TW201629216A - Method for inhibiting translation and inhibitors for translation - Google Patents

Abstract

Provided is a method for inhibiting, effectively and in a stable manner, the translation of mRNA into protein. Disclosed are: a target mRNA translation inhibitor that contains a first polynucleotide which may be hybridized to the 5'-untranslated region and/or translated region of target mRNA, and a second polynucleotide which can be hybridized to the 3'-untranslated region of said mRNA; and/or a method for inhibiting the translation from mRNA using said translation inhibitor. According to the present invention, it is possible to inhibit the translation from target mRNA in a stable manner.

Description

Translation inhibition method and translation inhibitor

The present invention relates to a method for inhibiting gene expression and an inhibitor thereof, and more particularly to a method for inhibiting translation of mRNA and a translation inhibitor.

The field of biopharmaceuticals is booming. Currently mainly protein preparations. In the future, development of a relatively low molecular weight, chemically synthesized nucleic acid pharmaceutical product is expected (Non-Patent Document 1).

Nucleic acid pharmaceuticals comprise DNA or RNA, or derivatives thereof. The size is smaller than the protein preparation and the lower molecular medicine. A nucleic acid pharmaceutical or a candidate thereof is a single or double-stranded molecule having a length of about 10 to 20 bases (Non-Patent Document 2). By the hydrogen bond formed between the bases, and the target nucleic acid, mainly mRNA, exhibits a double-stranded state. When a double strand is partially generated in a specific region of the mRNA, 1) the target mRNA is decomposed, and 2) the bond between the target mRNA and the specific functional protein is suppressed (Non-Patent Document 2). By exploiting the properties of 1) or 2), the performance of a particular gene can be inhibited. In other words, the gene expression inhibitory activity of the nucleic acid drug can be used for the treatment of diseases.

As a method for suppressing gene expression, an antisense method (Non-Patent Document 2), a ribozyme method (Non-Patent Document 3), or RNAi (using small interfering RNA) has been developed. ): siRNA) method (Non-Patent Document 4). In the antisense method, mainly DNA or a derivative thereof which will be in a single-strand state, for example, a polynucleotide or an MO containing LNA (locked nucleic acids) Nucleic acid derivatives of morpholino oligonucleotides are designed to form a double strand in the peripheral region of the translation initiation codon or the peripheral region of the splicing site. Thereby, the inhibition effect of gene expression is obtained by using translation to inhibit or inhibit the normal splicing reaction. However, by such an antisense method, there is a problem that it is difficult to stably obtain a sufficient suppression effect.

The ribonuclease method and the RNAi method mainly use RNA, and the gene expression inhibition is achieved by inducing the cleavage and decomposition of the mRNA of the target gene. Inhibition of gene expression by ribonuclease does not necessarily achieve sufficient inhibitory effects.

Based on the performance of the RNAi mechanism, short-chain double-stranded RNA and microRNA (miRNA) have been found to function under physiological conditions. The miRNA forms a double strand in the 3' untranslated region (UTR) of the mRNA. Thereby, the deadenylase is activated and the poly(A) chain is shortened (Non-Patent Document 5). At the same time, the activity of the translation initiation factor complex which acts on the 5'UTR of mRNA is suppressed (Non-Patent Document 6). As a result, the translation reaction was suppressed. The siRNA system forms a double strand comprising a base sequence that is completely complementary to the target mRNA, and induces decomposition of mRNA. On the other hand, a double strand containing a mismatch is formed between the miRNA system and the target mRNA, and no cleavage or decomposition of mRNA occurs (Non-Patent Document 5).

We have recently included a 25-base MO designed for the parental mRNA close to the upstream of the poly(A) chain junction, which occurs by microinjection into the zebrafish embryo or starfish egg. When the strand is shortened or cleaved, the result is a method for successfully developing a translation reaction capable of artificially suppressing the target gene (Patent Document 1 and Non-Patent Document 7). This method is considered to be a novel antisense method which is different from the conventional antisense method, ribonuclease method, or RNAi method because of the difference in position and inhibition mechanism on the double-stranded mRNA. However, its scope of application is limited and a method of translational inhibition that is efficient for a wide range of target mRNAs is being sought.

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] WO2013/015152

[Non-patent literature]

[Non-Patent Document 1] Mizuho Industry Focus/156, No. 12 (2014)

[Non-Patent Document 2] Trends in the development and regulation of nucleic acid pharmaceutical products (2014) HS Report No. 82.

[Non-Patent Document 3] Couture L. A., Stinchcomb D. T. (1996) Anti-gene therapy: the use of ribozymes to inhibit gene function. Trends Genet., 12, 12, 510-515.

[Non-Patent Document 4] Vaishnaw A. K., Gollob J., Gamba-Vitalo C., Hutabarat R., Sah D., Meyers R., de Fougerolles T., Maraganore J. (2010) A status reporton RNAi therapeutics. Silence, 1, 14.

[Non-Patent Document 5] Valinezhad Orang A., Safaralizadeh R., Kazemzadeh-Bavili M. (2014) Mechanisms of miRNA-mediated gene regulation from common downre gulation to mRNA-specific upregulation. Int J Genomics.doi: 10.1155/2014/ 970607.

[Non-Patent Document 6] Mathonnet G., Fabian M. R., Svitkin Y. V., Kazemzadeh-Bavili M. (2007) MicroRNA inhibition of translation initiation in vitro by targeting the cap-binding complex eIF4F. Science 317, 5845, 1764-1767.

[Non-Patent Document 7] Wada T., Hara M., Taneda T., Qingfu C., Takata R., Moro K., Takeda K., Kishimoto T., Handa H. (2012) Antisense morpholino targeting just upstream from a poly(A) tail junction ofmaternal mRNA removes the tail and inhibits translation. Nucleic Acid Res., doi:10.1093/nar/gks765, 1-10.

A translation inhibitor and a translation inhibition method that efficiently and stably inhibit translation from mRNA are provided.

According to the present invention, the following invention is provided.

(1) A target mRNA translation inhibitor comprising a first polynucleotide which hybridizes to a 5'-non-translated region and/or a translation region of a target mRNA, and a 3'-non-translated region compatible with the mRNA A second polynucleotide that hybridizes.

(2) A translational inhibitor of mRNA as defined in (1), wherein said first polynucleotide is a polynucleotide which hybridizes to a region of said target mRNA comprising a 5'-non-translated region.

(3) A translational inhibitor of mRNA as defined in (1) or (2), characterized in that said first polynucleotide is hybridized to a region of said target mRNA comprising a 5'-end or a translation initiation codon.

(4) The transcriptional inhibitor of the target mRNA according to any one of (1) to (3) wherein the second polynucleotide is hybridized to the immediately preceding poly-A sequence of the target mRNA.

