WO2014193611A1 - Bright/arid3a fonction/expression en tant que marqueur indiquant la gravité et l'intensité du lupus érythémateux disséminé - Google Patents

Bright/arid3a fonction/expression en tant que marqueur indiquant la gravité et l'intensité du lupus érythémateux disséminé Download PDF

Info

Publication number
WO2014193611A1
WO2014193611A1 PCT/US2014/036881 US2014036881W WO2014193611A1 WO 2014193611 A1 WO2014193611 A1 WO 2014193611A1 US 2014036881 W US2014036881 W US 2014036881W WO 2014193611 A1 WO2014193611 A1 WO 2014193611A1
Authority
WO
WIPO (PCT)
Prior art keywords
arid3a
cells
bright
subject
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2014/036881
Other languages
English (en)
Other versions
WO2014193611A8 (fr
Inventor
Carol Webb
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oklahoma Medical Research Foundation
Original Assignee
Oklahoma Medical Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Oklahoma Medical Research Foundation filed Critical Oklahoma Medical Research Foundation
Publication of WO2014193611A1 publication Critical patent/WO2014193611A1/fr
Publication of WO2014193611A8 publication Critical patent/WO2014193611A8/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]

Definitions

  • the present invention relates generally to the fields of autoimmune disease, immunology and molecular biology. More particularly, it concerns Bright/ARID3a activity in the context of systemit lupus erythematosus. 2. Description of Related Art
  • SLE Systemic lupus erythematosus
  • ANA antinuclear antibody
  • SLE systemic lupus erythematosus
  • a method of predicting an auto-antibody flare in a subject with systemtic lupus erythematosus comprising (a) obtaining a whole blood sample from a subject; and (b) assessing the level of Bright/ARID3a in B-cells of said sample, wherein an elevated level of Bright/ARID3a in said B-cells, as compared to control B-cells from a subject not undergoing a disease flare indicates that said subject will incur a disease flare.
  • the B-cells may be CD19+ B-cells.
  • the control B-cells may be from a health subject or a non-flaring SLE subject.
  • Assessing may comprise immunologic detection of Bright/ARID3a, such as flow cytometry (FACS), ELISA, RIA or Western blot, or detection of a Bright/ARID3a transcript, such as employing amplification of Bright/ARID3a mRNA, including RT-PCR.
  • the method may further comprise performing a SLED A Index analysis on said subject, and/or further assessing anti-nuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti- ENA) in a sample from said subject, and/or further comprising taking a medical history of said subject.
  • the method may further include treating the subject with an SLE therapy if a flare is predicted.
  • a method of determining the severity of systemtic lupus erythematosus (SLE) in a subject (a) obtaining a whole blood sample from a subject; and (b) assessing the level of Bright/ARID3a in B-cells of said sample, wherein an elevated level of Bright/ARID3a in said B-cells, as compared to B-cells from a subject having moderate SLE, indicates that said subject has severe SLE.
  • the B-cells may be CD19+ B-cells.
  • Assessing may comprise immunologic detection of Bright/ARID3a, such as flow cytometry (FACS), ELISA, RIA or Western blot, or detection of a Bright/ARID3a transcript, such as employing amplification of Bright/ARID3a mRNA, including RT-PCR.
  • the method may further comprise performing a SLEDA Index analysis on said subject, and/or further assessing anti-nuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti- ENA) in a sample from said subject, and/or further comprising taking a medical history of said subject.
  • the method may further include treating the subject with an SLE therapy if severity is predicted.
  • a method of assessing the progression of systemic lupus erythematosus (SLE) in a subject comprising (a) obtaining a whole blood sample from a subject; (b) assessing the level of Bright/ARID3a in B-cells of said first sample; (c) obtaining a second B-cell containing sample from a subject; and (d) assessing the level of Bright/ARID3a in B-cells of said second sample, wherein an elevated level of Bright/ARID3a in B-cells of said second sample, as compared to B-cells from said first sample, indicates that SLE in said subject is progressing.
  • SLE systemic lupus erythematosus
  • the B-cells may be CD 19+ B-cells.
  • Assessing may comprise immunologic detection of Bright/ARID3a, such as flow cytometry (FACS), ELISA, RIA or Western blot, or detection of a Bright/ARID3a transcript, such as employing amplification of Bright/ARID3a mRNA, including RT-PCR.
  • the method may further comprise performing a SLEDA Index analysis on said subject, and/or further assessing anti-nuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti- ENA) in a sample from said subject, and/or further comprising taking a medical history of said subject.
  • the method may further include treating the subject with an SLE therapy if progression is predicted.
  • a method of assessing the efficacy of a treatment for systemic lupus erythematosus (SLE) in a subject comprising (a) obtaining a first whole blood sample from a subject; (b) assessing the level of Bright/ARID3a in B-cells of said first sample; (c) treating said subject for with an SLE treatment; (d) obtaining a second whole blood sample from a subject; and (e) assessing the level of Bright/ARID3a in B-cells of said second sample, wherein a reduced level of Bright/ARID3a in B-cells of said second sample, as compared to B-cells from said first sample, indicates that said SLE treatment was effective.
  • the B-cells may be CD 19+ B-cells.
  • Assessing may comprise immunologic detection of Bright/ARID3a, such as flow cytometry (FACS), ELISA, RIA or Western blot, or detection of a Bright/ARID3a transcript, such as employing amplification of Bright/ARID3a mRNA, including RT-PCR.
  • the method may further comprise performing a SLEDA Index analysis on said subject, and/or further assessing anti-nuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti- ENA) in a sample from said subject, and/or further comprising taking a medical history of said subject.
  • ANA anti-nuclear antibody
  • anti- ENA anti-extractable nuclear antigen
  • a method of treating a subject with systemtic lupus erythematosus (SLE) in a subject (a) obtaining a whole blood sample from a subject; (b) assessing the level of Bright/ARID3a in B-cells of said sample; and (c) treating said subject when said B-cells exhibit an elevated level of Bright/ARID3a as compared to B- cells from a healthy control.
  • the B-cells may be CD 19+ B-cells.
  • Assessing may comprise immunologic detection of Bright/ARID3a, such as flow cytometry (FACS), ELISA, RIA or Western blot, or detection of a Bright/ARID3a transcript, such as employing amplification of Bright/ARID3a mRNA, including RT-PCR.
  • the method may further comprise performing a SLEDA Index analysis on said subject, and/or further assessing anti-nuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti- ENA) in a sample from said subject, and/or further comprising taking a medical history of said subject.
  • ANA anti-nuclear antibody
  • anti- ENA anti-extractable nuclear antigen
  • kits comprising (a) one or more reagents for assessing the expression of Bright/ARID3a in a biological sample; and (b) one or more reagents for assessing anti-nuclear antibody (ANA) testing and/or anti-extractable nuclear antigen (anti-ENA) in a biological sample.
  • the kit may further comprise an agent for treating systemic lupus erythematosus, such as a Bright/ARID3a inhibitor, including an interfering RNA, a dominant-negative Bright/ARID3a molecule, a Bright/ARID3a peptide or an expression vector coding therefor.
  • the agent may also be a corticosteroid or an anti-malarial drug.
  • FIGS. 1A-D ARID3a is over-expressed in B lineage cells in a subset of SLE patients.
  • FIG. 1A Immunofluorescent staining of ARID3a + cells in B (CD 19 ) and T (CD3 + ) lymphocytes from a representative control (Ctrl 6) and 2 lupus patients (SLE #15, #2) are shown relative to isotype controls.
  • FIG. IB Mononuclear cells from a representative control and 4 SLE patients were assessed by Western blotting. CLOl is a positive control. Data represent > 6 experiments.
  • FIG. 1A Immunofluorescent staining of ARID3a + cells in B (CD 19 ) and T (CD3 + ) lymphocytes from a representative control (Ctrl 6) and 2 lupus patients (SLE #15, #2) are shown relative to isotype controls.
  • FIG. IB Mononuclear cells from a representative control and 4 SLE patients were assessed by Western blotting.
  • FIGS 2A-B ARID3a expressing cells occur in multiple B cell subpopulations in SLE patients.
  • FIG. 2A Histograms show ARID3a expression in 6 B cell subsets for
  • ARID3a N , ARID3a H and control samples compared to isotype controls were set relative to the isotype controls.
  • FIG. 2B Total numbers of ARID3a + B cells/ml in each B cell subset from 42 SLE, 6 RA and 17 healthy controls were enumerated. Means ⁇ SEM are shown. Mann- Whitney calculated p values are given. Zero values were included in data analyses.
  • FIG. 4A Mean percentages of CD27 " , ARID3a + and ARID3a " B cells in CD86 and CD43 gates and
  • FIG. 4B mean percentages of CD27 " , CD86 + and CD86 " and CD43 + and CD43 " B cells in the ARID3a gate from 42 SLE, 15 controls and 4 RA patient samples are shown.
  • FIG. 5 Circulating IL-10 levels were increased in SLE patients with increased numbers of ARID3a + B cells. Plasma levels of TGF- ⁇ , IL-17, and IL-10 from ARID3a H (7-15) and ARID3a N (9-24) samples were measured and calculated relative to standard curves. Each symbol represents a different sample. Means and SEMs are indicated.
  • FIG. 6 Immunofluorescent gating of peripheral blood B lineage subpopulations. Representative data show surface markers and flow cytometric-gating strategies used to identify individual B lymphocyte subsets. Numbers correspond to common nomenclature for CD19 + B cell subsets defined as: naive IgD + B cells (IgD + IgM ⁇ CD27 ⁇ ), C5 class- switched (IgD + IgM " CD27 + ), naive (IgD + IgM + CD27 ⁇ CD38 +/ ⁇ ), marginal zone (MZ) memory (IgD + IgM + CD27 + CD38 "/+ ), memory IgM (IgDTgM + CD27 + ), class-switched memory (IgD " IgM " CD27 + CD38 +/ ), double negative (DN) cells (IgDTgM CD27 ⁇ CD38 ⁇ ), and plasmablast/plasma cells (IgDTgM ⁇ CD27 + CD38 + ).
  • FIG. 7 Increased numbers of ARID3a + cells are not due to expansions of specific B cell subsets.
  • CD19 + B cells from healthy control, ARID3a H and ARID3a N patient samples were stained and B cell subsets were gated as shown in FIG. 6. Each symbol represents one sample. Samples with ⁇ 1000 total B cells/ml were excluded because B cell subsets numbers were too few for valid enumeration. Means ⁇ SEM are shown. Zero values were included in analyses. Mann-Whitney analyses showed increased ARID3a H memory IgM cells versus controls (p — 0.0428).
  • FIGS. 8A-B Transitional B cells did not account for increased numbers of ARID3a + B cells. Quantification of transitional (CD10 + ) and naive (CD 10 " ) B cells (FIG. 8A) and numbers of ARID3a + cells (FIG. 8B) within those subsets were determined in peripheral blood of healthy controls, ARID3a H patients, and ARID3a N patients by flow cytometry. Each symbol represents a different sample. Means and SEMs are indicated.
  • FIG. 9 Increased SLEDAI scores are correlated with numbers of ARID3a + CD19 + B cells. Total numbers of ARID3a + CD 19 + B cells from each patient at each visit and the corresponding SLEDAI scores at the respective visits were subjected to Spearman's rank correlation coefficient analyses to determine their relationship. Rank correlation and p values are shown.
  • FIGS. 10A-B Plasma immunoglobulin levels are higher in ARID3a H SLE samples, but all samples had similar autoreactivities.
  • FIG. 10B Percentages of plasma from 16 ARID3a H and 26 ARID3a N samples positive for common cross-reactivities are presented.
  • mice that overexpress the DNA-binding protein Bright/ARID3a (B cell regulator of immunoglobulin heavy chain transcription/A+T rich interaction domain family protein 3 a) in all B lineage cells produced serum ANAs by four weeks of age (10, 11).
  • Bright/ARID3a B cell regulator of immunoglobulin heavy chain transcription/A+T rich interaction domain family protein 3 a
  • ARID3a Overexpression of ARID3a (or Bright as it was first called in the mouse (Webb et ah, 1991 ; Herscher et ah, 1995)) in B lineage cells of transgenic mice was also associated with autoantibody (antinuclear antigen, ANA) production in 100% of the mice as early as four weeks of age (Shankar et al, 2007; Oldham et al, 2011). These mice accumulated glomerular immune complexes with time. Furthermore, overexpression of ARID3a in mouse B lineage cells altered the gene expression pattern of the resulting mature follicular B cells so that they resembled marginal zone B cells, a subpopulation of B cells associated with autoantibody production (Oldham et al, 201 1). These data strongly suggest that ARID3a overexpression contributes to autoimmunity, possibly by driving the development of mature B cells that produce autoantibodies.
  • ARID3a expression in patients' B cells was not limited to the B cell subsets that normally express ARID3a, but that it was expressed in some cells at every stage of differentiation.
  • a longitudinal study of 44 patients was conducted over three separate visits and the numbers of B cells in each subpopulation quantitated, including the numbers of cells within those subpopulations that express ARID3a.
  • the inventor was also provided with SLEDAI scores to measure disease activity in these patients. Increased numbers of ARID3a+ B cells correlated with disease activity on one visit (FIG. 14).
  • ARID3a+ total B cells ARID3a+ plasmablasts (which are activated to secrete antibody) and ARID3a+ Marginal Zone memory cells (see Table 2). These cells develop from naive B cell subpopulations, and presumably from those that are ARID3a+. In healthy individuals, ARID3a+ cells are not usually observed in the na ' ive B cell subpopulation. These cells have not yet begun to secrete antibody.
  • the inventor proposes that identification of increased numbers of ARID3a+ cells, particularly in the naive B cell subpopulation, will help identify individuals who may be about to generate an inflammatory response that could correlate with disease activity. In other words, determining ARID3a could help predict disease flares. This would define a new biomarker for disease activity.
  • the transcription factor Bright (B cell regulator of IgH transcription) is a member of a growing family of proteins that interact with DNA through a highly conserved A+T-rich interaction domain, or ARID (Herrscher et al, 1995). Currently, Bright is the only member of this family for which target sequences have been identified, and which binds to DNA in a sequence-specific fashion.
