US20100316615A1 - Antisense Oligonucleotides Against VR1 - Google Patents
Antisense Oligonucleotides Against VR1 Download PDFInfo
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- US20100316615A1 US20100316615A1 US12/645,625 US64562509A US2010316615A1 US 20100316615 A1 US20100316615 A1 US 20100316615A1 US 64562509 A US64562509 A US 64562509A US 2010316615 A1 US2010316615 A1 US 2010316615A1
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/121—Hammerhead
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/323—Chemical structure of the sugar modified ring structure
- C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
Definitions
- the invention relates to antisense oligodeoxynucleotides against VR1, to corresponding nucleotide constructs, to cells, pharmaceutical preparations and diagnostic preparations containing these, to the use thereof in pain therapy and to methods for diagnosing symptoms associated with VR1 and for identifying pain-modulating substances.
- Acute and transient pain is an important bodily signal protecting people from severe injury by their environment or by overloading their body.
- chronic pain which lasts longer than the cause of the pain and the anticipated time frame for cure, has no known biological function and affects hundreds of millions of people worldwide. In the Federal Republic of Germany alone, some 7.5 million people suffer from chronic pain.
- pharmacological treatment of chronic pain is still unsatisfactory and thus remains a challenge to current medical research.
- existing analgesics often have an inadequate action and sometimes have severe side effects.
- the vanilloid receptor subtype 1 (VR1, also known as the capsaicin receptor) cloned by Caterina et al. (1997) is a promising starting point for the development of new analgesic drugs.
- This receptor is a cation channel which is predominantly expressed by primary sensory neurons (Catarina et al. 1997).
- VR1 is activated by capsaicin, a component of chillies, heat (>43° C.) and a low pH as a result of tissue injury, and brings about a calcium influx in primary afferents.
- VR1 knockout mice did not develop thermal hyperalgesia after tissue injury or inflammation (Caterina et al., 2000; Davis et al., 2000).
- Antisense oligodeoxynucleotides, ribozymes and other catalytic nucleic acids may be used for the treatment, in particular of chronic pain, by degrading or modifying the mRNA of selected targets, in the case of the present invention the above-described VR1, to down-regulate the expression thereof and thus reduce the number of receptors per cell.
- the ODN attach themselves to the mRNA, so firstly blocking translation and secondly initiating degradation of the mRNA by RNase H, which cleaves the DNA/RNA duplex.
- the object of the present document was accordingly to develop antisense oligodeoxynucleotides and catalytic nucleic acids together with corresponding ribozymes against the mRNA of the vanilloid receptor.
- the present invention accordingly provides an oligonucleotide containing or corresponding to a base sequence according to one of sequences (b) to (j) in each of FIG. 1 , 2 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 or 16 or a sequence differing therefrom by at most one different base, wherein the base difference is not located in the sequence domain shown in sequence (a).
- oligonucleotide means a molecule having between 2 and 40 nucleotides.
- the present invention also provides an oligonucleotide containing or corresponding to a base sequence according to one of sequences (b) to (j) in one of FIG. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 or 16 .
- the present invention also provides an oligonucleotide containing or corresponding to a base sequence according to (k) in one of FIG. 1 , 2 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 or 16 , or a sequence differing therefrom by at most two different bases, preferably one, wherein the base difference(s) is (are) not located in the sequence domain shown in (a).
- the present invention also provides an oligonucleotide containing or corresponding to a base sequence according to (k) in each of FIG. 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 or 16 .
- the present invention preferably also provides an oligonucleotide which has a length of 15 to 30, preferably of 15 to 25, in particular 17 to 19, or exactly 18, nucleotides.
- the present invention also preferably provides an oligonucleotide (hereinafter denoted oligonucleotide A) according to one of the above-stated oligonucleotide forms in which, optionally with one different base, the base sequence contained in the oligonucleotides or corresponding thereto may be found in one of FIG. 1 , 2 , 3 , 4 , 9 , 10 , 11 , 12 , 15 or 16 .
- oligonucleotides which have been found to be particularly effective or contain corresponding, optionally slightly different, sequences, which bind strongly to the mRNA of VR1 (see oligos V15, V30, V2, V16 and V4 or the corresponding human sequences).
- the present invention also preferably provides an oligonucleotide (hereinafter denoted oligonucleotide B) in which, optionally with one different base, the base sequence contained in the oligonucleotides or corresponding thereto may be found in one of FIG. 1 , 2 , 3 , 4 , 11 or 12 , preferably 1, 3 or 11, in particular 1 or 3.
- oligonucleotide B an oligonucleotide in which, optionally with one different base, the base sequence contained in the oligonucleotides or corresponding thereto may be found in one of FIG. 1 , 2 , 3 , 4 , 11 or 12 , preferably 1, 3 or 11, in particular 1 or 3.
- the present invention preferably provides an oligonucleotide according to the invention which comprises at least one modified or unmodified ribose, at least one modified or unmodified phosphodiester bond and/or at least one modified or unmodified base.
- the present invention preferably provides an oligonucleotide according to the invention, in which at least one of the nucleotides, in particular two or more of the nucleotides, are “locked nucleic acids” (LNAs) or at least one of the nucleotides, in particular all of the nucleotides, are phosphorothioates, preferably one in which two or more of the nucleotides are “locked nucleic acids (LNAs).
- LNAs locked nucleic acids
- “Locked nucleic acids” (LNAs) are ribonucleotides which contain a methylene bridge which joins the 2′ oxygen of the ribose with the 4′ carbon (see FIG. 27 ). Braasch D. A. and Corey, D. R.
- LNA Locked nucleic acids
- Chem. Biol. 8, 1-7 provide an overview of LNAs. This article is herein explicitly incorporated by reference in its entirety.
- LNAs are available commercially, for example, from the company Proligo, Boulder, Colo., USA.
- Phosphorothioates are also known to the person skilled in the art and may be ordered, for example, from MWG-Biotech AG, Ebersberg, Germany.
- oligonucleotides are those in which the LNAs are located at the 5′ and 3′ end of the oligonucleotide, preferably in each case the final 2-5 nucleotides, in particular in each case the final 3 or 4 nucleotides, on the 3′ and 5′ end of the oligonucleotide are LNAs.
- oligonucleotides of the invention have >6, in particular >8 contiguous nucleotides in the oligonucleotide that are not LNAs.
- sequence (a) of the oligonucleotide according to one of FIGS. 1 to 16 at most one or none of the nucleotides is in each case an LNA.
- the oligonucleotide is an oligonucleotide A according to the invention or an oligonucleotide B according to the invention, preferably an oligonucleotide B according to the invention (see above).
- the present invention specifically provides nucleic acids, in particular oligonucleotides, in which two or more of the nucleotides are “locked nucleic acids” (LNAs), in which the LNAs are on the 5′ and 3′ end of the oligonucleotide, preferably in each case the final 2-5 nucleotides, in particular in each case the final 3 or 4 nucleotides, on the 3′ and 5′ end of the oligonucleotide are LNAs, and/or in which >6, in particular >8 contiguous nucleotides in the oligonucleotide are not LNAs.
- LNAs locked nucleic acids
- the present invention also preferably provides a polynucleotide construct containing at least one oligonucleotide according to the invention.
- Polynucleotide construct should here be taken to have a very wide meaning. It includes RNA and DNA and nucleotides from a length of at least 20 nucleotides.
- a “recombinant” polynucleotide construct should here be taken to be a general designation for any kind of DNA or RNA molecules which have been obtained by in vitro linkage of DNA or RNA molecules.
- Polynucleotide is taken to have the following meaning: the underlying nucleotide is a fundamental nucleic acid building block essentially consisting of nucleic base, pentose and phosphoric acid.
- polynucleotide prepared from two or more nucleotides linked together by phosphoric acid/pentose esterification.
- the invention also includes modified polynucleotides which, while they retain the sequence of bases, have a modified backbone instead of phosphoric acid/pentose.
- the present invention also preferably provides a polynucleotide construct containing in two separate domains, two nucleotide subsequences Fragment I and Fragment III according to one of sequences (l)-(n) or the two nucleotide subsequences helix I and helix III according to one of sequences (o)-(q) in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 , or nucleotide subsequences differing from these nucleotide subsequences in each case by at most one base. Further details relating to the division of the two domains may be found in FIG.
- the present invention also preferably provides a polynucleotide construct which codes for at least one oligonucleotide according to the invention.
- This in particular comprises DNA to be read out or a vector containing DNA or RNA, the product of which is or may be an oligonucleotide according to the invention.
- the present invention also particularly preferably provides a polynucleotide construct according to the invention which comprises a ribozyme, a DNA enzyme, a vector, in particular an expression vector, or a peptide nucleic acid (PNA).
- a polynucleotide construct according to the invention which comprises a ribozyme, a DNA enzyme, a vector, in particular an expression vector, or a peptide nucleic acid (PNA).
- the present invention also preferably provides a polynucleotide construct containing in two separate domains two nucleotide subsequences, as described above, wherein said construct comprises a ribozyme, preferably a “hammerhead” ribozyme, or a DNA enzyme, preferably a type 10-23 or 12-32 DNA enzyme. It is particularly preferred and selected if said construct comprises a DNA enzyme containing at least the nucleotide subsequences Fragment I and Fragment III according to one of sequences (l) to (n), preferably (n), in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 or 13 ; preferably 1, 3, 7 or 11, in particular 1 or 3; or preferably 4, 6, 8 or 12, in particular 4 or 12.
- said polynucleotide construct comprises a ribozyme containing at least the nucleotide subsequences helix I and helix III according to one of sequences (o) to (q), preferably (o), in one of FIGS. 1, 3, 4, 5, 6, 8, 11 or 12; preferably 1, 3 or 11, in particular 11; or preferably 4, 6, 8 or 12, in particular 4 or 12.
- FIG. 24 is a general diagram of a “hammerhead” ribozyme after Vaish, N. K. et al. (1998), Nucl. Acid Res. 26, 5237-5242 with the “recognition arms” helix I and helix III, into which the helices I and III according to the invention according to sequences (o)-(q) in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 are inserted, in order to obtain the hammerhead ribozymes according to the invention.
- the fragment helix I here replaces in each case in one of FIG.
- nucleotides 24 towards the 5′ end are replaced by the nucleotides which are shown in one of sequences (o) to (q) of helix I in each case in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 .
- the nucleotides “A” and “C” on the 5′ end of helix III in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 in each case replace the nucleotides “A” and “C” in helix III in FIG. 24 , and the following desired nucleotides “N” in helix III, FIG.
- FIG. 24 shows a specific example.
- the hammerhead ribozyme V16 (7/7) is derived from FIG. 24 and FIG. 11 .
- ribozyme V16 (7/7) here means that the enzyme is oriented towards the GUC site of oligo V16 and contains 7 nucleotides in each of the “recognition arms” (helix I and helix III), in this case according to helix I and helix III of sequence (o) in FIG. 11 .
- FIG. 26 is a general diagram of a type “10-23” DNA enzyme according to Santoro et al., 1997, FIG. 2, p. 4264.
- the upper strand marked with an arrow is the RNA strand to be cleaved, the arrow showing the cleavage site, while the lower strand is a representation of the DNA enzyme.
- the cleavage site on the upper strand is thus a GUC site (see above).
- the second with the RNA base-paired fragment, the unpaired “A” on Fragment III is then directly followed from the 5′ direction 3′-wards by the nucleotides from Fragment III according to sequences (l to n) in each case in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 , i.e. 7 further nucleotides in sequence l, 8 further nucleotides in sequence m and 9 further nucleotides in sequence n.
- Fragment III and Fragment I are the “recognition arms” of the DNA enzyme (see Example 3 below).
- the type “10-23” DNA enzyme for sequence n according to FIG. 1 which is particularly preferred for the purposes of the invention would thus have the following sequence, wherein the underlined fragment would be base-paired with the RNA:
- This DNA enzyme was named V15 (9/9), wherein the name indicates that the enzyme is oriented towards the GUC site of oligo V15 and the “recognition arms” in each case contain 9 nucleotides (Fragment I and III), for example according to Fragment I and Fragment III of sequence (n) in FIG. 1 .
- the present invention also preferably provides a polynucleotide construct according to the invention, wherein it comprises at least one modified or unmodified ribose, at least one modified or unmodified phosphodiester bond and/or at least one modified or unmodified base.
- the present invention also preferably provides an oligonucleotide according to the invention or polynucleotide construct according to the invention, wherein it is bound on a support, in particular a protein, preferably tet-, transportin or ferritin, and/or is packaged in a liposome.
- a support in particular a protein, preferably tet-, transportin or ferritin, and/or is packaged in a liposome.
- the present invention also preferably provides a cell containing at least one oligonucleotide according to the invention and/or a polynucleotide construct according to the invention.
- the present invention also preferably provides a pharmaceutical preparation containing at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention, optionally together with a suitable auxiliary substance and/or additive.
- the pharmaceutical preparations according to the invention may be administered in the form of solutions for injection, drops or juice, as semi-solid dosage forms in the form of granules, tablets, pellets, patches, capsules, dressings or aerosols and, in addition to the at least one subject matter of the invention, they also optionally contain, depending upon the pharmaceutical presentation, excipients, fillers, solvents, diluents, colorants and/or binders.
- auxiliary substances and the quantities thereof which are to be used depends upon whether the pharmaceutical preparation is to be administered orally, perorally, parenterally, intravenously, intraperitoneally, intradermally, intramuscularly, intranasally, buccally, rectally or topically, for example onto infections of the skin, mucous membranes or eyes.