(5) The translation inhibitor of the target mRNA according to any one of (1) to (4) wherein the first polynucleotide comprises a polynucleotide of 18 to 30 bases.

(6) The translation inhibitor of the target mRNA according to any one of (1) to (5) wherein the second polynucleotide comprises a polynucleotide of 6 to 30 bases.

(7) The translation inhibitor of the target mRNA according to any one of (1) to (6) wherein the polynucleotide is DNA, RNA, a polynucleotide comprising LNA or Alkranyl oligonucleic acid.

(8) The translation inhibitor of the target mRNA according to any one of (1) to (7) wherein the polynucleotide comprises at least one nucleotide analog.

(9) The translation inhibitor of the target mRNA according to any one of (1) to (8), wherein the first polynucleotide and the second plurality are separated by at least one spacer. The polynucleotides of the nucleotides are each made to hybridize to a 5'-non-translated region and/or a translation region and a 3'-non-translated region of the aforementioned target mRNA.

(10) The translation inhibitor of the target mRNA according to any one of (1) to (9) wherein the target mRNA is an mRNA of a protein causing an inflammatory reaction.

The translation of the target mRNA according to any one of (1) to (10) wherein the target mRNA is NFκB mRNA.

(12) An anti-inflammatory agent and/or an anticancer agent comprising a translational inhibitor of the mRNA of (10) or (11).

(13) A cell into which a translational inhibitor of the target mRNA according to any one of (1) to (11) or an anti-inflammatory agent and/or an anticancer agent according to (12) is introduced.

(14) A kit comprising the translation inhibitor of the target mRNA according to any one of (1) to (11) and/or the anti-inflammatory agent and/or anticancer agent according to claim 12.

(15) A method for inhibiting translation of a target mRNA, which is a first polynucleotide which hybridizes to a 5'-non-translated region and/or a translation region of a target mRNA, and a 3'- to which the target mRNA can be A second polynucleotide that hybridizes to the non-translated region, hybridizes to the target mRNA.

(16) A method for inhibiting translation of a target mRNA according to (15), wherein said first polynucleotide is a polynucleotide which can hybridize to a region of said target mRNA comprising a 5'-non-translated region.

(17) A method of inhibiting translation of a target mRNA according to (15) or (16), wherein said first polynucleotide sequence hybridizes to a region of said target mRNA comprising a 5'-end or a translation initiation codon.

(18) The method for inhibiting translation of a target mRNA according to any one of (15) to (17), wherein the second polynucleotide is hybridized to the immediately preceding poly-A sequence of the target mRNA.

(19) The method for inhibiting translation of a target mRNA according to any one of (15) to (18) wherein the first polynucleotide comprises a polynucleotide of 18 to 30 bases.

(20) The method for inhibiting translation of a target mRNA according to any one of (15) to (19), wherein the second polynucleotide is a polynucleotide comprising 6 to 30 bases.

(21) A method for inhibiting translation of a target mRNA according to any one of (15) to (20), wherein the polynucleotide is DNA, RNA, a polynucleotide comprising LNA or Alkranyl oligonucleic acid.

(22) A method for inhibiting translation of a target mRNA according to any one of (15) to (21), wherein the polynucleotide is a polynucleotide comprising at least one nucleotide analog.

(23) The method for inhibiting translation of a target mRNA according to any one of (15) to (22), wherein the first polynucleotide and the second one are linked by separating at least one spacer The polynucleotides of the polynucleotides can each be hybridized to a 5'-non-translated region and/or a translation region and a 3'-non-translated region of the aforementioned target mRNA.

(24) A method for inhibiting translation of a target mRNA according to any one of (15) to (23), wherein said target mRNA is a protein which causes an inflammatory reaction Qualitative mRNA.

(25) A method for inhibiting translation of a target mRNA according to any one of (15) to (24), wherein the target mRNA is NFκB mRNA.

(26) A method for treating inflammatory diseases and/or a method for treating cancer, which comprises using a method of inhibiting translation of a target mRNA as in (24) or (25).

(27) A method of screening a substance having a translation inhibitory activity by screening a substance having a translation inhibitory activity using an in vitro translation system.

(28) The method according to (27), wherein the substance having the translation inhibitory activity is a polynucleotide.

(29) The method according to (28), wherein the polynucleotide is the first polynucleotide and/or the second polynucleotide as described in (1).

(30) The translation inhibitor of the target mRNA according to any one of (1) to (11), the anti-inflammatory agent and/or the anticancer agent of (12), the cell of (13), and/or (14) A kit comprising an anti-inflammatory agent and/or an anti-cancer agent, wherein the second polynucleotide is an siRNA.

(31) The method of inhibiting translation according to (15) to (25), and/or the method for treating inflammation and/or the method of treating cancer according to (26), wherein the second polynucleotide is siRNA.

According to the present invention, translation of self-labeled mRNA can be stably inhibited.

[Fig. 1] shows an analytical plastid containing NFκB and luciferase Figure.

[Fig. 2] A diagram showing the transcriptional control mechanism using the NFκB complex.

[Fig. 3] is a diagram showing the subject of antisense nucleic acids.

[Fig. 4] is a diagram showing the translation inhibitory activity of the translation inhibitory polynucleotide.

[Fig. 5] is a diagram showing the specificity of a translational repressor polynucleotide.

[Fig. 6] is a diagram showing the translation inhibitory activity of a polynucleotide having a mutation.

[Fig. 7] is a graph showing the length of the polynucleotide and the inhibitory activity.

[Fig. 8] is a graph showing the translation inhibitory activity of a polynucleotide linked by a spacer.

[Fig. 9] is a graph showing the amount of mRNA in the presence of a translation inhibitor.

[Fig. 10] is a graph showing the amount of mRNA in the presence of a translation inhibitor.

[Fig. 11] is a diagram showing a method of preparing a HeLa cell disrupting solution.

[Fig. 12] is a diagram showing an in vitro translation system.

[Fig. 13] is a diagram showing the effect of Antisense Oligo linked by a spacer and its effect.

[Fig. 14] is a diagram showing the translation inhibitory activity of the spacer type Antisense Oligo.

[Fig. 15] is a graph showing the translation inhibitory activity of the spacer type Antisense Oligo.

[Fig. 16] A graph showing translation inhibitory activity by a combination of a polynucleotide and siRNA.

[Fig. 17] A graph showing translation inhibitory activity by a combination of a polynucleotide and a gapmer (spacer).

[Fig. 18] is a graph showing the translation inhibitory activity of gapmers and mixers (mixtures).

[Fig. 19] is a diagram showing Western analysis of mouse RelA and JunB proteins.

[Fig. 20] is a diagram showing the Ponceau S staining of the film after the Western printing method.