  • ARID family proteins include the Drosophila proteins Dead ringer and eyelid that play important roles in lineage decisions in the gut and eyelid of the fruit fly, and are required for embryonic segmentation (Gregory et al, 1996; Treisman et al, 1997); retinoblastoma binding protein ( bpl) that interacts with retinoblastoma protein in a cell cycle-specific fashion (Fattaey et al, 1993); and BDP, a ubiquitously expressed human protein identified in a two-hybrid screen as a novel protein that also interacts with retinoblastoma protein (Rb) (Numata et al, 1999).
  • the yeast protein SWI/1 has homology to Bright, and is a component of a larger protein complex that serves to modulate chromatin organization in that organism (Peterson and Herskowitz, 1992; Burns and Peterson, 1997).
  • the human SWI-SNF complex contains a 270 kDa protein with non-sequence specific DNA binding activity that is also a member of the ARID family (Dallas et al, 2000).
  • members of this family may participate in lineage decisions, cell cycle control, tumor suppression and modulation of chromatin.
  • ARID3a Sequencing of the human genome identified fifteen members of this family, including the human Bright ortholog known as ARID3a (Wilsker et al, 2005).
  • ARID family proteins have diverse functions including, chromatin remodeling, binding to retinoblastoma protein, regulating X-Y chromosome functions and participation in embryonic development (Wilsker et al, 2005). Generally, these proteins are components of large protein complexes and are tightly regulated throughout development.
  • Human ARID3a can bind to E2F in cell lines of embryonic origin where its over-expression is controversial, as it has been associated with both tumor suppressor and oncogenic functions (Peeper et al, 2002; Suzuki et al, 1998; Ma et al, 2003; Fukuyo et al, 2004). Recent studies indicate that Bright activity is regulated tightly through intracellular partitioning and that it contributes to chromatin accessibility of the heavy chain enhancer (Kim and Tucker, 2006; Lin et al, 2007). It is likely that Bright/ARID3a can participate in a wide variety of regulatory functions in both embryonic and adult tissues because it functions as both a transcription factor and has a role in altering chromatin accessibility.
  • ARID family proteins are expressed ubiquitously.
  • murine Bright is expressed widely throughout embryonic development, but expression in the adult is largely limited to the B lymphocyte lineage where its expression is tightly regulated and is restricted at the mRNA level to the pre-B cell and peanut agglutinin-high germinal center cell populations (Herrscher et al, 1995; Webb et al, 1991 ; Webb et al, 1998).
  • Activated splenic B cells in the mouse can be induced to express Bright after antigen binding, but the protein is not present in the majority of peripheral IgM + B cells (Webb et al, 1991 ; Webb et al, 1998).
  • Bright binding sites associated with the intronic ⁇ enhancer also function as matrix- association regions, or MARs, A+T rich regions that have been proposed to organize chromatin into transcriptionally active domains (Herrscher et al 1995; Webb et al, 1991).
  • F ⁇ R nuclear factor ⁇ negative regulator
  • F ⁇ R contains the ubiquitously expressed CAAAT displacement protein (CDP/Cut/Cux) (Wang et al, 1999). While non-B cells in the mouse express F ⁇ R, B lymphocytes generally do not exhibit such protein complexes.
  • Bright and F ⁇ R play opposing roles in regulating the immunoglobulin locus (Webb et al, 1999). Transfection studies in which Bright and CDP were coexpressed showed repression of Bright (Wang et al, 1999). Therefore, Bright may activate transcription, directly or indirectly through chromatin remodeling or through more complex interactions with additional proteins. F ⁇ R may act in opposition to that activity (Wang et al, 1999).
  • Btk Bruton's tyrosine kinase, or Btk, associates with Bright in activated murine B lymphocytes (Webb et al, 2000).
  • Btk is an X-linked gene that encodes a tyrosine kinase critical for proper development and maintenance of B lymphocytes both in humans and in mice (reviewed in (Conley et al, 1994; Satterthwaite and Witte, 1996).
  • X-linked agammaglobulinemia an immunodeficiency state characterized by blocks at the pro-B cell stage of development and severely depressed serum antibody levels (Conley et al, 1994).
  • Btk is clearly the defective gene product in both human and murine diseases, the molecular mechanisms by which Btk deficiencies result in blocks in B cell development are currently unknown.
  • X-linked immunodeficient mice the mouse model for XLA, produce a mutated Btk protein that fails to form stable complexes with Bright (Webb et al, 2000). These data suggest that Bright may function as a component of the same signaling pathway(s) important in XLA.
  • the present invention may concerns Bright/ARID3a protein molecules.
  • a "protein” or “polypeptide” generally refers, but is not limited to, a protein of greater than about 100 amino acids or the full length endogenous sequence translated form of a gene.
  • a peptide is usually from about 3 to about 100 amino acids. All the “proteinaceous” terms described above may be used interchangeably herein.
  • a human ARID3a polypeptide sequence is provided in SEQ ID NO:2.
  • Proteins may be produced recombinantly or purified from natural sources. Shorter peptide molecules may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, e.g., Stewart and Young (1984); Tarn et al. (1983); Merrifield (1986); and Barany and Merrifield (1979), each incorporated herein by reference.
  • the size of the at least one proteinaceous molecule may comprise, but is not limited to, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86
  • amino acid refers to any amino acid, amino acid derivitive or amino acid mimic as would be known to one of ordinary skill in the art.
  • the residues of the proteinaceous molecule are sequential, without any non- amino molecule interrupting the sequence of amino molecule residues.
  • the sequence may comprise one or more non-amino molecule moieties.
  • the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.
  • the proteinaceous composition comprises at least one protein, polypeptide or peptide.
  • the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide.
  • biocompatible refers to a substance which produces no significant untoward effects when applied to, or administered to, a given organism according to the methods and amounts described herein. Such untoward or undesirable effects are those such as significant toxicity or adverse immunological reactions.
  • biocompatible protein, polypeptide or peptide containing compositions will generally be mammalian proteins or peptides or synthetic proteins or peptides each essentially free from toxins, pathogens and harmful immunogens.
  • Proteinaceous compositions may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials.
  • the nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art.
  • One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (world-wide-webe at ncbi.nlm.nih.gov).
  • Genbank and GenPept databases world-wide-webe at ncbi.nlm.nih.gov.
  • the coding regions for these known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art.
  • various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the
  • Peptides may also be fused to other proteinaceous compositions, thereby altering or supplementing their properties.
  • a targeting moiety may be provided which facilitate cellular transport of the Bright derived peptide or polypeptide.
  • sequences such as Tat can provide nuclear localization signals, thereby transporting peptides into the nucleus.
  • a proteinaceous compound may be purified.
  • purified will refer to a specific or protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, as would be known to one of ordinary skill in the art for the specific or desired protein, polypeptide or peptide.
  • nucleic acids derived from or encoding ARID3a are provided.
  • the nucleic acids may comprise wild-type or a mutant version of these genes.
  • the nucleic acid encodes for or comprises a transcribed nucleic acid.
  • the nucleic acid comprises a nucleic acid segment of SEQ ID NO: l, or a biologically functional equivalent thereof.
  • the nucleic acid encodes a protein, polypeptide, or peptide.
  • nucleic acid is well known in the art.
  • a “nucleic acid” as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase.
  • a nucleobase includes, for example, a naturally-occurring purine or pyrimidine base found in DNA (e.g., an adenine "A,” a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an "A,” a "G,” an uracil “U” or a "C”).
  • nucleic acid encompass the terms “oligonucleotide” and “polynucleotide,” each as a subgenus of the term “nucleic acid.”
  • oligonucleotide refers to a molecule of between about 3 and about 100 nucleobases in length.
  • polynucleotide refers to at least one molecule of greater than about 100 nucleobases in length.
  • a nucleic acid may encompass a double-stranded molecule or a triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a molecule.
  • a single-stranded nucleic acid may be denoted by the prefix "ss,” a double- stranded nucleic acid by the prefix "ds,” and a triple-stranded nucleic acid by the prefix "ts.”
  • a nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production or biological production.
  • a synthetic nucleic acid e.g., a synthetic oligonucleotide
  • Non-limiting examples of a synthetic nucleic acid include a nucleic acid made by in vitro chemically synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in EP 266 032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler e? al. (1986) and U.S. Patent 5,705,629, each incorporated herein by reference.
  • one or more oligonucleotide may be used.
  • oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5, 141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574, 146, 5,602,244, each of which is incorporated herein by reference.
  • a non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM (see for example, U.S. Patent 4,683,202 and U.S. Patent 4,682, 195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U.S. Patent 5,645,897, incorporated herein by reference.
  • a non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (i.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria (see for example, Sambrook et al. 2001, incorporated herein by reference).
  • a nucleic acid may be purified on polyacrylamide gels, cesium chloride centrifugation gradients, or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al, 2001, incorporated herein by reference).
  • the present invention concerns a nucleic acid that is an isolated nucleic acid.
  • isolated nucleic acid refers to a nucleic acid molecule (e.g., an RNA or DNA molecule) that has been isolated free of, or is otherwise free of, the bulk of the total genomic and transcribed nucleic acids of one or more cells.
  • isolated nucleic acid refers to a nucleic acid that has been isolated free of, or is otherwise free of, bulk of cellular components or in vitro reaction components such as for example, macromolecules such as lipids or proteins, small biological molecules, and the like. 3. Nucleic Acid Segments
  • the nucleic acid is a nucleic acid segment.
  • nucleic acid segment are smaller fragments of a nucleic acid, such as for non- limiting example, those that encode only part of ARID3a.
  • a “nucleic acid segment” may comprise any part of a gene sequence, of from about 2 nucleotides to the full length of ARID3a.
  • the nucleic acid segment may be a probe or primer.
  • a “probe” generally refers to a nucleic acid used in a detection method or composition.
  • a “primer” generally refers to a nucleic acid used in an extension or amplification method or composition. 4. Nucleic Acid Complements
  • the present invention also encompasses a nucleic acid that is complementary to a Bright-encoding nucleic acid.
  • the invention encompasses a nucleic acid or a nucleic acid segment complementary to the sequence set forth in SEQ ID O: l.
  • a nucleic acid is a "complement(s)" or is “complementary” to another nucleic acid when it is capable of base-pairing with another nucleic acid according to the standard Watson-Crick, Hoogsteen or reverse Hoogsteen binding complementarity rules.
  • another nucleic acid may refer to a separate molecule or a spatial separated sequence of the same molecule.
  • the term “complementary” or “complement(s)” also refers to a nucleic acid comprising a sequence of consecutive nucleobases or semiconsecutive nucleobases (e.g., one or more nucleobase moieties are not present in the molecule) capable of hybridizing to another nucleic acid strand or duplex even if less than all the nucleobases do not base pair with a counterpart nucleobase.
  • a "complementary" nucleic acid comprises a sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, to about 100%, and any range derivable therein, of the nucleobase sequence is capable of base-pairing with a single or double stranded nucleic acid molecule during hybridization.
  • the term "complementary" refers to a nucleic acid that may hybridize to another nucleic acid strand or duplex in stringent conditions, as would be understood by one of ordinary skill in the art.
  • a "partly complementary" nucleic acid comprises a sequence that may hybridize in low stringency conditions to a single or double stranded nucleic acid, or contains a sequence in which less than about 70% of the nucleobase sequence is capable of base-pairing with a single or double stranded nucleic acid molecule during hybridization.
  • hybridization As used herein, “hybridization,” “hybridizes” or “capable of hybridizing” is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature.
  • anneal as used herein is synonymous with “hybridize.”
  • hybridization “hybridize(s)” or “capable of hybridizing” encompasses the terms “stringent condition(s)” or “high stringency” and the terms “low stringency” or “low stringency condition(s).”
  • stringent condition(s) or “high stringency” are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but precludes hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.
  • Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.
  • low stringency or “low stringency conditions”
  • non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20°C to about 50°C.
  • hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20°C to about 50°C.
