the presence of ANA
autoantibodies. Such disorders include but not necessarily limited
to systemic autoimmune rheumatic diseases (SARDs). Kits/products
comprising reagents for use in detection of ANA autoantibodies are
also provided.
[0026] In general, the disclosure provides approaches to reducing
and/or eliminating the DFS pattern that is frequently
characteristic of IIF analysis of ANAs that in current testing
typically rely on HEp-2 cells as a substrate, and encompasses in
vitro compositions comprising modified mammalian cells that have
less LEDGF expression relative to unmodified HEp-2 cells, and
methods of using such modified cells to detect ANA autoantibodies.
However, rather than being limited to modified HEp-2 cells, the
present disclosure provides compositions comprising mammalian cells
of any origin, wherein the cells have been modified to be improved
substrates for ANA and/or LEDGF testing. Thus, in various
embodiments, the present disclosure involves mammalian cells that
comprise the PSIP1 gene, and to at least some degree express the
LEDGF protein encoded by the PSIP1 gene, but subsequent to being
modified as more fully described below, express less LEDGF protein
relative to unmodified cells of the same type, or do not express
any detectable LEDGF protein. It will be recognized by those
skilled in the art that most mammalian cells express LEDGF, and
thus it is expected that cells used in compositions and methods of
this disclosure can include cells of any mammalian cell line, or
cells derived from any mammalian cell line, or any other suitable
source. In embodiments, the cells are immortalized. In embodiments,
the cells are progeny of a cell line derived from cancer, such as a
tumor. In embodiments, the cells are multiploid and as such have
more than two copies of at least one chromosome. In embodiments,
the cells comprise more than two copies of a chromosome that
comprises the PSIP1 gene, which as described further below encodes
the LEDGF protein. In embodiments, the cells are aneuploid, and may
be pseudo hypotripoloid. In embodiments, the cells are derived from
human cells and comprise more than 23 distinct chromosomes. In
embodiments, the disclosure provides modified cell lines that are
altered such that expression of the LEDGF protein is reduced or
eliminated. In embodiments, the disclosure provides modifications
of known cell lines, such as cell lines that can be grown in
monolayers and fixed, including but not necessarily limited to
HEp-2 and HeLa cells. In embodiments, cell types that can be
modified for use with the present disclosure are commercially
available from sources, such as the American Type Culture
Collection (ATCC). In non-limiting embodiments the cells are HEp2
or HeLa cells. These cells are available from ATCC as catalog
#CCL-23 for HEp2, and CCL-2 or CCL-2.2 for HeLa adherent and
suspension cultures respectively. As described above, in
embodiments, the disclosure includes cells that are modified such
that they express no detectable LEDGF protein, or express less
LEDGF protein than cells of the same type. Thus, it will be
recognized that cells of the same type can comprise, as one
non-limiting example, HEp2 cells, wherein the modified HEp2 cells
express less LEDGF protein than unmodified HEp2 cells, wherein the
HEp2 cells are the "type" of cells that are described. The same
applies to any other mammalian cells, wherein the unmodified cells
express detectable LEDGF, including but not limited to HeLa cells,
and other cell lines derived from, for example, blood plasma cells,
monocytes, neutrophils, T-lymphocytes, platelets, T-cell leukemia
cells, myeloid leukemia cells, lymphoblastic leukemia cells, kidney
cells, kidney cancer cells, liver cells, liver cancer cells, lung
cells, lung cancer cells, colon cells, colon cancer cells, heart
cells, bone cells, bone cancer cells, brain cells, brain cancer
cells, ovary cells, ovarian cancer cells, prostate cells, prostate
cancer cells, cervical cells, cervical cancer cells, melanoma,
breast tissue cells, breast cancer cells, skin cells, melanoma
cells, pancreatic cells and pancreatic cancer cells, and
others.
[0027] Approaches to immunodiagnostic assays provided in this
disclosure involve modifying mammalian cells to cause
down-regulation or elimination of the PSIP1/LEDGF gene product, and
methods of using the modified cells in autoimmune assays. The
disclosure also includes the modified cells, and compositions
comprising the modified, such as cell cultures. Kits for use in the
assays are also provided.
[0028] As described above, the DFS pattern is well known in the art
and comprises a dense fine speckled pattern resulting from
autoantibodies that specifically bind to LEDGF protein (Ayaki,
Sueno et al. 1999). Autoantibodies to LEDGF protein were first
reported in association with atopic dermatitis and other conditions
such as Asthma, interstitial cystitis (Ochs, Muro et al. 2000),
alopecia areata (Okamoto, Ogawa et al. 2004) and in 0-20% of
healthy individuals (Watanabe, Kodera et al. 2004, Mahler, Parker
et al. 2012). LEDGF belongs to a selected group of autoantigens
that are targeted for cleavage during cell death, and it has been
proposed that the caspase-induced LEDGF cleavage and the generation
of autoantibodies to the protein might contribute to the
pathogenesis of various human atopic and inflammatory disorders
associated with deregulated apoptosis (Ganapathy, Daniels et al.
2003, Ganapathy and Casiano 2004). LEDGF protein has also been
implicated in HIV integration, and LEDGF has been knocked down both
transiently (using siRNA) and stably (using shRNA followed by
selection) resulting in a 3-5 fold reduction in HIV-1 replication
in HeLaP4 cells (Vandekerckhove, Christ et al. 2006).