[Formation of the Invention]

According to the present invention, a translation inhibitor and a translation inhibition method for inhibiting protein translation of a self-labeled mRNA can be provided. In the present specification, the target mRNA refers to an mRNA which is a target for inhibiting translation, and refers to an mRNA which is transcribed from a gene by RNA polymerase II and which has poly-A at the 3'-end. Preferably, the mRNA of a eukaryotic organism. The eukaryotic organism includes, for example, humans, animals other than humans, plants, eukaryotic microorganisms (eg, yeast, fungi, etc.), archaea, etc., but as long as the organism has a poly-A sequence at the 3'-end of the mRNA, It can be the object of the present invention. Further, cultured cells in vitro can also be the object of the present invention. All or part of these eukaryotes may be selected as applicable, as necessary.

The type of the target mRNA is not particularly limited, and any of the mRNAs to which the protein is translated may be the target. For example, mRNAs of proteins related to disease, mRNAs of translation- and metabolism-related proteins, mRNAs of proteins involved in translation and transcription, translation, translation, and the like, and mRNAs of proteins involved in translation and biosynthesis may be cited. Etc., but not limited to this. Further, it may be an mRNA which expresses an unknown function by translation.

Regarding the translation inhibitor and the translation inhibition method of the present invention, a first polynucleotide which can hybridize with a 5'-non-translated region and/or a translation region of the target mRNA, and a 3'-non-translation with the mRNA can be used. A second polynucleotide that hybridizes to the region. The term "hybridization" as used herein refers to the formation of a double strand by intracellular binding of the target mRNA to the polynucleotide of the present invention by hydrogen bonding.

In a preferred embodiment of the present invention, in the translation inhibitor and translation inhibition method of the present invention, a region comprising a 5'-non-translated region (5'-UTR) and a 3'-non-translation are used with the target mRNA. Region (3'-UTR) hybridized polynucleotide.

In the present specification, a non-translated region refers to a region other than a region (translation region) encoding a protein of the target mRNA. That is, the region encoding the protein in the mRNA refers to the region encoding the protein from the initiation codon AUG to one codon before the stop codon, and will be the 5'-upstream side and the stop codon of its initiation codon. The 3'-poly-A region on the downstream side is previously referred to as a 5'-non-translated region and a 3'-non-translated region. In the present invention, a first polynucleotide that hybridizes to the 5'-non-translated region and/or the translated region of the target mRNA, and a second polynucleotide that hybridizes to the 3'-non-translated region of the mRNA can be used. acid. For a combination of preferred polynucleotides, a polynucleotide that hybridizes to a region comprising a 5'-non-translated region and a polynucleotide that hybridizes to a 3'-non-translated region (3'-UTR) can be used. More preferred are polynucleotides that hybridize to the 5'-non-translated region and polynucleotides that hybridize to the 3'-non-translated region, particularly preferably in the 5'-non-translated region. A polynucleotide that hybridizes to the 5'-end region and a polynucleotide that hybridizes to the 3'-non-translated region, preferably a polynucleotide that hybridizes to the 5'-terminal region of the 5'-non-translated region and A polynucleotide that hybridizes to a region immediately adjacent to poly-A in the 3'-non-translated region.

One embodiment of the polynucleotide of the translational inhibitor of the present invention uses a multinucleus having a sequence which hybridizes to the 5'-terminal region of the target mRNA and the upstream sequence from the immediately preceding poly-A of the 3'-end. Glycosylate. In this case, there is an advantage that a polynucleotide which can mechanically and easily inhibit translation of all target mRNAs can be designed to be a translation inhibitor. In the conventional technology, in order to suppress gene expression by antisense technology, the region to be designed and used as an antisense strand is insufficient, and it is necessary to confirm whether or not there is a translation inhibitory effect by experiments. This is because even if an antisense nucleotide is introduced depending on the region, there is a case where no inhibitory activity is produced. The present invention solves this problem by easily designing a sequence of an antisense nucleotide having a translation inhibitory effect on all mRNAs, and stably inhibiting translation using an antisense nucleotide having the sequence. That is, the invention of the present embodiment does not particularly require a screening experiment, and can be applied mechanically to all mRNAs.

18 to 30 bases are preferred for hybridization with a region of the target mRNA comprising a 5'-non-translated region and/or a translation region of the present invention to inhibit the length of the first polynucleotide to be translated. Preferably, it is 18 to 22 bases, more preferably 19 to 21 bases, and most preferably 20 bases. Among these, a polynucleotide which hybridizes with 1 to 50 bases from the 5'-end in the 5'-non-translated region of the target mRNA is preferably used, and it is more preferably used from the 5'-end. The polynucleotide is 1 to 30 bases, more preferably 1 to 22 bases, and most preferably 1 to 20 bases are hybridized.

The effect of translation inhibition can be measured by using the plastid shown in Fig. 1 and using the transcription activation mechanism of NFκB in Fig. 2 . That is, it is used in a region having a region binding to NFκB upstream of the promoter, and having a plastid of the luciferase gene downstream of the promoter, and a polynucleotide of the Rel gene which will be a subunit of the NFκB transcription factor as a target polynucleotide. When the cells are introduced together, in Fig. 2, the transcriptional activation of luciferase is inhibited by the translation of Rel protein, and the activity of luciferase is lowered.

Hybridization with a region of the target mRNA comprising a 5'-non-translated region of the invention that inhibits translation does not require that all polynucleotide assignments hybridize only to the 5'-non-translated region, as long as a portion is 5'-non- The translation area can be hybridized. Namely, as long as it hybridizes to a part of the 5'-non-translated region of the target mRNA, it may hybridize to the start codon or the like which is the translation initiation site of the target mRNA. That is, the entire region of the polynucleotide of the present invention does not need to hybridize only to the non-translated region as long as a part hybridizes to the non-translated region. Alternatively, one of the sequences encoding the coding region of the protein may be hybridized (see Fig. 3).

The second polynucleotide sequence which hybridizes to the 3'-non-translated region of the subject mRNA of the present invention hybridizes to the sequence from the downstream of the stop codon to the poly-A chain. In terms of the length range of the polynucleotide which inhibits translation by hybridizing with the 3'-non-translated region, 10 to 50 bases are preferred, more preferably 10 to 30 bases, still more preferably 10 to 20 The base is preferably 10 to 19 bases. Among these, it is optimal to use the sequence from the 3'-end in the 3'-non-translated region of the target mRNA (before the poly-A region, from the 5'- side of AAA...) Figure) ~30 base hybridized polynucleotides.