  • wild-type refers to the naturally occurring sequence of a nucleic acid at a genetic locus in the genome of an organism, or a sequence transcribed or translated from such a nucleic acid.
  • wild-type also may refer to an amino acid sequence encoded by a nucleic acid.
  • a genetic locus may have more than one sequence or alleles in a population of individuals, the term “wild-type” encompasses all such naturally occurring allele(s).
  • polymorphic means that variation exists (i.e., two or more alleles exist) at a genetic locus in the individuals of a population.
  • mutant refers to a change in the sequence of a nucleic acid or its encoded protein, polypeptide or peptide that is the result of the hand of man.
  • the present invention also concerns the isolation or creation of a recombinant construct or a recombinant host cell through the application of recombinant nucleic acid technology known to those of skill in the art or as described herein.
  • a recombinant construct or host cell may comprise a Bright-encoding nucleic acid, and may express a Bright protein, peptide or peptide, or at least one biologically functional equivalent thereof.
  • a “gene” refers to a nucleic acid that is transcribed.
  • the gene includes regulatory sequences involved in transcription, or message production or composition.
  • the gene comprises transcribed sequences that encode for a protein, polypeptide or peptide.
  • this function term "gene” includes both genomic sequences, RNA or cDNA sequences or smaller engineered nucleic acid segments, including nucleic acid segments of a non-transcribed part of a gene, including but not limited to the non-transcribed promoter or enhancer regions of a gene.
  • Smaller engineered gene nucleic acid segments may express, or may be adapted to express using nucleic acid manipulation technology, proteins, polypeptides, domains, peptides, fusion proteins, mutants and/or such like.
  • isolated substantially away from other coding sequences means that the gene of interest forms the significant part of the coding region of the nucleic acid, or that the nucleic acid does not contain large portions of naturally-occurring coding nucleic acids, such as large chromosomal fragments, other functional genes, RNA or cDNA coding regions. Of course, this refers to the nucleic acid as originally isolated, and does not exclude genes or coding regions later added to the nucleic acid by the hand of man.
  • nucleic acid(s) of the present invention may be combined with other nucleic acid sequences, including but not limited to, promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, coding segments, and the like, to create one or more nucleic acid construct(s).
  • a "nucleic acid construct" is a nucleic acid engeneered or altered by the hand of man, and generally comprises one or more nucleic acid sequences organized by the hand of man.
  • one or more nucleic acid constructs may be prepared that include a contiguous stretch of nucleotides identical to or complementary to SEQ ID O: l .
  • a nucleic acid construct may be about 3, about 5, about 8, about 10 to about 14, or about 15, about 20, about 30, about 40, about 50, about 100, about 200, about 500, about 1,000, about 2,000, about 3,000, about 5,000, or about 10,000 nucleotides in length, as well as constructs of greater size, up to and including chromosomal sizes (including all intermediate lengths and intermediate ranges), given the advent of nucleic acids constructs such as a yeast artificial chromosome are known to those of ordinary skill in the art.
  • intermediate lengths and “intermediate ranges,” as used herein, means any length or range including or between the quoted values (i.e., all integers including and between such values).
  • Intermediate lengths include about 1 1, about 12, about 13, about 16, about 17, about 18, about 19, etc.; about 21, about 22, about 23, etc.; about 31, about 32, etc.; about 51, about 52, about 53, etc.; about 101, about 102, about 103, etc.; about 151, about 152, about 153, etc.; about 1,001, about 1002, etc. ; about 10,001, about 10,002, etc.
  • Non-limiting examples of intermediate ranges include about 3 to about 32, about 150 to about 500, or about 5,000 to about 15,000, etc.
  • nucleic acid sequences may include additional residues, such as additional 5' or 3' sequences, or various combinations thereof, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein, polypeptide or peptide activity where expression of a proteinaceous composition is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' and/or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
  • nucleic acid sequences encoding relatively small peptides or fusion peptides such as, for example, peptides of from about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about
  • SLE Systemic lupus erythematosus
  • SLE is a systemic autoimmune disease (or autoimmune connective tissue disease) that can affect any part of the body. As occurs in other autoimmune diseases, the immune system attacks the body's cells and tissue, resulting in inflammation and tissue damage. It is a Type III hypersensitivity reaction in which antibody- immune complexes precipitate and cause a further immune response. SLE most often harms the heart, joints, skin, lungs, blood vessels, liver, kidneys, and nervous system. The course of the disease is unpredictable, with periods of illness (called "flares”) alternating with remissions. A flare has been defined as a measurable increase in disease activity in one or more organ systems involving new or worse clinical signs and symptoms and/or laboratory measurements.
  • SLE is one of several diseases known as "the great imitators" because it often mimics or is mistaken for other illnesses.
  • SLE is a classical item in differential diagnosis, because SLE symptoms vary widely and come and go unpredictably. Diagnosis can thus be elusive, with some people suffering unexplained symptoms of untreated SLE for years. Common initial and chronic complaints include fever, malaise, joint pains, myalgias, fatigue, and temporary loss of cognitive abilities. Because they are so often seen with other diseases, these signs and symptoms are not part of the diagnostic criteria for SLE. When occurring in conjunction with other signs and symptoms (see below), however, they are considered suggestive.
  • Low platelet and white blood cell counts may be due to the disease or a side effect of pharmacological treatment.
  • People with SLE may have an association with antiphospholipid antibody syndrome (a thrombotic disorder), wherein autoantibodies to phospholipids are present in their serum.
  • Abnormalities associated with antiphospholipid antibody syndrome include a paradoxical prolonged partial thromboplastin time (which usually occurs in hemorrhagic disorders) and a positive test for antiphospholipid antibodies; the combination of such findings has earned the term "lupus anticoagulant-positive.”
  • Another autoantibody finding in SLE is the anticardiolipin antibody, which can cause a false positive test for syphilis.
  • a person with SLE may have inflammation of various parts of the heart, such as pericarditis, myocarditis, and endocarditis.
  • the endocarditis of SLE is characteristically noninfective (Libman-Sacks endocarditis), and involves either the mitral valve or the tricuspid valve.
  • Atherosclerosis also tends to occur more often and advances more rapidly than in the general population. Lung and pleura inflammation can cause pleuritis, pleural effusion, lupus pneumonitis, chronic diffuse interstitial lung disease, pulmonary hypertension, pulmonary emboli, pulmonary hemorrhage, and shrinking lung syndrome.
  • Painless hematuria or proteinuria may often be the only presenting renal symptom.
  • Acute or chronic renal impairment may develop with lupus nephritis, leading to acute or end- stage renal failure.
  • end-stage renal failure occurs in less than 5% of cases.
  • a histological hallmark of SLE is membranous glomerulonephritis with "wire loop" abnormalities. This finding is due to immune complex deposition along the glomerular basement membrane, leading to a typical granular appearance in immunofluorescence testing.
  • Neuropsychiatric syndromes can result when SLE affects the central or peripheral nervous systems.
  • the American College of Rheumatology defines 19 neuropsychiatric syndromes in systemic lupus erythematosus.
  • the diagnosis of neuropsychiatric syndromes concurrent with SLE is one of the most difficult challenges in medicine, because it can involve so many different patterns of symptoms, some of which may be mistaken for signs of infectious disease or stroke.
  • the most common neuropsychiatric disorder people with SLE have is headache, although the existence of a specific lupus headache and the optimal approach to headache in SLE cases remains controversial.
  • Other common neuropsychiatric manifestations of SLE include cognitive dysfunction, mood disorder, cerebrovascular disease, seizures, polyneuropathy, anxiety disorder, and psychosis.
  • intracranial hypertension syndrome characterized by an elevated intracranial pressure, papilledema, and headache with occasional abducens nerve paresis, absence of a space- occupying lesion or ventricular enlargement, and normal cerebrospinal fluid chemical and hematological constituents. More rare manifestations are acute confusional state, Guillain- Barre syndrome, aseptic meningitis, autonomic disorder, demyelinating syndrome, mononeuropathy (which might manifest as mononeuritis multiplex), movement disorder (more specifically, chorea), myasthenia gravis, myelopathy, cranial neuropathy and plexopathy. Neural symptoms contribute to a significant percentage of morbidity and mortality in patients with lupus.
  • NPSLE neuropsychiatric systemic lupus erythematosus
  • Lupus has a wide range of symptoms which span the body.
  • the neurological symptoms include headaches, depression, seizures, cognitive dysfunction, mood disorder, cerebrovascular disease, polyneuropathy, anxiety disorder, psychosis, and in some extreme cases, personality disorders.
  • Neonatal lupus is the occurrence of SLE symptoms in an infant born from a mother with SLE, most commonly presenting with a rash resembling discoid lupus erythematosus, and sometimes with systemic abnormalities such as heart block or hepatosplenomegaly. Neonatal lupus is usually benign and self-limited.
  • Fatigue in SLE is probably multifactorial and has been related to not only disease activity or complications such as anemia or hypothyroidism, but also to pain, depression, poor sleep quality, poor physical fitness and lack of social support.
  • Determining if a patient is in flare can be achieved by appliying the Systemic Lupus Erythematosus Disease Activity Index SELENA Modification (world-wide-web at rheumatology.org/Practice/Clinical/Indexes/Systemic_Lupus_Erythematosus_Disease_Activi ty_Index_SELENA_Modification/).
  • This scale uses a point system to calculate when the accumulated significance of recent changes in various indicators translates into a mild/moderate (SLEDA Index of 3-1 1 point change) or a severe (12 of more point change) flare.
  • Antinuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti-ENA) form the mainstay of serologic testing for SLE.
  • ANA Antinuclear antibody
  • anti-ENA anti-extractable nuclear antigen
  • Clinically the most widely used method is indirect immunofluorescence. The pattern of fluorescence suggests the type of antibody present in the patient's serum.
  • Direct immunofluorescence can detect deposits of immunoglobulins and complement proteins in the patient's skin. When skin not exposed to the sun is tested, a positive direct IF (the so-called Lupus band test) is an evidence of systemic lupus erythematosus.
  • ANA screening yields positive results in many connective tissue disorders and other autoimmune diseases, and may occur in normal individuals.
  • Subtypes of antinuclear antibodies include anti-Smith and anti-double stranded DNA (dsDNA) antibodies (which are linked to SLE) and anti-histone antibodies (which are linked to drug-induced lupus).
  • Anti- dsDNA antibodies are highly specific for SLE; they are present in 70% of cases, whereas they appear in only 0.5% of people without SLE.
  • the anti-dsDNA antibody titers also tend to reflect disease activity, although not in all cases.
  • ANA that may occur in SLE sufferers are anti-Ul RNP (which also appears in systemic sclerosis), SS-A (or anti-Ro) and SS-B (or anti-La; both of which are more common in Sj5gren's syndrome).
  • SS-A and SS-B confer a specific risk for heart conduction block in neonatal lupus.
  • LE cell test was commonly used for diagnosis, but it is no longer used because the LE cells are only found in 50-75% of SLE cases, and they are also found in some people with rheumatoid arthritis, scleroderma, and drug sensitivities. Because of this, the LE cell test is now performed only rarely and is mostly of historical significance.
  • SLE The treatment of SLE involves preventing flares and reducing their severity and duration when they occur.
  • Treatment can include corticosteroids and anti-malarial drugs.
  • Certain types of lupus nephritis such as diffuse proliferative glomerulonephritis require bouts of cytotoxic drugs. These drugs include cyclophosphamide and mycophenolate.
  • Hydroxychloroquine (HCQ) was approved by the FDA for lupus in 1955. Some drugs approved for other diseases are used for SLE Off-label.' In November 2010, an FDA advisory panel recommended approving belimumab (Benlysta) as a treatment for the pain and flare-ups common in lupus. The drug was approved by the FDA in March 2011. N- acetylcysteine has been shown to reverse depletion of the natural anti-oxidant glutathione and to safely improve disease activity in a double-blind placebo-controlled pilot study.
  • DMARDs Disease-modifying antirheumatic drugs
  • DMARDs Disease-modifying antirheumatic drugs
  • DMARDs are used preventively to reduce the incidence of flares, the process of the disease, and lower the need for steroid use; when flares occur, they are treated with corticosteroids.
  • DMARDs commonly in use are antimalarials such as Plaquenil and immunosuppressants (e.g., methotrexate and azathioprine).
  • Plaquenil hydroxychloroquine
  • Hydroxychloroquine has relatively few side effects, and there is evidence that it improves survival among people who have SLE.
  • Cyclophosphamide is used for severe glomerulonephritis or other organ-damaging complications.
  • Mycophenolic acid is also used for treatment of lupus nephritis, but it is not FDA-approved for this indication, and FDA is investigating reports that it may be associated with birth defects when used by pregnant women.
  • medications that modulate the immune system primarily corticosteroids and immunosuppressants
