EP1729570A1 - Ion channel - Google Patents
Ion channelInfo
- Publication number
- EP1729570A1 EP1729570A1 EP05731819A EP05731819A EP1729570A1 EP 1729570 A1 EP1729570 A1 EP 1729570A1 EP 05731819 A EP05731819 A EP 05731819A EP 05731819 A EP05731819 A EP 05731819A EP 1729570 A1 EP1729570 A1 EP 1729570A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- tφm8
- pain
- seq
- cell
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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Definitions
- This invention relates to newly identified nucleic acids, polypeptides encoded by them and to their production and use. More particularly, the nucleic acids and polypeptides of the present invention relate to an ion channel, hereinafter referred to as "TrpM8" or “TrpM8 ion channel”. The invention also relates to inhibiting or activating the action of such nucleic acids and polypeptides.
- TRP Transient receptor potential
- DRG Dorsal Root Ganglia
- CMRI cold- and menthol-sensitive receptor
- CNIR1 belongs in a member of the transient receptor potential (TRP) channel subfamily, which is similar to other thermoreceptors, -VR1 and NRL1, responding with a noxious heat and transfer the sensory information to the spinal cord and brain [ ⁇ agy L, Rang H.. Noxious heat activates all capsaicin-sensitive and also a sub-population of capsaicin insensitive dorsal root ganglion neurons. Neuroscience 88:995-997, 1999] [Cesare P., McNaughton P.: A novel heat-activated current in nociceptive neurons and its sensitizationbybradykinin. Proc. Natl. Acad. Sci. U.S.A. 93:1543 5-1543% 1996].
- TRP transient receptor potential
- TRPM8 a nonselective cationic channel expressed in a subpopulation of sensory neurons that is activated both by decreases in temperature and the cooling compound menthol.
- TRPM8 Combined fluorometric calcium imaging of cultured rat trigeminal neurons with single-cell RT-PCR has demonstrated that there are distinct subpopulations of cold responsive neurons and that TRPM8 likely contributes to cold transduction in one of them.
- TRPM8 is preferentially expressed within a subset of rapidly responsive, low- threshold (less than 30 degrees C), cold-sensitive neurons.
- mice TRPM8 The function of mouse TRPM8 was characterized as an ion channel gated by cold stimuli and menthol, and its expression was limited in a subpopulation of the pain- and temperature-sensing DRG neurons [Peiet A.M., Moqrich A., Hergarden A.C., Reeve A.J., Andersson D.A., Story G.M., Earley T J., Dragoni I., Mclntyre P., Bevan S., Patapoutian A.: A TRP Channel that Senses Cold Stimuli and Menthol. Cell 108:705-715, 2002].
- mice TrpM8 ion channel is also triggered by noxious heat stimuli and therefore may have an effect in pain.
- TrpM8 ion channel was also triggered by noxious heat stimuli and therefore may have an effect in pain.
- TrpM8 ion channel was also triggered by noxious heat stimuli and therefore may have an effect in pain.
- TrpM8 ion channel was also triggered by noxious heat stimuli and therefore may have an effect in pain.
- TrpM8 ion channel in stress and anxiety.
- TrpM8 gene comprises a nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4 or a sequence having at least 70% sequence identity thereto.
- the transgenic non-human animal has a deletion in a TrpM8 gene or a portion thereof.
- it displays any one or combination of the following phenotypes: (a) decreased sensitivity to pain, preferably as measured in a tail-flick test; (b) decreased stress, preferably as measured in an open field test; (c) decreased blood plasma corticosterone levels; as compared to a wild-type animal.
- transgenic non-human animal in which at least a portion or the whole of the TrpM8 gene of the animal is replaced with a sequence from the TrpM8 gene of another animal, preferably another species, more preferably a human.
- the transgenic non-human animal is a mouse.
- the transgenic non-human animal comprises a functionally disrupted TrpM8 gene, preferably a deletion in a TrpM8 gene, in which the TrpM8 gene comprises a nucleic acid sequence shown in SEQ ID NO: 4 or a sequence having at least 70% sequence identity thereto.
- TrpM8 gene comprises a nucleic acid sequence shown in SEQ ID NO: 4 or a sequence having at least 70% sequence identity thereto.
- a cell having a functionally disrupted endogenous TrpM8 gene in which the TrpM8 gene comprises a nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence having at least 70% sequence identity thereto.
- the TrpM8 gene comprises a nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a sequence having at least 70% sequence identity thereto.
- a non-hurnan transgenic animal an isolated cell or tissue thereof, or a cell, each as described, in a method of identifying an agonist or antagonist of a TrpM8 polypeptide comprising an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or a sequence having at least 70% sequence identity thereto.
- a method of identifying an agonist or antagonist of a TrpM8 polypeptide having an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or a sequence having at least 70% sequence identity thereto comprising administering a candidate compound to an animal, preferably a wild type animal or a transgenic non-human animal as set out above, and measuring a change in any of the following phenotypes: (a) sensitivity to pain, preferably as measured in a tail-flick test; (b) stress, preferably as measured in an open field test; and (c) blood plasma corticosterone levels.
- the method identifies an agonist of TrpM8 polypeptide according to by identifying a candidate compound capable of causing the animal to display a increase in any of the phenotypes (a)-(c).
- the method identifies an antagonist of TrpM8 polypeptide by identifying a candidate compound capable of causing the animal to display any of the phenotypes (a)-(c) or a decrease in such a phenotype.
- a method of identifying an agonist or antagonist of a TrpM8 polypeptide having an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or a sequence having at least 70% sequence identity thereto comprising exposing a candidate compound to a cell or tissue, preferably wild type cell or tissue or a cell or tissue or a cell as described above and measuring a change in conductance or intracellular calcium concentration of the cell or a cell of the tissue.
- the method identifies an agonist of TrpM8 polypeptide by identifying a candidate compound capable of increasing conductance or intracellular calcium concentration of the cell.
- the method of identifies an antagonist of TrpM8 polypeptide by identifying a candidate compound capable of decreasing conductance or intracellular calcium concentration of the cell.
- a method of identifying a compound suitable for the treatment or alleviation of pain or stress, preferably a TrpM8 associated disease comprising exposing a TrpM8 polypeptide comprising an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or a sequence having at least 70% sequence identity thereto to a candidate compound, and determining whether the candidate compound is an antagonist or antagonist of the TrpM8 polypeptide.
- TrpM8 polynucleotide comprising a nucleic acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4 or a sequence having at least 70% sequence identity thereto, for the identification of an agonist or antagonist thereof for the treatment of pain or stress, preferably a TrpM8 associated disease.
- TrpM8 polypeptide comprising an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or a sequence having at least 70% sequence identity thereto, for the identification of an agonist or antagonist thereof for the treatment of pain or stress, preferably a TrpM8 associated disease.
- TrpM8 polypeptide having an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or a sequence having at least 70% sequence identity thereto for use in a method of treatment of pain or stress, preferably a TrpM8 associated disease, in an individual.
- TrpM8 polypeptide having an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or a sequence having at least 70% sequence identity thereto for the preparation of a pharmaceutical composition for the treatment of pain or stress, preferably a TrpM8 associated disease, in an individual.
- a method of treating an individual suffering from pain or stress, preferably suffering from a TrpM8 associated disease comprising administering an antagonist of TrpM8 to the individual.
- a 16 th aspect of the present invention we provide a method of diagnosis of pain or stress, preferably a TrpM8 associated disease, in an individual, the method comprising detecting a change in expression, level or activity of TrpM8 in the individual or a cell or tissue thereof.
- the TrpM8 associated disease is selected from the group consisting of: Pain, cancer, inflammatory, inflammatory bowel disease, thermal hyperalgesia, viseral pain, migraine, post herpatic neuralgia, diabetic neuralgia, trigeminal neuralgia, post operative pain, osteoarthritis, rhuematoid arthritis, acute pain, chronic pain, cutaneous pain, somatic pain, visceral pain, referred pain, including myocardial ischaemia, phantom pain, neuropathic pain (neuralgia), pain arising from injuries, diseases, headaches, migraines, cancer pain, pain arising from neurological disorders such as Parkinson's disease, pain arising from spine and peripheral nerve surgery, brain tumors, traumatic brain injury (TBI), spinal cord trauma, chronic pain syndromes, chronic fatigue syndrome, neuralgias such as trigeminal neuralgia, glossopharyngeal neuralgia, postherpetic neuralgia and causalgia, pain arising from the TrpM
- TrpM8 polypeptide comprising the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof having at least 70% sequence identity thereto.
- such a nucleic acid comprises the nucleic acid sequence shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof having at least 70% sequence identity thereto.
- Figure 1 is a diagram showing the knockout vector.
- Figure 2 is a shows the gene expression results from the RT-PCR testing.
- Figure 3 is a graph showing the results of the tail flick studies for knockout mice (mutant) compared with wild-type mice (wt).
- Figures 4A-C are graphs showing the results of the open field test for knockout mice (mutant, -/-) compared with wild-type mice (wt, +/+).
- Figure 4A shows the permenance time in the central zone.
- Figure 4B shows the distance moved in the central zone.
- Figure 4C shows the total distance moved.
- Figure 5 is a graph showing the results of the assay for blood plasma corticosterone levels, for female knockout mice (mutant, white bar) compared to female wild-type mice (black bar) at 3 months.
- SEQ ID NO: 1 shows the cDNA sequence of human TrpM8.
- SEQ ID NO: 2 shows an open reading frame derived from SEQ ID NO: 1.
- SEQ ID NO: 3 shows the amino acid sequence of human TrpM8.
- SEQ ID NO: 4 shows the open reading frame of a cDNA for Mouse TrpM8.
- SEQ ID NO: 5 shows the amino acid sequence of Mouse TrpM8.
- SEQ ID No. 6-18 show the genotyping primers used to construct the knockout plamsid.
- SEQ ID No: 19 shows the knockout plasmid sequence. DETAILED DESCRIPTION
- TrpM8 ion channel we describe an ion channel, in particular, to TrpM8 ion channel, as well as homologues, variants or derivatives thereof, as well as their uses in treatment and diagnosis of diseases, including TrpM8 associated diseases.
- TrpM8 is structurally related to other proteins of the ion channel family, as shown by the results of sequencing the amplified cDNA products encoding human TrpM8.
- the cDNA sequence of SEQ ID NO: 1 contains an open reading flame (SEQ ID NO: 2, nucleotide numbers 41 to 3352) encoding a polypeptide of 1104 amino acids shown in SEQ ID NO: 3.
- Human TrpM8 is found to map to Homo sapiens chromosome 2q37.
- TrpM8 polypeptide SEQ ID NO: 3
- HMM structural prediction software of pfam http://www.sanger.ac.uk/Software/Pfam/search.shtml
- the mouse homologue of the human TrpM8 ion channel has been cloned, and its nucleic acid sequence and amino acid sequence are shown as SEQ ID NO: 4 and SEQ ID NO: 5 respectively.
- the mouse TrpM8 ion channel cDNA of SEQ ID NO: 4 shows a high degree of identity with the human TrpM8 ion channel (SEQ ID NO: 2) sequence, while the amino acid sequence (SEQ ID NO: 5) of mouse TrpM8 ion channel shows a high degree of identity and similarity with human TrpM8 ion channel (SEQ ID NO: 3).Human and mouse TrpM8 ion channel are therefore members of a large family of ion channel.
- TrpM8 cDNA Polymerase chain reaction (PCR) amplification of TrpM8 cDNA detects expression of TrpM8 to varying abundance in the prostate (+++), Liver (+++), Muscle (+), Testis (++), and Ovary (+).
- TrpM8 cDNA of SEQ ID NO: 1 to search the human EST data sources by BLASTN, identities are found in cDNA libraries. This indicates that TrpM8 is expressed in these normal or abnormal tissues.
- TrpM8 polypeptides, nucleic acids, probes, antibodies, expression vectors and ligands are useful for detection, diagnosis, treatment and other assays for diseases associated with over-, under- and abnormal expression of TrpM8 ion channel in these and other tissues.
- diseases may include the TrpM8 associated diseases set out below.
- TrpM8 ion channel is useful for treating and diagnosing a range of diseases, described in detail below. These diseases are referred to for convenience as TrpM8 associated diseases.
- TrpM8 ion channel may be used to treat or diagnose a disease which maps to this locus, chromosomal band, region, arm or the same chromosome.
- diseases which have been determined as being linked to the same locus, chromosomal band, region, arm or chromosome as the chromosomal location of T ⁇ M8 ion channel include prostate cancer where the ion channel has been found to be upregrulated.
- Knockout mice deficient in T ⁇ M8 display a range of phenotypes, as demonstrated in the Examples.
- T ⁇ M8 deficient mice are hypoalgesic, i.e., less sensitive to pain.
- T ⁇ M8 and modmlators of T ⁇ M8 activity may be used to treat or alleviate diseases or syndromes in which pain is a feature.
- activity or expression of T ⁇ M8 may be down-regulated in an individual, for example by administration of antagonists or blockers of T ⁇ M8, for analgesia / in order to reduce jpain.
- the methods and compositions described here including T ⁇ M8 ion channel and its modulators and antagonists may be used to diagnose or treat or relieve, by any means as described in this document, pain and cancer.
- pain include ⁇ es neuropathic, inflammatory, inflammatory bowel disease, thermal hyperalgesia, visexal pain, migraine, post he ⁇ atic neuralgia, diabetic neuralgia, trigeminal neuralgia, post operative pain, osteoarthritis, rhuematoid arthritis.