The first and second polynucleotides used in the translation inhibitor or translation inhibition method of the present invention may comprise a nucleic acid, an amino acid, a PEG (polyethylene glycol), an o-nitrobenzyl group, a methylene group. The group is bonded to a spacer such as a guanamine skeleton, a stilbene skeleton, or a honokiol having a physiologically active low molecular compound. The number of spacers to which the first and second polynucleotides are linked is at least one, and the first and second polynucleotides may be linked using (for example, spacers) a plurality of spacers. The length of the spacer is preferably from 3 to 50 bases, more preferably from 3 to 30 bases, still more preferably from 3 to 20 bases, most preferably from 3 to 18 bases, in the case of using a nucleic acid. . In the case where an amino acid is used as the spacer, 2 to 30 amino acids are preferred, more preferably 3 to 20 amino acids, and most preferably 3 to 18 amino acids. In the case of PEG, [-C-C-O-] is used as the unit, and 2 to 30 doses are preferred, more preferably 3 to 20 doses, and most preferably 3 to 18 doses. In the case of using a spacer containing a molecule other than these, it is preferred to use a spacer having a repeating unit or length corresponding thereto. The spacers are designed in such a way that the polynucleotides are each hybridized to the 5'-non-translated region and/or the translated region and the 3'-non-translated region of the target mRNA.

Further, the polynucleotide of the present invention may contain an analog of a nucleotide in place of a natural nucleotide. For example, it may be a polynucleotide or MO containing LNA (locked nucleic acids) a nucleic acid derivative of a morpholino oligo) nucleic acid, which can be a saccharide, a base, and/or a phosphate chemical, as long as it can inhibit translation of the above-mentioned target mRNA. Modified nucleotide analogs. As the base-modified nucleotide analog, for example, 5-position modified uridine or cytidine (for example, 5-propargyl uridine, 5-propargyl cytidine, 5-methyl group) Glycosides, 5-methyluridine, 5-(2-amino)propyluridine, 5-haloperytidine, 5-halouridine, 5-methoxyuridine, etc.; 8-modified adenosine or Guanosine (eg, 8-bromoguanosine, etc.); deaza nucleotides (eg, 7-deaza-adenosine, etc.); O- and N-alkylated nucleotides (eg, N6-methyladenosine) and many more.

Further, as the sugar-modified nucleotide analog, for example, LNA, MO, ribonucleotide 2'-OH via H, OR, R, halogen atom, SH, SR, NH ₂ , NHR NR ₂ or CN (wherein R represents an alkyl group having 1 to 6 carbon atoms, an alkenyl group or an alkynyl group), a substituted 2'-position sugar modification, and a 5'-end monophosphorylation 5' terminal phosphorylation modification.

In the case of a phosphate-modified nucleotide analog, a phosphate group in which a contiguous ribonucleotide is bonded is substituted with a phosphorothioate.

These nucleotide analogs can be introduced into a polynucleotide by a known method such as chemical synthesis.

The translated target mRNA translated by the translation inhibitor of the present invention may be an mRNA expressing a protein that causes an inflammatory response. Examples of the protein that causes the inflammatory reaction include, but are not limited to, inflammatory cytokine (IL-12, IL-6, TNF-α). The inflammatory cytokine gene is released from the NFκB complex by IκB, and is transcribed by RelA and P50 to transcribe mRNA and translate into cytokine protein (see Fig. 2). Therefore, the mRNA of the cytokine itself can also be used as a target to inhibit translation by the translation inhibitor of the present invention, but can also inhibit the expression of all or one of the transcription factors that promote the expression of the cytokine gene. The performance of cytokines to suppress inflammatory reactions. Examples of the transcription factor of the cytokine gene include NFκB, AP-1, and STAT3. Therefore, a translational inhibitor of the NFκB gene can be used as an anti-inflammatory agent.

The translational inhibitors of the invention may also be adapted for use in diseases and conditions mediated by the inflammatory cytokine cascade. The following diseases are included in the diseases and symptoms mediated by the inflammatory cytokine cascade, but are not limited thereto.

Systemic inflammatory response syndrome includes the following: sepsis syndrome, Gram-positive sepsis, Gram-negative sepsis, culture-negative sepsis, fungal sepsis, neutrophilic fever, urinary septicemia, meningococcalemia , traumatic bleeding, murmur (hum), exposure to free radiation, acute pancreatitis, adult respiratory obsessive-compulsive disorder (ARDS).

Reperfusion injuries include the following: Post-pump syndrome, ischemia-reperfusion injury.

Cardiovascular diseases include the following: palpitations, fainting syndrome, myocardial infarction, and septic heart failure.

Infectious diseases include the following: HIV infection/HIV neuropathy, meningitis, hepatitis, septic arthritis, peritonitis, pneumonia laryngotomy, coliform O157:H7, hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, malaria , dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis (human type tuberculosis), bird tuberculosis Mycobacterium aviun Intracellulare, Pneumocystis Carinii Pneumonia, inflammatory disease of pelvis, testicular/parallelitis, Legionnaires' disease, Lyme disease, influenza A, Epstein-Barr virus, viral hemophagocytosis (Viral associated hemiaphagocytic syndrome), viral encephalitis/aseptic meningitis.

Obstetrics/gynecology symptoms include the following: premature birth, miscarriage, infertility.

Inflammatory/autoimmune diseases include the following: rheumatoid arthritis/seronegative arthropathies, osteoarthritis, inflammatory bowel disease, systemic lupus erythematosus, iridocyclitis/ Uveitis optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener's granulomatosis, sarcoidosis, vasectomy reversal procedures.

Allergic/ectopic diseases include the following: asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, and allergic pneumonia.

Malignant diseases include the following: ALL, AML, CML, CLL, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, colorectal cancer, nasopharyngeal carcinoma, malignancy Tissue hyperplasia, paraneoplastic syndrome/hypercalcemia of malignant disease.

The graft contains the following: organ transplant rejection, graft versus host disease, cachexia.

Congenitality includes the following: cystic fibrosis (cystic Fibrosis), familial hematophagocytic lymphohistiocytosis, sickle cell anemia.

Dermatological science includes the following: dryness, hair loss.

Neurological diseases include the following: multiple sclerosis, migraine.

Renal septic diseases include the following: renal syndrome, hemodialysis, and uremia.

Toxic persons include the following: OKT3 therapy, anti-CD3 therapy, cytokine therapy, chemotherapy, radiation therapy, Chronic salicylate intoxication.

Metabolic/idiopathic diseases include the following: Wilson's disease, Hemachromatosis, alpha-1 antitrypsin deficiency, diabetes, Hashimoto's thyroiditis, osteoporosis , hypothalamus - pituitary gland - adrenal axis evaluation, primary biliary cirrhosis.

The mRNA encoding the protein causing the disease and symptoms mediated by such an inflammatory cytokine cascade is used as a target, and by applying the translation inhibitor of the present invention, inflammation can be alleviated and/or treated.

Further, the target mRNA which is inhibited by translation of the translation inhibitor of the present invention may be mRNA which expresses a protein causing a tumor disorder. "Tumor disorder" is not limited, but means: all kinds of cancers or tumors or malignant tumors that are found in humans such as leukemia, lymphoma, melanoma, carcinoma, and sarcoma. Such a tumor disorder can be included as an object of the anticancer agent and/or cancer treatment method of the present invention.