[0029] The amino acid sequence of the LEDGF protein is known in the
art and the canonical sequence is provided here as SEQ ID NO:1:
MTRDFKPGDLIFAKMKGYPHWPARVDEVPDGAVKPPTNKLPIFFFGTHETAFLGPKDIF
PYSENKEKYGKPNKRKGFNEGLWEIDNNPKVKFSSQQAATKQSNASSDVEVEEKETSV
SKEDTDHEEKASNEDVTKAVDITTPKAARRGRKRKAEKQVETEEAGVVTTATASVNLK
VSPKRGRPAATEVKIPKPRGRPKMVKQPCPSESDIITEEDKSKKKGQEEKQPKKQPKKD
EEGQKEEDKPRKEPDKKEGKKEVESKRKNLAKTGVTSTSDSEEEGDDQEGEKKRKGGR
NFQTAHRRNMLKGQHEKEAADRKRKQEEQMETEQQNKDEGKKPEVKKVEKKRETSM
DSRLQRIHAEIKNSLKIDNLDVNRCIEALDELASLQVTMQQAQKHTEMITTLKKIRRFKV
SQVIMEKSTMLYNKFKNMFLVGEGDSVITQVLNKSLAEQRQHEEANKTKDQGKKGPN
KKLEKEQTGSKTLNGGSDAQDGNQPQHNGESNEDSKDNHEASTKKKPSSEERETEISLK DSTLDN
(SEQ ID NO:1). Other isoforms and truncated versions which comprise
mutations that differ from the canonical sequence are known in the
art. In particular, GenBank (NCBI) entries NP_001121689.1,
NM_001128217.1, [O75475-1], NP_066967.3, NM_021144.3, [O75475-2],
NP_150091.2, NM_033222.3, [O75475-1], XP_005251413.1 provide LEDGF,
and the polynucleotide and amino acid sequences described in these
database entries are incorporated herein by reference as they exist
as of the filing of this application or patent XM_005251356.1,
[O75475-2], XP_005251415.1, XM_005251358.1 and [O75475-3].
[0030] The immunoreactive sequence of the SEQ ID NO:1 has been
reported (Ogawa, Sugiura et al. 2004) to be a polypeptide from
amino acid number 349-455 described in SEQ ID NO:2.
TABLE-US-00001 (SEQ ID NO: 2)
DSRLQRIHAEIKNSLKIDNLDVNRCIEALDELASLQVTMQQAQKHTEMIT
TLKKIRRFKVSQVIMEKSTMLYNKFKNMFLVGEGDSVITQVLNKSLAEQR QHEEANK.
[0031] The cDNA sequence encoding LEDGF is also known in the art
and is provided here as SEQ ID NO:3.
TABLE-US-00002 (SEQ ID NO: 3)
ATGACTCGCGATTTCAAACCTGGAGACCTCATCTTCGCCAAGATGAAAGG
TTATCCCCATTGGCCAGCTCGAGTAGACGAAGTTCCTGATGGAGCTGTAA
AGCCACCCACAAACAAACTACCCATTTTCTTTTTTGGAACTCATGAGACT
GCTTTTTTAGGACCAAAGGATATATTTCCTTACTCAGAAAATAAGGAAAA
GTATGGCAAACCAAATAAAAGAAAAGGTTTTAATGAAGGTTTATGGGAGA
TAGATAACAATCCAAAAGTGAAATTTTCAAGTCAACAGGCAGCAACTAAA
CAATCAAATGCATCATCTGATGTTGAAGTTGAAGAAAAGGAAACTAGTGT
TTCAAAGGAAGATACCGACCATGAAGAAAAAGCCAGCAATGAGGATGTGA
CTAAAGCAGTTGACATAACTACTCCAAAAGCTGCCAGAAGGGGGAGAAAG
AGAAAGGCAGAAAAACAAGTAGAAACTGAGGAGGCAGGAGTAGTGACAAC
AGCAACAGCATCTGTTAATCTAAAAGTGAGTCCTAAAAGAGGACGACCTG
CAGCTACAGAAGTCAAGATTCCAAAACCAAGAGGCAGACCCAAAATGGTA
AAACAGCCCTGTCCTTCAGAGAGTGACATCATTACTGAAGAGGACAAAAG
TAAGAAAAAGGGGCAAGAGGAAAAACAACCTAAAAAGCAGCCTAAGAAGG
ATGAAGAGGGCCAGAAGGAAGAAGATAAGCCAAGAAAAGAGCCGGATAAA
AAAGAGGGGAAGAAAGAAGTTGAATCAAAAAGGAAAAATTTAGCTAAAAC
AGGGGTTACTTCAACCTCCGATTCTGAAGAAGAAGGAGATGATCAAGAAG
GTGAAAAGAAGAGAAAAGGTGGGAGGAACTTTCAGACTGCTCACAGAAGG
AATATGCTGAAAGGCCAACATGAGAAAGAAGCAGCAGATCGAAAACGCAA
GCAAGAGGAACAAATGGAAACTGAGCAGCAGAATAAAGATGAAGGAAAGA
AGCCAGAAGTTAAGAAAGTGGAGAAGAAGCGAGAAACATCAATGGATTCT
CGACTTCAAAGGATACATGCTGAGATTAAAAATTCACTCAAAATTGATAA
TCTTGATGTGAACAGATGCATTGAGGCCTTGGATGAACTTGCTTCACTTC
AGGTCACAATGCAACAAGCTCAGAAACACACAGAGATGATTACTACACTG
AAAAAAATACGGCGATTCAAAGTTAGTCAGGTAATCATGGAAAAGTCTAC
AATGTTGTATAACAAGTTTAAGAACATGTTCTTGGTTGGTGAAGGAGATT
CCGTGATCACCCAAGTGCTGAATAAATCTCTTGCTGAACAAAGACAGCAT
GAGGAAGCGAATAAAACCAAAGATCAAGGGAAGAAAGGGCCAAACAAAAA
GCTAGAGAAGGAACAAACAGGGTCAAAGACTCTAAATGGAGGATCTGATG
CTCAAGATGGTAATCAGCCACAACATAACGGGGAGAGCAATGAAGACAGC
AAAGACAACCATGAAGCCAGCACGAAGAAAAAGCCATCCAGTGAAGAGAG
AGAGACTGAAATATCTCTGAAGGATTCTACACTAGATAACTAG
[0032] To provide compositions and methods for improved ANA and/or
LEDGF antibody detection for systemic and non-systemic autoimmune
diseases (organ specific autoimmune diseases, atopic dermatitis,
alopecia etc.,) and differentiation from disease free human
population, any suitable mammalian cells, including but not limited
to HEp-2 HeLa, HEK293, or a cell line suitable for culturing as
adherent (monolayer) or suspension format can be modified in a
variety of ways, given the benefit of the present disclosure. In
various embodiments LEDGF protein is reduced in the modified cells
by reducing mRNA encoding it. In another approach the disclosure
includes disrupting the PSIP1 gene from making a protein by via
knock-out or targeted mutation. The disclosure also includes making
and using modified cells characterized by reduced or eliminated
LEDGF protein. In embodiments, the modified cells are also
engineered to express a detectable marker, such as a fluorescent
protein or an immunoreactive protein that can further be detected
using a specific secondary antibody (including but not necessarily
limited to a Poly histidine tag, c-Myc tag, FLAG tag etc., which
are well characterized in the art).
