WO2010035725A1 - Ngfに対するアプタマー及びその使用 - Google Patents
Ngfに対するアプタマー及びその使用 Download PDFInfo
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- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- A61P11/06—Antiasthmatics
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/04—Centrally acting analgesics, e.g. opioids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Definitions
- the present invention relates to an aptamer for NGF and a method for using the aptamer.
- Nerve growth factor NGF nerve growth factor
- nerve growth factor NGF nerve growth factor
- TrkA As the NGF receptor, a high-affinity tyrosine kinase receptor TrkA and a low-affinity tumor necrosis factor receptor superfamily are known. These receptors act as homodimers or heterodimers and are deeply involved in the development and maintenance of the nervous system. TrkA is a single transmembrane receptor and has a tyrosine kinase structure in the intracellular domain. When NGF binds, tyrosine phosphorylation occurs and a signal is transmitted downstream, which promotes cell differentiation and sustains survival. TrkB and TrkC are known as TrkA family receptors. TrkB binds to BDNF and NT-4 / 5, and TrkC binds to NT-3.
- p75 has lower ligand specificity than TrkA and binds to BDNF, NT-3, and NT-4 / 5 in addition to NGF.
- p75 is a single-transmembrane receptor, but there is no tyrosine kinase domain on the cytoplasm side. Like TrkA, it is expressed not only in neurons but also in non-neurons. In addition to being involved in promoting cell differentiation and maintaining survival, this receptor is known to be involved in the induction of apoptosis and cell migration. From the results of crystal structure analysis, it was suggested that homodimeric NGF binds to TrkA at 2: 2, but binds to p75 at 2: 1. Homodimeric NGF may also bind to the TrkA and p75 heterodimers.
- NGF is produced by Schwann cells, keratinocytes, bronchial epithelial cells, fibroblasts, T lymphocytes, macrophages, mast cells, B lymphocytes, keratinocytes, smooth muscle cells, renal glomerular cells, skeletal muscle cells, and the like.
- TrkA is known to be expressed not only in neurons but also in monocytes other than neurons, T lymphocytes, B lymphocytes, and mast cells.
- p75 is expressed not only in nerve cells but also in non-neuronal cells.
- NGF plays an important role in the nervous system. It has been shown to have an effect of maintaining the survival of cholinergic neurons, and is considered to have some relation to Alzheimer's disease. In addition, when NGF is administered into the brain of aged rats, improvement in memory impairment is observed, and therefore, it is also expected as a therapeutic agent for senile dementia.
- NGF also acts on tissues and cells other than the nervous system and is involved in biological defense and tissue repair processes. For example, when NGF is administered to an animal, vascular permeability increases, T cell and B cell immune responses increase, lymphocyte differentiation induction, mast cell proliferation induction, and various cytokine release induction from mast cells occur. It is known.
- NGF is associated with inflammation, and increased expression of NGF has been observed in patients with inflammatory diseases and inflammatory animal models. For example, systemic lupus erythematosus, multiple sclerosis, psoriasis, arthritis, interstitial cystitis, asthma and the like. It has been reported that the concentration of NGF in the synovial fluid of patients with rheumatoid arthritis is elevated. Further, it has been reported that NGF expression is improved in rheumatoid arthritis model rats, and that mast cells are increased and NGF expression is improved in arthritis model mice.
- NGF is deeply related to pain.
- deep pain such as myalgia continues for several days, resulting in hyperalgesia at the injection site.
- NGF knockout mice and TrkA knockout mice lack a myelinated nerve and do not feel pain.
- Intraperitoneal administration of 1 mg / kg NGF to adult rats results in nociceptive fever and hyperalgesia to mechanical stimulation.
- NGF transgenic mice show hyperalgesia without inflammatory symptoms.
- CIPA congenital indolent anhidrosis
- NGF inhibitors can be used as therapeutic agents for pain such as nociceptive pain, inflammatory pain, neuropathic pain, cancer pain, fibromyalgia.
- Combination therapy of NGF antibody and NSAID International Publication WO04 / 073653 pamphlet
- Combination therapy of NGF antibody and opioid analgesic International Publication WO04 / 096122 pamphlet
- Postoperative pain therapy using NGF antibody International Published WO 04/032870 pamphlet, International published WO 05/000194 pamphlet, bone cancer pain treatment using NGF antibody (international published WO 05/1101077 pamphlet), osteoarthritis pain treatment using NGF antibody (International publication WO06 / 110883 pamphlet) has been reported.
- Tanezumab (PF-4383119 or RN624) is an antibody against NGF and has been shown to be effective in pain model experiments using osteoarthritis model animals, and is currently undergoing clinical trials. Moreover, although the presence or absence of the inhibitory activity of NGF and an NGF receptor is unknown, there exists a report regarding natural RNA couple
- RNA aptamers are an RNA that specifically binds to a target substance such as a protein, and can be prepared using the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment) (Patent Documents 1 to 3).
- the SELEX method is a method in which the RNA pools having different nucleotide sequences of 10 14 or so, come to selecting RNA that binds specifically to a target substance.
- the RNA used has a structure in which a random sequence of about 40 residues is sandwiched between primer sequences. This RNA pool is associated with the target substance, and only the RNA bound to the target substance is recovered using a filter or the like. The recovered RNA is amplified by RT-PCR and used as a template for the next round. By repeating this operation about 10 times, an RNA aptamer that specifically binds to the target substance may be obtained.
- Aptamer drugs can target extracellular factors in the same way as antibody drugs, but there are possibilities in many respects that exceed antibody drugs in reference to many published academic papers. For example, aptamers often have higher binding power and specificity than antibodies. Moreover, it is hard to receive immune exclusion, and side effects such as antibody-specific antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) do not occur. In terms of delivery, since aptamers are about 1/10 the size of antibodies, it is easier to deliver drugs to the target site. Since aptamers are produced by chemical synthesis, various modifications can be easily made, and the cost can be reduced by mass production. On the other hand, the blood half-life of aptamers is generally shorter than that of antibodies. However, this point may be a merit when toxicity is considered. From the above points, even if the drug targets the same molecule, the aptamer drug may be superior to the antibody drug.
- An object of the present invention is to provide an aptamer for NGF and a method for using the aptamer.
- the present invention provides the following inventions and the like: [1] An aptamer that binds to NGF and inhibits the binding between NGF and the NGF receptor; [2] An aptamer that binds to NGF and inhibits the neurite outgrowth activity of NGF; [3] The aptamer according to [2], wherein the 50% inhibitory concentration (IC50) is 100 nM or less; [4] The aptamer according to [2], wherein the 50% inhibitory concentration (IC50) is 10 nM or less; [5] The aptamer according to any one of [1] to [4], wherein at least one nucleotide is modified; [6] including a sequence represented by HGAANNNANCY (SEQ ID NO: 106), wherein N is any nucleotide, H is a nucleotide other than G, Y is a pyrimidine nucleotide, and at least one nucleotide of the sequence is modified The aptamer according to any one
- the aptamer and complex of the present invention may be useful as a medicine for diseases such as pain and inflammatory diseases, or as a diagnostic agent or reagent.
- the aptamers and complexes of the present invention may also be useful for NGF purification and enrichment, and NGF detection and quantification.
- the common sequence is indicated by a black circle. It is a figure which shows the secondary structure anticipated by the MFOLD program of the aptamer represented by sequence number 2. It is a figure which shows the secondary structure anticipated by the MFOLD program of the aptamer represented by sequence number 3. The common sequence is indicated by a black circle. It is a figure which shows the secondary structure anticipated by the MFOLD program of the aptamer represented by sequence number 4. The common sequence is indicated by a black circle. It is a figure which shows the secondary structure anticipated by the MFOLD program of the aptamer represented by sequence number 5. The common sequence is indicated by a black circle.
- TrkA TrkA
- Apt an analyte
- BIAcore2000 manufactured by BIAcore. It is a figure which shows a mode that the aptamer (Apt) represented by sequence number 6 inhibits the coupling
- 40N is RNA containing a random sequence of 40 nucleotides.
- the present invention provides an aptamer having binding activity to NGF.
- the aptamer of the present invention can inhibit the activity of NGF by binding to NGF and inhibiting the binding between NGF and the NGF receptor.
- Aptamer refers to a nucleic acid molecule having binding activity to a predetermined target molecule. Aptamers can inhibit the activity of a given target molecule by binding to the given target molecule.
- the aptamer of the present invention may be RNA, DNA, modified nucleic acid or a mixture thereof.
- the aptamer of the present invention can also be in a linear or cyclic form.
- NGF is a known neurotrophin and is an important secreted protein involved in the development and survival of peripheral and central neurons.
- NGF is an abbreviation for Nerve Growth Factor.
- NGF particularly means ⁇ -type NGF.
- the amino acid sequence of human ⁇ -NGF is represented by Accession Number NP002497, P01138, AAI26151, AAI26149, CAB75625, but may be a mutated one, its domain, or peptide. Moreover, not only a monomer but a dimer and a multimer may be sufficient.
- the aptamer of the present invention binds to NGF in a physiological buffer (for example, solution A: see Example 1).
- the aptamer of the present invention binds to NGF with an intensity that can be detected by the following test.
- BIAcore2000 manufactured by BIAcore is used for the measurement.
- the aptamer is immobilized on the sensor chip. The amount immobilized is 1000 RU.
- An NGF solution (0.5 ⁇ M) is prepared with a physiological buffer containing 0.3 M NaCl (Solution A: see Example 1). 20 ⁇ L of this NGF solution is injected, and the binding of NGF to the aptamer is detected.
- RNA containing a random nucleotide sequence consisting of 40 nucleotides is used as a negative control, and when NGF binds significantly more strongly to the aptamer than the control RNA, it is determined that the aptamer has the ability to bind to NGF.
- the aptamer of the present invention inhibits the activity of NGF by binding to NGF and inhibiting the binding between NGF and the NGF receptor.
- “inhibitory activity against NGF” means the ability to inhibit any activity possessed by NGF.
- the activity of inhibiting the binding of NGF to the NGF receptor include inhibition of signal transduction downstream of the NGF receptor (Ras-MAP kinase pathway, PI3 kinase pathway) caused by binding of NGF to the NGF receptor, TRPV1, SP And inhibition of increased expression of BDNF and the like, and inhibitory activity of expression of HA, BK, PG, NGF and other cytokines released from mast cells.
- the preferable “inhibitory activity against NGF” possessed by the aptamer of the present invention is an activity that inhibits the binding of NGF to the NGF receptor, and an activity that inhibits the neurite outgrowth activity induced by NGF.
- NGF receptor means a cell surface protein to which NGF binds. TrkA and p75 are known as NGF receptors.
- the NGF receptor referred to in the present invention may be a protein containing a natural amino acid sequence or a variant thereof.
- the “variant thereof” refers to an amino acid sequence obtained by substituting several amino acids of “NGF receptor” or a part of the amino acid sequence, having binding activity to NGF, and NGF and NGF receptor.
- the aptamer of the present invention is an aptamer that binds to NGF and inhibits the binding between NGF and the NGF receptor. Whether the aptamer inhibits the binding of NGF to the NGF receptor can be evaluated by the following test.
- BIAcore2000 manufactured by BIAcore is used for the measurement.
- a fusion protein of NGF receptor and Fc (for example, Trk A-Fc (175-TK, R & D systems)) or p75-Fc (R & D systems)) is immobilized on the CM5 sensor chip. The immobilization amount is 1100 RU.
- NGF (0.1 ⁇ M) and aptamer (0.33 ⁇ M) are mixed in a physiological buffer (solution A: see Example 1) and prepared for 30 minutes.
- the binding amount means the RU value at the peak top of the BIAcore sensorgram (the RU value immediately after the end of NGF injection).
- the aptamer of the present invention can inhibit both the binding between NGF and TrkA and the binding between NGF and p75.
- the aptamer of the present invention may have an inhibitory activity against NGF derived from any mammal.
- mammals include, for example, primates (eg, humans, monkeys), rodents (eg, mice, rats, guinea pigs), and pets, livestock and working animals (eg, dogs, cats, horses, cows). , Goats, sheep, pigs).
- the aptamer of the present invention is not particularly limited as long as it can bind to any part of NGF and inhibit the binding between NGF and the NGF receptor.
- the aptamer of the present invention has a sequence represented by HGAANNANNCY (SEQ ID NO: 106) (N is any nucleotide, H is a nucleotide other than G, and Y is a pyrimidine nucleotide).
- the nucleotide sequence represented by SEQ ID NO: 106 is contained in an aptamer that binds to NGF and inhibits the binding between NGF and the NGF receptor, which is obtained by the SELEX method described later.
- at least one nucleotide of the sequence is modified.
- the aptamer of the present invention is UGAAANNANCY (SEQ ID NO: 107), CGAANANAACY (SEQ ID NO: 108) or AGAANANAACY (SEQ ID NO: 109), where N is any nucleotide and Y is a pyrimidine nucleotide. It is an aptamer that contains the expressed consensus sequence and binds to NGF and inhibits the binding between NGF and NGF receptor.
- the nucleotide sequences represented by SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109 are included in aptamers that bind to NGF and inhibit the binding between NGF and the NGF receptor, which are obtained by the SELEX method described later. Preferably at least one nucleotide of these sequences is modified.
- the aptamer of the present invention is UGAAAAAAACY (SEQ ID NO: 110), UGAAAGAAACY (SEQ ID NO: 111), CGAACAAAACY (SEQ ID NO: 112) or CGAAAGAAAACY (SEQ ID NO: 113) (Y is a pyrimidine nucleotide). It is an aptamer that contains the expressed consensus sequence and binds to NGF and inhibits the binding between NGF and NGF receptor.
- the nucleotide sequences represented by SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113 are aptamers that are obtained by the SELEX method described below and bind to NGF and inhibit the binding between NGF and the NGF receptor. included. Preferably at least one nucleotide of these sequences is modified.
- the aptamer of the present invention may be an aptamer that binds to NGF and inhibits NGF's neurite outgrowth activity. Whether the aptamer inhibits the neurite outgrowth activity of NGF can be evaluated by the test described in Example 7 or Example 8.
- the aptamer concentration (IC50) when the neurite outgrowth activity is 50% can be determined by performing the test described in Example 8 while changing the aptamer concentration.
- the IC50 of the aptamer of the present invention is preferably 100 nM or less, more preferably 10 nM or less.
- the length of the aptamer of the present invention is not particularly limited and can generally be about 10 to about 200 nucleotides, but is, for example, about 100 nucleotides or less, preferably about 60 nucleotides or less, more preferably about 50 nucleotides or less. And most preferably may be about 45 nucleotides or less. If the total number of nucleotides is small, chemical synthesis and mass production are easier, and the merit in cost is also great. In addition, chemical modification is easy, the in vivo stability is high, and the toxicity is considered low.
- Each nucleotide contained in the aptamer of the present invention is the same or different and contains a hydroxyl group at the 2 ′ position of ribose (eg, ribose of pyrimidine nucleotide, ribose of purine nucleotide) (ie, nucleotide that is unsubstituted).
- ribose eg, ribose of pyrimidine nucleotide, ribose of purine nucleotide
- the hydroxyl group can be a nucleotide substituted with any atom or group.
- Examples of such an arbitrary atom or group include a hydrogen atom, a fluorine atom, —O-alkyl group (eg, —O—Me group), —O-acyl group (eg, —O—CHO group), amino And nucleotides substituted with groups (eg, —NH 2 groups).
- the aptamer of the present invention also has at least one (eg, 1, 2, 3 or 4) nucleotides at the 2 ′ position of ribose, a hydroxyl group, or any atom or group described above, eg, a hydrogen atom, It may be a nucleotide containing at least two kinds (for example, 2, 3 or 4 kinds) of groups selected from the group consisting of a fluorine atom, a hydroxyl group and an —O—Me group.
- all pyrimidine nucleotides are the same or different at the 2 ′ position of ribose and are substituted with fluorine atoms, or any atom or group described above, preferably hydrogen. It may be a nucleotide substituted with an atom or group selected from the group consisting of an atom, a hydroxy group and a methoxy group.
