WO2010141999A1 - Agents et méthodes de diagnostic et de traitement de la spondylarthrite ankylosante - Google Patents

Agents et méthodes de diagnostic et de traitement de la spondylarthrite ankylosante Download PDF

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WO2010141999A1
WO2010141999A1 PCT/AU2010/000729 AU2010000729W WO2010141999A1 WO 2010141999 A1 WO2010141999 A1 WO 2010141999A1 AU 2010000729 W AU2010000729 W AU 2010000729W WO 2010141999 A1 WO2010141999 A1 WO 2010141999A1
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polynucleotide
nr4a2
rora
risk
development
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PCT/AU2010/000729
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English (en)
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Matthew Arthur Brown
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The University Of Queensland
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Priority claimed from AU2009902698A external-priority patent/AU2009902698A0/en
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Publication of WO2010141999A1 publication Critical patent/WO2010141999A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates generally to methods and agents for diagnosing the presence or risk of development of ankylosing spondylitis (AS). More particularly, the present invention relates to the use of Nuclear Receptor subfamily 4, group A, member 2 (NR4A2), Tumour Necrosis Factor, Alpha-Induced Protein 3 (TNFAIP3), RAR-related orphan receptor A (RORA) and CD69 as diagnostic markers of AS.
  • the invention has practical use in early diagnosis of AS, and in enabling better treatment and management decisions to be made in clinically and sub-clinically affected subjects.
  • AS affects 1-9 per 1000 Caucasian individuals, making it one of the most common causes of inflammatory arthritis (Van der Linden, S. et al., 1983, Br J Rheumatol, 22: 18-19 and; Braun, J. et al, ⁇ 99S, Arthritis Rheum, 41: 58-67).
  • the condition principally affects the axial skeleton including the spine and sacroiliac joints, causing pain, stiffness, and eventually bony ankylosis.
  • Peripheral joints and tendon insertions (entheses) are commonly affected, and approximately one-third of patients develop acute anterior uveitis.
  • HLA-B27 human leukocyte antigen
  • NR4A2, TNFAIP3, RORA and CD69 are useful in diagnosing the presence or risk of development of AS.
  • the present inventors have determined that NR4A2, TNFAIP3, RORA and CD69 are down regulated in subjects with AS, as compared to their expression in subjects lacking AS.
  • the inventors have reduced these discoveries to practice in novel diagnostic assays that detect aberrant expression of NR4A2,
  • TNFA1P3, RORA and CD69 for diagnosing the presence or risk of development of AS.
  • the present invention represents a significant advance over current technologies by providing better surrogate markers of AS, as well as facile methods of diagnosing or detecting the presence or risk of developing AS.
  • the present invention discloses methods of diagnosing the presence or risk of development of AS in a subject through detecting gene expression.
  • Advantageous embodiments involve monitoring the expression of certain genes in peripheral leukocytes of the immune system, which may be reflected in changing patterns of RNA levels or protein production that correlate with the presence or risk of development of AS. As such, these methods are suitable for widespread screening of symptomatic and asymptomatic subjects.
  • the present invention provides methods for diagnosing the presence or risk of development of AS in a subject. These methods generally comprise detecting in the subject aberrant expression of an AS marker gene selected from NR4A2, TNFAIP3, RORA and CD69, which indicates the presence or risk of development of AS. Generally, the subject is one that is suspected of having AS or at risk of having AS.
  • the methods comprise detecting aberrant expression of an AS marker polynucleotide.
  • AS marker polynucleotides can be selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 17-23 or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence similarity with at least a portion of the
  • the methods comprise detecting aberrant expression of an AS marker polynucleotide(s) selected from the group consisting of; (a) a polynucleotide comprising a nucleotide sequence that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence identity with the sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 1 1, 13 and 17-23 or a complement thereof; (b) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14; (c) a polynucleotide comprising a nucleotide sequence that encodes a polypeptide that shares at least 80% (and at least 81% to at least 99% and all integer percentages in between) sequence similarity with at least a portion of the sequence set forth in SEQ ID NO: 2, 4,
  • the methods comprise detecting aberrant expression of a NR4A2 polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a TNFAIP3 polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a CD69 polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a RORA polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a NR4A2 polynucleotide and a TNFAIP3 polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a NR4A2 polynucleotide and a CD69 polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a NR4A2 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a TNFAIP 3 polynucleotide and a CD69 polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a TNFAlP 3 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a NR4A2 polynucleotide, a TNFAIP 3 polynucleotide and a CD69 polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a NR4A2 polynucleotide, a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a TNFAIP 3 polynucleotide, a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.
  • the methods comprise detecting aberrant expression of a NR4A2 polynucleotide, a TNFAIP3 polynucleotide, a CD69 polynucleotide and a RORA polynucleotide, which indicates the presence or risk of development of AS.
  • such aberrant expression is detected by: (1) providing a biological sample from the subject; (2) measuring in the biological sample the level or functional activity of at least one AS marker expression product; and (3) comparing the measured level or functional activity of the or each expression product to the level or functional activity of a corresponding expression product in a reference sample obtained from one or more subjects lacking AS, wherein a lower level or functional activity of the or each expression product in the biological sample as compared to the level or functional activity of the corresponding expression product in the reference sample is indicative of the presence or risk of development of AS in the subject.
  • the methods further comprise diagnosing the presence of AS in the subject when the measured level or functional activity of the, or each expression product is lower than the measured level or functional activity of the corresponding expression product.
  • the lower expression typically represents an at least about 9/10, 4/5, 7/10, 3/5, 1/2, 2/5, 3/10, 1/5, 1/10, 1/20, 1/50, 10 "2 , 10 "3 , 10 “4 , 10 “5 , 10 “6 , 10 '7 , 10- 8 , 10 "9 , 10 "10 , 10 ' “, 10 '12 , 10 “13 , 10 "14 , 10 "15 , 10- 16 , 10 "17 , 10 “18 , 10 “19 or 10 "20 of the level or functional activity of an individual corresponding expression product, which is referred to as "aberrant expression.”
  • the methods further comprise diagnosing the absence of AS when the measured level or functional activity of the or each expression product is the same as or similar to the measured level or functional activity of the corresponding expression product.
  • the measured level or functional activity of an individual expression product varies from the measured level or functional activity of an individual corresponding expression product by no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1 %, which is hereafter referred to as "normal expression.”
  • the biological sample comprises tissues, cells or cell lines, bodily fluids, blood or peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the expression product is selected from a RNA molecule or a polypeptide.
  • the expression product is the same as the corresponding expression product.
  • the expression product is a variant (e.g., an allelic variant) of the corresponding expression product.
  • the expression product or corresponding expression product is a target RNA (e.g., mRNA) or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under at least medium or high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 15 contiguous nucleotides of a polynucleotide.
  • the measured level or abundance of the target RNA or its DNA copy is normalized to the level or abundance of a reference RNA or a DNA copy of the reference RNA that is present in the same sample.
  • the nucleic acid probe is immobilized on a solid or semi-solid support.
  • the nucleic acid probe forms part of a spatial array of nucleic acid probes.
  • the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by hybridization (e.g., using a nucleic acid array).
  • the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nucleic acid amplification (e.g., using a polymerase chain reaction (PCR)).
  • PCR polymerase chain reaction
  • the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nuclease protection assay.
  • the expression product or corresponding expression product is an AS marker polypeptide whose level is measured using at least one antigen-binding molecule that is immuno-interactive with the AS marker polypeptide.
  • the measured level of the AS marker polypeptide is normalized to the level of a reference AS marker polypeptide that is present in the same sample.
  • the antigen-binding molecule is immobilized on a solid or semi-solid support.
  • the antigen-binding molecule forms part of a spatial array of antigen-binding molecules.
  • the level of antigen-binding molecule that is bound to the target polypeptide is measured by immunoassay (e.g., using an ELISA or RIA).
  • the diagnostic methods of the present invention further comprise detecting expression of at least one other AS marker, illustrative examples of which include HLA-B27.
  • the present invention provides methods for treating or inhibiting the development or progression of AS in a subject. These methods generally comprise detecting aberrant expression of at least one AS marker expression product in the subject, and administering to the subject at least one therapy that treats or ameliorates the symptoms or reverses or inhibits the development or progression of AS in the subject.
  • Representative examples of such therapies include nonsteroidal antiinflammatory drugs (NSAIDS) examples of which include Sulfasalazine (Azulfidine), medication, exercise, physical therapy and surgery.
  • the present invention provides probes for interrogating nucleic acid for the presence of an AS marker polynucleotide as described for example above for use in the diagnostic methods of the present invention.