[0033] In one aspect, the disclosure includes reducing LEDGF mRNA,
and as a result reducing the LEDGF protein, in modified mammalian
cells. In one approach this aspect comprises introducing into the
suitable mammalian cells a polynucleotide that can inhibit
translation of LEDGF mRNA, and/or can participate in and/or
facilitate RNAi-mediated reduction of LEDGF mRNA. In one
embodiment, an antisense polynucleotide is used to inhibit
translation of LEDGF mRNA. Antisense nucleic acids can be DNA or
RNA molecules that are complementary to at least a portion of the
LEDGF mRNA. In embodiments, oligomers of about fifteen nucleotides,
and/or those that hybridize to the AUG initiation codon will be
particularly efficient. The polynucleotides described herein for
use in targeting LEDGF mRNA can in certain embodiments be modified,
such as to be resistant to nucleases.
[0034] In embodiments, the present disclosure provides for
replacement of the PSIP1 gene with a sequence encoding a detectable
marker, such as a fluorescent protein, or integrating such a
sequence into the PSIP1 gene, thereby disrupting it, or integrating
such a sequence elsewhere in the genome of the cells. By replacing
PSIP1 or integrating a sequence encoding a detectable protein into
it the disclosure provides for marking modified mammalian cells
which do not express LEDGF. This is valuable in that those cells
which express the detectable protein can be selected for use in the
immunoassays of the invention, and for including in products that
are intended to be used in such immunoassay. In embodiments,
disrupting the PSIP1 gene with a sequence encoding a fluorescent
protein will allow for enriching a cell population with cells that
contain the LEDGF disruption, such as by using FACS to separate
cells that contain the disruption from those that do not, thereby
providing an isolated and/or purified population of modified cells
that do not express LEDGF. The detectable marker can be any protein
that can be detected, and is preferably a fluorescent protein. Any
fluorescent protein can be used. In embodiment, the fluorescent
protein is selected from GFP, eGFP, Red Fluorescent protein or
variants thereof such as tRFP, dsRED, mCherry, tdTomato etc.,) or
any fluorescent protein that does not interfere with the conjugates
used in, for example, an IIF method to detect autoantibodies. Thus,
in embodiments, the present disclosure includes cells characterized
by having the PSIP1 gene disrupted or knocked out. In embodiments,
the knock out comprises a disruption of the gene by introducing (a
knock in) of a detectable protein.
[0035] In embodiments, the disclosure includes introducing an
expression vector which can inhibit LEDGF protein, and which may
also express a detectable marker. For example, an expression vector
with two distinct promoters or a bidirectional promoter can be used
to express shRNA targeted to PSIP1 and express the detectable
marker. In alternative embodiments, two distinct expression vectors
can be used for this purpose. In embodiments, the vectors are
stably or transiently present in the cells. In embodiments, one or
both vectors, or the single vector encoding the shRNA and the
detectable marker, is integrated into at least one position in a
chromosome in a mammalian cell.
[0036] In another aspect the disclosure includes RNAi-mediated
reduction in LEDGF mRNA. RNAi-based inhibition can be achieved
using any suitable RNA polynucleotide that is targeted to LEDGF
mRNA. In embodiments, a single stranded or double stranded RNA,
wherein at least one strand is complementary to the LEDGF mRNA, can
be introduced into the cell to promote RNAi-based degradation of
LEDGF mRNA. In another embodiment, microRNA (miRNA) targeted to the
LEDGF mRNA can be used. In another embodiment, a ribozyme that can
specifically cleave LEDGF mRNA can be used. In yet another
embodiment, small interfering RNA (siRNA) can be used. siRNA (or
ribozymes) can be introduced directly, for example, as a double
stranded siRNA complex, or by using a modified expression vector,
such as a lentiviral vector, to produce an shRNA. As is known in
the art, shRNAs adopt a typical hairpin secondary structure that
contains a paired sense and antisense portion, and a short loop
sequence between the paired sense and antisense portions. shRNA is
delivered to the cytoplasm where it is processed by DICER into
siRNAs. siRNA is recognized by RNA-induced silencing complex
(RISC), and once incorporated into RISC, siRNAs facilitate cleavage
and degradation of targeted mRNA. In embodiments, an shRNA
polynucleotide used to suppress LEDGF expression can comprise or
consist of between 45-100 nucleotides, inclusive, and including all
integers between 45 and 100. The portion of the shRNA that is
complementary to the LEDGF mRNA mRNA can be from 21-29 nucleotides,
inclusive, and including all integers between 21 and 29.