  • flares recurrence of symptoms
  • people who require steroids may develop Cushing's syndrome, symptoms of which may include obesity, puffy round face, diabetes mellitus, increased appetite, difficulty sleeping and osteoporosis. These may subside if and when the large initial dosage is reduced, but long-term use of even low doses can cause elevated blood pressure and cataracts.
  • NSAIDs such as indomethacin and diclofenac are relatively contraindicated for patients with SLE because they increase the risk of kidney failure and heart failure.
  • Moderate pain is typically treated with mild prescription opiates such as dextropropoxyphene and co-codamol.
  • Moderate to severe chronic pain is treated with stronger opioids, such as hydrocodone or longer-acting continuous-release opioids, such as oxycodone, MS Contin, or methadone.
  • the fentanyl duragesic transdermal patch is also a widely used treatment option for the chronic pain caused by complications because of its long-acting timed release and ease of use.
  • opioids are used for prolonged periods, drug tolerance, chemical dependency, and addiction may occur.
  • Opiate addiction is not typically a concern, since the condition is not likely to ever completely disappear.
  • lifelong treatment with opioids is fairly common for chronic pain symptoms, accompanied by periodic titration that is typical of any long-term opioid regimen.
  • Intravenous immunoglobulins may be used to control SLE with organ involvement, or vasculitis. It is believed that they reduce antibody production or promote the clearance of immune complexes from the body, even though their mechanism of action is not well- understood. Unlike immunosuppressives and corticosteroids, IVIGs do not suppress the immune system, so there is less risk of serious infections with these drugs.
  • Avoiding sunlight is the primary change to the lifestyle of SLE sufferers, as sunlight is known to exacerbate the disease, as is the debilitating effect of intense fatigue. These two problems can lead to patients becoming housebound for long periods of time. Drugs unrelated to SLE should be prescribed only when known not to exacerbate the disease. Occupational exposure to silica, pesticides and mercury can also make the disease worsen.
  • Renal transplants are the treatment of choice for end-stage renal disease, which is one of the complications of lupus nephritis, but the recurrence of the full disease is common in up to 30% of patients.
  • Antiphospholipid syndrome is also related to the onset of neural lupus symptoms in the brain.
  • the cause is very different from lupus: thromboses (blood clots or "sticky blood”) form in blood vessels, which prove to be fatal if they move within the blood stream. If the thromboses migrate to the brain, they can potentially cause a stroke by blocking the blood supply to the brain.
  • brain scans are usually required for early detection. These scans can show localized areas of the brain where blood supply has not been adequate.
  • the treatment plan for these patients requires anticoagulation. Often, low-dose aspirin is prescribed for this purpose, although for cases involving thrombosis anticoagulants such as warfarin are used.
  • methods are provided for the assaying of expression of ARID3a.
  • the principle applications are to (a) determine if a patient has SLE as opposed to a distinct autoimmune condition, (b) to determine the severity of the disease, (c) to determine the current intensity of the inflammatory state, and (d) to predict or assess the efficacy of a.
  • the expression of ARID3a will be measured, and in some, the expression is measured multiple times to assess not only absolute values, but changes in these values overtime. Virtually any method of measuring gene expression may be utilized, and the following discussion is exemplary in nature and in no way limiting.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • antibody also refers to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • immunodetection methods are provided.
  • immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoradiometric assay fluoroimmunoassay
  • fluoroimmunoassay fluoroimmunoassay
  • chemiluminescent assay chemiluminescent assay
  • bioluminescent assay bioluminescent assay
  • Western blot to mention a few.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle & Ben-Zeev O, 1999; Gulbis & Galand, 1993; De Jager et al, 1993; and Nakamura e
  • the immunobinding methods include obtaining a sample suspected of containing a relevant polypeptide, and contacting the sample with a first antibody under conditions effective to allow the formation of immunocomplexes.
  • the biological sample analyzed may be any sample that is suspected of containing an antigen, such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, or even a biological fluid.
  • the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any antigens present.
  • the sample- antibody composition such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • the antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • One method of immunodetection designed by Charles Cantor uses two different antibodies.
  • a first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin.
  • the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex.
  • the antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex.
  • streptavidin or avidin
  • biotinylated DNA and/or complementary biotinylated DNA
  • the amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin.
  • This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
  • a conjugate can be produced which is macroscopically visible.
  • PCR Polymerase Chain Reaction
  • the PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls.
  • the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
  • immunoassays are in essence binding assays.
  • immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.
  • the antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA.” Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the antigen are immobilized onto the well surface and then contacted with the anti-ORF message and anti- ORF translated product antibodies of the invention. After binding and washing to remove non-specifically bound immune complexes, the bound anti-ORF message and anti-ORF translated product antibodies are detected. Where the initial anti-ORF message and anti-ORF translated product antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-ORF message and anti-ORF translated product antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which the antigens are immobilized involves the use of antibody competition in the detection.
  • labeled antibodies against an antigen are added to the wells, allowed to bind, and detected by means of their label.
  • the amount of an antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against the antigen during incubation with coated wells.
  • the presence of an antigen in the sample acts to reduce the amount of antibody against the antigen available for binding to the well and thus reduces the ultimate signal.
  • This is also appropriate for detecting antibodies against an antigen in an unknown sample, where the unlabeled antibodies bind to the antigen- coated wells and also reduces the amount of antigen available to bind the labeled antibodies.
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • the "suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25°C to 27°C, or may be overnight at about 4°C or so.
  • FACS Fluorescence- Activated Cell Sorting
  • the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured.
  • An electrical charging ring is placed just at the point where the stream breaks into droplets.
  • a charge is placed on the ring based on the immediately prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream.
  • the charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.
  • FAC Fluorescence-Activated Cell Sorting
  • mass spectrometry can resolved and confidently identified a wide variety of complex compounds, including proteins.
  • Traditional quantitative MS has used electrospray ionization (ESI) followed by tandem MS (MS/MS) while newer quantitative methods are being developed using matrix assisted laser desorption/ionization (MALDI) followed by time of flight (TOF) MS.
  • ESI electrospray ionization
  • MALDI matrix assisted laser desorption/ionization
  • TOF time of flight
  • mass spectrometry profiles that are useful for grading gliomas and predicting glioma patient survival, without regard for the identity of specific proteins.
  • mass spectrometry may be used to look for the levels of these proteins particularly.
  • ESI is a convenient ionization technique that is used to produce gaseous ions from highly polar, mostly nonvolatile biomolecules, including lipids.
  • the sample is injected as a liquid at low flow rates (1-10 ⁇ / ⁇ ) through a capillary tube to which a strong electric field is applied.
  • the field generates additional charges to the liquid at the end of the capillary and produces a fine spray of highly charged droplets that are electrostatically attracted to the mass spectrometer inlet.
  • the evaporation of the solvent from the surface of a droplet as it travels through the desolvation chamber increases its charge density substantially. When this increase exceeds the Rayleigh stability limit, ions are ejected and ready for MS analysis.
  • ESI tandem mass spectroscopy In ESI tandem mass spectroscopy (ESI/MS/MS), one is able to simultaneously analyze both precursor ions and product ions, thereby monitoring a single precursor product reaction and producing (through selective reaction monitoring (SRM)) a signal only when the desired precursor ion is present.
  • SRM selective reaction monitoring
  • the internal standard is a stable isotope-labeled version of the analyte, this is known as quantification by the stable isotope dilution method.
  • This approach has been used to accurately measure pharmaceuticals and bioactive peptides. Newer methods are performed on widely available MALDI-TOF instruments, which can resolve a wider mass range and have been used to quantify metabolites, peptides, and proteins.
  • Protein quantification has been achieved by quantifying tryptic peptides.
  • Complex mixtures such as crude extracts can be analyzed, but in some instances sample clean up is required.
  • Secondary ion mass spectroscopy is an analytical method that uses ionized particles emitted from a surface for mass spectroscopy at a sensitivity of detection of a few parts per billion.
  • the sample surface is bombarded by primary energetic particles, such as electrons, ions (e.g., O, Cs), neutrals or even photons, forcing atomic and molecular particles to be ejected from the surface, a process called sputtering. Since some of these sputtered particles carry a charge, a mass spectrometer can be used to measure their mass and charge. Continued sputtering permits measuring of the exposed elements as material is removed. This in turn permits one to construct elemental depth profiles. Although the majority of secondary ionized particles are electrons, it is the secondary ions which are detected and analysis by the mass spectrometer in this method.
  • Laser desorption mass spectroscopy involves the use of a pulsed laser, which induces desorption of sample material from a sample site - effectively, this means vaporization of sample off of the sample substrate. This method is usually only used in conjunction with a mass spectrometer, and can be performed simultaneously with ionization if one uses the right laser radiation wavelength.
  • LD-MS When coupled with Time-of-Flight (TOF) measurement, LD-MS is referred to as LDLPMS (Laser Desorption Laser Photoionization Mass Spectroscopy).
  • LDLPMS Laser Desorption Laser Photoionization Mass Spectroscopy
  • the LDLPMS method of analysis gives instantaneous volatilization of the sample, and this form of sample fragmentation permits rapid analysis without any wet extraction chemistry.
  • the LDLPMS instrumentation provides a profile of the species present while the retention time is low and the sample size is small.
  • an impactor strip is loaded into a vacuum chamber. The pulsed laser is fired upon a certain spot of the sample site, and species present are desorbed and ionized by the laser radiation. This ionization also causes the molecules to break up into smaller fragment-ions.
  • the positive or negative ions made are then accelerated into the flight tube, being detected at the end by a microchannel plate detector.
  • Signal intensity, or peak height, is measured as a function of travel time.
  • the applied voltage and charge of the particular ion determines the kinetic energy, and separation of fragments is due to different size causing different velocity. Each ion mass will thus have a different flight- time to the detector.
  • Positive ions are made from regular direct photoionization, but negative ion formation requires a higher powered laser and a secondary process to gain electrons. Most of the molecules that come off the sample site are neutrals, and thus can attract electrons based on their electron affinity. The negative ion formation process is less efficient than forming just positive ions. The sample constituents will also affect the outlook of a negative ion spectrum.
  • MALDI-TOF-MS The properties that make MALDI-TOF-MS a popular qualitative tool— its ability to analyze molecules across an extensive mass range, high sensitivity, minimal sample preparation and rapid analysis times— also make it a potentially useful quantitative tool.
  • MALDI-TOF-MS also enables non-volatile and thermally labile molecules to be analyzed with relative ease. It is therefore prudent to explore the potential of MALDI-TOF-MS for quantitative analysis in clinical settings, for toxicological screenings, as well as for environmental analysis.
  • the application of MALDI-TOF-MS to the quantification of peptides and proteins is particularly relevant. The ability to quantify intact proteins in biological tissue and fluids presents a particular challenge in the expanding area of proteomics and investigators urgently require methods to accurately measure the absolute quantity of proteins.
  • MALDI-TOF-MS Because of these difficulties, practical examples of quantitative applications of MALDI-TOF-MS have been limited. Most of the studies to date have focused on the quantification of low mass analytes, in particular, alkaloids or active ingredients in agricultural or food products, whereas other studies have demonstrated the potential of MALDI-TOF-MS for the quantification of biologically relevant analytes such as neuropeptides, proteins, antibiotics, or various metabolites in biological tissue or fluid. In earlier work it was shown that linear calibration curves could be generated by MALDI-TOF- MS provided that an appropriate internal standard was employed. This standard can "correct" for both sample-to-sample and shot-to-shot variability. Stable isotope labeled internal standards (isotopomers) give the best result.
