Classifications

• • 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
  • C12Q2600/158—Expression markers

Definitions

• Background of the Invention Present invention relates to Slfn 2 for use in the diagnosis or prognosis of a pain-related tissue status or a pain related-disease, methods of identifying or prognosing a pain- related tissue status or disease, a kit for use in the prognosis or diagnosis of a pain- related tissue status or a pain-related disease, a kit for detecting Slfn2, and a means for detecting Slfn2 for use in the prognosis or diagnosis of a pain-related tissue status or a pain-related disease.
• IASP International Association for the Study of Pain
• Pain of any type is the most common reason for physician consultation in the United States, prompting half of all Americans to seek medical care annually. It is a major symptom in many medical conditions, significantly interfering with a person's quality of life and general functioning. Diagnosis is based on characterizing pain in various ways, according to duration, intensity, type (dull, burning, throbbing or stabbing), source, or location in body. Usually pain stops without treatment or responds to simple measures such as resting or taking an analgesic, and it is then called 'acute' pain. But it may also become intractable and develop into a condition called chronic pain, in which pain is no longer considered a symptom but an illness by itself.
• Pain is part of the body's defense system, triggering a reflex reaction to retract from a painful stimulus, and helps adjust behavior to increase avoidance of that particular harmful situation in the future.
• Acute pain is 'normal' pain, it is felt when hurting a toe, breaking a bone, having a toothache, or walking after an extensive surgical operation.
• Chronic pain is a 'pain illness', it is felt day after day, month after month, and seems impossible to heal.
• Diagnosis of pain is mostly based on symptomatic description by the patient. However, this description is naturally very subjective and hardly quantifiable, so that the choice of the correct dosage and drug mostly relates to the patients age and body weight, together with some symptomatic analyses as to the nature of the pain-related tissue state (such as inflammation, necrosis etc.), pain-related disease (such as osteoarthritis, cancer, migraine etc) or pain (such as neuropathic pain or inflammatory pain). Especially in children, animals and mentally-disabled people, a proper diagnosis of pain is mostly difficult. Objective parameters allowing for a qualitative and quantitative assessment of pain e.g.
• neuropathic pain i.e. pain arising as a consequence of neuronal damage
• Several animal models of neuropathic pain are known in the art, which mimic many aspects of the clinical condition.
• One object of the present invention is thus to provide a target for diagnosis and prognosis for pain-related tissue states and pain-related diseases, especially pain.
• Slfn2 is involved in pain, which, in light of the then existing knowledge of possible functions of Slfn2 that rather hinted to completely different functions (e.g. in cell cycle control, see below) was not to be expected:
• a screening assay for the identification of genes involved in pain three different inbred mouse strains differing in their pain sensitivity were examined. The expression of various genes was correlated with the pain sensitivity of the mouse strains.
• Slfn2 is an interesting target for the identification and profiling of compounds concerning their analgesic effects. The above overview does not necessarily describe all problems solved by present invention.
• the invention relates to Slfn2 for use as an indicator of a pain-related tissue status or a pain related disease or as an indicator of the risk for developing a pain-related tissue status or disease.
• a second aspect of present invention relates to Slfn2 for use in the diagnosis or prognosis of a pain-related tissue status or of a pain-related disease.
• present invention relates to a method of identifying
• the method comprising detecting the level of Slfn2.
• present invention relates to a kit for use in a method of identifying a pain-related tissue status or the presence of a pain-related disease and/or the risk of developing a pain-related tissue status or a pain-related disease and/or the progression or a stage of a pain-related tissue status or a pain-related disease in an individual, the method comprising detecting the level of Slfn2 and the kit comprising one or means for detecting Slfn2.
• present invention relates to a kit for detecting Slfn2 comprising
• a data carrier comprising instructions for a method a pain-related tissue status or the presence of a pain-related disease and/or the risk of developing a pain-related tissue status or a pain-related disease and/or the progression or a stage of a pain-related tissue status or a pain-related disease in an individual, the method comprising detecting the level of Slfn2 (iii) a container.
• present invention relates to a means of detecting Slfn2 for use in diagnosis or prognosis of a tissue status or a disease.
• this invention relates to a method for diagnosing or prognosing a pain- related tissue status or a pain-related disease, comprising:
• an elevated level of Slfn2 in the individual as compared to the reference samples indicates the presence, increased risk of, or progression of the pain- related tissue status or pain-related disease.
• the Slfn2 is selected from the group consisting of SEQ ID NO: 1 , 2, 6, 7, 8, and 9.
• the detecting step comprises detecting the level of Slfn2 from a sample from an individual.
• the sample is selected from the group consisting of: cerebrospinal fluid, neural tissue, extraneural tissue, a neural cell, and an extraneural cell.
• the reference sample is selected from the group consisting of: a sample from a subject not having a pain-related tissue status or a pain- related disease, a sample from a subject having a pain-related tissue status or a pain- related disease, and a sample from the individual from an earlier point in time.
• the level of Slfn2 is either the level of Slfn2 nucleic acid or the level of Slfn2 protein.
• this invention also pertains to a kit for detecting Schlafen 2 (Slfn2) comprising:
• the means for detecting Slfn2 is a means for determining the expression level of Slfn2.
• the means for detecting is selected from the group consisting of: a means for detecting the expression level of the Slfn2 gene, a means for detecting the expression level of the Slfn2 RNA, and a means for detecting the expression level of the Slfn2 protein.
• said kit further comprises:
• the means comprises one or more nucleic acids or derivatives for detecting a Slfn2 gene or gene product or functionally active variant thereof, wherein the nucleic acid or derivative is selected from the group consisting of a nucleic acid probe, a polyamide or peptide nucleic acid (PNA), microRNA (miRNA), small interfering RNA (siRNA), primers for polymerase chain reaction (PCR), primers for reverse transcription (RT) reaction, and primers for DNA sequencing.
• PNA polyamide or peptide nucleic acid
• miRNA microRNA
• siRNA small interfering RNA
• PCR primers for reverse transcription
• RT reverse transcription
• the means comprises a peptide, polypeptide, or protein for detecting a Slfn2 gene or gene product or functionally active variant thereof, wherein the protein or polypeptide is a protein ligand, an antibody, a fragment or derivate thereof, a protein scaffold, a darpin or an anticalin, or wherein the polypeptide or peptide is a probe or a mass spectrometry probe.
• the means comprises:
• Slfn2 refers to murine Slfn2 comprising the nucleic acid sequence shown in SEQ ID NO: 1 , and the amino acid sequence shown in SEQ ID NO: 2, or to a functional variant thereof as defined below.
• functional variant of Slfn2 encompasses naturally occurring variants, e.g. the below listed homologs, especially the human and/or rat protein and/or nucleic acid as identified below.
• Slfn2 is a member of the (Schlafen) Slfn gene family comprising 10 family members in mouse and 7 family members in human (Schwarz et al., 1998 infra; Berger et al., Nature Immunology Vol.1 1 , Nr.4 (2010), p.335-343). Slfn2 is sometimes also referred to as schlafen family member 12 like (see e.g. accession number NP_035538 in the NCBI database that corresponds to the murine Slfn2 protein and coding sequence).
• Slfn 2 has strongest homology to human SLFN12L, rat slfn 2, orang utan SLFN12L and chimpanzee SLFN12L (see below).
• the gene of murine Slfn 2 is located on chromosome 4 (Ferguson et al., 2005).
• the reference sequence of the murine Slfn2 coding sequence is known and publicly available under the accession number: AF099973 from the NCBI (National Centre for Biotechnology Information; National Library of Medicine 38A, Bethesda, MD20894, USA; www.ncbi.nih.gov). Version AF099973.1 of the coding sequence (SEQ ID NO: 1 , positions 1 to 1684) is given in the following: 1 gctttaatgc agcaaggaac aaagaactca catgggctta gaggtgaaca cacaaccagg
• coding sequences comprised herein are indicated as cDNA sequences corresponding to the respective mRNA sequences.
• the above coding sequence (SEQ ID NO: 1 ) encodes a 378 amino acid protein the reference sequence of which is publicly available at the NCBI database under accession number AAC83826.
• the translated region starts at position 183 of SEQ ID NO: 1 and ends at position 1319.
• Version AAC83826.1 (SEQ ID NO: 2) is given in the following:
• the promoter region of murine Slfn2 has been cloned and analysed (see Wern-Joo et al., 2007). According to Wern-Joo et al., Molecular Immunology 44 (2007), 3273-3282 (the content of which is specifically incorporated herein by reference, see especially also page 3278, e.g. figure 3 of that article), the transcription start site is located 12 nucleotides upstream of the first nucleotide according to SEQ ID NO: 1 . In the sequence according to SEQ ID NO: 3 as given below, the nucleotides according to Wern-Joo infra (capital letters) are attached to the first part of SEQ ID NO: 1 (small letters):
• the translation start (methionin/atg) is underlined and typed bold.
• the promoter region of murine Slfn2 has been cloned and analysed (see Wern-Joo et al., 2007).
• the promoter of murine Slfn2 plus some downstream sequences according to Wern-Joo et al., 2007 is given in figure 4 (the sequence according to figure 4 includes some downstream positions incl. the translation start - Met).
• the upstream regulatory region, i.e. the promoter from position -1766 to +1 (+1 is underlined) is given in the following as SEQ ID NO: 4:
• Slfn2 sequence containing the upstream regulatory sequence according to Wern- Joo et al., 2007 (capital letters) and the complete coding sequence according to SEQ ID NO: 1 (small letters) comprises positions -1766 to +1696 and is given in the following:
• the upstream sequence comprises two AP-1 sites (-917 to -908 and -703 to -695) and one NF-kappa B binding site (-21 to -12). According to Wern-Joo et al., 2007 infra both AP-1 sites are important for maximal promoter activity upon LPS or CpG-DNA
• Slfn-2 promoter fragments usable for cloning of reporter-gene constructs are nucleic acids comprising or having the following positions according to SEQ ID NO:5: from -1766/-1265 to +1/+50/
• Slfn2 has been reported to be implicated in the regulation of quiescence in T-cells and monocytes (Berger et al., 2010 infra) and in interferon alpha-induced growth arrest in fibroblasts (Katsoulidis et al., Journal of Biological Chemistry Vol.284, Nr. 37 (2009)).
• a point mutation on position 135 of the 278 amino acid protein caused by a thymidine-to- adenosine transversion in the Slfn2 gene and leading to an isoleucine to asparagine substitution leads to a loss-of-f unction mutation causing lymphoid and myeloid immunodeficiency due to loss of quiescence of T-cells and monocytes in mice, and causes the "electra" phenotype, leaving natural killer cells or B cells unaffected (Berger et al., 2010).
• NFAT and NF-kB and Erk and Akt appear to be normally activated in electra mice, the mitogen-activated protein kinases p38 and Jnk are constitutively phosphorylated.
• expression levels of the anti-apoptotic Bcl-2 is reported to be lower in T-cells derived from electra mice, wherein transgenic expression of Bcl-2 rescues CD4+ and CD8+ T-cells derived from electra mice from apoptotic cell death (all Berger et al., 2010 infra, see also Horton and Powell, Nature Immunology Vol. 1 1 , Nr.4 (2010), p.281 -282).
• the reported mutation of Slfn2 appears to affect T- cell antigen receptor-induced signaling pathways, wherein other pathways are not affected.
• the isoleucine at position 135 of murine Slfn2 according to SEQ ID NO:2 (and corresponding to position 135 in rat Slfn2 according to SEQ ID NO:7 and position 123 in hsSLFN12L according to SEQ ID NO:9) is conserved among murine Slfn2, rat Slfn2 and homo sapiens SLFN12L hinting to an equal importance of said amino acid or surrounding protein region within the respective rat and homo sapiens proteins.
• Percent identity has been determined using the EMBL ClustalW program.
• the rat homolog of Slfn2 is schlafen family member 12-like (Slfn12L).
• the protein consists of 381 amino acids and the protein sequence is available under accession number NP_001 100501 .1 (SEQ ID NO: 7) and is encoded by the coding sequence NM_001 107031 .1 (from positions 193-1338).
• rat Slfn2 The coding sequence of rat Slfn2 according to NM_001 107031 .1 is given following (SEQ ID NO: 6):
• the divergent AAA domain spans positions 231 to 361 of the rat Slfn2 protein.
• the Human homolog of Slfn2 is schlafen family member 12-like (SLFN12L).
• the coding sequence can be retrieved from the NCBI database under accession number
• NM_001 195790.1 (SEQ ID NO: 8), coding for a 588 amino acid protein with sequence according to NCBI accession number NP_001 182719.1 (SEQ ID NO:9) .
• the divergent AAA domain spans positions 218 to 349 of human SLFN12L.
• SLFN2 homologs are known in Orang Utan (Pongo abelii, see Gene ID: 100190853 of NCBI database) and Chimpanzee (Pan troglodytes, see Gene ID: 454585 of NCBI database).
• Slfn2 protein refers e.g to a protein having or comprising
• amino acid sequence that is a fragment of the amino acid sequence according to (i) and/ or
• amino acid sequence that has at least 80%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence according to (i) or (ii) and/or
• amino acid sequence with at least 95% or at least 99% or 100% identity to at least 10, 1 1 , 12, 13, 14 or 15 consecutive amino acids of the Slfn2 protein according to SEQ ID NO:2, and/or
• amino acid sequence with at least 50% identity with the amino acid sequence according to SEQ ID NO:2 and/or 6.
• amino acid sequence with one of the following sequence motifs: YxxlxV, YxxlxVxxW, YxxlxVxxWS or YxxlxVxxWSxS with "x" standing for any amino acid and/or
• 7. carries a variant AAA domain, preferably with 50, 60, 70, 80, 90, 95 or more sequence identity with the variant AAA domain of the Slfn2 protein according to SEQ ID NO:2.
• the Slfn2 protein or the functionally active variant can comprise, consist of or have an amino acid sequence with the following features: An amino acid sequence having (1 ), (2), (3), (4), (5), (6) and/or (7).
• the protein can also have (1 ) and (2) or (1 ) and (3) or (1 ) and (4) or (1 ) and (5) or (1 ) and (6) or (1 ) and (7) or (2) and (3) or (2) and (6) or (2) and (5) or (2) and (6) or (2) and (7) or (3) and (4) or (3) and (5) or (3) and (6) or (3) and (7) or (6) and (5) or (4) and (6) or (4) and (7) or (5) and (6) or (5) and (7) or (6) and (7);
• the data carrier can also comprise (1 ) and (2) and (3) or (1 ) and (2) and (4) or (1 ) and (2) and (5) or (1 ) and
• the data carrier can also comprise (1 ) and (2) and (3) and (4) or (1 ) and (2) and (3) and (5) or (1 ) and (2) and (3) and (6) or (1 ) and (2) and (3) and (7) or (2) and (3) and (4) and (5) or (2) and (3) and (4) and (6) or (2) and (3) and (4) and (7) or (4) and (5) and (6) or (4) and (5) and (7) or (5) and (6) and (7);
• the data carrier can also comprise (1 ) and (2) and (3) and (4) or (1 ) and (2) and (3) and (5) or (1 ) and (2) and (3) and (6) or (1 ) and (2) and (3) and (7) or (2) and (3) and (4) and (5) or (2) and (3) and (4) and (6) or (2) and (3) and (4) and (7) or
• the data carrier can also comprise (1 ) and (2) and (3) and (4) and (5) or (1 ) and (2) and (3) and (4) and (6) or (1 ) and (2) and (3) and (4) and (7) or (2) and (3) and (4) and (5) and (6) or (2) and (3) and (4) and (5) and (7) or (3) and (4) and (5) and (6) and (7).