- Preparations in the form of tablets, coated tablets, capsules, granules, drops, succi and syrups are suitable for oral administration, while solutions, suspensions, easily reconstitutible dried preparations and sprays are suitable for parenteral, topical and inhalatory administration.
- Subject matters of the invention in a depot in dissolved form or in a dressing, optionally with the addition of skin penetration promoters, are suitable percutaneous administration preparations. Orally or percutaneously administrable formulations may release the subject matters of the invention in delayed manner.
- the quantity of active substance to be administered to the patient varies as a function of patient weight, mode of administration, the indication and the severity of the condition. 2 to 500 mg/kg of at least one subject matter of the invention are conventionally administered.
- suitable auxiliary substances and additives which it is advisable to use are, for example, a physiological saline solution, stabilizers, proteinase inhibitors, DNAse inhibitors etc.
- the present invention also preferably provides a diagnostic aid containing at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention, optionally together with suitable additives.
- the present invention also preferably provides the use of at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention for the production of a pharmaceutical preparation for the treatment of pain, in particular chronic pain, tactile allodynia, thermally induced pain and/or inflammatory pain.
- the present invention also preferably provides the use of at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention for the production of a pharmaceutical preparation for the treatment of urinary incontinence; also of neurogenic bladder symptoms; pruritus, tumors, inflammation; in particular VR1 receptor-associated inflammation with symptoms such as asthma; together with any disease symptoms associated with VR1.
- the present invention also preferably provides the use of at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention for gene therapy, preferably in vivo or in vitro gene therapy.
- Gene therapy is taken to be a type of therapy in which, by the introduction of nucleic acids into cells, an effector gene product, usually a protein, but also an antisense oligodeoxynucleotide, is expressed.
- a fundamental distinction is drawn between in vivo and in vitro methods. In in vitro methods, cells are removed from the organism and transfected with vectors ex vivo before subsequently being introduced back into the same organism or a different one. In in vivo gene therapy, vectors, for example for combating tumors, are administered systemically (for example via the bloodstream) or directly into the tumor.
- the present invention also preferably provides a process for the identification of pain-modulating substances, characterized in that identification is made by quantification of the binding of at least one, preferably labelled, oligonucleotide according to the invention or at least one polynucleotide construct onto an RNA.
- the present invention also preferably provides a process for the identification of pain-modulating substances comprising the following process steps:
- the term pain-modulating here refers to a potentially regulating influence upon physiological pain phenomena, in particular to an analgesic action.
- the term substance comprises any compound suitable as a pharmaceutical active substance, thus in particular low molecular weight active substances, but also others such as nucleic acids, fats, sugars, peptides or proteins such as antibodies.
- Incubation under suitable conditions should here be taken to mean that the substance to be investigated is capable of reacting in an aqueous medium for a defined period prior to measurement with the cell or the corresponding preparation.
- the aqueous medium may be temperature-controlled, for example between 4° C. and 40° C., preferably at room temperature or at 37° C.
- the incubation time may be varied between a few seconds and several hours, depending upon how the substance interacts with the receptor. Times of between 1 min and 60 min are, however, preferred.
- the aqueous medium may contain suitable salts and/or buffer systems, such that a pH of between 6 and 8, preferably pH 7.0-7.5 prevails in the medium during incubation. Suitable substances, such as coenzymes, nutrients etc., may additionally be added to the medium. Suitable conditions may readily be established by the person skilled in the art as a function of the substance-receptor interaction which is to be investigated on the basis of his/her experience, the literature or a few simple preliminary tests, so that the clearest possible measured value is obtained from the process.
- a cell which has synthesized a receptor is a cell which has already endogenously expressed this receptor or one which has been modified by genetic engineering in such a manner that it expresses this receptor and consequently contains the receptor prior to the beginning of the process according to the invention.
- the cells may be cells from possibly immortalized cell lines or be native cells originating and isolated from tissues, wherein cell aggregation has usually been broken down.
- the preparation made from these cells in particular comprises cell homogenates, the cytosol, a membrane fraction of the cells with membrane fragments, a suspension of isolated cell organelles etc.
- the yardstick which permits the identification of interesting substances is either binding to the receptor, which may, for example, be detected by displacement of a known ligand or by the extent of substance binding, or the modification of a functional parameter due to the interaction of the substance with the receptor.
- This interaction may in particular involve regulation, inhibition and/or activation of receptors, ion channels and/or enzymes, while modified functional parameters may be, for example, gene expression, ion concentration, pH or membrane potential or modification of enzyme activity or the concentration of secondary messengers.
- Another preferred embodiment of the present process provides that the cell has already been subjected to genetic engineering manipulation before process steps (a) and (a′).
- Another preferred embodiment of the present process provides that the genetic engineering manipulation permits the measurement of at least one of the functional parameters modified by the test substance.
- Another preferred embodiment of the present process provides that the genetic engineering manipulation results in the expression of a form, not endogenously expressed in the cell, of a member of the vanilloid receptor family, preferably the VR-1 receptor, or in the introduction of a reporter gene.
- Another preferred embodiment of the present process provides that binding is measured by the displacement of a known, labelled ligand of a member of the vanilloid receptor family, preferably the VR-1 receptor.
- Another preferred embodiment of the present process provides that there is an elapse of not less than 8 h, preferably not less than 12 h, in particular not less than 24 h between the parallel process steps (a) and (a′), and process step (b).
- the present invention also preferably provides a process for the diagnosis of clinical conditions which are associated with modified expression of genes of the vanilloid receptor family, which process is characterized in that the diagnosis is made by means of quantification of the binding of an oligonucleotide according to the invention and/or at least one polynucleotide construct onto an RNA.
- oligonucleotides and also the polynucleotide constructs are produced by processes known to the person ordinarily skilled in the art. Nucleotides, in particular also oligonucleotides, are synthesized in the manner of the Merrifield synthesis on an insoluble support (H. G. Gassen et al., Chemical and Enzymatic Synthesis of Gene Fragments (Verlag Chemie, Weinheim 1982)) or in another manner (Beyer/Walter; Lehrbuch der Organischen Chemie, 20th edition, (S. Hirzel Verlag, Stuttgart 1984), pp. 816 et seq.).
- the present invention also provides a process for the treatment, in particular treatment for pain, of a non-human mammal or human requiring treatment for pain, in particular chronic pain, by administration of a pharmaceutical preparation according to the invention, in particular such a preparation containing an oligonucleotide according to the invention and/or a polynucleotide construct according to the invention.
- a pharmaceutical preparation according to the invention in particular such a preparation containing an oligonucleotide according to the invention and/or a polynucleotide construct according to the invention.
- the invention also provides corresponding processes for the treatment of pruritus and/or urinary incontinence.
- FIG. 1 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V15, oligonucleotide no. 15 or V15. Sequences or subtypes (a)-(j) show truncated fragments from this antisense oligodeoxynucleotide sequence. Sequence (k) shows the full-length antisense oligodeoxynucleotide sequence with which the “ messengerger walk screening” was carried out.
- Sequences (l)-(n) each shows two nucleotide subsequences (Fragment I and Fragment III) starting from the full-length antisense oligodeoxynucleotide sequence, which each comprises non-overlapping subdomains of this sequence or corresponding sequence thereto, is usually divided at or in the GAC region and, in certain polynucleotide constructs, in particular DNA enzymes, and occurs in two separate domains, the “recognition arms.”
- Subtypes (o)-(q) each shows two nucleotide subsequences (helix I and helix III) starting from the full-length antisense oligodeoxynucleotide sequence (in this case as RNA), which each comprises non-overlapping subdomains of this sequence or a corresponding sequence thereto, is usually divided at or in the GAC region and, in certain polynucleotide constructs, in particular ribozymes, and occurs in two separate domains, the “recognition
- FIG. 2 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V15 in FIG. 1 .
- subtypes or sequences (a)-(k) correspond to that already described in relation to FIG. 1 .
- FIG. 3 shows a list of general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V30, oligonucleotide no. 30 or V30.
- the type and general content of the subtypes correspond to that already described in relation to FIG. 1 .
- FIG. 4 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V30 in FIG. 3 .
- subtypes (a)-(q) correspond to that already described in relation to FIG. 1 .
- FIG. 5 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V32, oligonucleotide no. 32 or V32.
- the type and general content of the subtypes correspond to that already described in relation to FIG. 1 .
- FIG. 6 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V32 in FIG. 5 .
- subtypes (a)-(q) correspond to that already described in relation to FIG. 1 .
- FIG. 7 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V26, oligonucleotide no. 26 or V26.
- the type and general content of the subtypes correspond to that already described in relation to FIG. 1 .
- FIG. 8 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V26 in FIG. 7 .
- subtypes (a)-(q) correspond to that already described in relation to FIG. 1 .
- FIG. 9 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V2, oligonucleotide no. 2 or V2.
- the type and general content of the subtypes correspond to that already described in relation to FIG. 1 .
- FIG. 10 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V2 in FIG. 9 .
- subtypes (a)-(k) correspond to that already described in relation to FIG. 1 .
- FIG. 11 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V16, oligonucleotide no. 16 or V16.
- the type and general content of the subtypes correspond to that already described in relation to FIG. 1 .
- FIG. 12 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V16 in FIG. 11 .
- subtypes (a)-(q) correspond to that already described in relation to FIG. 1 .
- FIG. 13 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V28, oligonucleotide no. 28 or V28.
- the type and general content of the subtypes correspond to that already described in relation to FIG. 1 .
- FIG. 14 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V28 in FIG. 13 .
- subtypes (a)-(k) correspond to that already described in relation to FIG. 1 .
- FIG. 15 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V4, oligonucleotide no. 4 or V4.
- the type and general content of the subtypes correspond to that already described in relation to FIG. 1 .
- FIG. 16 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V4 in FIG. 15 .
- subtypes (a)-(k) correspond to that already described in relation to FIG. 1 .
- FIG. 17 shows the result of messenger walk screening.
- each track there can be seen, in addition to the upper band of the uncut substrate, the two product bands of the cut mRNA together with several non-specific bands.
- the figure shows the VR1 mRNA after degradation by RNase H in the presence in each case of one of 33 antisense oligonucleotides (oligo V1 to oligo V33).
- Track 1 VR1 mRNA
- tracks 2-34 RNase H assay with antisense oligodeoxynucleotides against the 33 GUC sites of VR1 mRNA (oligo V1 to oligo V33).
- FIG. 18 shows quantitative evaluation of messenger walk screening.
- FIG. 18 is a plot of the percentage of the uncut mRNA after the RNase H assay with the individual ODNs. Each value is the mean of at least two experiments, such that the standard deviation does not ever exceed 10%.
- the antisense oligodeoxynucleotides against the 15th and 30th GUC sites bind most efficiently to the VR1 mRNA, such that the latter is respectively 88 ⁇ 4 and 97 ⁇ 1% degraded by the RNase H (oligo V15 and oligo V30).
- FIG. 19 is an image of a gel after RNase H assay with oligonucleotides V15, V15ctrl. (mismatch), V30 and V30ctrl. (mismatch).
- the Figure shows VR1 mRNA (track 1) and RNase H assay with oligodeoxynucleotides V15, V15ctrl., V30 and V30ctrl. (tracks 2-5).
- FIG. 20 shows the quantitative evaluation of cleavage of mRNA by ribozymes and DNA enzymes under “single turnover” conditions.
- FIG. 21 shows the kinetics of VR1 mRNA cleavage by ribozymes and DNA enzymes under “single turnover” conditions.
- FIG. 22 shows the kinetics of VR1 mRNA cleavage by ribozymes and DNA enzymes under “multiple turnover” conditions.
- FIG. 23 shows an estimation of tactile allodynia during treatment by VR1-antisense (AS) oligodeoxynucleotides and mismatch (MS) oligodeoxynucleotides (V15 and V15ctrl.).
- AS VR1-antisense
- MS mismatch
- FIG. 24 is a general diagram of a “hammerhead” ribozyme with the “recognition arms” helix I and helix III, into which the helices I and III according to subtypes (o)-(q) in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 are inserted, in order to obtain the inventive hammerhead ribozymes (see description relating to FIG. 1 ).
- the fragment helix I here replaces in each case in one of FIG.
- nucleotides 24 towards the 5′ end are replaced by the nucleotides which are shown in one of sequences (o) to (q) of helix I in each case in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 .
- the nucleotides “A” and “C” on the 5′ end of helix III in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 in each case replace the nucleotides “A” and “C” in helix III in FIG. 24 and the following desired nucleotides “N” in helix III, FIG.
- nucleotides which are shown in one of sequences (o)-(q), helix III, in each case in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 .
- FIG. 25 shows a specific example of the preparation of the (particularly preferred) “hammerhead” ribozyme V16 (7/7) according to FIG. 24 and FIG. 11 .
- the designation ribozyme V16 (7/7) here means that the enzyme is oriented towards the GUC site of oligo V16 and contains 7 nucleotides in each of the “recognition arms” (helix I and helix III), in this case according to helix I and helix III of sequence (o) in FIG. 11 .
- FIG. 26 is a diagram of a type “10-23” DNA enzyme according to Santoro et al., 1997, FIG. 2, p. 4264; the upper strand marked with an arrow is the RNA strand to be cleaved, the arrow showing the cleavage site, while the lower strand is a representation of the DNA enzyme.
- the cleavage site on the upper strand is thus a GUC site (see above).