[0037] For delivering siRNA via shRNA, modified lentiviral vectors
can be made and used according to standard techniques, given the
benefit of the present disclosure. Further, lentiviral vectors
expressing shRNAs targeted to many human mRNAs are commercially
available. Additionally, custom siRNAs or shRNA can be obtained
from, for example Thermo-Dharmacon for transient transfection
resulting in temporary reduction in LEDGF levels. Alternatively,
lentiviral constructs expressing human PSIP11 targeted shRNA can be
obtained from Thermo Dharmacon. These lentiviruses are capable of
stably and permanently infecting target cells, such as by
integrating into a chromosome in the cells. However, as will be
apparent from the following description, RNAi-mediated approaches
for disrupting expression of the LEDGF protein may not be optimal.
For example, we introduced DNA sequences encoding shRNAs designed
against the PSIP1 gene and cloned them into lenti-viral vectors
downstream of a U6 promoter. Lentivirus with the DNA insert capable
of producing either target shRNA or negative control were used to
infect HEp2 cells. Viral infectivity and titer was measured by an
integrated RFP (Red fluorescent protein) marker that is expressed
in all the infected cells. The RFP marker was also fused to
puromycin (antibiotic) resistance factor which is used for
selection of cells that stably incorporated the construct into the
genome of HEp2 cells. Examples of tested sequences are below, where
the DNA equivalent of the
TABLE-US-00003 shRNA sequence is provided: shRNA(h PSIP1) example
#1 sequence: (SEQ ID NO: 4) AGACAGCATGAGGAAGCGA. Cloned shRNA
hairpin sequence: (SEQ ID NO: 5)
AGACAGCATGAGGAAGCGAttcaagagaTCGCTTCCTCATGCTGTCT shRNA(h PSIP1)
example #2 sequence: (SEQ ID NO: 6) AGTTCCTGATGGAGCTGTAAA Cloned
shRNA hairpin sequence: (SEQ ID NO: 7)
AGTTCCTGATGGAGCTGTAAAcgagTTTACAGCTCCATCAGGAACT hPSIP1) example #3
sequence: (SEQ ID NO: 8) GCAATGAAGACAGCAAAGACA Cloned shRNA hairpin
sequence: (SEQ ID NO: 9)
GCAATGAAGACAGCAAAGACAcgagTGTCTTTGCTGTCTTCATTGC shRNA-Neg-Control:
(SEQ ID NO: 10) GTCTCCACGCGCAGTACATTT Cloned shRNA-Neg hairpin
sequence: (SEQ ID NO: 11)
GTCTCCACGCGCAGTACATTTcgagAAATGTACTGCGCGTGGAGAC
[0038] IIF analysis using the above sequences (lentiviral
transduction procedure followed by selection for resistant
colonies) using DFS70 specific antisera indicated a low level
decrease in PSIP1/LEDGF levels. Negative controls did not show any
reduction in the PSIP1/LEDGF levels using the same procedure.
Further, while siRNA can produce an intense reduction in mRNA
levels, the effects are usually transient. Thus, even though shRNA
technology is compatible with selection processes and allows the
isolation of colonies stably expressing the short hairpin RNA,
which further aids in the degradation of specific complementary
mRNA in subsequent generation of cells, as observed in the
aforementioned approaches, the reduction of PSIP1 levels at the
mRNA level were not adequate to provide an improved substrate for
IIF analysis for use in detecting autoantibodies directed to ANA.
Thus, the disclosure includes alternative approaches for disrupting
LEDGF protein production. In this regard, the disclosure also
includes disrupting the PSIP1 gene with a mutation such that LEDGF
mRNA and protein are not expressed. In one embodiment, the PSIP1
gene can be disrupted by targeted mutagenesis. In embodiments,
targeted mutagenesis can be achieved by, for example, targeting a
CRISPR (clustered regularly interspaced short palindromic repeats)
site in the PSIP1 gene. So-called CRISPR systems designed for
targeting specific genomic sequences are known in the art and can
be adapted to disrupt the PSIP1 gene for making modified cells
encompassed by this disclosure. In general, the CRISPR system
includes one or more expression vectors encoding at least a
targeting RNA and a polynucleotide sequence encoding a
CRISPR-associated nuclease, such as Cas9, but other Cas nucleases
can be used. CRISPR systems for targeted disruption of mammalian
chromosomal sequences are commercially available and can be adapted
to disrupt the PSIP1 gene in HEp-2 cells given the benefit of this
disclosure.
[0039] In embodiments, a targeting RNA encoded by the CRISPR system
can be a CRISPR RNA (crRNA) or a guide RNA, such as sgRNA. The
sequence of the targeting RNA has a segment that is the same as or
complementarity to any CRISPR site in the PSIP1 gene. In this
regard, the target sequence comprises a specific sequence on its 3'
end referred to as a protospacer adjacent motif or "PAM". In an
embodiment a CRISPR Type II system is used, and the target
sequences therefore conform to the well-known N12-20NGG motif,
wherein the NGG is the PAM sequence. Thus, in embodiments, a target
RNA will comprise or consist of a segment that is from 12-20
nucleotides in length which is the same as or complementary to a
DNA target sequence (a spacer) in the PSIP1 gene. The 12-20
nucleotides directed to the spacer sequence will be present in the
targeting RNA, regardless of whether the targeting RNA is a crRNA
or a guide RNA. In embodiments, a separate trans-activating crRNA
(tracrRNA) can be used to assist in maturation of a crRNA targeted
to the PSIP1 gene. Introduction a CRISPR system into HEp-2 cells
will result in binding of a targeting RNA/Cas9 complex to the PSIP1
target sequence so that the Cas9 can cut both strands of DNA
causing a double strand break. The double stranded break can be
repaired by non-homologous end joining DNA repair, or by a homology
directed repair pathway, which will result in either insertions or
deletions at the break site, or by using a repair template to
introduce mutations, respectively. Double-stranded breaks can also
be introduced into the PSIP1 gene by expressing Transcription
activator-like effector nucleases (TALENs) in the cells. TALENs are
artificial restriction enzymes generated by fusing a TAL effector
DNA binding domain to a DNA cleavage domain and are known in the
art and can be adapted for use in embodiments of this disclosure.