• pain refers to a complex subjective sensation reflecting real or potential tissue damage and the affective response to it.
• the term “pain” encompasses acute pain, subacute pain and chronic pain. It further encompasses disease-associated pain, algesia and migraine.
• Acute pain is a physiological signal indicating a possible or actual injury. Acute pain starts abruptly and is generally sharp in quality. It serves as a warning of disease or a threat to the body. Acute pain might be mild and last just a moment, or it might be severe and last for weeks. In most cases, acute pain disappears when the underlying cause of pain has been treated or healed. Acute pain might be somatogenetic (organic).
• Organic pain might be any pain resulting from a disorder, abnormality or chemical imbalance in an organ system, for example, the human or animal body.
• organic pain is a term covering pain causes that range in diversity from heartburn to multiples sclerosis.
• any system or body organ can be affected by organic pain including, but not limited to, the circulatory system, musculoskeletal system, or neurological system.
• Further examples of organic pain are constipation, diarrhoea, gastrointestinal tract infections, enteritis, colitis, or urinary tract infections.
• Unrelieved acute pain might lead to chronic pain. Subacute pain can be classified as a status between acute pain and chronic pain.
• Chronic pain can either be somatogenetic (organic) (see above) or psychogenic (psychosomatic). Chronic pain is frequently accompanied or followed by vegetative signs, which often result in depression.
• acute pain is pain that lasts less than 30 days
• chronic pain is pain of more than six months duration
• subacute pain is pain that lasts from one to six months
• acute pain may refer to pain that lasts less than 30 days (about 1 month)
• chronic pain may refer to pain that lasts more than six months
• subacute pain may refer to pain that lasts from one to six months.
• acute pain may refer to pain that does not extend beyond the expected period of healing and chronic pain may refer to pain that extends beyond the expected period of healing.
• pain means pain of nociceptive, inflammatory or neuropathic origin.
• Nociceptive pain is part of a rapid warning relay instructing the motor neurons of the central nervous system to minimize detected physical harm.
• Nociceptive pain is caused by activation of nociceptors. These nociceptors are free nerve endings that terminate just below the skin, in tendons, joints, and in body organs.
• Nociceptive pain may be divided into “superficial somatic” pain and “deep” pain.
• “Deep” pain in turn, may be divided into “deep somatic” pain and "visceral” pain.
• Superficial somatic pain is initiated by activation of nociceptors in the skin or superficial tissues, while “deep somatic” pain is initiated by stimulation of nociceptors in ligaments, tendons, bones, blood vessels, fasciae and muscles, and is dull, aching, poorly-localized pain.
• "Visceral” pain originates in the viscera (organs).
• “Visceral” pain may be well-localized, but often it is extremely difficult to locate, and several visceral regions produce "referred” pain when injured, where the sensation is located in an area distant from the site of pathology or injury. Examples of nociceptive pain include, but are not limited to, sprains, bone fractures, burns or obstructions.
• nociceptive pain can be associated with nerve damage caused by trauma, diseases such as diabetes, shingles, irritable bowel syndrome, late-stage cancer or the toxic effects of chemotherapy.
• Nociceptive pain is generally time-limited. This means that when the tissue damage heals, the pain typically resolves. Thus, nociceptive pain is usually acute pain. Another characteristic of nociceptive pain is that it tends to respond well to treatment with opioids.
• Neuropathic pain or neuralgia can be defined as non-nociceptive pain or as pain that is not related to activation of pain receptor cells in any part of the body.
• Neuropathic pain or neuralgia is rather caused by damage to or malfunction of the nervous system.
• neuropathic pain exists with no continuous nociceptive input.
• Neuralgia or neuropathic pain can be defined as non-nociceptive pain, or in other words, pain that is not related to activation of pain receptor cells in any part of the body. It is believed that neuralgia is pain produced by a change in neurological structure or function. Unlike nociceptive pain, neuralgia exists with no continuous nociceptive input.
• Neuralgia falls into two categories: central neuralgia and peripheral neuralgia. This unusual pain is thought to be linked to four possible mechanisms: ion gate malfunctions; the nerve becomes mechanically sensitive and creates an ectopic signal; cross signals between large and small fibers; and malfunction due to damage in the central processor.
• Neuralgia falls into two categories: central neuropathic pain or neuralgia (originating in the brain or spinal cord) and peripheral neuropathic pain or neuralgia (originating in the peripheral nervous system).
• Peripheral neuropathic pain can be described as burning, tingling, stabbing or as pins and needles. Bumping the "funny bone", for example, elicits peripheral neuropathic pain.
• Neuropathic pain is often difficult to diagnose, and most treatments show little or no effectiveness. Diagnosis typically involves locating the damaged nerve by identifying missing sensory or motor function. This may involve tests such as an EMG test or a nerve conduction test. Neuralgia is more difficult to treat than other types of pain because it does not respond well to normal pain medications. Examples of neuropathic pain include, but are not limited to, reflex sympathetic dystrophy, nerve trauma, components of cancer pain, phantom limb pain, entrapment neuropathy (e.g.
• peripheral neuropathy widespread nerve damage
• bony hyperostosis casts crutches, prolonged cramped postures; haemorrhage into a nerve; exposure to cold or radiation; collagen-vascular disorders; infectious diseases such as Lyme disease and HIV; toxins such as emetine, hexobarbital, barbital, chlorobutanol, sulfonamides, phenytoin, nitrofurantoin, the vinca alkaloids, heavy metals, carbon monoxide, triorthocresylphosphate, orthodinitrophenol, and other solvents and industrial poisons; autoimmune reactions; nutritional deficiency, and vitamin B deficiency in particular; and metabolic disorders such as hypothyroidism, porphyria, sarcoidosis, amyloidosis, uremia and diabetes (see e.g.
• Peripheral neuropathy may have several causes, for example, diabetes, chronic alcohol use, exposure to toxins (applied in chemotherapies), or vitamin deficiencies. Neuropathic pain is frequently chronic. It tends to have a less robust response to treatment with opioids. However, it may respond well to other drugs such as anti-seizure and anti-depressant drugs.
• the pain may also be a mixed category pain.
• the pain appears to be caused by a complex mixture of nociceptive pain and neuropathic pain.
• an initial nervous system dysfunction or injury may trigger the neural release of inflammatory mediators and subsequent neurogenic inflammation.
• Migraine headaches for example, probably represent a mixture of nociceptive pain and neuropathic pain.
• Inflammatory pain is associated with tissue damage and the resulting inflammatory process.
• Chronic pain may involve a mix of both inflammatory and neuropathic components, whereas inflammation may cause damage to the neurons and produce neuropathic pain or neuronal injury may cause an inflammatory reaction that contributes to the inflammatory pain.
• Algesia from the Greek word algesis, is the sensitivity to pain.
• Hyperalgesia is an increased sensitivity to pain, which may be caused by damage to nociceptors or peripheral nerves. Hyperalgesia can be experienced in focal, discrete areas, or as a more diffuse, body-wide form. Conditioning studies have established that it is possible to experience a learned hyperalgesia of the latter, diffuse form.
• the focal form is typically associated with injury, and is divided into two subtypes: (i) primary hyperalgesia which describes pain sensitivity that occurs directly in the damaged tissues or (ii) secondary hyperalgesia which describes pain sensitivity that occurs in surrounding undamaged tissues.
• disease-associated pain examples include, but are not limited to, pain associated with arthritis, back pain, cancer, epilepsy, lumbago, sciatica, lumbar spinal stenosis, cervical spinal stenosis, (clinical) depression, Fibromyalgia, Chronic Fatigue Syndrome, Complex Regional Pain Syndrome, Irritable Bowel Syndrome, Myofascial Pain Syndrome, Post-Vasectomy Pain Syndrome or Restless Leg Syndrome.
• Disease- associated pain may also be pain associated with diabetes, e.g. diabetic neuropathy or the diabetic foot, pain associated with Parkinson's Disease, pain associated with viral infections, e.g. influenza or HIV, or bacterial infections, pain associated with fever, pain associated with levodopa, e.g.
• Parkinson's disease pain associated with coronary heart disease, pain associated with shingles, pain associated with headache, pain associated with migraine, pain associated with muscular tension, especially chronic muscular tension, osteoarthritis as well as the pain-associated diseases listed under causes of neuropathic pain above.
• Migraine (from the Greek words hemi, meaning half, and kranion, meaning skull) is a debilitating condition characterized by moderate to severe headaches. Migraine headache is typically associated with unilateral pain (affecting one half of the head) and pulsating in nature. It usually lasts between 2 to 70 hours. Symptoms of migraine include, but are not limited to, nausea, vomiting, photophobia (increased sensitivity to light), and phonophobia (increased sensitivity to sound). The cause of migraine headache is unknown. The most common theory is a disorder of the serotonergic control system. Migraine is usually treated with analgesics for the headache and antimetics for the nausea. In addition, triggering conditions such as noise or bright light should be avoided.
• migraine is a disease that is associated with pain but is a disease that comprises more and different symptoms than symptoms of pain.
• Said types of disease that are intimately connected with symptoms of pain but that have in addition symptoms outside of pain/algesia, are referred to, herein, as pain-associated diseases.
• Some pain associated diseases are listed above in connection with disease-associated pain states and display pain as one characteristic disease symptom (e.g. migraine).
• Other pain associated diseases may be increased, provoked or even caused by pain, such as depression or epilepsy.
• pain also comprises equivalent states, stages, sensations and signalling cascades in lower animals or organisms, such as c. elegans.
• pain also comprises pre-stages of pain.
• tissue refers to an ensemble of cells of the same origin which fulfil a specific function concertedly.
• tissue include but are not limited to bone, cartilage, connective tissue, muscle tissue, nervous tissue, and epithelial tissue. Multiple tissues together form an "organ” to carry out a specific function.
• organ include but are not limited to brain, spinal cord, joint, skeleton, muscle, blood, brain, heart, liver, kidney, stomach, and skin.
• a joint is formed of many different tissues such as but not limited to bone, cartilage, synovium, muscle, ligament, and tendon.
• tissue furthermore relates to body liquids such as blood, liquor cerebrospinalis, saliva and lymph.
• tissue status means the condition of a tissue.
• the state of a tissue may be characterized by a specific morphology of such tissue or may be characterised by the expression of one or more specific molecules such as but not limited to peptides, proteins, and nucleic acids, or combinations thereof.
• the status of a tissue may be regarded as "healthy” or “normal” in case it resembles the condition of such tissue when being free from illness or injury and efficiently fulfilling its specific function.
• the status of a tissue may be regarded as "degenerative”, “diseased” or “abnormal” in case such tissue fails to fulfil its function due to an illness or injury.
• the status of a tissue may be regarded as "degenerative”, “diseased” or “abnormal” in case the morphology of the tissue or its molecule expression pattern is “altered” or “changed” in comparison to normal tissue.
• the morphology of a tissue or the expression pattern of specific molecules in a tissue may be an indicator for the state of a tissue.
• a tissues status include but are not limited to tissue degradation such as cartilage degradation, bone degradation, and degradation of the synovium, tissue inflammation such as cartilage inflammation, or inflammation of the synovium, tissue remodelling such as bon remodelling or cartilage remodelling, sclerosis, liquid accumulation, or proliferative tissue such as proliferation in wound healing processes, cystic formations, or in cancer.
• an altered (pathological) tissue status is preferably characterised by the tissue exhibiting a different (higher) Slfn2 level than normal (healthy) tissue, wherein the normal tissue can be tissue from another individual that is known not to have a pain-related tissue status or disease (e.g. not to experience pain) or the same tissue at an earlier time point (preferably before onset or worsening/progressing of the pain-related tissue status or disease, e.g. of pain).
• a disease status is preferably characterised by an organ or individual exhibiting different slfn2 level in comparison to a normal organ or individual or in comparison to the same organ or individual at an earlier time point.
• pain-related or pain-associated tissue status refers to a status of a tissue typically associated with pain or a pain-associated disease, typically a diseased or abnormal tissue state such as but not limited to an infection of tissue, infarction, necrosis or other states of tissue associated with pain.
• tissue refers to an ensemble of cells of the same origin which concertedly fulfil a specific function.
• tissue include but are not limited to bone, cartilage, connective tissue, muscle tissue, nervous tissue, and epithelial tissue. Multiple tissues together form an "organ” to carry out a specific function.
• organ include but are not limited to joint, skeleton, muscle, blood, brain, heart, liver, kidney, stomach, and skin.
• a joint is formed of many different tissues such as but not limited to bone, cartilage, synovium, muscle, ligament, and tendon.
• disease and “disorder” are used interchangeably herein, referring to an abnormal condition, especially an abnormal medical condition such as an illness or injury, wherein a tissue, an organ or an individual is not able to efficiently fulfil its function anymore.
• a disease is associated with specific symptoms or signs indicating the presence of such disease. The presence of such symptoms or signs may thus, be indicative for a tissue, an organ or an individual suffering from a disease. An alteration of these symptoms or signs may be indicative for the progression of such a disease.
• a progression of a disease is typically characterized by an increase or decrease of such symptoms or signs which may indicate a "worsening" or “bettering” of the disease.
• tissue, an organ or an individual being at "risk of developing" a disease is in a healthy state but shows potential of a disease emerging.
• the risk of developing a disease is associated with early or weak signs or symptoms of such disease. In such case, the onset of the disease may still be prevented by treatment.
• a disease include but are not limited to traumatic diseases, inflammatory diseases, infectious diseases, cutaneous conditions, endocrine diseases, intestinal diseases, neurological disorders, joint diseases, genetic disorders, autoimmune diseases, and various types of cancer.
• Symptoms of a disease are implication of the disease noticeable by the tissue, organ or organism having such disease and include but are not limited to pain, weakness, tenderness, strain, stiffness, and spasm of the tissue, an organ or an individual.
• “Signs” or “signals” of a disease include but are not limited to the change or alteration such as the presence, absence, increase or elevation, decrease or decline, of specific indicators such as biomarkers or molecular markers, or the development, presence, or worsening of symptoms.
• Symptoms of pain include, but are not limited to an unpleasant sensation that may be felt as a persistent or varying burning, throbbing, itching or stinging ache.
• biomarker or “indicator” are used interchangeably herein.
• a biomarker can be defined as a substance within a biological system that is used as an indicator of a biological state of said system.
• biomarker is sometimes also applied to means for the detection of said endogenous substances (e.g. antibodies, nucleic acid probes etc, imaging systems). In the context of present invention, however, the term “biomarker” shall be only applied for the substance, not for the detection means.
• biomarkers can be any kind of molecule present in a living organism, such as a nucleic acid (DNA, mRNA, miRNA, rRNA etc.), a protein (cell surface receptor, cytosolic protein etc.), a metabolite or hormone (blood sugar, insulin, estrogen, etc.), a molecule characteristic of a certain modification of another molecule (e.g. sugar moieties or phosphoryl residues on proteins, methyl-residues on genomic DNA) or a substance that has been internalized by the organism or a metabolite of such a substance.
• Slfn2 can thus be used as an indicator of pain, i.e. qualifies as biomarker for pain.
• the indicator or biomarker refers to a sign or signal for a condition or is used to monitor a condition.
• a condition refers to the biological status of a cell, tissue or organ or to the health and/or disease status of an individual.