- pain includes, but is not limited to pain arising from injuries,, diseases, headaches, migraines, cancer pain, pain arising from neurological disorders such as Parkinson's disease, pain arising from spine and peripheral nerve sitrgery, brain tumors, traumatic brain injury (TBI), spinal cord trauma, chronic pain syndromes, chronic fatigue syndrome, neuralgias such as trigeminal neuralgia, glossopharyngeal neuralgia, posthe ⁇ etic neuralgia and causalgia, pain arising from lupus, sarcoidosis, arachnoiditis, arthritis, rheumatic disease, period pain, back pain, lower back pain, joint pain, abdominal pain, chest pain, labour pain, musculoskeletal and skin diseases, diabetes, head trauma, and f ⁇ bromyalgia.
- Example 5 describes an Open Field test, in which T ⁇ M8 knockout mice are shown to be less anxious than their wild type counte ⁇ arts. Furthennore, blood plasma levels of corticosterone, an indicator of stress and anxiety, are seen to be lower in T ⁇ M8 knockout mice than corresponding wild type mice (Example 6). A deficit of T ⁇ M8 activity is therefore correlated with a decrease in stress.
- T ⁇ M8 we therefore disclose a method of lowering stress or anxiety or both in an individual, the method comprising decreasing the level or activity of T ⁇ M8 in that individual. As noted elsewhere, this can be achieved by down-regulating the expression of T ⁇ M8, or by use of antagonists to T ⁇ M8.
- T ⁇ M8 may be used to treat or alleviate diseases or syndromes in which stress and anxiety feature.
- diseases include social anxiety, post traumatic stress disorder, phobias, social phobia, special phobias, panic disorder, obsessive compulsive disorder, acute stress, disorder, separation anxiety disorder, generalised anxiety disorder, major depression, dysthymia, bipolar disorder, seasonal affective disorder, post natal depression, manic depression, bipolar depression.
- the T ⁇ M8 associated disease comprises a disease in which stress or anxiety is a symptom.
- the T ⁇ M8 disease comprises the above list of anxiety and stress related diseases.
- T ⁇ M8 ion channel may be used to diagnose and/or treat any of these specific diseases using any of the methods and compositions described here.
- nucleic acids vectors comprising T ⁇ M8 ion channel nucleic acids, polypeptides, including homologues, variants or derivatives thereof, pharmaceutical compositions, host cells, and -transgenic animals comprising T ⁇ M8 ion channel nucleic acids and/or polypeptides, for the treatment or diagnosis of the specific diseases listed above.
- T ⁇ M8 ion ctiannel preferably antagonists of T ⁇ M8, preferably a compound capable decreasing the conductance of the channel, antibodies against T ⁇ M8 ion channel, as well as methods of making or identifying these, in diagnosis or treatment of the specific diseases mentioned above.
- T ⁇ M8 ion ctiannel preferably antagonists of T ⁇ M8, preferably a compound capable decreasing the conductance of the channel, antibodies against T ⁇ M8 ion channel, as well as methods of making or identifying these, in diagnosis or treatment of the specific diseases mentioned above.
- diagnostic kits for the detection of the specific diseases in an individual are also disclose diagnostic kits for the detection of the specific diseases in an individual.
- T ⁇ M8 associated diseases Anxiety and stress, as well as disorders in which these are manifested, including T ⁇ M8 associated diseases, are well known in the art. A summary description follows:
- Anxiety and stress are also referred to as feeling uptight, tension, jitters, and apprehension. Stress can come from any situation or thought that makes an individual feel frustrated, angry, or anxious. What is stressful to one person is not necessarily stressful to another. Anxiety is a feeling of apprehension or fear. The source of this uneasiness is not always known or recognized, which can add to the distress the individual feels. Stress is a normal part of life. In small quantities, stress is may be benefiicaL - it can motivate an individual and him to be more productive. However, too much stress, or a strong response to stress, is harmful. It can set the individual up for general poor health as well as specific physical or psychological illnesses like infection, heart disease, or depression. Persistent and unrelenting stress often leads to anxiety and unhealthy behaviors like overeating and abuse of alcohol or drugs.
- Emotional states like grief or depression and health conditions like an overactive thyroid, low blood sugar, or heart attack can also cause stress.
- Anxiety is often accompanied by physical symptoms, including: twitching or trembling, muscle tension, headaches, sweating, dry mouth, difficulty swallowing, abdominal pain (this may be the only symptom of stress, especially in a child)
- T ⁇ M8 and its modulators may be used to treat or alleviate any of these symptoms.
- Anxiety disorders are a group of psychiatric conditions that involve excessive anxiety. They include generalized anxiety disorder, specific phobias, obsessive- compulsive disorder, and social phobia. See also T ⁇ M8 associated diseases set omt above.
- Certain drugs can lead to symptoms of anxiety due to either side effects or withdrawal from the drug.
- Such drugs include caffeine, alcohol, nicotine, cold remedies, decongestants, bronchodilators for asthma, tricyclic antidepressants, cocaine, amphetamines, diet pills, ADHD medications, and thyroid medications.
- T ⁇ M8 and its modulators in combination with such drugs to alleviate their stress and/or anxiety inducing effects.
- a poor diet can also contribute to stress or anxiety — for example, low levels of vitamin B 12 .
- Performance anxiety is related to specific situations, like taking a test or making a presentation in public.
- Post-traumatic stress disorder PTSD is a stress disorder that develops after a traumatic event like war, physical or sexual assault, or a natural disaster.
- a tumor of the adrenal gland may be the cause of anxiety. This happens because of an ove ⁇ roduction of hormones responsible for the feelings and symptoms of anxiety.
- Acute Pain is defined as short-term pain or pain with an easily identifiable cause. Acute pain is the body's warning of present damage to tissue or disease. It is often fast and sha ⁇ followed by aching pain. Acute pain is centralized in one area before becoming somewhat spread out.
- Chronic Pain Chronic pain is medically defined as pain that has lasted 6 months or longer.
- the experience of physiological pain can be grouped into four categories according to the source and related nociceptors (pain detecting nerves). Cutaneous Pain
- Cutaneous pain is caused by injury to the skin or superficial tissues. Cutaneous nociceptors terminate just below the skin, and due to the high concentration of nerve endings, produce a well-defined, localised pain of short duration. Example injuries that produce cutaneous pain include paper cuts, minor (first degree) burns and lacerations. Somatic Pain
- Somatic pain originates from ligaments, tendons, bones, blood vessels, and even nerves themselves, and are detected with somatic nociceptors.
- the scarcity of pain receptors in these areas produces a dull, poorly-localised pain of longer duration than cutaneous pain; examples include sprained ankle and broken bones.
- Visceral pain originates from body organs visceral nociceptors are located within body organs and internal cavities. The even greater scarcity of nociceptors in these areas produces a pain usually more aching and of a longer duration than somatic pain. Visceral pain is extremely difficult to localise, and several injuries to visceral tissue exhibit "referred" pain, where the sensation is localised to an area completely unrelated to the site of injury. Myocardial ischaemia (the loss of blood flow to a part of the heart muscle tissue) is possibly the best known example of referred pain; the sensation can occur in the upper chest as a restricted feeling, or as an ache in the left shoulder, arm or even hand. Other Types of Pain
- Phantom limb pain is the sensation of pain from a limb that one no longer has or no longer gets physical signals from - an experience almost universally reported by amputees and quadriplegics.
- Neuropathic pain (“neuralgia”) can occur as a result of injury or disease to the nerve tissue itself. This can disrupt the ability of the sensory nerves to transmit correct information to the thalamus, and hence the brain inte ⁇ rets painful stimuli even though there is no obvious or documented physiologic cause for the pain.
- Trigeminal neuralgia (“tic douloureux”) refers to pain caused by injury or damage to the trigeminal nerve.
- the trigeminal nerve has 3 branches: VI gives sensation to the area of the forehead and eye and V2 gives sensation to the nose and face and V3 gives sensation to the jaw and chin area. Each side of the face has a trigeminal nerve that gives sensation The one-sided pain of trigeminal neuralgia may extend through the cheek, mouth, nose and/or jaw muscles.
- Trigeminal neuralgia generally affects older people, although younger people or those with multiple sclerosis may also experience trigeminal neuralgia.
- the primary symptom of trigeminal neuralgia is pain in either the forehead, cheek, chin or jawline. Severe cases may involve all three areas or both left and right sides. Pain episodes are severe, spastic and short, and are described as similar to what would be felt as electrical shock. The pain can be triggered by common daily activities such as brushing the teeth, talking, chewing, drinking, shaving or even kissing. The frequency of the pain episodes increases over time, becoming more disruptive and disabling.
- Glossopharyngeal neuralgia is a clinical entity characterized by bursts of pain in the sensory distribution of the ninth cranial nerve. Except for the location of the pain and the stimulus for the pain the attacks are identical to trigeminal neuralgia.
- the typical pain is a severe lancinating, repetitive series of electrical-like stabs in the region of the tonsils or the back of the tongue, on one side. In addition, the pain may radiate to or originate in the ear. .
- the sensory stimulus which induces the pain is swallowing, and during severe attacks the patient may sit motionless, head flexed forward, allowing saliva to freely drool from the mouth.
- Cardiac arrest, syncope (fainting), and seizures have been associated with attacks of glossopharyngeal neuralgia.
- the cause of glossopharyngeal neuralgia in most cases is unknown. However, a certain number of cases have been ascribed to tumors, compression of the ninth nerve by the vertebral artery, and vascular malformations.
- Posthe ⁇ etic neuralgia refers to chronic pain continuing after an infection of he ⁇ es zoster virus.
- He ⁇ es zoster also known as shingles, is a recurrent infection of varicella-zoster (chickenpox) viral infection.
- the virus lies dormant within nerves until the patient's immunity wanes.
- the acute lesion of shingles causes pain which usually goes away.
- the pain continues chronically - posthe ⁇ etic neuralgia.
- the symptoms of he ⁇ es zoster include a lancinating, deep, continuous pain: the pain is in the thoracic region 65% and the face 20%.
- the virus shows a predilection for the ophthalmic division of the trigeminal nerve (top of the face above the eyebrows).
- the pain usually resolves spontaneously in 2 to 4 weeks.
- a few patients will have persistent pain.
- the pain is in the region of the previous rash and is exacerbated by gently stroking the affected skin and is relieved by applying pressure to the area.
- the rubbing of clothing is often very painful. This continuing pain is called posthe ⁇ etic neuralgia.
- Symptoms include Pain: usually burning, and prominent in hand or foot. Onset in the majority is within 24 hours of injury. The median, ulnar and sciatic nerves are the most commonly involved. Almost any sensory stimulation worsens the pain.
- Vascular changes Either increased blood by vasodilatation (warm and pink) or decreased blood by vasoconstriction (cold, mottled blue).
- Trophic changes dry/scaly skin, stiff joints, tapering fingers, ridged uncut nails, either long/coarse hair or loss of hair, sweating alteration,
- T ⁇ M8 ion channel polypeptide is intended to refer to a polypeptide comprising the amino acid sequence shown in SEQ ED No. 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof.
- the polypeptide comprises or is a homologue, variant or derivative of the sequence shown in SEQ ID NO: 3.
- Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
- Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.
- Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
- Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylmositol, cross-inking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-inks, formation of cystine, formation of pyro glutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
- variants include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to a sequence.
- references to “T ⁇ M8” and “T ⁇ M8 ion channel” include references to such variants, homologues, derivatives and fragments of T ⁇ M8.
- the resultant amino acid sequence has ion channel activity, more preferably having at least the same activity of the T ⁇ M8 ion channel shown as SEQ ID NO: 3 or SEQ ID NO: 5.
- homologue covers identity with respect to structure and/or function providing the resultant amino acid sequence has ion channel activity.
- sequence identity i.e. similarity
- sequence identity preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
- sequence identity preferably there is at least 70%, more preferably at least 75%, more preferably at least 85%, even more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
- channel activity or “biological activity” of an ion channel such as T ⁇ M8 ion channel
- these terms are intended to refer to the metabolic or physiological function of the T ⁇ M8 ion channel, including similar activities or improved activities or these activities with decreased undesirable side effects.
- antigenic and immunogenic activities of the T ⁇ M8 ion channel are also included. Examples of ion channel activity, and methods of assaying and quantifying these activities, are known in the art, and are described in detail elsewhere in this document.
- a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
- an “insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring substance.
- substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
- T ⁇ M8 polypeptides as described here may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent amino acid sequence.
- Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues.
- negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
- T ⁇ M8 polypeptides may further comprise heterologous amino acid sequences, typically at the N-terminus or C-te ⁇ ninus, preferably the N-terminus.
- Heterologous sequences may include sequences that affect intra or extracellular protein targeting (such as leader sequences).
- Heterologous sequences may also include sequences that increase the immunogenicity of the polypeptide and/or which facilitate identification, extraction and/or purification of the polypeptides.
- Another heterologous sequence that is particularly preferred is a polyamino acid sequence such as polyhistidine which is preferably N-terminal.
- a polyhistidine sequence of at least 10 amino acids, preferably at least 17 amino acids but fewer than 50 amino acids is especially preferred.
- the T ⁇ M8 ion channel polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
- T ⁇ M8 polypeptides are advantageously made by recombinant means, using known techniques. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis.
- the polypeptides described here may also be produced as fusion proteins, for example to aid in extraction and purification.
- fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and ⁇ -galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences, such as a thrombin cleavage site.