In yet another approach, zinc-finger nucleases (ZFNs) can be
expressed in the cells to target the PSIP1 gene. ZFNs are
artificial restriction enzymes produced by fusing a zinc finger
DNA-binding domain to a DNA-cleavage domain. ZF domains can be
designed to target PSIP1 gene DNA sequences where the zinc-finger
nucleases cleave the sequence, thereby disrupting the gene. In
another embodiment, site-specific gene integration or targeted
integration of a sequence into specific integration sites within
the gene can be accomplished by using commercial systems such as
Jump-In.TM. or Flp-In.TM. systems commercially available from
Thermo Fisher Scientific Inc. Multiple integration sites may be
targeted by PhiC31 in the Jump-In.TM. Fast system. As will be
recognized by those skilled in the art, a FRT site (34 bp) in the
target genome is needed for gene integration, and is provided by
specific commercial cell lines derived from Flp-In.TM.
technology.
[0040] In a non-limiting reduction to practice, we used a
CRISPR-CAS-9 system to design specific constructs with guide RNA
(gRNA) and a complimentary region upstream of Protospacer Adjacent
Motif (PAM) sequences to create double strand breaks in the target
PSIP1 gene. We then selected colonies with homozygous disruption of
the PSIP1 gene at the break site. A cell line such as HEp2 may have
multiple copies of PSIP1 gene and it is thus important to isolate a
clone where all copies of the PSIP1 gene have been disrupted,
thereby eliminating the LEDGF protein from cells. Five CRISPR-CAS9
examples for PSIP1 gene disruption are described below. There are
numerous PAM sites spanning across the exons and introns of the
PSIP1 gene, but exons are preferred targets for CRISPR-CAS9 induced
mutations or disruptions in the coding region.
[0041] Each representative sequence below includes U6 promoter
sequence, gRNA targeting site and a gRNA scaffold upstream of PAM
sequence which in combination with a CAS9 enzyme supplied to the
cell either as part of the same vector or a different vector will
create a functional CRISPR complex capable of creating a double
stranded break in the targeted area of the genome.
TABLE-US-00004 Example Sequence 1: (SEQ ID NO: 12):
5'GTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCG
GTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATAT
TTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGAC
TGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAAT
TTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCAT
ATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT
GTGGAAAGGACGAAACACCGTAATCAGCCACAACATAACGTTTTAGAGCT
AGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTG
GCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTG GCATTA 3'
Example Sequence 2: (SEQ ID NO: 13):
5'TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCC
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATA
TTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGA
CTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAA
TTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCA
TATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCT
TGTGGAAAGGACGAAACACCGACGCCTCTGCGGCAGCTGGGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT
GGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTT GGCATTA 3'
Example Sequence 3: (SEQ ID NO: 14)
5'TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCC
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATA
TTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGA
CTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAA
TTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCA
TATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCT
TGTGGAAAGGACGAAACACCGAGGTAGACGAAGTTCCTGAGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT
GGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTT GGCATTA 3'
Example Sequence 4 (SEQ ID NO: 15):
5'TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCC
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATA
TTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGA
CTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAA
TTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCA
TATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCT
TGTGGAAAGGACGAAACACCGAACTACCCATTTTCTTTTTGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT
GGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTT GGCATTA 3'
Example Sequence 5: (SEQ ID NO: 16)
5'TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCC
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATA
TTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGA
CTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAA
TTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCA
TATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCT
TGTGGAAAGGACGAAACACCGAGTGCTTTTTTAGGACCAAGTTTTAGAGC
TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGT
GGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTT GGCATTA 3'
[0042] For Example Sequence 3, the disruption was targeted into the
exon 1 of the PSIP1 gene, thereby eliminating of the potential to
make a partial LEDGF protein. Further, we isolated a single colony
of HEp2 cells where all copies of PSIP1 gene were disrupted as
confirmed by DNA sequencing. Following confirmation by DNA
sequencing, the cells from the colony were mixed with WT cells in
1:1 ratio and IIF analysis was performed using a panel of anti-sera
that were specifically positive for DFS70 pattern and confirmed by
LIA (Line Immunoassay) or Line blot assay for DFS70 antisera
(ImmcoStripe ANA-Advanced LIA, Immco Diagnostics Inc., Buffalo,
N.Y.). The results are depicted in FIG. 1. Thus, the disclosure
includes mammalian cell cultures which comprise mammalian cells
wherein every copy of the PSIP1 gene in the cells is disrupted, and
thus the cells do not express LEDGF protein. In an embodiment, the
cells do not express detectable LEDGF protein, wherein the
detection is by IIF. In embodiments, when the PSIP1 gene is
disrupted using a CRISPR approach, the cells can further comprise a
Cas9 protein coding region integrated into one or more locations in
the chromosome(s) of the cells, and can further comprise a sequence
encoding the gRNA integrated in the chromosome(s) of the cells.
[0043] In another aspect the disclosure includes a method for
detecting ANA antibodies, and/or LEDGF antibodies. The method
comprises obtaining a biological sample from an individual, mixing
the sample with modified cells described herein, and performing an
immuno-assay, such as an IIF assay to determine the antibodies. The
presence of the antibodies is a diagnosis or aids in the diagnosis
of an autoimmune disease, such as SARDS, and the absence of the ANA
antibodies indicate the lack of an autoimmune disease. Thus, the
disclosure provides diagnostic methods using novel agents in the
steps of the method. In embodiments, the presence of antibodies to
LEDGF in LEDGF+ cells, is indicative that further diagnostic
testing of the individual is warranted.