  • These probes generally comprise a nucleotide sequence that hybridizes under at least medium or high stringency conditions to a NR4A2, TNFAIP3, RORA or CD69 polynucleotide.
  • the probes consist essentially of a nucleic acid sequence which corresponds or is complementary to at least a portion of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, wherein the portion is at least 15 nucleotides in length.
  • the probes comprise a nucleotide sequence which is capable of hybridizing to at least a portion of a nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14 under at least medium or high stringency conditions, wherein the portion is at least 15 nucleotides in length.
  • the probes comprise a nucleotide sequence that is capable of hybridizing to at least a portion of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 17-23 under at least medium or high stringency conditions, wherein the portion is at least 15 nucleotides in length.
  • the invention provides a solid or semi-solid support for use with the diagnostic methods of the present invention, wherein the solid or semisolid support comprises at least one nucleic acid probe as broadly described above immobilized thereon.
  • the solid or semi-solid support comprises a spatial array of nucleic acid probes immobilized thereon.
  • Still a further aspect of the present invention provides an antigen- binding molecule that is immuno-interactive with an AS marker polypeptide as described for example above for use in the diagnostic methods of the present invention.
  • the invention provides a solid or semi-solid support comprising at least one antigen-binding molecule as broadly described above immobilized thereon.
  • the solid or semi-solid support comprises a spatial array of antigen-binding molecules immobilized thereon.
  • Still another aspect of the invention provides the use of one or more
  • AS marker polynucleotides as described for example above, or the use of one or more probes as broadly described above, or the use of one or more AS marker polypeptides as described for example above, or the use of one or more antigen-binding molecules as broadly described above, in the manufacture of a kit for diagnosing the presence of AS in a subject.
  • Figure 1 is a graphical representation of unsupervised hierarchical clustering of 34 samples based on expression levels detected in the microarray experiment described in the Examples using 17,308 probes flagged as present. Samples are clustered on the horizontal axis with the vertical axis representing the degree of correlation between samples. The lengths of the branches are indicative of the similarities between samples or genes.
  • Figure 2 is a graphical representation of unsupervised hierarchical clustering of 34 samples based on expression levels detected in the microarray experiment using the 485 probes differentially expressed between AS and control samples.
  • AS samples are in red and control samples are blue.
  • Samples are clustered on the horizontal axis and genes clustered on the vertical axis.
  • the lengths of the branches in the dendrograms represent the degrees of correlation between samples or genesets. For expression levels, yellow represents overexpressed genes and red underexpressed genes.
  • Figure 3 is a graphical representation showing quantitative RT-PCR analysis of NR4A2, TNFAIP3, RORA and CD69 expression in subjects without AS (control) and patients with AS.
  • Figure 4 is a graphical representation illustrating a Receiver Operator Curve(ROC) showing predictive power of the identified gene sets.
  • Graph (A) illustrates a ROC curve generated from array expression values for the 452 genes significantly different between AS and control samples.
  • Graph (B) illustrates ROC curves generated from the combined quantitative RT-PCR data for NR4A2, TNFAIP3 and CD69 from the discovery and confirmation sample sets.
  • Area under curve (AUC) represents overall ability of the test to discriminate between those individuals with the disease and those without the disease.
  • Inset in (B) are the AUC values from the ROC curves for the individual candidate genes.
  • NR4A2, TNFAIP3, RORA or CD69 refers to the down regulation of at least one NR4A2, TNFAIP3, RORA or CD69 gene relative to a 'normal' level of expression of at least one NR4A2, TNFAIP 3, RORA or CD69 gene or allelic variant thereof in healthy or normal cells or in cells obtained from a subject lacking AS, and/or to a level of at least one NR4A2, TNFAIP 3, RORA or CD69 gene product (e.g., transcript or polypeptide) in a healthy tissue sample or body fluid or in a tissue sample or body fluid obtained from at least one subject lacking AS.
  • TNFAIP 3, RORA or CD69 gene product e.g., transcript or polypeptide
  • a gene of the present invention is aberrantly- or under-expressed if its level of expression is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or 0.0001% of the level of expression of the corresponding gene in healthy or normal cells or in cells obtained from a subject without AS, or in a healthy tissue sample or body fluid or in a tissue sample or body fluid obtained from a healthy subject or from a subject lacking AS.
  • amplicon refers to a target sequence for amplification, and/or the amplification products of a target sequence for amplification. In certain other embodiments an “amplicon” may include the sequence of probes or primers used in amplification.
  • amplicon may include the sequence of probes or primers used in amplification.
  • the term "ankylosing spondylitis (AS)" as used herein includes subjects suffering from undifferentiated spondylarthritis (USpA).
  • a subject is generally considered as suffering from USpA if he/she fulfils the criteria for spondylitis as defined in the European Spondyloarthropathy Study Group (ESSG) classification, the Amor criteria or the ASAS Criteria.
  • ESSG classification includes inflammatory spinal pain or synovitis (asymmetrical, predominantly in lower limbs) and any one or more of the following: positive family history, psoriasis, inflammatory bowel disease, alternate buttock pain and enthesopathy.
  • Amor criteria includes clinical symptoms or past history of any one or more of: lumbar or dorsal pain at night or lumbar or dorsal morning stiffness; asymmetrical oligoarthritis; buttock pain; sausage-like finger or toe; heel pain; ulceris; non-gonococcal urethritis or cervicitis accompanying, or within 1 month before, the onset of arthritis; acute diarrhea accompanying, or within 1 month before, the onset of arthritis; presence of history of psoriasis and/or balanitis and/or of inflammatory bowel disease (ulcerative colitis, Crohn's disease). Radiological findings; Sacroiliitis (grade >2 if bilateral, grade >3 if unilateral).
  • Amor criteria may further include genetic background; presence of HLA-B27 and/or family history of ankylosing spondylitis, reactive arthritis, uveitis, psoriasis or chronic inflammatory bowel disease. Amor criteria may further include response to therapy; definite improvement of musculoskeletal complaints with non-steroidal anti-inflammatory drugs (NSAIDs) in less than 48 h or relapse of the pain in less than 48 h if NSAIDs discontinued.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • the ASAS criteria comprises axial spondyloarthritis as either sacroiliitis on magnetic resonance imaging (MRI) or on x-ray, plus one or more clinical feature of spondyloarthritis, or carriage of HLA-B27 plus two or more clinical features of spondyloarthritis.
  • MRI magnetic resonance imaging
  • HLA-B27 carriage of HLA-B27 plus two or more clinical features of spondyloarthritis.
  • Clinical features of spondyloarthritis may include presence of inflammatory back pain, arthritis, enthesitis of the heel, uveitis, dactylitis, psoriasis, Crohn's disease or ulcerative colitis, marked reduction in back pain with 24-48 hours of a full dose of a non-steroidal anti-inflammatory drug, a family history of spondyloarthritis, carriage of HLA-B27, or an elevated c-reactive protein level.
  • AS is classified by the modified New York Criteria (1984) which includes any one or more of: low back pain and stiffness for more than 3 months, which improves with exercise, but is not relieved by rest; limitation of motion of the lumbar spine in both the sagittal and frontal planes; limitation of chest expansion relative to normal values correlated for age and sex.
  • AS may further include radiological criterion Sacroiliitis grade 2 bilaterally or grade 3—4 unilaterally. AS is generally present if the radiological criterion is associated with at least one clinical criterion.
  • antigen-binding molecule a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
  • immuno-interactive and the like when referring to an antigen-binding molecule refers to a binding reaction which is determinative of the presence of an antigen in the presence of a heterogeneous population of proteins and other biologies.
  • the specified antigen-binding molecules bind to a particular antigen and do not bind in a significant amount to other proteins or antigens present in the sample.
  • Specific binding to an antigen under such conditions may require an antigen-binding molecule that is selected for its specificity for a particular antigen.
  • antigen-binding molecules can be raised to a selected protein antigen, which bind to that antigen but not to other proteins present in a sample.
  • a variety of immunoassay formats may be used to select antigen-binding molecules specifically immuno-interactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immuno-interactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • biological sample refers to a sample that may be extracted, untreated, treated, diluted or concentrated from an animal.
  • the biological sample may include a biological fluid such as peripheral blood and the like.
  • the biological sample comprises cells from a tissue biopsy.
  • Cells and the like are terms that not only refer to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • complementarity refers to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • a polynucleotide having a nucleotide sequence that is substantially identical (e.g., at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity) to all or a portion of a reference polynucleotide sequence or of a complement thereof.
  • an effective amount in the context of treating or preventing a condition is meant the administration of that amount of active to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition.
  • the effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • the terms "expression” or “gene expression” refer to either production of RNA message or translation of RNA message into proteins or polypeptides. Detection of either types of gene expression in use of any of the methods described herein are part of the invention.