- the second with the RNA base-paired fragment, the unpaired “A” on Fragment III is then directly followed from the 5′ direction 3′-wards by the nucleotides from Fragment III according to sequences (l) to (n) in each case in one of FIG. 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 11 , 12 , 13 or 15 , i.e. 7 further nucleotides in sequence l, 8 further nucleotides in sequence m and 9 further nucleotides in sequence n.
- Fragment III and Fragment I are the “recognition arms” of the DNA enzyme (see Example 3).
- the type “10-23” DNA enzyme for sequence (n) according to FIG. 1 would thus have the following sequence, wherein the underlined fragment would be base-paired with the RNA:
- This DNA enzyme would be named V15 (9/9), wherein the name indicates that the enzyme is oriented towards the GUC site of oligo V15 and the “recognition arms” in each case contain 9 nucleotides (Fragment I and III), for example according to Fragment I and Fragment III of sequence (n) in FIG. 1 .
- FIG. 27 is a schematic diagram of a “locked nucleic acid” (LNA).
- LNA locked nucleic acid
- the first step in the antisense and ribozyme strategy is to identify accessible sites on the mRNA for binding oligonucleotides, in particular ribozymes.
- the VR1 mRNA had to be investigated for such restriction sites. Analysis of the VR1 mRNA revealed the following potential recognition sites for ribozymes in the coding domain:
- the mRNA was systematically screened with ODNs in an RNase H assay (messenger walk screening).
- the ODNs were 18 nucleotides in length and contained a central GAC sequence, which is reverse complementary to GUC sequences in the mRNA. This triplet was selected as the target as it provided good results and may be used in a second step to develop hammerhead ribozymes and DNA enzymes.
- 33 ODNs, designated V1 to V33 were tested against all the GUC sites of the VR1 mRNA.
- the ODNs were systematically screened for their suitability by the addition in each case of one ODN and RNase H to the mRNA.
- RNase H cuts formed DNA/RNA duplexes wherever an oligonucleotide can bind to the mRNA ( FIG. 17 ).
- the cDNA of the vanilloid receptor was cloned into the vector pcDNA3.1 (+) from Invitrogen. Then, in vitro transcription of the mRNA was performed using the RiboMAX Large Scale RNA Production System—T7 from Promega in accordance with the manufacturer's instructions.
- RNase H assay was performed to test whether an antisense oligodeoxynucleotide had bound to the mRNA.
- the VR1 mRNA 100 mM was incubated with a five-fold excess of the ODNs in a total volume of 10 ⁇ l in 40 mM tris/HCl pH 7.2, 4 mM MgCl 2 , 1 mM DTT and 150 mM NaCl for 7.5 minutes at 37° C. in the presence of 0.4 unit of RNase H (from Promega).
- the reactions were terminated by addition of EDTA (65 mM final concentration).
- the samples were separated on a 1.5% agarose gel and stained with ethidium bromide (1 ⁇ g/ml) for 20 minutes. The gels were photographed with the Gel Doc 2000 Gel Documentation System from Biorad and evaluated with Quantity One software.
- FIG. 17 shows the result of messenger walk screening. In each track there can be seen, in addition to the upper band of the uncut substrate, the two product bands of the cut mRNA together with several non-specific bands.
- RNA degradation revealed that the most effective antisense oligonucleotide (oligo no. 30 (V30)) was able specifically to cut/cleave more than 90% of the VR1 mRNA when used in a five-fold excess relative to the target mRNA ( FIG. 18 ).
- FIG. 18 is a plot of the percentage of the uncut mRNA for the individual ODNs after the RNase H assay.
- the antisense oligodeoxynucleotides against the 15th and 30th GUC sites bind most efficiently to the VR1 mRNA, such that the latter is respectively 88 ⁇ 4 and 97 ⁇ 1% degraded by the RNase H.
- FIGS. 1 , 3 , 5 , 7 , 9 , 11 , 13 and 15 The sequences of the antisense oligodeoxynucleotides most effective in this test are shown in FIGS. 1 , 3 , 5 , 7 , 9 , 11 , 13 and 15 , wherein inter alia preferential attention was paid to those designated oligo V15 ( FIG. 1 ) and oligo V30 ( FIG. 3 ).
- Oligodeoxynucleotides in which every fifth and six base (or in the event that these are identical, two adjacent bases) are swapped were synthesized and used in the following experiments as mismatch controls for these antisense oligodeoxynucleotides.
- the sequences of the control (mismatch) oligodeoxynucleotides are:
- Oligo V15ctrl. CAT GCT ATG AGC GTT GAG (SEQ ID NO: 250)
- the RNase H assay was performed with the oligonucleotides V15, V15ctrl., V30 and V30ctrl. for control purposes (see FIG. 19 ). As anticipated, the RNA is degraded only with the antisense ODNs, but not with the mismatch controls, as the latter do not bind to the mRNA.
- FIG. 24 is a schematic representation of a ribozyme with the helices I and III, while FIG. 25 shows a specific example. This may be seen for a 10-23 DNA enzyme (5′-end (Fragment I) and 3′-end (Fragment III) from the catalytic motif) in Santoro et al. (1997, p. 4264, FIG. 2) (see also FIG. 26 ) with the description of the Figures).
- ribozyme V15 (7/7), for example, here means that the enzyme is oriented towards the GUC site of oligo V15 and contains 7 nucleotides in each of the “recognition arms” (helix I and helix III), for example according to Fragment I and Fragment III of subtype (l) in FIG. 1 .
- FIG. 22 shows the cleavage of the mRNA under “multiple turnover” conditions (10-fold substrate excess). Again DNA enzyme V15 (9/9) has a higher apparent rate (rate constant) (by a factor of 3.4) than ribozyme V16 (7/7) (Table 2).
- oligonucleotides according to the invention were produced, in particular according to subgroup (k) of FIG. 1 and, in some cases, also FIG. 3 . Most of these were LNA constructs which were obtained from PROLIGO, in Boulder, Colo., and in which LNAs were located at various sites (see Table 3). An unmodified oligonucleotide according to subgroup (k) of FIG. 1 was also synthesized and a phosphorothioate corresponding in terms of base sequence was obtained from MWG Biotech AG in Ebersberg, Germany.
Abstract
Antisense oligodeoxynucleotides against VR1, corresponding nucleotide constructs, cells containing said nucleotide constructs, pharmaceutical and diagnostic substances, uses thereof in pain therapy, and methods for diagnosing symptoms related to VR1 and for identifying pain-modulating substances.
Description
- The present application is a division of application Ser. No. 10/376,341, filed Mar. 3, 2003, now U.S. patent Ser. No. ______, which in turn is a continuation of International Patent Application No. PCT/EP01/10081, filed Aug. 31, 2001, designating the United States of America and published in German as WO 02/18407 A2, the entire disclosure of which is incorporated herein by reference. Priority is claimed based on Federal Republic of Germany Patent Application Nos. 100 43 674.9, filed Sep. 2, 2000, and 100 43 702.8, filed Sep. 4, 2000.
- The invention relates to antisense oligodeoxynucleotides against VR1, to corresponding nucleotide constructs, to cells, pharmaceutical preparations and diagnostic preparations containing these, to the use thereof in pain therapy and to methods for diagnosing symptoms associated with VR1 and for identifying pain-modulating substances.
- The effective treatment of pain is a major challenge to molecular medicine. Acute and transient pain is an important bodily signal protecting people from severe injury by their environment or by overloading their body. In contrast, chronic pain, which lasts longer than the cause of the pain and the anticipated time frame for cure, has no known biological function and affects hundreds of millions of people worldwide. In the Federal Republic of Germany alone, some 7.5 million people suffer from chronic pain. Unfortunately, pharmacological treatment of chronic pain is still unsatisfactory and thus remains a challenge to current medical research. Currently existing analgesics often have an inadequate action and sometimes have severe side effects.
- The search is thus now on for new targets or endogenous body structures which appear to provide a way of exerting pain-modulating action, for example using low molecular weight active substances or other compounds such as antisense oligodeoxynucleotides (ODN), in particular for the treatment of chronic pain.
- The vanilloid receptor subtype 1 (VR1, also known as the capsaicin receptor) cloned by Caterina et al. (1997) is a promising starting point for the development of new analgesic drugs. This receptor is a cation channel which is predominantly expressed by primary sensory neurons (Catarina et al. 1997). VR1 is activated by capsaicin, a component of chillies, heat (>43° C.) and a low pH as a result of tissue injury, and brings about a calcium influx in primary afferents. VR1 knockout mice did not develop thermal hyperalgesia after tissue injury or inflammation (Caterina et al., 2000; Davis et al., 2000).
- Antisense oligodeoxynucleotides, ribozymes and other catalytic nucleic acids may be used for the treatment, in particular of chronic pain, by degrading or modifying the mRNA of selected targets, in the case of the present invention the above-described VR1, to down-regulate the expression thereof and thus reduce the number of receptors per cell. The ODN attach themselves to the mRNA, so firstly blocking translation and secondly initiating degradation of the mRNA by RNase H, which cleaves the DNA/RNA duplex. Porreca et al. (1999) were able to demonstrate that intrathecally administered ODNs against the PN3/SNS channel in rats prevent the development of hyperalgesia and allodynia due to chronic nerve or tissue damage.
- Even once the sequence of VR1 is known, effective blocking and cleavage of the mRNA in particular depends upon the selection of the correct antisense oligodeoxynucleotides, ribozymes and other catalytic nucleic acids. The target's mRNA is usually folded and only a few sites are accessible for attachment and subsequent cleavage. Nothing is known in the prior art about how to select the ODN correctly.
- The object of the present document was accordingly to develop antisense oligodeoxynucleotides and catalytic nucleic acids together with corresponding ribozymes against the mRNA of the vanilloid receptor. The present invention accordingly provides an oligonucleotide containing or corresponding to a base sequence according to one of sequences (b) to (j) in each of
FIG. 1 , 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 or a sequence differing therefrom by at most one different base, wherein the base difference is not located in the sequence domain shown in sequence (a). For the purposes of the present invention, oligonucleotide means a molecule having between 2 and 40 nucleotides. - The present invention also provides an oligonucleotide containing or corresponding to a base sequence according to one of sequences (b) to (j) in one of
FIG. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. - The present invention also provides an oligonucleotide containing or corresponding to a base sequence according to (k) in one of
FIG. 1 , 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence differing therefrom by at most two different bases, preferably one, wherein the base difference(s) is (are) not located in the sequence domain shown in (a). - The present invention also provides an oligonucleotide containing or corresponding to a base sequence according to (k) in each of
FIG. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. - The present invention preferably also provides an oligonucleotide which has a length of 15 to 30, preferably of 15 to 25, in particular 17 to 19, or exactly 18, nucleotides.
- The present invention also preferably provides an oligonucleotide (hereinafter denoted oligonucleotide A) according to one of the above-stated oligonucleotide forms in which, optionally with one different base, the base sequence contained in the oligonucleotides or corresponding thereto may be found in one of
FIG. 1 , 2, 3, 4, 9, 10, 11, 12, 15 or 16. These in particular comprise oligonucleotides which have been found to be particularly effective or contain corresponding, optionally slightly different, sequences, which bind strongly to the mRNA of VR1 (see oligos V15, V30, V2, V16 and V4 or the corresponding human sequences). - The present invention also preferably provides an oligonucleotide (hereinafter denoted oligonucleotide B) in which, optionally with one different base, the base sequence contained in the oligonucleotides or corresponding thereto may be found in one of
FIG. 1 , 2, 3, 4, 11 or 12, preferably 1, 3 or 11, in particular 1 or 3. - The present invention preferably provides an oligonucleotide according to the invention which comprises at least one modified or unmodified ribose, at least one modified or unmodified phosphodiester bond and/or at least one modified or unmodified base.
- The present invention preferably provides an oligonucleotide according to the invention, in which at least one of the nucleotides, in particular two or more of the nucleotides, are “locked nucleic acids” (LNAs) or at least one of the nucleotides, in particular all of the nucleotides, are phosphorothioates, preferably one in which two or more of the nucleotides are “locked nucleic acids (LNAs). “Locked nucleic acids” (LNAs) are ribonucleotides which contain a methylene bridge which joins the 2′ oxygen of the ribose with the 4′ carbon (see
FIG. 27 ). Braasch D. A. and Corey, D. R. (2001), Locked nucleic acids (LNA); fine-tuning the recognition of DNA and RNA. Chem. Biol. 8, 1-7, provide an overview of LNAs. This article is herein explicitly incorporated by reference in its entirety. LNAs are available commercially, for example, from the company Proligo, Boulder, Colo., USA. Phosphorothioates are also known to the person skilled in the art and may be ordered, for example, from MWG-Biotech AG, Ebersberg, Germany. - Preferred oligonucleotides are those in which the LNAs are located at the 5′ and 3′ end of the oligonucleotide, preferably in each case the final 2-5 nucleotides, in particular in each case the final 3 or 4 nucleotides, on the 3′ and 5′ end of the oligonucleotide are LNAs. Preferably, oligonucleotides of the invention have >6, in particular >8 contiguous nucleotides in the oligonucleotide that are not LNAs. Preferably, of the nucleotides shown in the sequence domain according to the particular, sequence (a) of the oligonucleotide according to one of
FIGS. 1 to 16 , at most one or none of the nucleotides is in each case an LNA. - In the case of oligonucleotides modified with LNAs or phosphorothioates, it is particularly preferred if the oligonucleotide is an oligonucleotide A according to the invention or an oligonucleotide B according to the invention, preferably an oligonucleotide B according to the invention (see above).