[0044] As noted above, IIF assays using HEp-2 cells as a substrate
to detect ANA antibodies are well known in the art and can be used
with the modified cells of the present disclosure without modifying
such well known protocols. The biological sample that is used in
the assay can be any biological sample, including but not limited
to blood, serum, semen, pleural fluid, cerebrospinal fluid, saliva,
urine, exosomes, or tissue. The biological sample can be used
directly, or it can be subjected to a processing step before being
exposed to the cells. The amount of antibodies, if any, can be
compared to any suitable reference for, for instance, correcting
for background, or for staging the degree and/or severity of an
autoimmune disease that is positively correlated with the
antibodies. In embodiments, the disclosure includes testing
combinations of LEDGF+ and LEDGF- cells to determine whether or not
a sample comprises antibodies that bind to LEDGF, and thus can
provide for correction of background that complicates previously
available approaches which frequently result in false positive
results for ANA autoantibodies.
[0045] In another aspect, the disclosure includes kits and articles
of manufacture for use in detecting ANA antibodies. The kit can
comprise at least one container in which the modified cells of this
disclosure are kept. The cells can be preserved using any suitable
reagents, and can be provided, for example, in the form of a
pellet. The kit can include reagents for use in IIF assays, and
instruction which describe the modified cells, such as by providing
a description of how or that they have been modified to reduce DFS,
and instructions for using the cells in the IIF assays. In
embodiments, the kits comprise LEDGF+ and LEDGF- cells which are
fixed to one or more suitable solid substrates. The cells may be
permeabilized using any suitable approach, many of which are well
known in the art, and are thus killed cells. In embodiments, the
fixed cells that are immobilized on the solid substrate are
dried.
[0046] It will be apparent form the foregoing that the present
disclosure includes the modified cells described herein, the
methods for making the modified cells, cell cultures comprising the
modified cells, and all methods for using the modified cells in any
assay designed to detect any one, or any combination of the
antibodies that are comprised by the ANA antibody profile.
REFERENCES
[0047] Ayaki, M., T. Sueno, D. P. Singh, L. T. Chylack, Jr. and T.
Shinohara (1999). "Antibodies to lens epithelium-derived growth
factor (LEDGF) kill epithelial cells of whole lenses in organ
culture." Exp Eye Res 69(1): 139-142.
[0048] Ganapathy, V. and C. A. Casiano (2004). "Autoimmunity to the
nuclear autoantigen DFS70 (LEDGF): what exactly are the
autoantibodies trying to tell us?" Arthritis Rheum 50(3):
684-688.
[0049] Ganapathy, V., T. Daniels and C. A. Casiano (2003).
"LEDGF/p75: a novel nuclear autoantigen at the crossroads of cell
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[0050] Mahler, M. and M. J. Fritzler (2012). "The Clinical
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[0051] Mahler, M., T. Parker, C. L. Peebles, L. E. Andrade, A.
Swart, Y. Carbone, D. J. Ferguson, D. Villalta, N. Bizzaro, J. G.
Hanly and M. J. Fritzler (2012). "Anti-DFS70/LEDGF Antibodies Are
More Prevalent in Healthy Individuals Compared to Patients with
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[0052] Ochs, R. L., Y. Muro, Y. Si, H. Ge, E. K. Chan and E. M. Tan
(2000). "Autoantibodies to DFS 70 kd/transcription coactivator p75
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105(6 Pt 1): 1211-1220.
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Mishima, Y. Tomita and Y. Muro (2004). "Autoantigenicity of DFS70
is restricted to the conformational epitope of C-terminal
alpha-helical domain." J Autoimmun 23(3): 221-231.
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Shimomura, N. Aoki, T. Nagasaka, Y. Tomita and Y. Muro (2004).
"Autoantibodies to DFS70/LEDGF are increased in alopecia areata
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[0055] Vandekerckhove, L., F. Christ, B. Van Maele, J. De Rijck, R.
Gijsbers, C. Van den Haute, M. Witvrouw and Z. Debyser (2006).
"Transient and stable knockdown of the integrase cofactor LEDGF/p75
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[0057] While the disclosure has been particularly shown and
described with reference to specific embodiments, it should be
understood by those having skill in the art that various changes in
form and detail may be made therein without departing from the
spirit and scope of the present disclosure as disclosed herein.