The term "sarcoma" generally refers to a tumor comprising a substance such as embryonic connective tissue and consisting essentially of densely packed cells embedded in a fibrous substance or a homogeneous substance. Examples of sarcomas that can be treated by the anticancer agent and/or cancer treatment method of the present invention are not limited, but include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanoma, mucinous sarcoma, osteosarcoma, and benenett's sarcoma ( Abernethy's sarcoma), liposarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, grape sarcoma, green sarcoma, Villous carcinoma, embryonal sarcoma, Will Wilms' Tumor, endometrial sarcoma, interstitial sarcoma, Ewing sarcoma, sarcoma sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's Sarcoma (Hodgkin's sarcoma), idiopathic multiple pigmented hemorrhagic sarcoma, B cell immunoblast sarcoma, lymphoma, T cell immunoblast sarcoma, Jensen's sarcoma, Kaposi's sarcoma , Kupffer cell sarcoma, angiosarcoma, leukemia sarcoma (leukosarcoma), malignant mesenchymal sarcoma, parosteal sarcoma , reticulum cell sarcoma, Rous' sarcoma, serous sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

The term "melanoma" is interpreted to mean a tumor produced by the melanocyte system of the skin and other organs. The melanoma which can be treated by the anticancer agent and/or cancer treatment method of the present invention is not limited, but includes, for example, terminal melanoma type melanoma, melanoma-deficient melanoma, benign juvenile melanoma, and Claudeman melanin. Tumor (Cloudman melanoma), S91 melanoma, Harding-Passey melanoma, juvenile melanoma, malignant melanoma, malignant melanoma, nodular melanoma, subungual melanoma ), and superficial spreading melanoma (superficial spreading melanoma).

The term "carcinoma" refers to a malignant tumor that includes epithelial cells that infiltrate into surrounding tissues and tend to cause metastasis. The term "carcinoma" refers to a malignant tumor that includes epithelial cells that infiltrate into surrounding tissues and tend to cause metastasis. The cancer treatable by the anticancer agent and/or cancer treatment method of the present invention is not limited, but includes, for example, acinar carcinoma, acinar cell carcinoma, adenoctic carcinoma, adenoid cyst Adenoid cystic carcinoma, adenoma, paracancerous, lung cancer, lung cell carcinoma, basal cell carcinoma, basal cell carcinoma, basal cell carcinoma, basal squamous cell carcinoma, Bronchopulmonary cell epithelial carcinoma, bronchial carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocarcinoma, villus cancer, colloidal carcinoma, vesicular cancer, endometrial cancer, squamous carcinoma, squamous carcinoma (carcinoma) En cuirasse), skin cancer, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, hard cancer, embryonal carcinoma, brain-like carcinoma, epiermoid carcinoma, adenoid epithelial carcinoma, outward developmental cancer, Ulcer cancer, fibrosum carcinoma, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, gigantocellulare carcinoma, adenocarcinoma, granule Cell carcinoma, carcinoma of the hair matrix (hair matrix carcinoma), Hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, pediatric embryonal carcinoma, intraepithelial carcinoma, epidermis Cancer, intraepithelial carcinoma, Krompecher carcinoma, Kultschitzky cell carcinoma, large cell carcinoma, lenticular carcinoma, lenticulare carcinoma, fatty cancer , lymphoid epithelial cancer, carcinoma medullare, medullary carcinoma, black cancer, cancer molle, mucinous carcinoma, cancer muciparum, mucinous cell carcinoma, mucinous Epidermal cancer, cancer mucosum, mucous carcinoma, carcinoma myxomatodes, upper pharyngeal carcinoma, oat cells, ossifying carcinoma, bone cancer, papillary carcinoma, periportal carcinoma , pre-invasive carcinoma, echinocytic carcinoma, pultaceous carcinoma, renal cell carcinoma, reserve cell carcinoma, sarcomatoid carcinoma, Schneiderian carcinoma, hard cancer, Carcinoma, signet ring cell carcinoma, simple cancer, small cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, sponge-like carcinoma, squamous cell carcinoma , squamous cell carcinoma, string carcinoma, vasodilator carcinoma, telangiecta-like carcinoma, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and Chorionic cancer.

As the cancer-associated gene, for example, myc, src, ras, abl, bcl, rb, p53, apc, brca1, brca2, akt2, braf , hras, kras, kit, msh2, cdk4, pten, egfr, erbb2, fgfr1, fgfr3, flt3, jak2, pdgfra, plk3ca, ret gene, etc., by inhibiting the expression of these genes, can exert anticancer effects.

Further, according to the present invention, a cell into which a translation inhibitor is introduced can be obtained. For the introduction of a polynucleotide related to the present invention to a cell, a usual gene introduction method can be used, and a calcium phosphate method, a liposome method, an electroporation method, a gene gun, a whisker method can be used. (whisker method), microinjection method, laser injection method, protoplast method (plant, yeast), Agrobacterium method (plant), lithium chloride method (yeast), etc., but not limited thereto Wait. The introduction into the individual can also be administered to the affected part by injection or a gene gun, an external preparation or the like. The translation inhibitor of the present invention can be used in vitro or in vivo. In other words, it is possible to study the function of the gene, regulate metabolism, and use it for substance production, etc., by suppressing translation of cells in a culture container such as a test tube.

Further, in accordance with the present invention, a kit comprising a translation inhibitor is provided. Examples of the kit include a kit comprising the above-described gene-introducing reagent, a selected reagent, and a polynucleotide of the present invention, a kit to which a medium is added, and the like.

Further, according to the present invention, a first polynucleotide which hybridizes to a 5'-non-translated region and/or a translation region of a target mRNA, and a second hybridizable to a 3'-non-translated region of the mRNA can be provided A method of hybridizing a polynucleotide to a target mRNA while inhibiting translation of the target mRNA. According to the method for inhibiting the translation of the target mRNA according to the present invention, it is not necessary to make a complex antisense for each target mRNA to measure the inhibition efficiency, and the labor of the most appropriate one can be selected, and the gene expression can be mechanically suppressed.

The polynucleotide of the present invention can also be administered continuously. DDS technology can also be used to slowly release cells and tissues to continuously suppress gene expression, and can also be used for the prevention and treatment of chronic diseases.

According to the present invention, there is provided a method of screening a substance having a translational inhibitory activity using an invotro translation system. An in vitro translation system refers to a system for translating proteins in a cell-free extract or in a solution containing a translational enzyme and a substrate required for translation. For the in vitro translation system, for example, a Rabbit Reticulocyte Lysate System, a Wheat Germ Extract, an insect culture cell extract, and a human culture cell extract are used (refer to Example 8). And a cell extract of a coliform extract or the like; a mixture of those purified by using an individual to produce a protein or the like, but any system can be used in the present invention. Moreover, not only the translation system but also a system that can be transcribed and translated in the same system has been developed, and these can also be used in the present invention.