- In general, the present invention specifically provides nucleic acids, in particular oligonucleotides, in which two or more of the nucleotides are “locked nucleic acids” (LNAs), in which the LNAs are on the 5′ and 3′ end of the oligonucleotide, preferably in each case the final 2-5 nucleotides, in particular in each case the final 3 or 4 nucleotides, on the 3′ and 5′ end of the oligonucleotide are LNAs, and/or in which >6, in particular >8 contiguous nucleotides in the oligonucleotide are not LNAs. The embodiments previously explained with regard to the LNAs also apply to this subject matter of the invention.
- The present invention also preferably provides a polynucleotide construct containing at least one oligonucleotide according to the invention. Polynucleotide construct should here be taken to have a very wide meaning. It includes RNA and DNA and nucleotides from a length of at least 20 nucleotides. A “recombinant” polynucleotide construct should here be taken to be a general designation for any kind of DNA or RNA molecules which have been obtained by in vitro linkage of DNA or RNA molecules. Polynucleotide is taken to have the following meaning: the underlying nucleotide is a fundamental nucleic acid building block essentially consisting of nucleic base, pentose and phosphoric acid. This corresponds to a high molecular weight polynucleotide prepared from two or more nucleotides linked together by phosphoric acid/pentose esterification. However, the invention also includes modified polynucleotides which, while they retain the sequence of bases, have a modified backbone instead of phosphoric acid/pentose.
- The present invention also preferably provides a polynucleotide construct containing in two separate domains, two nucleotide subsequences Fragment I and Fragment III according to one of sequences (l)-(n) or the two nucleotide subsequences helix I and helix III according to one of sequences (o)-(q) in one of
FIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15, or nucleotide subsequences differing from these nucleotide subsequences in each case by at most one base. Further details relating to the division of the two domains may be found inFIG. 24 (helix I and helix III/ribozyme) and in Santoro et al. (1997) FIG. 2, p. 4264 (Fragment I and Fragment III/DNA enzyme), the entire content of the latter is herein explicitly incorporated by reference. - The present invention also preferably provides a polynucleotide construct which codes for at least one oligonucleotide according to the invention. This in particular comprises DNA to be read out or a vector containing DNA or RNA, the product of which is or may be an oligonucleotide according to the invention.
- The present invention also particularly preferably provides a polynucleotide construct according to the invention which comprises a ribozyme, a DNA enzyme, a vector, in particular an expression vector, or a peptide nucleic acid (PNA).
- The following definitions apply for the purposes of the present invention:
-
- Cloning vector: A general name for nucleic acid molecules acting as vectors or carriers for foreign genes or parts thereof during cloning.
- Expression vector: A name for specially constructed cloning vectors which, once inserted in a suitable host cell, permit the transcription and translation of the foreign gene cloned into the vector.
- PNA: Standard international abbreviation for peptide nucleic acids. Peptide-linked amino acids here form a chain, the amino acids bearing as side chain a base capable of hybridisation with DNA or RNA.
- Sequence: A succession of nucleotides or amino acids. For the purposes of the present invention, sequence is intended to mean nucleic acid sequence.
- Ribozyme: A name for a catalytically active ribonucleic acid (for example ligase, endonuclease, polymerase, exonuclease), see for example hammerhead ribozyme according to
FIG. 24 or 25 and the description of the Figures or see Vaish, N. K. et al. (1998), Nucl. Acid Res. 26, 5237-5242. - DNA enzyme: A name for a DNA molecule which exhibits catalytic activity (for example ligase, endonuclease, polymerase, exonuclease), see for example DNA enzyme 10-23 according to
FIG. 26 and the description of the Figure, or see Santoro and Joyce (1997) Proc. Natl.Acad. Sci. USA 94, 4262-4266. - Catalytic RNA/DNA: A general name for ribozymes or DNA enzymes (see above).
- The present invention also preferably provides a polynucleotide construct containing in two separate domains two nucleotide subsequences, as described above, wherein said construct comprises a ribozyme, preferably a “hammerhead” ribozyme, or a DNA enzyme, preferably a type 10-23 or 12-32 DNA enzyme. It is particularly preferred and selected if said construct comprises a DNA enzyme containing at least the nucleotide subsequences Fragment I and Fragment III according to one of sequences (l) to (n), preferably (n), in one of
FIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12 or 13; preferably 1, 3, 7 or 11, in particular 1 or 3; or preferably 4, 6, 8 or 12, in particular 4 or 12. - It is particularly preferred and selected if said polynucleotide construct comprises a ribozyme containing at least the nucleotide subsequences helix I and helix III according to one of sequences (o) to (q), preferably (o), in one of FIGS. 1, 3, 4, 5, 6, 8, 11 or 12; preferably 1, 3 or 11, in particular 11; or preferably 4, 6, 8 or 12, in particular 4 or 12.
- Specifically, a preferred embodiment may in particular be found in
FIGS. 24 and 25 .FIG. 24 is a general diagram of a “hammerhead” ribozyme after Vaish, N. K. et al. (1998), Nucl. Acid Res. 26, 5237-5242 with the “recognition arms” helix I and helix III, into which the helices I and III according to the invention according to sequences (o)-(q) in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15 are inserted, in order to obtain the hammerhead ribozymes according to the invention. The fragment helix I here replaces in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15 the desired nucleotides in helix I according toFIG. 24 in such a manner that the first nucleotide on the 3′ end of helix I in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15 replaces the first desired nucleotide “N” on the 3′ end of helix I ofFIG. 24 and the following desired nucleotides “N” in helix I,FIG. 24 towards the 5′ end are replaced by the nucleotides which are shown in one of sequences (o) to (q) of helix I in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15. The nucleotides “A” and “C” on the 5′ end of helix III in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15 in each case replace the nucleotides “A” and “C” in helix III inFIG. 24 , and the following desired nucleotides “N” in helix III,FIG. 24 are replaced in the 5′ end direction by the nucleotides which are shown in one of sequences (o)-(q), helix III, in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15.FIG. 25 shows a specific example. The hammerhead ribozyme V16 (7/7) is derived fromFIG. 24 andFIG. 11 . The designation ribozyme V16 (7/7) here means that the enzyme is oriented towards the GUC site of oligo V16 and contains 7 nucleotides in each of the “recognition arms” (helix I and helix III), in this case according to helix I and helix III of sequence (o) inFIG. 11 . The same applies to all ribozymes according to sequences (o to q) in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15. - Specifically, a preferred embodiment may in particular be found in
FIG. 26 .FIG. 26 is a general diagram of a type “10-23” DNA enzyme according to Santoro et al., 1997, FIG. 2, p. 4264. The upper strand marked with an arrow is the RNA strand to be cleaved, the arrow showing the cleavage site, while the lower strand is a representation of the DNA enzyme. With regard to the present application, in the upper strand the “Y”=“U” and the “R”=“G”, a “C” being located 3′-wards from “Y”. The cleavage site on the upper strand is thus a GUC site (see above). Correspondingly, “R” in the lower strand=“A”, a “G” correspondingly being located 5′-wards from “R” in the lower strand. This is followed 5′-wards by the further nucleotides from Fragment I according to sequences (l to n) in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15, i.e. 5 further nucleotides in sequence l, 6 further nucleotides in sequence m and 7 further nucleotides in sequence n. In Fragment III according toFIG. 25 , the second with the RNA base-paired fragment, the unpaired “A” on Fragment III is then directly followed from the 5′direction 3′-wards by the nucleotides from Fragment III according to sequences (l to n) in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15, i.e. 7 further nucleotides in sequence l, 8 further nucleotides in sequence m and 9 further nucleotides in sequence n. Fragment III and Fragment I are the “recognition arms” of the DNA enzyme (see Example 3 below). - The type “10-23” DNA enzyme for sequence n according to
FIG. 1 which is particularly preferred for the purposes of the invention would thus have the following sequence, wherein the underlined fragment would be base-paired with the RNA: -
(SEQ ID NO: 249) ATGTCATGA(=R)-GGCTAGCTACAACGA-GGTTAGGGG - This DNA enzyme was named V15 (9/9), wherein the name indicates that the enzyme is oriented towards the GUC site of oligo V15 and the “recognition arms” in each case contain 9 nucleotides (Fragment I and III), for example according to Fragment I and Fragment III of sequence (n) in
FIG. 1 . The same applies to all DNA enzymes according to subpoints (l to n) in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15. - The present invention also preferably provides a polynucleotide construct according to the invention, wherein it comprises at least one modified or unmodified ribose, at least one modified or unmodified phosphodiester bond and/or at least one modified or unmodified base.
- The present invention also preferably provides an oligonucleotide according to the invention or polynucleotide construct according to the invention, wherein it is bound on a support, in particular a protein, preferably tet-, transportin or ferritin, and/or is packaged in a liposome.
- The present invention also preferably provides a cell containing at least one oligonucleotide according to the invention and/or a polynucleotide construct according to the invention.
- The present invention also preferably provides a pharmaceutical preparation containing at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention, optionally together with a suitable auxiliary substance and/or additive. The pharmaceutical preparations according to the invention may be administered in the form of solutions for injection, drops or juice, as semi-solid dosage forms in the form of granules, tablets, pellets, patches, capsules, dressings or aerosols and, in addition to the at least one subject matter of the invention, they also optionally contain, depending upon the pharmaceutical presentation, excipients, fillers, solvents, diluents, colorants and/or binders. Selection of the auxiliary substances and the quantities thereof which are to be used depends upon whether the pharmaceutical preparation is to be administered orally, perorally, parenterally, intravenously, intraperitoneally, intradermally, intramuscularly, intranasally, buccally, rectally or topically, for example onto infections of the skin, mucous membranes or eyes. Preparations in the form of tablets, coated tablets, capsules, granules, drops, succi and syrups are suitable for oral administration, while solutions, suspensions, easily reconstitutible dried preparations and sprays are suitable for parenteral, topical and inhalatory administration. Subject matters of the invention in a depot in dissolved form or in a dressing, optionally with the addition of skin penetration promoters, are suitable percutaneous administration preparations. Orally or percutaneously administrable formulations may release the subject matters of the invention in delayed manner. The quantity of active substance to be administered to the patient varies as a function of patient weight, mode of administration, the indication and the severity of the condition. 2 to 500 mg/kg of at least one subject matter of the invention are conventionally administered. Especially if the pharmaceutical preparation is to be used for gene therapy, suitable auxiliary substances and additives which it is advisable to use are, for example, a physiological saline solution, stabilizers, proteinase inhibitors, DNAse inhibitors etc.
- The present invention also preferably provides a diagnostic aid containing at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention, optionally together with suitable additives.
- The following definitions apply:
-
- Pharmaceutical preparation: A substance as defined in
Article 1 §2 of the German Drug Law (Arzneimittelgesetz, AMG). In other words, substances or preparations made from substances which, by application on or in the human or animal body are intended - 1. to cure, alleviate, prevent or diagnose diseases, suffering, bodily injury or sickness symptoms,
- 2. to diagnose the nature, the state or the functions of the body or mental health conditions,
- 3. to replace active substances or body fluids produced in the human or animal body,
- 4. to ward off pathogens, parasites or substances alien to the body or to destroy them or to render them harmless or
- 5. to influence either the nature, the state or the functions of the body or mental health conditions.
- Diagnostic aid: A compound or method which may be used to diagnose a disease.
- Pharmaceutical preparation: A substance as defined in
- The present invention also preferably provides the use of at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention for the production of a pharmaceutical preparation for the treatment of pain, in particular chronic pain, tactile allodynia, thermally induced pain and/or inflammatory pain.
- The present invention also preferably provides the use of at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention for the production of a pharmaceutical preparation for the treatment of urinary incontinence; also of neurogenic bladder symptoms; pruritus, tumors, inflammation; in particular VR1 receptor-associated inflammation with symptoms such as asthma; together with any disease symptoms associated with VR1.
- The present invention also preferably provides the use of at least one oligonucleotide according to the invention, a polynucleotide construct according to the invention and/or a cell according to the invention for gene therapy, preferably in vivo or in vitro gene therapy. Gene therapy is taken to be a type of therapy in which, by the introduction of nucleic acids into cells, an effector gene product, usually a protein, but also an antisense oligodeoxynucleotide, is expressed. A fundamental distinction is drawn between in vivo and in vitro methods. In in vitro methods, cells are removed from the organism and transfected with vectors ex vivo before subsequently being introduced back into the same organism or a different one. In in vivo gene therapy, vectors, for example for combating tumors, are administered systemically (for example via the bloodstream) or directly into the tumor.
- The present invention also preferably provides a process for the identification of pain-modulating substances, characterized in that identification is made by quantification of the binding of at least one, preferably labelled, oligonucleotide according to the invention or at least one polynucleotide construct onto an RNA.
- The present invention also preferably provides a process for the identification of pain-modulating substances comprising the following process steps:
-
- (a) genetic engineering manipulation of at least one cell (test cell) with at least one oligonucleotide according to the invention and/or a polynucleotide construct according to the invention,
- (a′) parallel genetic engineering manipulation, of at least one identical cell (control cell), which manipulation is either
- not carried out,
- carried out in parallel with the oligonucleotide or polynucleotide construct, or
- carried out with a modified oligonucleotide or polynucleotide construct which is other than one according to the invention,
- (b) parallel incubation of a substance to be tested under suitable conditions with at least one test cell and at least one control cell and/or a preparation made from such a cell, which has synthesized at least one receptor protein selected from the vanilloid receptor family, preferably the VR-1 receptor,
- (c) measurement of the binding of the test substance onto the protein synthesized by the cells or measurement of at least one functional parameter modified by binding of the test substance onto the receptor protein,
- (d) identification of the substances by the extent of the difference between the measured value for the test cell and that for the control cell.