- the fusion protein will not hinder the function of the protein of interest sequence.
- T ⁇ M8 polypeptides may be in a substantially isolated form. This term is intended to refer to alteration by the hand of man from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
- a polynucleotide, nucleic acid or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide, nucleic acid or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.
- T ⁇ M8 ion channel protein may be mixed with carriers or diluents which will not interfere with the intended pu ⁇ ose of the protein and still be regarded as substantially isolated.
- a T ⁇ M8 polypeptide may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 90%, for example, 95%, 98% or 99% of the protein in the preparation is a T ⁇ M8 polypeptide.
- peptides comprising a portion of a T ⁇ M8 polypeptide.
- the peptides may be between 2 and 200 amino acids, preferably between 4 and 40 amino acids in length.
- the peptide may be derived from a T ⁇ M8 polypeptide as disclosed here, for example by digestion with a suitable enzyme, such as trypsin.
- the peptide, fragment, etc may be made by recombinant means, or synthesised synthetically,
- the term "peptide” includes the various synthetic peptide variations known in the art, such as a retroinverso D peptides.
- the peptide may be an antigenic determinant and/or a T-cell epitope.
- the peptide may be immunogenic in vivo.
- the peptide is capable of inducing neutralising antibodies in vivo.
- the T ⁇ M8 polypeptides may therefore comprise a sequence which corresponds to at least part of a homologous region.
- a homologous region shows a high degree of homology between at least two species.
- the homologous region may show at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% identity at the amino acid level using the tests described above.
- Peptides which comprise a sequence which corresponds to a homologous region may be used in therapeutic strategies as explained in further detail below.
- the T ⁇ M8 ion chamiel peptide may comprise a sequence which corresponds to at least part of a heterologous region.
- a heterologous region shows a low degree of homology between at least two species.
- T ⁇ M8 polynucleotides T ⁇ M8 nucleotides and T ⁇ M8 nucleic acids, methods of production, uses of these, etc, as described in further detail elsewhere in this document.
- the terms "T ⁇ M8 polynucleotide”, “T ⁇ M8 nucleotide” and “T ⁇ M8 nucleic acid” may be used interchangeably, and are intended to refer to a polynucleotide/nucleic acid comprising a nucleic acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof.
- the polynucleotide/nucleic acid comprises or is a homologue, variant or derivative of the nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2, most preferably, SEQ ID NO: 2.
- T ⁇ M8 ion channel polynucleotides and nucleic acids comprise a nucleotide sequence capable of encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof.
- the T ⁇ M8 ion channel polynucleotides and nucleic acids comprise a nucleotide sequence capable of encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, or a homologue, variant or derivative thereof.
- Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
- Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
- polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
- Modified bases include, for example, tritylated bases and unusual bases such as inosine.
- polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
- Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
- nucleotide sequence refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof).
- the nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin which may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
- the term nucleotide sequence may be prepared by use of recombinant DNA techniques (for example, recombinant DNA).
- nucleotide sequence means DNA.
- variants include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acids from or to the sequence of a T ⁇ M8 nucleotide sequence.
- references to "T ⁇ M8" and “T ⁇ M8 ion channel” include references to such variants, homologues, derivatives and fragments of T ⁇ M8.
- the resultant nucleotide sequence encodes a polypeptide having ion channel activity, preferably having at least the same activity of the ion channel shown as SEQ ID NO: 3 or SEQ ID NO: 5.
- the term "homologue” is intended to cover identity with respect to structure and/or function such that the resultant nucleotide sequence encodes a polypeptide which has ion channel activity.
- sequence identity i.e. similarity
- sequence identity preferably there is at least 70% 0 , more preferably at least 75%, more preferably at least 85%>, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
- sequence identity i.e. similarity
- sequence identity preferably there is at least 70% 0 , more preferably at least 75%, more preferably at least 85%>, more preferably at least 90% sequence identity. More preferably there is at least 95%, more preferably at least 98%, sequence identity.
- Sequence identity with respect to any of the sequences presented here can be determined by a simple "eyeball” comparison (i.e. a strict comparison) of any one or more of the sequences with another sequence to see if that other sequence has, for example, at least 70% sequence identity to the sequence(s).
- Relative sequence identity can also be determined by commercially available computer programs that can calculate % identity between two or more sequences using any suitable algorithm for determining identity, using for example default parameters.
- a typical example of such a computer program is CLUSTAL.
- Other computer program methods to determine identify and similarity between the two sequences include but are not limited to the GCG program package (Devereux et al 1984 Nucleic Acids Research 12: 387) and FASTA (A-tschul et al 1990 J Molec Biol 403-410).
- % homology may be calculated o"ver contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time.
- ungapped alignment This is called an "ungapped" alignment.
- ungapped alignments are performed only over a relatively short number of residues.
- this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology.
- a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
- An example of such a matrix commonly used is the BLOSU 62 matrix - the default matrix for the BLAST suite of programs.
- GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied. It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
- the BLAST algorithm is employed, with parameters set to default values.
- the BLAST algorithm is described in detail at http://www.ncbi.nih.gov/BLAST/blast_help.html, which i s inco ⁇ orated herein by reference. Search parameters can be defined and can be advantageously set over the defined default parameters.
- substantially identical when asses sed by BLAST equates to sequences which match with an EXPECT value of at least about 7, preferably at least about 9 and most preferably 10 or more.
- the default thresliold for EXPECT in BLAST searching is usually 10.
- BLAST Basic Local Alignment Search Tool
- blastp, blastn, blastx, tblastn, and tblastx these programs ascribe significance to their findings using the statistical methods of Karlin and Altschul (Karlin and Altschul 1990, Proc. Natl. Acad. Sci. USA 87:2264-68; Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-7; see http://www.ncbi.nih.gov/BLAST/blast_help.html) with a few enhancements.
- the BLAST programs are tailored for sequence similarity searching, for example to identify homologues to a query sequence. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al (1994) Nature Genetics 6:119-129.
- blastp compares an amino acid query sequence against a protein sequence database
- blastn compares a nucleotide query sequence against a nucleotide sequence database
- blastx compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database
- tblastn compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands)
- tblastx compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
- BLAST uses the following search parameters:
- HISTOGRAM - Display a histogram of scores for each search; default is yes. (See parameter H in the BLAST Manual).
- DESCRIPTIONS Restricts the number of short descriptions of matching sequences reported to the number specified; default limit is 100 descriptions. (See parameter V in the manual page).
- EXPECT The statistical significance threshold for reporting matches against database sequences; the default value is 10, such that 10 matches are expected to be found merely by chance, according to the stochastic model of Karlin and Altschul (1990). If the statistical significance ascribed to a match is greater than the EXPECT threshold, the match will not be reported. Lower EXPECT thresholds are more stringent, leading to fewer chance matches being reported. Fractional values are acceptable. (See parameter E in the BLAST Manual).
- CUTOFF - Cutoff score for reporting high-scoring segment pairs.
- the default value is calculated from the EXPECT value (see above).
- HSPs are reported for a database sequence only if the statistical significance ascribed to them is at least as higli as would be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher CUTOFF values are more stringent, leading to fewer chance matches being reported. (See parameter S in the BLAST Manual). Typically, significance thresholds can be more intuitively managed using EXPECT.
- ALIGNMENTS Restricts database sequences to the number specified for which high-scoring segment pairs (HSPs) are reported; the default limit is 50. If more database sequences than this happen to satisfy the statistical significance threshold for reporting (see EXPECT and CUTOFF below), only the matches ascribed the greatest statistical significance are reported. (See parameter B in the BLAST Manual).
- MATRIX - Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and TBLASTX.
- the default matrix is BLOSUM62 (Henikoff & Henikoff, 1992).
- the valid alternative choices include: PAM40, PAM120, PAM250 and
- IDENTITY No alternate scoring matrices are available for BLASTN; specifying the MATRIX directive in BLASTN requests returns an error response.
- FILTER - Mask off segments of the query sequence that have low compositional complexity, as determined by the SEG program of Wootton & Federhen (1993) Computers and Chemistry 17:149-163, or segments consisting of short- periodicity internal repeats, as determined by the XNU program of Claverie & States (1993) Computers and Chemistry 17 : 191 -201 , or, for BLASTN, by the DUST program of Tatusov and Lipman (see http://www.ncbi.nlm.nih.gov).
- Filtering can eliminate statistically significant but biologically uninteresting reports from the blast output (e.g., hits against common acidic-, basic- or proline-rich regions), leaving the more biologically interesting regions of the query sequence available for specific matching against database sequences.
- Low complexity sequence found by a filter program is substituted using the letter "N” in nucleotide sequence (e.g., "NNNNNNNNNNNNN”) and the letter "X” in protein sequences (e.g., "XXXXXXXXX").
- Filtering is only applied to the query sequence (or its translation products), not to database sequences. Default filtering is DUST for BLASTN, SEG for other programs.
- NCBI-gi causes NCBI gi identifiers to be shown in the output, in addition to the accession and/or locus name.
- sequence comparisons are conducted using the simple BLAST search algorithm provided at http://www.ncbi.nlm.nih.gov/BLAST. In some embodiments, no gap penalties are used when determining sequence identity.
- Hybridization means a "process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY) as well as the process of amplification as carried out in polymerase chain reaction technologies as described in Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY).
- Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
- Nucleotide sequences capable of selectively hybridising to the nucleotide sequences presented herein, or to their complement will be generally at least 70%, preferably at least 75%, more preferably at least 85 or 90% and even more preferably at least 95% or 98%> homologous to the corresponding nucleotide sequences presented herein over a region of at least 20, preferably at least 25 or 30, for instance at least 40, 60 or 100 or more contiguous nucleotides.
- Preferred nucleotide sequences will comprise regions homologous to SEQ ID NO: 1, 2 or 4, preferably at least 70%, 80% or 90% and more preferably at least 95% homologous to one of the sequences.
- the term "selectively hybridizable" means that the nucleotide sequence used as a probe is used under conditions where a target nucleotide sequence is found to hybridize to the probe at a level significantly above background.
- the background hybridization may occur because of other nucleotide sequences present, for example, in the cDNA or genomic DNA library being screened.
- background implies a level of signal generated by interaction between the probe and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target DNA.
- the intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with P.
- nucleotide sequences that are capable of hybridizing to the nucleotide sequences presented herein under conditions of intermediate to maximal stringency.
- Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency” as explained below.
- Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm.
- a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related nucleotide sequences.
- nucleotide sequence is double-stranded, both strands of the duplex, either individually or in combination, are encompassed.
- nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also of use.
- nucleotide sequences that are capable of hybridising to the sequences that are complementary to the sequences presented herein, or any fragment or derivative thereof.
- our disclosure encompasses nucleotide sequences that are complementary to sequences that are capable of hybridising to the sequence.
- variant nucleotide sequences are examples of variant nucleotide sequences.
- variant encompasses sequences that are complementary to sequences that are capable of hydridising to the nucleotide sequences presented herein.
- the term “variant” encompasses sequences that are complementary to sequences that are capable of hydridising under stringent conditions (eg.
- the present disclosure encompasses nucleotide sequences that are complementary to the sequences presented here, or any fragment or derivative thereof. If the sequence is complementary to a fragment thereof then that sequence can be used as a probe to identify and clone similar ion channel sequences in other organisms etc.
- T ⁇ M8 ion channel This enables the cloning of T ⁇ M8 ion channel, its homologues and other structurally or functionally related genes from human and other species such as mouse, pig, sheep, etc to be accomplished.
- Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate partial or full-length cDNAs and genomic clones encoding T ⁇ M8 ion channel from appropriate libraries.
- Such probes may also be used to isolate cDNA and genomic clones of other genes (including genes encoding homologues and orthologues from species other than human) that have sequence similarity, preferably high sequence similarity, to the T ⁇ M8 ion channel gene.
- Hybridization screening, cloning and sequencing techniques are known to those of skill in the art and are described in, for example, Sambrook et al (supra).
- nucleotide sequences suitable for use as probes are 70% identical, preferably 80% identical, more preferably 90% identical, even more preferably 95%> identical to that of the referent.
- the probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 150 and 500 nucleotides, more particularly about 300 nucleotides.
- to obtain a polynucleotide encoding a T ⁇ M8 polypeptide, including homologues and orthologues from species other than human comprises the steps of screening an appropriate library under stringent hybridization conditions with a labelled probe having the SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 or a fragment thereof and isolating partial or full-length cDNA and genomic clones containing said polynucleotide sequence.
- stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42 degrees C.
- T ⁇ M8 ion channel polynucleotides may be verified by sequence analysis or functional assays.
- the conductance of Xenopus oocytes tranfected as described above may be detected as a means of guaging and quantifying T ⁇ M8 activity, useful for screening assays described below.
- Such a conductance assay is referred to for convenience as a "Functional Assay of T ⁇ M8 (Conductance)".
- the putative T ⁇ M8 ion channel or homologue may be assayed for activity in a "Functional Assay of T ⁇ M8 (Conductance)" as follows.
- Capped RNA transcripts from linearized plasmid templates encoding the T ⁇ M8 ion channel cDNAs are synthesized in vitro with RNA polymerases in accordance with standard procedures.
- In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a microinjection apparatus.
- Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response to agonist exposure. Recordings are made in 96mM NaCl, 2mM KCl, lmMMgCl 2 , 0. lmMCaCl 2 , 5mM Hepes, pH7.4 and supplemented with 200mM mannitol at room temperature. OSM is 210mOsm.
- the Xenopus system may also be used to screen known ligands and tissue/cell extracts for activating ligands, as described in further detail below.