- all purine nucleotides are the same or different nucleotides substituted at the 2 ′ position of ribose with a hydroxy group, or any atom or group described above, preferably hydrogen. It may be a nucleotide substituted with an atom or group selected from the group consisting of an atom, a methoxy group and a fluorine atom.
- the aptamer of the present invention also has a group in which all nucleotides are, at the 2 ′ position of ribose, a hydroxyl group, or any atom or group described above, such as a hydrogen atom, a fluorine atom, a hydroxyl group, and an —O—Me group. It may be a nucleotide containing the same group selected from.
- the mode of modification to the sugar group in the nucleotide will be described. Does not mean that DNA is excluded from the nucleotides constituting, and is appropriately read as a modification to DNA.
- the nucleotide constituting the aptamer is DNA
- the replacement of the hydroxyl group at the 2'-position of ribose with X is read as the replacement of one hydrogen atom at the 2'-position of deoxyribose with X.
- the aptamer of the present invention is also (A) an aptamer comprising a nucleotide sequence selected from any of SEQ ID NOs: 1-9, 12, 24-55, 57-90 (provided that uracil may be thymine); (B) In a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 9, 12, 24 to 55, 57 to 90 (provided that uracil may be thymine), one or several nucleotides are substituted or missing.
- Aptamers comprising nucleotide sequences that have been deleted, inserted or added; (C) a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 9, 12, 24 to 55, 57 to 90 (provided that uracil may be thymine) and 70% or more (preferably 80% or more, More preferably 90% or more, most preferably 95% or more) an aptamer comprising a nucleotide sequence having the identity; or (d) a plurality of ligations of (a) above, a plurality of ligations of (b) above, It may be a connected product selected from the group consisting of a plurality of connected products of (c) and a plurality of connected products of (a), (b) and (c) above.
- the aptamers (b) to (d) above can bind to NGF and / or inhibit the activity of NGF (such as the binding activity with NGF receptor).
- the aptamers of (b) to (d) above bind to NGF and inhibit the binding between NGF and the NGF receptor, and / or bind to NGF, and exhibit NGF neurite outgrowth activity.
- the aptamers (b) to (d) are aptamers having an inhibitory concentration of NGF on neurite outgrowth of 100 nM or less, more preferably an inhibitory concentration of NGF on neurite outgrowth of 10 nM or less. Is an aptamer.
- the number of substitutions, deletions, insertions or additions is not particularly limited as long as the aptamer can bind to NGF and inhibit NGF activity (binding activity with NGF receptor, etc.)
- it may be about 30 or less, preferably about 20 or less, more preferably about 10 or less, even more preferably 5 or less, and most preferably 4, 3, 2 or 1.
- identity means an optimal alignment when two nucleotide sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is an optimal alignment). The percentage of identical nucleotide residues relative to all overlapping nucleotide residues), which may allow for the introduction of gaps into one or both of the sequences.
- NCBI BLAST-2 National Center for Biotechnology Information Information Basic Local Alignment Search Tool
- connection can be made by tandem coupling.
- a linker may be used for the connection.
- the linker a nucleotide chain (eg, 1 to about 20 nucleotides), a non-nucleotide chain (eg, — (CH 2 ) n-linker, — (CH 2 CH 2 O) n-linker, hexaethylene glycol linker, TEG linker , A linker containing a peptide, a linker containing a —S—S— bond, a linker containing a —CONH— bond, and a linker containing a —OPO 3 — bond).
- the number of the plurality of connected objects is not particularly limited as long as it is 2 or more, but may be 2, 3, or 4, for example.
- Each nucleotide in the above (a) to (d) is the same or different and is a nucleotide containing a hydroxyl group at the 2 ′ position of ribose (eg, ribose of pyrimidine nucleotide), or at the 2 ′ position of ribose,
- the hydroxyl group can be a nucleotide substituted with any group (eg, a hydrogen atom, a fluorine atom or an —O—Me group).
- the aptamer of the present invention is a sugar residue of each nucleotide in order to enhance binding to NGF, binding inhibitory activity between NGF and NGF receptor, NGF neurite outgrowth inhibitory activity, aptamer stability, drug delivery, etc.
- ribose may be modified.
- the site modified in the sugar residue include those in which the oxygen atom at the 2′-position, 3′-position and / or 4′-position of the sugar residue is replaced with another atom. Examples of modifications include fluorination, O-alkylation (eg, O-methylation, O-ethylation), O-allylation, S-alkylation (eg, S-methylation, S-ethylation).
- Such modification of sugar residues can be performed by a method known per se (for example, Sproat et al., (1991) Nucle. Acid. Res. 19, 733-738; Cotton et al., (1991)). Nucl. Acid. Res. 19, 2629-2635; Hobbs et al., (1973) Biochemistry 12, 5138-5145).
- the aptamer of the present invention also has a modified nucleobase (eg, purine, pyrimidine) (eg, purine, pyrimidine) in order to enhance binding to NGF, NGF and NGF receptor binding inhibitory activity, NGF neurite outgrowth inhibitory activity, etc.
- a modified nucleobase eg, purine, pyrimidine
- Chemical substitution may be used. Examples of such modifications include 5-position pyrimidine modification, 6- and / or 8-position purine modification, modification with exocyclic amine, substitution with 4-thiouridine, substitution with 5-bromo or 5-iodo-uracil. Can be mentioned.
- the phosphate group contained in the aptamer of the present invention may be modified so as to be resistant to nuclease and hydrolysis.
- the P (O) O group may be P (O) S (thioate), P (S) S (dithioate), P (O) NR 2 (amidate), P (O) R, R (O) OR ′. , CO or CH 2 (formacetal) or 3'-amine (-NH-CH 2 -CH 2 - ) wherein each R or R 'may be substituted [wherein the independently comprise H Or substituted or unsubstituted alkyl (eg, methyl, ethyl)].
- the linking group include —O—, —N—, and —S—, which can be bonded to adjacent nucleotides through these linking groups. Modifications may also include 3 ′ and 5 ′ modifications such as capping.
- Modifications are further made of polyethylene glycol, amino acids, peptides, inverted dT, nucleic acids, nucleosides, Myristoy, Lithocholic-oleyl, Docosanyl, Lauroyl, Stearoyl, Palmitoyl, Oleoyl, Linoleyl, other lipids, vitamins, steroids, cholesterol, steroids It can be performed by adding a fluorescent substance, an anticancer agent, a toxin, an enzyme, a radioactive substance, biotin or the like to the terminal. See US Pat. Nos. 5,660,985 and 5,756,703 for such modifications.
- the aptamer of the present invention can be chemically synthesized by the disclosure in the present specification and a method known per se in the technical field.
- Aptamers bind to a target substance by various binding modes such as ionic bonds using the negative charge of the phosphate group, hydrophobic bonds and hydrogen bonds using ribose, hydrogen bonds using nucleobases and stacking bonds.
- the ionic bond utilizing the negative charge of the phosphate group that exists in the number of constituent nucleotides is strong and binds to the positive charge of lysine or arginine present on the surface of the protein. For this reason, nucleobases that are not involved in direct binding to the target substance can be substituted.
- the stem structure portion is already base-paired and faces the inside of the double helix structure, so that the nucleobase is difficult to bind directly to the target substance. Therefore, the activity of the aptamer is often not reduced even if the base pair is replaced with another base pair. Even in a structure that does not form a base pair, such as a loop structure, base substitution is possible when the nucleobase is not involved in direct binding to the target molecule.
- the modification of the 2 'position of ribose in rare cases, the functional group at the 2' position of ribose may directly interact with the target molecule, but in many cases it is irrelevant and can be replaced with other modified molecules. Is possible.
- aptamers often retain their activity unless functional groups involved in direct binding to the target molecule are replaced or deleted. It is also important that the overall three-dimensional structure does not change significantly.
- Aptamers are produced by using the SELEX method and its improved methods (for example, Ellington et al., (1990) Nature, 346, 818-822; Tuerk et al., (1990) Science, 249, 505-510). can do.
- the SELEX method by increasing the number of rounds or using a competitive substance, aptamers having a stronger binding force to the target substance are concentrated and selected. Therefore, by adjusting the number of rounds of SELEX and / or changing the competition state, aptamers with different binding strength, aptamers with different binding forms, aptamers with the same binding power or binding form but different base sequences You may be able to get it.
- the SELEX method includes an amplification process by PCR. By introducing a mutation by using manganese ions in the process, it becomes possible to perform SELEX with more diversity.
- An aptamer obtained by SELEX is a nucleic acid having a high affinity for a target substance, which does not mean binding to the active site of the target substance. Therefore, the aptamer obtained by SELEX does not necessarily affect the function of the target substance.
- NGF is a basic protein, and it is considered that nucleic acids are likely to bind nonspecifically. Aptamers that do not bind to the active site do not affect the activity of the target substance. In fact, the RNA used as a control did not inhibit the binding of NGF and NGF receptor.
- SELEX can be carried out by further changing the primer in order to obtain an aptamer having higher activity.
- a specific method is to prepare a template in which a part of an aptamer whose sequence is determined is a random sequence or a template in which a random sequence of about 10 to 30% is doped, and perform SELEX again.
- the aptamer obtained by SELEX has a length of about 80 nucleotides, and it is difficult to make it as a medicine as it is. Therefore, it is necessary to repeat trial and error to shorten the length to about 50 nucleotides or less that can be easily chemically synthesized.
- the aptamers obtained by SELEX vary in ease of subsequent minimization work depending on the primer design. If the primer is not designed well, even if active aptamers can be selected by SELEX, subsequent development becomes impossible. In the present invention, an aptamer having activity even at 38 nucleotides could be obtained.
- Aptamers are easy to modify because they can be chemically synthesized. Aptamers use the MFOLD program to predict secondary structure, and to predict conformation by X-ray analysis or NMR analysis, which nucleotides can be replaced or deleted, and where new nucleotides are inserted It can be predicted to some extent whether it is possible. Aptamers of the predicted new sequence can be easily chemically synthesized, and whether or not the aptamer retains activity can be confirmed by existing assay systems.
- the activity often changes even if a new sequence is added to both ends of the sequence. do not do.
- the length of the new sequence is not particularly limited.
- the aforementioned HGAANNANCY (SEQ ID NO: 106), UGAAANNANCY (SEQ ID NO: 107), CGAANNANAACY (SEQ ID NO: 108), AGAANNAAACY (SEQ ID NO: 109), UGAAAAAAACY (SEQ ID NO: 110), UGAAAGAAACY (SEQ ID NO: 111), CGAACAAAAAC (SEQ ID NO: 112), the sequence represented by CGAAAGAAAAC (SEQ ID NO: 113) is an important part for the aptamer of the present invention to bind to NGF and inhibit the binding between NGF and the NGF receptor. Adding new sequences at both ends often does not change activity.
- the modification can be designed or altered to a high degree as well as the sequence.
- aptamers can be highly designed or modified.
- the present invention also includes a predetermined sequence (eg, a sequence corresponding to a portion selected from a stem portion, an internal loop portion, a hairpin loop portion, and a single-stranded portion: hereinafter, abbreviated as a fixed sequence if necessary).
- a predetermined sequence eg, a sequence corresponding to a portion selected from a stem portion, an internal loop portion, a hairpin loop portion, and a single-stranded portion: hereinafter, abbreviated as a fixed sequence if necessary.
- the method for producing such aptamer is as follows:
- (N) a represents a nucleotide chain consisting of a N
- (N) b represents a nucleotide chain consisting of b N
- N is the same or different, respectively, A, G
- It is a nucleotide selected from the group consisting of C, U and T (preferably A, G, C and U).
- a and b are the same or different and may be any number, for example, 1 to about 100, preferably 1 to about 50, more preferably 1 to about 30, even more preferably 1 to about There may be 20 or 1 to about 10.
- Each of a single type of nucleic acid molecule or a plurality of types of nucleic acid molecules eg, a library of nucleic acid molecules having different numbers of a, b, etc.
- primer sequences eg, a library of nucleic acid molecules having different numbers of a, b, etc.
- primer sequences i
- ii Producing an aptamer comprising a fixed sequence using the corresponding primer pair.
- the present invention also provides a complex comprising the aptamer of the present invention and a functional substance bound thereto.
- the bond between the aptamer and the functional substance in the complex of the present invention can be a covalent bond or a non-covalent bond.
- the complex of the present invention may be a conjugate of the aptamer of the present invention and one or more (eg, 2 or 3) of the same or different functional substances.
- the functional substance is not particularly limited as long as it newly adds some function to the aptamer of the present invention or can change (eg, improve) some property that can be retained by the aptamer of the present invention.
- Examples of the functional substance include proteins, peptides, amino acids, lipids, carbohydrates, monosaccharides, polynucleotides, and nucleotides.
- Examples of functional substances include, for example, affinity substances (eg, biotin, streptavidin, polynucleotides having affinity for target complementary sequences, antibodies, glutathione sepharose, histidine), labeling substances (eg, fluorescent substances, Luminescent substances, radioisotopes), enzymes (eg, horseradish peroxidase, alkaline phosphatase), drug delivery vehicles (eg, liposomes, microspheres, peptides, polyethylene glycols), drugs (eg, calicheamicin and duocarmycin) Used in missile therapy, nitrogen mustard analogs such as cyclophosphamide, melphalan, ifosfamide or trophosphamide, ethyleneimines such as thiotepa, nitrosourea such as carmustine, temozo
- ⁇ унк ⁇ онент may eventually be removed. Furthermore, it may be a peptide that can be recognized and cleaved by an enzyme such as thrombin, matrix metalloprotease (MMP), FactorX, or a polynucleotide that can be cleaved by a nuclease or a restriction enzyme.
- an enzyme such as thrombin, matrix metalloprotease (MMP), FactorX, or a polynucleotide that can be cleaved by a nuclease or a restriction enzyme.
- the aptamer and complex of the present invention can be used, for example, as a pharmaceutical or diagnostic agent, test agent, reagent, drinking water or food additive, enhancer, or relaxation agent.
- the aptamer and complex of the present invention may have an activity of inhibiting the function of NGF by binding to NGF and inhibiting the binding between NGF and the NGF receptor.
- NGF is deeply related to pain and inflammation. Therefore, the aptamer and complex of the present invention are useful as a medicament (anti-pain agent, anti-inflammatory agent, etc.) for treating or preventing diseases accompanied by pain and inflammation.
- nociceptive pain muscle pain, back pain, upper limb pain, lashing injury, joint pain, osteoarthritis, gout, rheumatoid arthritis, headache, migraine, tension headache, cluster headache, secondary sexual headache, oral and facial pain, toothache, causalgia after extraction, phantom tooth pain, visceral pain, heart pain, abdominal pain, intermediate pain, dysmenorrhea, labor pain, kidney pain, ureteral pain, bladder pain, etc.), inflammatory pain, neurogenic Pain (diabetic neuropathy, toxic neuropathy, postoperative pain, phantom limb pain, fragment pain, reflex sympathetic dystrophy, causalgia, postherpetic pain, trigeminal neuralgia, central pain), cancer pain (visceral) Pain caused by cancer infiltration into organs, pain caused by vascular occlusion due to blood vessel infiltration of cancer tissue, pain due to bone metastasis, pain due to intracerebral metastasis, pain due to peripheral nerve invasion of cancer tissue), fibro
- the diseases associated with inflammation are not particularly limited, but include systemic lupus erythematosus, multiple sclerosis, psoriasis, osteoarthritis, rheumatoid arthritis, interstitial cystitis, asthma and the like.
- the above cancer is not particularly limited, but esophageal cancer, thyroid cancer, bladder cancer, colon cancer, stomach cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, non-small cell lung cancer, breast cancer, neuroblastoma , Neuroblastoma, glioblastoma, uterine cancer, cervical cancer, ovarian cancer, Wilms tumor, prostate cancer and the like.
- the aptamer and complex of the present invention can be used as a medicine or diagnostic agent, test agent, or reagent for diseases associated with activation of these signal transduction pathways. Examples of the diseases related to activation of these signal transduction pathways include the above pain and cancer.
- the subject to which it is administered is not particularly limited.
- primates eg, humans, monkeys
- rodents Eg, mouse, rat, guinea pig
- livestock and working animals eg, dogs, cats, horses, cows, goats, sheep, pigs.