- The term pain-modulating here refers to a potentially regulating influence upon physiological pain phenomena, in particular to an analgesic action. The term substance comprises any compound suitable as a pharmaceutical active substance, thus in particular low molecular weight active substances, but also others such as nucleic acids, fats, sugars, peptides or proteins such as antibodies. Incubation under suitable conditions should here be taken to mean that the substance to be investigated is capable of reacting in an aqueous medium for a defined period prior to measurement with the cell or the corresponding preparation. The aqueous medium may be temperature-controlled, for example between 4° C. and 40° C., preferably at room temperature or at 37° C. The incubation time may be varied between a few seconds and several hours, depending upon how the substance interacts with the receptor. Times of between 1 min and 60 min are, however, preferred. The aqueous medium may contain suitable salts and/or buffer systems, such that a pH of between 6 and 8, preferably pH 7.0-7.5 prevails in the medium during incubation. Suitable substances, such as coenzymes, nutrients etc., may additionally be added to the medium. Suitable conditions may readily be established by the person skilled in the art as a function of the substance-receptor interaction which is to be investigated on the basis of his/her experience, the literature or a few simple preliminary tests, so that the clearest possible measured value is obtained from the process. A cell which has synthesized a receptor is a cell which has already endogenously expressed this receptor or one which has been modified by genetic engineering in such a manner that it expresses this receptor and consequently contains the receptor prior to the beginning of the process according to the invention. The cells may be cells from possibly immortalized cell lines or be native cells originating and isolated from tissues, wherein cell aggregation has usually been broken down. The preparation made from these cells in particular comprises cell homogenates, the cytosol, a membrane fraction of the cells with membrane fragments, a suspension of isolated cell organelles etc.
- The yardstick which permits the identification of interesting substances is either binding to the receptor, which may, for example, be detected by displacement of a known ligand or by the extent of substance binding, or the modification of a functional parameter due to the interaction of the substance with the receptor. This interaction may in particular involve regulation, inhibition and/or activation of receptors, ion channels and/or enzymes, while modified functional parameters may be, for example, gene expression, ion concentration, pH or membrane potential or modification of enzyme activity or the concentration of secondary messengers. The following definitions apply:
-
- subjected to genetic engineering manipulation: manipulation of cells, tissues or organisms in such a manner that genetic material is introduced or altered therein
- endogenously expressed: expression of a protein exhibited by a cell line under suitable culture conditions, without expression of this corresponding protein having been effected by genetic engineering manipulation.
- Another preferred embodiment of the present process provides that the cell has already been subjected to genetic engineering manipulation before process steps (a) and (a′).
- Another preferred embodiment of the present process provides that the genetic engineering manipulation permits the measurement of at least one of the functional parameters modified by the test substance.
- Another preferred embodiment of the present process provides that the genetic engineering manipulation results in the expression of a form, not endogenously expressed in the cell, of a member of the vanilloid receptor family, preferably the VR-1 receptor, or in the introduction of a reporter gene.
- Another preferred embodiment of the present process provides that binding is measured by the displacement of a known, labelled ligand of a member of the vanilloid receptor family, preferably the VR-1 receptor.
- Another preferred embodiment of the present process provides that there is an elapse of not less than 8 h, preferably not less than 12 h, in particular not less than 24 h between the parallel process steps (a) and (a′), and process step (b).
- The present invention also preferably provides a process for the diagnosis of clinical conditions which are associated with modified expression of genes of the vanilloid receptor family, which process is characterized in that the diagnosis is made by means of quantification of the binding of an oligonucleotide according to the invention and/or at least one polynucleotide construct onto an RNA.
- The oligonucleotides and also the polynucleotide constructs are produced by processes known to the person ordinarily skilled in the art. Nucleotides, in particular also oligonucleotides, are synthesized in the manner of the Merrifield synthesis on an insoluble support (H. G. Gassen et al., Chemical and Enzymatic Synthesis of Gene Fragments (Verlag Chemie, Weinheim 1982)) or in another manner (Beyer/Walter; Lehrbuch der Organischen Chemie, 20th edition, (S. Hirzel Verlag, Stuttgart 1984), pp. 816 et seq.).
- The present invention also provides a process for the treatment, in particular treatment for pain, of a non-human mammal or human requiring treatment for pain, in particular chronic pain, by administration of a pharmaceutical preparation according to the invention, in particular such a preparation containing an oligonucleotide according to the invention and/or a polynucleotide construct according to the invention. The invention also provides corresponding processes for the treatment of pruritus and/or urinary incontinence.
- The following Examples and Figures are intended to illustrate the invention, but without restricting the subject matter of the invention thereto.
- Figures, Figure and Figure should be regarded as synonymous. In relation to the Figures, the terms subtype and sequence are likewise synonymous. An “X” in the sequences shown denotes any desired complementary nucleotide to the corresponding base on the mRNA of VR1.
-
FIG. 1 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V15, oligonucleotide no. 15 or V15. Sequences or subtypes (a)-(j) show truncated fragments from this antisense oligodeoxynucleotide sequence. Sequence (k) shows the full-length antisense oligodeoxynucleotide sequence with which the “messenger walk screening” was carried out. Sequences (l)-(n) each shows two nucleotide subsequences (Fragment I and Fragment III) starting from the full-length antisense oligodeoxynucleotide sequence, which each comprises non-overlapping subdomains of this sequence or corresponding sequence thereto, is usually divided at or in the GAC region and, in certain polynucleotide constructs, in particular DNA enzymes, and occurs in two separate domains, the “recognition arms.” Subtypes (o)-(q) each shows two nucleotide subsequences (helix I and helix III) starting from the full-length antisense oligodeoxynucleotide sequence (in this case as RNA), which each comprises non-overlapping subdomains of this sequence or a corresponding sequence thereto, is usually divided at or in the GAC region and, in certain polynucleotide constructs, in particular ribozymes, and occurs in two separate domains, the “recognition arms.” -
FIG. 2 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V15 inFIG. 1 . With regard to type and general content, subtypes or sequences (a)-(k) correspond to that already described in relation toFIG. 1 . -
FIG. 3 shows a list of general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V30, oligonucleotide no. 30 or V30. The type and general content of the subtypes correspond to that already described in relation toFIG. 1 . -
FIG. 4 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V30 inFIG. 3 . With regard to type and general content, subtypes (a)-(q) correspond to that already described in relation toFIG. 1 . -
FIG. 5 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V32, oligonucleotide no. 32 or V32. The type and general content of the subtypes correspond to that already described in relation toFIG. 1 . -
FIG. 6 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V32 inFIG. 5 . With regard to type and general content, subtypes (a)-(q) correspond to that already described in relation toFIG. 1 . -
FIG. 7 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V26, oligonucleotide no. 26 or V26. The type and general content of the subtypes correspond to that already described in relation toFIG. 1 . -
FIG. 8 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V26 inFIG. 7 . With regard to type and general content, subtypes (a)-(q) correspond to that already described in relation toFIG. 1 . -
FIG. 9 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V2, oligonucleotide no. 2 or V2. The type and general content of the subtypes correspond to that already described in relation toFIG. 1 . -
FIG. 10 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V2 inFIG. 9 . With regard to type and general content, subtypes (a)-(k) correspond to that already described in relation toFIG. 1 . -
FIG. 11 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V16, oligonucleotide no. 16 or V16. The type and general content of the subtypes correspond to that already described in relation toFIG. 1 . -
FIG. 12 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V16 inFIG. 11 . With regard to type and general content, subtypes (a)-(q) correspond to that already described in relation toFIG. 1 . -
FIG. 13 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V28, oligonucleotide no. 28 or V28. The type and general content of the subtypes correspond to that already described in relation toFIG. 1 . -
FIG. 14 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V28 inFIG. 13 . With regard to type and general content, subtypes (a)-(k) correspond to that already described in relation toFIG. 1 . -
FIG. 15 lists general nucleotide sequences starting from an antisense oligodeoxynucleotide sequence against VR1 mRNA from the rat, here frequently designated oligo V4, oligonucleotide no. 4 or V4. The type and general content of the subtypes correspond to that already described in relation toFIG. 1 . -
FIG. 16 shows the sequence of an antisense deoxyoligonucleotide against human VR1 corresponding in position on the mRNA to oligo V4 inFIG. 15 . With regard to type and general content, subtypes (a)-(k) correspond to that already described in relation toFIG. 1 . -
FIG. 17 shows the result of messenger walk screening. In each track there can be seen, in addition to the upper band of the uncut substrate, the two product bands of the cut mRNA together with several non-specific bands. The figure shows the VR1 mRNA after degradation by RNase H in the presence in each case of one of 33 antisense oligonucleotides (oligo V1 to oligo V33). In each track there can be seen, in addition to the upper band of the uncut substrate, the two product bands of the cut mRNA together with several non-specific bands. Track 1: VR1 mRNA, tracks 2-34: RNase H assay with antisense oligodeoxynucleotides against the 33 GUC sites of VR1 mRNA (oligo V1 to oligo V33). -
FIG. 18 shows quantitative evaluation of messenger walk screening.FIG. 18 is a plot of the percentage of the uncut mRNA after the RNase H assay with the individual ODNs. Each value is the mean of at least two experiments, such that the standard deviation does not ever exceed 10%. The antisense oligodeoxynucleotides against the 15th and 30th GUC sites bind most efficiently to the VR1 mRNA, such that the latter is respectively 88±4 and 97±1% degraded by the RNase H (oligo V15 and oligo V30). -
FIG. 19 is an image of a gel after RNase H assay with oligonucleotides V15, V15ctrl. (mismatch), V30 and V30ctrl. (mismatch). The Figure shows VR1 mRNA (track 1) and RNase H assay with oligodeoxynucleotides V15, V15ctrl., V30 and V30ctrl. (tracks 2-5). -
FIG. 20 shows the quantitative evaluation of cleavage of mRNA by ribozymes and DNA enzymes under “single turnover” conditions. -
FIG. 21 shows the kinetics of VR1 mRNA cleavage by ribozymes and DNA enzymes under “single turnover” conditions. -
FIG. 22 shows the kinetics of VR1 mRNA cleavage by ribozymes and DNA enzymes under “multiple turnover” conditions. -
FIG. 23 shows an estimation of tactile allodynia during treatment by VR1-antisense (AS) oligodeoxynucleotides and mismatch (MS) oligodeoxynucleotides (V15 and V15ctrl.). -
FIG. 24 is a general diagram of a “hammerhead” ribozyme with the “recognition arms” helix I and helix III, into which the helices I and III according to subtypes (o)-(q) in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15 are inserted, in order to obtain the inventive hammerhead ribozymes (see description relating toFIG. 1 ). The fragment helix I here replaces in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15 the desired nucleotides in helix I according toFIG. 24 in such a manner that the first nucleotide on the 3′ end of helix I in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15 replaces the first desired nucleotide “N” on the 3′ end of helix I ofFIG. 24 and the following desired nucleotides “N” in helix I,FIG. 24 towards the 5′ end are replaced by the nucleotides which are shown in one of sequences (o) to (q) of helix I in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15. The nucleotides “A” and “C” on the 5′ end of helix III in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15 in each case replace the nucleotides “A” and “C” in helix III inFIG. 24 and the following desired nucleotides “N” in helix III,FIG. 24 are replaced in the 5′ end direction by the nucleotides which are shown in one of sequences (o)-(q), helix III, in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15. -
FIG. 25 shows a specific example of the preparation of the (particularly preferred) “hammerhead” ribozyme V16 (7/7) according toFIG. 24 andFIG. 11 . The designation ribozyme V16 (7/7) here means that the enzyme is oriented towards the GUC site of oligo V16 and contains 7 nucleotides in each of the “recognition arms” (helix I and helix III), in this case according to helix I and helix III of sequence (o) inFIG. 11 . The same applies to all ribozymes according to sequences (o to q) in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15. -
FIG. 26 is a diagram of a type “10-23” DNA enzyme according to Santoro et al., 1997, FIG. 2, p. 4264; the upper strand marked with an arrow is the RNA strand to be cleaved, the arrow showing the cleavage site, while the lower strand is a representation of the DNA enzyme. With regard to the present application, in the upper strand the “Y”=“U” and the “R”=“G”, a “C” being located 3′-wards from “Y”. The cleavage site on the upper strand is thus a GUC site (see above). Correspondingly, “R” in the lower strand=“A”, a “G” correspondingly being located 5′-wards from “R” in the lower strand. This is followed 5′-wards by the further nucleotides from Fragment I according to sequences (l) to (n) in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15, i.e. 5 further nucleotides in sequence l, 6 further nucleotides in sequence m and 7 further nucleotides in sequence n. In Fragment III according toFIG. 25 , the second with the RNA base-paired fragment, the unpaired “A” on Fragment III is then directly followed from the 5′direction 3′-wards by the nucleotides from Fragment III according to sequences (l) to (n) in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15, i.e. 7 further nucleotides in sequence l, 8 further nucleotides in sequence m and 9 further nucleotides in sequence n. Fragment III and Fragment I are the “recognition arms” of the DNA enzyme (see Example 3). The type “10-23” DNA enzyme for sequence (n) according toFIG. 1 would thus have the following sequence, wherein the underlined fragment would be base-paired with the RNA: -
(SEQ ID NO: 249) ATGTCATGA(=R)-GGCTAGCTACAACGA-GGTTAGGGG - This DNA enzyme would be named V15 (9/9), wherein the name indicates that the enzyme is oriented towards the GUC site of oligo V15 and the “recognition arms” in each case contain 9 nucleotides (Fragment I and III), for example according to Fragment I and Fragment III of sequence (n) in
FIG. 1 . The same applies to all DNA enzymes according to sequences (l) to (n) in each case in one ofFIG. 1 , 3, 4, 5, 6, 7, 8, 9, 11, 12, 13 or 15. -
FIG. 27 is a schematic diagram of a “locked nucleic acid” (LNA). - The first step in the antisense and ribozyme strategy is to identify accessible sites on the mRNA for binding oligonucleotides, in particular ribozymes. To this end, the VR1 mRNA had to be investigated for such restriction sites. Analysis of the VR1 mRNA revealed the following potential recognition sites for ribozymes in the coding domain:
- 33×GT(U)C sequences,
- 28×GT(U)T sequences, and
- 12×GT(U)A sequences.