- Alternative functional assays include whole cell electrophysiology, fluorescence resonance energy transfer (FRET) analysis and FLIPR analysis.
- T ⁇ M8 ion channel associated diseases
- methods known in the art may be used to determine the organs, tissues and cell types (as well as the developmental stages) in which T ⁇ M8 is expressed.
- traditional or “electronic" Northerns may be conducted.
- Reverse-transcriptase PCR RT-PCR
- More sensitive methods for determining the expression profile of T ⁇ M8 include RNAse protection assays, as known in the art.
- Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labelled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound.
- Analogous computer techniques (“electronic Northerns") applying BLAST may be used to search for identical or related molecules in nucleotide databases such as GenBank or the LIFESEQ database (Incyte Pharmaceuticals). This type of analysis has advantages in that they may be faster than multiple membrane-based hybridizations.
- the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
- polynucleotides and polypeptides described here may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease, as explained in further detail elsewhere in this document.
- the method comprises in general culturing a host cell comprising a nucleic acid encoding T ⁇ M8 ion channel polypeptide, or a homologue, variant, or derivative thereof, under suitable conditions (i.e., conditions in which the T ⁇ M8 ion channel polypeptide is expressed).
- the nucleotide sequences encoding T ⁇ M8 ion channel or homologues, variants, or derivatives thereof are inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
- a variety of expression vector/host systems may be utilized to contain and express sequences encoding T ⁇ M8 ion channel. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus (
- control elements are those non-translated regions of the vector (i.e., enhancers, promoters, and 5' and 3' untranslated regions) which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid
- the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding T ⁇ M8 ion channel, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
- a number of expression vectors may be selected depending upon the use intended for T ⁇ M8 ion channel. For example, when large quantities of T ⁇ M8 ion channel are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be used.
- Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding T ⁇ M8 ion channel may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced, pIN vectors (Van Heeke, G. and S. M.
- pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST).
- GST glutathione S- transferase
- fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion to glutathione-agarose beads followed by elution in the presence of free glutathione.
- Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
- plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
- plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used.
- These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews. (See, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.).
- An insect system may also be used to express T ⁇ M8 ion channel.
- Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
- the sequences encoding T ⁇ M8 ion channel may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
- Successful insertion of T ⁇ M8 ion channel will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
- the recombinant viruses may then be used to infect, for example, S.
- a number of viral-based expression systems may be utilized.
- sequences encoding T ⁇ M8 ion channel may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome maybe used to obtain a viable virus which is capable of expressing T ⁇ M8 ion channel in infected host cells.
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
- RSV Rous sarcoma virus
- the T ⁇ M8 ion channels may be expressed in either human embryonic kidney 293 (HEK293) cells or adherent CHO cells.
- HEK293 human embryonic kidney 293
- adherent CHO cells typically all 5' and 3' untranslated regions (UTRs) are removed from the T ⁇ M8 cDNA prior to insertion into a pCDN or pCDNA3 vector.
- the cells are transfected with individual cDNAs by lipofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis.
- HEK293 or CHO cells transfected with the vector alone serve as negative controls.
- T ⁇ M8 ion channel mRNAs are generally detectable in about 50% of the G - 18-resistant clones analyzed.
- HACs Human artificial chromosomes
- HACs Human artificial chromosomes
- HACs Human artificial chromosomes
- HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic pu ⁇ oses.
- Specific initiation signals may also be used to achieve more efficient translation of sequences encoding T ⁇ M8 ion channel. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding T ⁇ M8 ion channel and its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
- exogenous translational control signals including the ATG initiation codon should be provided.
- the initiation codon should be in the correct reading frame to ensure translation of the entire insert.
- Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular cell system used, such as those described in the literature. (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
- a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
- modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
- Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding, and/or function.
- Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure the correct modification and processing of the foreign protein.
- ATCC American Type Culture Collection
- cell lines capable of stably expressing T ⁇ M8 ion channel can be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The pmpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully/ express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems may be used to recover transformed cell lines.
- he ⁇ es simplex virus thymidine kixiase genes (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltrans ⁇ erase genes (Lowy, I. et al. (1980) Cell 22:817-23), which can be employed in tk " or apr " cells, respectively.
- antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
- dhfr confers resistance to methotrexate
- npt confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, t ⁇ B, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. (Hartman, S. C.
- T ⁇ M8 ion channel is inserted within a marker gene sequence, transformed cells containing sequences encoding; T ⁇ M8 ion channel can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding T ⁇ M8 ion channel under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
- host cells which contain the nucleic acid sequence encoding T ⁇ M8 ion chamiel and express T ⁇ M8 ion channel may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
- the presence of polynucleotide sequences encoding T ⁇ M8 ion channel can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding T ⁇ M8 ion channel.
- Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding T ⁇ M8 ion channel to detect transformants containing DNA or RNA encoding T ⁇ M8 ion channel.
- T ⁇ M8 ion channel A variety of protocols for detecting and measuring the expression of T ⁇ M8 ion channel, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
- ELISAs enzyme-linked immunosorbent assays
- RIAs radioimmunoassays
- FACS fluorescence activated cell sorting
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on T ⁇ M8 ion channel is preferred, but a competitive binding assay may be employed.
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding T ⁇ M8 ion channel include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
- the sequences encoding T ⁇ M8 ion channel, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
- RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
- T7, T3, or SP6 RNA polymerase
- Suitable reporter molecules or labels which may be used for ease of detection include radionuchdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host cells transformed with nucleotide sequences encoding T ⁇ M8 subunits may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
- the protein produced by a transformed cell may be located in the cell membrane, secreted or contained intracellularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode T ⁇ M8 subunits may be designed to contain signal sequences which direct secretion of T ⁇ M8 subunits through a prokaryotic or eukaryotic cell membrane.
- purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension affinity purification system (Immunex Co ⁇ ., Seattle, Wash.).
- cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, Calif) between the purification domain and the T ⁇ M8 subunit encoding sequence may be used to facilitate purification.
- One such expression vector provides for expression of a fusion protein containing T ⁇ M8 subunit and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on immobilized metal ion affinity chromatography (IMIAC; described in Porath, J. et al. (1992) Prot. Exp. Purif.
- IIMIAC immobilized metal ion affinity chromatography
- enterokinase cleavage site provides a means for purifying T ⁇ M8 subunits from the fusion protein.
- Fragments of T ⁇ M8 subunits may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques.
- Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the Applied Biosystems 431 A peptide synthesizer (Perkin Elmer). Various fragments of T ⁇ M8 subunits may be synthesized separately and then combined to produce the full length molecule.
- T ⁇ M8 polypeptides, nucleic acids, probes, antibodies, expression vectors and ligands are useful as (and for the production of) biosensors.
- a biosensor is defined as being a unique combination of a receptor for molecular recognition, for example a selective layer with immobilized antibodies or ion channels such as a T ⁇ M8, and a transducer for transmitting the values measured.
- a receptor for molecular recognition for example a selective layer with immobilized antibodies or ion channels such as a T ⁇ M8, and a transducer for transmitting the values measured.
- One group of such biosensors will detect the change which is caused in the optical properties of a surface layer due to the interaction of the receptor with the sunounding medium.
- Among such techniques may be mentioned especially ellipso-metry and surface plasmon resonance.
- Biosensors inco ⁇ orating T ⁇ M8 may be used to detect the presence or level of T ⁇ M8 ligands. The construction of such biosensors is well known in the art.
- cell lines expressing T ⁇ M8 subunits may be used as reporter systems for detection of ligands such as ATP via receptor-promoted formation of [3H]inositol phosphates or other second messengers (Watt et al., 1998, JBiol Chem May 29;273(22): 14053-8).
- ligands such as ATP via receptor-promoted formation of [3H]inositol phosphates or other second messengers
- Receptor-ligand biosensors are also described in Hoffman et al., 2000, Proc Natl Acad Sci USA Oct 10;97(21):11215-20.
- Optical and other biosensors comprising T ⁇ M8 may also be used to detect the level or presence of interaction with G-proteins and other proteins, as described by, for example, Figler et al, 1997, Biochemistiy Dec 23;36(51):16288-99 and Sarrio et al., 2000, Mol Cell Biol 2000 Jul;20(14):5164-74). Sensor units for biosensors are described in, for example, US 5,492,840.
- the T ⁇ M8 polypeptide may be employed in a screening process for compounds which bind the T ⁇ M8 ion channel and which activate (agonists) or inhibit activation of (antagonists or blockers) of T ⁇ M8.
- such polypeptides may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
- substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991).
- T ⁇ M8 ion channel polypeptides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate T ⁇ M8 ion channels on the one hand and which can inhibit the function of T ⁇ M8 ion channels on the other hand.
- agonists and antagonists are employed for therapeutic and prophylactic pu ⁇ oses for such conditions as anxiety, stress, depression cancer or pain.
- Rational design of candidate compounds likely to be able to interact with T ⁇ M8 ion channel proteins may be based upon structural studies of the molecular shapes of a polypeptide.
- One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., X-ray crystallography or two- dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions.
- X-ray crystallography or two- dimensional NMR techniques.
- T ⁇ M8 ion channel polypeptide on the surface thereof.
- Such cells include cells from animals, yeast, Drosophila or E. coli.
- Cells expressing T ⁇ M8 are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
- Xenopus oocytes may be injected with T ⁇ M8 mRNA or polypeptide, and currents induced by exposure to test compounds measured by use of voltage clamps measured, as described in further detail elsewhere.
- a library or bank of candidate ligands may advantageously be produced and screened.
- a bank of over 200 putative ligands has been assembled for screening.
- the bank comprises: transmitters, hormones and chemokines known to act via an ion channel; naturally occurring compounds which may be putative agonists for an ion channel, non-mammalian, biologically active peptides for which a mammalian counte ⁇ art has not yet been identified; and compounds not found in nature, but which activate ion channels with unknown natural ligands.
- This bank may be used to screen the T ⁇ M8 ion channel for known ligands, using both functional (i.e.
- T ⁇ M8 may also be functionally screened (using calcium, microphysiometer, ooyte electrophysiology, etc., functional screens) against tissue extracts to identify natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated, with the fractions being assayed as described here, until an activating ligand is isolated and identified.
- Ion channels which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimuation or inhibition.
- One screening technique therefore includes the use of cells which express T ⁇ M8 (for example, transfected Xenopus oocytes, CHO or HEK293 cells) in a system which measures extracellular pH or intracellular calcium changes caused by channel activity.
- T ⁇ M8 for example, transfected Xenopus oocytes, CHO or HEK293 cells
- compounds may be contacted with cells expressing the T ⁇ M8 polypeptide.
- a second messenger response e.g., signal transduction, pH changes, or preferably changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the channel.
- the response is a change in calcium level; such an assay is referred to for convenience as a "Functional Assay for T ⁇ M8 (Calcium Concentration)".
- basal calcium levels in the HEK 293 cells in transfected or vector control cells are observed to be in the nonnal, 100 nM to 200 nM, range.
- HEK 293 cells expressing homomeric or heteromeric T ⁇ M8 ion channels or recombinant homomeric or heteromeric T ⁇ M8 ion channels are loaded with fura 2 and in a single day more than 150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization.
- HEK 293 cells expressing T ⁇ M8 ion channel or recombinant T ⁇ M8 ion channel are evaluated for the increase or decrease of Ca flux.
- Agonists presenting a calcium transient are tested in vector control cells to determine if the response is unique to the transfected cells expressing the ion channel.
- Another method involves screening for ion channel inhibitors by determining inhibition or stimulation of T ⁇ M8 ion channels.
- Such a method involves transfecting a eukaryotic cell with the T ⁇ M8 subunits either alone to form a homomeric channel or with other T ⁇ channel subunits to form a heteromeric channel to express the ion channel on the cell surface. The cell is then exposed to potential antagonists in the presence of the T ⁇ M8 ion channel. The cell can be tested using whole cell electrophsysiology to determine the changes in the conductance or kinetics of the current.
- T ⁇ M8 Another method for detecting agonists or antagonists of T ⁇ M8 is the yeast based technology as described in U.S. Pat. No. 5,482,835, inco ⁇ orated by reference herein.
- the screen employs detection of a change in conductance to screen for agonists and antagonists of T ⁇ M8.
- antagonists of T ⁇ M8 lower the conductance of a suitably transfected cell.
- the conductance is lowered by 10%, 20%>, 30%>, 40%>, 50%), 60%), 70%) or more in the presence of an antagonist of T ⁇ M8.
- the conductance is lowered by 1 pS, 2 pS, 3 pS, 4pS, 5pS, lOpS, 15pS, 25pS, 35pS, 45pS, 60pS, 70pS or more in the presence of an antagonist of T ⁇ M8.
- agonists of T ⁇ M8 increase the conductance of a suitably transfected cell.
- the conductance is increased by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more in the presence of an agonist of T ⁇ M8.
- the conductance is increased by 1 pS, 2 pS, 3 pS, 4pS, 5pS, lOpS, 15pS, 25pS, 35pS, 45pS, 60pS, 70pS or more in the presence of an agonist of T ⁇ M8.
- the screen employs detection of a change in intracellular calcium concentration to screen for agonists and antagonists of T ⁇ M8.
- the screen employs a function assay as set out above under "Functional Assay of T ⁇ M8 (Calcium Concentration)" Specifically, we disclose a method in which antagonists of T ⁇ M8 lower the calcium concentration of a suitably transfected cell.