  • the term “functional activity” generally refers to the ability of a molecule (e.g., a transcript or polypeptide) to perform its designated function including a biological, enzymatic, or therapeutic function. In certain embodiments, the functional activity of a molecule corresponds to its specific activity as determined by any suitable assay known in the art.
  • the term "gene” as used herein refers to any and all discrete coding regions of a host genome, or regions that code for a functional RNA only (e.g., tRNA, rRNA, regulatory RNAs such as ribozymes, post-transcription gene silencing- (PTGS) associated RNAs etc) as well as associated non-coding regions and optionally regulatory regions.
  • a functional RNA e.g., tRNA, rRNA, regulatory RNAs such as ribozymes, post-transcription gene silencing- (PTGS) associated RNAs etc
  • PTGS post-transcription gene silencing-
  • the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • the gene sequences may be cDNA or genomic DNA or a fragment thereof.
  • the gene may be introduced into an appropriate vector for extra-chromosomal maintenance or for integration into the host.
  • hybridization is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.
  • Complementary base sequences are those sequences that are related by the base- pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G.
  • match and “mismatch” as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands.
  • Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently.
  • the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds.
  • nucleobases complementary nucleoside or nucleotide bases
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Hybridization can occur under varying circumstances as known to those of skill in the art.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • immuno-interactive includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
  • isolated is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an
  • isolated polynucleotide refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.
  • a "naturally-occurring" nucleic acid molecule refers to a RNA or DNA molecule having a nucleotide sequence that occurs in nature.
  • a naturally-occurring nucleic acid molecule can encode a protein that occurs in nature.
  • a sample such as, for example, a nucleic acid extract is isolated from, or derived from, a particular source.
  • the extract may be isolated directly from a biological tissue or fluid of the subject.
  • oligonucleotide refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof, including nucleotides with modified or substituted sugar groups and the like) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof).
  • oligonucleotide typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally-occurring
  • the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphorothioate, phosphorodithioate, phophoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like.
  • PNAs peptide nucleic acids
  • phosphorothioate phosphorodithioate
  • phophoroselenoate phosphorodiselenoate
  • phosphoroanilothioate phosphoraniladate
  • phosphoroamidate methyl phosphonates
  • 2-O-methyl ribonucleic acids 2-O-methyl rib
  • Oligonucleotides are a polynucleotide subset with 200 bases or fewer in length. Preferably, oligonucleotides are 10 to 60 bases in length and most preferably 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the construction of a variant nucleic acid sequence. Oligonucleotides of the invention can be either sense or antisense oligonucleotides.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages ⁇ e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages ⁇ e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties ⁇ e.g., polypeptides), intercalators ⁇ e.g., acridine, psoralen, etc.), chelators, alkylators and modified linkages ⁇ e.g., ⁇ -anomeric nucleic acids, etc.).
  • internucleotide modifications such as uncharged linkages ⁇ e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages ⁇ e.g., phospho
  • RNA forms of the genetic molecules of the present invention are generally mRNA or iRNA including siRNAs.
  • the genetic form may be in isolated form or integrated with other genetic molecules such as vector molecules and particularly expression vector molecules.
  • nucleotide sequence used herein interchangeably and encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.
  • polynucleotide variant and “variant” refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide.
  • polynucleotide variant and “variant” also include naturally-occurring allelic variants.
  • Polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • polypeptide variant refers to polypeptides which are distinguished from a reference polypeptide by the addition, deletion or substitution of at least one amino acid residue.
  • one or more amino acid residues of a reference polypeptide are replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter.
  • primer an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent.
  • the primer is typically single-stranded for maximum efficiency in amplification but may alternatively be double-stranded.
  • a primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15 to 35 or more nucleotides, although it may contain fewer nucleotides.
  • Primers can be large polynucleotides, such as from about 200 nucleotides to several kilobases or more. Primers may be selected to be “substantially complementary” to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By “substantially complementary,” it is meant that the primer is sufficiently complementary to hybridize with a target nucleotide sequence. Suitably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-complementary nucleotides may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template.
  • non-complementary nucleotides or a stretch of non- complementary nucleotides can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.
  • Probe refers to a molecule that binds to a specific sequence or sub- sequence or other moiety of another molecule. Unless otherwise indicated, the term “probe” typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly and include primers within their scope. As used herein, the term “probe” encompasses primers which can be used for example in template- dependent nucleic acid extension, ligation or amplification reactions.
  • promoter is meant a region of DNA, which controls at least in part the initiation and level of transcription.
  • Reference herein to a “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including a TATA box and CCAAT box sequences, as well as additional regulatory elements (i. e. , activating sequences, enhancers and silencers) that alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner.
  • a promoter is usually, but not necessarily, positioned upstream or 5', of a transcribable sequence (e.g., a coding sequence or a sequence encoding a functional RNA), the expression of which it regulates.
  • the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. Promoters according to the invention may contain additional specific regulatory elements, located more distal to the start site to further enhance expression in a cell, and/or to alter the timing or inducibility of expression of a structural gene to which it is operably connected.
  • promoter also includes within its scope inducible, repressible and constitutive promoters as well as minimal promoters.
  • Minimal promoters typically refer to minimal expression control elements that are capable of initiating transcription of a selected DNA sequence to which they are operably linked.
  • a minimal promoter is not capable of initiating transcription in the absence of additional regulatory elements (e.g., enhancers or other cis-acting regulatory elements) above basal levels.
  • a minimal promoter frequently consists of a TATA box or TATA-like box.
  • Numerous minimal promoter sequences are known in the literature.
  • minimal promoters may be selected from a wide variety of known sequences, including promoter regions from fos, CMV, SV40 and IL-2, among many others. Illustrative examples are provided which use a minimal CMV promoter or a minimal IL2 gene promoter (-72 to +45 with respect to the start site; Siebenlist, 1986).
  • recombinant polynucleotide refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature.
  • the recombinant polynucleotide may be in the form of an expression vector.
  • expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.
  • recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i. e. , the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity will be understood to mean the "match percentage” calculated by an appropriate method.
  • sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.
  • similarity refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table A infra. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12, 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.
  • references to describe sequence relationships between two or more polynucleotides include “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides, in length.
  • two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0,
  • Stringent conditions refers to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably having exact complementarity, will hybridize.
  • the stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization, and is greatly changed when nucleotide analogues are used.
  • stringent conditions are selected to be about 10° C to 20° C less than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe.
  • subject or “individual” or “patient”, used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian male subject, for whom therapy or prophylaxis is desired.
  • a preferred subject is a human in need of diagnosis of the presence or absence of AS. However, it will be understood that the aforementioned terms do not imply that symptoms are present.
  • template refers to a nucleic acid that is used in the creation of a complementary nucleic acid strand to the "template” strand.
  • the template may be either RNA and/or DNA, and the complementary strand may also be RNA and/or DNA.
  • the complementary strand may comprise all or part of the complementary sequence to the "template,” and/or may include mutations so that it is not an exact, complementary strand to the "template”. Strands that are not exactly complementary to the template strand may hybridize specifically to the template strand in detection assays described here, as well as other assays known in the art, and such complementary strands that can be used in detection assays are part of the invention.
  • transcribable polynucleotides or "transcribed nucleic acid sequence” excludes the non-transcribed regulatory sequence that drives transcription.
  • the transcribable sequence may be derived in whole or in part from any source known to the art, including an animal, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesised DNA.
  • a transcribable sequence may contain one or more modifications in either the coding or the untranslated regions, which could affect the biological activity or the chemical structure of the expression product, the rate of expression or the manner of expression control. Such modifications include, but are not limited to, insertions, deletions and substitutions of one or more nucleotides.
  • the transcribable sequence may contain an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions.
  • the transcribable sequence may also encode a fusion protein.
  • the transcribable sequence comprises non-coding regions only.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i. e. , causing regression of the disease.
  • vector is meant a nucleic acid molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned.
  • a vector typically contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a closed circular plasmid, an extra- chromosomal element, a mini-chromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may also include a marker such as an antibiotic resistance gene that can be used for identification of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.
  • underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing.
  • underscoring or italicizing shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing.
  • NR4A2 shall mean the "NR4A2” gene shall mean the NR4A2 gene, whereas "NR4A2" shall indicate the protein product or products generated from transcription and translation.
  • the present invention concerns the detection or diagnosis of AS.
  • Markers of AS in the form of RNA molecules and polypeptides of specified sequences, of subjects with or susceptible to AS, are disclosed. These markers are indicators of AS and, when aberrantly expressed as compared to their expression in normal subjects or in subjects lacking AS, are diagnostic for the presence or risk of development of AS in tested subjects. Such markers provide considerable advantages over the prior art in this field.