• An indicator may be the presence or absence of a molecule, including but not limited to peptide, protein, and nucleic acid, or may be a change in the expression level or pattern of such molecule in a cell, or tissue, organ or individual.
• An indicator may be a sign for the onset, development or presence of a disease in an individual or for the further progression of such disease.
• An indicator may also be a sign for the risk of developing a disease in an individual.
• Known indicators of pain include but are not limited to: Cystatin C (Pain 102 (2003) p.251 -256), Cathepsin H and Cathepsin C and the circulatory pain-indicators beta endorphin, serotonin, 5-hydroxy indole acetic acid, anandamide, N-palmitoylethanolamide (for an overview see e.g. Journal of the American Osteopathic Association (2007) 107; p.387- 400).
• level of Slfn2 refers to the expression level or the level of gene copies (also absence of Slfn2 or parts thereof or presence of fragments) present in a cell, tissue, organ or individual.
• expression level refers to the amount of gene product present in the body or a sample at a certain point of time.
• the expression level can e.g. be measured/quantified/detected by means of the protein or mRNA expressed from the gene.
• the expression level can for example be quantified by normalizing the amount of gene product of interest (e.g. Slfn2 mRNA or protein) present in a sample with the total amount of gene product of the same category (total protein or mRNA) in the same sample or a reference sample (e.g.
• the expression level can be measured or detected by means of any method as known in the art, e.g. methods for the direct detection and quantification of the gene product of interest (such as mass spectrometry) or methods for the indirect detection and measurement of the gene product of interest that usually work via binding of the gene product of interest with one or more different molecules or detection means (e.g. primer(s), probes, antibodies, protein scaffolds) specific for the gene product of interest, here Slfn2.
• methods for the direct detection and quantification of the gene product of interest such as mass spectrometry
• methods for the indirect detection and measurement of the gene product of interest that usually work via binding of the gene product of interest with one or more different molecules or detection means (e.g. primer(s), probes, antibodies, protein scaffolds) specific for the gene product of interest, here Slfn2.
• the determination of the level of gene copies of Slfn2 comprising also the determination of the the absence or presence of one or more fragments (e.g. via nucleic acid probes or primers, e.g. quantitative PCR, Multiplex ligation-dependent probe amplification (MLPA) PCR (http://www.mlpa.com/) is also within the knowledge of the skilled artisan.
• nucleic acid probes or primers e.g. quantitative PCR, Multiplex ligation-dependent probe amplification (MLPA) PCR (http://www.mlpa.com/)
• the term "variant" is to be understood as a polynucleotide or protein which differs in comparison to the polynucleotide or protein from which it is derived by one or more changes in the sequence and comprises fragments or derivatives.
• the polypeptide or polynucleotide from which a protein or nucleic acid variant is derived is also known as the parent polypeptide or nucleic acid.
• a variant is constructed artificially, preferably by gene-technological means.
• the parent polypeptide or polynucleotide is a wild-type protein or nucleic acid.
• variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent molecule or from artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent molecule, i.e. is functionally active, if the variant is meant to be a functional variant.
• a "variant" as used herein can be characterized by a certain degree of sequence identity to the parent polypeptide or parent nucleic acid from which it is derived. More precisely, a protein variant in the context of the present invention exhibits 80% or more sequence identity to its parent polypeptide. A nucleic acid variant in the context of the present invention exhibits 80% or more sequence identity to its parent nucleic acid. The term "80% or more sequence identity” is used throughout the specification with regard to polypeptide and nucleic acid sequence comparisons.
• This expression preferably refers to a sequence identity of 80% or more, 81 % or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91 % or more, 92% or more, 93% or more, 94% or more, 95% or more 96% or more, 97% or more, 98% or more, or 99% or more to the respective reference polypeptide or to the respective reference nucleic acid.
• sequence alignments can be carried out with several art-known algorithms, e.g. with the mathematical algorithm of Karl in and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673- 80) available e.g.
• sequence identity may be calculated using e.g.
• BLAST, BLAT or BlastZ (or BlastX).
• BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410.
• Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.
• Sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle- LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1 :154-162) or Markov random fields.
• Shuffle- LAGAN Brudno M., Bioinformatics 2003b, 19 Suppl 1 :154-162
• Markov random fields a homology mapping technique like Shuffle- LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1 :154-162) or Markov random fields.
• Hybridization can also be used as a measure of sequence identity or homology between two nucleic acid sequences.
• a nucleic acid sequence encoding miRNAs or a portion thereof can be used as a "hybridization probe” according to standard hybridization techniques.
• the hybridization of a Slfn2 probe to DNA or RNA from a test source is an indication of the presence of the Slfn2 DNA or RNA in the test source.
• Hybridization conditions are known to those skilled in the art and can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 6.3.1 - 6.3.6, 1991 .
• Mode hybridization conditions are defined as equivalent to hybridization in 2X sodium chloride/sodium citrate (SSC) at 30°C, followed by a wash in 1 X SSC, 0.1 % SDS at 50°C.
• Highly stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.
• SSC sodium chloride/sodium citrate
• Lowly stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C.
• protein and “polypeptide” are used interchangeably herein and refer to any peptide-linked chain of amino acids, regardless of length or post-translational modification.
• Proteins usable in the present invention can be further modified by chemical or biological modification.
• This means such a biologically or chemically modified polypeptide comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids, phosphorylated amino acids or covalent attachment of amino-acid chains e.g. for stabilization of the protein/polypeptide (such as attachment of, e.g. rPEG, XTEN or PAS).
• Modification of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility.
• Chemical modifications applicable to the variants usable in the present invention include without limitation: PEGylation, glycosylation of non-glycosylated parent polypeptides, or the modification of the glycosylation pattern present in the parent polypeptide, rPEGylation, XTENylation or PASylation.
• peptide refers to a short polymer of amino acids linked by peptide bonds. It has the same chemical (peptide) bonds as proteins, but is commonly shorter in length.
• the shortest peptide is a dipeptide, consisting of two amino acids joined by a single peptide bond. There can also be a tripeptide, tetrapeptide, pentapeptide, etc.
• the peptide has a length of up to 8, 10, 12, 15, 18 or 20 amino acids.
• a peptide has an amino end and a carboxyl end, unless it is a cyclic peptide.
• polypeptide refers to a single linear chain of amino acids bonded together by peptide bonds and preferably comprises at least about 21 amino acids.
• a polypeptide can be one chain of a protein that is composed of more than one chain or it can be the protein itself if the protein is composed of one chain.
• protein refers to a molecule comprising one or more polypeptides that resume a secondary and tertiary structure and additionally refers to a protein that is made up of several polypeptides, i.e. several subunits, forming quaternary structures.
• the protein has sometimes non- peptide groups attached, which can be called prosthetic groups or cofactors.
• the primary structure of a protein or polypeptide is the sequence of amino acids in the polypeptide chain.
• the secondary structure in a protein is the general three-dimensional form of local segments of the protein. It does not, however, describe specific atomic positions in three-dimensional space, which are considered to be tertiary structure.
• the secondary structure is defined by patterns of hydrogen bonds between backbone amide and carboxyl groups.
• the tertiary structure of a protein is the three-dimensional structure of the protein determined by the atomic coordinates.
• the quaternary structure is the arrangement of multiple folded or coiled protein or polypeptide molecules molecules in a multi-subunit complex.
• amino acid chain and “polypeptide chain” are used synonymously in the context of present invention.
• Slfn2 or Slfn2 protein also encompasses naturally occurring variants such as homologs and orthologs in same or different species, in particular in human.
• the different aspects of present invention also relate to functional variants of Slfn2 protein, either naturally occurring or non-naturally occurring.
• variant of a Slfn2 protein comprises fragments and derivatives of a protein.
• a fragment is a protein or polypeptide that carries one or more end-terminal (n- and/or c-terminal) or internal deletions of one, two or more amino acids, when compared to the full-length protein.
• a functional fragment of the protein is any fragment of this protein having at least one and preferably two or more of the functional characteristics of the full-length protein.
• the term derivative of a protein comprises any type of modification of the protein in comparison to the naturally-occurring form, and in the context of present invention especially in comparison to the Slfn2 protein according to SEQ IDs no. 2 and 9, which is not a deletion.
• a functional derivative of the protein is any derivative of this protein having at least one and preferably two or more of the functional characteristics of the unmodified protein.
• the variants of Slfn2 of present invention also comprise functional derivatives of fragments of Slfn2.
• Non-naturally occurring variants may be obtained by a limited number of amino acid deletions, insertions and/or substitutions, particularly deletions, insertions and/or substitutions of at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid(s).
• a Slfn2 variant of the present invention can e.g. be a functionally active variant, i.e. the variant maintains at least one of the biological functions of Slfn2 as described herein, e.g. its function in the context of pain (e.g. as manifestation of the pain phenotype "mechanic hyperalgesia") or as known in the literature.
• maintenance of biological function is defined as having 50 % or more, 60 % or more, 70 % or more, 80 % or more or 90 % or more or 95 % or more of the activity of the natural occurring Slfn2.
• a non-functional variant of a Slfn2 protein may also be subject of one of the aspects of present invention, e.g. as negative control.
• the biological activity may be determined as known to the skilled person.
• the manifestation of the pain phenotype "mechanic hyperalgesia" can be determined as detailed in the Examples and in Persson et al., 2009, Molecular Pain 5:7.
• the Slfn2 variant may be modified in order to comprise a further component. Accordingly, the variant may be a molecule having a domain composed of a naturally occurring Slfn2 protein or a variant thereof as detailed herein and at least one further component.
• variant may be a fusion protein comprising (i) a Slfn2 protein or functionally active variant and (ii) a further protein component.
• the protein may be coupled to a marker, such as a tag used for purification purposes (e.g. 6 His (or HexaHis) tag, Strep tag, HA tag, c-myc tag or glutathione S-transferase (GST) tag).
• a marker such as a tag used for purification purposes (e.g. 6 His (or HexaHis) tag, Strep tag, HA tag, c-myc tag or glutathione S-transferase (GST) tag).
• a marker such as a tag used for purification purposes (e.g. 6 His (or HexaHis) tag, Strep tag, HA tag, c-myc tag or glutathione S-transferase (GST) tag).
• GST glutathione S-transferas
• the proteins are purified in two or more separation chromatography steps, in each case utilizing the affinity of a first and then of a second tag.
• double or tandem tags are the GST-His-tag (glutathione-S-transferase fused to a polyhistidine- tag), the 6xHis-Strep-tag (6 histidine residues fused to a Strep-tag), the 6XH is-tag 100- tag (6 histidine residues fused to a 12-amino-acid protein of mammalian MAP-kinase 2), 8xHis-HA-tag (8 histidine residues fused to a hemagglutinin-epitope-tag), His-MBP (His- tag fused to a maltose-binding protein, FLAG-HA-tag (FLAG-tag fused to a hemagglutinin-epitope-tag), and the FLAG-Strep-tag.
• GST-His-tag glutas
• the marker could be used in order to detect the tagged protein, wherein specific antibodies could be used. Suitable antibodies include anti-HA (such as 12CA5 or 3F10), anti-6 His, anti-c-myc and anti- GST. Furthermore, the Slfn2 protein could be linked to a marker of a different category, such as a fluorescence marker or a radioactive marker, which allows for the detection of Slfn2. In a further embodiment, Slfn2 could be part of a fusion protein, wherein the second part could be used for detection, such as a protein component having enzymatic activity.
• a marker of a different category such as a fluorescence marker or a radioactive marker
• the Slfn2 variant could be a Slfn2 fragment, wherein the fragment is still functionally active.
• This may include Slfn2 proteins with short internal and/or C- and/or N-terminal deletions (e.g. deletions of at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6 5, 4, 3, 2, or 1 amino acid).
• the Slfn2 fragment may be further modified as detailed above for the Slfn2 protein.
• the Slfn2 variant may comprise one or more amino acid substitution(s).
• Semi-conservative and especially conservative amino acid substitutions wherein an amino acid is substituted with a chemically related amino acid are preferred.
• Typical substitutions are among the aliphatic amino acids, among the amino acids having aliphatic hydroxyl side chain, among the amino acids having acidic residues, among the amide derivatives, among the amino acids with basic residues, or the amino acids having aromatic residues.
• Typical semi-conservative and conservative substitutions are:
• the Slfn2 protein or fragment or variant with substitution may be modified as detailed above for the Slfn2 protein or fragment or variant.
• all details given with respect to Slfn2 protein also relate to functionally active variants thereof, unless stated otherwise.
• the above modifications of the Slfn2 protein may be combined.
• the variant of the present invention may be e.g. fragment of Slfn2 having a marker fused to it, or a Slfn2 protein fragment comprising one or more amino acid substitutions.
• a derivative of Slfn2 protein or a Slfn2 protein fragment is a Slfn2 protein or fragment carrying any kind of modification of the amino acid sequence that is not a deletion and/or carrying any other kind of modification, such as a chemical or biological modification.
• Derivatives comprise proteins carrying one or more modifications e.g. leading to the stabilization of the polypeptide, or enabling a specific targeting of the polypeptide to certain cells or facilitating its entry into or uptake by cells (such as cell- permeant phosphopeptides ortho coupling to cell-permeant peptide vectors, e.g.
• the Slfn2 protein is a naturally occurring Slfn2 protein as detailed above, such as a naturally occurring mouse Slfn2 protein such as comprising or having SEQ ID NO: 2, or a naturally occurring human orthologous protein, such as comprising or having SEQ ID NO: 9.
• the terms "nucleic acid” or “nucleic acid molecule” are used synonymously and are understood as single or double-stranded oligo- or polymers of deoxyribonucleotide or ribonucleotide bases or both.
• nucleic acid is formed through phosphodiester bonds between the individual nucleotide monomers.
• nucleic acid includes but is not limited to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• the depiction of a single strand of a nucleic acid also defines (at least partially) the sequence of the complementary strand.
• the nucleic acid may be single or double stranded, or may contain portions of both double and single stranded sequences.
• the nucleic acid may be obtained by biological, biochemical or chemical synthesis methods or any of the methods known in the art.
• nucleic acid comprises the terms "polynucleotide” and "oligonucleotide”.
• nucleic acid comprises cDNA, genomic DNA, recombinant DNA, cRNA and mRNA.
• a nucleic acid may consist of an entire gene, or a portion thereof, the nucleic acid may also be a microRNA (miRNA) or small interfering RNA (siRNA).
• miRNAs are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long, found in all eukaryotic cells.
• MircoRNAs miRNAs
• miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing.
• RNA molecules short ribonucleic acid
• RNAi RNA interference
• the nucleic acid can also be an artificial nucleic acid.
• Artificial nucleic acids include polyamide or peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule.
• nucleic acids can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1460) Chemical Reviews, 90, 543-584).
• Aptamers are nucleic acids which bind with high affinity to a polypeptide, here Slfn2.
• Aptamers can be isolated by selection methods such as SELESIfn2 (see e.g. Jayasena (1469) Clin. Chem., 45, 1628-50; Klug and Famulok (1464) M. Mol. Biol. Rep., 20, 97-107; US 5,582,981 ) from a large pool of different single-stranded RNA molecules.
• Aptamers can also be synthesized and selected in their mirror-image form, for example as the L-ribonucleotide (Nolte et al. (1466) Nat. Biotechnol., 14, 1 1 16-9; Klussmann et al. (1466) Nat. Biotechnol., 14, 1 1 12-5).