- In order to determine the accessible sites on the VR1 mRNA, in a first step, three independent nucleotide mixtures with the following sequence were synthesized:
-
- Mixture 1: NNNAACNNN “GUU library,”
- Mixture 2: NNNCACNNN “GUA library,” and
- Mixture 3: NNNGACNNN “GUC library.”
- These were used consecutively in an RNase H experiment and it was found that appreciable degradation of the VR1 mRNA was observed only with the GUC library. Thus, of the potential target sequences for ribozymes, the 33 GUC sites in the VR1 mRNA are the most readily accessible and they were used for further analyses.
- Messenger Walk Screening
- In order to identify mRNA domains which are accessible to antisense oligodeoxynucleotides, the mRNA was systematically screened with ODNs in an RNase H assay (messenger walk screening). The ODNs were 18 nucleotides in length and contained a central GAC sequence, which is reverse complementary to GUC sequences in the mRNA. This triplet was selected as the target as it provided good results and may be used in a second step to develop hammerhead ribozymes and DNA enzymes. In total, 33 ODNs, designated V1 to V33, were tested against all the GUC sites of the VR1 mRNA. The ODNs were systematically screened for their suitability by the addition in each case of one ODN and RNase H to the mRNA. RNase H cuts formed DNA/RNA duplexes wherever an oligonucleotide can bind to the mRNA (
FIG. 17 ). - In Vitro Transcription of VR1 mRNA
- First of all, the cDNA of the vanilloid receptor was cloned into the vector pcDNA3.1 (+) from Invitrogen. Then, in vitro transcription of the mRNA was performed using the RiboMAX Large Scale RNA Production System—T7 from Promega in accordance with the manufacturer's instructions.
- RNase H Assay
- An RNase H assay was performed to test whether an antisense oligodeoxynucleotide had bound to the mRNA. To this end, the VR1 mRNA (100 mM) was incubated with a five-fold excess of the ODNs in a total volume of 10 μl in 40 mM tris/HCl pH 7.2, 4 mM MgCl2, 1 mM DTT and 150 mM NaCl for 7.5 minutes at 37° C. in the presence of 0.4 unit of RNase H (from Promega). The reactions were terminated by addition of EDTA (65 mM final concentration). The samples were separated on a 1.5% agarose gel and stained with ethidium bromide (1 μg/ml) for 20 minutes. The gels were photographed with the Gel Doc 2000 Gel Documentation System from Biorad and evaluated with Quantity One software.
-
FIG. 17 shows the result of messenger walk screening. In each track there can be seen, in addition to the upper band of the uncut substrate, the two product bands of the cut mRNA together with several non-specific bands. - Quantification of RNA degradation revealed that the most effective antisense oligonucleotide (oligo no. 30 (V30)) was able specifically to cut/cleave more than 90% of the VR1 mRNA when used in a five-fold excess relative to the target mRNA (
FIG. 18 ). - The intensities of the individual bands were evaluated.
FIG. 18 is a plot of the percentage of the uncut mRNA for the individual ODNs after the RNase H assay. The antisense oligodeoxynucleotides against the 15th and 30th GUC sites (oligo V15 and oligo V30) bind most efficiently to the VR1 mRNA, such that the latter is respectively 88±4 and 97±1% degraded by the RNase H. - The sequences of the antisense oligodeoxynucleotides most effective in this test are shown in
FIGS. 1 , 3, 5, 7, 9, 11, 13 and 15, wherein inter alia preferential attention was paid to those designated oligo V15 (FIG. 1 ) and oligo V30 (FIG. 3 ). - Oligodeoxynucleotides in which every fifth and six base (or in the event that these are identical, two adjacent bases) are swapped were synthesized and used in the following experiments as mismatch controls for these antisense oligodeoxynucleotides. The sequences of the control (mismatch) oligodeoxynucleotides are:
-
Oligo V15ctrl.: CAT GCT ATG AGC GTT GAG (SEQ ID NO: 250) Oligo V30ctrl.: ATC TGT TTG AGC GTC TAC (SEQ ID NO: 251) - The RNase H assay was performed with the oligonucleotides V15, V15ctrl., V30 and V30ctrl. for control purposes (see
FIG. 19 ). As anticipated, the RNA is degraded only with the antisense ODNs, but not with the mismatch controls, as the latter do not bind to the mRNA. - By using the RNase H assay with antisense oligodeoxynucleotides against all the GUC triplets of the VR1 mRNA, it was possible to identify oligodeoxynucleotides against the GUC triplets (2, 4, 15, 16, 26, 28, 30 and 32), in particular the 15th and 30th which were the ODNs with the best binding properties. These oligodeoxynucleotides and the two mismatch controls are thus available for testing in an animal model and for other uses.
- The human sequences according to
FIGS. 2 , 4, 6, 8, 10, 12, 14, 16, which correspond in position to those found in the rat sequences, are of particular interest. Rat and human mRNAs are highly homologous (probably also in terms of folding) and it is thus clear that restriction sites identified as readily accessible on rat mRNA are also of interest for humans. Particularly preferred sequences here are those which also exhibit the GUC triplet,FIGS. 4 , 6, 8 and 12, and, due to the location similar to that in rats, also those corresponding to V15 and V30 according toFIGS. 2 and 4 . - On the basis of the identified most effective binding sites, corresponding ribozymes and DNA enzymes were also investigated.
- “Hammerhead” ribozymes and type “10-23” DNA enzymes (Santoro et al., 1997) were constructed against the mRNA sites which were accessible to ODNs. The length of the “recognition arms” was 7 or 9 nucleotides on each side. Quantitative evaluation of mRNA cleavage/restriction under “single turnover” conditions (10-fold excess of ribozymes and DNA enzymes) revealed after 20 minutes at 37° C. that ribozymes with shorter “recognition arms” (helix I and helix III) are more active, while in turn in DNA enzymes, those with longer “arms” (Fragment I and Fragment III) are more active (
FIG. 20 ).FIG. 24 is a schematic representation of a ribozyme with the helices I and III, whileFIG. 25 shows a specific example. This may be seen for a 10-23 DNA enzyme (5′-end (Fragment I) and 3′-end (Fragment III) from the catalytic motif) in Santoro et al. (1997, p. 4264, FIG. 2) (see alsoFIG. 26 ) with the description of the Figures). - Experiments with ribozymes and DNA enzymes were carried out in 50 mM tris/HCl, pH 7.5 and 10 mM MgCl2 at 37° C. In the “single turnover” experiments, the ribozymes and DNA enzymes were used in a 10-fold excess. In “multiple turnover” experiments, the substrate mRNA was used in a 10-fold excess.
- “Single Turnover” Kinetics
- A kinetic analysis under “single turnover” conditions was carried out for the two most effective ribozymes and DNA enzymes (
FIG. 21 ). The data are shown in Table 1. DNA enzyme V15 (9/9) (see description ofFIG. 26 ), which cuts the mRNA with biphasic kinetics, has the highest rate (rate constant), followed by ribozyme V16 (7/7) (seeFIG. 25 ), DNA enzyme V30 (9/9) and the slowest ribozyme V15 (7/7). The designation ribozyme V15 (7/7), for example, here means that the enzyme is oriented towards the GUC site of oligo V15 and contains 7 nucleotides in each of the “recognition arms” (helix I and helix III), for example according to Fragment I and Fragment III of subtype (l) inFIG. 1 . -
TABLE 1 Kinetics data for ribozymes and DNA enzymes against VR1 mRNA under “single turnover” conditions. k1 k2 A1 [min−1] A2 [min−1] A∞ DNAzyme V15 (9/9) 0.43 ± 0.05 2.3 ± 0.5 0.34 ± 0.04 0.07 ± 0.01 0.21 ± 0.03 DNAzyme V30 (9/9) 0.90 ± 0.02 0.042 ± 0.002 — — 0.08 ± 0.02 Ribo V15 (7/7) 0.51 ± 0.09 0.023 ± 0.007 — — 0.49 ± 0.09 Ribo V16 (7/7) 0.58 ± 0.02 0.077 ± 0.008 — — 0.34 ± 0.03 - “Multiple Turnover” Kinetics
-
FIG. 22 shows the cleavage of the mRNA under “multiple turnover” conditions (10-fold substrate excess). Again DNA enzyme V15 (9/9) has a higher apparent rate (rate constant) (by a factor of 3.4) than ribozyme V16 (7/7) (Table 2). -
TABLE 2 Kinetics data for ribozymes and DNA enzymes against VR1 mRNA under “multiple turnover” conditions k [min−1] DNAzyme V15 (9/9) (6.5 ± 0.6) * 10−3 Ribo V16 (7/7) (1.9 ± 0.2) * 10−3 - Spinal nerve ligatures were placed as described by Kim & Chung (1992) on the left L5/L6 spinal nerves of 20 male Sprague-Dawley rats. At the same time, spinal catheters were implanted as described by Pogatzki et al. (2000). Four to six days after the operation, the tactile threshold baseline (withdrawal thresholds) was measured on the ipsilateral and contralateral hind paw using an electronic von Frey anaesthesiometer (IITC Life Science, USA). Correct positioning of the spinal catheter was confirmed by administering lidocaine (10 μl, 2%), which resulted in transient paralysis of both hind limbs. After the test and measurement of the baseline, 45 μg of VR-1 antisense oligonucleotides (AS, n=10) or mismatch oligonucleotides (MS, n=10) in 0.9% NaCl were in each case given once on the first day and b.i.d. on the following 4 days. The tactile withdrawal thresholds were measured 30 minutes after the first daily administration of oligo. The results are stated as a percentage of the maximum possible effect (% MPE) on the ipsilateral side, the base line being taken as 0% and the withdrawal threshold of a control group as 100% MPE. The antisense oligo used was V15 (
FIG. 1 , subtype k), while the mismatch oligo was V15ctrl., as already described above. - Treating mononeuropathic rats with antisense, but not with mismatch, oligodeoxynucleotides brought about a reduction in tactile allodynia beginning on the third day of treatment, with a plateau being reached on
days - Various different oligonucleotides according to the invention were produced, in particular according to subgroup (k) of
FIG. 1 and, in some cases, alsoFIG. 3 . Most of these were LNA constructs which were obtained from PROLIGO, in Boulder, Colo., and in which LNAs were located at various sites (see Table 3). An unmodified oligonucleotide according to subgroup (k) ofFIG. 1 was also synthesized and a phosphorothioate corresponding in terms of base sequence was obtained from MWG Biotech AG in Ebersberg, Germany. -
TABLE 3 List of oligonucleotides used % mRNA % LNA DNA Name Sequence cleavage content gap DNA 1 catgtcatgacggttagg 90 ± 1 0 — PS CATGTCATGACGGTTAGG 71 ± 7 0 — mixed LNA 1 catgTcaTgacggTtagg* 12 ± 2 16 5 LNA 2 CatgTcaTgacggTtagg 11 ± 9 22 5 LNA 3 catgTcaTgacggTTagg 4 ± 1 22 5 LNA 4 catgTcaTgaCggTtagG 5 ± 9 27 2 LNA 5 CaTgTcaTgaCggTTagG 2 ± 3 44 2 LNA 6 catgTcatGacggTtagg 12 ± 2 16 4 LNA 7 CatgTcatGacggTtagg 8 ± 4 22 4 LNA 8 catgTcatGacggTtagG 9 ± 1 22 4 LNA 9 CatgTcatGacggTtagG 7 ± 2 27 4 LNA 10 CatgTcaTgaCggTtagG 7 ± 1 33 2 LNA 11 CaTgTcatgacggTTagG 81 ± 5 33 8 gaps LNA 12 CatgTcaTgacggTtagG 6 ± 4 27 5 LNA 13 CatgTcAtgacggTtagG 49 ± 12 27 6 LNA 14 CatgTCatgacggTtagG 83 ± 6 27 7 LNA 15 CatgTcatgacggTtagG 91 ± 2 22 8 end block oligonucleotides LNA 16 CATGTcatgacggTTAGG 85 ± 6 55 8 LNA 17 CATGtcatgacggtTAGG 94 ± 3 44 10 LNA 18 CATgtcatgacggttAGG 93 ± 2 33 12 LNA 19 CatgtcatgacggttaGG 87 ± 12 22 14 LNA 20 CatgtcatgacggttagG 85 ± 6 11 16 control sequences DNA 2 atcttgttgacggtctca 97 ± 2 0 — LNA 21 AtctTgttGacggTctcA 0 ± 0 27 4 LNA 22 AtctTgttgacggTctcA 95 ± 3 22 8 LNA 23 ATCTTgttgacggTCTCA 97 ± 1 55 8 *Note: Lower case = DNA monomers; italic and underlined = phosphorothioates; bold characters = LNA monomers. - Various tests were carried out with these oligonucleotides:
- a) Firstly, the percentage of RNA cleavage of VR1 by RNase H initiated by the oligonucleotide was investigated, the test conditions substantially matching those stated in Example 2.