  • cells of the immune system e.g., leukocytes and leukocyte-containing populations such as peripheral blood mononuclear cells
  • NR4A2, TNFAIP 3, RORA and CD69 as compared to the expression of those genes in normal subjects or in subjects lacking AS, is diagnostic for the presence or risk of AS in tested subjects.
  • NR4A2, TNFAIP3, RORA and CD69 expression products will find utility in a variety of applications in detection, diagnosis and treatment of AS.
  • Examples of such applications within the scope of the present disclosure include amplification O ⁇ NR4A2, TNFAIP 3, RORA and/or CD69 transcripts using specific primers, detection of NR4A2, TNFAIP 3, RORA and/or CD69 transcripts by hybridization with oligonucleotide probes and detection of NR4A2, TNFAIP3, RORA and/or CD69 polypeptides.
  • NR4A2, TNFAIP3, RORA and CD69 nucleic acid sequences find utility inter alia as hybridization probes or amplification primers. These nucleic acids may be used, for example, in diagnostic evaluation of biological samples.
  • these probes and primers represent oligonucleotides, which are of sufficient length to provide specific hybridization to a RNA or DNA sample extracted from the biological sample.
  • the sequences typically will be about 10-20 nucleotides, but may be longer. Longer sequences, e.g., of at least about 30, 40, 50, 100, 500, 1000, 5000, 10000, 15000 and even up to full-length, are desirable for certain embodiments.
  • Nucleic acid molecules having contiguous stretches of at least about 10, 15, 17, 20, 30, 40, 50, 60, 75 or 100 or 500 or 1000 or 5000 or 10000 or 15000 nucleotides of a sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 1 1, 13 and 17-23 are contemplated. Molecules that are complementary to the above mentioned sequences and that bind to these sequences under at least medium or high stringency conditions are also contemplated. These probes are useful in a variety of hybridization embodiments, such as Southern and northern blotting.
  • probes may be used that hybridize to multiple target sequences (e.g., allelic variants and/or single nucleotide polymorphisms) without compromising their ability to effectively diagnose the presence or risk of development of AS.
  • target sequences e.g., allelic variants and/or single nucleotide polymorphisms
  • the hybridization probes described herein are useful both as reagents in solution hybridization, as in PCR, for detection or quantification of NR4A2, TNFAIP 3, RORA or CD69 expression, as well as in embodiments employing a solid phase.
  • probes and primers may be designed around the disclosed nucleotide sequences.
  • the sequences used to design probes and primers may include repetitive stretches of adenine nucleotides (poly- A tails) normally attached at the ends of the RNA for the identified marker genes.
  • probes and primers may be specifically designed to not include these or other segments from the identified marker genes, as one of ordinary skilled in the art may deem certain segments more suitable for use in the detection methods disclosed.
  • the choice of primer or probe sequences for a selected application is within the realm of the ordinary skilled practitioner.
  • Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is desirable. Probes, while perhaps capable of priming, are designed to bind to a target DNA or RNA and need not be used in an amplification process.
  • the probes or primers are labeled with radioactive species 32 P, 14 C, 35 S, 3 H, or other label), with a fluorophore (e.g., rhodamine, fluorescein) or with a chemillumiscent label (e.g., luciferase).
  • a fluorophore e.g., rhodamine, fluorescein
  • chemillumiscent label e.g., luciferase
  • the invention also contemplates detection or quantification of naturally-occurring NR4A2, TNFAIP3, RORA and/or CD69 nucleic acid sequences, inclusive of NR4A2, TNFAIP3, RORA or CD69 allelic variants (same locus), homologues (different locus), and orthologues (different organism).
  • NR4A2, TNFAIP3, RORA and CD69 nucleic acid sequences may therefore contain variations such as nucleotide substitutions, deletions, inversions and insertions, relative to the sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17-23. Variation can occur in either or both the coding (e.g., SEQ ID NO: 17-23) and non-coding regions.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the same amino acid sequence.
  • NR4A2, TNFAIP3, RORA and CD69 polynucleotides will have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and usually at least about 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to reference NR4A2, TNFAIP3, RORA or CD69 nucleotide sequences, as set forth for example in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17-23, or to their complements, as determined by sequence alignment programs described elsewhere herein using default parameters.
  • NR4A2, TNFAIP3, RORA or CD69 polynucleotides will generally hybridize to reference NR4A2, TNFAIP3, RORA or CD69 polynucleotides, as set forth for example in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17-23, or to a complement thereof, under low stringency, medium stringency, high stringency, or very high stringency conditions.
  • the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Ausubel et al, (1998, supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used.
  • Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C.
  • Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at room temperature.
  • BSA Bovine Serum Albumin
  • 1 mM EDTA 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C
  • 2 x SSC 0.1% SDS
  • 0.5% BSA 1
  • One embodiment of low stringency conditions includes hybridization in 6 x sodium chloride/sodium citrate (SSC) at about 45° C, followed by two washes in 0.2 x SSC, 0.1% SDS at least at 50° C (the temperature of the washes can be increased to 55° C for low stringency conditions).
  • SSC sodium chloride/sodium citrate
  • Another embodiment of low stringency conditions includes conditions equivalent to binding or hybridization at 42° C in a solution consisting of 5 x SSPE (43.8 g/L NaCl, 6.9 g/L NaH 2 PO 4 H 2 O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5 x Denhardt's reagent [50 x Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ⁇ g/mL denatured salmon sperm DNA followed by washing in a solution comprising 5 x SSPE, 0.1% SDS at 42° C when a probe of about 500 nucleotides in length is employed.
  • 5 x SSPE 43.8 g/L NaCl, 6.9 g/L NaH 2 PO 4 H 2 O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH
  • Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C.
  • Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 5% SDS for washing at 60-65° C.
  • BSA Bovine Serum Albumin
  • medium stringency conditions includes hybridizing in 6 x SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 60° C.
  • Another embodiment of medium stringency conditions includes conditions equivalent to binding or hybridization at 42° C in a solution consisting of 5 x SSPE (43.8 g/L NaCl, 6.9 g/L NaH 2 PO 4 H 2 O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5 x Denhardt's reagent and 100 ⁇ g/mL denatured salmon sperm DNA followed by washing in a solution comprising 1.0 x SSPE, 1.0% SDS at 42° C when a probe of about 500 nucleotides in length is employed.
  • High stringency conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42° C, and about 0.01 M to about 0.02 M salt for washing at 55° C.
  • High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO 4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C.
  • high stringency conditions includes hybridizing in 6 x SSC at about 45° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65° C.
  • Another embodiment of high stringency conditions includes conditions equivalent to binding or hybridization at 42° C in a solution consisting of 5 x SSPE (43.8 g/L NaCl, 6.9 g/L NaH 2 PO 4 H 2 O and 1.85 g/L EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5 x Denhardt's reagent and 100 ⁇ g/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1 x SSPE, 1.0% SDS at 42° C when a probe of about 500 nucleotides in length is employed.
  • a NR4A2, TNFAIP3, RORA or CD69 polynucleotide hybridizes to a disclosed nucleotide sequence, as for example set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 17-23, or to a complement thereof, under very high stringency conditions.
  • very high stringency conditions includes hybridizing 0.5 M sodium phosphate, 7% SDS at 65° C, followed by one or more washes at 0.2 x SSC, 1% SDS at 65° C.
  • Tm 81.5 + 16.6 (loglO M) + 0.41 (%G+C) - 0.63 (% formamide) - (600/length)
  • M is the concentration of Na+, preferably in the range of 0.01 molar to 0.4 molar; %G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases, within the range between 30% and 75% G+C; % formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex.
  • Tm of a duplex DNA decreases by approximately 1° C with every increase of 1% in the number of randomly mismatched base pairs. Washing is generally carried out at Tm - 15° C for high stringency, or Tm - 30° C for moderate stringency.
  • a membrane e.g., a nitrocellulose membrane or a nylon membrane
  • immobilized DNA is hybridized overnight at 42° C in a hybridization buffer (50% deionized formamide, 5 x SSC, 5 x Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA) containing labeled probe.
  • a hybridization buffer 50% deionized formamide, 5 x SSC, 5 x Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA
  • the membrane is then subjected to two sequential medium stringency washes (i.e., 2 x SSC, 0.1% SDS for 15 min at 45° C, followed by 2 x SSC, 0.1% SDS for 15 min at 50° C), followed by two sequential higher stringency washes (i.e., 0.2 x SSC, 0.1% SDS for 12 min at 55° C followed by 0.2 x SSC and 0.1%SDS solution for 12 min at 65-68° C.