• L-ribonucleotide Nolte et al. (1466) Nat. Biotechnol., 14, 1 1 16-9; Klussmann et al. (1466) Nat. Biotechnol., 14, 1 1 12-5).
• Forms which have been isolated in this way enjoy the advantage that they are not degraded by naturally occurring ribonucleases and, therefore, possess greater stability.
• Nucleic acids may be degraded by endonucleases or exonucleases, in particular by DNases and RNases which can be found in the cell.
• nucleic acids it is, therefore, advantageous to modify the nucleic acids in order to stabilize them against degradation, thereby ensuring that a high concentration of the nucleic acid is maintained in the cell over a long period of time (Beigelman et al. (1465) Nucleic Acids Res. 23:3989-94; WO 95/1 1910; WO 98/37240; WO 97/291 16).
• stabilization can be obtained by introducing one or more internucleotide phosphorus groups or by introducing one or more non-phosphorus internucleotides.
• Suitably modified internucleotides are compiled in Uhlmann and Peyman (1460), supra (see also Beigelman et al. (1465) Nucleic Acids Res.
• Modified internucleotide phosphate radicals and/or non-phosphorus bridges in a nucleic acid which can be employed in one of the uses according to the invention contain, for example, methyl phosphonate, phosphorothioate, phosphoramidate, phosphorodithioate and/or phosphate esters, whereas non-phosphorus internucleotide analogues contain, for example, siloxane bridges, carbonate bridges, carboxymethyl esters, acetamidate bridges and/or thioether bridges. It is also the intention that this modification should improve the durability of a pharmaceutical composition which can be employed in one of the uses according to the invention.
• oligonucleotide when used in the context of one of the different aspects of present invention, refers to a nucleic acid of up to about 50 nucleotides, e.g. 2 to about 50 nucleotides in length.
• polynucleotide when used in the context of one of the different aspects of present invention, refers to a nucleic acid of more than about 50 nucleotides in length, e.g. 51 or more nucleotides in length.
• means for detection or means for detecting as used herein refers to any means suitable for specific detection of Slfn or nucleic acid, especially in a probe, in isolated organic matter (e.g. isolated protein or nucleic acid), a tissue, an organ or an animal body. The detection is usually mediated by a specific binding to the target molecule, here Slfn2 nucleic acid or polypeptide.
• Probes and primers are short polynucleotides or oligonucleotides for the detection of nucleic acids in a sample or in vivo by hybridizing (probe) to the target nucleic acid or by hybridization to and amplification of the target nucleic acid.
• Nucleic acids suitable for use as probe or primer comprise e.g. a polynucleotide probe, one or more primers (e.g.
• a primer pair preferably one or more primers for polymerase chain reaction (PCR), reverse transcription (RT) reaction, or DNA sequencing, a peptide- or polyamid- nucleic acid (PNA), a locked nucleic acid (LNA), a glycol nucleic acid (GNA), a threose nucleic acid (TNA), a microRNA (miRNA), and a small interfering RNA (siRNA).
• the one or more primers e.g. a primer pair
• RT-PCR real time polymerase chain reaction
• qRT-PCR quantitative real time polymerase chain reaction
• probe refers to a nucleic acid which is typically used for the detection of target RNA and/or DNA sequences that is complementary to the sequence of the probe.
• a probe hybridizes to single-stranded nucleic acid (DNA or RNA) whose nucleotide sequence allows for nucleotide pairing due to complementarity between the probe and the target sequence.
• the length of a probe depends on the intended use as well as the required specificity of the probe. Typically, a probe is 20-500 (i.e.
• Probes are used in various experimental set ups such as but not limited to Southern and Northern Blots, for real-time PCR and In Situ Hybridization (ISH) as well as for microarray experiments.
• a probe may be unlabeled, directly labelled, or indirectly labelled, such as with biotin to which a streptavidin complex may later bind.
• Said label may be a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
• suitable labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which are or can be made detectable.
• a label may be incorporated into nucleic acids at any position, e.g. at the 3' end, at the 5' end or internally.
• probe also encompasses nucleic acids differing in the composition of their backbone such as but not limited to peptide nucleic acids (PNAs), locked nucleic acids (LNAs), glycol nucleic acids (GNAs) and threose nucleic acids (TNAs).
• PNAs peptide nucleic acids
• LNAs locked nucleic acids
• GNAs glycol nucleic acids
• TAAs threose nucleic acids
• Said nucleic acid as a probe may be unlabeled, directly labeled, or indirectly labeled, such as with biotin to which a streptavidin complex may later bind.
• Said label may be a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
• suitable labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which are or can be made detectable.
• a label may be incorporated into nucleic acids at any position, e.g. at the 3' end, at the 5' end or internally.
• primer refers to a single-strand nucleic acid which typically serves as a starting point for DNA-replicating enzymes.
• a primer binds to or hybridises with a DNA template and typically comprises a sequence being complementary to the DNA sequence to which it is supposed to bind.
• a primer may also comprise additional sequences e.g. sequences serving as nuclease cleavage sites (e.g. Bam H1 , Hind III, etc.).
• the length of a primer is chosen depending on the intended use. For instance, primers used for the amplification of DNA in Polymerase-Chain Reactions (PCR) typically have a length of at least 10 nucleotides, preferably between 10 to 50 (i.e.
• primers are used for sequencing of DNA templates.
• primer also encompassed in the term "primer” are "degenerate primers", which are a mixture of similar but not identical primers.
• a primer may be tagged or labeled with a marker molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
• Probes and primers for the detection of Slfn2 are known in the art (see e.g. Wern-Joo et al, 2007) and can be ordered custom-made by commercial vendors against any known sequence including that of Slfn2. Examples include (all taken from Wern-Joo et al., 2007):
• 5'-CTCACCTCAGAAAACAGGAGAATGC-3' (sense murine Slfn2; SEQ ID NO:10), 5'-CAGAAGTGAGTGACAGGCAGCTG-3' (anti-sense, murine Slfn2; SEQ ID NO:1 1 ), 5'-CGGCTAGCTTGGTGGCTCCTAGCGRR-3' (sense Slfn2 -1766; SEQ ID NO.12), 5'-CGGCTAGCGGAACTGAACTGGAGCT-3' (sense Slfn2 -1265; SEQ ID NO.13), 5'-CCCTCGAGCCATGTCAGCAGTTGGTAAAGC-3' (anti-sense SIfn 2 +198; SEQ ID NO:14).
• the skilled artisan knows how to select suitable primers.
• peptide nucleic acids PNAs
• locked nucleic acids LNAs
• glycol nucleic acids GAAs
• threose nucleic acids TAAs
• the above-mentioned nucleic acids are e.g. for detecting the SIfn nucleic acid sequences of present invention, e.g. one or more of the SIfn nucleic acid sequences of embodiment 20 and are e.g.
• nucleic acids may allow the determination of the expression level of Slfn2 on the Slfn2 gene or Slfn2 mRNA level, e.g. in a sample, such as in a sample from a subject or in an assay, such as a biochemical or cellular assay (e.g. in a mixture in the context of a biochemical assay or in a cell in the context of a cellular assay).
• variant of a nucleic acid comprises fragments and derivatives of a nucleic acid.
• the variants of present invention also comprise functional derivatives of fragments of the nucleic acid.
• Non-naturally occurring variants may be obtained by a limited number of nucleotide deletions, insertions and/or substitutions, particularly deletions, insertions and/or substitutions of at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide(s).
• a fragment of a nucleic acid is a nucleic acid that carries one or more terminal (5' (upstream) and/or 3' (downstream)) or internal deletions of one, two or more
• a functional fragment of the nucleic acid is any fragment of the Slfn2 nucleic acid having at least one and preferably two or more of the functional characteristics of the full-length nucleic acid (e.g. the ability to confer Slfn2-characteristic transcriptional regulation of downstream elements or the characteristic to code for a Slfn2 protein).
• derivative of a nucleic acid comprises any type of modification (e.g. a chemical or biological modification) of the nucleic acid in comparison to the naturally- occurring form, and in the context of present invention especially in comparison to the Slfn2 nucleic acid according to SEQ ID NOs. 1 , 3, 4, 6 or 8 that is not a deletion.
• Derivatives comprise Slfn2 nucleic acids carrying one or more modifications leading to the stabilization of the nucleic acid (such as phosphoorothioate modification or modification of the nucleic acid backbone).
• a functional derivative of the nucleic acid is any derivative of this nucleic acid having at least one and preferably two or more of the functional characteristics of the unmodified nucleic acid.
• Slfn2 nucleic acid encompasses nucleic acids coding for the above Slfn2 protein and regulatory nucleic acids of the Slfn2 gene as well as naturally occurring and non-naturally occurring variants thereof (as defined herein).
• the term relates to coding or non-coding regions of the Slfn2 gene, wherein these sections are of a relevant size in order to be specific for that gene. Examples of those regions are introns, exons or regulatory elements such as a Slfn2 promoter.
• Slfn2 or Slfn2 nucleic acid also encompasses naturally occurring variants such as homologs and orthologs in same or different species, in particular in human (e.g.
• Slfn2 nucleic acids according to SEQ IDs NO. 1 (mouse), 6 (rat) or 8 (human).
• the different aspects of present invention also relate to functional variants of Slfn2 nucleic acid, either naturally occurring or non- naturally occurring.
• Preferred aspects of a Slfn2 nucleic acid relate to the Slfn2 gene, promoter, DNA, cDNA or mRNA or oligonucleotides or polynucleotides able to specifically hybridize with one of these nucleic acids.
• Slfn2 nucleic acids are e.g. a Slfn2 nucleic acid coding for the naturally occurring Slfn2 protein as detailed above (e.g.
• a protein comprising or having an amino acid sequence according to SEQ ID NO: 2, 7 or 9) such as a nucleic acid comprising or having a nucleotide sequence according to SEQ ID NO: 1 , 3, 4, 6, or 8).
• Slfn2 nucleic acid is a Slfn2 nucleic acid able to drive expression of a downstream element such as a nucleic acid derived from the Slfn2 gene and comprising regulatory elements thereof but lacking the Slfn2 coding sequence, e.g. a nucleic acid comprising or consisting of a nucleotide sequence according to SEQ ID NO:4.
• a Slfn2 nucleic acid variant of the present invention can be a functionally active variant, i.e. the variant maintains at least one of the biological functions of Slfn2 nucleic acid as described herein, e.g. its function in the context of pain (e.g. as manifestation of the pain phenotype "mechanic hyperalgesia", the ability to confer Slfn2-characteristic transcriptional regulation of downstream elements or the characteristic to code for a Slfn2 protein) or as known in the literature.
• maintenance of biological function is defined as having 50 % or more, 60 % or more, 70 % or more, 80 % or more or 90 % or more or 95 % or more of the activity of the natural occurring Slfn2.
• a non- functional variant of a Slfn2 nucleic acid may also be subject of one of the aspects of present invention, e.g. as negative control.
• the biological activity may be determined as known to the skilled person, for example, the manifestation of the transcriptional activity as detailed in Wern-Joo et al., 2007; examples of functional and non-functional variants (fragments and derivatives) of nucleic acids, especially different promoter variants are disclosed in Wern-Joo et al, 2007 the disclosure of which is explicitly included herein, as reference.
• nucleic acids comprising a Slfn2 promoter comprise a Slfn2 nucleic acid from -1256 to +1 or from -1265 to +198 or from +1699 to +1 or from -1699 to +198 with respect to the sequence according to SEQ ID NO: 5.
• antibody or fragment thereof refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that specifically binds an antigen. Also comprised are immunoglobulin-like proteins that are selected through techniques including, for example, phage display to specifically bind to a target molecule or target protein.
• the immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass of immunoglobulin molecule.
• antibodies and fragments thereof include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (in particular CDR-grafted), deimmunized, or chimeric antibodies, single chain antibodies (e.g. scFv), Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, diabodies or tetrabodies (Holliger P. et al., 1993), nanobodies, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
• the antibody fragments are mammalian, preferably human antigen-binding antibody fragments and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VH domain.
• Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable domain(s) alone or in combination with the entirety or a portion of the following: hinge region, CL, CH1 , CH2, and CH3 domains.
• the antigen-binding fragments may also comprise any combination of variable domain(s) with a hinge region, CL, CH1 , CH2, and CH3 domains.
• Antibodies usable in the invention may be from any animal origin including birds and mammals.
• the antibodies are human, simian (e.g. chimpanzee, bonobo, macaque), rodent (e.g. mouse and rat), donkey, sheep rabbit, goat, guinea pig, camel, horse, or chicken. It is particularly preferred that the antibodies are of human or murine origin.
• "human antibodies” include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins.
• Antibodies or antibody fragments specific for the Slfn2 protein may be used in a variety of assays in order to quantitate the Slfn2 protein.
• Well known methods include, for example, immunoprecipitation, antibody sandwich assays, ELISA and affinity chromatography methods (see above).
• the antibody that recognizes the target antigen here Slfn2 or a functionally active variant thereof is generally called the "primary antibody".
• Said antibody may be labeled with a detectable tag/label in order to allow direct detection of the target antigen.
• Said detectable tag/label may be an enzymatic, fluorescent or radioisotope tag/label.
• the primary antibody is not labeled for direct detection. Instead a "secondary antibody” that has been labeled with a detectable tag/label (e.g. enzymatic, fluorescent or radioisotope tag/label) is applied in a second step to probe for the primary antibody, which is bound to the target antigen.
• the primary antibody or the secondary antibody may be labeled with an affinity tag such as biotin.
• an affinity tag such as biotin.
• Antibodies against Slfn2 are known in the art (see e.g. Katsoulidis et al., 2009) and can be purchased from commercial vendors such as antibodies-online.com (http://www.antibodies-online.com).
• An example of an n-terminal anti Slfn2 antibody suitable for use in e.g. Western Blot or ELISA is a polyclonal rabbit anti-mouse antibody that has been generated using a synthetic peptide corresponding to the n-terminal residues of mouse Slfn2 and that can be purchased from antibodies-online under ABIN393095.
• protein scaffold in the context of present invention refers to non-antibody recognition proteins derived from one or more of structurally different protein families and able to bind with high specificity to a selected target, thereby mimeting the binding principle of immunoglobulins to varying degrees (see e.g. Hey, T., et al., 2005: Artificial, non-antibody binding proteins for pharmaceutical and industrial applications, Trends in Biotechnology, Vol. 23, No. 10, the disclosure of which is incorporated herein, by reference).
• Examples comprise lipocalin-derived repeat proteins such as darpins, ankyrin-repeat proteins such as anticalins, nanobodies, affibodies, maxibodies, trans- bodies, tetranectin, iMabs, Adnectin, domain antibodies, Kunitz-type domains, Evibodies, Affilins, Microbodies).
• lipocalin-derived repeat proteins such as darpins, ankyrin-repeat proteins such as anticalins, nanobodies, affibodies, maxibodies, trans- bodies, tetranectin, iMabs, Adnectin, domain antibodies, Kunitz-type domains, Evibodies, Affilins, Microbodies).
• biochip and “microarray” are interchangeably used herein
• the terms “attached” or “immobilized”, as used herein, refer to the binding between the nucleic acid(s), protein, polypeptide, or peptide and the solid support and may mean that the binding between the nucleic acid, protein, polypeptide, or peptide and the solid support is sufficient to be stable under conditions of binding, washing, analysis and removal.
• the binding may be covalent or non-covalent. Covalent bonds may be formed directly between the nucleic acid, protein, polypeptide, or peptide and the solid support or may be formed by a cross linker or by inclusion of specific reactive groups on either the solid support or the nucleic acid, protein, polypeptide, or peptide, or both.