  • the properties of the matrix material used in the MALDI method are critical. Only a select group of compounds is useful for the selective desorption of proteins and polypeptides. A review of all the matrix materials available for peptides and proteins shows that there are certain characteristics the compounds must share to be analytically useful. Despite its importance, very little is known about what makes a matrix material "successful" for MALDI. The few materials that do work well are used heavily by all MALDI practitioners and new molecules are constantly being evaluated as potential matrix candidates. With a few exceptions, most of the matrix materials used are solid organic acids. Liquid matrices have also been investigated, but are not used routinely.
  • an indirect method for detecting protein expression is to detect mRNA transcripts from which the proteins are made. The following is a discussion of such methods, which are applicable particularly to calcyclin, calpactin I light chain, astrocytic phosphoprotein PEA- 15 and tubulin-specific chaperone A in the context of the present invention.
  • Hybridization is defined as the ability of a nucleic acid to selectively form duplex molecules with complementary stretches of DNAs and/or RNAs. Depending on the application envisioned, one would employ varying conditions of hybridization to achieve varying degrees of selectivity of the probe or primers for the target sequence.
  • a probe or primer of between 13 and 100 nucleotides preferably between 17 and 100 nucleotides in length up to 1-2 kilobases or more in length will allow the formation of a duplex molecule that is both stable and selective.
  • Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and selectivity of the hybrid molecules obtained.
  • Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
  • relatively high stringency conditions For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids.
  • relatively low salt and/or high temperature conditions such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • lower stringency conditions may be used. Under these conditions, hybridization may occur even though the sequences of the hybridizing strands are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Hybridization conditions can be readily manipulated depending on the desired results.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl 2 , 1.0 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , at temperatures ranging from approximately 40°C to about 72°C.
  • nucleic acids of defined sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected.
  • enzyme tags colorimetric indicator substrates are known that can be employed to provide a detection means that is visibly or spectrophotometrically detectable, to identify specific hybridization with complementary nucleic acid containing samples.
  • the probes or primers described herein will be useful as reagents in solution hybridization, as in PCRTM, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase.
  • the test DNA or R A
  • R A is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions.
  • the conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art.
  • hybridization After washing of the hybridized molecules to remove non-specifically bound probe molecules, hybridization is detected, and/or quantified, by determining the amount of bound label.
  • Representative solid phase hybridization methods are disclosed in U.S. Patents 5,843,663, 5,900,481 and 5,919,626.
  • Other methods of hybridization that may be used in the practice of the present invention are disclosed in U.S. Patents 5,849,481, 5,849,486 and 5,851,772. The relevant portions of these and other references identified in this section of the Specification are incorporated herein by reference.
  • nucleic acid amplification greatly enhances the ability to assess expression.
  • the general concept is that nucleic acids can be amplified using paired primers flanking the region of interest.
  • primer as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded and/or single- stranded form, although the single-stranded form is preferred.
  • Pairs of primers designed to selectively hybridize to nucleic acids corresponding to selected genes are contacted with the template nucleic acid under conditions that permit selective hybridization.
  • high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers.
  • hybridization may occur under reduced stringency to allow for amplification of nucleic acids contain one or more mismatches with the primer sequences.
  • the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.
  • the amplification product may be detected or quantified.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemilluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals.
  • PCRTM polymerase chain reaction
  • a reverse transcriptase PCRTM amplification procedure may be performed to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known (see Sambrook et al, 1989).
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641.
  • Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Patent 5,882,864.
  • MPCR multiplex-PCR
  • MPCR buffers contain a Taq Polymerase additive, which decreases the competition among amplicons and the amplification discrimination of longer DNA fragment during MPCR.
  • MPCR products can further be hybridized with gene-specific probe for verification. Theoretically, one should be able to use as many as primers as necessary.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, may also be used as an amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence which may then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha- thio] -triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention (Walker et ah, 1992).
  • Strand Displacement Amplification (SDA) disclosed in U.S. Patent 5,916,779, is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et ah, 1989; Gingeras et ah, PCT Application WO 88/10315, incorporated herein by reference in their entirety).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et ah, 1989; Gingeras et ah, PCT Application WO 88/10315, incorporated herein by reference in their entirety.
  • European Application No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence.
  • This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "race” and "one-sided PCR” (Frohman, 1990; Ohara et al, 1989).
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et al, 1989). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.
  • Separation of nucleic acids may also be effected by chromatographic techniques known in art.
  • chromatographic techniques There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.
  • the amplification products are visualized.
  • a typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et al, 2001).
  • U.S. Patent 5,279,721, incorporated by reference herein discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids.
  • the apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • Other methods of nucleic acid detection that may be used in the practice of the instant invention are disclosed in U.S.
  • Microarrays comprise a plurality of polymeric molecules spatially distributed over, and stably associated with, the surface of a substantially planar substrate, e.g., biochips.
  • Microarrays of polynucleotides have been developed and find use in a variety of applications, such as screening and DNA sequencing.
  • One area in particular in which microarrays find use is in gene expression analysis.
  • an array of "probe" oligonucleotides is contacted with a nucleic acid sample of interest, i.e., target, such as polyA mRNA from a particular tissue type. Contact is carried out under hybridization conditions and unbound nucleic acid is then removed. The resultant pattern of hybridized nucleic acid provides information regarding the genetic profile of the sample tested. Methodologies of gene expression analysis on microarrays are capable of providing both qualitative and quantitative information.
  • the probe molecules of the arrays which are capable of sequence specific hybridization with target nucleic acid may be polynucleotides or hybridizing analogues or mimetics thereof, including: nucleic acids in which the phosphodiester linkage has been replaced with a substitute linkage, such as phophorothioate, methylimino, methylphosphonate, phosphoramidate, guanidine and the like; nucleic acids in which the ribose subunit has been substituted, e.g., hexose phosphodiester; peptide nucleic acids; and the like.
  • the length of the probes will generally range from 10 to 1000 nts, where in some embodiments the probes will be oligonucleotides and usually range from 15 to 150 nts and more usually from 15 to 100 nts in length, and in other embodiments the probes will be longer, usually ranging in length from 150 to 1000 nts, where the polynucleotide probes may be single- or double-stranded, usually single-stranded, and may be PCR fragments amplified from cDNA.
  • the probe molecules on the surface of the substrates will correspond to selected genes being analyzed and be positioned on the array at a known location so that positive hybridization events may be correlated to expression of a particular gene in the physiological source from which the target nucleic acid sample is derived.
  • the substrates with which the probe molecules are stably associated may be fabricated from a variety of materials, including plastics, ceramics, metals, gels, membranes, glasses, and the like.
  • the arrays may be produced according to any convenient methodology, such as preforming the probes and then stably associating them with the surface of the support or growing the probes directly on the support. A number of different array configurations and methods for their production are known to those of skill in the art and disclosed in U.S.
  • a washing step is employed where unhybridized labeled nucleic acid is removed from the support surface, generating a pattern of hybridized nucleic acid on the substrate surface.
  • wash solutions and protocols for their use are known to those of skill in the art and may be used.
  • the array now comprising bound target
  • the other member(s) of the signal producing system that is being employed.
  • the label on the target is biotin
  • streptavidin-fluorescer conjugate under conditions sufficient for binding between the specific binding member pairs to occur.
  • any unbound members of the signal producing system will then be removed, e.g., by washing.
  • the specific wash conditions employed will necessarily depend on the specific nature of the signal producing system that is employed, and will be known to those of skill in the art familiar with the particular signal producing system employed.
  • the resultant hybridization pattern(s) of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the nucleic acid, where representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement and the like.
  • the array of hybridized target/probe complexes may be treated with an endonuclease under conditions sufficient such that the endonuclease degrades single stranded, but not double stranded DNA.
  • endonucleases include: mung bean nuclease, SI nuclease, and the like.
  • the endonuclease treatment will generally be performed prior to contact of the array with the other member(s) of the signal producing system, e.g., fluorescent-streptavidin conjugate. Endonuclease treatment, as described above, ensures that only end-labeled target/probe complexes having a substantially complete hybridization at the 3' end of the probe are detected in the hybridization pattern.
  • the resultant hybridization pattern is detected.
  • the intensity or signal value of the label will be not only be detected but quantified, by which is meant that the signal from each spot of the hybridization will be measured and compared to a unit value corresponding the signal emitted by known number of end-labeled target nucleic acids to obtain a count or absolute value of the copy number of each end-labeled target that is hybridized to a particular spot on the array in the hybridization pattern.
  • the present invention contemplates the treatment of SLE using either standard therapeutic approaches, and/or ARID3a inhibition. Advantages accruing to the present invention include earlier intervention, when ARID3a levels are rising or have risen, but the symptoms of excessive B cell activity have not appeared. Also, the present disclosure is the first connection between ARID3a and SLE, thus opening a new possible therapeutic target in this challenging disease.
  • compositions When given in combination, these compositions one would generally be administered in a combined amount effective to achieve a reduction in one or more SLE disease parameter.
  • This process may involve contacting the subject with the both agents/therapies at the same time, e.g., using a single composition or pharmacological formulation that includes both agents, or by contacting the subject with two distinct compositions or formulations, at the same time.
  • one treatment may precede or follow the other treatment by intervals ranging from minutes to weeks.
  • compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
  • pharmaceutically or pharmacologically acceptable refesr to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. Such routes include oral, nasal, buccal, rectal, vaginal or topical route. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences,” 15 th Ed., 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • the present invention contemplates the use of any composition that will inhibit Bright/ARID3a function.
  • Biological inhibitors as described below, may be utilized to interfere with Bright/ARID3a function.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
  • complementary it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • ribozyme e.g., ribozyme; see below
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • Ribozymes Another general class of inhibitors is ribozymes. Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA -protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et ah, 1987; Forster and Symons, 1987).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).
  • This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al, 1981). For example, U.S.
  • Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990).