- the calcium concentration is lowered by 10%, 20%, 30%, 40%, 50%, 60%, 70% or more in the presence of an antagonist of T ⁇ M8.
- the calcium concentration is lowered by 1 pS, 2 pS, 3 pS, 4pS, 5pS, lOpS, 15pS, 25pS, 35pS, 45pS, 60pS, 70pS or more in the presence of an antagonist of T ⁇ M8.
- agonists of T ⁇ M8 increase the calcium concentration of a suitably transfected cell.
- the conductance is increased by 10%), 20%, 30%, 40%, 50%), 60%, 70% or more in the presence of an agonist of T ⁇ M8.
- the calcium concentration is increased by 1 pS, 2 pS, 3 pS, 4pS, 5pS, lOpS, 15pS, 25pS, 35pS, 45pS, 60pS, 70pS or more in the presence of an agonist ofT ⁇ M8.
- Phage display is a protocol of molecular screening which utilises recombinant bacteriophage.
- the technology involves transforming bacteriophage with a gene that encodes one compound from the library of candidate compounds, such that each phage or phagemid expresses a particular candidate compound.
- the transformed bacteriophage (which preferably is tethered to a solid support) expresses the appropriate candidate compound and displays it on their phage coat.
- Specific candidate compounds which are capable of binding to a T ⁇ M8 polypeptide or peptide are enriched by selection strategies based on affinity interaction.
- the successful candidate agents are then characterised.
- Phage display has advantages over standard affinity ligand screening technologies.
- the phage surface displays the candidate agent in a three dimensional configuration, more closely resembling its naturally occurring conformation. This allows for more specific and higher affinity binding for screening pu ⁇ oses.
- Another method of screening a library of compounds utilises eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing a library of compounds.
- Such cells either in viable or fixed form, can be used for standard binding-partner assays. See also Parce et al. (1989) Science 246:243- 247; and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA 87;4007-4011, which describe sensitive methods to detect cellular responses.
- This separation step could typically involve a procedure such as adhesion to filters followed by washing, adhesion to plastic following by washing, or centrifugation of the cell membranes.
- Still another approach is to use solubilized, unpurified or solubilized purified polypeptide or peptides, for example extracted from transformed eukaryotic or prokaryotic host cells.
- This allows for a "molecular" binding assay with the advantages of increased specificity, the ability to automate, and high drug test throughput.
- Another technique for candidate compound screening involves an approach which provides high throughput screening for new compounds having suitable binding affinity, e.g., to a T ⁇ M8 polypeptide, and is described in detail in International Patent application No. WO 84/03564 (Commonwealth Serum Labs.), published on September 13 1984.
- Ligand binding assays provide a direct method for ascertaining pharmacology and are adaptable to a high throughput format.
- the purified ligand may be radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its target.
- Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell receptor or ion channel sources.
- specific binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.
- the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the receptor or ion channel is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the target, using detection systems appropriate to the cells bearing the target at their surfaces. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
- the assays may simply comprise the steps of mixing a candidate compound with a solution containing a T ⁇ M8 polypeptide to form a mixture, measuring T ⁇ M8 ion channel activity in the mixture, and comparing the T ⁇ M8 ion channel activity of the mixture to a standard.
- the T ⁇ M8 subunit cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of T ⁇ M8 subunit mRNA and protein in cells.
- an ELISA may be constructed for measuring secreted or cell associated levels of T ⁇ M8 subunit protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of T ⁇ M8 subunit (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. Standard methods for conducting screening assays are well understood in the art.
- T ⁇ M8 ion channel antagonistsand blockers examples include antibodies or, in some cases, nucleotides and their analogues, including purines and purine analogues, oligonucleotides or proteins which are closely related to the ligand of the T ⁇ M8 ion chaimel, e.g., a fragment of the ligand, or small molecules which bind to the ion channel but do not elicit a response, so that the activity of the channel is prevented.
- the term "compound” refers to a chemical compound (naturally occurring or synthesised), such as a biological macromolecule (e.g., nucleic acid, protein, non- peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
- a biological macromolecule e.g., nucleic acid, protein, non- peptide, or organic molecule
- an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or even an inorganic element or molecule.
- the compound is an antibody.
- the materials necessary for such screening to be conducted may be packaged into a screening kit. Such a screening kit is useful for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc.
- the screening kit comprises: (a) a T ⁇ M8 polypeptide; (b) a recombinant cell expressing a T ⁇ M8 polypeptide; (c) a cell membrane expressing a T ⁇ MS polypeptide; or (d) antibody to a T ⁇ M8 polypeptide.
- the screening kit may optionally comprise instructions for use.
- transgenic animals capable of expressing natural or recombinant T ⁇ M8 ion channel, or a homologue, variant or derivative, at normal, elevated or reduced levels compared to the normal expression level.
- a transgenic animal is a non-human mammal, such as a pig, a sheep or a rodent.
- the transgenic animal is a mouse or a rat.
- transgenic animals in which all or a portion of the native T ⁇ M8 gene is replaced by T ⁇ M8 sequences from another organism. Preferably this organism is another species, most preferably a human. In highly preferred embodiments, we disclose a mouse which has substantially its entire T ⁇ M8 gene replaced with a human T ⁇ M8 gene. Such transgenic animals, as well as animals wliich are wild type for T ⁇ M8, may be used for screening agonists and/or antagonists of T ⁇ M8.
- such assays may involve exposing the wild type or transgenic animal, or a portion thereof, preferably a cell, tissue or organ of the transgenic animal, to a candidate substance, and assaying for a T ⁇ M8 associated phenotype such as pain or stress.
- a candidate substance preferably a cell, tissue or organ of the transgenic animal
- Cell-based screens employing cells derived from Che relevant animal and assaying for effects on conductance or intracellular calcium concentration may also be conducted.
- transgenic animals comprising f ⁇ mctionally disrupted
- T ⁇ M8 gene in which any one or more of the functions of T ⁇ M8 as disclosed in this document is partially or totally abolished. Included are transgenic animals ("T ⁇ M8 knockouf's) which do not express functional T ⁇ M8 ion channel as a result of one or more loss of function mutations, including a deletion, of the T ⁇ M8 gene.
- partial loss-of-function mutants e.g., an incomplete knockout, which may for example have deletions in selected portions of the T ⁇ M8 gene.
- Such animals may be generated by selectively replacing or deleting relevant portions of the T ⁇ M8 sequence, for example, functionally important protein domains.
- Such complete or partial loss of function mutants are useful as models for T ⁇ M8 related diseases, particularly pain or stress related diseases.
- An animal displaying partial-loss-of-function may be exposed to a candidate substance to identify substances which enhance the phenotype, that is to say, to increase (in the case of T ⁇ M8) the hypoalgesia or reduction of stress level phenotype observed.
- Other parameters such as reduction in conductance or reduction in intracellular calcium levels may also be detected using the methods identified elsewhere in this document.
- Partial and complete knockouts may also be used to identify selective agonists and/or antagonists of T ⁇ M8. For example, an agonist and/or antagonist may be administered to a wild type and a T ⁇ M8 deficient animal (knockout).
- a selective agonist or antagonist of T ⁇ M8 will be seen to have an effect on the wild type animal but not in the T ⁇ M8 deficient animal.
- a specific assay is designed to evaluate a potential drug (a candidate ligand or compound) to determine if it produces a physiological response in the absence of T ⁇ M8 ion channel. This may be accomplished by administering the drug to a transgenic animal as discussed above, and then assaying the animal for a particular response. Analogous cell-based methods employing cells derived from the relevant animal and assaying for effects on conductance or intracellular calcium concentration may also be conducted. Such animals may also be used to test for efficacy of drugs identified by the screens described in this document.
- a transgenic animal having a partial loss-of-function phenotype is employed for screening.
- the screen may involve assaying for partial or complete restoration or reversion to the wild type phenotype.
- Cell-based screens employing cells derived from the relevant animal and assaying for effects on conductance or intracellular calcium concentration may also be conducted .
- a candidate compound which is found to be capable of such can be regarded as a T ⁇ M8 agonist or analogue.
- Such agonists may be used for example to restore or increase sensitivity to stimuli, for example pain, or to increase stress levels in an individual.
- the transgenic T ⁇ M8 animals display the phenotypes set out in the Examples, preferably as measured by the tests set out therein.
- the T ⁇ M8 animals, particularly T ⁇ M8 knockouts preferably display any one or more of the following: decreased grip strength, propensity to drink more then wild-type mice, lower sensitivity to pain (hypoalgesia), lowered stress, decreased blood plasma corticosterone levels.
- the transgenic T ⁇ M8 animals display at least 10%, preferably at least 20%, more preferably at least30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher or lower (as the case may be) of the measured parameter as compared to the corresponding wild-type mice.
- T ⁇ M8 knockouts have an increased pain threshold in response to the Tail Flick test set out in the examples, of 1 second, 2 seconds, 5 seconds, 10 seconds, 30 seconds or more, or 5%, 10%, 20%>, 50% or more when compared to wi Id type mice.
- T ⁇ M8 deficient mice preferably have an increased pennance time for central zone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 seconds or more, or an increased distance moved in central zone of 15, 20, 25, 30, 50 cm or more, when compared to wild type mice.
- T ⁇ M8 deficient transgenic animals may be usefully employed in a screen using wild type animals, to detect compounds which cause similar effects to loss-of-function of T ⁇ M8.
- a wild type animal may be exposed to a candidate compound, and a change in a relevant T ⁇ M8 phenotype observed, such as hypoalgesia, reduction in sensitivity to pain, reduction in stress levels, reduction in corticosterone levels, etc, to identify modulators of T ⁇ M8 function, particularly antagonists.
- Cellular phenotypes such a.s reduction in conductance or reduction in intracellular calcium levels may also be detected using the methods identified elsewhere in this document.
- a compound identified by such a screen could be used as an antagonist of
- T ⁇ M8 e.g, as an analgesic or a stress reliever, particularly for the treatment or relief of a T ⁇ M8 associated disease.
- the screens described above may involve observation of any suitable parameter, such as a behavioural, physiological or biochemical response.
- Preferred responses include physiological responses and may comprise one or more of the following: changes to disease resistance; altered inflammatory responses; altered tumour susceptability: a change in blood pressure; neovascularization; a change in eating behavior; a change in body weight; a change in bone density; a change in body temperature; insulin secretion; gonadotropin secretion; nasal and bronchial secretion; vasoconstriction; loss of memory; anxiety; changed anxiety state; hyporeflexia or hyperreflexia; pain or stress responses.
- Biochemical parameters may also be employed, such as a change in conductance or intracellular calcium concentration.
- the conductance is measured using the "Functional Assay for T ⁇ M8 (Conductance)" and the intracelli lar calcium concentration is measured using the “Functional Assay for T ⁇ M8 (Calcium Concentration”. This is particularly useful in cell-based screens.
- the conductance of a cell (for example a wild type or partial loss-of-function cell) exposed to a T ⁇ M8 agonist is increased by at least 10%), preferably at least 20%, more preferably at least +30% > , more preferably at least 40%), more preferably at least 50%>, more preferably at least 60%>, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%). In preferred embodiments, this is measured using the "Functional Assay for T ⁇ M8 (Conductance)" described elsewhere in this document.
- the intracellular calcium concentration of a cell (for example a wild type or partial loss-of-function cell) exposed to a T ⁇ M8 agonist is increased by at least 10%, preferably at least 20%, more preferably at least +30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%), more preferably at least 70%>, more preferably at least 80%>, more preferably at least 90%. In preferred embodiments, this is measured using the "Functional Assay for T ⁇ M8 (Intracellular Calcium Concentration)" described elsewhere in this document.
- the corticosterone levels of a wild type or T ⁇ M8 partial knockout animal exposed to a T ⁇ M8 agonist is increased by at least 10%, preferably at least 20%>, more preferably at least +30%>, more preferably at least 40%>, more preferably at least 50%, more preferably at least 60%>, more preferably at least 70%), more preferably at least 80%>, more preferably at least 90%.
- antagonists of T ⁇ M8 are such that wild type or partial loss-of-function animals exposed to such antagonists exhibit at least partial identity of phenotype, to at least a partial degree, as T ⁇ M8 partial or complete loss-of- function mutants.
- preferred antagonists are those which cause hypoalgesia, or reduction of stress, or reduction in serum corticosterone levels, or reduction in conductance, or reduction in intracellular calcium levels, or any combination of the above.
- the relevant phenotype is expressed to the same degree as a T ⁇ M8 knock-out animal.
- the conductance of a wild type or partial loss-of- function cell exposed to a T ⁇ M8 antagonist is within +80%, preferably within +70%, more preferably within +60%, more preferably within +50%, more preferably within +40%), more preferably within +30%, more preferably within +20%, more preferably within +10%, more preferably within +5%, of the conductance of a T ⁇ M8 deficient cell. In preferred embodiments, this is measured using the "Functional Assay for T ⁇ M8 (Conductance)" described elsewhere in this document.
- the intracellular calcium concentration of a wild type or T ⁇ M8 partial loss-of-function cell exposed to a T ⁇ M8 antagonist is within +80%, preferably within +70%, more preferably within +60%>, more preferably within +50%), more preferably within +40%>, more preferably within +30%>, more preferably within +20%), more preferably within +10%, more preferably within +5%, of the intracellular calcium concentration of a T ⁇ M8 deficient cell. In preferred embodiments, this is measured using the "Functional Assay for T ⁇ M8 (Intracellular Calcium Concentration)" described elsewhere in this document.