- The results are shown in Table 3. It was found that oligonucleotides with LNA only exhibited cleavage comparable with the native oligonucleotide if at least 6, or especially at least 8 contiguous nucleotides were not LNAs.
- b) The melting temperature of the LNA/RNA:DNA hybrids were then measured by standard methods (Table 4). Surprisingly, the LNAs did not exhibit a raised melting temperature in comparison with the native oligonucleotides and the phosphorothioates. This is very favorable for stability.
-
TABLE 4 Melting temperature Tm of LNA/DNA:RNA hybrids Tm Oligonucleotide No. of LNAs ° C. DNA 1 0 58 PS 0 49 LNA 202 61 LNA 194 66 LNA 186 73 LNA 173 79 LNA 1610 ~85 - c) The kinetics of RNase H cleavage were then investigated under identical conditions to those above, except that equimolar quantities of RNA and antisense oligonucleotide (100 nM each) were used. The results are shown in Table 5. Surprisingly, the oligonucleotide with LNA exhibited a distinct increase in activity in comparison with the native oligonucleotides and phosphorothioates.
-
TABLE 5 Rate constants for RNase H cleavage of “full-length” VR1 mRNA by antisense oligonucleotides. Oligonucleotide k [min−1] DNA 10.17 ± 0.01 LNA 171.1 ± 0.6 PS 0.07 + 0.01 - d) Finally, the half-life of the radioactively labelled oligonucleotides with LNA of the native oligonucleotide and of the phosphorothioate was determined at 37° C. in human serum over a period of up to 2 days. The results are shown in Table 6. While the half-life of the native DNA is 1.5 h and that of the phosphorothioate is 10 h, the half-life of nucleotides with LNA, on the 3 or 4 ends, were significantly long with a t1/2 of approximately 17 h.
-
TABLE 6 Half-life of native, phosphorothioate and end block LNA/DNA oligonucleotides in human serum. Oligonucleotide nt end block t1/2 [h] DNA 10 1.5 ± 0.3 PS 0 10 ± 2 LNA 201 4 ± 2 LNA 192 6 ± #? # LNA 18 3 17 + 2 LNA 174 15 ± 1 LNA 165 15 + 2 - The best restriction properties were thus achieved by the nucleotides with LNAs which had approximately 8 contiguous nucleotides without LNAs and had 3 or 4 LNAs on the 3′ and 5′ ends.
- The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof.
-
- Caterina, M. J.; Schumacher, M. A.; Tominaga, T. A.; Rosen, T. A.; Levine, J. D.; Julius, D. (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816-824.
- Caterina, M. J.; Leffler, A.; Malmberg, A. B.; Martin, W. J.; Trafton, J.; Petersen-Zeitz, K. R.; Koltzenburg, M; Basbaum, A. L; Julius, D. (2000) Impaired nociception and pain Sensation in mice lacking the capsaicin receptor. Science 288, 306-313.
- Davis, J. B.; Gray, J.; Gunthorpe, M. J.; Hatcher, J. P.; Davey, P. T.; Overend, P.; Harries, M. H.; Latcham, J.; Clapham, C.; Atkinson, K.; Hughes, S. A.; Rance, K.; Grau, E.; Harper, A. J.; Pugh, P. L.; Rogers, D. C.; Bingham, S.; Randall, A.; Sheardown, S. A. (2000) Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 405, 183-187.
- Kim, S. H.; Chung, J. M. (1992) An experimental model for peripheral mononeuropathy produced by segmental spinal nerve ligation in the rat.
Pain 50, 355-363. - Pogatzki, E. M.; Zahn, P. K.; Brennan, T. J. (2000) Lumbar catheterization of the subarachnoid space with a 32-gauge polyurethane catheter in the rat.
Eur. J. Pain 4, 111-113. - Porreca, F.; Lai, J.; Bian, D.; Wegert, S.; Ossipov, M. H.; Eglen, R. M.; Kassotakis, L.; Novakovic, S.; Rabert, D. K.; Sangameswaran, L.; Hunter, J. C. (1999) A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain. Proc. Natl. Acad. Sei.
USA 96, 7640-7644. - Santoro, S. W.; Joyce, G. F. (1997) A general purpose RNA-cleaving DNA enzyme. Proc. Natl.
Acad. Sci. USA 94, 4262-4266. - Vaish, N. K. et al. (1998), Nucl. Acid Res. 26, 5237-5242.
Claims (31)
1. An oligonucleotide comprising the sequence according to one of SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239.
2. An oligonucleotide according to claim 1 , comprising
(1) a base sequence according to SEQ ID NOs 28, 56, 73, 90, 107, 124, 141, 158, 169, 187, 204, 221, 232, or 249, or
(2) a sequence differing from (1) by at most two different bases not located in the sequence according to SEQ ID NO 18, 46, 63, 80, 97, 114, 131, 148, 159, 177, 194, 211, 222, or 239, respectively.
3. An anti-sense oligonucleotide 15 to 30 nucleotides in length and comprising
(1) the sequence according to one of SEQ ID NOs 19-27, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, or
(2) a sequence differing from (1) by one different base not located in the sequence according to SEQ ID NO 18, 46, 63, 80, 97, 114, 131, 148, 159, 177, 194, 211, 222 or 239, respectively.
4. An anti-sense oligonucleotide according to claim 2 , wherein the oligonucleotide is SEQ ID NO 28, 56, 73, 90, 107, 124, 141, 158, 169, 187, 204, 221, 232, or 249.
5. An anti-sense oligonucleotide selected from the group consisting of SEQ ID NOs 12-17, SEQ ID NOs 18-28, SEQ ID NOs 29-45, SEQ ID NOs 46-62, SEQ ID NOs 131-147, SEQ ID NOs 148-158, SEQ ID NOs 159-176, SEQ ID NOs 177-193, SEQ ID NOs 222-238, and SEQ ID NOs 239-249.
6. An oligonucleotide according to claim 5 , wherein the oligonucleotide is selected from the group consisting of SEQ ID NOs 12-17, SEQ ID NOs 18-28, SEQ ID NOs 29-45, SEQ ID NOs 46-62, SEQ ID NOs 159-176 and SEQ ID NOs 177-193.
7. An oligonucleotide according to claim 5 , wherein the oligonucleotide is selected from the group consisting of SEQ ID NOs 12-17, SEQ ID NOs 29-45 and SEQ ID NOs 159-176.
8. An oligonucleotide according to claim 5 , wherein the oligonucleotide is selected from the group consisting of SEQ ID NOs 12-17 and SEQ ID NOs 29-45.
9. An oligonucleotide according to claim 1 , wherein at least one of the nucleotides is a “locked nucleic acid” (LNA), and wherein more than 6 contiguous nucleotides in the oligonucleotide are not LNA's, and wherein the more than 6 contiguous nucleotides that are not LNAs are in the sequence domain of SEQ ID NO 18, 29, 46, 63, 80, 97, 114, 131, 148, 159, 177, 194, 211, 222 or 239.
10. A polynucleotide construct comprising at least one oligonucleotide according to claim 1 .
11. A polynucleotide construct comprising, in two separate domains, Fragment I and Fragment III according to one of SEQ ID NOs 12-14, SEQ ID NOs 40-42, SEQ ID NOs 57-59, SEQ ID NOs 74-76, SEQ ID NOs 91-93, SEQ ID NOs 108-110, SEQ ID NOs 125-127, SEQ ID NOs 1452-144, SEQ ID NOs 170, 172 and 173, SEQ ID NOs 188-190, SEQ ID NOs 205-207, or SEQ ID NOs 233-235, or helix I and helix III according to one of SEQ ID NOs 15-17, SEQ ID NOs 43-45, SEQ ID NOs 60-62, SEQ ID NOs 77-79, SEQ ID NOs 94-96, SEQ ID NOs 11-113, SEQ ID NOs 128-130, SEQ ID NOs 145-147, SEQ ID NOs 174-176, SEQ ID NOs 191-193, SEQ ID NOs 208-210 or SEQ ID NOs 236-238.
12. A ribozyme comprising an oligonucleotide of claim 1 , comprising helix I and helix III according to one of SEQ ID NOs 15-17, SEQ ID NOs 43-45, SEQ ID NOs 60-62, SEQ ID NOs 77-79, SEQ ID NOs 94-96, SEQ ID NOs 128-130, SEQ ID NOs 174-176 or SEQ ID NOs 191-193.
13. A ribozyme according to claim 12 , comprising helix I and helix III according to SEQ ID NO 15, 43, 60, 77, 94, 128, 174 or 191.
14. A ribozyme according to claim 12 , comprising helix I and helix III according to one of SEQ ID NOs 15-17, SEQ ID NOs 43-45 or SEQ ID NOs 174-176.
15. A ribozyme according to claim 12 , comprising helix I and helix III according to one of SEQ ID NOs 60-62, SEQ ID NOs 94-96, SEQ ID NOs 128-130 or SEQ ID NOs 191-193.
16. A DNA enzyme comprising an oligonucleotide of claim 1 , comprising Fragment I and Fragment III according to one of SEQ ID NOs 12-14, SEQ ID NOs 40-42, SEQ ID NOs 57-59, SEQ ID NOs 74-76, SEQ ID NOs 91-93, SEQ ID NOs 108-110, SEQ ID NOs 125-127, SEQ ID NOs 1452-144, SEQ ID NOs 170, 172 and 173, SEQ ID NOs 188-190 or SEQ ID NOs 205-207.
17. A DNA enzyme according to claim 16 , comprising Fragment I and Fragment III according to one of SEQ ID NOs 12-14, SEQ ID NOs 40-42, SEQ ID NOs 108-110 or SEQ ID NOs 170, 172 and 173.
18. A DNA enzyme according to claim 16 , comprising Fragment I and Fragment III according to one of SEQ ID NOs 57-59, SEQ ID NOs 91-93, SEQ ID NOs 125-127 or SEQ ID NOs 188-190.
19. A DNA enzyme according to claim 16 , comprising Fragment I and Fragment III according to SEQ ID NO 14, 42, 59, 76, 93, 110, 127, 144, 173, 190 or 207.
20. A method for the treatment of pain, comprising administering an effective amount of a pharmaceutical composition comprising at least one oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239, and a pharmaceutically acceptable carrier, to a patient in need thereof.
21. A method according to claim 20 , for the treatment of chronic pain selected from the group consisting of tactile allodynia, thermally induced pain and inflammatory pain.
22. A method for the treatment of urinary incontinence, neurogenic bladder symptoms; pruritus; or tumors, comprising administering an effective amount of a pharmaceutical composition comprising at least one oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239, and a pharmaceutically acceptable carrier, to a patient in need thereof.
23. A method for the treatment of vanilloid receptor subtype 1 (VR1)-associated disease, comprising administering an effective amount of a pharmaceutical composition comprising at least one oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239, and a pharmaceutically acceptable carrier, to a patient in need thereof.
24. A method according to claim 23 , wherein the VR1 receptor-associated disease is inflammation.
25. A gene therapy method comprising administering to a patient in need thereof an effective amount of an oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239.
26. A gene therapy method according to claim 25 , which is an in vitro gene therapy.
27. A gene therapy method according to claim 25 , which is an in vivo gene therapy.
28. A gene therapy method according to claim 25 , wherein the oligonucleotide is comprised in an expression vector.
29. A gene therapy method according to claim 25 , wherein the expression vector is comprised in a cell.
30. A process for the identification of pain-modulating substances comprising the following process steps:
(a) providing a test cell which comprises at least one oligonucleotide comprising the sequence according to one of SEQ ID NOs 2-10, SEQ ID NOs 19-27, SEQ ID NOs 30-38, SEQ ID NOs 47-55, SEQ ID NOs 64-72, SEQ ID NOs 81-89, SEQ ID NOs 98-106, SEQ ID NOs 115-123, SEQ ID NOs 132-140, SEQ ID NOs 149-157, SEQ ID NOs 160-168, SEQ ID NOs 178-186, SEQ ID NOs 195-203, SEQ ID NOs 212-220, SEQ ID NOs 223-231 or SEQ ID NOs 240-248, wherein each of SEQ ID NOs 2-10 comprises a sequence domain of SEQ ID NO 1, each of SEQ ID NOs 19-27 comprises a sequence domain of SEQ ID NO 18, each of SEQ ID NOs 30-38 comprises a sequence domain of SEQ ID NO 29, each of SEQ ID NOs 47-55 comprises a sequence domain of SEQ ID NO 46, each of SEQ ID NOs 64-72 comprises a sequence domain of SEQ ID NO 63, each of SEQ ID NOs 81-89 comprises a sequence domain of SEQ ID NO 80, each of SEQ ID NOs 98-106 comprises a sequence domain of SEQ ID NO 97, each of SEQ ID NOs 115-123 comprises a sequence domain of SEQ ID NO 114, each of SEQ ID NOs 132-140 comprises a sequence domain of SEQ ID NO 131, each of SEQ ID NOs 149-157 comprises a sequence domain of SEQ ID NO 148, each of SEQ ID NOs 160-168 comprises a sequence domain of SEQ ID NO 159, each of SEQ ID NOs 178-186 comprises a sequence domain of SEQ ID NO 177, each of SEQ ID NOs 195-203 comprises a sequence domain of SEQ ID NO 194, each of SEQ ID NOs 212-220 comprises a sequence domain of SEQ ID NO 211, each of SEQ ID NOs 223-231 comprises a sequence domain of SEQ ID NO 222, or each of SEQ ID NOs 240-248 comprises a sequence domain of SEQ ID NO 239,
(b) providing a control cell that does not comprise said oligonucleotide,
(c) incubating a candidate substance under suitable conditions with the test cell, or a preparation made from the test cell, which has synthesized at least one vanilloid receptor,
(d) measuring the binding of the test substance to the receptor synthesized by the test cell, or measuring at least one functional parameter of the receptor modified by binding of the test substance onto the receptor,
(e) measuring the binding of the test substance to the receptor synthesized by the control cell, or measuring at least one functional parameter of the receptor modified by binding of the test substance onto the receptor,
(d) identifying a substance by a difference between the measured value for the test cell and that for the control cell.