  • ribozymes can elicit genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV.
  • Most of this work involved the modification of a target mR A, based on a specific mutant codon that was cleaved by a specific ribozyme.
  • RNA interference also referred to as "RNA-mediated interference” or RNAi
  • RNAi Double- stranded RNA
  • dsRNA double- stranded RNA
  • dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp et al, 2000; Tabara et al, 1999). Activation of these mechanisms targets mature, dsRNA-complementary mRNA for destruction.
  • RNAi offers major experimental advantages for study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp, 1999; Sharp et al, 2000; Tabara et al, 1999). Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C. elegans, Trypanasoma, Drosophila, and mammals (Grishok et al, 2000; Sharp, 1999; Sharp et al, 2000; Elbashir et al, 2001). It is generally accepted that RNAi acts post- transcriptionally, targeting RNA transcripts for degradation, and possibly by inhibiting translation. It appears that both nuclear and cytoplasmic RNA can be targeted (Bosher et al, 2000).
  • siRNAs must be designed so that they are specific and effective in suppressing the expression of the genes of interest. Methods of selecting the target sequences, i.e. those sequences present in the gene or genes of interest to which the siRNAs will guide the degradative machinery, are directed to avoiding sequences that may interfere with the siRNA's guide function while including sequences that are specific to the gene or genes. Typically, siRNA target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs as described above (Montgomery et al, 1998). Of particular interest are those siRNAs that span an exon-intron junction.
  • siRNAs has been mainly through direct chemical synthesis; through processing of longer, double stranded RNAs through exposure to Drosophila embryo lysates; or through an in vitro system derived from S2 cells. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, 21-23 nucleotide siRNAs from the lysate, etc., making the process somewhat cumbersome and expensive.
  • Chemical synthesis proceeds by making two single stranded RNA-oligomers followed by the annealing of the two single stranded oligomers into a double stranded RNA. Methods of chemical synthesis are diverse. Non-limiting examples are provided in U.S. Patents 5,889, 136, 4,415,732, and 4,458,066, expressly incorporated herein by reference, and in Wincott et al. (1995).
  • RNA sequences having di-nucleotide overhangs may provide the greatest level of suppression.
  • These protocols primarily use a sequence of two (2'-deoxy) thymidine nucleotides as the di- nucleotide overhangs. These dinucleotide overhangs are often written as dTdT to distinguish them from the typical nucleotides incorporated into RNA.
  • the literature has indicated that the use of dT overhangs is primarily motivated by the need to reduce the cost of the chemically synthesized RNAs. It is also suggested that the dTdT overhangs might be more stable than UU overhangs, though the data available shows only a slight ( ⁇ 20%) improvement of the dTdT overhang compared to an siRNA with a UU overhang.
  • siRNAs are found to work optimally when they are in cell culture at concentrations of 25-100 nM. This had been demonstrated by Elbashir et al. (2001) wherein concentrations of about 100 nM achieved effective suppression of expression in mammalian cells. siRNAs have been most effective in mammalian cell culture at about 100 nM. In several instances, however, lower concentrations of chemically synthesized siRNA have been used (Caplen et al, 2000; Elbashir et al, 2001).
  • RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference.
  • the enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6) via the use and production of an expression construct as is known in the art. See U.S. Patent 5,795,715.
  • the contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene.
  • the length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length.
  • RNA single strands of RNA can be produced enzymatically or by partial/total organic synthesis.
  • single stranded RNA is enzymatically synthesized from the PCRTM products of a DNA template, preferably a cloned cDNA template and the RNA product is a complete transcript of the cDNA, which may comprise hundreds of nucleotides.
  • WO 01/36646 places no limitation upon the manner in which the siRNA is synthesized, providing that the RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures.
  • RNA polymerase e.g., T3, T7, SP6
  • RNA interference no distinction in the desirable properties for use in RNA interference is made between chemically or enzymatically synthesized siRNA.
  • U.S. Patent 5,795,715 reports the simultaneous transcription of two complementary DNA sequence strands in a single reaction mixture, wherein the two transcripts are immediately hybridized.
  • the templates used are preferably of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence.
  • the templates can be attached to a solid surface. After transcription with RNA polymerase, the resulting dsRNA fragments may be used for detecting and/or assaying nucleic acid target sequences.
  • the inventor proposes to inhibit ARID3a expression in adult tissues in vitro using siRNA or shRNA in a lentiviral vector.
  • a GFP marker can be utilized to determine that cells took up the vector, and thus permit checking for appropriate inhibition of ARID3a production.
  • the B cell line BCg3R-ld and/or over-expressing transgenic mouse spleen cells can be utilized. After the inventor confirms that inhibition of ARID3a occurs in these cells, they will inhibit ARID3a expression in mouse embryo fibroblasts and culture the GFP+ cells to confirm that pluripotent stem cells develop.
  • the use of an inducible promoter discussed below that allow induction of the siRNA or shRNA only under specific growth conditions permit reversible inhibition of ARID3a.
  • cells can be induced to dedifferentiate into a pluripotent and self-renewing state in vitro, and can then be induced to differentiate into mature lineage cells under different growth conditions without inhibition of ARID3a.
  • These methods offer considerable advantages over current methodologies, which involve introduction of multiple viral copies and genes, some of which are known to be oncogenic. Self-deleting vectors may also be used. 4. Antibodies
  • antibodies may find use as inhibitors of ARID3a.
  • antibody is intended to refer broadly to any appropriate immunologic binding agent such as IgG, IgM, IgA, IgD and IgE.
  • IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • antibody also refers to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single chain Fv
  • scFv single chain Fv
  • Monoclonal antibodies are recognized to have certain advantages, e.g., reproducibility and large-scale production, and their use is generally preferred.
  • the invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.
  • Single- chain Mabs are described in U.S. Patents 4,946,778 and 5,888,773, each of which are hereby incorporated by reference. The present invention would most likely utilize single-chain antibodies expressed from expression vectors, as described below.
  • Humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. Methods for the development of antibodies that are "custom-tailored” to the patient's dental disease are likewise known and such custom-tailored antibodies are also contemplated. 5. Inhibitory Peptides
  • Peptides may prove to be useful inhibitors of Bright/ARID3a function by competiting with or mimicking Bright domains that bind or interact with DNA, Btk, TFII-I or other molecules, or compete with Bright dimerization sequences. Also contemplated are regions of Bright that comprise nuclear shuttling sequences. Bright-derived peptides are therefore a particular type of compound that may prove useful in inhibiting Bright function.
  • the peptides may be designed around an existing structure, i.e., portions of Bright, or they may be selected for function from a randomized library.
  • SEQ ID NO: l Of particular interest is a region of SEQ ID NO: l from about residue 444 to residue 549, and more particularly from 449-544. This region has been shown to be involved in Brigth/ARID3a dimerization, and also to contain nuclear shuttling sequences. Within these regions, all possible peptides of 8 to about 40 residues are contemplated. Other more particular regions include residues 444-483, 449-488, 510-549, 505-544, 444-473, 449-473, 531-549 and 531-544.
  • the peptides will be less than 50 residues, and comprising at least about 10 consecutive residues of Bright/ARID3a.
  • the number of consecutive Bright/ARID3a residues may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, with additional non-Bright sequences attached thereto.
  • Ranges of peptide length of 10-50 residues, 10-40 residues, 15-50 residues 15-40, residues, 15-35 residues, 15-30 residues, 15-25 residues, 15- 20 residues and 20-25 residues are contemplated.
  • the number of additional non- Bright/ARID3 a residues may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more residues.
  • the overall size of the peptides may therefore range from 8 reisudes to 75 or more residues, with 10-70 residues, 10-60 residues, 10-50 residues, 10-40 residues, 10-30 residues and 15-70 residues, 15-60 residues, 15-50 residues, 15-40 residues, 15-30 residues, 20-70 residues, 20-60 residues, 20-50 residues, 20-40 residues, and 20-30 residues being specifically contemplated ranges.
  • Peptides may be produced by cleavage of polypeptides, such as Bright, with proteolytic enzymes (trypsin, chymotrypsin, etc.), or chemicals. They may also be produced recombinantly using vectors and techniques described supra. However, it may be be most advantageous to produce peptides using the solid-phase synthetic techniques (Merrifield, 1963). Other peptide synthesis techniques are well known to those of skill in the art (Bodanszky et al, 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984). Appropriate protective groups for use in such syntheses will be found in the above texts, as well as in Protective Groups in Organic Chemistry, 1973.
  • the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group.
  • the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted with the residue already attached to the solid support.
  • the protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide.
  • the peptides of the invention are preferably devoid of benzylated or methylbenzylated amino acids.
  • Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.
  • non-standard amino acids Aside from the twenty standard amino acids can be used, there are a vast number of "non-standard” amino acids. Two of these can be specified by the genetic code, but are rather rare in proteins. Selenocysteine is incorporated into some proteins at a UGA codon, which is normally a stop codon. Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG. Examples of non-standard amino acids that are not found in proteins include lanthionine, 2- aminoisobutyric acid, dehydroalanine and the neurotransmitter gamma-aminobutyric acid.
  • Non-standard amino acids often occur as intermediates in the metabolic pathways for standard amino acids - for example ornithine and citrulline occur in the urea cycle, part of amino acid catabolism.
  • Non-standard amino acids are usually formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine, while hydroxyproline is made by a posttranslational modification of proline.
  • the present invention may utilize L-configuration amino acids, D-configuration amino acids, or a mixture thereof.
  • L-amino acids represent the vast majority of amino acids found in proteins
  • D-amino acids are found in some proteins produced by exotic sea- dwelling organisms, such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria. D-serine may act as a neurotransmitter in the brain.
  • the L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which that amino acid can theoretically be synthesized (D-glyceraldehyde is dextrorotary; L- glyceraldehyde is levorotary).
  • Retro-inverso modification of naturally occurring polypeptides involves the synthetic assemblage of amino acids with a- carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D-amino acids in reverse order with respect to the native peptide sequence.
  • a retro-inverso analogue thus has reversed termini and reversed direction of peptide bonds (NH-CO rather than CO- NH) while approximately maintaining the topology of the side chains as in the native peptide sequence. See U.S. Patent 6,261,569, incorporated herein by reference.
  • Peptides may advantageously be attached or fused to certain additional peptide segments for beneficial properties associated therewith.
  • such domains are cell delivery domains (also called a cell delivery vector, or cell transduction domain).
  • cell delivery domains also called a cell delivery vector, or cell transduction domain.
  • These types of domains are well known in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides.
  • poly-D-Arg and poly-D-Lys sequences e.g., dextrorotary residues, eight residues in length. Others are listed below in Table A. TABLE 0
  • Linkers or cross-linking agents may be used to fuse peptides to other proteinaceous sequences.
  • Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies.
  • Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites.
  • Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially.
  • the bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino, sulfhydryl, guanidino, indole, carboxyl specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied.
  • a majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group.
  • heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described in U.S. Patent 5,889, 155, specifically incorporated herein by reference in its entirety.
  • the cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example, of aldehydes to free thiols.
  • the cross-linking reagent can be modified to cross-link various functional groups and is thus useful for cross-linking polypeptides. In instances where a particular peptide does not contain a residue amenable for a given cross-linking reagent in its native sequence, conservative genetic or synthetic amino acid changes in the primary sequence can be utilized.
  • Peptides modified for in vivo use by the addition, at the amino- and/or carboxyl- terminal ends, of a blocking agent to facilitate survival of the peptide in vivo are contemplated. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake.
  • blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. These agents can be added either chemically during the synthesis of the peptide, or by recombinant DNA technology by methods familiar in the art. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues.