- the corticosterone levels of a wild type or partial T ⁇ M8 knockout animal exposed to a T ⁇ M8 antagonist is within +80%>, preferably within +70%), more preferably within +60%, more preferably within +50%>, more preferably within +40%>, more preferably within +30%, more preferably within +20%, more preferably within +10%, more preferably within +5%>, of the corticosterone levels of a T ⁇ M8 deficient transgenic animal.
- Tissues derived from the T ⁇ M8 knockout animals may be used in binding assays to determine whether the potential drug (a candidate ligand or compound) binds to the T ⁇ M8.
- assays can be conducted by obtaining a first ion channel preparation from the transgenic animal engineered to be deficient in T ⁇ M8 ion channel production and a second ion channel preparation from a source known to bind any identified T ⁇ M8 ligands or compounds.
- the first and second ion channel preparations will be similar in all respects except for the source from which they are obtained.
- a transgenic animal such as described above and below
- comparable brain tissue from a normal (wild type) animal is used as the source of the second ion channel preparation.
- Each of the ion chamiel preparations is incubated with a ligand known to bind to T ⁇ M8 ion channels, both alone and in the presence of the candidate ligand or compound.
- the candidate ligand or compound will be examined at several different concentrations.
- Tissues derived from transgenic animals may be used in assays directly or the tissues may be processed to isolate membranes or membrane proteins, which are themselves used in the assays.
- a preferred transgenic animal is the mouse.
- the ligand may be labeled using any means compatible with binding assays. This would include, without limitation, radioactive, enzymatic, fluorescent or chemiluminescent labeling (as well as other labelling techniques as described in further detail above).
- T ⁇ M8 ion channel may be identified by administering candidate compounds, etc, to wild type animals expressing functional T ⁇ M8, and animals identified which exhibit any of the phenotypic characteristics associated with reduced or abolished expression of T ⁇ M8 function.
- Transgenic gene constructs can be introduced into the germ line of an animal to make a transgenic mammal. For example, one or several copies of the construct may be inco ⁇ orated into the genome of a mammalian embryo by standard transgenic techniques.
- the transgenic non-human animals are produced by introducing transgenes into the germline of the non-human animal.
- Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell. The specific line(s) of any animal are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness. In addition, the haplotype is a significant factor. Introduction of the transgene into the embryo can be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
- the T ⁇ M8 transgene can be introduced into a mammal by microinjection of the construct into the pronuclei of the fertilized mammalian egg(s) to cause one or more copies of the construct to be retained in the cells of the developing mammal(s).
- the egg may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity may also be conducted.
- the progeny of the transgenically manipulated embryos can be tested for the presence of the construct by Southern blot analysis of the segment of tissue. If one or more copies of the exogenous cloned construct remains stably integrated into the genome of such transgenic embryos, it is possible to establish permanent transgenic mammal lines carrying the transgenically added construct.
- the litters of transgenically altered mammals can be assayed after birth for the inco ⁇ oration of the construct into the genome of the offspring.
- this assay is accomplished by hybridizing a probe corresponding to the DNA sequence coding for the desired recombinant protein product or a segment thereof onto chromosomal material from the progeny. Those mammalian progeny found to contain at least one copy of the construct in their genome are grown to maturity.
- a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.
- the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes.
- the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism.
- a euploid zygote is preferred. If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
- the biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
- the number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. There will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences. Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane or other existing cellular or genetic structures. The exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.
- Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.
- Transgenic offspring of the surrogate host maybe screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
- DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene.
- the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
- transgenic animals include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents. Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.
- suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.
- Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
- Progeny of the transgenic animals may be obtained by mating the trans
- the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
- the partner may be a parental line.
- in vitro fertilization the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both.
- the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
- the transgenic animals produced in accordance with the methods described here will include exogenous genetic material.
- the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of a T ⁇ M8 ion channel. Further, in such embodiments the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
- a transcriptional control element e.g., a promoter
- Retroviral infection can also be used to introduce transgene into a non-human animal.
- the developing non-human embryo can be cultured in vitro to the blastocyst stage.
- the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).
- Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
- the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al.
- the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
- transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
- ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448).
- Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
- transgenic animals where the transgenic animal is characterized by having an altered T ⁇ M8 gene, preferably as described above, as models for T ⁇ M8 ion channel function. Alterations to the gene include deletions or other loss of function mutations, introduction of an exogenous gene having a nucleotide sequence with targeted or random mutations, introduction of an exogenous gene from another species, or a combination thereof.
- the transgenic animals may be either homozygous or heterozygous for the alteration.
- the animals and cells derived therefrom are useful for screening biologically active agents that may modulate T ⁇ M8 ion channel function.
- the screening methods are of particular use for determining the specificity and action of potential therapies for pain and cancer, particularly prostate cancer.
- the animals are useful as a model to investigate the role of T ⁇ M8 ion channels in normal tissues and organs such as the brain, heart, spleen and liver and the effect on their function.
- Another aspect pertains to a transgenic nonhuman animal having a functionally disrupted endogenous T ⁇ M8 gene but which also carries in its genome, and expresses, a transgene encoding a heterologous T ⁇ M8 protein (i.e., a T ⁇ M8 from another species).
- the animal is a mouse and the heterologous T ⁇ M8 is a human T ⁇ M8.
- An animal, or cell lines derived from such an animal, which has been reconstituted with human T ⁇ M8, can be used to identify agents that inhibit human T ⁇ M8 in vivo and in vitro.
- a stimulus that induces signalling through human T ⁇ M8 can be administered to the animal, or cell line, in the presence and absence of an agent to be tested and the response in the animal, or cell line, can be measured.
- An agent that inhibits human T ⁇ M8 in vivo or in vitro can be identified based upon a decreased response in the presence of the agent compared to the response in the absence of the agent.
- T ⁇ M8 deficient transgenic non-human animal a "T ⁇ M8 subunit knock-out"
- Such an animal is one which expresses lowered or no T ⁇ M8 ion channel activity, preferably as a result of an endogenous T ⁇ M8 ion channel genomic sequence being disrupted or deleted.
- a T ⁇ M8 subunit knock-out Such an animal expresses no ion channel activity. More preferably, the animal expresses no activity of the T ⁇ M8 ion channel shown as SEQ ID NO: 3 or SEQ ID NO: 5.
- T ⁇ M8 ion channel knock-outs may be generated by various means known in the art, as described in further detail below.
- the present disclosure also pertains to a nucleic acid construct for functionally disrupting a T ⁇ M8 gene in a host cell.
- the nucleic acid construct comprises: a) a non- homologous replacement portion; b) a first homology region located upstream of the non-homologous replacement portion, the first homology region having a nucleotide sequence with substantial identity to a first T ⁇ M8 gene sequence; and c) a second homology region located downstream of the non-homologous replacement portion, the second homology region having a nucleotide sequence with substantial identity to a second T ⁇ M8 gene sequence, the second T ⁇ M8 gene sequence having a location downstream of the first T ⁇ M8 gene sequence in a naturally occurring endogenous T ⁇ M8 gene.
- the first and second homology regions are of sufficient length for homologous recombination between the nucleic acid construct and an endogenous T ⁇ M8 gene in a host cell when the nucleic acid molecule is introduced into the host cell.
- the non-homologous replacement portion comprises an expression reporter, preferably including lacZ and a positive selection expression cassette, preferably including a neomycin phosphotransferase gene operatively linked to a regulatory element(s).
- the first and second T ⁇ M8 gene sequences are derived from SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof.
- Another aspect pertains to recombinant vectors into which the nucleic acid construct described above has been inco ⁇ orated. Yet another aspect pertains to host cells into which the nucleic acid construct has been introduced to thereby allow homologous recombination between the nucleic acid construct and an endogenous T ⁇ M8 gene of the host cell, resulting in functional disruption of the endogenous T ⁇ M8 gene.
- the host cell can be a mammalian cell that normally expresses T ⁇ M8 from the liver, brain, spleen or heart, or a pluripotent cell, such as a mouse embryonic stem cell.
- an embryonic stem cell into which the nucleic acid construct has been introduced and homologously recombined with the endogenous T ⁇ M8 gene produces a transgenic nonhuman animal having cells that are descendant from the embryonic stem cell and thus carry the T ⁇ M8 gene disruption in their genome. Animals that carry the T ⁇ M8 gene disruption in their germline can then be selected and bred to produce animals having the T ⁇ M8 gene disruption in all somatic and germ cells. Such mice can then be bred to homozygosity for the T ⁇ M8 gene disruption.
- ANTIBODIES ANTIBODIES
- Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab') 2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
- the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
- Neutralizing antibodies i.e., those which inhibit biological activity of the substance amino acid sequences, are especially preferred for diagnostics and therapeutics.
- Antibodies may be produced by standard techniques, such as by immunisation or by using a phage display library.
- a polypeptide or peptide of may be used to develop an antibody by known techniques. Such an antibody may be capable of binding specifically to the T ⁇ M8 ion channel protein or homologue, fragment, etc.
- a selected mammal e.g., mouse, rabbit, goat, horse, etc.
- an immunogenic composition comprising a relevant polypeptide or peptide.
- various adjuvants may be used to increase immunological response.
- adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
- BCG Bacilli Calmette-Guerin
- Corynebacterium parvum are potentially useful human adjuvants which may be employed if purified the substance amino acid sequence is administered to immunologically compromised individuals for the pu ⁇ ose of stimulating systemic defence.
- Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an epitope obtainable from a T ⁇ M8 polypeptide contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, we also provide amino acid sequences of T ⁇ M8 or fragments thereof haptenised to another amino acid sequence for use as immunogens in animals or humans. Monoclonal antibodies directed against epitopes obtainable from a T ⁇ M8 polypeptide or peptide can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known.
- Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.
- Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by
- Antibodies both monoclonal and polyclonal, whichi are directed against epitopes obtainable from a T ⁇ M8 polypeptide or peptide are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy.
- Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
- Anti-idiotype antibodies are immunoglobulins which cany an "internal image" of the substance and/or agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful in therapy.
- Antibodies may also be produced by inducing in vi ⁇ vo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293- 299).
- Antibody fragments which contain specific binding sites for the polypeptide or peptide may also be generated.
- such fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
- Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-128 1). Techniques for the production of single chain antibodies (U.S. Pat. No.
- 4,946,778 can also be adapted to produce single chain antibodies to T ⁇ M8 polypeptides.
- transgenic mice, or other organisms including other mammals may be used to express humanized antibodies.
- the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
- Antibodies against T ⁇ M8 ion channel polypeptides may also be employed to treat pain and cancer, particularly neuropathic pain and prostate cancer.
- This disclosure also relates to the use of T ⁇ M8 ion chamiel polynucleotides and polypeptides (as well as homologues, variants and derivatives thereof) for use in diagnosis as diagnostic reagents or in genetic analysis.
- Nucleic acids complementary to or capable of hybridising to T ⁇ M8 ion channel nucleic acids (including homologues, variants and derivatives), as well as antibodies against T ⁇ M8 polypeptides are also useful in such assays.
- Detection of a mutated form of the T ⁇ M8 ion channel gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over- expression or altered expression of T ⁇ M8 ion channel.
- Individuals carrying mutations in the T ⁇ M8 ion channel gene may be detected at the DNA level by a variety of techniques.
- DNA may be isolated from a patient and the DNA polymo ⁇ hism pattern of T ⁇ M8 determined. The identified pattern is compared to controls of patients known to be suffering from a disease associated with over-, under- or abnormal expression of T ⁇ M8. Patients expressing a genetic polymo ⁇ hism pattern associated with T ⁇ M8 associated disease may then be identified. Genetic analysis of the T ⁇ M8 ion channel gene may be conducted by any technique known in tfcie art. For example, individuals may be screened by determining DNA sequence of a ⁇ M8 allele, by RFLP or SNP analysis, etc.
- Patients may be identified as having a genetic predisposition for a disease associated with the over-, under-, or abnormal expression of T ⁇ M8 by detecting the presence of a DNA polymo ⁇ hism in Che gene sequence for T ⁇ M8 or any sequence controlling its expression.
- T ⁇ M8 associated diseases include stress, anxiety, depression, pain and cancer, particularly neuropathic pain and prostate cancer.
- kits for the identification of a patient's genetic polymo ⁇ hism pattern associated with T ⁇ M8 associated disease includes DNA sample collecting means and means for determining a genetic polymo ⁇ hism pattern, which is then compared to control samples to determine a patient's susceptibility to T ⁇ M8 associated disease. Kits for diagnosis of a T ⁇ M8 associated disease comprising T ⁇ M8 polypeptide and/or an antibody against such a polypeptide (or fragment of it) are also provided.
- Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
- the DNA is obtained from blood cells obtained from a finger prick of the patient with the blood collected on absorbent paper.
- the blood will be collected on an AmpliCard.TM. (University of Sheffield, Department of Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, England S10 2JF).
- the DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques pri or to analysis.
- Oligonucleotide DNA primers that target the specific polymo ⁇ hic DNA region within the genes of interest may be prepared so that in the PCR reaction amplification of the target sequences is achieved.
- RNA or cDNA may also be used as templates in similar fashion.
- the amplified DNA sequences from the template DNA may then be analyzed using restriction enzymes to determine the genetic polymo ⁇ hisms present in the amplified sequences and thereby provide a genetic polymo ⁇ hism profile of the patient. Restriction fragments lengths may be identified by gel analysis.