31. A method according to claim 30 , wherein the vanilloid receptor is a VR-1 receptor.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9427415B2 (en) | 2014-04-01 | 2016-08-30 | The Procter & Gamble Company | Methods and compositions for modifying sensorial perception |
US11879215B2 (en) | 2016-10-25 | 2024-01-23 | The Procter & Gamble Company | Fibrous structures |
Families Citing this family (66)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030105044A1 (en) * | 2001-10-17 | 2003-06-05 | Isis Pharmaceuticals Inc. | Antisense modulation of matrix metalloproteinase 1 expression |
US7737264B2 (en) | 2002-04-05 | 2010-06-15 | Enzon Pharmaceuticals, Inc. | Oligomeric compounds for the modulation HIF-1α expression |
DE10322662A1 (en) * | 2002-11-06 | 2004-10-07 | Grünenthal GmbH | New DNA type 10-23 enzyme, useful for treating e.g. pain, and related short interfering RNA, directed against the vanillin receptor or picorna viruses, contains specific nucleotide modifications for improved stability |
DE10257421A1 (en) * | 2002-12-09 | 2004-07-08 | Grünenthal GmbH | Regulatory elements in the 5 'region of the VR1 gene |
US20040115634A1 (en) * | 2002-12-11 | 2004-06-17 | Isis Pharmaceuticals, Inc. | Modulation of stat 6 expression |
US7480382B2 (en) * | 2003-09-30 | 2009-01-20 | Microsoft Corporation | Image file container |
US9447138B2 (en) | 2004-11-09 | 2016-09-20 | Roche Innovation Center Copenhagen A/S | Potent LNA oligonucleotides for the inhibition of HIF-1a expression |
DE602005022768D1 (en) * | 2004-11-09 | 2010-09-16 | Santaris Pharma As | EFFECTIVE LNA OLIGONUCLEOTIDES FOR INHIBITING HIF-1A |
JP5713377B2 (en) | 2005-12-28 | 2015-05-07 | ザ スクリプス リサーチ インスティテュート | Natural antisense and non-coding RNA transcripts as drug targets |
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US8927511B2 (en) * | 2008-12-04 | 2015-01-06 | Curna, Inc. | Treatment of vascular endothelial growth factor (VEGF) related diseases by inhibition of natural antisense transcript to VEGF |
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WO2010148050A2 (en) | 2009-06-16 | 2010-12-23 | Curna, Inc. | Treatment of collagen gene related diseases by inhibition of natural antisense transcript to a collagen gene |
WO2010151671A2 (en) | 2009-06-24 | 2010-12-29 | Curna, Inc. | Treatment of tumor necrosis factor receptor 2 (tnfr2) related diseases by inhibition of natural antisense transcript to tnfr2 |
CA2765815A1 (en) | 2009-06-26 | 2010-12-29 | Opko Curna, Llc | Treatment of down syndrome gene related diseases by inhibition of natural antisense transcript to a down syndrome gene |
CA2768947C (en) | 2009-07-24 | 2018-06-19 | Opko Curna, Llc | Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to a sirtuin (sirt) |
US8635223B2 (en) * | 2009-07-28 | 2014-01-21 | Fti Consulting, Inc. | System and method for providing a classification suggestion for electronically stored information |
CA2769665A1 (en) | 2009-08-05 | 2011-02-10 | Opko Curna, Llc | Treatment of insulin gene (ins) related diseases by inhibition of natural antisense transcript to an insulin gene (ins) |
ES2599986T3 (en) | 2009-08-11 | 2017-02-06 | Curna, Inc. | Treatment of adiponectin-related diseases (ADIPOQ) by inhibiting a natural antisense transcript of an adiponectin (ADIPOQ) |
EP2982755B1 (en) | 2009-08-21 | 2020-10-07 | CuRNA, Inc. | Treatment of 'c terminus of hsp70-interacting protein' (chip) related diseases by inhibition of natural antisense transcript to chip |
KR101892760B1 (en) | 2009-08-25 | 2018-08-28 | 큐알엔에이, 인크. | Treatment of 'iq motif containing gtpase activating protein' (iqgap) related diseases by inhibition of natural antisense transcript to iqgap |
CA2775111C (en) | 2009-09-25 | 2019-12-31 | Opko Curna, Llc | Treatment of filaggrin (flg) related diseases by modulation of flg expression and activity |
NO2513310T3 (en) | 2009-12-16 | 2018-03-31 | ||
US9068183B2 (en) | 2009-12-23 | 2015-06-30 | Curna, Inc. | Treatment of uncoupling protein 2 (UCP2) related diseases by inhibition of natural antisense transcript to UCP2 |
CN102869776B (en) | 2009-12-23 | 2017-06-23 | 库尔纳公司 | HGF relevant diseases are treated by suppressing the natural antisense transcript of HGF (HGF) |
KR101853508B1 (en) | 2009-12-29 | 2018-06-20 | 큐알엔에이, 인크. | TREATMENT OF TUMOR PROTEIN 63 (p63) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO p63 |
CA2785173A1 (en) | 2009-12-29 | 2011-07-28 | Curna, Inc. | Treatment of nuclear respiratory factor 1 (nrf1) related diseases by inhibition of natural antisense transcript to nrf1 |
ES2677044T3 (en) | 2009-12-31 | 2018-07-27 | Curna, Inc. | Treatment of diseases related to insulin receptor substrate 2 (IRS2) by inhibition of natural antisense transcript for IRS2 and transcription factor E3 (TFE3) |
US8946181B2 (en) | 2010-01-04 | 2015-02-03 | Curna, Inc. | Treatment of interferon regulatory factor 8 (IRF8) related diseases by inhibition of natural antisense transcript to IRF8 |
RU2612161C2 (en) | 2010-01-06 | 2017-03-02 | Курна, Инк. | Treatment of pancreatic developmental gene related diseases by inhibition of natural antisense transcript to pancreatic developmental gene |
WO2011085347A2 (en) | 2010-01-11 | 2011-07-14 | Opko Curna, Llc | Treatment of sex hormone binding globulin (shbg) related diseases by inhibition of natural antisense transcript to shbg |
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CA2790506A1 (en) | 2010-02-22 | 2011-08-25 | Curna, Inc. | Treatment of pyrroline-5-carboxylate reductase 1 (pycr1) related diseases by inhibition of natural antisense transcript to pycr1 |
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TWI531370B (en) | 2010-05-14 | 2016-05-01 | 可娜公司 | Treatment of par4 related diseases by inhibition of natural antisense transcript to par4 |
DK2576784T3 (en) | 2010-05-26 | 2018-02-26 | Curna Inc | TREATMENT OF METHIONIN SULPHOXIDE REDUCTASE A (MSRA) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENCE TRANSCRIPTION TO MSRA |
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NO2593547T3 (en) | 2010-07-14 | 2018-04-14 | ||
RU2624048C2 (en) | 2010-10-06 | 2017-06-30 | Курна, Инк. | Treatment of diseases associated with the sialidase 4 (neu4) gene, by gene neu4 natural antisense transcript inhibition |
JP6049623B2 (en) | 2010-10-22 | 2016-12-21 | カッパーアールエヌエー,インコーポレイテッド | Treatment of IDUA-related diseases by inhibition of natural antisense transcripts to α-L-iduronidase (IDUA) |
US10000752B2 (en) | 2010-11-18 | 2018-06-19 | Curna, Inc. | Antagonat compositions and methods of use |
JP6071893B2 (en) | 2010-11-23 | 2017-02-01 | カッパーアールエヌエー,インコーポレイテッド | Treatment of NANOG-related diseases by inhibition of natural antisense transcripts to NANOG |
CN103620036B (en) | 2011-06-09 | 2016-12-21 | 库尔纳公司 | FXN relevant disease is treated by the natural antisense transcript of suppression Frataxin (FXN) |
KR101991980B1 (en) | 2011-09-06 | 2019-06-21 | 큐알엔에이, 인크. | TREATMENT OF DISEASES RELATED TO ALPHA SUBUNITS OF SODIUM CHANNELS, VOLTAGE-GATED (SCNxA) WITH SMALL MOLECULES |
ES2694592T3 (en) | 2012-03-15 | 2018-12-21 | Curna, Inc. | Treatment of diseases related to brain-derived neurotrophic factor (BDNF) by inhibition of the natural antisense transcript of BDNF |
GB201410693D0 (en) | 2014-06-16 | 2014-07-30 | Univ Southampton | Splicing modulation |
WO2016054615A2 (en) | 2014-10-03 | 2016-04-07 | Cold Spring Harbor Laboratory | Targeted augmentation of nuclear gene output |
SG11201802870RA (en) | 2015-10-09 | 2018-05-30 | Univ Southampton | Modulation of gene expression and screening for deregulated protein expression |
US11096956B2 (en) | 2015-12-14 | 2021-08-24 | Stoke Therapeutics, Inc. | Antisense oligomers and uses thereof |
WO2017106377A1 (en) | 2015-12-14 | 2017-06-22 | Cold Spring Harbor Laboratory | Antisense oligomers for treatment of autosomal dominant mental retardation-5 and dravet syndrome |
GB2599884B (en) | 2017-08-25 | 2022-08-31 | Stoke Therapeutics Inc | Antisense oligomers for treatment of conditions and diseases |
US20230287410A1 (en) | 2020-05-11 | 2023-09-14 | Stoke Therapeutics, Inc. | Opa1 antisense oligomers for treatment of conditions and diseases |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6278038B1 (en) * | 1992-04-10 | 2001-08-21 | Oregon Health And Science University | Mammalian melanocortin receptors and uses |
US5985558A (en) * | 1997-04-14 | 1999-11-16 | Isis Pharmaceuticals Inc. | Antisense oligonucleotide compositions and methods for the inibition of c-Jun and c-Fos |
FR2724934B1 (en) * | 1994-09-26 | 1997-01-24 | Bio Merieux | CHIMERIC OLIGONUCLEOTIDE AND ITS USE IN OBTAINING NUCLEIC ACID TRANSCRIPTS |
US6346398B1 (en) * | 1995-10-26 | 2002-02-12 | Ribozyme Pharmaceuticals, Inc. | Method and reagent for the treatment of diseases or conditions related to levels of vascular endothelial growth factor receptor |
DE69819345T2 (en) * | 1997-08-20 | 2004-07-15 | The Regents Of The University Of California, Oakland | NUCLEIC ACID SEQUENCES ENCODING THE CAPSAICIN RECEPTOR AND POLYPEPTIDES SIMILAR TO THE CAPSAICIN RECEPTOR AND THEIR USE |
CA2309903C (en) * | 1998-01-22 | 2012-03-27 | The Regents Of The University Of California | Nucleic acid sequences encoding capsaicin receptor |
EP0943683A1 (en) | 1998-03-10 | 1999-09-22 | Smithkline Beecham Plc | Human vanilloid receptor homologue Vanilrep1 |
GB9822124D0 (en) * | 1998-10-09 | 1998-12-02 | Univ London | Ion channels |
CA2348479A1 (en) * | 1998-11-13 | 2000-05-25 | Millennium Pharmaceuticals, Inc. | Novel members of the capsaicin/vanilloid receptor family of proteins and uses thereof |
GB9826359D0 (en) | 1998-12-01 | 1999-01-27 | Glaxo Group Ltd | Novel receptors |
GB9827016D0 (en) | 1998-12-08 | 1999-02-03 | Merck Sharp & Dohme | Receptor protein |
EP1175504A4 (en) | 1999-04-15 | 2002-07-31 | Ortho Mcneil Pharm Inc | Dna encoding the human vanilloid receptor vr1 |
US6482611B1 (en) * | 1999-09-23 | 2002-11-19 | Neurogen Corporation | Human capsaicin receptor and uses thereof |
EP1423427A2 (en) * | 2001-08-27 | 2004-06-02 | Novartis AG | Nogo receptor homologues and their use |
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- 2001-08-31 AT AT01976176T patent/ATE385505T1/en active
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- 2003-03-03 US US10/376,341 patent/US7662948B2/en not_active Expired - Fee Related
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9427415B2 (en) | 2014-04-01 | 2016-08-30 | The Procter & Gamble Company | Methods and compositions for modifying sensorial perception |
US11879215B2 (en) | 2016-10-25 | 2024-01-23 | The Procter & Gamble Company | Fibrous structures |
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US7662948B2 (en) | 2010-02-16 |
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EP1313768A2 (en) | 2003-05-28 |
PL366039A1 (en) | 2005-01-24 |
WO2002018407A2 (en) | 2002-03-07 |
ES2300366T3 (en) | 2008-06-16 |
WO2002018407A9 (en) | 2003-05-15 |
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