• Non- covalent binding may be electrostatic, hydrophilic and hydrophobic interactions or combinations thereof. Immobilization or attachment may also involve a combination of covalent and non-covalent interaction.
• a marker is any kind of substance which is able to indicate the presence of another substance or complex of substances.
• the marker can be a substance that is linked to or introduced in the substance to be detected.
• Detectable markers are used in molecular biology and biotechnology to detect e.g. a protein, a product of an enzymatic reaction, a second messenger, DNA, interactions of molecules etc.
• suitable marker or labels include a fluorophore, a chromophore, a radiolabel, a metal colloid, an enzyme, or a chemiluminescent or bioluminescent molecule.
• fluorophores include fluorescein, rhodamine, and sulfoindocyanine dye Cy5.
• radiolabels examples include 3 H, 14 C, 32 P, 33 P, 35 S, 99m Tc or 125 l.
• enzymes include horseradish peroxidase, alkaline phosphatase, glucose oxidase, and urease. Further examples and preferred embodiments are detailed herein.
• Radioisotopes were used extensively in the past, but they are expensive, have a short shelf-life, offer no improvement in signal:noise ratio and require special handling and disposal.
• Enzymes and fluorophores have largely replaced radioactive isotopes as detectable tags for assays. A number of advancements in reagents and instrumentation make these newer technologies more versatile and powerful.
• Enzymatic tags such as horseradish peroxidase (HRP) are most commonly used for blotting, immunoassays and immunohistochemistry methods. Fluorescent tags are used predominately for cellular imaging, nucleic acid amplification and sequencing and microarrays; however, fluorescence technology is developing rapidly for application in all types of assays.
• Slfn2 protein or a variant thereof may include a specific Slfn2 antibody or protein scaffold.
• antibodies can be raised using well established techniques for immunizing animals with prepared forms of the antigen.
• a variety of reagents is available to assist in antibody production and purification, and various companies specialize in antibody production services.
• different levels of purity and types of specificity are needed in a supplied primary antibody.
• antibodies may be monoclonal or polyclonal, supplied as antiserum or affinity-purified solution, and validated for native protein or denatured protein detection.
• An antibody that recognizes the target antigen here Slfn2 or fragment or variant thereof, is called the "primary antibody.” If this antibody is labeled with a tag, direct detection of the antigen is possible. Usually, however, the primary antibody is not labeled for direct detection. Instead a "secondary antibody” that has been labeled with a detectable tag is applied in a second step to probe for the primary antibody, which is bound to the target antigen. Thus, the antigen is detected indirectly. Another form of indirect detection involves using a primary or secondary antibody that is labeled with an affinity tag such as biotin.
• a secondary (or tertiary) probe such as streptavidin that is labeled with the detectable enzyme or fluorophore tag
• streptavidin that is labeled with the detectable enzyme or fluorophore tag
• a primary antibody without a detectable label and some sort of secondary (indirect) detection method is required in assay methods. Nevertheless, nearly any antibody can be labeled with biotin, HRP enzyme or one of several fluorophores if needed.
• Most primary antibodies are produced in mouse, rabbit or one of several other species. Nearly all of these are antibodies of the IgG class. Therefore, it is relatively easy and economical for manufacturers to produce and supply ready-to-use, labeled secondary antibodies for most applications and detection systems. Even so, several hundred options are available, differing in the level of purity, IgG- and species-specificity, and detection label.
• the choice of secondary antibody depends upon the species of animal in which the primary antibody was raised (the host species). For example, if the primary antibody is a mouse monoclonal antibody then the secondary antibody must be an anti-mouse antibody obtained from a host other than the mouse.
• biotin-binding proteins As probes, the highly specific affinity interaction between biotin and avidin or streptavidin protein is the basis for many kinds of detection and affinity-purification methods. Biotin is very small (244 Daltons), so its covalent attachment to antibodies or other probes rarely interferes with their functions. Yet its presence as a label on a probe allows efficient and specific secondary detection with either avidin or streptavidin. Both kinds of biotin-binding proteins are available in purified forms labeled with enzymatic or fluorescent tags that enable detection in many kinds of assays systems. Enzymatic labels are most commonly used as secondary antibody (or streptavidin) tags for detection in blotting and immunoassays.
• Enzymes provide detectable signal via their activity; reaction with a specific substrate chemical yields a colored, light-emitting, or fluorescent product. While reporter enzymes like beta-galactosidase and luciferase have been successfully used to make probes, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme.
• Alkaline phosphatase is a large (140 kDa) protein that catalyzes the hydrolysis of phosphate groups from a substrate molecule resulting in a colored or fluorescent product or the release of light as a byproduct of the reaction.
• AP has optimal enzymatic activity at a basic pH (pH 8-10) and can be inhibited by cyanides, arsenate, inorganic phosphate and divalent cation chelators, such as EDTA.
• pH 8-10 basic pH
• AP offers a distinct advantage over other enzymes. Because its reaction rate remains linear, detection sensitivity can be improved by simply allowing a reaction to proceed for a longer time period.
• Horseradish peroxidase is a 40 kDa protein that catalyzes the oxidation of substrates by hydrogen peroxide, resulting in a colored or fluorescent product or the release of light as a byproduct of the reaction.
• HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides.
• Antibody-HRP conjugates are superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody. In addition, its high turnover rate, good stability, low cost and wide availability of substrates makes HRP the enzyme of choice for most applications.
• Fluorescent labels for detection were historically used in a small number of cell biology applications such as flow cytometry (FC), fluorescence-activated cell sorting (FACS) and immunohistochemistry (IHC) using fluorescence microscopy.
• FC flow cytometry
• FACS fluorescence-activated cell sorting
• IHC immunohistochemistry
• fluorescent proteins such as the various forms of green fluorescent protein (GFP) and the phycobiliproteins (allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin). While having the ability to produce an intense fluorescent signal for detection, fluorescent proteins can be difficult to optimize for conjugation purposes and may create steric hindrance or background signal issues in binding assays.
• GFP green fluorescent protein
• phycobiliproteins allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin
• fluorophore-conjugated probes require fewer steps compared to the use of enzymatic labels because there is no substrate development step to perform. While the protocol is shorter, fluorescent detection requires special equipment and the sensitivity is not a high as that which can be obtained with enzymatic chemiluminescent systems. Although not as sensitive as enzymatic detection, fluorescent detection methods reduce chemical waste and have the added advantage of multiplex compatibility (using more than one fluorophore in the same experiment).
• two markers may be used in order to detect proximity of two substances, e.g. the test compound or the known Slfn2 ligand and the Slfn2 protein.
• Papain and cathepsin L and H are known ligands for Slfn2.
• the markers may be, e.g. one radioactive or fluorescent marker and one scintillator (e.g. for a scintillation proximity assay) or two fluorescent markers may be used (e.g. for FRET).
• the Slfn2 protein and the test substance could be labeled with a first and a second marker.
• test substance is bound to the protein, and the labels are therefore in close proximity, energy could be transferred from the first to the second label, thus detecting the interacting of Slfn2 protein and test substance.
• This test could be designed as a competition binding test, wherein a known Slfn2 ligand carries one of the labels.
• suitable marker combinations include:
• radiolabels 3 H, 33 P, 35 S or 14 C, 125 l combined with scintillator such as Yttrium silicate or polyvinyl-toluene, e.g. compartmented in a microparticle or
• a donor fluorescent markers such as fluorescein, Lucifer Yellow, B-phycoerythrin, 9- acridineisothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothiocyanatostilbene- 2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1 -pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'- disulfonic acid derivatives combined with a acceptor fluorescent marker such as LC- Red 610, LC -Red 640, LC-Red 670, LC -Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate
• Detection of Slfn2 can also refer to detection of Slfn2 mRNA:
• RNA samples may be first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe.
• Nonisotopic or high specific activity radio labeled probes can be used including random- primed, nick-translated, or PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides.
• sequences with only partial homology e.g., cDNA from a different species or genomic DNA fragments that might contain an exon may be used as probes.
• the Nuclease Protection Assay is an extremely sensitive method for the detection and quantitation of specific mRNAs.
• the basis of the NPA is solution hybridization of an antisense probe (radio labeled or non-isotopic) to an RNA sample. After hybridization, single-stranded, unhybridized probe and RNA are degraded by nucleases. The remaining protected fragments are separated e.g. on an acrylamide gel. Solution hybridization is typically more efficient than membrane-based hybridization, and it can accommodate up to 100 g of sample RNA, compared with the 20-30 g maximum of blot hybridizations.
• NPAs are also less sensitive to RNA sample degradation than Northern analysis since cleavage is only detected in the region of overlap with the probe (probes are usually about 100-400 bases in length).
• RT-PCR an RNA template is copied into a complementary DNA (cDNA) using a retroviral reverse transcriptase. The cDNA is then amplified exponentially by PCR.
• Relative quantitative RT-PCR involves amplifying an internal control simultaneously with the gene of interest. The internal control is used to normalize the samples. Once normalized, direct comparisons of relative abundance of a specific mRNA can be made across the samples.
• Competitive RT-PCR is used for absolute quantitation.
• This technique involves designing, synthesizing, and accurately quantitating a competitor RNA that can be distinguished from the endogenous target by a small difference in size or sequence.
• Known amounts of the competitor RNA are added to experimental samples and RT-PCR is performed. Signals from the endogenous target are compared with signals from the competitor to determine the amount of target present in the sample.
• the above methods may include nucleic acid labeling.
• a series of techniques are known to the skilled person allowing for labeling of DNA, RNA or oligonuleotides. These include for example Nick translational labeling, random primed DNA labeling, PCR labeling of DNA probes and oligonucleotide 375' end labeling, transcriptional labeling of RNA probes, oligonucleotide 375' end labeling and oligonucleotide tailing.
• the nick translation method is based on the ability of DNase I to introduce randomly distributed nicks into DNA.
• DNA polymerase I synthesizes DNA complementary to the intact strand in a 5' ⁇ 3' direction using the 3'-OH termini of the nick as a primer.
• the 5' ⁇ 3' exonucleolytic activity of DNA Polymerase I simultaneously removes nucleotides in the direction of synthesis.
• the polymerase activity sequentially replaces the removed nucleotides with isotope-labeled or hapten-labeled deoxyribonucleoside triphosphates.
• the unlabeled DNA in the reaction is thus replaced by newly synthesized labeled DNA.
• Common labels include digoxigenin-, biotin-, or fluorochromes such as fluorescein or tetramethylrhodamin.
• the method of "random primed" DNA labeling is based on the hybridization of a mixture of all possible hexanucleotides to the DNA to be labeled. All sequence combinations are represented in the hexanucleotide primer mixture, which leads to binding of primer to the template DNA in a statistic manner. Thus an equal degree of labeling along the entire length of the template DNA is guaranteed.
• the complementary strand is synthesized from the 3' OH termini of the random hexanucleotide primer using Klenow enzyme, labeling grade. Modified deoxyribonucleoside triphosphates (e.g.
• PCR polymerase chain reaction
• digoxigenin a steroid hapten
• the digoxigenin is usually coupled to dUTP via an alkali-labile ester bond.
• the labeled dUTP can be easily incorporated by enzymatic nucleic-acid synthesis using DNA polymerases.
• Oligonucleotides may enzymatically be labeled at their 3 ' -end with terminal transferase either by incorporation of a label such as single digoxigenin-labeled dideoxyuridine- triphosphate (DIG-ddUTP) or by the addition of a longer nucleotide tail.
• Terminal Transferase catalyzes the template independent addition of deoxy- and dideoxynucleoside triphosphates to the 3 ⁇ ends of double and single-stranded DNA fragments and oligonucleotides.
• Terminal transferase incorporates digoxigenin-, biotin-, and fluorochrome-labeled deoxy- and dideoxynucleotides as well as radioactive labeled deoxy-and dideoxynucleotides.
• oligonucleotides may be labeled at the 5 ' -terminus, e.g. by reacting with a phosphoramidite in a final step according to the classical solid phase phosphoramidite synthesis method. By this process a 5 ' -terminal amino function is created. Treatment with ammonia releases the oligonucleotide from the support and cleaves the protecting groups. In the subsequent step the digoxigenin moiety is introduced at the 5 ' -position.
• Different labels are known which may be used in the above labeling methods. Some of them including their detection are exemplarily described in the following:
• Biotin-labeled compounds can be detected for example by anti-biotin antibodies or by streptavidin conjugates.
• Anti-biotin antibodies e.g. monoclonal anti-biotin antibody or Fab-fragment, conjugated with alkaline phosphatase (AP)
• AP alkaline phosphatase
• Biotin-labeled compounds can be detected for example by anti-biotin antibodies or by streptavidin conjugates.
• Anti-biotin antibodies e.g. monoclonal anti-biotin antibody or Fab-fragment, conjugated with alkaline phosphatase (AP)
• AP alkaline phosphatase
• This method of detection may be employed for detection of biotin labeled nucleic acids on membranes (e.g. Southern blots, dot blots), in cells and tissues (e.g. in situ hybridization), immunoblotting, immunohistochemistry or ELISA.
• Streptavidin conjugates are used for the detection of biotin-labeled substances (e.g., biotinylated antibodies) which can be used for several immunological detection systems.
• biotin-labeled substances e.g., biotinylated antibodies
• streptavidin e.g. from Streptomyces avidinii could be coupled to alkaline phosphatase or to ⁇ -peroxidase. This method of detection may be employed with immunoblotting, immunohistochemistry or ELISA.
• Probe-target hybrids may be detected with an enzyme-linked immunoassay. This immunochemical detection step is usually more sensitive than radioactive detection procedures. In this assay, the membrane may be blocked to prevent non-specific interaction of the antibody with the filter. Alkaline phosphatase-conjugated antibody, specific for digoxigenin, recognizes the digoxigenin molecule on the labeled hybrid. Addition of an alkaline phosphatase substrate allows the visualization of the hybrids.
• suitable substrates for alkaline phosphatase such as disodium 3-(4-methoxyspiro ⁇ 1 ,2-dioxetane-3,2-(5-chloro)tricyclo [3.3.1 .1 3,7 ]decan ⁇ -4- yl)phenyl phosphate or disodium 4-chloro-3-(methoxyspiro ⁇ 1 ,2-dioxetane-3,2-(5- chloro)tricyclo [3.3.1 .1 3,7 ]decan ⁇ -4-yl)phenyl phosphate belong to the group of the dioxetane phenyl phosphates.
• an intermediate Upon dephosphorylation by alkaline phosphatase, an intermediate is formed whose decomposition results in light emission which can be recorded e.g. on X-ray film.
• Colorimetric detection of DIG-labeled probes is usually performed with colorless substrates which form a redox system.
• Examples are like 5-bromo-4-chloro-3-indolyl- phosphate and 4-Nitro-blue-tetrazolium-chloride.
• 5-bromo-4-chloro-3-indolyl-phosphate is oxidized by the alkaline phosphatase to indigo by release of a phosphate group.
• 4-Nitro-blue-tetrazolium-chloride is reduced to diformazan. The reaction products form a water insoluble dark blue to brownish precipitate, depending on the type of membrane.
• reporter molecules can be coupled to detecting antibodies to visualize the specific probe-target hybridization including, but not limited to, enzyme-coupled antibodies, fluorochrome-labeled antibodies (detection by fluorescent microscope and specific filters which allow visualization of the wavelength emitted by the fluorescent dye) and antibodies coupled to colloidal gold (detection by electron microscope on cryostatic sections).