- the aptamer and complex of the present invention can specifically bind to NGF. Therefore, the aptamer and complex of the present invention are useful as a probe for NGF detection.
- the probe is useful for in vivo imaging of NGF, blood concentration measurement, tissue staining, ELISA, and the like.
- the probe is useful as a diagnostic agent, a test agent, a reagent and the like for diseases involving NGF (such as diseases associated with pain and inflammation).
- the aptamer and complex of the present invention can be used as a ligand for separation and purification of NGF.
- the aptamer and the complex of the present invention can be used as a test agent for examining a mental state such as romance, a pharmaceutical agent for adjusting the mental state, a regulator, an enhancer, and a relaxation agent.
- the aptamer and complex of the present invention can also be used as a drug delivery agent.
- the medicament of the present invention may be formulated with a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier include excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Lubricant such as sodium lauryl sulfate, Citric acid, Menthol, Glycyllysine / Ammonium salt, Glycine, Orange powder and other fragrances, Sodium benzoate Preservatives such as sodium, sodium bisulfite, methylparaben, propylparab
- Preparations suitable for oral administration include a solution in which an effective amount of a ligand is dissolved in a diluent such as water, physiological saline, orange juice, a capsule containing an effective amount of the ligand as a solid or a granule, a sachet or Examples thereof include tablets, suspensions in which an effective amount of an active ingredient is suspended in a suitable dispersion medium, and emulsions in which a solution in which an effective amount of an active ingredient is dissolved is dispersed in an appropriate dispersion medium and emulsified.
- a diluent such as water, physiological saline, orange juice
- a capsule containing an effective amount of the ligand as a solid or a granule a sachet or Examples thereof include tablets, suspensions in which an effective amount of an active ingredient is suspended in a suitable dispersion medium, and emulsions in which a solution in which an effective amount of an active ingredient is dissolved is dispersed
- the medicament of the present invention can be coated by a method known per se for the purpose of taste masking, enteric solubility or sustainability, if necessary.
- the coating agent used for coating include hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, Eudragit (manufactured by Rohm, Germany, methacrylic acid / acrylic acid copolymer) and pigments (eg, Bengala, titanium dioxide, etc.) are used.
- the medicine may be either an immediate release preparation or a sustained release preparation.
- the sustained release substrate include liposomes, atelocollagen, gelatin, hydroxyapatite, and PLGA.
- Suitable formulations for parenteral administration are aqueous and non-aqueous isotonic.
- parenteral administration eg, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, nasal, pulmonary, etc.
- aqueous and non-aqueous isotonic are aqueous and non-aqueous isotonic.
- sterile injection solutions which may contain antioxidants, buffers, antibacterial agents, isotonic agents and the like.
- aqueous and non-aqueous sterile suspensions can be mentioned, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
- the preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials.
- the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile solvent immediately before use.
- Sustained release preparations can also be mentioned as suitable preparations.
- Sustained release formulations include artificial bones, biodegradable or non-degradable sponges, bags, drug pumps, osmotic pumps, sustained release forms from carriers or containers embedded in the body, or continuous or intermittent from outside the body. And the like delivered to the body or locally.
- biodegradable base material examples include liposomes, cationic liposomes, poly (lactide-co-glycolic) acid (PLGA), atelocollagen, gelatin, hydroxyapatite, and polysaccharide schizophyllan.
- inhalants and ointments are also possible.
- an inhalant the active ingredient in a lyophilized state is refined and administered by inhalation using an appropriate inhalation device.
- conventionally used surfactants, oils, seasonings, cyclodextrins or derivatives thereof can be appropriately blended as necessary.
- surfactant examples include oleic acid, lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl trioleate, glyceryl monolaurate, glyceryl monooleate, glyceryl monostearate.
- oil examples include corn oil, olive oil, cottonseed oil and sunflower oil.
- an appropriate pharmaceutically acceptable base yellow petrolatum, white petrolatum, paraffin, plastibase, silicone, white ointment, beeswax, pig oil, vegetable oil, hydrophilic ointment, hydrophilic petrolatum, purified lanolin, hydrolyzed lanolin , Water-absorbing ointment, hydrophilic plastibase, macrogol ointment, etc.
- an appropriate pharmaceutically acceptable base yellow petrolatum, white petrolatum, paraffin, plastibase, silicone, white ointment, beeswax, pig oil, vegetable oil, hydrophilic ointment, hydrophilic petrolatum, purified lanolin, hydrolyzed lanolin , Water-absorbing ointment, hydrophilic plastibase, macrogol ointment, etc.
- Inhalants can be manufactured according to conventional methods. That is, the aptamer and the complex of the present invention can be produced by making a powder or liquid, blending in an inhalation propellant and / or carrier, and filling an appropriate inhalation container.
- the aptamer and complex of the present invention are powder, a normal mechanical powder inhaler can be used, and when it is liquid, an inhaler such as a nebulizer can be used.
- the propellant conventionally known ones can be widely used.
- the dose of the medicament of the present invention varies depending on the type / activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age, etc.
- the amount of active ingredient may be about 0.0001 to about 100 mg / kg, such as about 0.0001 to about 10 mg / kg, preferably about 0.005 to about 1 mg / kg.
- the present invention also provides a solid phase carrier on which the aptamer and complex of the present invention are immobilized.
- the solid phase carrier include a substrate, a resin, a plate (eg, multiwell plate), a filter, a cartridge, a column, and a porous material.
- the substrate may be one used for DNA chips, protein chips, etc., for example, nickel-PTFE (polytetrafluoroethylene) substrate, glass substrate, apatite substrate, silicon substrate, alumina substrate, etc. And the like coated with a polymer or the like.
- the resin examples include agarose particles, silica particles, copolymers of acrylamide and N, N′-methylenebisacrylamide, polystyrene-crosslinked divinylbenzene particles, particles obtained by crosslinking dextran with epichlorohydrin, cellulose fibers, and allyldextran.
- examples include N, N'-methylenebisacrylamide cross-linked polymers, monodisperse synthetic polymers, monodisperse hydrophilic polymers, sepharose, and Toyopearl.
- resins in which various functional groups are bonded to these resins. .
- the solid phase carrier of the present invention can be useful for, for example, purification of NGF and detection and quantification of NGF.
- the aptamer and complex of the present invention can be immobilized on a solid support by a method known per se.
- an affinity substance for example, one described above
- a predetermined functional group is introduced into the aptamer and complex of the present invention, and then immobilized on a solid phase carrier using the affinity substance or the predetermined functional group.
- a method is mentioned.
- the present invention also provides such a method.
- the predetermined functional group may be a functional group that can be subjected to a coupling reaction, and examples thereof include an amino group, a thiol group, a hydroxyl group, and a carboxyl group.
- the present invention also provides an aptamer having such a functional group introduced therein.
- the present invention also provides methods for purification and concentration of NGF.
- the present invention can separate NGF from other family proteins.
- the purification and concentration method of the present invention can include adsorbing NGF to the solid phase carrier of the present invention and eluting the adsorbed NGF with an eluent.
- Adsorption of NGF to the solid phase carrier of the present invention can be carried out by a method known per se. For example, a sample containing NGF (eg, bacterial or cell culture or culture supernatant, blood) is introduced into the solid phase carrier of the present invention or a content thereof. NGF can be eluted using an eluent such as a neutral solution.
- NGF eg, bacterial or cell culture or culture supernatant, blood
- the neutral eluate is not particularly limited, and may be, for example, a pH of about 6 to about 9, preferably about 6.5 to about 8.5, more preferably about 7 to about 8.
- Neutral solutions can also contain, for example, urea, chelating agents (eg, EDTA), potassium salts (eg, KCl), magnesium salts (eg, MgCl 2 ), surfactants (eg, Tween 20, Triton, NP40), glycerin. Can be included.
- the purification and concentration method of the present invention may further comprise washing the solid phase carrier with a washing solution after NGF adsorption.
- the cleaning liquid examples include urea, a chelating agent (eg, EDTA), a surfactant such as Tris, acid, alkali, Transfer RNA, DNA, Tween 20, and a salt containing a salt such as NaCl.
- a chelating agent eg, EDTA
- a surfactant such as Tris, acid, alkali, Transfer RNA, DNA, Tween 20, and a salt containing a salt such as NaCl.
- the purification and concentration method of the present invention may further include heat-treating the solid phase carrier. By this process, the solid phase carrier can be regenerated and sterilized.
- the present invention also provides a method for detecting and quantifying NGF.
- the present invention can detect and quantify NGF separately from other family proteins.
- the detection and quantification method of the present invention can include measuring NGF utilizing the aptamer of the present invention (eg, by using the complex of the present invention and a solid phase carrier).
- the method for detecting and quantifying NGF can be performed by the same method as the immunological method except that the aptamer of the present invention is used instead of the antibody.
- enzyme immunoassay eg, direct competitive ELISA, indirect competitive ELISA, sandwich ELISA
- radioimmunoassay (RIA) radioimmunoassay
- fluorescent immunoassay Detection and quantification can be performed by the same method as the method (FIA), Western blot method, immunohistochemical staining method, cell sorting method and the like. It can also be used as a molecular probe such as PET.
- a method may be useful, for example, for measuring the amount of NGF in a biological or biological sample, or diagnosing a disease associated with NGF.
- RNA aptamer that specifically binds to NGF was prepared using the SELEX method. SELEX was performed with reference to the method of Ellington et al. (Ellington and Szostak, Nature 346, 818-822, 1990) and the method of Tuerk et al. (Tuerk and Gold, Science 249, 505-510, 1990). Human NGF (R & D Systems) was used as a target substance.
- the RNA (40N-RNA) used in the first round was obtained by transcription of DNA obtained by chemical synthesis using a DuraScript TM T7 Transcription Kit (manufactured by Epicentre).
- RNA obtained by this method is one in which the 2 ′ position of ribose of the pyrimidine nucleotide is fluorinated.
- a DNA template 79-nucleotide DNA having a primer sequence at both ends of a 40-nucleotide random sequence shown below was used. DNA templates and primers were prepared by chemical synthesis.
- DNA template 5'-ccagttgttggtgacaatgc-40N-gcagctccacaggcttccc-3 '(SEQ ID NO: 114)
- Primer Fwd 5'-taatacgactcactatagggaagcctgtggagctgc-3 '(SEQ ID NO: 115)
- Primer Rev 5'-ccagttgttggtgacaatgc-3 '(SEQ ID NO: 116)
- N represents any one of A, G, C, and T.
- Primer Fwd contains the promoter sequence for T7 RNA polymerase. Variations of the RNA pool used in the first round was theoretically 10 14.
- SEQ ID NO: 1 gggaagc (F) c (F) u (F) gu (F) ggagc (F) u (F) gc (F) aggau (F) gaaaaaac (F) c (F) c (F) aaaac (F) aaagac ( F) aau (F) gau (F) u (F) gagu (F) agc (F) au (F) u (F) gu (F) c (F) ac (F) c (F) ac (F) c (F) aac (F) aac (F) u (F) gg SEQ ID NO: 2: gggaagc (F)
- the binding activity of the nucleic acids represented by SEQ ID NOs: 1 to 9 and 12 to NGF was evaluated by the surface plasmon resonance method.
- BIAcore2000 manufactured by BIAcore was used for the measurement.
- the sensor chip used was an SA chip on which streptavidin was immobilized. To this, approximately 1500 RU of 16 nucleotide Poly dT having biotin bound to the 5 'end was bound.
- a nucleic acid to be a ligand was added with 16 nucleotides of Poly A at the 3 'end and immobilized on the SA chip by the binding of T and A.
- the immobilization amount was about 1000 RU.
- NGF for analyte was prepared to 0.5 ⁇ M, and 20 ⁇ L of a final concentration of 0.3 M NaCl added to reduce non-specific adsorption was injected.
- Solution A was used as the running buffer.
- the solution A is a mixed solution of 145 mM sodium chloride, 5.4 mM potassium chloride, 1.8 mM calcium chloride, 0.8 mM magnesium chloride, 20 mM Tris (pH 7.6), 0.05% Tween 20.
- SEQ ID NOs: 1 to 9 and 12 bound to NGF significantly more than 40N of the control.
- 40N is a nucleic acid pool used in the first round including a random sequence of 40 nucleotides.
- a sensorgram showing how the aptamer represented by SEQ ID NO: 6 binds to NGF is shown in FIG. From the above, these nucleic acids were shown to be aptamers that bind to NGF.
- Example 2 Preparation of RNA aptamer that specifically binds to NGF 2 SELEX similar to Example 1 was performed using a template in which the random sequence was 30 nucleotides and the primer sequence was changed from that used in Example 1.
- the template and primer sequences used are shown below. A 30 nucleotide random sequence was used as a template. DNA templates and primers were prepared by chemical synthesis.
- DNA template 5'-tgaggatccatgtatgcgcacata-30N-cttctggtcgaagttctccc-3 '(SEQ ID NO: 117)
- Primer Fwd 5'-cggaattctaatacgactcactatagggagaacttcgaccagaag-3 '(SEQ ID NO: 118)
- Primer Rev 5′-tgaggatccatgtatgcgcacata-3 ′ (SEQ ID NO: 119)
- SEQ ID NO: 56 When the sequence was examined again after the SELEX round 19 was completed, there were 3 sequences represented by SEQ ID NO: 56, and 2 sequences each represented by SEQ ID NOs: 54, 57, and 67. The other sequences did not converge. Many of these sequences contained consensus sequences of UGAAAAAAAACC (SEQ ID NO: 91), UGAAAGAAAACC (SEQ ID NO: 92) and UGAAAGAAACC (SEQ ID NO: 95). Some of these sequences are shown in SEQ ID NOs: 54-59.
- sequences represented by SEQ ID NO: 67 and 3 sequences represented by SEQ ID NO: 68 included sequences of CGAACAAAACU (SEQ ID NO: 103) and CGAAAGAAACU (SEQ ID NO: 104), which are very similar to the consensus sequence represented by SEQ ID NO: 91.
- Other sequences did not converge, but many sequences contained a consensus sequence of UGAAAAAAAACC (SEQ ID NO: 91) and UGAAAGAAACC (SEQ ID NO: 92). Some of these sequences are shown in SEQ ID NOs: 60-68.
- SEQ ID NO: 37 gggagaac (F) u (F) u (F) c (F) gac (F) c (F) agaagu (F) u (F) u (F) gaaagaaac (F) c (F) c (F) aaaggu ( F) gaaac (F) aac (F) aau (F) au (F) gu (F) gc (F) gc (F) au (F) ac (F) au (F) ac (F) au (F) ggau (F) c (F) c (F) u (F) c (F) a SEQ ID NO: 38: gggagaac (F) u (F) u (F) c (F) a SEQ ID NO: 38: gggagaac (F) u (F) u (F) c (F) a SEQ ID NO: 38: ggg
- the binding activity of the nucleic acids represented by SEQ ID NOs: 37 to 68 to NGF was evaluated by the surface plasmon resonance method. In the experiment, the same method as that shown in Example 1 was used. As a result, all the sequences were found to bind to NGF significantly more than the control 30N.
- Example 3 Preparation of RNA-DNA mosaic aptamer that specifically binds to NGF
- a mosaic aptamer having purine nucleotides as RNA and pyrimidine nucleotides as DNA was prepared by SELEX method.
- a template containing a random sequence of 40 nucleotides and a primer different from that used in Examples 1 and 2 was used.
- the RNA-DNA mosaic nucleic acid pool used in the first round was obtained by transcribing rATP, rGTP, dCTP, and dTTP as substrates using DNA obtained by chemical synthesis as a template.
- rNTP represents ribonucleotide
- dNTP represents deoxyribonucleotide.
- Other experimental methods are almost the same as those shown in the first embodiment.
- the template and primer sequence used are shown below.
- DNA-RNA template 5'-tcctaatgtctcttctcttcac-40N-gccctattcttgcctctccc-3 '(SEQ ID NO: 120)
- Primer Fwd 5'-taatacgactcactatagggagaggcaagaatagggc-3 '(SEQ ID NO: 121)
- Primer Rev 5'-tcctaatgtctcttctctcttcac-3 '(SEQ ID NO: 122)
- nucleotide sequences corresponding to SEQ ID NOs: 69 to 74 are shown below.