  • 2 x SSC 0.1% SDS for 15 min at 45° C
  • 2 x SSC 0.1% SDS for 15 min at 50° C
  • two sequential higher stringency washes i.e., 0.2 x SSC, 0.1% SDS for 12 min at 55° C followed by 0.2 x SSC and 0.1%SDS solution for 12 min at 65-68° C.
  • the present invention contemplates detection or quantification of naturally-occurring NR4A2, TNFAIP3, RORA or CD69 polypeptides, inclusive of NR4A2, TNFAIP3, RORA or CD69 allelic variants, homologues, and orthologues.
  • NR4A2, TNFAIP3, RORA or CD69 polypeptide sequences may therefore contain variations such as amino acid substitutions, deletions and insertions, relative to the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14.
  • NR4A2, TNFAIP3, RORA or CD69 polypeptides will generally have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and usually at least about 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity or identity with a reference NR4A2, TNFAIP3, RORA or CD69 polypeptide, as set forth for example in SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a NR4A2, TNFAIP3, RORA or CD69 polypeptide sequence may differ from a reference NR4A2, TNFAIP3, RORA or CD69 polypeptide sequence generally by as much as 60, 50, 40, 30 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • the present invention also contemplates the use of antigen-binding molecules that are specifically immuno-interactive with a NR4A2, TNFAIP3, RORA or CD69 polypeptide for diagnosing the presence of AS.
  • the antigen-binding molecule is a whole polyclonal antibody.
  • Such antibodies may be prepared, for example, by injecting a NR4A2, TNFAIP3, RORA or CD69 polypeptide or portion thereof into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art.
  • monoclonal antibodies may be produced using the standard method as described, for example, by Kohler and Milstein (1975, Nature 256, 495-497), or by more recent modifications thereof as described, for example, in Coligan et al, (1991, supra) by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or more of NR4A2, TNFAIP3, RORA or CD69 polypeptides.
  • the invention also contemplates as antigen-binding molecules Fv, Fab, Fab 1 and F(ab') 2 immunoglobulin fragments.
  • the antigen-binding molecule may comprise a synthetic stabilized Fv fragment.
  • Exemplary fragments of this type include single chain Fv fragments (sFv, frequently termed scFv) in which a peptide linker is used to bridge the N terminus or C terminus of a V # domain with the C terminus or N-terminus, respectively, of a Vj, domain.
  • sFv single chain Fv fragments
  • ScFv lack all constant parts of whole antibodies and are not able to activate complement.
  • ScFvs may be prepared, for example, in accordance with methods outlined in Kreber et al (Kreber et al.
  • the synthetic stabilized Fv fragment comprises a disulfide stabilized Fv (dsFv) in which cysteine residues are introduced into the W H and V / , domains such that in the fully folded Fv molecule the two residues will form a disulfide bond between them.
  • dsFv disulfide stabilized Fv
  • Suitable methods of producing dsFv are described for example in (Glockscuther et al.
  • Phage display and combinatorial methods for generating anti-NR4A2, TNFAIP3, RORA or CD69 antigen-binding molecules are known in the art (as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al.
  • the antigen-binding molecule can be coupled to a compound, e.g., a label such as a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.
  • a label such as a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.
  • An anti-NR4A2, TNFAIP3, RORA or CD69 antigen-binding molecule e.g., monoclonal antibody
  • AS marker polypeptides e.g., in a cellular lysate or cell supernatant
  • such antigen-binding molecules can be used to monitor NR4A2, TNFAIP3, RORA or CD69 polypeptide levels in biological samples (including tissues, cells and fluids) for diagnosing the presence or absence of AS. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance (i.e., antibody labeling).
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 1, 35 S or 3 H.
  • the label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu 34 ), a radioisotope and a direct visual label.
  • a direct visual label use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, ⁇ -galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like.
  • the enzyme label may be used alone or in combination with a second enzyme in solution. 3.
  • the present invention is predicated in part on the determination that NR4A2, TNFAIP3, RORA and CD69 transcripts are down regulated in AS. Accordingly, in certain embodiments, the invention features a method for diagnosing or detecting the presence or absence of AS or the risk of developing AS in a subject, by determining the expression status of NR4A2, TNFAIP 3, RORA and/or CD69 transcripts in a biological sample obtained from the subject and diagnosing the presence or absence of AS in the subject based on the expression status.
  • the presence of AS in the patient is diagnosed when a NR4A2, TNFAIP3, RORA and/or CD69 gene product is expressed at a detectably lower level in the biological sample as compared to the level at which that gene is expressed in a reference sample obtained from normal subjects or from subjects lacking AS.
  • the corresponding expression product is generally selected from the same gene product, an alternate gene product including splice variants or expression products produced from alternate promoters of the gene, a gene product expressed from a variant gene (e.g., an homologous or orthologous gene) including an allelic variant, or protein products thereof.
  • the method comprises measuring the level or functional activity of at least one NR4A2, TNFAIP 3, RORA or CD69 expression product.
  • expression is measured directly (e.g., at the RNA or protein level). In some embodiments, expression is detected in tissue samples. In other embodiments, expression is detected in bodily fluids (e.g., including but not limited to blood and PMBCs). In specific embodiments, the biological sample comprises cells.
  • NR4A2, TNFAIP 3, RORA and/or CD69 expression may be detected along with other AS markers, especially, in a multiplex or panel format. Such markers are selected for their predictive value alone or in combination with NR4A2, TNFAIP3, RORA and/or CD69.
  • the diagnostic methods of the present invention further comprise detecting expression of at least one other AS marker gene, illustrative examples of which include HLA-B27.
  • Nucleic acids used in polynucleotide-based assays can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, et al., 1989, supra; and Ausubel et al., 1994, supra).
  • the nucleic acid is typically fractionated (e.g., poly A + RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA.
  • the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the NR4A2, TNFAIPS, RORA and CD69 sequences present in a given template sample.
  • PCR polymerase chain reaction
  • NR4A2, TNFAIP 3, RORA or CD69 sequence is present in a sample
  • the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art.
  • the template-dependent amplification involves the quantification of transcripts in real-time.
  • RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al, Biotechnology 10: 413-417).
  • the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells.
  • LCR ligase chain reaction
  • Q ⁇ Replicase described in PCT Application No. PCT/US87/00880, may also be used.
  • 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 that can 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' ⁇ -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 al. , (1992, Proc. Natl. Acad. ScI U.S. A 89: 392-396).
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • CPR cyclic probe reaction
  • modified primers are used in a PCR-like, template- and enzyme- dependent synthesis.
  • the primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription- based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al, PCT Application WO 88/10315).
  • TAS transcription- based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al, 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al, PCT Application WO 88/10315.
  • NASBA the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • the RNAs are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • T7 or SP6 an isothermal cyclic reaction
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • Vincent and Kong disclose a method termed helicase-dependent isothermal DNA amplification (HDA) (Vincent and Kong, EMBO Reports, 5(8):795- 800, 2004). This method uses DNA helicase to separate DNA strands and hence does not require thermal cycling. The entire reaction can be carried out at one temperature and this method should have broad application to point-of-care DNA diagnostics.
  • HDA helicase-dependent isothermal DNA amplification
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al. in PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/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, M. A., In: “PCR Protocols: A Guide to Methods and Applications", Academic Press, N. Y., 1990; Ohara et al, 1989, Proc. Natl Acad. Sci. U.S.A., 86: 5673-567).
  • the NR4A2, TNFAIP3, RORA or CD69 nucleic acid of interest is identified in the sample directly using a template-dependent amplification as described, for example, above, or with a second, known nucleic acid following amplification.
  • the identified product is detected.
  • the detection may be performed by visual means (e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994, JMacromol. Sci. Pure, Appl. Chem., A31(l): 1355-1376).
  • amplification products or "amplicons" are visualized in order to confirm amplification of the NR4A2, TNFAIP 3, RORA or CD69 sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly. Following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified NR4A2, TNFAIP3, RORA or CD69 sequence.
  • the probe is suitably conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antigen-binding molecule, or biotin, and the other member of the binding pair carries a detectable moiety or reporter molecule.
  • a binding partner such as an antigen-binding molecule, or biotin
  • the other member of the binding pair carries a detectable moiety or reporter molecule.
  • the techniques involved are well known to those of skill in the art and can be found in many standard texts on molecular protocols (e.g., see Sambrook et al, 1989, supra and Ausubel et al. 1994, supra).
  • chromophore or radiolabel probes or primers identify the target during or following amplification.
  • target nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art.
  • Southern blotting involves the use of DNA as a target
  • Northern blotting involves the use of RNA as a target.
  • cDNA blotting is analogous, in many aspects, to blotting or RNA species.
  • a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter.
  • the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.