• Multiple simultaneous hybridizations can be performed by using combinations of digoxigenin-, biotin- and fluorochrome-labeled probes to localize different chromosomal regions or different RNA sequences in one preparation.
• Such multiprobe experiments are made possible by the availability of different fluorescent dyes coupled to antibodies. These include fluorescein or FITC (fluorescein isothiocyanate; yellow), rhodamine or TRITC (tetramethylrhodamine isothiocyanate; red) and AMCA (amino-methylcoumarin acetic acid; blue).
• Suitable diagnostic assays comprise the detection of Slfn2 protein:
• Suitable methods for detecting a protein include e.g. detection of protein immunostaining, protein immunoprecipitation, Immunoelectrophoresis, immunoblotting, Western blotting, spectrophotometry, enzyme assays etc..
• the method may require protein purification prior to the detection, which could involve protein isolation (e.g. by chromatography methods, protein extraction, protein solubilization, gel electrophoresis, and electrofocusing).
• Protein immunostaining is an antibody-based method to detect a specific protein in a sample.
• the term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections. Now however, immunostaining encompasses a broad range of techniques used in histology, cell biology, and molecular biology that utilize antibody-based staining methods. Immunohisto- or -cytochemistry of tissue sections or cells which are preserved by fixation.
• Western blotting allows the detection of specific proteins (native or denatured) from extracts made from cells or tissues, before or after any purification steps. Proteins are generally separated by size using gel electrophoresis before being transferred to a synthetic membrane (typically nitrocellulose or PVDF) via dry, semi-dry, or wet blotting methods. The membrane can then be probed using antibodies using methods similar to immunohistochemistry, but without a need for fixation. Detection is typically performed using peroxidase linked antibodies to catalyze a chemiluminescent reaction.
• Western blotting is a routine molecular biology method that can be used to semi quantitatively or quantitatively compare protein levels between extracts.
• Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions).
• the enzyme-linked immunosorbent assay or ELISA is a diagnostic method for quantitatively or semi-quantitatively determining protein concentrations from blood plasma, serum or cell/tissue extracts in a multi-well plate format (usually 96-wells per plate). Broadly, proteins in solution are adsorbed to ELISA plates. Antibodies specific for the protein of interest are used to probe the plate. Background is minimized by optimizing blocking and washing methods (as for IHC), and specificity is ensured via the presence of positive and negative controls. Detection methods are usually colorimetric or chemiluminescence based.
• Electron microscopy or EM can be used to study the detailed micro architecture of tissues or cells.
• Immuno-EM allows the detection of specific proteins in ultrathin tissue sections.
• Antibodies labeled with heavy metal particles e.g. gold
• transmission electron microscopy While powerful in detecting the sub- cellular localization of a protein, immuno-EM can be technically challenging, expensive, and require rigorous optimization of tissue fixation and processing methods.
• an “individual” refers to a human or a non-human animal (e.g. a mammal, avian, reptile, fish, amphibian or invertebrate; preferably an individual that can either benefit from one of the different aspects of present invention (e.g. a method of treatment or a drug identified by present methods) or that can be used as laboratory animal for the identification or characterisation of a drug or a method of treatment.
• the individual can e.g. be a human, a wild-animal, domestic animal or laboratory animal; examples comprise: mammal, e.g.
• human, non-human primate chimpanzee, bonobo, gorilla
• dog, cat rodent
• rodent e.g. mouse, guinea pig, rat, hamster or rabbit, horse, donkey, cow, sheep, goat, pig, camel
• avian such as duck, dove, turkey, goose or chick
• reptile such as: turtle, tortoise, snake, lizard, amphibian such as frog (e.g. Xenopus laevis); fish such as koy or zebrafish
• invertebrate such as a worm (e.g. c.elegans) or an insect (such as a fly, e.g. drosophila melanogaster).
• the term individual also comprises the different morphological developmental stages of avian, fish, reptile or insects, such as egg, pupa, larva or imago.
• the individual may be an individual which is suspected to experience pain.
• the individual may be diagnosed and/or prognosed to experience pain.
• the individual as mentioned in the method above may also be an individual which is experiencing pain.
• the individual may be retested for experiencing pain and may be diagnosed and/or prognosed to still experiencing pain.
• sample preferably refers to a biologically sample.
• sample or “sample of interest” are used interchangeably herein, referring to a small part intended to represent the whole of a tissue, an organ or an individual.
• samples include but are not limited to fluid samples such as cerebrospinal fluid, blood, serum, plasma, synovial fluid, urine, saliva, and lymphatic fluid, or solid samples such as biopsy samples, tissue, and tissue-extracts, e.g. taken from nervous tissue (e.g from the spinal cord), skin, muscle, cartilage, bone, synovium, perichondrium, capsule, and connective tissue.
• cell cultures or tissue cultures such as but not limited to cultures of neural cells.
• Analysis of a sample may be accomplished on a visual or chemical basis.
• Visual analysis includes but is not limited to microscopic imaging or radiographic scanning of a tissue, organ or individual allowing for morphological evaluation of a sample.
• Chemical analysis includes but is not limited to the detection of the presence or absence of specific indicators or alterations in their amount or level.
• a tissue sample may be removed from a subject by conventional biopsy techniques or a blood sample may be taken from a subject by conventional blood collection techniques.
• the sample e.g. tissue or blood sample, may be obtained from a subject prior to initiation of the therapeutic treatment, during the therapeutic treatment, and/or after the therapeutic treatment, e.g. with a pain reducing compound.
• the sample is a body fluid sample, a tissue sample, a cell colony sample, a single cell sample or a cell culture sample. More preferably, the tissue sample is a section or an explant sample, e.g. an explant sample of dorsal root ganglia or spinal cord.
• body fluid sample refers to a liquid sample derived from the body of a subject. Said body fluid sample may be a blood, urine, cerebrospinal fluid, cerumen (earwax), endolymph, perilymph, gastric juice, mucus, peritoneal fluid, pleural fluid, saliva, or sebum (skin oil) sample including components or fractions thereof. Said body fluid samples may be mixed or pooled.
• a body fluid sample may be a mixture of a blood sample and anurine sample or a mixture of a blood sample and cerebrospinal fluid sample.
• a "body fluid sample” may be provided by removing a body liquid from a subject, but may also be provided by using previously isolated body fluid sample material.
• the blood sample of a subject is whole blood or a blood fraction such as serum or plasma. It is also particularly preferred to use blood cells also known as hemopoietic cells.
• the tissue sample has a weight of between 0.1 and 500 mg, more preferably of between 0.5 and 250 mg, and most preferably of between 1 and 50 mg, i.e.
• the cell sample e.g. cell colony sample or cell culture sample
• the cell sample consists of between 100 and 1000 cells, more preferably of between 200 and 800 cells, and most preferably of between 400 and 600 cells.
• the body fluid sample has a volume of between 0.1 and 20 ml, more preferably of between 0.5 and 10 ml, more preferably between 1 and 8 ml and most preferably between 2 and 5 ml, i.e. 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml. More preferably, the blood sample has a volume of between 0.1 and 20 ml, more preferably of between 0.5 and 10 ml, and most preferably of between 1 and 5 ml, i.e. 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml.
• a reference value is a standard value for a certain parameter (e.g. expression level of a certain indicator/biomarker molecule) that is typical for a certain status (e.g. a disease status or health status)
• a reference individual is an individual that has been selected for comparison and has a certain health state or disease
• a reference sample can e.g. be a sample from a reference individual or an artificial sample with a characteristic level of a certain indicator or biomarker typical for a disease state or health state.
• reference sample refers to a sample which is analysed in a substantially identical manner as the sample of interest and whose information is compared to that of the sample of interest. A reference sample thereby provides a standard allowing for the evaluation of the information obtained from the sample of interest.
• a reference sample may be derived from a healthy or normal tissue, organ or individual, thereby providing a standard of a healthy status of a tissue, organ or individual. Differences between the status of the normal reference sample and the status of the sample of interest may be indicative of the risk of disease development or the presence or further progression of such disease or disorder.
• a reference sample may be derived from an abnormal or diseased tissue, organ or individual thereby providing a standard of a diseased status of a tissue, organ or individual. Differences between the status of the abnormal reference sample and the status of the sample of interest may be indicative of a lowered risk of disease development or the absence or bettering of such disease or disorder.
• a reference sample may also be derived from the same tissue, organ, or individual as the sample of interest but has been taken at an earlier time point. Differences between the status of the earlier taken reference sample and the status of the sample of interest may be indicative of the progression of the disease, i.e. a bettering or worsening of the disease over time.
• a reference sample was taken at an earlier or later time point in case a period of time has lapsed between taking of the reference sample and taking of the sample of interest.
• Such period of time may represent years (e.g. 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 years), months (1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 months), weeks (e.g. 1 , 2, 3, 4, 5, 6, 7, 8 weeks), days (e.g. 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 days), hours (1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 hours), minutes (e.g. 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 minutes), or seconds (e.g. 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 seconds).
• years e.g. 1
• the reference sample representative for a status or stage of pain may be from a control subject known to experience pain.
• the control sample is from a control subject known to experience algesia, particularly hyperalgesia.
• the control subject may be a mammal such as a human, rodent (e.g. rat, hamster, or mouse) or monkey, or may be another animal than a mammal such as an avian.
• both the sample or value and the reference sample or value are from subjects of the same species (e.g. human), more preferably of the same gender (e.g. female or male) and/or of a similar age or phase of life (e.g. infant, young child, juvenile, adult, or elderly).
• the reference or reference sample in the different aspects and embodiments of present invention is preferably derived from a healthy individual, a diseased individual, or from the same individual as the sample of interest.
• the reference (e.g. reference value) or reference sample was taken from the same individual as the sample of interest, the reference (e.g. reference value) or reference sample was preferably taken at an earlier or later time point then the sample of interest.
• the time period which has lapsed between taking of the reference (e.g. reference value) or reference sample and taking of the reference (e.g. reference value) or sample or value of interest preferably represents years (e.g.
• the reference sample is a reference sample with a level of Slfn2 representative for a healthy individual or representative for the presence or absence of an altered tissue status or disease or representative for an increased or decreased risk of developing an altered tissue status or disease.
• an elevated level of Slfn2 in the reference or value or sample of interest in comparison to said reference (value) or reference sample indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
• a similar level of Slfn2 indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
• an elevated level of Slfn2 in the individual/value/sample of interest indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
• a lowered level of Slfn2 in the sample of interest indicates (a) an alteration of the tissue status or an improvement or absence of a disease and/or (b) a decreased risk to develop a deteriorated tissue status or a disease and/or (c) a declined progression of the deteriorated tissue status or the disease.
• a similar level of Slfn2 in the sample of interest indicates (a) a similar risk to develop a deteriorated tissue status or a disease and/or (b) a stagnation in the progression of a deteriorated tissue status or a disease, and/or (c) a persistence of the deteriorated tissue status or the disease in the individual.
• the reference (value) or reference sample is derived from a healthy individual or from an individual with a decreased risk of developing an altered tissue status or disease or comprises a level of Slfn2 representative of a healthy individual or of a status of disease-absence or for a decreased risk of developing an altered tissue status or disease, wherein an elevated level of Slfn2 indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
• the reference (value) or reference sample is derived from a diseased individual or from an individual with an increased risk of developing an altered tissue status or disease or comprises a level or amount of Slfn2 representative for a diseased individual or for a status of disease-presence or for an increased risk of developing an altered tissue status or disease, wherein a similar level of Slfn2 indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
• an elevated level of Slfn2 in the sample of interest indicates (a) the presence of a deteriorated tissue status or a disease and/or (b) an increased risk to develop a deteriorated tissue status or a disease and/or (c) the progression of a deteriorated tissue status or a disease in the individual.
• a lowered level of Slfn2 in the sample of interest indicates (a) an alteration of the tissue status or an improvement or absence of a disease and/or (b) a decreased risk to develop a deteriorated tissue status or a disease and/or (c) a declined progression of the deteriorated tissue status or the disease.
• a similar level of Slfn2 in the sample of interest indicates (a) a similar risk to develop a deteriorated tissue status or a disease and/or (b) a stagnation in the progression of a deteriorated tissue status or a disease, and/or (c) a persistence of the deteriorated tissue status or the disease in the individual.
• lowered or “decreased”, especially in the context of the level of Slfn2 refer to the level of Slfn2 in the sample being reduced in comparison to the reference or reference sample.
• elevated or “increased”, especially in the context of the level of Slfn2 refer to the level of Slfn2 in the sample being higher in comparison to the reference or reference sample.
• a miRNA that is detectable in higher amounts in cerebrospinal fluid or spinal cord tissue of one individual suffering from pain than in cerebrospinal fluid or spinal cord tissue of individuals not suffering from pain has an elevated level.
• an elevated level in a patient sample indicates the presence of a pain-associated disease or pain-associated tissue state or pain or an increased susceptibility or increased probability to develop a pain-associated disease or pain-associated tissue state or pain, especially the presence or an increased susceptibility to neuropathic algesia or neuropathic pain.
• treat means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in an individual that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in individuals that were previously symptomatic for the disorder(s).
• prevent means preventing that such disease or disorder occurs in patient.
• a "patient” means any mammal, reptile or bird that may benefit from a treatment with the compounds described herein.
• a “patient” is selected from the group consisting of laboratory animals (e.g. mouse or rat), domestic animals (including e.g. guinea pig, rabbit, chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog), or primates including chimpanzees and human beings. It is particularly preferred that the "patient” is a human being.
• laboratory animals e.g. mouse or rat
• domestic animals including e.g. guinea pig, rabbit, chicken, turkey, pig, sheep, goat, camel, cow, horse, donkey, cat, or dog
• primates including chimpanzees and human beings. It is particularly preferred that the "patient” is a human being.
• present invention relates to Slfn2 for use as an indicator of a pain- related tissue status or a pain related disease or as an indicator of the risk for developing a pain-related tissue status or disease.
• Slfn2 correlates with pain, especially neuropathic pain in mice leads to the conclusion that Slfn2 can be used as indicator/biomarker for tissue statuses or diseases that correlate with pain and for pain itself.
• Slfn2 is indicative of a pain-related tissue status or a pain-related disease.
• the presence, absence or altered amounts of Slfn2 are thus a sign for the presence, absence or progression of a pain-related tissue status or disease.
• a second aspect of present invention relates to Slfn2 for use in the diagnosis or prognosis of a pain-related tissue status or of a pain-related disease.
• Slfn2 is used as an indicator of a tissue status or a disease in the context of present invention.
• the level of Slfn2 is indicative of a pain-related tissue status or the presence of a pain-related disease and/or the risk of developing a pain-related tissue status or a pain-related disease and/or the progression or a stage of a pain-related tissue status or a pain-related disease in an individual.
• the level of Slfn2 in the first and second aspect of present invention is the expression level of Slfn2 or the level or the amount (number) of Slfn2 gene (copies), wherein the level can be the level of Slfn2 in an individual (wherein Slfn2 needs to be determined by a method able to directly determine and quantify Slfn present in an individual without a sample needing to be taken) or in a sample of an individual.
• the sample to be used in the first and second aspect of present invention can be any suitable sample, e.g. such as herein defined wherein cerebrospinal fluid is one of the preferred embodiments.
• the reference(s) or reference sample(s) can be any suitable reference(s) or reference sample(s), e.g. such as herein defined.