- Uppercase letters T and C indicate deoxyribonucleotides
- lowercase letters a and g indicate ribonucleotides.
- SEQ ID NO: 69 gggagaggCaagaaTagggCCCagCTgaaaaaaCCTggaCgTaCaCCgTTCgCCgagCgggTgaagagaagagaCaTTagga
- SEQ ID NO: 70 gggagaggCaagaaTagggCTggaaaTagaaCCgCgCTgTCTTCaTTaagCCgCCCaaCggTgaagagaagagaCaTTagga
- SEQ ID NO: 71 gggagaggCaagaaTagggCaCTTgaaaaaaaCCCaaaTTTaCCgTCTTCagCgTCgggTgTgaagagaCaTTagga
- SEQ ID NO: 72 gggagaggCaagaaTagggCTggaTgggCagTaaCCTgaaaaaaaCCTgaaaaCCTgaaagagaC
- the binding activity of the nucleic acids represented by SEQ ID NOs: 69 to 74 to NGF was evaluated by the surface plasmon resonance method. In the experiment, the same method as that shown in Example 1 was used. As a result, it was found that all of them bound to NGF significantly more than 40N of the control.
- Example 4 Production of Higher Activity NGF Aptamer SELEX was performed using an RNA pool in which 30% random sequence was doped to the sequence represented by SEQ ID NO: 36 and new primer sequences were added to both ends thereof. SELEX was performed in substantially the same manner as in Example 1. The template and primer sequences are shown below.
- SEQ ID NO: 75 gggagaac (F) u (F) u (F) c (F) gac (F) c (F) agaagu (F) gaaagaau (F) c (F) u (F) c (F) c (F) aaagac ( F) aagau (F) aaaac (F) aac (F) c (F) gu (F) au (F) gu (F) gc (F) gc (F) au (F) ac (F) au (F) ac (F) au (F) ggau (F) c (F) c (F) u (F) c (F) SEQ ID NO: 76: gggagaac (F) u (F) u (F) c (F) gac (F) c (F) agaaggau (F) aaac (F) gc (F) au (F)
- Example 5 Preparation of DNA aptamer that specifically binds to NGF
- a DNA aptamer that specifically binds to NGF was prepared using the SELEX method.
- the SELEX method was carried out by improving the method of Fitzwater and Polsky et al. (Fitzwater and Polsky, Methods Enzymol. 267, 275-301, 1996).
- Human NGF used in Example 1 was used as a target substance.
- biotin (bio) was added to the 5 ′ end of the primer Rev.
- SEQ ID NO: 87 GGGATCGACAGGGCTGCAGCACTGGCGTAGGTTGGAATATGGGTATTTTTGTGGTCCGAGTCGTGCCATCT
- Example 6 Aptamer that inhibits binding between NGF and NGF receptor Aptamers represented by SEQ ID NOs: 1 to 9, 12, 37 to 55, and 57 to 87 inhibit binding between NGF and NGF receptors (TrkA and P75). It was investigated using surface plasmon resonance. In accordance with the protocol of BIAcore, Protein A (21181, PIERCE) was immobilized on a CM5 sensor chip. There, about 1100 RU of human Trk A-Fc (175-TK, R & D systems) fused with the Fc portion of IgG was immobilized. As an analyte, NGF (0.1 ⁇ M) and aptamer (0.33 ⁇ M) mixed and retained for 30 minutes were injected.
- SEQ ID NOs: 1 to 9, 12, 37 to 55, and 57 to 87 inhibit binding between NGF and NGF receptors (TrkA and P75). It was investigated using surface plasmon resonance. In accordance with the protocol of BIAcore, Protein A (211
- the aptamer inhibits the binding of NGF and TrkA, the signal of the sensorgram does not increase, but if it does not inhibit, a tripartite complex is formed and the signal is expected to increase.
- the aptamer may be detached and NGF may bind to the receptor. It was confirmed that NGF bound to TrkA before starting the inhibition experiment.
- the binding amount of NGF and NGF receptor not containing an aptamer was defined as 100, and the binding amount of NGF and NGF receptor when an aptamer was added was determined as a correction value.
- the binding amount was the RU value at the peak top of the BIAcore sensorgram (the RU value immediately after the end of NGF injection). A value obtained by subtracting this correction value from 100 was defined as% inhibition activity, and 60% or more was regarded as having inhibition activity.
- SEQ ID NOs: 1 to 9, 12, 37 to 55, and 57 to 87 inhibit the binding between NGF and TrkA (Table 1).
- FIG. 12 shows a state in which the aptamer represented by SEQ ID NO: 6 inhibits the binding between NGF and TrkA. Similar experiments were performed for another receptor, P75 (p75-Fc; R & D systems).
- FIG. 13 shows a state in which the aptamer represented by SEQ ID NO: 6 inhibits the binding between NGF and P75.
- Table 1 shows aptamers that inhibit the binding between TrkA or p75 and NGF. “+” Indicates that the inhibition activity% is 60% or more, and “ ⁇ ” indicates that the inhibition activity is less than 60%.
- the same inhibition experiment as described above was performed under the condition that the molar ratio of NGF to aptamer was 1: 1 (0.1 ⁇ M).
- the same preparation solution was used in the experiments of TrkA and P75, and the experiment was performed without the influence of sample preparation errors.
- the aptamer represented by SEQ ID NO: 87 inhibited the binding between NGF and TrkA by 93%, but the binding between NGF and p75 only inhibited by 29%.
- PC-12 cells which are a cell line derived from rat adrenal pheochromocytoma, are model cells of the nervous system, and their processes are elongated by NGF stimulation and differentiated into nerve cells. It was evaluated whether aptamers inhibit this protrusion extension.
- PC-12 cells were seeded in a collagen-coated 96-well flat bottom plate, and a mixed solution of NGF (final concentration 25 ng / mL or 1.9 nM) and aptamer (final concentration 500 nM) pre-reacted at 37 ° C. for 1 hour there.
- the cell culture was started after addition. Thereafter, the same amount of aptamer was added twice every 24 hours, and the degree of protrusion extension was observed and evaluated on the third day under a microscope. Scores 0 to 3 were used for evaluation. Score 0 was no protrusion extension, score 1 was slightly protrusion extension, score 2 was a protrusion extending to a nearby cell, and score 3 was a reticulated extension. A system in which PC-12 cells were cultured for 3 days with addition of NGF alone was used as a negative control, and a system in which the cells were cultured for 3 days without addition of NGF was used as a positive control.
- the aptamer represented by SEQ ID NO: 2 containing no consensus sequence did not show inhibitory activity.
- aptamers represented by SEQ ID NOs: 5 and 6 including a common sequence showed inhibitory activity.
- the aptamer represented by SEQ ID NO: 8 did not contain a common sequence, but showed inhibitory activity. From the above, it was shown that the aptamers represented by SEQ ID NOs: 1, 3 to 9 and 12 can be inhibitors of NGF.
- Table 2 shows aptamers that inhibit neurite outgrowth of PC12 cells. Those having an inhibitory activity of 50% or more were designated as “+”, and those less than 50% were designated as “ ⁇ ”.
- Example 8 Evaluation of Aptamer Physiological Activity Using Neuroscreen-1 Cells
- Neuroscreen-1 cells a subclone of PC12 cells, were used to evaluate aptamer neurite outgrowth inhibitory activity.
- RPMI-1640 medium containing 2.5% horse serum and 1.25% fetal bovine serum for 1 day.
- a mixed solution of NGF (final concentration 15 ng / mL or 1.1 nM) and aptamer (final concentration 500-3 nM) previously reacted in serum-free RPMI-1640 medium at room temperature or 37 ° C. for 30 minutes to 1 hour. was added.
- aptamers are UGAAAAAAAACC (SEQ ID NO: 91), UGAAAGAAAACC (SEQ ID NO: 92), UGAAAGAAAACC (SEQ ID NO: 93), UGAAAGAAACC (SEQ ID NO: 95), UGAAAAAACCC (SEQ ID NO: 96), UGAAAGGAACC (SEQ ID NO: 105), CGAACAAAC (SEQ ID NO: 105) 103), CGAAAGAAACU (SEQ ID NO: 104), and AGAAUGAAACU (SEQ ID NO: 102).
- Table 3 shows the inhibitory activity (%) and 50% inhibitory concentration (IC50) when the aptamer concentrations inhibiting the neurite outgrowth of Neuroscreen-1 cells are 100 nM and 10 nM.
- Example 9 Shortening of an aptamer
- the aptamers represented by SEQ ID NOs: 2, 5, 6, and 8 were shortened.
- the aptamers represented by SEQ ID NOs: 5 and 6 contain a common sequence of UGAAAAAAAACC (SEQ ID NO: 91).
- SEQ ID NOs: 2 and 8 are aptamers that do not contain these consensus sequences.
- the sequence of the variant is as follows.
- SEQ ID NO: 24 69-nucleotide aptamer variant of the aptamer represented by SEQ ID NO: 2 gggaagc (F) c (F) u (F) gu (F) ggagc (F) u (F) gc (F) c (F) u (F) ac (F) ac (F) u (F) u (F) u (F) agu (F) au (F) gac (F) aaac (F) c (F) u (F) agagu (F) gu (F) aaau (F) gc (F) u (F) u (F) c (F) gc (F) au (F) u (F) gu (F) c (F) ac (F) c (F) SEQ ID NO: 25: A modified aptamer represented by SEQ ID NO: 2 and having an aptamer length of 47
- the secondary structure prediction of the aptamer represented by SEQ ID NOs: 31 and 36 is shown in FIG. 14 and FIG.
- the common sequence is indicated by a black circle.
- Aptamers with a length of 40 nucleotides or more were prepared by transcription, and aptamers with a length shorter than that were prepared by chemical synthesis. Whether these nucleic acids inhibit the binding of NGF and NGF receptor was examined by the surface plasmon resonance method in the same manner as in Example 6. As a result, it was found that all these nucleic acids have inhibitory activity (Table 1). Further, the neurite outgrowth inhibitory activity of PC12 cells was examined in the same manner as in Example 7. Strong inhibitory activities were observed in SEQ ID NOs: 28-30, 32, and 35 (Table 2). The aptamer represented by SEQ ID NO: 32 is a product shortened to 38 nucleotides, leaving the common sequence of the aptamer represented by SEQ ID NO: 5.
- the aptamer represented by SEQ ID NO: 35 is a chain shortened to 38 nucleotides, leaving the common sequence of the aptamer represented by SEQ ID NO: 6. From the above, it was shown that the consensus sequence is important for at least SEQ ID NOs: 5 and 6. On the other hand, the aptamer represented by SEQ ID NO: 30 was obtained by shortening the aptamer represented by SEQ ID NO: 8 which does not contain a common sequence, and its activity was confirmed with a length of 41 nucleotides. These aptamers have been shown to be usable as NGF inhibitors.
- Example 10 Modification of shortened aptamer
- the hydroxyl group at the 2 ′ position of ribose was replaced with an o-methyl group. A variant was produced.
- all of these aptamers were found to have strong inhibitory activity.
- the following shows the sequence of each modified product.
- the parentheses in the nucleotide indicate the modification at the 2 'position, F indicates a fluorine atom, M indicates an o-methyl group, and idT indicates inverted dT.
- Sequence number 30 (1): idT-gggau (F) aaaaau (F) a (M) g (M) a (M) g (M) u (F) u (F) u (F) g (M) a (M) a (M) c (F) a (M) c (F) a (M) c (F) c (F) u (F) gu (F) au (F) u (F) aaaac (F) c (F) c (F) -idT SEQ ID NO: 30 (2): gggau (F) aaaa (M) a (M) u (F) agagu (F) u (F) u (F) gau (F) aaac (F) ac (F) c (F) u (F) gu ( F) au (F) u (F) aaaac (F) c (F)
- Example 11 Identification of aptamer NGF binding site by footprinting method
- an enzyme digestion experiment was performed in the absence and presence of NGF. If the consensus sequence is an NGF binding site, it should be enzymatically digested in the absence of NGF, but in the presence of NGF, the nuclease cannot bind to the consensus sequence and should result in no enzymatic digestion.
- Experiments were performed using an aptamer in which a fluorescent substance (FAM6) was bound to the 5 ′ end or 3 ′ end of the aptamer represented by SEQ ID NO: 62.
- FAM6 fluorescent substance
- the aptamer represented by SEQ ID NO: 62 contains a common sequence of UGAAAGAAACC (SEQ ID NO: 92).
- S1 nuclease manufactured by Takara Bio Inc.
- V1 nuclease manufactured by Ambion
- G that selectively cleaves a single strand.
- T1 nuclease Three types were used. Each enzyme reaction was performed under the conditions shown in Table 4 with reference to the attached specifications. 0.833 mM ZnCl 2 was added to the reaction solution of S1 nuclease.
- the aptamer to NGF molar ratio was 1: 2, and dissolved in Solution B, which is a binding buffer.
- the solution B is a mixed solution of 145 mM sodium chloride, 5.4 mM potassium chloride, 1.8 mM calcium chloride, 0.8 mM magnesium chloride, 20 mM Tris (pH 7.6).
- the enzyme reaction the reaction was stopped by treatment with phenol / chloroform, and the water-soluble fraction was collected and concentrated, and then the terminal phosphate was removed with alkaline phosphatase (manufactured by Takara Bio Inc.). The enzyme treatment with alkaline phosphatase was performed at 37 ° C. for 1.5 hours with reference to the attached specifications.
- Example 12 Activity change by introduction of mutation into common sequence portion Mutation was introduced into the common sequence portion, and the activity change was evaluated using Neuroscreen-1 cells in the same manner as in Example 8.
- an aptamer containing the consensus sequence UGAAAAAAAACC SEQ ID NO: 91
- the one having a length of 38 nucleotides represented by SEQ ID NO: 35 was used as an aptamer containing the consensus sequence UGAAAAAAAACC (SEQ ID NO: 91).
- the results are shown in Table 5.
- 300 nM of an aptamer not mutated was added, 92% inhibition of neurite outgrowth was observed.
- 1 nucleotide mutation was introduced, the activity was completely lost.
- the 3rd to 5th A in UGAAAAAAAACC SEQ ID NO: 91
- was replaced with a DNA type the activity was completely lost. From the above, it was shown that the common sequence is important in inhibiting the activity of NGF.
- Table 5 shows the results of the neurite outgrowth inhibition experiment using Neuroscreen-1 cells.
- the aptamer represented by SEQ ID NO: 35 and its mutant were added at 300 nM.
- Pyrimidine nucleotides are fluoro-modified products, purine nucleotides indicate RNA type, and lower case letters indicate DNA type. Mutations were introduced in the underlined part.
- Example 13 Examination of common sequences In this experiment, common sequences always appeared with high frequency in SELEX in which conditions such as pools and primer sequences were changed (Examples 1 to 4).
- SELEX 59 aptamers containing a common sequence of UGAAANNANCY (SEQ ID NO: 107) were present.
- N may be any nucleotide of A, G, C, and U, and U may be T.
- Y represents a pyrimidine nucleotide.
- there were 29 sequences of UGAAAAAAACY SEQ ID NO: 110
- 13 sequences of UGAAAGAAACY (SEQ ID NO: 111).
- Example 14 Mutagenesis into shortened aptamer When mutation was introduced into a shortened aptamer, it was confirmed whether the activity could be retained.
- the aptamer of SEQ ID NO: 30 (6) is 41 nucleotides in length and does not contain a consensus sequence. Inverted dT is added to the 5 ′ and 3 ′ ends.
- the activity was evaluated using Neuroscreen-1 cells as in Example 8. The results are shown in Table 6. When the G1: C41, A10: U33, A12: U31 base pairs in the stem portion predicted by the MFOLD program were replaced with C1: G41, U10: A33, U12: A31, no significant decrease in activity was observed.