  • a probe usually labeled
  • a control reaction e.g., a statistically significant reference group of normal subjects or of subjects lacking AS; or a statistically significant reference group of subjects with AS.
  • a control reaction e.g., a statistically significant reference group of normal subjects or of subjects lacking AS; or a statistically significant reference group of subjects with AS.
  • genotyping methods and allelic discrimination methods and technologies such as those described by Kristensen et al. (Biotechniques 30(2): 318-322), including the use of single nucleotide polymorphism analysis, high performance liquid chromatography, TaqMan®, liquid chromatography, and mass spectrometry.
  • biochip-based technologies such as those described by Hacia et al. (1996, Nature Genetics 14: 441-447) and Shoemaker et al. (1996, Nature Genetics 14: 450-456). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994, Proc. Natl. Acad. Sci. U.S.A. 91: 5022-5026); Fodor et al. (1991, Science 251: 767-773).
  • nucleic acid probes to AS marker polynucleotides are made and attached to biochips to be used in screening and diagnostic methods, as outlined herein.
  • the nucleic acid probes attached to the biochip are designed to be substantially complementary to specific expressed NR4A2, TNFA1P3, RORA or CD69 nucleic acids, i.e., the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs.
  • This complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the nucleic acid probes of the present invention.
  • the sequence is not a complementary target sequence.
  • more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target.
  • the probes can be overlapping (i.e. have some sequence in common), or separate.
  • immobilized and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below.
  • the binding can be covalent or non-covalent.
  • non-covalent binding and grammatical equivalents herein is meant one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin.
  • covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
  • the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art.
  • the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
  • the biochip comprises a suitable solid or semi-solid substrate or solid support.
  • substrate or “solid support” is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method.
  • the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc.
  • the substrates allow optical detection and do not appreciably fluoresce.
  • the substrate is planar, although as will be appreciated by those of skill in the art, other configurations of substrates may be used as well.
  • the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
  • the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
  • oligonucleotides probes are synthesized on the substrate, as is known in the art.
  • photoactivation techniques utilizing photopolymerization compounds and techniques can be used.
  • the nucleic acids are synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited within; these methods of attachment form the basis of the Affymetrix GeneChipTM technology.
  • oligonucleotide probes on the biochip are exposed to or contacted with a nucleic acid sample suspected of containing one or more NR4A2, TNFAIP3, RORA or CD69 polynucleotides under conditions favoring specific hybridization.
  • Sample extracts of DNA or RNA may be prepared from fluid suspensions of biological materials, or by grinding biological materials, or following a cell lysis step which includes, but is not limited to, lysis effected by treatment with SDS (or other detergents), osmotic shock, guanidinium isothiocyanate and lysozyme.
  • Suitable DNA which may be used in the method of the invention, includes cDNA.
  • Such DNA may be prepared by any one of a number of commonly used protocols as for example described in Ausubel, et al, 1994, supra, and Sambrook, et al., et al, 1989, supra.
  • RNA which may be used in the method of the invention, includes messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or subgenomic RNA.
  • cRNA complementary RNA transcribed from DNA
  • genomic or subgenomic RNA Such RNA may be prepared using standard protocols as for example described in the relevant sections of Ausubel, et al. 1994, supra and Sambrook, et al. 1989, supra).
  • cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases.
  • cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilized oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridization conditions.
  • fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.
  • the target NR4A2, TNFAIP3, RORA or CD69 polynucleotides are detectably labeled so that their hybridization to individual probes can be determined.
  • the target polynucleotides are typically detectably labeled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu 34 ), a radioisotope and a direct visual label.
  • a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu 34 ), a radioisotope and a direct visual label.
  • a direct visual label use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide.
  • an enzyme is used as a direct visual label
  • biotinylated bases are incorporated into a target polynucleotide. Hybridization is detected by incubation with streptavidin-reporter molecules.
  • Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RPE R-Phycoerythrin
  • Texas Red Texas Red
  • Other exemplary fluorochromes include those discussed by Dower et al. (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al), 5,326,692 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos.
  • fluorescent labels include, for example, fluorescein phosphoramidites such as FluoreprimeTM (Pharmacia), FluorediteTM (Millipore) and FAM (Applied Biosystems International)
  • Radioactive reporter molecules include, for example, P, which can be detected by an X-ray or phosphoimager techniques.
  • the hybrid-forming step can be performed under suitable conditions for hybridizing oligonucleotide probes to test nucleic acid including DNA or RNA.
  • suitable conditions for hybridizing oligonucleotide probes to test nucleic acid including DNA or RNA.
  • whether hybridization takes place is influenced by the length of the oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid-forming region, the viscosity of the medium and the possible presence of denaturants.
  • Such variables also influence the time required for hybridization.
  • the preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation
  • high discrimination hybridization conditions are used.
  • a hybridization reaction can be performed in the presence of a hybridization buffer that optionally includes a hybridization-optimizing agent, such as an isostabilizing agent, a denaturing agent and/or a renaturation accelerant.
  • a hybridization-optimizing agent such as an isostabilizing agent, a denaturing agent and/or a renaturation accelerant.
  • isostabilizing agents include, but are not restricted to, betaines and lower tetraalkyl ammonium salts.
  • Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules.
  • Denaturing agents include, but are not restricted to, formamide, formaldehyde, dimethylsulfoxide, tetraethyl acetate, urea, guanidium isothiocyanate, glycerol and chaotropic salts.
  • Hybridization accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol.
  • CAB cetyltrimethylammonium bromide
  • DTAB dodecyl trimethylammonium bromide
  • polylysine polylysine
  • spermine spermine
  • spermidine single stranded binding protein
  • SSB single
  • Hybridization buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM.
  • a hybridization mixture containing the target AS marker polynucleotides is placed in contact with the array of probes and incubated at a temperature and for a time appropriate to permit hybridization between the target sequences in the target polynucleotides and any complementary probes.
  • Contact can take place in any suitable container, for example, a dish or a cell designed to hold the solid support on which the probes are bound.
  • incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20° C and about 75° C, example, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 65° C.
  • a sample of target polynucleotides is incubated with the probes for a time sufficient to allow the desired level of hybridization between the target sequences in the target polynucleotides and any complementary probes.
  • the hybridization may be carried out at about 45° C +/-10° C in formamide for 1-2 days.
  • the probes are washed to remove any unbound nucleic acid with a hybridization buffer, which can typically comprise a hybridization optimizing agent in the same range of concentrations as for the hybridization step. This washing step leaves only bound target polynucleotides.
  • the probes are then examined to identify which probes have hybridized to a target polynucleotide.
  • the hybridization reactions are then detected to determine which of the probes has hybridized to a corresponding target sequence.
  • a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a colored colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography.
  • a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light.
  • a charge couple device or a photocell can be used to scan for emission of light from a probe:target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer.
  • electronic detection of the signal may not be necessary.
  • the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile.
  • oligonucleotide probes specific for different NR4A2, TNFAIP 3, RORA or CD69 gene products are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'.
  • a detection system that can be used by a 'chip reader' is described for example by Pirrung et al (U.S. Patent No. 5,143,854).
  • the chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal.
  • Exemplary chip readers are described for example by Fodor et al (U.S. Patent No., 5,925,525).
  • the reaction may be detected using flow cytometry.
  • the aberrant expression of a NR4A2, TNFAIP3, RORA and CD69 protein is indicative of the presence or risk of development of AS.
  • NR4A2, TNFAIP3, RORA or CD69 protein levels in biological samples can be assayed using any suitable method known in the art.
  • antibody-based techniques may be employed, such as, for example, immunohistological and immunohistochemical methods for measuring the level of a protein of interest in a tissue sample.
  • Specific recognition may be provided, for example, by a primary antibody (polyclonal or monoclonal) and a secondary detection system is used to detect presence (or binding) of the primary antibody.
  • Detectable labels can be conjugated to the secondary antibody, such as a fluorescent label, a radiolabel, or an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) which produces a quantifiable, e.g., coloured, product.
  • the primary antibody itself can be detectably labeled.
  • immunohistological labeling of a tissue section is provided.
  • a protein extract is produced from a biological sample (e.g., tissue, cells) for analysis.
  • Such an extract e.g., a detergent extract
  • Other useful antibody-based methods include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • a protein-specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify a NR4A2, TNFAIP3, RORA or CD69 protein.
  • the amount of such protein present in a sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm (see Lacobilli et al, 1988, Breast Cancer Research and Treatment 11: 19-30).
  • two different monoclonal antibodies to the protein of interest can be employed, one as the immunoadsorbent and the other as an enzyme-labeled probe.
  • recent developments in the field of protein capture arrays permit the simultaneous detection and/or quantification of a large number of proteins.