Sequence CWU 1
1
161530PRTHomo sapien 1Met Thr Arg Asp Phe Lys Pro Gly Asp Leu Ile
Phe Ala Lys Met Lys1 5 10 15Gly Tyr Pro His Trp Pro Ala Arg Val Asp
Glu Val Pro Asp Gly Ala 20 25 30Val Lys Pro Pro Thr Asn Lys Leu Pro
Ile Phe Phe Phe Gly Thr His 35 40 45Glu Thr Ala Phe Leu Gly Pro Lys
Asp Ile Phe Pro Tyr Ser Glu Asn 50 55 60Lys Glu Lys Tyr Gly Lys Pro
Asn Lys Arg Lys Gly Phe Asn Glu Gly65 70 75 80Leu Trp Glu Ile Asp
Asn Asn Pro Lys Val Lys Phe Ser Ser Gln Gln 85 90 95Ala Ala Thr Lys
Gln Ser Asn Ala Ser Ser Asp Val Glu Val Glu Glu 100 105 110Lys Glu
Thr Ser Val Ser Lys Glu Asp Thr Asp His Glu Glu Lys Ala 115 120
125Ser Asn Glu Asp Val Thr Lys Ala Val Asp Ile Thr Thr Pro Lys Ala
130 135 140Ala Arg Arg Gly Arg Lys Arg Lys Ala Glu Lys Gln Val Glu
Thr Glu145 150 155 160Glu Ala Gly Val Val Thr Thr Ala Thr Ala Ser
Val Asn Leu Lys Val 165 170 175Ser Pro Lys Arg Gly Arg Pro Ala Ala
Thr Glu Val Lys Ile Pro Lys 180 185 190Pro Arg Gly Arg Pro Lys Met
Val Lys Gln Pro Cys Pro Ser Glu Ser 195 200 205Asp Ile Ile Thr Glu
Glu Asp Lys Ser Lys Lys Lys Gly Gln Glu Glu 210 215 220Lys Gln Pro
Lys Lys Gln Pro Lys Lys Asp Glu Glu Gly Gln Lys Glu225 230 235
240Glu Asp Lys Pro Arg Lys Glu Pro Asp Lys Lys Glu Gly Lys Lys Glu
245 250 255Val Glu Ser Lys Arg Lys Asn Leu Ala Lys Thr Gly Val Thr
Ser Thr 260 265 270Ser Asp Ser Glu Glu Glu Gly Asp Asp Gln Glu Gly
Glu Lys Lys Arg 275 280 285Lys Gly Gly Arg Asn Phe Gln Thr Ala His
Arg Arg Asn Met Leu Lys 290 295 300Gly Gln His Glu Lys Glu Ala Ala
Asp Arg Lys Arg Lys Gln Glu Glu305 310 315 320Gln Met Glu Thr Glu
Gln Gln Asn Lys Asp Glu Gly Lys Lys Pro Glu 325 330 335Val Lys Lys
Val Glu Lys Lys Arg Glu Thr Ser Met Asp Ser Arg Leu 340 345 350Gln
Arg Ile His Ala Glu Ile Lys Asn Ser Leu Lys Ile Asp Asn Leu 355 360
365Asp Val Asn Arg Cys Ile Glu Ala Leu Asp Glu Leu Ala Ser Leu Gln
370 375 380Val Thr Met Gln Gln Ala Gln Lys His Thr Glu Met Ile Thr
Thr Leu385 390 395 400Lys Lys Ile Arg Arg Phe Lys Val Ser Gln Val
Ile Met Glu Lys Ser 405 410 415Thr Met Leu Tyr Asn Lys Phe Lys Asn
Met Phe Leu Val Gly Glu Gly 420 425 430Asp Ser Val Ile Thr Gln Val
Leu Asn Lys Ser Leu Ala Glu Gln Arg 435 440 445Gln His Glu Glu Ala
Asn Lys Thr Lys Asp Gln Gly Lys Lys Gly Pro 450 455 460Asn Lys Lys
Leu Glu Lys Glu Gln Thr Gly Ser Lys Thr Leu Asn Gly465 470 475
480Gly Ser Asp Ala Gln Asp Gly Asn Gln Pro Gln His Asn Gly Glu Ser
485 490 495Asn Glu Asp Ser Lys Asp Asn His Glu Ala Ser Thr Lys Lys
Lys Pro 500 505 510Ser Ser Glu Glu Arg Glu Thr Glu Ile Ser Leu Lys
Asp Ser Thr Leu 515 520 525Asp Asn 5302107PRTHomo sapien 2Asp Ser
Arg Leu Gln Arg Ile His Ala Glu Ile Lys Asn Ser Leu Lys1 5 10 15Ile
Asp Asn Leu Asp Val Asn Arg Cys Ile Glu Ala Leu Asp Glu Leu 20 25
30Ala Ser Leu Gln Val Thr Met Gln Gln Ala Gln Lys His Thr Glu Met
35 40 45Ile Thr Thr Leu Lys Lys Ile Arg Arg Phe Lys Val Ser Gln Val
Ile 50 55 60Met Glu Lys Ser Thr Met Leu Tyr Asn Lys Phe Lys Asn Met
Phe Leu65 70 75 80Val Gly Glu Gly Asp Ser Val Ile Thr Gln Val Leu
Asn Lys Ser Leu 85 90 95Ala Glu Gln Arg Gln His Glu Glu Ala Asn Lys
100 10531593DNAHomo sapien 3atgactcgcg atttcaaacc tggagacctc
atcttcgcca agatgaaagg ttatccccat 60tggccagctc gagtagacga agttcctgat
ggagctgtaa agccacccac aaacaaacta 120cccattttct tttttggaac
tcatgagact gcttttttag gaccaaagga tatatttcct 180tactcagaaa
ataaggaaaa gtatggcaaa ccaaataaaa gaaaaggttt taatgaaggt
240ttatgggaga tagataacaa tccaaaagtg aaattttcaa gtcaacaggc
agcaactaaa 300caatcaaatg catcatctga tgttgaagtt gaagaaaagg
aaactagtgt ttcaaaggaa 360gataccgacc atgaagaaaa agccagcaat
gaggatgtga ctaaagcagt tgacataact 420actccaaaag ctgccagaag
ggggagaaag agaaaggcag aaaaacaagt agaaactgag 480gaggcaggag
tagtgacaac agcaacagca tctgttaatc taaaagtgag tcctaaaaga
540ggacgacctg cagctacaga agtcaagatt ccaaaaccaa gaggcagacc
caaaatggta 600aaacagccct gtccttcaga gagtgacatc attactgaag
aggacaaaag taagaaaaag 660gggcaagagg aaaaacaacc taaaaagcag
cctaagaagg atgaagaggg ccagaaggaa 720gaagataagc caagaaaaga
gccggataaa aaagagggga agaaagaagt tgaatcaaaa 780aggaaaaatt
tagctaaaac aggggttact tcaacctccg attctgaaga agaaggagat
840gatcaagaag gtgaaaagaa gagaaaaggt gggaggaact ttcagactgc
tcacagaagg 900aatatgctga aaggccaaca tgagaaagaa gcagcagatc
gaaaacgcaa gcaagaggaa 960caaatggaaa ctgagcagca gaataaagat
gaaggaaaga agccagaagt taagaaagtg 1020gagaagaagc gagaaacatc