- Example 15 Comparison with NGF aptamers described in prior literatures Aptamers represented by SEQ ID NOs: 30, 32, and 35 and described in prior literatures (Binkley J et al., (1995) Nucleic Acids Res. 23, 3198). Of NGF aptamers of NGF were compared with NGF binding activity, NGF and NGF receptor binding inhibitory activity, and neurite outgrowth inhibitory activity. All aptamers described in the prior art are unmodified RNAs and none match the sequences described herein. As aptamers described in the prior literature, H1, L2, and L6 having high binding activity were selected and prepared by transcription with T7 polymerase.
- the binding activity was evaluated in the same manner as in Example 1, the binding inhibition activity of NGF and NGF receptor was evaluated in Example 6, and the neurite outgrowth inhibitory activity was evaluated in the same manner as in Example 8.
- H1, L2, and L6 bind to NGF but have lower activity than the aptamers represented by SEQ ID NOs: 30, 32, and 35 (Table 7).
- H1, L2, and L6 did not inhibit the binding of NGF and NGF receptor, and did not show inhibitory activity even when 500 nM was added in the protrusion elongation inhibition experiment (Table 7). From the above, it was shown that the aptamer described in the present specification has higher activity than the aptamer described in the prior literature.
- Table 7 shows the binding activity of the aptamers represented by SEQ ID NOs: 30, 32, and 35 and the aptamers H1, L2, and L6 described in Non-Patent Document 1 to NGF, inhibition of binding between NGF and NGF receptor, and NGF. Inhibiting activity of neurite outgrowth.
- the binding activity with NGF the maximum RU value when SEQ ID NO: 35 was bound to NGF was evaluated as 100%. “+” Indicates 80% or more, “+” indicates 50% or more, and “ ⁇ ” indicates 50% or less.
- the inhibition of binding between NGF and the NGF receptor “+” represents that the% inhibition activity is 60% or more, and “ ⁇ ” represents less than 60%.
- the inhibitory activity of neurite outgrowth by NGF indicates the inhibitory activity (%) when the final concentration of each aptamer is 500 nM and 250 nM.
- the aptamer and complex of the present invention may be useful as a medicine for diseases such as pain and inflammatory diseases, or as a diagnostic agent or reagent.
- the aptamers and complexes of the present invention may also be useful for NGF purification and enrichment, and NGF detection and quantification.
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Abstract
Description
TrkAのファミリー受容体としてTrkBとTrkCが知られている。TrkBはBDNFおよびNT-4/5と結合し、TrkCはNT-3と結合する。p75はTrkAに比べてリガンド特異性が低く、NGF以外にもBDNF、NT-3、NT-4/5と結合する。p75は一回膜貫通型受容体であるが、細胞質側にチロシンキナーゼドメインはない。TrkA同様、神経細胞だけでなく非神経細胞にも発現している。この受容体は細胞の分化促進や生存維持に関与しているほか、アポトーシスの誘導や細胞遊走とも関係していることが知られている。結晶構造解析の結果から、ホモ二量体のNGFはTrkAと2:2で結合するが、p75とは2:1で結合することが示唆された。ホモ二量体のNGFがTrkAとp75のヘテロ二量体に結合することもある。
Tanezumab(PF-4383119またはRN624)はNGFに対する抗体で、変形性関節炎モデル動物を用いた疼痛モデル実験で効果を示し、現在臨床試験が行われている。また、NGFとNGF受容体の阻害活性の有無は不明であるが、NGFに結合する天然のRNAに関する報告がある(非特許文献1)。
[1] NGFに結合し、NGFとNGF受容体の結合を阻害するアプタマー;
[2] NGFに結合し、NGFの神経細胞突起伸長活性を阻害するアプタマー;
[3] 50%阻害濃度(IC50)が100nM以下である、[2]に記載のアプタマー;
[4] 50%阻害濃度(IC50)が10nM以下である、[2]に記載のアプタマー;
[5] 少なくとも1つのヌクレオチドが修飾されている、[1]~[4]のいずれか一に記載のアプタマー;
[6] HGAANNNANCY(配列番号106)で表される配列を含み、ここでNは任意のヌクレオチド、HはGを除くヌクレオチド、Yはピリミジンヌクレオチドであり、前記配列の少なくとも一つのヌクレオチドが修飾されている、[1]~[4]のいずれか一に記載のアプタマー;
[7] UGAAANNANCY(配列番号107)、CGAANNAAACY(配列番号108)又はAGAANNAAACY(配列番号109)で表される配列を含み、ここでNは任意のヌクレオチド、Yはピリミジンヌクレオチドであり、前記配列の少なくとも一つのヌクレオチドが修飾されている、[1]~[4]のいずれか一に記載のアプタマー;
[8] UGAAAAAAACY(配列番号110)、UGAAAGAAACY(配列番号111)、CGAACAAAACY(配列番号112)又はCGAAAGAAACY(配列番号113)で表される配列を含み、ここでYはピリミジンヌクレオチドであり、前記配列の少なくとも一つのヌクレオチドが修飾されている、[1]~[4]のいずれか一に記載のアプタマー;
[9] 各ピリミジンヌクレオチドのリボースの2’位のヒドロキシル基が、同一又は異なって、無置換であるか、水素原子、フッ素原子及びメトキシ基からなる群より選ばれる原子又は基で置換されている、[5]~[8]のいずれか一に記載のアプタマー;
[10] 各プリンヌクレオチドのリボースの2’位のヒドロキシル基が、同一又は異なって、無置換であるか、水素原子、フッ素原子及びメトキシ基からなる群より選ばれる原子又は基で置換されている、[5]~[8]のいずれか一に記載のアプタマー;
[11] 以下の(a)、(b)又は(c)のいずれかのヌクレオチド配列を含む、[1]に記載のアプタマー:
(a)配列番号1~9、12、24~55、57~90のいずれかから選択されるヌクレオチド配列(但し、ウラシルはチミンであってもよい);
(b)配列番号1~9、12、24~55、57~90のいずれかから選択されるヌクレオチド配列(但し、ウラシルはチミンであってもよい)において、1又は数個のヌクレオチドが置換、欠失、挿入又は付加されたヌクレオチド配列;又は
(c)配列番号1~9、12、24~55、57~90のいずれかから選択されるヌクレオチド配列(但し、ウラシルはチミンであってもよい)と70%以上の同一性を有するヌクレオチド配列;
[12] 少なくとも1つのヌクレオチドが修飾されている、[11]に記載のアプタマー;
[13] 各ピリミジンヌクレオチドのリボースの2’位のヒドロキシル基が、同一又は異なって、無置換であるか、水素原子、フッ素原子及びメトキシ基からなる群より選ばれる原子又は基で置換されている、[12]に記載のアプタマー;
[14] 各プリンヌクレオチドのリボースの2’位のヒドロキシル基が、同一又は異なって、無置換であるか、水素原子、フッ素原子及びメトキシ基からなる群より選ばれる原子又は基で置換されている、[12]に記載のアプタマー;
[15] [1]~[14]のいずれか一に記載のアプタマー及び機能性物質を含む複合体;
[16] 機能性物質が、親和性物質、標識用物質、酵素、薬物送達媒体又は薬物である、[15]に記載の複合体;
[17] [1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体を含む医薬;
[18] [1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体を含む抗疼痛剤;
[19] [1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体を含む抗炎症剤;
[20] [1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体を含む診断薬;
[21] [1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体を含むNGF検出用プローブ;
[22] [1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体を含む、NGF精製用固相担体;
[23] [1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体を用いることを特徴とする、NGFの検出方法;
[24] [1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体を用いることを特徴とする、NGFの精製方法;
[25] [1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体を、それを必要とする対象に投与することを特徴とする、疼痛や炎症を伴う疾患を治療又は予防する方法;
[26] 疼痛や炎症を伴う疾患の治療又は予防用医薬のための、[1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体の使用;
[27] 疼痛や炎症を伴う疾患の治療又は予防用医薬としての使用のための、[1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体;
[28] 疼痛や炎症を伴う疾患を治療又は予防するための、[1]~[14]のいずれか一に記載のアプタマーあるいは[15]又は[16]に記載の複合体の使用;
[29] 疼痛や炎症を伴う疾患に対する治療剤又は予防剤の製造のための、請求項1~14のいずれか一項に記載のアプタマーあるいは請求項15又は16に記載の複合体の使用。
測定にはBIAcore社製のBIAcore2000を用いる。センサーチップにアプタマーを固定化する。固定化量は1000RUとする。0.3MのNaClを含有する生理的な緩衝液(溶液A:実施例1参照)によりNGF溶液(0.5μM)を調製する。このNGF溶液を20μL注入し、NGFのアプタマーへの結合を検出する。40ヌクレオチドからなるランダムなヌクレオチド配列を含むRNAをネガティブコントロールとし、該コントロールRNAと比較してNGFが有意に強くアプタマーに結合した場合、該アプタマーはNGFへの結合能を有すると判定する。
また、他の「NGFに対する阻害活性」としては、NGFがNGF受容体に結合することによって生じる、NGF受容体の下流のシグナル伝達(Ras-MAPキナーゼ経路、PI3キナーゼ経路)の阻害、TRPV1、SP、BDNFなどの発現上昇の阻害、肥満細胞などから放出されるHA、BK、PG、NGF、その他サイトカインの発現の阻害活性などが挙げられる。
更に、NGFにより誘導される神経細胞の分化、生存、神経突起伸長、血管透過性の増大、T細胞やB細胞の免疫応答の増強、リンパ球の分化、肥満細胞や赤白血病細胞、癌細胞など各種細胞の増殖などの阻害、疼痛、痛覚過敏の軽減などが挙げられる。
本発明のアプタマーが有する好ましい「NGFに対する阻害活性」は、NGFがNGF受容体に結合することを阻害する活性であり、NGFにより誘導される神経突起伸長活性を阻害する活性である。
測定にはBIAcore社製のBIAcore2000を用いる。CM5センサーチップにNGF受容体とFcとの融合タンパク質(例えば、Trk A-Fc(175-TK,R&D systems))又はp75-Fc(R&D systems))を固定化する。固定化量は1100RUとする。生理的な緩衝液(溶液A:実施例1参照)中でNGF(0.1μM)とアプタマー(0.33μM)を混合し、30分調製する。この混合液20μLを注入し、NGFのNGF受容体への結合を検出する。阻害活性%が60%以上だった場合、該アプタマーはNGFのNGF受容体への結合を阻害すると判定する。阻害活性%は、アプタマーを含まないNGFとNGF受容体の結合量を0、NGFを含まない溶液をインジェクションした場合の結合量を100として計算される。ここで結合量は、BIAcoreのセンサーグラムのピークトップでのRU値(NGFのインジェクション終了直後のRU値)を意味する。
(a)配列番号1~9、12、24~55、57~90のいずれかから選択されるヌクレオチド配列(但し、ウラシルはチミンであってもよい)を含むアプタマー;
(b)配列番号1~9、12、24~55、57~90のいずれかから選択されるヌクレオチド配列(但し、ウラシルはチミンであってもよい)において1又は数個のヌクレオチドが置換、欠失、挿入又は付加されたヌクレオチド配列を含むアプタマー;
(c)配列番号1~9、12、24~55、57~90のいずれかから選択されるヌクレオチド配列(但し、ウラシルはチミンであってもよい)と70%以上(好ましくは80%以上、より好ましくは90%以上、最も好ましくは95%以上)の同一性を有するヌクレオチド配列を含むアプタマー;或いは
(d)上記(a)の複数の連結物、上記(b)の複数の連結物、上記(c)の複数の連結物、上記(a)、(b)及び(c)の複数の連結物からなる群より選ばれる連結物
であり得る。
また好ましくは、上記(b)~(d)のアプタマーは、NGFに結合し、NGFとNGF受容体との結合を阻害するアプタマー、および/またはNGFに結合し、NGFの神経細胞突起伸長活性を阻害するアプタマーである。
さらに好ましくは、上記(b)~(d)のアプタマーは、NGFの神経細胞突起伸長の阻害濃度が100nM以下であるアプタマーであり、より好ましくは、NGFの神経細胞突起伸長の阻害濃度が10nM以下であるアプタマーである。
連結基としては、-O-、-N-又は-S-が例示され、これらの連結基を通じて隣接するヌクレオチドに結合し得る。
改変はまた、キャッピングのような3’及び5’の改変を含んでもよい。
NGFに特異的に結合するRNAアプタマーはSELEX法を用いて作製した。SELEXはEllingtonらの方法(Ellington and Szostak,Nature 346,818-822,1990)及びTuerkらの方法(Tuerk and Gold,Science 249,505-510,1990)を参考にして行った。標的物質としてヒトNGF(R&D Systems社製)を用いた。
最初のラウンドで用いたRNA(40N-RNA)は、化学合成によって得られたDNAをDuraScribeTMT7 Transcription Kit(Epicentre社製)を用いて転写して得た。この方法によって得られたRNAはピリミジンヌクレオチドのリボースの2’位がフルオロ化されたものである。DNA鋳型として以下に示す40ヌクレオチドのランダム配列の両端にプライマー配列を持った長さ79ヌクレオチドのDNAを用いた。DNA鋳型とプライマーは化学合成によって作製した。
プライマーFwd:5’-taatacgactcactatagggaagcctgtggagctgc-3’(配列番号115)
プライマーRev:5’-ccagttgttggtgacaatgc-3’(配列番号116)
配列番号1:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaaaac(F)aaagac(F)aau(F)gau(F)u(F)gagu(F)agc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号2:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)c(F)u(F)ac(F)ac(F)u(F)u(F)u(F)agu(F)au(F)gac(F)aaac(F)c(F)u(F)agagu(F)gu(F)aaau(F)gc(F)u(F)u(F)c(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号3:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaaau(F)aagu(F)agaaau(F)gac(F)agaau(F)ggc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号4:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaau(F)au(F)gac(F)aaau(F)aaaac(F)ggc(F)aac(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号5:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)u(F)u(F)aaac(F)aagc(F)aagu(F)gaaaaaaac(F)c(F)ac(F)agc(F)aaau(F)gu(F)aaaaagc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号6:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaaau(F)u(F)aaau(F)aaaaaau(F)agac(F)ggu(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号7:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaaau(F)u(F)agau(F)aaaaaau(F)agac(F)ggu(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号8:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)ggau(F)aaaaau(F)agagu(F)u(F)u(F)gau(F)aaac(F)ac(F)c(F)u(F)gu(F)au(F)u(F)aaaac(F)c(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号9:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)u(F)c(F)c(F)ac(F)aaggau(F)gaaaaaaac(F)c(F)c(F)aaau(F)aau(F)au(F)au(F)u(F)u(F)aau(F)c(F)agc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号10:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaau(F)u(F)aaagagc(F)u(F)u(F)gac(F)aaaac(F)au(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号11:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)u(F)c(F)c(F)ac(F)aaggau(F)gaaaaaaaac(F)c(F)c(F)aaau(F)aau(F)au(F)au(F)u(F)u(F)aau(F)c(F)agc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号12:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)gaaac(F)agu(F)gaaac(F)aaac(F)c(F)ac(F)agac(F)u(F)gagaaagc(F)agu(F)aac(F)agc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号13:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaaau(F)u(F)aaau(F)aaaaaaaaau(F)ggac(F)ggu(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号14:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)u(F)gaau(F)u(F)ggau(F)ac(F)agau(F)agu(F)u(F)gaaaaaaac(F)c(F)aau(F)gau(F)c(F)agc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号15:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)u(F)c(F)c(F)ac(F)aaggau(F)gaaaaaaac(F)c(F)c(F)aaau(F)aau(F)au(F)au(F)u(F)u(F)gau(F)c(F)agc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号16:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)u(F)c(F)c(F)ac(F)aaggau(F)gaaaaaaac(F)c(F)c(F)c(F)aaau(F)aau(F)gu(F)au(F)u(F)u(F)aau(F)c(F)agc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号17:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)ggaaaaaac(F)c(F)c(F)aaaau(F)aagu(F)agaaau(F)gac(F)agaau(F)ggc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号18:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)c(F)gaaau(F)ggac(F)u(F)gu(F)aaagc(F)au(F)gaaaaaaac(F)c(F)au(F)u(F)c(F)aau(F)c(F)gaggc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号19:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaac(F)u(F)aaagu(F)u(F)u(F)aaaac(F)u(F)gau(F)ac(F)gagc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号20:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaau(F)u(F)aaaaac(F)u(F)u(F)gc(F)c(F)gagc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号21:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaaac(F)c(F)c(F)aaaaac(F)aaagac(F)aac(F)gau(F)u(F)gagu(F)agc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号22:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaaau(F)u(F)gu(F)c(F)c(F)ac(F)agaaaau(F)ggau(F)u(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
配列番号23:
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)gaaac(F)agu(F)gaaac(F)aaac(F)c(F)ac(F)agac(F)u(F)gagaaagc(F)agu(F)aaaagc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)aac(F)aac(F)u(F)gg
ランダム配列が30ヌクレオチドでプライマー配列を実施例1で用いたものと変えた鋳型を用いて、実施例1と同様のSELEXをおこなった。使用した鋳型とプライマーの配列を以下に示す。鋳型には30ヌクレオチドのランダム配列を用いた。DNA鋳型とプライマーは化学合成によって作製した。
プライマーFwd:5’-cggaattctaatacgactcactatagggagaacttcgaccagaag-3’(配列番号118)
プライマーRev:5’-tgaggatccatgtatgcgcacata-3’(配列番号119)
また、一次配列は少し異なるが、MFOLDプログラムにより同じようなバルジ構造が予想された配列としてAGAAUGAAACU(配列番号102)が存在した。
それらの一部の配列を配列番号37~42に示す。
それらの配列の一部を配列番号43~53に示す。
これらの配列の一部を配列番号54~59に示す。
これらの配列の一部を配列番号60~68に示す。
配列番号37:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaagaaac(F)c(F)c(F)aaaggu(F)gaaac(F)aac(F)aau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号38:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagagaau(F)gaaac(F)u(F)c(F)c(F)ac(F)aaagu(F)ac(F)au(F)aaaac(F)au(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号39:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gu(F)gaaaagaac(F)c(F)c(F)aaau(F)aaaac(F)aac(F)aau(F)gu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号40:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaaaaaac(F)c(F)c(F)aggaaaau(F)ggaagac(F)gu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号41:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaaggaac(F)c(F)c(F)aaagc(F)gaaac(F)aaaac(F)gu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号42:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaaaaaac(F)c(F)c(F)aaaagagc(F)agc(F)agagau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号43:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagc(F)u(F)u(F)gaaaaaac(F)c(F)c(F)c(F)aau(F)au(F)gagaau(F)c(F)au(F)au(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号44:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaagaaac(F)c(F)c(F)aaaau(F)u(F)agc(F)ac(F)c(F)au(F)aau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号45:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagagaau(F)gaaac(F)u(F)c(F)c(F)c(F)aaau(F)c(F)aaggac(F)aau(F)gau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号46:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaac(F)aaac(F)c(F)c(F)aaagu(F)u(F)ac(F)gc(F)ac(F)aaaau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号47:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagaagu(F)u(F)u(F)gaaaagaac(F)c(F)c(F)aaaau(F)gagc(F)aaaau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号48:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaaagaac(F)c(F)c(F)gaaaaac(F)gc(F)au(F)aau(F)aau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号49:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gaaagaaac(F)u(F)c(F)c(F)c(F)aagac(F)ggu(F)aac(F)gaaagu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号50:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gaaaaaac(F)c(F)u(F)c(F)c(F)c(F)aau(F)ac(F)aaac(F)ac(F)aaaaau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号51:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaagaaac(F)c(F)c(F)aaaaaaac(F)aac(F)au(F)au(F)gaac(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号52:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaagaaac(F)c(F)c(F)aaau(F)au(F)ac(F)aaaac(F)ac(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号53:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaaggaac(F)c(F)c(F)aaaaac(F)ac(F)aaaau(F)gu(F)c(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号54:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)c(F)gaaagu(F)gaaagaaac(F)u(F)c(F)c(F)aac(F)gaaagc(F)au(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号55:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaagaaac(F)c(F)c(F)aaaaau(F)gaau(F)gc(F)aac(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggac(F)c(F)u(F)c(F)a
配列番号56:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gaaagaaac(F)u(F)c(F)c(F)c(F)aac(F)ac(F)aaau(F)gc(F)ac(F)aac(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号57:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaaaaaac(F)c(F)c(F)aaac(F)ac(F)c(F)gaagc(F)ac(F)aaau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号58:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaaagaac(F)c(F)c(F)aaau(F)ac(F)agaau(F)aaau(F)gu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号59:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)c(F)gaaac(F)gu(F)u(F)u(F)gaaaaaaac(F)c(F)c(F)aaggaggau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号60:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagau(F)u(F)u(F)gaaaaaaac(F)c(F)c(F)gaau(F)aaagau(F)aac(F)agu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号61:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaaggu(F)c(F)gu(F)aac(F)gaau(F)aaaac(F)u(F)c(F)c(F)u(F)gc(F)ac(F)aaaaau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号62:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaagaaac(F)c(F)c(F)aaau(F)u(F)aaagu(F)gaac(F)agu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号63:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagau(F)u(F)u(F)gaaagaaac(F)c(F)c(F)aaac(F)u(F)aagc(F)ac(F)aaaau(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号64:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaagaaac(F)c(F)c(F)aaaac(F)au(F)u(F)agc(F)ac(F)ac(F)au(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号65:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gu(F)gaaaaaaac(F)c(F)c(F)aaau(F)c(F)gagc(F)ac(F)aaaau(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号66:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)u(F)gaaaaaaac(F)c(F)c(F)aaagc(F)aagc(F)ac(F)aac(F)au(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号67:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)c(F)gau(F)aac(F)gaac(F)aaaac(F)u(F)c(F)c(F)c(F)aaaggaau(F)au(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
配列番号68:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)c(F)gagagc(F)gaaagaaac(F)u(F)c(F)c(F)c(F)aaaac(F)ac(F)agu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)a
プリンヌクレオチドがRNA、ピリミジンヌクレオチドがDNAのモザイクアプタマーをSELEX法により作製した。鋳型は40ヌクレオチドのランダム配列を含みプライマーが実施例1と2で用いたものと異なるものを使用した。最初のラウンドで用いたRNA-DNAモザイク核酸のプールは、化学合成によって得られたDNAを鋳型とし、rATP、rGTP、dCTP、dTTPを基質として転写して得た。ここでrNTPはリボヌクレオチド、dNTPはデオキシリボヌクレオチドを示す。その他の実験方法は実施例1で示した方法とほぼ同じである。用いた鋳型とプライマー配列を以下に示す。
プライマーFwd:5’-taatacgactcactatagggagaggcaagaatagggc-3’(配列番号121)
プライマーRev:5’-tcctaatgtctcttctcttcac-3’(配列番号122)
配列番号69:
gggagaggCaagaaTagggCCCagCTgaaaaaaaCCTggaCgTaCaCCgTTCgCCgagCgggTgaagagaagagaCaTTagga
配列番号70:
gggagaggCaagaaTagggCTggaaaTagaaCCgCgCTgTCTTCaTTaagCCgCCCaaCggTgaagagaagagaCaTTagga
配列番号71:
gggagaggCaagaaTagggCaCTTgaaaaaaaCCCaaaTTTaCCgTCTTCagCgTCgggTgTgaagagaagagaCaTTagga
配列番号72:
gggagaggCaagaaTagggCTggaTgggCagTaaCCTgaaaaaaaCCaCCCaCCTCTaCCgTgaagagaagagaCaTTagga
配列番号73:
gggagaggCaagaaTagggCaCTTgaaaaaaaCCCaaagaaagaaTaCTTaCCCggCgCgTgaagagaagagaCaTTagga
配列番号74:
gggagaggCaagaaTagggCaTagTgTagaCCCCTCTCaagaTaCCCCaTgaaTTgCCCCgTgaagagaagagaCaTTagga
配列番号36で表される配列に30%のランダム配列をドープし、その両端に新しいプライマー配列を付加したRNAプールを用いてSELEXをおこなった。SELEXは実施例1とほぼ同様におこなった。その鋳型とプライマーの配列を以下に示す。
5’- GAGGATCCATGTATGCGCACATAgggtttttttcatcctgcagctccacaggcttcccCTTCTGGTCGAAGTTCT-3’
a:a(70%), g(10%), c(10%), t(10%)
g:g(70%), a(10%), c(10%), t(10%)
c:c(70%), a(10%), g(10%), t(10%)
t:t(70%), a(10%), c(10%), g(10%)
(配列番号123)
プライマーFwd:
5’-CGGAATTCTAATACGACTCACTATAGGGAGAACTTCGACCAGAAG-3’(配列番号124)
プライマーRev:5’-GAGGATCCATGTATGCGCACATA-3’ (配列番号125)
また、UGAAAAAAACC(配列番号91)の共通配列に変異が入ったUGAAAGAAACC(配列番号92)、UGAAAGAAACU(配列番号93)、UGAAAACAACC(配列番号98)、UGAAAUAAACC(配列番号99)、UGAAAUAAACU(配列番号100)、UGAAAAAAUCU(配列番号101)なども存在した。
その中から12配列をランダムに選び、表面プラズモン共鳴法でNGFに対する結合活性を調べた。測定方法は実施例1に示した通りである。測定の結果、これら12配列の全てが、30%のランダム配列をドープした最初の鋳型よりも有意にNGFに結合することがわかった。以下に各配列番号に対応する実際に得られたヌクレオチド配列を示す。