  • the inventor also contemplated that certain non-natural amino acids that satisfy the structural constraints of the inhibitory peptides of the present invention without a loss, and perhaps with an improvement in, biological function.
  • structurally similar compounds may be formulated to mimic the key portions of Bright/ ARTD3 a.
  • Such compounds which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and, hence, also are functional equivalents.
  • Certain mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993).
  • peptide mimetics The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and/or antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
  • a-helix mimetics are disclosed in U.S. Patents 5,446, 128; 5,710,245; 5,840,833; and 5,859, 184.
  • Methods for generating conformationally restricted ⁇ -turns and ⁇ -bulges are described, for example, in U.S. Patents 5,440,013; 5,618,914; and 5,670, 155.
  • Other types of mimetic turns include reverse and ⁇ -turns.
  • Reverse turn mimetics are disclosed in U.S. Patents 5,475,085 and 5,929,237, and ⁇ -turn mimetics are described in U.S. Patents 5,672,681 and 5,674,976.
  • molecular modeling is meant quantitative and/or qualitative analysis of the structure and function of protein- protein physical interaction based on three-dimensional structural information and protein-protein interaction models. This includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure-based constraint models. Molecular modeling typically is performed using a computer and may be further optimized using known methods. Computer programs that use X-ray crystallography data are particularly useful for designing such compounds. Programs such as RasMol, for example, can be used to generate three dimensional models.
  • INSIGHT Error Light
  • GRASP Alignment-Proty Nicholls
  • Dock Molecular Design Institute, University of California at San Francisco
  • Auto-Dock Unmanned Equipment Deformation
  • the methods can involve the additional step of outputting to an output device a model of the 3-D structure of the compound.
  • the 3-D data of candidate compounds can be compared to a computer database of, for example, 3-D structures.
  • Compounds of the invention also may be interactively designed from structural information of the compounds described herein using other structure-based design/modeling techniques (see, e.g., Jackson, 1997; Jones et ah, 1996). Candidate compounds can then be tested in standard assays familiar to those skilled in the art. Exemplary assays are described herein.
  • the 3-D structure of biological macromolecules can be determined from data obtained by a variety of methodologies. These methodologies, which have been applied most effectively to the assessment of the 3-D structure of proteins, include: (a) x-ray crystallography; (b) nuclear magnetic resonance (NMR) spectroscopy; (c) analysis of physical distance constraints formed between defined sites on a macromolecule, e.g., intramolecular chemical crosslinks between residues on a protein (e.g., PCT/USOO/14667, the disclosure of which is incorporated herein by reference in its entirety), and (d) molecular modeling methods based on a knowledge of the primary structure of a protein of interest, e.g., homology modeling techniques, threading algorithms, or ab initio structure modeling using computer programs such as MONSSTER (Modeling Of New Structures from Secondary and Tertiary Restraints) (see, e.g., International Application No.
  • MONSSTER Modeling Of New Structures from Secondary and Tertiary Restraints
  • Dominant-negative proteins are defective proteins with can negate the effects of normal, functional proteins when both are present in the same environment. In many cases, dominant-negative proteins homo-multimerize and are thus able to "poison" a complex that contains one or more functional proteins. Dominant-negative forms of Bright have been produced which act in just this manner. In designing dominant-negative Bright molecules, several regions present useful points for mutation. First, changes in the DNA binding domain (ARID) that block DNA binding produce dominant-negative effects. Second, alterations in the nuclear localization sequence which block nuclear translocation result in a dominant- negative form of Bright/ARID3a. Third, manipulation of the interaction and dimerization domains causes a dominant-negative function. Other dominant-negative proteins may be produced by interfering with the amino-terminal domain. Dominant-negative forms of Bright are described in Nixon et al. (2004a; 2004b).
  • kits are also within the scope of the invention.
  • kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, in particular, a Bright inhibitor.
  • the kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial end user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • kits according to the present invention contemplate the assemblage of agents for assessing ARID3a levels along with one or more of an SLE therapeutic and/or a reagent for assessing antinuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti-ENA), as well as controls for assessing the same.
  • ANA antinuclear antibody
  • anti-ENA anti-extractable nuclear antigen
  • RA rheumatoid aifhritis
  • DAS28 Disease activity index 28
  • Mononuclear cells were isolated from heparinized peripheral blood ( ⁇ 15 ml) with Ficoll-Paque Plus (GE Healthcare) and stained with the following fluorochrome-labeled antibodies: CD 19 PE-Cy5, CD24 APC, IL-10 PE, CD 10 Pacific Blue (BioLegend), IgD PerCP-Cy5.5, CD27 PE-Cy7, CD3 Pacific Blue (BD Biosciences), CD38 Alexa Fluor 700 (BD Pharmingen), and IgM APC (Southern Biotech).
  • Western Blots and ELISAs Western Blots and ELISAs.
  • Western blots for ARID3a were performed as previously described (17) using 20,000 peripheral blood mononuclear cells per lane, ⁇ -actin (Santa Cruz Biotechnology) was detected as a loading control.
  • Immunoglobulin levels were determined from serially diluted plasma samples using alkaline phosphatase-conjugated anti- human IgG and IgM (Southern Biotech) and a Biotech EL800 plate reader at 405/490nm with GEN5 Biotech software. Autoantibody testing was determined using the Reichlin method as described (18).
  • Levels of plasma TGF- ⁇ , IL-17, IL-10, TNFa and ⁇ ⁇ were detected using a Single- Analyte ELISArray Kit (QIAGEN) according to the manufacturer's directions.
  • a flow cytometry assay for intracellular ARID3a was developed, validated and used to assess expression in SLE patient B cells.
  • Mononuclear peripheral blood cells from SLE patients and healthy controls were gated for B (CD19 + ) or T (CD3 + ) lymphocyte surface markers (FIG. 1A).
  • healthy control samples contained very low numbers of B cells expressing intracellular ARID3a ( ⁇ 15%).
  • ARID3a expression in T cells was not above background in either control or SLE patients.
  • numbers of ARID3a + B cells were either greatly increased (50 of 1 15 patients) in a cross section of lupus patient samples, or were similar to healthy controls (e.g., FIG. 1A, SLE # 2 vs SLE#15).
  • Numbers of ARID3a + CD19 + B cells in ARID3a H patients ranged from 3- to > 40-fold higher than average numbers of ARID3a + cells in controls or ARID3a N patients.
  • RA patient samples resembled healthy controls and did not show increased numbers of ARID3a + B cells.
  • SLEDAI SLE Disease Activity Index
  • ARID3a mRNA transcripts were predominantly limited to pre-B, antibody-secreting and memory B cell subpopulations, but were not detectable in immature or naive mature B cells in healthy individuals (14). Because circulating B cell subpopulations in SLE patients may be skewed compared to healthy populations (20), the inventor examined the possibility that increased numbers of ARID3a cells might reflect disproportionate numbers of memory B or plasma cells in the ARID3a H versus the ARID3a N samples. B cell numbers were quantified within eight different B cell subpopulations (gated as in FIG. 6) from ARID3a N and ARID3a H samples (FIG. 7) by flow cytometry.
  • ARID3a H samples FIG.
  • ARID3a + B cells occurred in both naive and transitional subsets (FIGS. 8A-B), indicating that increased numbers of ARID3a + transitional B cells within the IgM + IgD + CD27 " subset did not account for increases in ARID3a + naive B cells in ARID3a H patient samples.
  • ARID3a H and ARID3a N subsets were divided into ARID3a H and ARID3a N subsets based on analyses of CD19 + ARID3a + B cell numbers during their first visit and ARID3a + B cell numbers were plotted for subsequent visits (FIG. 3).
  • Ten of 15 ARID3a H patients showed normal numbers of ARID3a + cells on at least one visit, and several of those were designated ARID3a N on both of their subsequent visits.
  • Eight of 22 patients with normal numbers of ARID3a + B cells at their first visit showed increased numbers of ARID3a + cells at a single subsequent visit. Percentages of ARID3a + cells within individual B cell subsets also varied over time in all individuals (not shown).
  • ARID3a H or ARID3a N SLE phenotype Table 4 - Relationship between ARID3a + cells with SLEDAI for each B cell subset
  • ARID3a CD19 0.06624 0.02351 0.0039 **
  • ARID3a Plasmablasts 5.32660 2.64755 0.0474 *
  • ARID3a Naive 0.15942 0.09129 0.0862
  • ARID3a DN Memory 1.34904 0.79118 0.0990
  • ARID3a C delta Class-switched 1.18269 0.80329 0.1232
  • ARID3a H samples had increased plasma IgM and IgG levels compared to healthy control and ARID3a N samples (FIG. 10A), and there was little difference in reactivity of the plasma with a large number of common autoantigens (FIG. 10B). These data suggest that increased numbers of total ARID3a + peripheral blood B cells do not correlate with autoantibody reactivity patterns.
  • the inventor explored expression of the activation marker CD86 and the pre-plasmablast marker CD43 (22-24). Naive B cells are enriched in the CD27- subset which had larger numbers of ARID3a cells in many SLE patients, as shown in FIG. 2B. Therefore, the inventor examined ARID3a expression in both CD27 " CD86 + and CD27 " CD43 + B cell subpopulations of SLE, healthy control and RA patient samples (FIG. 4A). As expected, a portion of activated CD27 " B cells expressed ARID3a ( ⁇ 30%), with larger percentages occurring in both SLE and RA patient samples compared to healthy controls. However, ARID3a + cells did not segregate with these markers.
  • ARID3a + peripheral blood B lymphocytes revealed increased numbers of ARID3a + peripheral blood B lymphocytes in 43% of lupus patients.
  • ARID3a expression was not limited to B cell differentiation stages that express ARID3a in healthy individuals, but occurred in all patient B cell subsets examined.
  • Total numbers of ARID3a + B cells in 44 patients assessed at multiple time points varied over time, and showed a strong association with SLEDAI scores at each visit.
  • Increased disease activity correlated with total numbers of ARID3a + B cells and with ARID3a + marginal zone memory and plasmablast/plasma cells.
  • ARID3a/Bright enhances transcription of some Ig heavy chains (17), recent data suggest it can also repress transcription of other genes (26,27). Many ARID family proteins are members of large epigenetic regulatory complexes (28), although it is not clear if ARID3a serves similar types of functions in B lymphocytes. Perhaps the most surprising finding was that ARID3a H patient samples displayed bimodal ARID3a expression in most B cell subsets. Because the inventor's data show strong associations between numbers of ARID3a B cells and disease activity, it will be important to determine if ARID3a expression alters gene expression profiles in those cells that express ARID3a versus those that do not.
  • Increased plasma IL-10 levels have been associated with inflammatory responses.
  • ARID3a + cells clearly expressed the activation marker CD86 ARID3a expression did not segregate with expression of this marker (FIGS. 4A-B), an activation marker previously shown to be increased in SLE B lymphocytes (22).
  • ARID3a expression of ARID3a in SLE naive B lymphocytes that only rarely show detectable ARID3a expression in healthy controls was unexpected.
  • B 1 cells in the mouse may constitute a distinct B cell lineage (29).
  • Mouse BI B cells constitutively express Bright/ARID3a (21), and these cells were missing in Bright knockout mice (30).
  • ARID3a expression in SLE B cells may be a consequence, or a contributing factor of SLE.
  • >40% of SLE patient samples showed large numbers of ARID3a + B cells, and increased numbers of these cells were associated with disease activity, RA blood samples did not exhibit expansion of ARID3a + cells.
  • the inventor cannot rule out the possibility that examination of larger numbers of RA samples might show expansion of ARID3a + cells.
  • Lupus is a polygenic disease, which is thought to arise due to imbalances at multiple regulatory levels of tolerance or immune activity.
  • ARID3a expression in several mature B cell subsets was strongly associated with increased disease activity (Table 4), many patients also had dramatically increased numbers of ARID3a + naive B cells that are likely precursors to those mature cells. The inventor cannot conclude from the study that numbers of ARID3a + naive B cells might be predictive of future disease flares, but it will be important to further explore that idea.
  • the current results showing strong associations with increased numbers of ARID3a + B cells and SLEDAI scores suggest ARID3a may either be useful as a novel biomarker for disease activity or perhaps eventually be targeted for preventative treatment.
  • Bright/ARID3a acts as a barrier to somatic reprogramming through direct regulation of Oct4, Sox2 and Nanog. Stem Cell Reports 2, 26-35. 2014. (28) Kortschak RD, Tucker PW, Saint R. ARID proteins come in from the desert. Trends Biochem Sci 2000; 25(6):294-9.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Rehabilitation Therapy (AREA)
  • Biotechnology (AREA)
  • Rheumatology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne l'identification de taux élevés de Bright/ARID3a dans les lymphocytes B de patients atteints de lupus érythémateux disséminé en tant que marqueur pour empêcher une poussée de la maladie et atténuer la gravité de la maladie. Des méthodes pour traiter des patients ainsi identifiés sont également décrites.
PCT/US2014/036881 2013-05-29 2014-05-06 Bright/arid3a fonction/expression en tant que marqueur indiquant la gravité et l'intensité du lupus érythémateux disséminé Ceased WO2014193611A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361828473P 2013-05-29 2013-05-29
US61/828,473 2013-05-29