- SNP single nucleotide polymo ⁇ hisms
- Deletions and insertions can be detected by a change in size of the amplifi ed product in comparison to the normal genotype.
- Point mutations can be identified by hybridizing amplified DNA to labeled T ⁇ M8 ion channel nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, eg., IVdyers et al, Science (1985)230:1242.
- Sequence changes at specific locations may also b e revealed by nuclease protection assays, such as RNAse and Slprotection or the chemical cleavage method. See Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401.
- an array of oligonucleotides probes comprising the T ⁇ M8 ion channel nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
- Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
- Single strand conformation polymo ⁇ hism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79).
- Single-stranded DNA fragments of sample and control T ⁇ M8 nucleic acids maybe denatured and allowed to renature.
- the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
- the DNA fragments may be labelled or detected with labelled probes.
- RNA rather than DNA
- the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
- the diagnostic assays offer a process for diagnosing or determining a susceptibility to infections such as pain and cancer, particularly neuropathic pain and prostate cancer through detection of mutation in the T ⁇ M8 gene by the methods described.
- the presence of T ⁇ M8 polypeptides and nucleic acids may be detected in a sample.
- infections and diseases as listed above can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of the T ⁇ M8 polypeptide or T ⁇ M8 ion channel mRNA.
- the sample may comprise a cell or tissue sample from an organism suffering or suspected to be suffering from a disease associated with increased, reduced or otherwise abnormal T ⁇ M8 expression, including spatial or temporal changes in level or pattern of expression.
- the level or pattern of expression of T ⁇ M8 in an organism suffering from or suspected to be suffering from such a disease may be usefully compared with the level or pattern of expression in a normal organism as a means of diagnosis of disease.
- a method of detecting the presence of a nucleic acid comprising a T ⁇ M8 nucleic acid in a sample by contacting the sample with at least one nucleic acid probe which is specific for said nucleic acid and monitoring said sample for the presence of the nucleic acid.
- the nucleic acid probe may specifically bind to the T ⁇ M8 subunit nucleic acid, or a portion of it, and binding between the two detected; the presence of the complex itself may also be detected.
- we encompasse a method of detecting the presence of a T ⁇ M8 polypeptide by contacting a cell sample with an antibody capable of binding the polypeptide and monitoring said sample for the presence of the polypeptide.
- Methods of detecting binding between two entities include FRET (fluorescence resonance energy transfer), surface plasmon resonance, etc.
- Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNAse protection, Northern blotting and other hybridization methods.
- Assay techniques that can be used to determine levels of a protein, such as T ⁇ M8, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
- the diagnostic kit for a disease or susceptibility to a disease (including an infection), for example, pain and cancer, particularly neuropathic pain and prostate cancer.
- the diagnostic kit comprises a T ⁇ M8 polynucleotide or a fragment thereof; a complementary nucleotide sequence; a T ⁇ M8 polypeptide or a fragment thereof, or an antibody to a T ⁇ M8 polypeptide.
- CHROMOSOME ASSAYS The nucleotide sequences described here are also valuable for chromosome identification.
- the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
- human T ⁇ M8 ion channel is found to map to Homo sapiens chromosome 2q37.
- mapping of relevant sequences to chromosomes is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian heritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
- the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
- T ⁇ M8 ion channel If the activity of T ⁇ M8 ion channel is in excess, several approaches are available.
- One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the T ⁇ M8 ion channel, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
- T ⁇ M8 polypeptides still capable of binding the ligand in competition with endogenous T ⁇ M8 ion channel may be administered.
- Typical embodiments of such competitors comprise fragments of the T ⁇ M8 polypeptide.
- expression of the gene encoding endogenous T ⁇ M8 ion channels can be inhibited using expression blocking techniques.
- Known such techniques involve the use of antisense sequences, either internally generated or separately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxvnucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).
- oligonucleotides which form triple helices with the gene can be supplied.
- oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
- T ⁇ M8 ion channel For treating abnormal conditions related to an under-expression of T ⁇ M8 ion channel and its activity, several approaches are also available.
- One approach comprises administering to a subject a therapeutically effective amount of a compound which activates T ⁇ M8 ion channel, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition.
- gene therapy may be employed to effect the endogenous production of T ⁇ M8 ion channel by the relevant cells in the subject.
- a T ⁇ M8 polynucleotide may be engineered for expression in a replication defective retroviral vector, as discussed above.
- the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a T ⁇ M8 polypeptide such that the packaging cell now produces infectious viral particles containing the gene of interest.
- These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
- gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996).
- Peptides such as the soluble form of T ⁇ M8 ion channel polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier.
- suitable pharmaceutical carrier include but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Formulation should suit the mode of administration, and is well within the skill of the art.
- the disclosure further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions.
- T ⁇ M8 polypeptides and other compounds may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
- systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
- Other injection routes such as subcutaneous, intramuscular, or intraperitoneal, can be used.
- Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
- penetrants such as bile salts or fusidic acids or other detergents.
- oral administration may also be possible. Administration of these compounds may also be topical and/or localize, in the form of salves, pastes, gels and the like.
- the dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
- Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
- cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
- a polynucleotide such as a DNA or RNA
- PHARMACEUTICAL COMPOSITIONS We further disclose a pharmaceutical composition comprising administering a therapeutically effective amount of the T ⁇ M8 polypeptide, polynucleotide, peptide, vector or antibody thereof and optionally a phannaceutically acceptable carrier, diluent or excipients (including combinations thereof).
- the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
- Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
- the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
- the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
- Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
- preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
- Antioxidants and suspending agents may be also used.
- the pharmaceutical composition described here may be formulated to be delivered using a a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
- the formulation may be designed to be delivered by both routes.
- the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
- compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously.
- compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
- compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
- Another embodiment relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with the T ⁇ M8 ion channel polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from anxiety, stress, depression, pain and cancer, particularly prostate cancer, among others.
- Yet another embodiment relates to a method of inducing immunological response in a mammal which comprises delivering a T ⁇ M8 polypeptide via a vector directing expression of T ⁇ M8 polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
- a further embodiment relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a T ⁇ M8 polypeptide wherein the composition comprises a T ⁇ M8 polypeptide or T ⁇ M8 gene.
- the vaccine formulation may further comprise a suitable carrier.
- T ⁇ M8 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
- parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
- the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
- the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
- Vaccines may be prepared from one or more T ⁇ M8 polypeptides or peptides.
- vaccines which contain an immunogenic polyp eptide(s) or peptide(s) as active ingredient(s), is known to one skilled in the art.
- such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
- the preparation may also be emulsified, or the protein encapsulated in liposomes.
- the active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
- the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
- auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
- adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)- ethylamine (CGP 19835 A,
- adjuvants and other agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvum (Propionobacterium acnes), Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
- aluminum hydroxide aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide
- Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan).
- adjuvants such as Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel are used. Only aluminum hydroxide is approved for human use.
- the proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
- aluminum hydroxide can be present in an amount of about 0.5%> of the vaccine mixture (Al 2 O basis).
- the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 ⁇ g/ml, preferably 5 to 50 ⁇ g/ml, most preferably 15 ⁇ g/ml.
- the vaccine may be inco ⁇ orated into a sterile container which is then sealed and stored at a low temperature, for example 4°C, or it may be freeze-dried. Lyophilisation permits long-term storage in a stabilised form.
- the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
- Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
- suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%>, preferably 1% to 2%.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
- compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%>.
- the lyophilised material maybe reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S”, Eudragit "L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
- the T ⁇ M8 polypeptides may be formulated into the vaccine as neutral or salt forms.
- Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.
- a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
- the dosages below are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.
- compositions described here may be administered by direct injection.
- the composition may be formulated for parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration.
- each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
- the term "administered” includes delivery by viral or non-viral techniques.
- Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, he ⁇ es viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.
- Non- viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.
- the routes for such delivery mechanisms include but are not limited to mucosal, nasal, oral, parenteral, gastrointestinal, topical, or sublingual routes.
- administered includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.
- co-administered means that the site and time of administration of each of for example, the T ⁇ M8 polypeptide and an additional entity such as adjuvant are such that the necessary modulation of the immune system is achieved.
- polypeptide and the adjuvant may be administered at the same moment in time and at the same site, there may be advantages in administering the polypeptide at a different time and to a different site from the adjuvant.
- the polypeptide and adjuvant may even be delivered in the same delivery vehicle - and the polypeptide and the antigen may be coupled and/or uncoupled and/or genetically coupled and/or uncoupled.
- the T ⁇ M8 polypeptide, polynucleotide, peptide, nucleotide, antibody thereof and optionally an adjuvant may be administered separately or co-administered to the host subject as a single dose or in multiple doses.
- the vaccine composition and pharmaceutical compositions may be administered by a number of different routes such as injection (which includes parenteral, subcutaneous and intramuscular injection) intranasal, mucosal, oral, intra- vaginal, urethral or ocular administration.
- the vaccines and pharmaceutical compositions of described here may be conventionally administered parenterally, by injection, for ex: ample, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
- binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%>, may be 1% to 2%.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95%> of active ingredient, preferably 25% to 70%.
- the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer.
- Paragraph 1 A T ⁇ M8 polypeptide comprising the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO: 5, or a homologue, variant or derivative thereof.
- Paragraph 2. A nucleic acid encoding a polypeptide according to Paragraph 1.
- Paragraph 3 A nucleic acid according to Paragraph 2, comprising the nucleic acid sequence shown in SEQ ID No. 1, SEQ ID No.2 or SEQ ID NO: 4, or a homologue, variant or derivative thereof.
- Paragraph 4 A polypeptide comprising a fragment of a polypeptide according to Paragraph 1.
- Paragraph 5 A polypeptide according to Paragraph 3 which comprises one or more regions which are homologous between SEQ ID No. 3 and SEQ ID No. 5, or which comprises one or more regions which are heterologous between SEQ ID No. 3 and SEQ ID No. 5.
- Paragraph 6 A nucleic acid encoding a polypeptide according to Paragraph 4 or 5.
- Paragraph 7 A vector comprising a nucleic acid according to Paragraph 2, 3, or 6.
- Paragraph 8 A host cell comprising a nucleic acid according to Paragraph 2,
- Paragraph 9 A transgenic non-human animal comprising a nucleic acid according to Paragraph 2, 3 or 6, or a vector according to Paragraph 7.
- Paragraph 10 A transgenic non-human animal according to Paragraph 9 which is a mouse.
- Paragraph 11 Use of a polypeptide according to Paragraph 1 , 4 or 5 in a method of identifying a compound which is capable of interacting specifically with a ion channel.
- Paragraph 12 Use of a transgenic non-human animal according to Paragraph 9 or 10 in a method of identifying a compound which is capable of interacting specifically with a ion channel.
- Paragraph 13 A method for identifying an antagonist of T ⁇ M8, the method comprising contacting a cell which expresses T ⁇ M8 receptor with a candidate compound and determining whether the level of cyclic AMP (cAMP) in the cell is lowered as a result of said contacting.
- cAMP cyclic AMP
- Paragraph 14 A method for identifying a compound capable of lowering the endogenous level of cyclic AMP in a cell which method comprises contacting a cell which expresses T ⁇ M8 with a candidate compound and determining whether the level of cyclic AMP (cAMP) in the cell is lowered as a result of said contacting.
- Paragraph 15 A method of identifying a compound capable of binding to
- T ⁇ M8 polypeptide the method comprising contacting a T ⁇ M8 polypeptide with a candidate compound and determining whether the candidate compound binds to the T ⁇ M8 ion channel polypeptide.
- Paragraph 16 A compound identified by a method according to any of Paragraphs 11 to 15.
- Paragraph 17 A compound capable of binding specifically to a polypeptide according to Paragraph 1, 4 or 5.
- Paragraph 18 Use of a polypeptide according to Paragraph 1, 4 or 5, or part thereof or a nucleic acid according to Paragraph 2, 3 or 6, in a method for producing antibodies.
- Paragraph 19 An antibody capable of binding specifically to a polypeptide according to Paragraph 1 , 4 or 5, or part thereof or a polypeptide encoded by a nucleotide according to Paragraph 2, 3 or 6, or part thereof.
- Paragraph 20 A pharmaceutical composition comprising any one or more of the following: a polypeptide according to Paragraph 1, 4 or 5, or part thereof; a nucleic acid according to Paragraph 2, 3 or 6, or part thereof; a vector according to Paragraph 7; a cell according to Paragraph 8; a compouind according to Paragraph 16 or 17; and an antibody according to Paragraph 19, together with a pharmaceutically acceptable carrier or diluent.
- a vaccine composition comprising any one or more of the following: a polypeptide according to Paragraph 1, 4 or 5, or part thereof; a nucleic acid according to Paragraph 2, 3 or 6, or part thereof; a vector according to Paragraph 7; a cell according to Paragraph 8; a compound according to Paragraph 16 or 17; and an antibody according to Paragraph 19.
- Paragraph 22 A diagnostic kit for a disease or susceptibility to a disease comprising any one or more of the following : a polypeptide according to Paragraph 1, 4 or 5, or part thereof; a nucleic acid according to Paragraph 2, 3 or 6, or part thereof; a vector according to Paragraph 7; a cell according to Paragraph 8; a compound according to Paragraph 16 or 17; and an antibody according to Paragraph 19.
- Paragraph 23 A method of treating a patient suffering from a disease associated with enhanced activity of T ⁇ M8, "which method comprises administering to the patient an antagonist of T ⁇ M8 ion channel.
- Paragraph 24 A method of treating a patient suffering from a disease associated with reduced activity of T ⁇ M8, which method comprises administering to the patient an agonist of T ⁇ M8 ion channel.