In order to screen a translational inhibitory substance by an in vitro translation system, a candidate substance having a possibility of translational inhibitory activity is introduced into an in vitro translation system together with the target mRNA or, if necessary, staggered into the introduction period, and the self-labeled mRNA is measured. The synthesis activity of the protein can be. The measurement of the synthetic activity of a protein, if the protein has an enzyme activity, the amount of protein synthesized can be measured based on its enzyme activity. Examples of the enzyme activity include luminescence by luciferase, coloration by cleavage by a substrate using β-galactosidase, luminescence, and phosphorylation. Determination of phosphorylated radioisotopes, determination of matrix transfer activity by transferase, etc., but not limited This is the case. When the protein does not have an enzyme activity, for example, electrophoresis by SDS-PAGE (including Western blotting), HPLC, MASS, quantification using an antibody (ELISA, dot blot, etc.) Method, but not limited to this. Further, a plurality of such quantitative methods can be combined to measure the translational inhibitory activity.

The method for screening a translation inhibitory substance by the in vitro translation system of the present invention is not particularly limited as the translation inhibitory substance to be used, and examples thereof include a polynucleotide of an antisense strand, a ribonuclease, and an aptamer. (aptamer), antibiotics, antibodies, proteins, etc., but not limited to these. Further, in order to measure the activity of the translation inhibitor of the present invention, a combination of a polynucleotide having a higher screening activity and/or a combination of spacers may be used, and an in vitro translation system may be suitably used.

The translation inhibitor of the present invention, particularly the antisense oligonucleotide comprising a spacer, is not necessarily introduced into the cell, and is used by using an in vitro translation system that does not include a step of introducing the cell. Translational inhibitors with low cell introduction rates can also rapidly measure translational inhibitory activity, and the use of thorough screening with high throughput screening is also a possible advantage.

The translation inhibitor of the present invention may comprise siRNA. In this case, in terms of the sequence of the siRNA, siRNA which targets the 3'-end of the mRNA is preferred. In this case, the number of bases of the siRNA is 15 to 40, more preferably 19 to 30, further preferably 19 to 25 bases, particularly preferably 19 bases, at the 3'-end. An overhang of thymine may preferably be used.

The siRNA-containing translation inhibitor of the present invention may be anti-inflammatory The agent and/or the anticancer agent may be in the form of a kit comprising the anti-inflammatory agent and/or the anticancer agent. Further, cells comprising the translation inhibitor are also included in the scope of the invention.

In the method of inhibiting translation of the present invention, siRNA can be used, and the siRNA is preferably used in combination with the immediately preceding 3'-end of the target mRNA. A method of inhibiting translation using siRNA can also be used as a treatment for inflammation and/or a treatment for cancer.

[Examples]

<Example 1>

The HeLa cell line was cultured in DMEM (Sigma-Aldrich) containing 10% deactivated FBS (Gibco), penicillin-streptomycin (Life technologies), and cultured at 37 ° C under 5% CO ₂ . In this HeLa cell, the transcription factor RelA shown in Figure 1 was introduced as the target mRNA, and the antisense combined with the 5'-non-translated region, the start codon region, and the region immediately adjacent to poly-A of RelA was synthesized. Nucleic acid (polynucleotide containing LNA).

Transfection of HeLa cells was performed as follows. HeLa cells were suspended in DMEM medium containing 10% deactivated FBS in a manner of about 1×10 ⁶ cells/mL, and dispersed into a 24-well plate at 0.5 mL each. After 24 hr, when the cells were in a dense state of about 40 to 50%, pGL4.32 was added (see Fig. 1 for the cis-element + promoter + luciferase gene of NFκB; Promega) 1 μg, pGL4 .75 (Promega) 20 ng, LNA ASO and 1 μL Lipofectamine 2000 (Invitrogen) were mixed in 100 μL OptiMEM (Invitrogen). The translational inhibitory activity of RelA was measured by measuring the degree of translational repression of RelA and measuring the change in the expression of the luciferase gene linked to the promoter downstream of RelA.

ASO (Antisense Oligo) was used at a concentration of 3.3, 5, and 10 nM alone, and 5 nM was used for each of the two combinations, and 3.3 nM was used for each of the three combinations.

5'-end: The 5' end 20mer of the RelA mRNA (SEQ ID NO: 1:5'-TCGCGCGTCCGCGCCGGCCT-3') was designated as the target ASO.

First AUG: 20 mer (sequence number 2: 5'-CTGGGGCCGGTACCTGCTTG-3') containing the start codon was used as the target ASO.

3'-end/19mer: The 3' end 19mer of RelA mRNA (SEQ ID NO: 3: 5'-GACAACGGTTCGACCGATC-3') was designated as the target ASO.

3'-end/10mer: ReelA mRNA 3' end 10mer (SEQ ID NO: 4: 5'-TCGACCGATC-3') was used as the target ASO.

The sequence of ASO used is as follows.

5'-end: ASO at the 5' end of the RelA mRNA 20 mer (SEQ ID NO: 12: 5'-TCCGGCCGCGCCTGCGCGCT-3')

First AUG: 20mer ASO containing the start codon (sequence identification number 13:5'-GTTCGTCCATGGCCGGGGTC-3')

3'-end/19mer: ASO of the 3mer 19mer of RelA mRNA (SEQ ID NO: 14: 5'-CTAGCCAGCTTGGCAACAG-3')

3'-end/10mer: ASO at the 3' end of the RelA mRNA 10 mer (SEQ ID NO: 15: 5'-CTAGCCAGCT-3')

The stimulation using TNF-α was carried out as follows. At 22 hr post-transfection, the medium in which HeLa cells were cultured was exchanged into DMEM medium containing 10% deactivated FBS and 20 ng/mL TNF-α. Thereafter, HeLa cells were recovered at 2 hr. The luciferase test was carried out as follows. The recovered cells were washed with ice-cold PBS 0.5 mL and then 150 Ml Passive Lysis Buffer (Promega) dissolves. The Dual-Luciferase Reporter Assay System (Promega) was used in the analysis.

The results are shown in Fig. 4. The luciferase activity in the case where no ASO was added was taken as 100, and the inhibitory activity with respect to the same was expressed as a relative value (the same applies hereinafter). 5'-end (5'-end), start codon (First AUG), 3'-end 19mer (3'-end/19mer), 10mer (3'-end/10mer) In the case of glycosidic acid, there is about 65% residual viability even with the addition of 10 nM. However, at 5'+F(5'-end+First AUG), 5'+19(5'-end+3'-end/19mer), 5'+10(5'-end+3'-end/ 10mer), F+19 (First AUG+3'-end/19mer), F+10 (First AUG+3'-end/10mer), 5'+F+19 (5'-end+First AUG+3' -end/19mer), a combination of 5'+F+10 (5'-end+First AUG+3'-end/10mer), which is 39~23% of luciferase activity, achieving 77-61% inhibition .