Publications (2)

Publication Number Publication Date
WO2014193611A1 true WO2014193611A1 (fr) 2014-12-04
WO2014193611A8 WO2014193611A8 (fr) 2014-12-31

Family

ID=51989312

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/036881 Ceased WO2014193611A1 (fr) 2013-05-29 2014-05-06 Bright/arid3a fonction/expression en tant que marqueur indiquant la gravité et l'intensité du lupus érythémateux disséminé

Country Status (1)

Country Link
WO (1) WO2014193611A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018013912A1 (fr) * 2016-07-15 2018-01-18 The Board Of Regents Of The University Of Oklahoma Traitements anti-arid3a pour des troubles inflammatoires
CN113196059A (zh) * 2018-10-18 2021-07-30 俄克拉荷马医学研究基金会 表征疾病活动性的系统性红斑狼疮(sle)疾病活动性免疫指数的生物标志物
WO2024026407A1 (fr) * 2022-07-28 2024-02-01 The Board Of Regents Of The University Of Oklahoma Biomarqueurs, méthodes de diagnostic, traitements et agents thérapeutiques pour maladies et troubles auto-immuns

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050271651A1 (en) * 2004-01-23 2005-12-08 Oklahoma Medical Research Foundation Inhibition of bright function as a treatment for excessive immunoglobulin production
US20090169559A1 (en) * 2003-01-14 2009-07-02 Teva Pharmaceutical Industries, Ltd. Parenteral formulations of peptides for the treatment of systemic lupus erythematosus
WO2012109592A1 (fr) * 2011-02-11 2012-08-16 Exagen Diagnostics, Inc. Procédés de diagnostic de lupus érythémateux disséminé
US20120225790A1 (en) * 2009-06-24 2012-09-06 Fundacio Institut De Recerca De L'hospital Universitari Vall D'hebron Vitro method for the prognosis or prediction of the response in patients with rheumatoid arthritis treated with agents that recognize the cd20 membrane receptor in b lymphocytes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090169559A1 (en) * 2003-01-14 2009-07-02 Teva Pharmaceutical Industries, Ltd. Parenteral formulations of peptides for the treatment of systemic lupus erythematosus
US20050271651A1 (en) * 2004-01-23 2005-12-08 Oklahoma Medical Research Foundation Inhibition of bright function as a treatment for excessive immunoglobulin production
US20120225790A1 (en) * 2009-06-24 2012-09-06 Fundacio Institut De Recerca De L'hospital Universitari Vall D'hebron Vitro method for the prognosis or prediction of the response in patients with rheumatoid arthritis treated with agents that recognize the cd20 membrane receptor in b lymphocytes
WO2012109592A1 (fr) * 2011-02-11 2012-08-16 Exagen Diagnostics, Inc. Procédés de diagnostic de lupus érythémateux disséminé

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHANKAR ET AL.: "Anti-Nuclear Antibody Production and Autoimmunity in Transgenic Mice That Overexpress the Transcription Factor Bright", THE JOURNAL OF IMMUNOLOGY, vol. 178, 1 March 2007 (2007-03-01), pages 2996 - 3006, XP002536717 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018013912A1 (fr) * 2016-07-15 2018-01-18 The Board Of Regents Of The University Of Oklahoma Traitements anti-arid3a pour des troubles inflammatoires
US10876116B2 (en) 2016-07-15 2020-12-29 The Board Of Regents Of The University Of Oklahoma Anti-ARID3a treatments for inflammatory disorders
CN113196059A (zh) * 2018-10-18 2021-07-30 俄克拉荷马医学研究基金会 表征疾病活动性的系统性红斑狼疮(sle)疾病活动性免疫指数的生物标志物
WO2024026407A1 (fr) * 2022-07-28 2024-02-01 The Board Of Regents Of The University Of Oklahoma Biomarqueurs, méthodes de diagnostic, traitements et agents thérapeutiques pour maladies et troubles auto-immuns

Also Published As

Publication number Publication date
WO2014193611A8 (fr) 2014-12-31

Similar Documents

Publication Publication Date Title
US7595159B2 (en) Prediction of Parkinson's disease using gene expression levels of peripheral blood samples
EP3183582B1 (fr) Biomarqueurs pour le diagnostic et le suivi de maladies neuro-immunologiques
US20160368995A1 (en) Identification of cxcr8, a novel chemokine receptor
US12140592B2 (en) Biomarkers for assessing risk of transitioning to systemic lupus erythematosus classification and disease pathogenesis
EP3250599B1 (fr) Biomarqueur
KR102742415B1 (ko) 척수성 근위축증의 치료 방법
EP3110976B1 (fr) Procédé d'évaluation du risque de lemp
AU2018392780B2 (en) Biomarkers of methotrexate-induced immune tolerance
US20170108514A1 (en) Diagnosis of multiple sclerosis in human and animal subjects
US20140037618A1 (en) Method of treating autoimmune inflammatory disorders using il-23r loss-of-function mutants
Goodier et al. C9orf72-associated SMCR8 protein binds in the ubiquitin pathway and with proteins linked with neurological disease
Clarke et al. Autoantibody repertoire characterization provides insight into the pathogenesis of monogenic and polygenic autoimmune diseases
WO2014193611A1 (fr) Bright/arid3a fonction/expression en tant que marqueur indiquant la gravité et l'intensité du lupus érythémateux disséminé
Han et al. Dysbindin-associated proteome in the p2 synaptosome fraction of mouse brain
US20180313830A1 (en) Peptide and biomarker associated with inflammatory disorders, and uses thereof
TWI465720B (zh) 預測神經發育、神經的或神經精神性病症治療之臨床效益之方法
WO2021138273A2 (fr) Auto-anticorps en tant que biomarqueurs pour le syndrome polyglandulaire auto-immun de type 1
US9945843B2 (en) Methods for identifying compounds that inhibit G protein-coupled receptor (GPR84) agonist-stimulated chemotaxis
WO2020167715A1 (fr) Biomarqueurs de la leucoencéphalopathie multifocale progressive
David TLR mediated inflammatory disease and other type I interferonopathies
US10288619B2 (en) Biomarkers for human monocyte myeloid-derived suppresor cells
WO2014113291A1 (fr) Procédé pour identifier des patients susceptibles de répondre à une thérapie anti-tnf
US20250354998A1 (en) Methods of diagnosing and treating multiple sclerosis by detecting a biomarker in the cerebrospinal fluid
US20150191786A1 (en) Genetic Polymorphisms Associated With Advanced Lung Diseases
Leon et al. Fatigue in Sjögren’s syndrome: a search for

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14803914

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14803914

Country of ref document: EP

Kind code of ref document: A1