- Paragraph 25 A method according to Paragraph 23 or 24, in which the T ⁇ M8 ion channel comprises a polypeptide having the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5.
- Paragraph 26 A method for treating and/or preventing a disease in a patient, which comprises the step of administering any one or more of the following to the patient: a polypeptide according to Paragraph 1, 4 or 5, or part thereof; a nucleic acid according to Paragraph 2, 3 or 6, or part thereof; a vector according to Paragraph 7; a cell according to Paragraph 8; a compound according to Paragraph 16 or 17; an antibody according to Paragraph 19; a pharmaceutical composition according to Paragraph 20; and a vaccine according to Paragraph 20.
- Paragraph 27 An agent comprising a polypeptide according to Paragraph 1, 4 or 5, or part thereof; a nucleic acid according to Paragraph 2, 3 or 6, or part thereof; a vector according to Paragraph 7; a cell according to Paragraph 8; a compound according to Paragraph 16 or 17; and/or an antibody according to Paragraph 19, said agent for use in a method of treatment or prophylaxis of disease.
- Paragraph 28 Use of a polypeptide according to Paragraph 1, 4 or 5, or part thereof; a nucleic acid according to Paragraph 2, 3 or 6, or part thereof; a vector according to Paragraph 7; a cell according to Paragraph 8; a compound according to Paragraph 16 or 17; and an antibody according to Paragraph 19, for the preparation of a pharmaceutical composition for the treatment or prophylaxis of a disease.
- Paragraph 29 A non-human transgenic animal, characterised in that the transgenic animal comprises an altered T ⁇ M8 gene.
- Paragraph 30 A non-human transgenic animal according; to Paragraph 29, in which the alteration is selected from the group consisting of: a deletion of T ⁇ M8, a mutation in T ⁇ M8 resulting in loss of function, introduction of an exogenous gene having a nucleotide sequence with targeted or random mutations into T ⁇ M8, introduction of an exogenous gene from another species into T ⁇ lM8, and a combination of any of these.
- Paragraph 31 A non-human transgenic animal having a functionally disrupted endogenous T ⁇ M8 gene, in which the transgenic animal compris es in its genome and expresses a transgene encoding a heterologous T ⁇ M8 protein.
- Paragraph 32 A non-human transgenic animal having a functionally disrupted endogenous T ⁇ M8 gene, in which the transgenic animal compris es in its genome and expresses a transgene encoding a heterologous T ⁇ M8 protein.
- a nucleic acid construct for functionally disrupting a T ⁇ M8 gene in a host cell comprising: (a) a non-homologous replacement portion; (b) a first homology region located upstream, of the non- homologous replacement portion, the first homology region haviirg a nucleotide sequence with substantial identity to a first T ⁇ M8 gene sequence * and (c) a second homology region located downstream of the non-homologous replacement portion, the second homology region having a nucleotide sequence with substantial identity to a second T ⁇ M8 gene sequence, the second T ⁇ M8 gene sequence having a location downstream of the first T ⁇ M8 gene sequence in a naturally occurring endogenous T ⁇ M8 gene.
- Paragraph 33 A process for producing a T ⁇ M8 polypeptide, the method comprising culturing a host cell according to Paragraph 8 under conditions in which a nucleic acid encoding a T ⁇ M8 polypeptide is expressed.
- Paragraph 34 A method of detecting the presence of a nucleic acid according to Paragraph 2, 3 or 6 in a sample, the method comprising contacting the sample with at least one nucleic acid probe which is specific for said nucleic acid and monitoring said sample for the presence of the nucleic acid.
- Paragraph 35 A method of detecting the presence of a polypeptide according to Paragraph 1, 4 or 5 in a sample, the method comprising contacting the sample with an antibody according to Paragraph 19 and monitoring said sample for the presence of the polypeptide.
- Paragraph 36 A method of detecting the presence of a nucleic acid according to Paragraph 2, 3 or 6 in a sample, the method comprising contacting the sample with at least one nucleic acid probe which is specific for said nucleic acid and monitoring said sample for the presence of the nucleic acid.
- Paragraph 37 A kit, method, agent or use according to any of Paragraphs 22 to
- the disease is selected from the group consisting of: social anxiety, post traumatic stress disorder, phobias, social phobia, special phobias, panic disorder, obsessive compulsive disorder, acute stress, disorder, separation anxiety disorder, generalised anxiety disorder, major depression, dysthymia, bipolar disorder, seasonal affective disorder or post natal depression.
- the T ⁇ M8 gene was identified bio-informatically using homology searches of genome databases.
- a 87kb gapped genomic contig was assembled from various databases. This contig provided sufficient flanking sequence information to enable the design of homologous arms to clone into the targeting vector.
- the murine T ⁇ M8 gene has 23 coding exons.
- the targeting strategy is designed to remove part of the 15th coding exon.
- a 4.0kb 5' homologous arm and a 1.6kb 3 ' homologous arm flanking the region to be deleted are amplified by PCR and the fragments are cloned into the targeting vector.
- the 5' end of each oligonucleotide primer used to amplify the amis is synthesised to contain a different recognition site for a rare-cutting restriction enzyme, compatible with the cloning sites of the vector polylinkers and absent from the arms themselves.
- the primers are designed as listed in the primer table below, with 5' arm cloning sites of Agel/Notl and 3'arm cloning sites of Ascl/Fsel (the structure of the targeting vector used, including the relevant restriction sites, is shown in Figure X).
- further primers specific to the T ⁇ M8 locus are designed for the following pu ⁇ oses: 5' and 3 ' probe primer pairs (5 'prF/5 'prR and 3 'prF/3 'prR) to amplify two short 150- 300bp fragments of non-repetitive genomic DNA external to and extending beyond each arm, to allow Southern analysis of the targeted locus, in isolated putative targeted clones; a mouse genotyping primer pair (hetF and hetR) which allows differentiation between wild-type, heterozygote and homozygous mice, when used in a multiplex PCR with a vector specific primer, in this case, Asc306; and lastly, a target screening primer (3 'ser) which anneals upstream of the end of the 3' arm region, and which produces a target event specific 1.7kb amplimer when paired with a primer specific to the 3' end of
- This amplimer can only be derived from template DNA from cells where the desired genomic alteration has occurred and allows the identification of correctly targeted cells from the background of clones containing randomly integrated copies of the vector.
- the location of these primers and the genomic structure of the regions of the T ⁇ M8 locus used in the targeting strategy is shown.
- TrpM8 Primer Sequences musTr ⁇ M8 5'prF GGCTGTGTCCCTGTTTGCATGTACTTG - Seq ID No. 6 musTrpMS 5'prR GTGCTAGGGATCAAACCTAAGACCTTG - Seq ID No. 7 musTrpMS 5'armF Age tttaccggtGAATCTATGGATACCTGTGCTTCTGTC - Seq ID No. S musTrpM ⁇ 5'armR Not aaagcggccgcGGGAAATCTCTCCATACCATTGCTTAG - S eq ID No.
- a targeting vector is prepared where the T ⁇ M8 region to be deleted is replaced with non-homologous sequences composed of an endogenous gene expression reporter (a frame independent lacZ gene) upstream of a selection cassette composed of a promoted neomycin phosphotransferase (neo) gene arranged in the same orientation as the T ⁇ M8 gene.
- endogenous gene expression reporter a frame independent lacZ gene
- selection cassette composed of a promoted neomycin phosphotransferase (neo) gene arranged in the same orientation as the T ⁇ M8 gene.
- Clones are picked into 96 well plates, replicated and expanded before being screened by PCR (using primers 3 'ser and Asc 146, as described above) to identify clones in which homologous recombination has occurred between the endogenous T ⁇ M8 gene and the targeting construct. Positive clones can be identified at a rate of 1 to 5%>. These clones are expanded to allow replicas to be frozen and sufficient high quality DNA to be prepared for Southern blot confirmation of the targeting event using the external 5' and 3' probes prepared as described above, all using standard procedures (Russ et al, Nature 2000 Mar 2;404(6773):95-99).
- C57BL/6 female and male mice are mated and blastocysts are isolated at 3.5 days of gestation. 10-12 cells from a chosen clone are injected per blastocyst and IS blastocysts are implanted in the uterus of a pseudopregnant FI female. A litter of chimeric pups are bora containing several high level (up to 100%) agouti males (tlie agouti coat colour indicates the contribution of cells descended from the targeted clone). These male chimeras are mated with female MF1 and 129 mice, and germline transmission is determined by the agouti coat colour and by PCR genotyping respectively.
- PCR Genotyping is carried out on lysed tail clips, using the primers hetF and hetR with a third, vector specific primer (Asc306).
- This multiplex PCR allows amplification from the wild-type locus (if present) from primers hetF and hetR giving a 230bp band.
- the site for hetF is deleted in the knockout mice, so this amplification will fail from a targeted allele.
- the Asc306 primer will amplify a 338 bp band from the targeted locus, in combination with the hetR primer which anneals to a region just inside the 3' arm.
- this multiplex PCR reveals the genotype o_f the litters as follows: wild-type samples exhibit a single 230 bp band; heterozygous D fA samples yield two bands at 230 bp and 338bp; and the homozygous samples will show only the target specific 338 bp band.
- the X gal staining of dissected tissues is performed in the following manner.
- tissue slices are made of large organs. Whole small organs and tubes are sliced open, so fixative and stain will penetrate. Tissues are rinsed thoroughly in PBS (phosphate buffered saline) to remove blood or gut contents. Tissues are placed in fixative (PBS containing 2% formaldehyde, 0.2% glutaraldehyde, 0.02% NP40, ImM MgC12, Sodium deoxycholate 0.23mM) for 30-45 minutes. Following three 5 minute washes in PBS, tissues are placed in Xgal staining solution (4mM K Ferrocyanide, 4mMKFerricyanide, 2mM MgC12, Img/mlX-gal in PBS) for 18 hours at 30C. Tissues are PBS washed 3 times, postfixed for 24 hours in 4% formaldehyde, PBS washed again before storage in 70%> ethanol.
- PBS phosphate buffered saline
- fixative PBS containing 2% formaldehyde
- T ⁇ M8 is found to be expressed in the DRG and in particularly in the neurons.
- Example 4 Biological Data: Behaviour: Tail Flick Test A tail flick analgesia test is performed using a Tail-Flick Analgesia Meter. This equipment provides an easy to use method to determine pain sensitivity accurately and reproducibly in rodents (D'Amour, F.E. and D.L. Smith, 1941, Expt. Clin. Pharmacol., 16: 179-184).
- the instrument has a shutter-controlled lamp as a heat source. The lamp is located below the animal to provide a less confining environment. Tail flick is detected by the automatic detection circuitry, which leaves the user's hands free to handle the animal. The animal is restrained in a ventilated tube and its tail placed on a sensing groove on top of the equipment.
- Activation of an intense light beam to the tail through opening of the shutter results in discomfort at some point when the animal will flick its tail out of the beam.
- a photo-detector detects the tail motion causing the clock to stop and the shutter to close. The total time elapsed between the shutter opening and the animal's reaction is recorded.
- mutant transgenic mice are compared with age and sex matched wild-type mice.
- a single animal may be subjected to different heat settings to produce an increase in tail temperature no greater than 55°C.
- This test is used as an indication of knockout mice response to nociceptive pain.
- the mutants are less sensitive to heat induced pain showing hypoalgesia.
- the mutants are observed to have a longer latency to flick their tails away from the heat source compared to the age and sex matched wildtype mice ( Figure 3). Accordingly, this demonstrates that T ⁇ M8 is involved in the sensation of pain.
- mice Knockout and wild-type control mice are tested in an Open Field Test. Those skilled in the art will be familiar with such test and how it is preformed. Briefly, the mice are placed in the centre of a Perspex box with clear sides and the movement of the mice over a period of time is recorded on video. The mice are analysed for distance travelled and location of the mouse at any time. Control animals usually spend most of the time moving around the periphery of the arena. Variations from this normal pattern are recorded in particular the amount of time spent in the central areas of the arena, an increase of which can mean that the animal is less anxious.
- Example 6 Biological Data: Physiology: Blood Plasma Corticosterone Corticosterone is a hormone that is released in response to stress and anxiety.
- corticosterone The lower the levels of corticosterone the lower the levels of anxiety and stress the animal is likely to be undergoing.
- Blood plasma corticosterone are analysed from T ⁇ M8 -/- and age matched +/+ mice using an ELISA assay. Corticosterone levels are found to be lower in the mutant animals than in the wildtypes ( Figure 5) indicating that T ⁇ M8 mice are less anxious and stressed than their wildtype controls.
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EP2029614B1 (en) | 2006-05-10 | 2013-01-23 | Janssen Pharmaceutica, N.V. | Cold menthol receptor-1 antagonists |
US8629281B2 (en) | 2011-01-14 | 2014-01-14 | Janssen Pharmaceutica, Nv | Cold menthol receptor-1 antagonists |
GB0615136D0 (en) * | 2006-07-29 | 2006-09-06 | Univ Edinburgh | Induction of analgesia in neuropathic pain |
US8558011B2 (en) | 2010-08-31 | 2013-10-15 | Janssen Pharmaceutica, Nv | Cold menthol receptor-1 antagonists |
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See also references of WO2005094569A1 * |
VOGT B.A.: "Knocking out the DREAM to study pain", NEJM, vol. 347, no. 5, 1 August 2002 (2002-08-01), pages 362 - 364 * |
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