• the reference is selected from the group consisting of
• an individual having a pain-related tissue status or disease especially one that is known to be associated with a certain level of Slfn2
• a pain-related tissue status or disease especially one that is known to be associated with a certain level of Slfn2
• the reference is (derived from) an individual with a decreased
• the reference is a sample that is selected from the group consisting of a) a reference sample derived from an individual not having a pain-related tissue status or disease,
• a reference sample from an individual having a pain-related tissue status or disease b) a reference sample from an individual having a pain-related tissue status or disease, c) a reference sample from the same individual as the sample to be tested taken at an earlier or later time point or
• the reference sample is derived from an individual with a decreased risk of developing a pain-related tissue status or disease or wherein the reference sample comprises a level or amount of Slfn2 representative for an individual not having a pain-related tissue status or disease or for a status of disease-absence or for a decreased risk of developing a pain-related tissue status or disease, wherein an elevated level of Slfn2 in the tested sample indicates
• the reference is the same individual at an earlier time point or the sample is derived from the same individual as the tested sample and was taken at an earlier time point, wherein
• the reference is the same individual as the individual of interest at an earlier time point or the sample is derived from the same individual as the individual of interest, wherein the sample is derived from the same individual as the tested sample and was taken at an earlier time point, and wherein
• the sample e.g. the reference or tested sample of the first, second, third and fourth aspect of present invention can be any suitable sample, e.g. such as disclosed herein e.g. tissue such as extraneural or neural tissue or one or more extraneural or neural cells and/or body fluid such as cerebrospinal fluid. It can also be suitable to take samples of different origin such as a tissue sample and a body fluid sample.
• the Slfn2 according to one of the embodiments of the first and second aspect of present invention can e.g. a Slfn2 gene or gene product, preferably a Slfn2 mRNA, or Slfn2 protein or a variant (such as a functionally active variant) thereof.
• Slfn2 is a gene and the gene
• a. is a naturally occurring or non-naturally occurring Slfn2 gene variant, or
• amino acid sequence that has at least 80%, at least 90% at least 95% or at least 99% sequence identity to the amino acid sequence according to (i) or (ii) and/or
• vii. carries a variant AAA domain, preferably with 50, 60, 70, 80, 90, 95 or more sequence identity with the variant AAA domain of the Slfn2 protein according to SEQ ID NO:2.
• Slfn2 is a gene product, wherein the gene product
• a) is a naturally occurring Slfn2 protein variant, preferably a homolog or
• b) is a Slfn2 protein or a functionally active variant thereof having
• amino acid sequence i. an amino acid sequence according to SEQ ID NO: 2, 7 or 9, ii. an amino acid sequence that is a fragment of the amino acid sequence according to (i) and/ or
• amino acid sequence that has at least 80%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence according to (i) or (ii) and/or
• c) is a Slfn2 nucleic acid encoding a protein as defined in b), or
• d) is a Slfn2 nucleic acid having
• nucleic acid sequence that has at least 80%, at least 90%, at least
• the first or second aspects of present invention are e.g. suitable for diagnosing and/or prognosing pain in an in vitro or in vivo test model (i.e. a test animal, such as a non- human test animal, e.g. in the context of clinical studies) or in a patient (such as a human or non-human animal).
• an in vitro or in vivo test model i.e. a test animal, such as a non- human test animal, e.g. in the context of clinical studies
• a patient such as a human or non-human animal
• Another embodiment of the first and second aspect of present invention comprises determining the expression level of Slfn2 and preferably comprising determining the expression level of Slfn2 on the gene, mRNA or protein level.
• the tissue status is a pain-associated tissue status, such as a tissue damage, trauma, inflammation, infarction, necrosis, oedema, neoplasia of any tissue, the tissue preferably being selected from the group consisting of bone, cartilage, muscle, skin, neural tissue, such as a nerve, connective tissue such as perichondrium, periosteum, tendon, capsule, skin and and/or wherein the tissue status is characterized by an altered level of pain-related regulatory molecules or signaling molecules, indicators of pain and/or degenerative molecules present in the tissue.
• a pain-associated tissue status such as a tissue damage, trauma, inflammation, infarction, necrosis, oedema, neoplasia of any tissue
• the tissue preferably being selected from the group consisting of bone, cartilage, muscle, skin, neural tissue, such as a nerve, connective tissue such as perichondrium, periosteum, tendon, capsule, skin and and/or wherein the tissue status is characterized by
• the pain-related or pain-associated disease is a pain, e.g.
• nociceptive pain nociceptive pain, neuropathic pain, inflammatory pain, or a mixture thereof, (iii) disease-associated pain, or
• the acute pain is somatogenetic (organic) pain
• the chronic pain is somatogenetic (organic) pain or psychogenic
• the nociceptive pain is superficial somatic pain or deep pain, preferably deep somatic pain or visceral pain
• the neuropathic pain is central neuropathic pain or peripheral neuropathic pain
• the mixed pain is a mixture of nociceptive pain and neuropathic pain
• the disease-associated pain is pain associated with depression, migraine, epilepsy, cancer, or diabetes, or
• the algesia is hyperalgesia.
• the pain-related or pain-associated disease is characterized by an altered level of pain- related regulatory molecules or signaling molecules, indicators of pain and/or degenerative molecules present in the tissue.
• the prognosis comprises predicting or estimating the occurrence or reoccurrence, preferably the severity of occurrence or reoccurrence, of pain.
• present invention relates to a method of identifying the occurrence or reoccurrence, preferably the severity of occurrence or reoccurrence, of pain.
• the method comprising detecting the level of Slfn2.
• present invention relates to a method of identifying
• the method comprising detecting the level of Slfn2.
• the individual of the fourth aspect of present invention can be any suitable individual such as herein defined.
• the fourth aspect of present invention further comprises comparing the level of Slfn2 (e.g. the level of Slfn2 in the individual of interest (i.e. the test animal or the patient)) to the level of Slfn2 in one or more references or reference samples.
• the level of Slfn2 can e.g. be the level of Slfn2 in an individual (wherein Slfn2 needs to be determined by a method able to directly determine and quantify Slfn present in an individual without a sample needing to be taken) or in a sample of an individual.
• the level of Slfn2 is the expression level of Slfn2 or the level or the amount (number) of Slfn2 gene (copies).
• the reference is selected from the group consisting of an individual not having a pain-related tissue status or disease, an individual having a pain-related tissue status or disease or the same individual as the individual to be tested at an earlier or later time point or a value representative for the level of slfn2 in absence of the a pain-related tissue status or disease, in presence of a pain-related altered tissue status or disease or for an increased or decreased risk of developing a pain-related altered tissue status or disease.
• the reference sample is selected from the group consisting of a reference sample derived from an not having a pain-related tissue status or disease individual, a reference sample from an individual having a pain-related tissue status or disease, and a reference sample from the same individual as the sample to be tested taken at an earlier or later time point or a reference sample with a level or amount of slfn2 representative for an individual having or not having a pain-related tissue status or disease or for the absence or presence of the pain-related disease or altered tissue status or representative for an increased or decreased risk of developing a pain-related tissue status or disease.
• the reference is an individual not having a pain-related tissue status or disease or an individual with a decreased risk of developing a pain-related tissue status or disease or a value representative of the absence of a pain-related tissue status or disease, or the Slfn2 or method according to embodiment 8, wherein the reference sample is derived from an individual not having a pain-related tissue status or disease or from an individual with a decreased risk of developing a pain-related tissue status or disease or wherein the reference sample comprises a level or amount of Slfn2 representative for an individual not having a pain-related tissue status or disease or for a status of disease-absence or for a decreased risk of developing a pain-related tissue status or disease, wherein an elevated level of Slfn2 in the tested sample indicates
• the reference is an individual having a pain-related tissue status or disease or an individual with an increased risk of developing a pain-related tissue status or a disease or a value representative of the presence of a pain-related tissue status or disease, or Slfn2 or method according to embodiment 8, wherein the reference sample is derived from an individual having a pain-related tissue status or disease or from an individual with an increased risk of developing a pain-related tissue status or disease or wherein the reference sample comprises a level or amount of Slfn2 representative for an individual having a pain-related tissue status or disease or for a status of disease-presence or for an increased risk of developing a pain-related tissue status or disease, wherein a similar or higher level of Slfn2 in the tested sample indicates
• the reference is the same individual at an earlier time point or Slfn2 or method of embodiment 8, wherein the sample is derived from the same individual as the tested sample and was taken at an earlier time point, wherein
• the tested sample is tissue and/or body fluid, wherein the tissue sample can e.g. be extraneural or neural tissue or an extraneural or a neural cell, and wherein the body fluid sample can e.g. be cerebrospinal fluid.
• the pain- related disease is pain or a pain-associated disease, preferably a pain such as defined above and below.
• Slfn2 is a Slfn2 gene or gene product, preferably a Slfn2 mRNA, or Slfn2 protein or a functionally active variant thereof.
• Slfn2 is a gene, wherein the gene
• a. is a naturally occurring or non-naturally occurring Slfn2 gene variant, or
• amino acid sequence i. an amino acid sequence according to SEQ ID NO: 2, 7 or 9, ii. an amino acid sequence that is a fragment of the amino acid sequence according to (i) and/ or
• amino acid sequence that has at least 80%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence according to (i) or (ii) and/or
• amino acid sequence with at least 95% or at least 99% or 100% identity to at least 10, 1 1 , 12, 13, 14 or 15 consecutive amino acids of the Slfn2 protein according to
• vii. carries a variant AAA domain, preferably with 50, 60, 70, 80, 90, 95 or more sequence identity with the variant AAA domain of the Slfn2 protein according to SEQ ID NO:2.
• Slfn2 is a gene product, wherein the gene product
• a) is a naturally occurring Slfn2 protein variant, preferably a homolog or
• b) is a Slfn2 protein or a functionally active variant thereof having
• amino acid sequence i. an amino acid sequence according to SEQ ID NO: 2, 7 or 9, ii. an amino acid sequence that is a fragment of the amino acid sequence according to (i) and/ or
• amino acid sequence that has at least 80%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence according to (i) or (ii) and/or
• c) is a Slfn2 nucleic acid encoding a protein as defined in b), or
• d) is a Slfn2 nucleic acid having
• nucleic acid sequence that has at least 80%, at least 90%, at least
• the fourth aspect and its different embodiments are especially suitable for diagnosing and/or prognosing pain in an in vitro or in vivo test model (i.e. a test animal, such as a non-human test animal, e.g. in the context of clinical studies) or in a patient (such as a human or non-human animal).
• an in vitro or in vivo test model i.e. a test animal, such as a non-human test animal, e.g. in the context of clinical studies
• a patient such as a human or non-human animal
• the fourth aspect of present invention comprises determining the expression level of Slfn2 and preferably comprises determining the expression level of Slfn2 on the gene, mRNA or protein level.
• the tissue status is a pain-associated tissue status, such as a tissue damage, trauma, inflammation, infarction, necrosis, oedema, neoplasia of any tissue, the tissue preferably being selected from the group consisting of bone, cartilage, muscle, skin, neural tissue, such as a nerve, connective tissue such as perichondrium, periosteum, tendon, capsule, skin and and/or wherein the tissue status is characterized by an altered level of pain-related regulatory molecules or signaling molecules, indicators of pain and/or degenerative molecules present in the tissue.
• a pain-associated tissue status such as a tissue damage, trauma, inflammation, infarction, necrosis, oedema, neoplasia of any tissue
• the tissue preferably being selected from the group consisting of bone, cartilage, muscle, skin, neural tissue, such as a nerve, connective tissue such as perichondrium, periosteum, tendon, capsule, skin and and/or wherein the tissue status is characterized by
• the pain- related or pain-associated disease is a pain.
• the pain is (i) acute pain, subacute pain, or chronic pain
• the acute pain is somatogenetic (organic) pain
• the chronic pain is somatogenetic (organic) pain or psychogenic
• the nociceptive pain is superficial somatic pain or deep pain, preferably deep somatic pain or visceral pain
• the neuropathic pain is central neuropathic pain or peripheral neuropathic pain
• the mixed pain is a mixture of nociceptive pain and neuropathic pain
• the disease-associated pain is pain associated with depression, migraine, epilepsy, cancer, or diabetes, or
• the algesia is hyperalgesia.
• the disease is characterized by an altered level of pain-related regulatory molecules or signaling molecules, indicators of pain and/or degenerative molecules present in the tissue.
• the method relates to diagnosis, wherein the diagnosis comprises
• the method relates to prognosis, wherein the prognosis comprises predicting or estimating the occurrence or reoccurrence, preferably the severity of occurrence or reoccurrence, of pain.
• the kit for use in a method according to the fourth aspect and any of its embodiments, wherein the kit comprises one or more means of detecting Slfn2.
• present invention relates to a kit for detecting Slfn2 comprising
• a data carrier comprising instructions for a method according to the fourth aspect of present invention and any one its embodiments and/or
• kit is an article of manufacture that comprises at least the components as defined above and optionally one or more further components, the term kit in the context of present invention is understood as herein defined.
• the kit according to the fifth and sixth aspect of present invention comprises at least one or more means for detecting Slfn2, it can in addition, comprise one or more means for detecting another indicator of a pain-related tissue status or a pain-related disease such as pain, e.g. as listed above.
• the kit comprises a means for detecting Slfn2 or a means for detecting Slfn2 and a data carrier comprising instructions for a method according to the fourth aspect of present invention and any one its embodiments, it can also comprise a means for detecting Slfn2 and a container, it can also comprise a means for detecting Slfn2 and a means for detecting another indicator of a pain-related tissue status or pain-related disease such as pain, it can also comprise a means for detecting Slfn2 and a data carrier comprising instructions for a method according to the fourth aspect of present invention and any one its embodiments and a container it can also comprise a means for detecting Slfn2 and a data carrier comprising instructions for a method according to the fourth aspect of present invention and any one its embodiments and another indicator of a pain-related tissue status or pain-related disease such as pain, it can also comprise a means for detecting Slfn2 and another indicator of a pain-related tissue status or pain-related disease such as pain
• the means for detecting Slfn2 is a means for determining the expression level of Slfn2, preferably on the gene, protein or RNA level.
• the term means of detecting Slfn2 or means of detection of Slfn2 is understood as herein defined.
• the means for detecting Slfn2 is selected from the group consisting of nucleic acid, preferably DNA, RNA or PNA, peptide and protein, preferably monoclonal or polyclonal antibody or protein scaffold.
• the kit according to the fifth and sixth aspect of present invention can also comprise more than one means, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 means of detection.
• the kit comprises
• quality information such as information about the lot/batch number of the means for detecting Slfn2 and/or of the kit, the manufacturing or assembly site or the expiry or sell-by date, information concerning the correct storage or handling of the kit,
• the data carrier can thus comprise information according to (1 ), (2), (3), (4), (5), (6) and/or (7).
• the data carrier comprises at least information according to (1 ) or information according to (2) or information according to (3) or information according to (4) or information according to (5) or information according to (6) or information according to (7);
• the data carrier can also comprise (1 ) and (2) or (1 ) and (3) or (1 ) and (4) or (1 ) and (5) or (1 ) and (6) or (1 ) and (7) or (2) and (3) or (2) and (6) or (2) and (5) or (2) and (6) or (2) and (7) or (3) and (4) or (3) and (5) or (3) and (6) or (3) and (7) or (6) and (5) or (4) and (6) or (4) and (7) or (5) and (6) or (5) and (7) or (6) and (7);
• the data carrier can also comprise (1 ) and (2) and (3) or (1 ) and (2) and (4) or (1 ) and (2) and (5) or (1 ) and (2) and (6) or (1 ) and
• the kit is for diagnosing and/or prognosing pain by determination of Slfn2 and preferably determining the level of Slfn2 in a sample.