  • low-density protein arrays on filter membranes such as the universal protein array system (Ge, 2000 Nucleic Acids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector.
  • Immuno-sensor arrays have also been developed that enable the simultaneous detection of clinical analytes. It is now possible using protein arrays, to profile protein expression in bodily fluids, such as in sera of healthy or diseased subjects, as well as in subjects pre- and post-drug treatment.
  • Protein capture arrays typically comprise a plurality of protein-capture agents each of which defines a spatially distinct feature of the array.
  • the protein-capture agent can be any molecule or complex of molecules which has the ability to bind a protein and immobilize it to the site of the protein-capture agent on the array.
  • the protein-capture agent may be a protein whose natural function in a cell is to specifically bind another protein, such as an antibody or a receptor.
  • the protein- capture agent may instead be a partially or wholly synthetic or recombinant protein which specifically binds a protein.
  • the protein-capture agent may be a protein which has been selected in vitro from a mutagenized, randomized, or completely random and synthetic library by its binding affinity to a specific protein or peptide target.
  • the selection method used may optionally have been a display method such as ribosome display or phage display, as known in the art.
  • the protein- capture agent obtained via in vitro selection may be a DNA or RNA aptamer which specifically binds a protein target (see, e.g., Potyrailo et al., 1998 Anal. Chem. 70:3419- 3425; Cohen et al., 1998, Proc. Natl. Acad.
  • aptamers are selected from libraries of oligonucleotides by the SelexTM process and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; universal fluorescent protein stains can be used to detect binding.
  • the in vitro selected protein-capture agent may be a polypeptide (e.g., an antigen) (see, e.g., Roberts and Szostak, 1997 Proc. Natl. Acad. ScL USA, 94:12297-12302).
  • an alternative to an array of capture molecules is one made through
  • peptides e.g., from the C-terminal regions of proteins
  • the cavities can then specifically capture (denatured) proteins which have the appropriate primary amino acid sequence (e.g., available from ProteinPrintTM and Aspira Biosystems).
  • Exemplary protein capture arrays include arrays comprising spatially addressed antigen-binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of numerous proteins defining a proteome or subproteome.
  • Antibody arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma). Various methods for the preparation of antibody arrays have been reported (see, e.g., Lopez et al, 2003 J. Chromatogr. B 787:19-27; Cahill, 2000 Trends in Biotechnology 7:47-51; U.S. Pat. App. Pub.
  • the antigen-binding molecules of such arrays may recognise at least a subset of proteins expressed by a cell or population of cells, illustrative examples of which include growth factor receptors, hormone receptors, neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, antibodies, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras- like GTPases, hydrolases, steroid hormone receptors, transcription factors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, intracellular signal transduction modulators and effectors, apoptosis-related factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, cell-surface antigens, hepatitis C virus (HCV) proteases and HIV proteases.
  • HCV hepatitis C virus
  • Antigen-binding molecules for antibody arrays are made either by conventional immunization (e.g., polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage display or ribosome display libraries (e.g., available from Cambridge Antibody Technology, Biolnvent, Aff ⁇ tech and Biosite).
  • 'combibodies' comprising non-covalent associations of VH and VL domains, can be produced in a matrix format created from combinations of diabody -producing bacterial clones (e.g., available from Domantis).
  • Exemplary antigen-binding molecules for use as protein-capture agents include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab' and F(ab') 2 immunoglobulin fragments, synthetic stabilized Fv fragments, e.g., single chain Fv fragments (scFv), disulfide stabilized Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents.
  • Individual spatially distinct protein-capture agents are typically attached to a support surface, which is generally planar or contoured. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.
  • microdrops of protein delivered onto planar surfaces are widely used, related alternative architectures include CD centrifugation devices based on developments in microfluidics (e.g., available from Gyros) and specialized chip designs, such as engineered microchannels in a plate (e.g., The Living ChipTM, available from Biotrove) and tiny 3D posts on a silicon surface (e.g., available from Zyomyx).
  • microfluidics e.g., available from Gyros
  • chip designs such as engineered microchannels in a plate (e.g., The Living ChipTM, available from Biotrove) and tiny 3D posts on a silicon surface (e.g., available from Zyomyx).
  • Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g., QDotsTM, available from Quantum Dots), and barcoding for beads (UltraPlexTM, available from Smartbeads) and multimetal microrods (NanobarcodesTM particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions).
  • color coding for microbeads e.g., available from Luminex, Bio-Rad and Nanomics Biosystems
  • semiconductor nanocrystals e.g., QDotsTM, available from Quantum Dots
  • barcoding for beads UltraPlexTM, available from Smartbeads
  • NanobarcodesTM particles available
  • individual protein-capture agents are typically attached to an individual particle to provide the spatial definition or separation of the array.
  • the particles may then be assayed separately, but in parallel, in a compartmentalized way, for example in the wells of a microtiter plate or in separate test tubes.
  • a protein sample which is optionally fragmented to form peptide fragments (see, e.g., U.S. Pat. App. Pub. 2002/0055186) is delivered to a protein-capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components of the sample from the array.
  • the presence or amount of protein or peptide bound to each feature of the array is detected using a suitable detection system.
  • the amount of protein bound to a feature of the array may be determined relative to the amount of a second protein bound to a second feature of the array.
  • the amount of the second protein in the sample is already known or known to be invariant.
  • a protein sample of a first cell or population of cells is delivered to the array under conditions suitable for protein binding.
  • a protein sample of a second cell or population of cells to a second array is delivered to a second array which is identical to the first array.
  • Both arrays are then washed to remove unbound or non-specifically bound components of the sample from the arrays.
  • the amounts of protein remaining bound to the features of the first array are compared to the amounts of protein remaining bound to the corresponding features of the second array.
  • the amount of protein bound to individual features of the first array is subtracted from the amount of protein bound to the corresponding features of the second array.
  • fluorescence labeling can be used for detecting protein bound to the array.
  • the same instrumentation as used for reading DNA microarrays is applicable to protein-capture arrays.
  • capture arrays e.g. antibody arrays
  • fluorescently labeled proteins from two different cell states, in which cell lysates are labeled with different fiuorophores (e.g., Cy-3 and Cy-5) and mixed, such that the color acts as a readout for changes in target abundance.
  • Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (e.g., available from PerkinElmer
  • Planar waveguide technology e.g., available from Zeptosens
  • High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (e.g., available from Luminex) or the properties of semiconductor nanocrystals (e.g., available from Quantum Dot).
  • Fluorescence resonance energy transfer has been adapted to detect binding of unlabelled ligands, which may be useful on arrays (e.g., available from Affibody).
  • the techniques used for detection of AS marker expression products will include internal or external standards to permit quantitative or semi-quantitative determination of those products, to thereby enable a valid comparison of the level or functional activity of these expression products in a biological sample with the corresponding expression products in a reference sample or samples.
  • standards can be determined by the skilled practitioner using standard protocols.
  • absolute values for the level or functional activity of individual expression products are determined.
  • in vivo imaging techniques are used to visualize the expression of NR4A2, TNFAIP3, RORA or CD69 and optionally one or more other AS markers in a patient (e.g., a human or non-human mammal).
  • AS marker mRNA or protein is labeled using a labeled antigen-binding molecule (e.g., mAb) specific for the AS marker.
  • mAb labeled antigen-binding molecule
  • a specifically bound and labeled antigen-binding molecule can be detected in an individual using an in vivo imaging method, including, but not limited to, radionuclide imaging, positron emission tomography, computerized axial tomography, X-ray or magnetic resonance imaging method, fluorescence detection, and chemiluminescent detection.
  • an in vivo imaging method including, but not limited to, radionuclide imaging, positron emission tomography, computerized axial tomography, X-ray or magnetic resonance imaging method, fluorescence detection, and chemiluminescent detection.
  • the in vivo imaging methods of the present invention are useful in the diagnosis of AS.
  • In vivo imaging is used to visualize the presence and/or amount/level of a NR4A2, TNFAIP3, RORA or CD69 expression product (e.g., NR4A2, TNFAIP3, RORA or CD69 protein).
  • a NR4A2, TNFAIP3, RORA or CD69 expression product e.g., NR4A2, TNFAIP3, RORA or CD69 protein.
  • Such techniques allow for diagnosis without the use of an unpleasant biopsy or blood collection and are also useful for providing diagnosis to AS patients.
  • NR4A2, TNFAIP3, RORA and CD69 levels that are indicative of AS can be detected.
  • the in vivo imaging methods of the present invention can further be used to detect AS in other parts of the body.
  • reagents e.g., antibodies
  • NR4A2, TNFAIP3, RORA or CD69 and optionally one or more other AS markers are fluorescently labeled.