<Example 2>

Next, in order to demonstrate that the inhibitory effect of the combination of the 3'-end of RelA mRNA and the 5'-end as the target ASO is specific, the luciferase assay is used to measure the junB mRNA (sequence recognition). No. 5:5'-GCTGAGCGGCTGGACCTTGA-3') as the target ASO (SEQ ID NO: 16:5'-TCAAGGTCCAGCCGCTCAGC-3') combined with ASO (SEQ ID NO: 15) which uses the 3'-end of RelA mRNA as the target The inhibitory effect when it acts. The ASO was used in a concentration of 5, 10 nM alone, and 5 nM was used in each of the two combinations.

5'-end: 20 mer of the 5' end of RelA mRNA was used as the target ASO.

3'-end/10mer: 10 mer of the 3' end of RelA mRNA was used as the target ASO.

junB-AS: The 5 mer of the junB mRNA was used as the target ASO.

junB-S: The 5 mer 20 mer of junB mRNA was used as the target Sense strand.

The results of the luciferase analysis are shown in Fig. 5. It was found that whether it is junB-AS or junB-S, even with the 3'-end/10mer combination, about 70% of the activity remains rather than the specific inhibition.

<Example 3>

In order to demonstrate that the inhibitory effect of the combination of the 3'-end of RelA mRNA and the 5'-end as the target ASO is specific, the luciferase assay is used to determine the 5'- of the RelA mRNA. End is used as the target ASO and the 3'-end is used as the target ASO in combination with the mismatcher to make it work. The ASO was used in a concentration of 5, 10 nM alone, and 5 nM was used in each of the two combinations.

5'-end: 20 mer of the 5' end of RelA mRNA was used as the target ASO.

3'-end/10mer: 10 mer of the 3' end of RelA mRNA was used as the target ASO.

3'-end/10mer 3MM: A 3 base matcher (SEQ ID NO: 6: 5'-CTCTCCATCT-3') was placed in the ASO of the 3' end of RelA mRNA as the target ASO.

3'-end/10mer 4MM: A 4 base matcher was placed in the 3 mer of the RelA mRNA 3' end as the target ASO (SEQ ID NO: 7: 5'-TTCTCCATCT-3').

The results are shown in Fig. 6. It can be seen that when there is a mismatch, the suppression effect is lowered.

<Example 4>

The luciferase assay was used to measure the translational inhibition effect of shortening the 3'-end designed ASO of RelA mRNA to 8 mer and 6 mer. The ASO was used at a concentration of 5, 10 nM alone, and 5 nM was used for each of the two combinations.

5'-end: 20 mer of the 5' end of RelA mRNA was used as the target ASO.

3'-end/10mer: 10 mer of the 3' end of RelA mRNA was used as the target ASO.

3'-end/8mer: 8 mer of the 3' end of RelA mRNA was used as the target ASO.

3'-end/6mer: 6 mer of the 3' end of RelA mRNA was used as the target ASO.

The results are shown in Figure 7. The shorter the sequence of 3'-end is, the lower the translation suppression effect is.

<Example 5>

The ASO designed for the 5'-end of RelA mRNA and the ASO designed for 3'-end with a PEG-type spacer (manufactured by GeneDesign) were introduced into HeLa cells in the same manner as above, and the translation was investigated. Inhibition activity.

5'-end: 20 mer of the 5' end of RelA mRNA was used as the target ASO.

3'-end/10mer: the 3' end 10mer of RelA mRNA was used as the target ASO.

5'/20-Sp9-3'/10: Inserting a 9-molecular spacer

5'/20-Sp18-3'/10: Inserting a spacer of 18 molecules

The results are shown in Fig. 8.

<Example 6>

Total RNA was prepared by introducing cells with Full LNA ASO containing RelA mRNA as a target, and the amount of RelA mRNA was quantified using qReal-Time PCR. Total RNA was extracted from recovered cells using Sepasol-RNA I super (Nacalai Tesque). RT-PCR was performed using SuperScript III transcriptase (Invitrogen). Oligo(dT)Primers (Invitrogen) was used as a primer for the reverse transcription reaction.

Oligo (dT) Primers 50 ng and water were added to 100 ng of total RNA extracted, and the total amount was 7 μL. The mixture was incubated at 65 ° C for 5 minutes and allowed to stand on ice for 5 minutes. After ice-cooling, 4 μL of 5×First strand buffer, 1 mL of 0.1 M DTT, 1 μL of 10 mM dNTP, 0.5 μL of RNasin Plus RNase Inhibitor (Promega), 0.5 μL of Superscript III reverse transcriptase, and water were added to prepare a total amount of 20 μL. The reaction solution was incubated at 50 ° C for 60 minutes, and then incubated at 70 ° C for 30 minutes to synthesize cDNA. qReal-Time PCR system using ^{StepOnePlus TM Real-Time PCR System (} Life Technologies) is carried out as follows. GoTaq qPCR Master Mix (Promega) 10 μL, 5 pmol of gene-specific Forward Primer and Reverse Primer were added to the cDNA and the cycle was started with the following profile. The number of cycles of the PCR reaction was 50.

Initial heat denaturation 95 ° C 5 minutes

PCR reaction (50 cycles) 95 ° C 30 seconds (thermal denaturation)

55 ° C 60 seconds (annealing)

72°C 30 seconds (extension)

The gene-specific primers used in qReal-Time PCR are described below.

RelA forward: 5'-GCAGTTTGATGATGAAGACC-3' (sequence identification number 8)

RelA reverse: 5'-CTGTCACTAGGCGAGTTA-3' (sequence identification number 9)

--actin forward: 5'-GATAGCATTGCTTTCGTGTA-3' (SEQ ID NO: 10)

--actin reversal: 5'-TTCAACTGGTCTCAAGTCAG-3' (SEQ ID NO: 11)

The ASO was used in a concentration of 3.3, 5, and 10 nM alone, and 5 nM was used for each of the two combinations, and 3.3 nM was used for each of the three combinations.

5'-end: 20 mer of the 5' end of RelA mRNA was used as the target ASO.

First AUG: The 20 mer containing the start codon is used as the target ASO.

3'-end/19mer: The 3' end 19mer of RelA mRNA was used as the target ASO.

3'-end/10mer: 10 mer of the 3' end of RelA mRNA was used as the target ASO.