• the sample can be any suitable sample, e.g such as defined herein, e.g. in the definitions or above under the first and second aspect of present invention.
• the kit comprises a means as defined below for the eighth aspect of present invention.
• present invention refers to a means of detecting Slfn2 for use in the diagnosis or prognosis of a tissue status or a disease.
• the means comprises one or more nucleic acids or derivatives for detecting a Slfn2 gene or gene product or a functionally active variant thereof, wherein the nucleic acid or derivative is preferably selected from the group consisting of a nucleic acid probe, a polyamide or peptide nucleic acid (PNA), microRNA (miRNA), small interfering RNA (siRNA), primers for polymerase chain reaction (PCR), primers for reverse transcription (RT) reaction, and primers for DNA sequencing.
• PNA polyamide or peptide nucleic acid
• miRNA microRNA
• siRNA small interfering RNA
• PCR primers for polymerase chain reaction
• RT reverse transcription
• the means comprises a peptide, polypeptide or protein for detecting a Slfn2 gene or gene product or functionally active variant thereof, wherein the protein or polypeptide is preferably a protein ligand (such as an antibody) a fragment or derivate thereof, a protein scaffold (such as a darpin or an anticalin) or wherein the polypeptide or peptide is a probe, preferably a mass spectrometry probe.
• the tissue status is a pain-associated tissue status, such as a tissue damage, trauma, inflammation, infarction, necrosis, oedema, neoplasia of any tissue, the tissue preferably being selected from the group consisting of bone, cartilage, muscle, skin, neural tissue, such as a nerve, connective tissue such as perichondrium, periosteum, tendon, capsule, skin and and/or wherein the tissue status is characterized by an altered level of pain-related regulatory molecules or signaling molecules, indicators of pain and/or degenerative molecules present in the tissue.
• a pain-associated tissue status such as a tissue damage, trauma, inflammation, infarction, necrosis, oedema, neoplasia of any tissue
• the tissue preferably being selected from the group consisting of bone, cartilage, muscle, skin, neural tissue, such as a nerve, connective tissue such as perichondrium, periosteum, tendon, capsule, skin and and/or wherein the tissue status is characterized by
• the disease is a pain wherein the pain is preferably
• nociceptive pain nociceptive pain, neuropathic pain, inflammatory pain, or a mixture thereof, (iii) disease-associated pain, or
• the acute pain is somatogenetic (organic) pain
• the chronic pain is somatogenetic (organic) pain or psychogenic
• the nociceptive pain is superficial somatic pain or deep pain, preferably deep somatic pain or visceral pain
• the neuropathic pain is central neuropathic pain or peripheral neuropathic pain
• the mixed pain is a mixture of nociceptive pain and neuropathic pain
• the disease-associated pain is pain associated with depression, migraine, epilepsy, cancer, or diabetes, or
• the algesia is hyperalgesia.
• the pain- associated or pain-related disease is characterized by an altered level of pain-related regulatory molecules or signaling molecules, indicators of pain and/or degenerative molecules present in the tissue.
• the means is for use in the diagnosis or prognosis or a pain-related tissue status or a pain- related disease, preferably in a method of the third aspect of present invention.
• the diagnosis comprises
• the prognosis comprises predicting or estimating the occurrence or reoccurrence, preferably the severity of occurrence or reoccurrence, of pain.
• preferred embodiments of present invention are listed but are not to be understood as limiting present invention:
• Slfn2 for use as an indicator of a pain-related tissue status or a pain related disease or as an indicator of the risk for developing a pain-related tissue status or disease.
• Slfn2 for use in the diagnosis or prognosis of a pain-related tissue status or of a pain- related disease.
• the method comprising detecting the level of Slfn2. 5.
• the reference sample is selected from the group consisting of a reference sample derived from an not having a pain-related tissue status or disease individual, a reference sample from an individual having a pain-related tissue status or disease, and a reference sample from the same individual as the sample to be tested taken at an earlier or later time point or a reference sample with a level or amount of slfn2 representative for an individual having or not having a pain- related tissue status or disease or for the absence or presence of the pain-related disease or altered tissue status or representative for an increased or decreased risk of developing a pain-related tissue status or disease.
• the reference sample is derived from an individual not having a pain-related tissue status or disease or from an individual with a decreased risk of developing a pain- related tissue status or disease or wherein the reference sample comprises a level or amount of Slfn2 representative for an individual not having a pain-related tissue status or disease or for a status of disease-absence or for a decreased risk of developing a pain-related tissue status or disease, wherein an elevated level of Slfn2 in the tested sample indicates
• tissue sample is extraneural or neural tissue or an extraneural or a neural cell
• body fluid sample is cerebrospinal fluid
• Slfn2 according to one of the embodiments 1 -3 or 5-14 or the method according to one of the embodiments 4-14, wherein Slfn2 is a Slfn2 gene or gene product, preferably a Slfn2 mRNA, or Slfn2 protein or a functionally active variant thereof. 16. The Slfn2 or method according to embodiment 15, wherein Slfn2 is a gene and wherein the gene
• a. is a naturally occurring or non-naturally occurring Slfn2 gene variant, or b. codes for a Slfn2 protein or a functionally active variant thereof that has
• amino acid sequence i. an amino acid sequence according to SEQ ID NO: 2, 7 or 9, ii. an amino acid sequence that is a fragment of the amino acid sequence according to (i) and/ or
• amino acid sequence that has at least 80%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence according to (i) or (ii) and/or
• vii. carries a variant AAA domain, preferably with 50, 60, 70, 80, 90, 95 or more sequence identity with the variant AAA domain of the Slfn2 protein according to SEQ ID NO:2.
• Slfn2 or method according to embodiment 16, wherein Slfn2 is a gene product and wherein the gene product
• a) is a naturally occurring Slfn2 protein variant, preferably a homolog or
• b) is a Slfn2 protein or a functionally active variant thereof having
• amino acid sequence i. an amino acid sequence according to SEQ ID NO: 2, 7 or 9, ii. an amino acid sequence that is a fragment of the amino acid sequence according to (i) and/ or
• iii an amino acid sequence that has at least 80%, at least 90%, at least 95% or at least 99% sequence identity to the amino acid sequence according to (i) or (ii) and/or iv. an amino acid sequence with at least 95% or at least 99% or 100% identity to at least 10, 1 1 , 12, 13, 14 or 15 consecutive amino acids of the Slfn2 protein according to SEQ ID NO:2, and/or
• vii. carries a variant AAA domain, preferably with 50, 60, 70, 80,
• c) is a Slfn2 nucleic acid encoding a protein as defined in b), or
• d) is a Slfn2 nucleic acid having
• nucleic acid sequence that has at least 80%, at least 90%, at least
• the Slfn2 according to one of the embodiments 1 -3 or 5-18 or the method according to one of the embodiments 4-18 comprising determining the expression level of Slfn2 and preferably comprising determining the expression level of Slfn2 on the gene, mRNA or protein level.
• a kit for detecting Slfn2 comprising (i) a means for detecting Slfn2,
• a data carrier comprising instructions for a method according to one of the embodiments 4-19 and optionally
• kits according to embodiment 20 or 21 wherein the means for detecting Slfn2 is a means for determining the expression level of Slfn2, preferably on the gene, protein or RNA level.
• the means for detecting Slfn2 is selected from the group consisting of nucleic acid, preferably DNA, RNA or PNA, peptide and protein, preferably monoclonal or polyclonal antibody or protein scaffold.
• kit according to any of embodiments 20-23, wherein the kit comprises
• quality information such as information about the lot/batch number of the means for detecting Slfn2 and/or of the kit, the manufacturing or assembly site or the expiry or sell-by date, information concerning the correct storage or handling of the kit,
• kits according to any of embodiments 20-24, wherein the kit is for diagnosing and/or prognosing pain by determination of Slfn2 and preferably the level of Slfn2 in a sample.
• kit according to any of embodiments 20-25, wherein the kit comprises a means according to any of the embodiments 27-39.
• the means according to embodiment 28 comprising one or more nucleic acids or derivatives for detecting a Slfn2 gene or gene product or functionally active variant thereof.
• nucleic acid or derivative is selected from the group consisting of a nucleic acid probe, a polyamide or peptide nucleic acid (PNA), microRNA (miRNA), small interfering RNA (siRNA), primers for polymerase chain reaction (PCR), primers for reverse transcription (RT) reaction, and primers for DNA sequencing.
• PNA polyamide or peptide nucleic acid
• miRNA microRNA
• siRNA small interfering RNA
• PCR primers for reverse transcription (RT) reaction
• RT reverse transcription
• the means according to embodiment 28 comprising a peptide, polypeptide or protein for detecting a Slfn2 gene or gene product or functionally active variant thereof.
• the protein or polypeptide is a protein ligand, preferably an antibody, a fragment or derivate thereof, a protein scaffold, such as a darpin or an anticalin, or wherein the polypeptide or peptide is a probe, preferably a mass spectrometry probe.
• tissue status is a pain-associated tissue status, such as a tissue damage, trauma, inflammation, infarction, necrosis, oedema, neoplasia of any tissue, the tissue preferably being selected from the group consisting of bone, cartilage, muscle, skin, neural tissue, such as a nerve, connective tissue such as perichondrium, periosteum, tendon, capsule, skin and and/or wherein the tissue status is characterized by an altered level of pain-related regulatory molecules or signaling molecules, indicators of pain and/or degenerative molecules present in the tissue. 36. The Slfn2 according to any of embodiments 1 -3 or 5
• nociceptive pain nociceptive pain, neuropathic pain, inflammatory pain, or a mixture thereof, (iii) disease-associated pain, or
• the acute pain is somatogenetic (organic) pain
• the chronic pain is somatogenetic (organic) pain or psychogenic
• the nociceptive pain is superficial somatic pain or deep pain, preferably deep somatic pain or visceral pain
• the neuropathic pain is central neuropathic pain or peripheral neuropath pain
• the mixed pain is a mixture of nociceptive pain and neuropathic pain
• the disease-associated pain is pain associated with depression, migraine, epilepsy, cancer, or diabetes, or
• the algesia is hyperalgesia. 39.
• FIG. 1 ClustalW 2.1 (available by EMBL-EBI (EMBL-European Bioinformatics
• sequence 1 is murine (mus musculus) Slfn2 according to
• sequence 2 is rat (rattus norwegicus) Slfn2 according to NP_001 100501/NM_001 107031 .1 (SEQ ID NO:7)
• sequence 3 is homo sapiens SLFN12L according to NM_001 195790.1/NP_001 182719.1 (SEQ ID NO:9).
• the identity score with ClustalW is 83% between mSlfn2 and ratSlfn2, 50% between mSlfn2 and hsSLFN12L and 50% between rat Slfn2 and hsSLFN12L.
• the conserved variant AAA domain is shown underlined.
• EMBOSS needle 6.3.1 program working on Needleman-Wunsch algorithm of the EMBL EBI database .
• the divergent AAA domains (from position 231 to 355 of murine Slfn2 and from position 231 to 361 of rat Slfn2) as outlined in the NCBI database (see respective accession number) have been marked underlined.
• FIG. 1 Sequence alignment of murine Slfn2 (SEQ ID NO:2, sequence 1 of
• Figure 4 Promoter structure of murine Slfn2 according to Wern-Joo et al, 2009.
• Figure 5 shows for every individual mouse its neuropathic pain phenotype (mechanical hypersensitivity, X-axis) and the corresponding gene expression (signal intensity, Y-axis) in the L5 DRG. Mouse data are colour-coded depending on the used strain. A Pearson correlation analysis has been performed and revealed a significant positive correlation of the two parameters pain phenotype and Slfn2 gene expression. This means for individual mice that the higher the L5 DRG expression of Slfn2 in Chung-operated neuropathic mice was, the more pronounced the mechanical hyperalgesia as exhibited in the behavioral test.
• Figure 6 shows exemplary intensity data for Slfn2 of L5 DRG (3d p.o.).
• RNA samples of dorsal root ganglia (DRGs) of inbred mouse strains AKR/J (AKR), C57BL/6J (C57/B6) and CBA J (CBA) were examined.
• the left sciatic nerve was exposed at its trifurcation and the two major branches (tibial and common peroneal nerves) were ligated and transected distal to the ligation. Sham surgery was identical, the nerves were exposed but not ligated and transected (see Bourquin et al. 2006).
• Paw withdrawal thresholds were assessed using a dynamic plantar aesthesiometer (see Szabo et al. 2005). After acclimation in a compartment with metal mesh floor, the stimulator was positioned under the animal's hindpaw, a straight metal filament driven by an electrodynamic actuator touched the plantar surface and exerted an increasing upward force until the animal removed the paw (paw withdrawal threshold, PWT). PWTs were assessed for hindpaws of the ipsilateral, operated side and of the contralateral side. RNA isolation from dorsal root ganglia:
• RNA extraction for TaqMan and microarray analysis a method for isolating total RNA of murine DRGs was developed (Persson et al., supra, particularly section "RNA extraction for TaqMan and microarray analysis"), wherein the method provided for RNA in a sufficient amount (> 300 ng) and quality.
• Total RNA from DRGs was isolated with the PicoPure RNA Isolation Kit (Arcturus) following the manufacturer's instructions. RNA quality was assessed using the 2100 Bioanalyzer and RNA 6000 Nano LabChip kit (Agilent). After having extracted RNA from L5 DRGs of the three mouse strains, either Chung-operated or sham-operated control animals, the RNA probes were hybridized on Affymetrix microarrays (MOE430 2.0).
• Samples were profiled with Affymetrix microarrays (MOE430 2.0). At least five animals of each group were tested.
• First-strand cDNA synthesis was performed using 500ng total RNA with a 100pM T7-(dT)24 oligomer (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-dT 24 ) (SEQ ID NO: 15) according to Baugh, L.R, Hill, A.A., Brown, E.L. and Hunter, CP. (2001 ) Nucleic Acids Res. 29, e29 and Superscript II Reverse Transcriptase following the manufacturer's instructions. Double-stranded cDNA was synthesized and then extracted using phenol- chloroform followed by an ethanol precipitation step.
• RNA Transcription Labeling kit Enzo
• Transcription reactions were incubated at 37°C for 16h.
• cRNA was purified using the RNeasy Mini kit (Qiagen) protocol for RNA cleanup and quantified by a spectrophotometer.
• the biotin-labeled cRNA was fragmented using a RNA fragmentation buffer (200mM Tris-acetate, 500mM KOAc, 150mM MgOAc, pH 8.1 ).
• Hybridization and staining of mouse MOE430_2 GeneChipsTM was performed according to the manufacturer's instructions.
• the microarrays were scanned using a GeneChip 3000 Scanner, and the scanned data were imported and analyzed using Resolver v5.1 expression data analysis software (Rosetta Biosoftware).
• the Affymetrix gene expression data were statistically analyzed and filtered prior to a correlation analysis.
• the following filter criteria were used: - Abs. fold-change in at least 60 % of all Chung-operated animals > 1 .5 or
• S1 mean a ii sham anima is with in same strain ln(ipsilateral PWT / contralateral PWT).
• corresponding gene expression had to be detectable ("present") in at least five animals as defined by a respective intensity p-value ⁇ 0.001 .

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