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gaaagaau(F)c(F)u(F)c(F)c(F)aaagac(F)aagau(F)aaaaac(F)aac(F)c(F)gu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号76:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaaggau(F)aaac(F)gc(F)au(F)gu(F)au(F)u(F)u(F)gc(F)agu(F)au(F)u(F)aaaaau(F)gc(F)c(F)u(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号77:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gaaaaaau(F)c(F)u(F)c(F)c(F)agu(F)u(F)gc(F)aagac(F)gaaac(F)aaac(F)c(F)u(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号78:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gu(F)gu(F)au(F)u(F)gu(F)u(F)c(F)agggu(F)gu(F)gc(F)c(F)c(F)agc(F)c(F)u(F)au(F)aac(F)c(F)au(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号79:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaaggau(F)agc(F)c(F)au(F)gu(F)ggaggu(F)gaagac(F)u(F)gaaau(F)aaac(F)c(F)au(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号80:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gaaaac(F)aac(F)c(F)u(F)c(F)c(F)c(F)aau(F)aau(F)gau(F)c(F)ac(F)agaaau(F)c(F)c(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号81:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaaggagau(F)gac(F)u(F)gu(F)gu(F)aac(F)c(F)ac(F)agu(F)au(F)gaaau(F)aaac(F)u(F)c(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号82:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagaggau(F)gc(F)u(F)u(F)gu(F)u(F)u(F)ggu(F)u(F)ac(F)aagc(F)u(F)gaaagaaac(F)c(F)u(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号83:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)u(F)gaagc(F)u(F)u(F)gaaaaaaac(F)c(F)c(F)aggau(F)u(F)aaac(F)agac(F)agu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号84:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagu(F)gaaagaaac(F)u(F)c(F)c(F)c(F)gau(F)gaaagau(F)gu(F)aac(F)aaac(F)c(F)au(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号85:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaagc(F)ggaagc(F)c(F)u(F)gc(F)gu(F)aac(F)c(F)gc(F)aggau(F)gaaaac(F)aac(F)c(F)gu(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
配列番号86:
gggagaac(F)u(F)u(F)c(F)gac(F)c(F)agaaggagu(F)agc(F)c(F)agu(F)gaac(F)c(F)u(F)ggaau(F)au(F)gaaaaaaac(F)c(F)u(F)u(F)au(F)gu(F)gc(F)gc(F)au(F)ac(F)au(F)ggau(F)c(F)c(F)u(F)c(F)
NGFに特異的に結合するDNAアプタマーはSELEX法を用いて作製した。SELEX法はFitzwaterとPoliskyらの方法(Fitzwater and Polisky,Methods Enzymol.267,275-301,1996)を改良して行った。標的物質として実施例1で用いたヒトNGFを用いた。最初のラウンドのプールには、40ヌクレオチドのランダム配列の両端にプライマー配列を付加した長さ71のDNA(40N-DNA)を用いた。一本鎖DNAを得るために、プライマーRevの5’末端にはビオチン(bio)を付加した。
プライマーFwd:5’-GGGATCGACAGGGCT-3’(配列番号127)
プライマーRev:bio-AGATGGCACGACTCGG-3’(配列番号128)
GGGATCGACAGGGCTGCAGCACTGGCGTAGGTTGGAATATGGGTATTTTTGTGGTCCGAGTCGTGCCATCT
配列番号1~9、12、37~55、57~87で表されるアプタマーがNGFとNGF受容体(TrkAおよびP75)の結合を阻害するかどうか表面プラズモン共鳴法を用いて調べた。BIAcore社のプロトコールに従って、CM5センサーチップにProtein A(21181,PIERCE)を固定化した。そこに、IgGのFc部分が融合したヒトTrk A-Fc(175-TK,R&D systems)を約1100RU固定化した。アナライトとしてNGF(0.1μM)とアプタマー(0.33μM)を混合して30分保持したものをインジェクションした。もしアプタマーがNGFとTrkAの結合を阻害する場合はセンサーグラムのシグナルは上がらないが、もし阻害しない場合は三者複合体を形成しシグナルが上がることが予想される。また、NGFがアプタマーよりも受容体に強く結合する場合は、アプタマーがはずれて、NGFが受容体と結合する場合もある。阻害実験を開始する前にTrkAにNGFが結合することを確認した。アプタマーを含まないNGFとNGF受容体の結合量を100とし、アプタマーを添加した場合のNGFとNGF受容体の結合量を補正値として求めた。ここで結合量は、BIAcoreのセンサーグラムのピークトップでのRU値(NGFのインジェクション終了直後のRU値)とした。100からこの補正値を引いた値を阻害活性%とし、60%以上を阻害活性ありとした。実験の結果、配列番号1~9、12、37~55、57~87で表されるアプタマーの全てがNGFとTrkAの結合を阻害することがわかった(表1)。一例として配列番号6で表されるアプタマーがNGFとTrkAの結合を阻害している様子を図12に示す。もう一つの受容体P75(p75-Fc;R&D systems)に対しても同様な実験をおこなった。その結果、配列番号1~9、12、37~55、57~87で表されるアプタマーの全てがNGFとP75の結合を60%以上阻害することがわかった(表1)。一例として配列番号6で表されるアプタマーがNGFとP75の結合を阻害している様子を図13に示す。
PC-12細胞を用いた突起伸長抑制実験でアプタマーの生理活性を評価した。ラット副腎褐色細胞腫由来の細胞株であるPC-12細胞は、神経系のモデル細胞であり、NGF刺激によって突起を伸長し、神経細胞様に分化する。この突起伸長をアプタマーが阻害するか否か評価した。PC-12細胞をコラーゲンコートした96ウェル平底プレートに播種し、そこに1時間37℃で予め反応させたNGF(最終濃度25ng/mLまたは1.9nM)とアプタマー(最終濃度500nM)の混合溶液を添加し細胞培養を開始した。その後24時間おきに2回、同量のアプタマーを添加し、3日目に突起伸長の程度を顕微鏡で観察し評価した。評価にはスコア0~3を用い、スコア0は突起伸長なし、スコア1はわずかに突起伸長あり、スコア2は近傍の細胞まで突起が伸長、スコア3は突起伸長が著しく網状、とした。NGFのみの添加でPC-12細胞を3日間培養する系をネガティブコントロールとし、NGF無添加で同細胞を3日間培養する系をポジティブコントロールとした。また、NGF阻害剤によって突起伸長が抑制されることを確認するために、コントロールのNGF阻害剤として133nMの抗NGF抗体(MAB2561,R&D Systems)をNGFと共に添加して同細胞を3日間培養し、突起伸長が抑制されることを確認した。ネガティブコントロールのスコアを阻害活性0%、ポジティブコントロールのスコアを阻害活性100%と設定し、アプタマーの阻害活性%を算出した。その結果を表2に示す。阻害活性が50%以上を+、50%未満を-と表記した。配列番号1、3~9および12で表されるアプタマーが突起伸長を顕著に阻害することが明らかとなった(表2)。共通配列を含まない配列番号2で表わされるアプタマーは阻害活性を示さなかった。一方、共通配列を含む配列番号5や6などで表わされるアプタマーは阻害活性を示した。配列番号8で表わされるアプタマーは共通配列を含んでいないが阻害活性を示した。以上より、配列番号1、3~9および12で表されるアプタマーはNGFの阻害剤となり得ることが示された。
PC12細胞のサブクローンであるNeuroscreen-1細胞を用いて、アプタマーの神経突起伸長阻害活性を評価した。コラーゲンタイプIVでコートした96ウェル平底プレートに1ウェルあたり2500個の細胞を2.5%ウマ血清と1.25%胎児ウシ血清を含むRPMI-1640培地で1日培養した。そこに室温もしくは37℃にて30分間から1時間無血清のRPMI-1640培地中で予め反応させたNGF(最終濃度15ng/mLまたは1.1nM)とアプタマー(最終濃度500~3nM)の混合溶液を添加した。2日後もしくは3日後にCellomics Neurite Outgrowth Kits(Thermo Scientific社製)を使用して細胞質と核を染色し、Cellomics ArrayScan VTI(Thermo Scientific社製)によって1細胞あたりの神経突起長を測定した。NGFのみの添加で細胞を2日間培養して得られた1細胞あたりの神経突起長を阻害活性0%、NGF無添加で2日間培養して得られた1細胞あたりの神経突起長を阻害活性100%として、NGFとアプタマーを混合添加した場合に得られた1細胞あたりの神経突起長から、アプタマーの阻害活性を算出した。アプタマー濃度が100nMと10nMの場合の阻害活性と50%阻害濃度(IC50)を表3に示す。阻害活性が0%以下の場合は0%とした。100%以上の場合は100%とした。50%阻害濃度は、50%阻害活性を挟む上下二点の濃度より求めた。IC50値を<と記載したものは、測定最低濃度においても阻害活性が50%以上であった場合で、表示数字は最低測定濃度を示す。実験の結果、IC50が10nM以下の高い活性を有するアプタマーが存在することがわかった。
これらのアプタマーはUGAAAAAAACC(配列番号91)、UGAAAGAAACC(配列番号92)、UGAAAGAAACU(配列番号93)、UGAAAAGAACC(配列番号95)、UGAAAAAACCC(配列番号96)、UGAAAGGAACC(配列番号105)、CGAACAAAACU(配列番号103)、CGAAAGAAACU(配列番号104)、AGAAUGAAACU(配列番号102)、の共通配列を含んでいた。UGAAAAAAACC(配列番号91)とUGAAAGAAACC(配列番号92)の共通配列を含むアプタマーはそれぞれ6種類および5種類存在した。
配列番号2、5、6、8で表されるアプタマーの短鎖化を行った。配列番号5、6で表されるアプタマーはUGAAAAAAACC(配列番号91)の共通配列を含む。配列番号2および8はこれらの共通配列を含まないアプタマーである。改変体の配列は以下の通りである。
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)c(F)u(F)ac(F)ac(F)u(F)u(F)u(F)agu(F)au(F)gac(F)aaac(F)c(F)u(F)agagu(F)gu(F)aaau(F)gc(F)u(F)u(F)c(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)
配列番号25:配列番号2で表されるアプタマーの改変体で47ヌクレオチドの長さのアプタマー
ggagc(F)u(F)gc(F)c(F)u(F)ac(F)ac(F)u(F)u(F)u(F)agu(F)au(F)gac(F)aaac(F)c(F)u(F)agagu(F)gu(F)aaau(F)gc(F)u(F)u(F)c(F)
配列番号26:配列番号5で表されるアプタマーの改変体で46ヌクレオチドの長さのアプタマー
gggc(F)u(F)gu(F)ggagc(F)u(F)gc(F)u(F)u(F)aaac(F)aagc(F)aagu(F)gaaaaaaac(F)c(F)ac(F)agc(F)c(F)c(F)
配列番号27:配列番号6で表されるアプタマーの改変体で45ヌクレオチドの長さのアプタマー
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaaau(F)u(F)aaau(F)
配列番号28:配列番号6で表されるアプタマーの改変体で40ヌクレオチドの長さのアプタマー
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaaau(F)
配列番号29:配列番号8で表されるアプタマーの改変体で61ヌクレオチドの長さのアプタマー
ggu(F)ggagc(F)u(F)gc(F)ggau(F)aaaaau(F)agagu(F)u(F)u(F)gau(F)aaac(F)ac(F)c(F)u(F)gu(F)au(F)u(F)aaaac(F)c(F)gc(F)au(F)u(F)gu(F)c(F)ac(F)c(F)
配列番号30:配列番号8で表されるアプタマーの改変体で41ヌクレオチドの長さのアプタマー
gggau(F)aaaaau(F)agagu(F)u(F)u(F)gau(F)aaac(F)ac(F)c(F)u(F)gu(F)au(F)u(F)aaaac(F)c(F)c(F)
配列番号31:配列番号26で表されるアプタマーの改変体で34ヌクレオチドの長さのアプタマー
gggagc(F)u(F)gc(F)u(F)u(F)aaac(F)aagc(F)aagu(F)gaaaaaaac(F)c(F)c(F)
配列番号32:配列番号26で表されるアプタマーの改変体で38ヌクレオチドの長さのアプタマー
u(F)gu(F)ggagc(F)u(F)gc(F)u(F)u(F)aaac(F)aagc(F)aagu(F)gaaaaaaac(F)c(F)ac(F)a
配列番号33:配列番号26で表されるアプタマーの改変体で36ヌクレオチドの長さのアプタマー
u(F)gu(F)ggagc(F)u(F)gc(F)u(F)aaac(F)agc(F)aagu(F)gaaaaaaac(F)c(F)ac(F)a
配列番号34:配列番号26で表されるアプタマーの改変体で34ヌクレオチドの長さのアプタマー
gu(F)ggagc(F)u(F)gu(F)u(F)aaac(F)aac(F)aagu(F)gaaaaaaac(F)c(F)ac(F)
配列番号35:配列番号28で表されるアプタマーの改変体で38ヌクレオチドの長さのアプタマー
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)aaa
配列番号36:配列番号28で表されるアプタマーの改変体で35ヌクレオチドの長さのアプタマー
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)
配列番号88:配列番号36で表されるアプタマーの改変体で33ヌクレオチドの長さのアプタマー
gggaagc(F)c(F)gu(F)ggagc(F)u(F)gc(F)ggau(F)gaaaaaaac(F)c(F)c(F)
配列番号89:配列番号36で表されるアプタマーの改変体で34ヌクレオチドの長さのアプタマー
gggaagc(F)c(F)u(F)gu(F)aaac(F)agc(F)aggau(F)gaaaaaaac(F)c(F)c(F)
配列番号90:配列番号36で表されるアプタマーの改変体で32ヌクレオチドの長さのアプタマー
gggagc(F)c(F)u(F)gu(F)aaac(F)agc(F)aggu(F)gaaaaaaac(F)c(F)c(F)
また、PC12細胞の神経突起伸長阻害活性を実施例7と同様に調べたが、配列番号28~30、32、35に強い阻害活性が認められた(表2)。
配列番号32で表わされるアプタマーは配列番号5で表わされるアプタマーの共通配列を残して38ヌクレオチドまで短鎖化したものである。また、配列番号35で表わされるアプタマーは配列番号6で表わされるアプタマーの共通配列を残して38ヌクレオチドまで短鎖化したものである。以上より、少なくとも配列番号5および6に関しては共通配列が重要であることが示された。
一方、配列番号30で表わされるアプタマーは、共通配列を含まない配列番号8で表わされるアプタマーを短鎖化したものであり、41ヌクレオチドの長さで活性が確認された。これらのアプタマーはNGF阻害剤として使用可能であることが示された。
配列番号30、32、35で表されるアプタマーの血液中での安定性を高めるために、リボースの2’位の水酸基をo-メチル基に置き換えた改変体を作製した。実施例7と同様に、PC12細胞の神経突起伸長阻害を調べたところ、これらのアプタマー全てに強い阻害活性があることがわかった。
idT-gggau(F)aaaaau(F)a(M)g(M)a(M)g(M)u(F)u(F)u(F)g(M)a(M)u(F)a(M)a(M)a(M)c(F)a(M)c(F)c(F)u(F)gu(F)au(F)u(F)aaaac(F)c(F)c(F)-idT
配列番号30(2):
gggau(F)aaaa(M)a(M)u(F)agagu(F)u(F)u(F)gau(F)aaac(F)ac(F)c(F)u(F)gu(F)au(F)u(F)aaaac(F)c(F)c(F)
配列番号30(3):
gggau(F)aaaaau(F)agagu(F)u(F)u(F)gau(F)aaac(F)ac(F)c(F)u(F)gu(F)au(F)u(F)a(M)a(M)aac(F)c(F)c(F)
配列番号30(4):
idT-gggau(F)aaaa(M)a(M)u(F)a(M)g(M)a(M)g(M)u(F)u(F)u(F)g(M)a(M)u(F)a(M)a(M)a(M)c(F)a(M)c(F)c(F)u(F)gu(F)au(F)u(F)a(M)a(M)aac(F)c(F)c(F)-idT
配列番号30(5):
idT-gggau(F)aaaa(M)a(M)u(F)a(M)g(M)a(M)g(M)u(F)u(F)u(F)g(M)a(M)u(F)a(M)a(M)a(M)c(F)a(M)c(F)c(F)u(F)gu(F)a(F)u(F)u(F)a(M)a(M)a(F)a(F)c(F)c(F)c(F)-idT
配列番号30(6):
idT-g(M)g(M)g(M)au(F)a(M)aa(M)a(M)a(M)u(F)a(M)g(M)a(M)g(M)u(F)u(F)u(F)g(M)a(M)u(F)a(M)a(M)a(M)c(F)a(M)c(F)c(F)u(F)gu(F)a(M)u(F)u(F)a(M)a(M)a(F)a(F)c(F)c(F)c(F)-idT
配列番号32(1):
idT-u(F)gu(F)ggagc(F)u(F)g(M)c(F)u(F)u(F)a(M)a(M)a(M)c(F)a(M)a(M)g(M)c(F)a(M)a(M)g(M)u(F)gaaaaaaac(F)c(F)ac(F)a-idT
配列番号32(2):
u(F)g(M)u(F)ggagc(F)u(F)gc(F)u(F)u(F)aaac(F)aagc(F)aagu(F)gaaaaaaac(F)c(F)ac(F)a(M)
配列番号32(3):
u(F)gu(F)ggagc(F)u(F)gc(F)u(F)u(F)aaac(F)aagc(F)aagu(F)gaaaa(M)a(M)aac(F)c(F)ac(F)a
配列番号32(4):
u(F)gu(F)gga(M)gc(F)u(F)gc(F)u(F)u(F)aaac(F)aagc(F)aagu(F)gaaaaaaac(F)c(F)ac(F)a
配列番号32(5):
idT-u(F)g(M)u(F)gga(M)gc(F)u(F)g(M)c(F)u(F)u(F)a(M)a(M)a(M)c(F)a(M)a(M)g(M)c(F)aagu(F)gaaaa(M)a(M)a(M)a(M)c(F)c(F)ac(F)a-idT
配列番号32(6):
idT-u(F)g(M)u(F)g(M)ga(M)gc(F)u(F)g(M)c(F)u(F)u(F)a(M)a(M)a(M)c(F)a(M)a(M)g(M)c(F)a(M)a(M)g(M)u(F)gaaaa(M)a(M)a(M)a(M)c(F)c(F)ac(F)a-idT
配列番号35(1):
idT-ggga(M)a(M)g(M)c(F)c(F)u(F)g(M)u(F)g(M)g(M)a(M)g(M)c(F)u(F)g(M)c(F)a(M)g(M)g(M)au(F)gaaaaaaac(F)c(F)c(F)aaa-idT
配列番号35(2):
idT-ggga(M)a(M)g(M)c(F)c(F)u(F)g(M)u(F)ggagc(F)u(F)g(M)c(F)a(M)g(M)g(M)au(F)gaaaa(M)a(M)a(M)a(M)c(F)c(F)c(F)aaa-idT
配列番号35(3):
gggaagc(F)c(F)u(F)gu(F)ggagc(F)u(F)gc(F)aggau(F)gaaaaaaac(F)c(F)c(F)a(M)a(M)a(M)
配列番号35(4):
idT-ggga(M)a(M)g(M)c(F)c(F)u(F)g(M)u(F)g(M)g(M)a(M)g(M)c(F)u(F)g(M)c(F)a(M)g(M)g(M)au(F)gaaaa(M)a(M)a(M)a(M)c(F)c(F)c(F)a(M)a(M)a(M)-idT
配列番号35(5):
idT-g(M)g(M)ga(M)a(M)g(M)c(F)c(F)u(F)g(M)u(F)g(M)g(M)a(M)g(M)c(F)u(F)g(M)c(F)a(M)g(M)g(M)au(F)gaaaa(M)a(M)a(M)a(M)c(F)c(F)c(F)a(M)a(M)a(M)-idT
配列番号35(6):
idT-g(M)g(M)ga(M)a(M)g(M)c(F)c(F)u(F)g(M)u(F)g(M)g(M)a(M)g(M)c(F)u(F)g(M)c(F)a(M)g(M)g(M)a(F)u(F)gaaaa(M)a(M)a(M)a(M)c(F)c(F)c(F)a(M)a(M)a(M)-idT
共通配列がNGFの結合部位であることを確認するために、NGF非存在下および存在下で酵素消化実験を行った。共通配列がNGFの結合部位である場合、NGF非存在下では酵素消化されるが、NGF存在下ではヌクレアーゼが共通配列に結合することができないので、酵素消化されないという結果を得るはずである。配列番号62で表わされるアプタマーの5’末端または3’末端に蛍光物質(FAM6)を結合したアプタマーを用いて実験を行った。配列番号62で表わされるアプタマーはUGAAAGAAACC(配列番号92)の共通配列を含んでいる。ヌクレアーゼとしては、一本鎖を選択的に切断するS1ヌクレアーゼ(タカラバイオ社製)、二本鎖を選択的に切断するV1ヌクレアーゼ(アンビオン社製)、一本鎖のGを選択的に切断するT1ヌクレアーゼ(アンビオン社製)の3種類を用いた。各酵素反応は添付の仕様書を参考にして、表4の条件で行った。S1ヌクレアーゼの反応溶液には0.833mMのZnCl2を添加した。