  • the labeled reagents are introduced into a subject (e.g., orally or parenterally). Fluorescently labeled antibodies are detected using any suitable method (e.g., using the apparatus described in U.S. Pat. No. 6,198,107, herein incorporated by reference).
  • antibodies are radioactively labeled.
  • the use of antibodies for in vivo diagnosis is well known in the art. Sumerdon et al. (1990, Nucl. Med. Biol, 17:247-254) have described an optimized antibody-chelator for the radioimmunoscintigraphy imaging of tumors using Indium- 11 1 as the label. Griffin et al. (1991, J Clin One 9:631-640) have described the use of this agent in detecting tumors in patients suspected of having recurrent colorectal cancer. The use of similar agents with paramagnetic ions as labels for magnetic resonance imaging is known in the art (Lauffer, 1991, Magnetic Resonance in Medicine 22:339-342).
  • Radioactive labels such as Indium- 111, Technetium-99m, or Iodine-131 can be used for planar scans or single photon emission computed tomography (SPECT).
  • Positron emitting labels such as Fluorine- 19 can also be used for positron emission tomography (PET).
  • PET positron emission tomography
  • paramagnetic ions such as Gadolinium (III) or Manganese (II) can be used.
  • Radioactive metals with half-lives ranging from 1 hour to 3.5 days are available for conjugation to antibodies, such as scandium-47 (3.5 days) gallium-67 (2.8 days), gallium-68 (68 minutes), technetiium-99m (6 hours), and indium- 1 1 1 (3.2 days), of which gallium-67, technetium-99m, and indium- 1 1 1 are desirable for gamma camera imaging, gallium-68 is desirable for positron emission tomography.
  • An illustrative method of labeling antibodies with such radiometals is by means of a bifunctional chelating agent, such as diethylenetriaminepentaacetic acid (DTPA), as described, for example, by Khaw et al.
  • DTPA diethylenetriaminepentaacetic acid
  • a suitable method of labeling antibodies with Tc-99m which does not use chelation with DPTA is the pretinning method of Crockford et al (U.S. Pat. No. 4,323,546).
  • An exemplary method of labeling immunoglobulins with Tc-99m is that described by Wong et al. (1981, J. Nucl. Med., 23:229) for labeling antibodies.
  • in vivo biophotonic imaging (Xenogen, Almeda, Calif.) is utilized for in vivo imaging.
  • This real-time in vivo imaging utilizes luciferase.
  • the luciferase gene is incorporated into cells, microorganisms, and animals (e.g., as a fusion protein with an AS marker of the present invention). When active, it leads to a reaction that emits light.
  • a CCD camera and software is used to capture the image and analyze it.
  • kits All the essential materials and reagents required for detecting and/or quantifying NR4A2, TNFAIP3, RORA or CD69 gene expression products, and optionally other AS marker gene products may be assembled together in a kit.
  • the kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microliter plates dilution buffers and the like.
  • a nucleic acid-based detection kit may include: (i) a NR4A2, TNFAIP3, RORA and/or CD69 polynucleotide, and optionally one or more other AS marker polynucleotides, which may be used as a positive control; (ii) a primer or probe that specifically hybridizes to a NR4A2, TNFAIP3, RORA or CD69 polynucleotide, and optionally primers or probes that specifically hybridize to one or more other AS marker polynucleotides.
  • kits may comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
  • enzymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq, SequenaseTM DNA ligase etc depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
  • a protein-based detection kit may include: (i) a NR4A2, TNFAIP3, RORA and/or CD69 polypeptide, and optionally one or more other AS marker polypeptides, which may be used as a positive control; (ii) an antigen-binding molecule that is immuno-interactive with a NR4A2, TNFAIP3, RORA or CD69 polypeptide and optionally antigen-binding molecules that are immuno-interactive with one or more other AS marker polypeptides.
  • the kit can also feature various devices and reagents for performing one of the assays described herein; and/or printed instructions for using the kit to quantify the expression of a NR4A2, TNFAIP 3, RORA and/or CD69 AS marker polynucleotide and optionally the expression of one or more other AS marker polynucleotides.
  • the present invention also extends to the management of AS, or prevention of further progression of AS, or assessment of the efficacy of therapies in subjects following positive diagnosis for the presence of AS in the subjects.
  • AS often includes a treatment regime involving medication, exercise, physical therapy and if necessary surgery.
  • effective medications include but are not restricted to nonsteroidal anti-inflammatory drugs (NSAIDS) such as Sulfasalazine (Azulfidine), Methotrexate (Rheumatrex or Trexall) and Corticosteroids (cortisone); TNF blockers such as etanerce (Enbrel), infliximab (Remicade) and adalimumab (Humira);
  • AS-ameliorating agents will be administered in pharmaceutical (or veterinary) compositions together with a pharmaceutically acceptable carrier and in an effective amount to achieve their intended purpose.
  • the dose of active compounds administered to a subject should be sufficient to achieve a beneficial response in the subject over time such as a reduction in, or relief from, the symptoms of AS and the prevention of the disease from developing further.
  • the quantity of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the active compound(s) for administration will depend on the judgement of the practitioner.
  • the physician or veterinarian may evaluate severity of any symptom associated with the presence of AS including symptoms related to AS such as for example characterized by acute, painful episodes followed by temporary periods of remission.
  • those of skill in the art may readily determine suitable dosages of the AS-ameliorating agents and suitable treatment regimens without undue experimentation.
  • Exemplary subjects for treatment with the methods of the present invention are vertebrates, especially mammals.
  • the subject is selected from the group consisting of humans, sheep, cattle, horses, bovine, pigs, dogs and cats.
  • the subject is a human.
  • PBMCs peripheral blood mononuclear cells
  • RNA NanoDrop Spectrophotometer Normal size distribution of the cRNA species was verified with RNA Nano Chips on an Agilent 2100 BioAnalyser.
  • Illumina HumanHT-12 V3 Expression BeadChips (Illumina, CA) according to the manufacturer's protocol. The three chips with the 36 samples were processed in parallel for hybridization using the Direct Hyb Assay and read on an Illumina BeadArray Reader.
  • cDNA was generated from l ⁇ g of total RNA using the Bioline cDNA synthesis Kit (Bioline, London, UK) according to manufacturer's instructions. Both candidate and housekeeping gene expression levels were measured in triplicate by quantitative RT-PCR in Micro Amp 384- well optical plates on the ABI TaqMan 7900 Real-time Quantitative PCR platform (both Applied Biosystems, CA). Candidate genes were assayed using the pre-designed TaqMan assays which utilise MGB probes with
  • FAM dye FAM dye.
  • expression levels of the housekeeping gene, RPL32 Kriegova, E et al., 2008, BMC MoI Biol. 9: 69
  • SYBR green based quantitative RT-PCR using specific forward and reverse primers (see below). All assays were carried out using SensiMix dT RT-PCR reagent (Quantace, Sydney, Australia) under the following conditions; 50°C for 2 min, 95 0 C for 10 min, and 40 cycles of 95°C for 15 s and 6O 0 C for 60 s.
  • the relative amounts of mRNA for genes of interest were determined using the relative standard curve method, quantitative RT-PCR results were analysed with Mann- Whitney test, /"-values ⁇ 0.05 were considered significant.
  • Array data were processed using the Illumina BeadStudio software then the processed data were assessed for quality control and normalised in Lumi (Du, P et al., 2008, Bioinformatics, 24: 1547-1548). Analysis of gene expression patterns was performed in BRB-Array Tools (Simon, R et al., 2007, Cancer Inform, 3: 1 1-17). For quality control scanned images of the arrays were visually inspected for artefacts in Illumina BeadStudio followed by the graphical analysis of density plots in Lumi. One control sample was excluded due to problems during array hybridisation, and one AS sample was a biological outlier probably due to ethnicity (this patient was of Indonesian origin with the other patients and controls being Caucasian), thus statistical analyses were performed on 17 control and 17 AS samples.
  • VST variance stabilization transformation
  • RSN robust spline normalization

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Abstract

La présente invention concerne des méthodes et des agents qui permettent de diagnostiquer la présence d'une spondylarthrite ankylosante (AS) ou le risque de développer une telle affection. Plus particulièrement, la présente invention concerne l'utilisation de NR4A2, de TNFAIP3, de RORA et de CD69 en tant que marqueurs de diagnostic de l'AS. L'invention peut être utilisée dans le diagnostic précoce de l'AS, et pour permettre un meilleur traitement et de meilleures décisions en matière de gestion, qui doivent être prises pour des sujets atteints sur les plans clinique ou subclinique.
PCT/AU2010/000729 2009-06-12 2010-06-11 Agents et méthodes de diagnostic et de traitement de la spondylarthrite ankylosante WO2010141999A1 (fr)

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