aatggattct cgacttcaaa ggatacatgc tgagattaaa 1080aattcactca
aaattgataa tcttgatgtg aacagatgca ttgaggcctt ggatgaactt
1140gcttcacttc aggtcacaat gcaacaagct cagaaacaca cagagatgat
tactacactg 1200aaaaaaatac ggcgattcaa agttagtcag gtaatcatgg
aaaagtctac aatgttgtat 1260aacaagttta agaacatgtt cttggttggt
gaaggagatt ccgtgatcac ccaagtgctg 1320aataaatctc ttgctgaaca
aagacagcat gaggaagcga ataaaaccaa agatcaaggg 1380aagaaagggc
caaacaaaaa gctagagaag gaacaaacag ggtcaaagac tctaaatgga
1440ggatctgatg ctcaagatgg taatcagcca caacataacg gggagagcaa
tgaagacagc 1500aaagacaacc atgaagccag cacgaagaaa aagccatcca
gtgaagagag agagactgaa 1560atatctctga aggattctac actagataac tag
1593419DNAartificial sequenceDNA equivalent of shRNA sequences
4agacagcatg aggaagcga 19547DNAartificial sequenceDNA equivalent of
shRNA sequences 5agacagcatg aggaagcgat tcaagagatc gcttcctcat
gctgtct 47621DNAartificial sequenceDNA equivalent of shRNA
sequences 6agttcctgat ggagctgtaa a 21746DNAartificial sequenceDNA
equivalent of shRNA sequences 7agttcctgat ggagctgtaa acgagtttac
agctccatca ggaact 46821DNAartificial sequenceDNA equivalent of
shRNA sequences 8gcaatgaaga cagcaaagac a 21946DNAartificial
sequenceDNA equivalent of shRNA sequences 9gcaatgaaga cagcaaagac
acgagtgtct ttgctgtctt cattgc 461021DNAartificial sequenceDNA
equivalent of shRNA sequences 10gtctccacgc gcagtacatt t
211146DNAartificial sequenceDNA equivalent of shRNA sequences
11gtctccacgc gcagtacatt tcgagaaatg tactgcgcgt ggagac 4612454DNAHomo
sapien 12gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac tggatccggt
accaaggtcg 60ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga
tacaaggctg 120ttagagagat aattagaatt aatttgactg taaacacaaa
gatattagta caaaatacgt 180gacgtagaaa gtaataattt cttgggtagt
ttgcagtttt aaaattatgt tttaaaatgg 240actatcatat gcttaccgta
acttgaaagt atttcgattt cttggcttta tatatcttgt 300ggaaaggacg
aaacaccgta atcagccaca acataacgtt ttagagctag aaatagcaag
360ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg
tgcttttttt 420ctagacccag ctttcttgta caaagttggc atta 45413455DNAHomo
sapien 13tgtacaaaaa agcaggcttt aaaggaacca attcagtcga ctggatccgg
taccaaggtc 60gggcaggaag agggcctatt tcccatgatt ccttcatatt tgcatatacg
atacaaggct 120gttagagaga taattagaat taatttgact gtaaacacaa
agatattagt acaaaatacg 180tgacgtagaa agtaataatt tcttgggtag
tttgcagttt taaaattatg ttttaaaatg 240gactatcata tgcttaccgt
aacttgaaag tatttcgatt tcttggcttt atatatcttg 300tggaaaggac
gaaacaccga cgcctctgcg gcagctgggt tttagagcta gaaatagcaa
360gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
gtgctttttt 420tctagaccca gctttcttgt acaaagttgg catta
45514455DNAHomo sapien 14tgtacaaaaa agcaggcttt aaaggaacca
attcagtcga ctggatccgg taccaaggtc 60gggcaggaag agggcctatt tcccatgatt
ccttcatatt tgcatatacg atacaaggct 120gttagagaga taattagaat
taatttgact gtaaacacaa agatattagt acaaaatacg 180tgacgtagaa
agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
240gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
atatatcttg 300tggaaaggac gaaacaccga ggtagacgaa gttcctgagt
tttagagcta gaaatagcaa 360gttaaaataa ggctagtccg ttatcaactt
gaaaaagtgg caccgagtcg gtgctttttt 420tctagaccca gctttcttgt
acaaagttgg catta 45515455DNAHomo sapien 15tgtacaaaaa agcaggcttt
aaaggaacca attcagtcga ctggatccgg taccaaggtc 60gggcaggaag agggcctatt
tcccatgatt ccttcatatt tgcatatacg atacaaggct 120gttagagaga
taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg
180tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
ttttaaaatg 240gactatcata tgcttaccgt aacttgaaag tatttcgatt
tcttggcttt atatatcttg 300tggaaaggac gaaacaccga actacccatt
ttctttttgt tttagagcta gaaatagcaa 360gttaaaataa ggctagtccg
ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 420tctagaccca
gctttcttgt acaaagttgg catta 45516455DNAHomo sapien 16tgtacaaaaa
agcaggcttt aaaggaacca attcagtcga ctggatccgg taccaaggtc 60gggcaggaag
agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
120gttagagaga taattagaat taatttgact gtaaacacaa agatattagt
acaaaatacg 180tgacgtagaa agtaataatt tcttgggtag tttgcagttt
taaaattatg ttttaaaatg 240gactatcata tgcttaccgt aacttgaaag
tatttcgatt tcttggcttt atatatcttg 300tggaaaggac gaaacaccga
gtgctttttt aggaccaagt tttagagcta gaaatagcaa 360gttaaaataa
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
420tctagaccca gctttcttgt acaaagttgg catta 455
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