酵素反応後、フェノール・クロロホルム処理により反応を停止し、水溶性画分を回収して濃縮した後、アルカリフォスファターゼ(タカラバイオ社製)により末端のリン酸を取り除いた。アルカリホスファターゼによる酵素処理は、添付の仕様書を参考にして、37℃で1.5時間行った。これらのサンプルは20%の変性ポリアクリルアミド電気泳動法により分析した。蛍光検出にはStorm850(GEヘルスケアー社製)を用いた。実験の結果、NGF非存在下ではUGAAAGAAACC(配列番号92)のGAAAGAがS1およびT1ヌクレアーゼにより切断された。一方、NGF存在下ではこれらの切断は顕著に抑制された。ここで、ピリミジンヌクレオチドはフルオロ修飾体である。以上より、共通配列部分はNGFの結合部位であることが示された。
共通配列の部分に変異を導入し、活性の変化を実施例8と同様にNeuroscreen-1細胞を用いて評価した。共通配列UGAAAAAAACC(配列番号91)を含むアプタマーとして配列番号35で表わされる38ヌクレオチド長のものを用いた。その結果を表5に示す。
変異導入していないアプタマーを300nM添加した場合、92%の神経突起伸長の阻害が見られた。一方、1ヌクレオチドの変異を導入すると活性は完全に消失した。また、UGAAAAAAACC(配列番号91)の3~5番目のAをDNAタイプに置換すると活性が完全に消失した。以上より、共通配列はNGFの活性を阻害する上で重要であることが示された。
本実験において、プールやプライマー配列など、条件を変えたSELEXで常に高頻度に共通配列が出現した(実施例1~4)。SELEXで得られた74配列中、UGAAANNANCY(配列番号107)の共通配列を含むアプタマーは59配列存在した。ここで、NはA、G、C、Uの任意のヌクレオチドで、UはTであってもよい。Yはピリミジンヌクレオチドを示す。このうち、UGAAAAAAACY(配列番号110)は29配列、UGAAAGAAACY(配列番号111)は13配列存在した。
一方、一般式CGAANNAAACY(配列番号108)およびAGAANNAAACY(配列番号109)で表わされる共通配列もそれぞれ3個と2個存在していた。このうち、CGAACAAAACY(配列番号112)は1配列、CGAAAGAAACY(配列番号113)は1配列存在した。これらはHGAANNNANCY(配列番号106)という一般式で表示できる。
これらの共通配列は、38ヌクレオチド長の短鎖化体に必要であること(実施例9)、酵素消化の実験でNGFを添加することで保護されること(実施例11)、変異導入で生理活性が大きく低下すること(実施例12)からも、NGFの機能を阻害する上で重要であることがわかる。
短鎖化したアプタマーに変異を導入した場合、活性が保持できるかどうか確認した。配列番号30(6)のアプタマーは41ヌクレオチドの長さで共通配列を含んでいない。5’及び3’末端はinverted dTが付加している。活性は実施例8と同様にNeuroscreen-1細胞を用いて評価した。結果を表6に示す。
MFOLDプログラムで予測されたステム部分のG1:C41、A10:U33、A12:U31塩基対をC1:G41、U10:A33、U12:A31に入れ替えたところ、活性の顕著な低下は見られなかった。また、ループ部分のG20とG23をA20とA23に置換したところ、同様に活性の顕著な低下は見られなかった。以上より、配列番号30(6)で表わされるアプタマーは数個の変異が入っても活性が保持されていることが示された。
配列番号30、32、35で表わされるアプタマーと、先行文献(Binkley J et al.,(1995)Nucleic Acids Res.23, 3198)に記載のNGFアプタマーの、NGFへの結合活性、NGFとNGF受容体との結合阻害活性、神経突起伸長阻害活性の比較を行った。
先行文献記載のアプタマーは全て未修飾RNAであり、本明細書記載の配列と一致するものはない。先行文献記載のアプタマーとして結合活性が高いH1、L2、L6を選び、T7ポリメラーゼで転写して作製した。結合活性は実施例1、NGFとNGF受容体の結合阻害活性は実施例6、神経突起伸長阻害活性は実施例8と同様な方法で評価した。
その結果、H1、L2、L6はNGFと結合するものの配列番号30、32、35で表わされるアプタマーよりも活性が低いことがわかった(表7)。また、H1、L2、L6はNGFとNGF受容体の結合を阻害せず、突起伸長阻害実験において500nM添加した場合でも阻害活性を示さなかった(表7)。以上より、本明細書記載のアプタマーは先行文献記載のアプタマーよりも高い活性を有していることが示された。
NGFとの結合活性については、NGFに配列番号35が結合した場合の最大RU値を100%として評価した。80%以上の場合は“++”、50%以上の場合は“+”、50%以下の場合は“-”で表す。NGFとNGF受容体との結合阻害については、“+”は阻害活性%が60%以上であること、“-”は60%未満であることを表す。
NGFによる神経突起伸張の阻害活性は、それぞれのアプタマーの最終濃度が500nMおよび250nMである場合の阻害活性(%)を示す。
Claims (24)
- NGFに結合し、NGFとNGF受容体の結合を阻害するアプタマー。
- NGFに結合し、NGFの神経細胞突起伸長活性を阻害するアプタマー。
- 50%阻害濃度(IC50)が100nM以下である、請求項2に記載のアプタマー。
- 50%阻害濃度(IC50)が10nM以下である、請求項2に記載のアプタマー。
- 少なくとも1つのヌクレオチドが修飾されている、請求項1~4のいずれか一項に記載のアプタマー。
- HGAANNNANCY(配列番号106)で表される配列を含み、ここでNは任意のヌクレオチド、HはGを除くヌクレオチド、Yはピリミジンヌクレオチドであり、前記配列の少なくとも一つのヌクレオチドが修飾されている、請求項1~4のいずれか一項に記載のアプタマー。
- UGAAANNANCY(配列番号107)、CGAANNAAACY(配列番号108)又はAGAANNAAACY(配列番号109)で表される配列を含み、ここでNは任意のヌクレオチド、Yはピリミジンヌクレオチドであり、前記配列の少なくとも一つのヌクレオチドが修飾されている、請求項1~4のいずれか一項に記載のアプタマー。
- UGAAAAAAACY(配列番号110)、UGAAAGAAACY(配列番号111)、CGAACAAAACY(配列番号112)又はCGAAAGAAACY(配列番号113)で表される配列を含み、ここでYはピリミジンヌクレオチドであり、前記配列の少なくとも一つのヌクレオチドが修飾されている、請求項1~4のいずれか一項に記載のアプタマー。
- 各ピリミジンヌクレオチドのリボースの2’位のヒドロキシル基が、同一又は異なって、無置換であるか、水素原子、フッ素原子及びメトキシ基からなる群より選ばれる原子又は基で置換されている、請求項5~8のいずれか一項に記載のアプタマー。
- 各プリンヌクレオチドのリボースの2’位のヒドロキシル基が、同一又は異なって、無置換であるか、水素原子、フッ素原子及びメトキシ基からなる群より選ばれる原子又は基で置換されている、請求項5~8のいずれか一項に記載のアプタマー。
- 以下の(a)、(b)又は(c)のいずれかのヌクレオチド配列を含む、請求項1に記載のアプタマー:
(a)配列番号1~9、12、24~55、57~90のいずれかから選択されるヌクレオチド配列(但し、ウラシルはチミンであってもよい);
(b)配列番号1~9、12、24~55、57~90のいずれかから選択されるヌクレオチド配列(但し、ウラシルはチミンであってもよい)において、1又は数個のヌクレオチドが置換、欠失、挿入又は付加されたヌクレオチド配列;又は
(c)配列番号1~9、12、24~55、57~90のいずれかから選択されるヌクレオチド配列(但し、ウラシルはチミンであってもよい)と70%以上の同一性を有するヌクレオチド配列。 - 少なくとも1つのヌクレオチドが修飾されている、請求項11に記載のアプタマー。
- 各ピリミジンヌクレオチドのリボースの2’位のヒドロキシル基が、同一又は異なって、無置換であるか、水素原子、フッ素原子及びメトキシ基からなる群より選ばれる原子又は基で置換されている、請求項12に記載のアプタマー。
- 各プリンヌクレオチドのリボースの2’位のヒドロキシル基が、同一又は異なって、無置換であるか、水素原子、フッ素原子及びメトキシ基からなる群より選ばれる原子又は基で置換されている、請求項12に記載のアプタマー。
- 請求項1~14のいずれか一項に記載のアプタマー及び機能性物質を含む複合体。
- 機能性物質が、親和性物質、標識用物質、酵素、薬物送達媒体又は薬物である、請求項15に記載の複合体。
- 請求項1~14のいずれか一項に記載のアプタマーあるいは請求項15又は16に記載の複合体を含む医薬。
- 請求項1~14のいずれか一項に記載のアプタマーあるいは請求項15又は16に記載の複合体を含む抗疼痛剤。
- 請求項1~14のいずれか一項に記載のアプタマーあるいは請求項15又は16に記載の複合体を含む抗炎症剤。
- 請求項1~14のいずれか一項に記載のアプタマーあるいは請求項15又は16に記載の複合体を含む診断薬。
- 請求項1~14のいずれか一項に記載のアプタマーあるいは請求項15又は16に記載の複合体を含むNGF検出用プローブ。
- 請求項1~14のいずれか一項に記載のアプタマーあるいは請求項15又は16に記載の複合体を含む、NGF精製用固相担体。
- 請求項1~14のいずれか一項に記載のアプタマーあるいは請求項15又は16に記載の複合体を用いることを特徴とする、NGFの検出方法。
- 請求項1~14のいずれか一項に記載のアプタマーあるいは請求項15又は16に記載の複合体を用いることを特徴とする、NGFの精製方法。
Priority Applications (13)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES09816140.9T ES2543222T3 (es) | 2008-09-24 | 2009-09-18 | Aptámero para NGF y uso del mismo |
US13/120,650 US10260070B2 (en) | 2008-09-24 | 2009-09-18 | Aptamer for NGF and use thereof |
KR1020117009135A KR101694559B1 (ko) | 2008-09-24 | 2009-09-18 | Ngf에 대한 압타머 및 이의 용도 |
CN200980137647.6A CN102171339B (zh) | 2008-09-24 | 2009-09-18 | 针对ngf的适体及其用途 |
JP2010530841A JP5602020B2 (ja) | 2008-09-24 | 2009-09-18 | Ngfに対するアプタマー及びその使用 |
MX2011003144A MX2011003144A (es) | 2008-09-24 | 2009-09-18 | Aptamero para factor de crecimiento de nervio y uso del mismo. |
AU2009297626A AU2009297626B2 (en) | 2008-09-24 | 2009-09-18 | Aptamer for NGF and use thereof |
CA2738129A CA2738129C (en) | 2008-09-24 | 2009-09-18 | Aptamer for ngf and use thereof |
EP20090816140 EP2354225B1 (en) | 2008-09-24 | 2009-09-18 | Aptamer for ngf and use thereof |
DK09816140.9T DK2354225T3 (en) | 2008-09-24 | 2009-09-18 | APPLICATIONS TO NGF AND USE THEREOF |
BRPI0919268A BRPI0919268B8 (pt) | 2008-09-24 | 2009-09-18 | aptâmero que se liga ao ngf e medicamento |
IL211929A IL211929A0 (en) | 2008-09-24 | 2011-03-24 | Ngf aptamer, compositions comprising the same and uses thereof |
ZA2011/02682A ZA201102682B (en) | 2008-09-24 | 2011-04-11 | Aptamer for agf and use thereof |
Applications Claiming Priority (2)
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JP2008-244982 | 2008-09-24 | ||
JP2008244982 | 2008-09-24 |
Publications (1)
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WO2010035725A1 true WO2010035725A1 (ja) | 2010-04-01 |
Family
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PCT/JP2009/066457 WO2010035725A1 (ja) | 2008-09-24 | 2009-09-18 | Ngfに対するアプタマー及びその使用 |
Country Status (16)
Country | Link |
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US (1) | US10260070B2 (ja) |
EP (1) | EP2354225B1 (ja) |
JP (1) | JP5602020B2 (ja) |
KR (1) | KR101694559B1 (ja) |
CN (1) | CN102171339B (ja) |
AU (1) | AU2009297626B2 (ja) |
BR (1) | BRPI0919268B8 (ja) |
CA (1) | CA2738129C (ja) |
DK (1) | DK2354225T3 (ja) |
ES (1) | ES2543222T3 (ja) |
HU (1) | HUE026595T2 (ja) |
IL (1) | IL211929A0 (ja) |
MX (1) | MX2011003144A (ja) |
RU (1) | RU2011116175A (ja) |
WO (1) | WO2010035725A1 (ja) |
ZA (1) | ZA201102682B (ja) |
Cited By (5)
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JP2011229452A (ja) * | 2010-04-27 | 2011-11-17 | Up Well:Kk | Par−2活性化阻害物質 |
JP2012024026A (ja) * | 2010-07-23 | 2012-02-09 | Tdk Corp | Sm菌特異的アプタマー、Sm菌増殖抑制剤及びSm菌の検出方法 |
US20130052176A1 (en) * | 2010-03-24 | 2013-02-28 | Shionogi & Co., Ltd | Aptamer for ngf and use thereof |
WO2013047844A1 (ja) | 2011-09-28 | 2013-04-04 | 株式会社リボミック | Ngfに対するアプタマー及びその用途 |
JP2013523177A (ja) * | 2010-04-12 | 2013-06-17 | ソマロジック,インコーポレイテッド | β−NGFに対するアプタマー及びβ−NGF介在疾患及び障害の治療におけるその使用 |
Families Citing this family (3)
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US8975026B2 (en) | 2007-01-16 | 2015-03-10 | Somalogic, Inc. | Method for generating aptamers with improved off-rates |
AU2011223527B2 (en) | 2010-03-03 | 2014-11-13 | Somalogic Operating Co., Inc. | Aptamers to 4-1BB and their use in treating diseases and disorders |
PT2611775E (pt) | 2010-08-31 | 2016-06-07 | Consejo Superior De Investig Científicas | Agonistas dos recetores de neurotrofina e sua utilização como medicamentos |
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US9175292B2 (en) * | 2010-03-24 | 2015-11-03 | Fujimoto Pharmaceutical Corporation | Aptamer for NGF and use thereof |
US20130052176A1 (en) * | 2010-03-24 | 2013-02-28 | Shionogi & Co., Ltd | Aptamer for ngf and use thereof |
AU2011230388B2 (en) * | 2010-03-24 | 2016-09-08 | Fujimoto Pharmaceutical Corporation | Aptamer for NGF and use thereof |
JP2013523177A (ja) * | 2010-04-12 | 2013-06-17 | ソマロジック,インコーポレイテッド | β−NGFに対するアプタマー及びβ−NGF介在疾患及び障害の治療におけるその使用 |
US8598140B2 (en) | 2010-04-12 | 2013-12-03 | Somalogic, Inc. | Aptamers to β-NGF and their use in treating β-NGF mediated diseases and disorders |
JP2011229452A (ja) * | 2010-04-27 | 2011-11-17 | Up Well:Kk | Par−2活性化阻害物質 |
JP2012024026A (ja) * | 2010-07-23 | 2012-02-09 | Tdk Corp | Sm菌特異的アプタマー、Sm菌増殖抑制剤及びSm菌の検出方法 |
WO2013047844A1 (ja) | 2011-09-28 | 2013-04-04 | 株式会社リボミック | Ngfに対するアプタマー及びその用途 |
JPWO2013047844A1 (ja) * | 2011-09-28 | 2015-03-30 | 株式会社リボミック | Ngfに対するアプタマー及びその用途 |
KR20140069216A (ko) | 2011-09-28 | 2014-06-09 | 가부시키가이샤 리보믹 | Ngf에 대한 압타머 및 그의 용도 |
US9567589B2 (en) | 2011-09-28 | 2017-02-14 | Ribomic Inc. | NGF aptamer and application thereof |
AU2012317323B2 (en) * | 2011-09-28 | 2017-10-05 | Fujimoto Pharmaceutical Corporation | NGF aptamer and application thereof |
RU2633510C2 (ru) * | 2011-09-28 | 2017-10-12 | Рибомик Инк. | Аптамер против ngf и его применение |
Also Published As
Publication number | Publication date |
---|---|
DK2354225T3 (en) | 2015-06-29 |
RU2011116175A (ru) | 2012-10-27 |
ES2543222T3 (es) | 2015-08-17 |
AU2009297626A1 (en) | 2010-04-01 |
CN102171339B (zh) | 2017-06-09 |
CA2738129A1 (en) | 2010-04-01 |
EP2354225A1 (en) | 2011-08-10 |
EP2354225A9 (en) | 2012-06-27 |
AU2009297626B2 (en) | 2016-01-14 |
US20110251266A1 (en) | 2011-10-13 |
BRPI0919268A2 (pt) | 2017-11-07 |
KR20110059888A (ko) | 2011-06-07 |
EP2354225A4 (en) | 2012-03-21 |
BRPI0919268B1 (pt) | 2021-01-12 |
BRPI0919268A8 (pt) | 2018-02-06 |
IL211929A0 (en) | 2011-06-30 |
KR101694559B1 (ko) | 2017-01-09 |
EP2354225B1 (en) | 2015-04-22 |
AU2009297626A2 (en) | 2011-05-19 |
ZA201102682B (en) | 2012-06-27 |
CN102171339A (zh) | 2011-08-31 |
BRPI0919268B8 (pt) | 2021-05-25 |
HUE026595T2 (hu) | 2016-06-28 |
JP5602020B2 (ja) | 2014-10-08 |
JPWO2010035725A1 (ja) | 2012-02-23 |
CA2738129C (en) | 2017-11-14 |
MX2011003144A (es) | 2011-05-19 |
US10260070B2 (en) | 2019-04-16 |
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