The results are shown in Figure 9. The decrease in RelA mRNA caused by the addition of ASO was not observed except for the 3'-end/10mer, but instead It is observed that the mRNA is increased.

<Example 7>

Total RNA was prepared by introducing cells in which RelA mRNA was used as the target ASO, and the amount of RelA mRNA was quantified using qReal-Time PCR in the same manner as in Example 6.

5'-end: 20 mer of the 5' end of RelA mRNA was used as the target ASO.

3'-end/10mer: 10 mer of the 3' end of RelA mRNA was used as the target ASO.

5'/20-Sp9-3'/10: Inserting a 9-molecular spacer

5'/20-Sp18-3'/10: Inserting a spacer of 18 molecules

The results are shown in Fig. 10. The amount of mRNA of RelA is not inhibited by ASO, but rather the relative amount of mRNA is increased.

<Example 8>

Modulation of cell disruption for in vitro translation

The cell disruption was prepared according to the method of Rakotondrafara & Hentze (refer to Nature protocol 6, 563-571 (2011) An efficient factor-depleted mammalian in vitro translation system, Fig. 11). The HeLa cells cultured in a 10 cm culture dish were peeled off by trypsin treatment, recovered by centrifugation, suspended in a low volume of the same volume as the precipitate (Fig. 11), and allowed to stand at 4 ° C for 45 minutes. After standing, the cells were disrupted by inhalation ejection of a plurality of suspensions using a 1 mL syringe loaded with a 27G injection needle. After centrifugation, the supernatant was used as a cell disrupting solution.

<Example 9>

Translation inhibition by Antisense Oligo using an in vitro translation system test

The in vitro translation was carried out by the method described in Fig. 12. Modulation consisted of 4 μL of cell disrupted solution (20 mg protein/mL), 1 μL of translation buffer (16 mM HEPES, pH 7.6, 20 mM creatine phosphate, 0.1 μg/μl creatine kinase, 0.1 mM spermidine) , 100 μM of various amino acid mixture), 0.4 μL of 1 M KOAc, 0.2 mL of 100 mM Mg(OAc) 2, 1 μL of RNasin Plus RNase inhibitor (Promega), 0.25 pmol of mRNA template, and 10 μL of reaction solution of Antisense Oligo. A 5-hour translation reaction was carried out at 30 °C. After the completion of the translation reaction, the reaction was stopped by adding 100 μL of 1 × Passive Lysate Buffer, and the activity of the fluorescent enzyme was measured as a sample for the fluorescent enzyme test.

Antisense Oligo is made up of 20 mer Antisense Oligo (SEQ ID NO: 12) from the 5'-end of RelA mRNA or 20 mer Antisense Oligo (SEQ ID NO: 16: 5'-TCAAGGTCCAGCCGCTCAGC-3' from the Jun B5'-end And 10 mer Antisense Oligo (SEQ ID NO: 15) from the 3'-end is used by a PEG-type spacer. The PEG type spacer system uses ginseng glycol.

The gene system to be the translation template is the DNA fragment (RelA 5'-UTR-luciferase ORF-RelA 3'-UTR: SEQ ID NO: 17) shown in Fig. 14 or the DNA fragment described in Fig. 15 (JunB 5'- The UTR-luciferase ORF-RelA 3'-UTR: SEQ ID NO: 18) was each ligated to the position of the HindIII-EcoRI vector of the vector pcDNA3.1+ by total synthesis. The synthetic vectors are each cleaved at the restriction enzyme EcoRI position downstream of the RelA 3'-UTR by T7 RNA polymerase. The mRNA was transcribed, and the translation activity was measured by adding CAP and polyA in a usual manner and adding it together with Antisense Oligo in an in vitro translation system. The results are shown in Figures 14 and 15.

The graph in Figure 14 shows the relative activity of the luciferase. In the case where no treatment was taken as 1, the translation inhibition was less caused by 5'-LNA and 3'-LNA, but about 40% of translational inhibition was observed in the case of 5'+3'.

Further, in the case of using Antisense Oligo having the same sequence at the 5'-end, the translation was inhibited in an amount-dependent manner. On the other hand, in the case of using JunB-SP9-RelA having a different 5'-end, translation inhibition was only produced by 15%. Thus, translational inhibition can be considered to be sequence dependent.

Next, the 5'-end of the translation template was made into the JunB 5'-UTR gene in the same manner as described above, and the inhibitory activity of the translation was measured. As a result, the interpretation of the amount dependence of Juns' Antisense Oligo was suppressed, but even with the addition of RelA-SP9-RelA, the translation was reduced less. The sequence specificity of Antisense Oligo was also observed by this experiment.

Further, even if the gene was transformed, the Antisense Oligo of the present invention was shown to be effective. That is, it is shown that 10 bases at the 5'-end and 10 bases at the 3'-end of the 5'-end are synthesized, and each of them is added or linked by a spacer, thereby inhibiting translation of the gene. .

With respect to the following Examples 10 to 12, the following methods were carried out.

Cell culture

The HeLa cell line was cultured in DMEM (Dulbecco's-modified Eagle's medium; Sigma) containing 10% deactivated FBS (Gibco), penicillin-streptomycin (Life technologies) at 37 ° C, 5% CO ₂ .

Transfection

HeLa cells were suspended in DMEM medium containing 10% deactivated FBS in a manner of about 1×10 ⁶ cells/mL, and dispersed into a 24-well plate at 0.5 mL each. After 24 hr, the cells were transfected in a dense state of about 40 to 50%. Using pGL4.32 [luc2P/NF-κB-RE/Hygro] plastid, pGL4.75[hRluc/CMV] plastid, 0.8 μg pGL4.32 [luc2P/NF-κB-RE/Hygro] plastid 20 ng of pGL4.75 [hRluc/CMV] plastid, siRNA or Antisense Oligo, 1 μL of Lipofectamine 2000 (Invitrogen) was mixed in 100 μL of OptiMEM (Invitrogen), and allowed to stand at room temperature for 20 minutes. After standing, the mixture was added to the culture solution and transfected.

Stimulation with TNF-α

In the case of transfection of pGL4.32 [luc2P/NF-κB-RE/Hygro] plastid, for the activation of NF-κB, the medium in which HeLa cells were cultured was removed by aspirator and exchanged for 22 hours after transfection. 500 μL of DMEM medium containing 10% deactivated FBS, 20 ng/mL TNF-α. After the medium was exchanged, it was returned to the incubator (37 ° C, 5% CO ₂ ) and allowed to stand for 2 hours.

Fluorescent enzyme test

Fluorescent enzyme activity was determined in the following manner using a Dual-Luciferase Reporter Assay System (Promega). After the transfected HeLa cells were washed twice with 500 μL of ice-cold phosphate buffered saline (PBS) (pH 7.4), 150 μL of Passive Lysis Buffer (Promega) was added and allowed to cool at room temperature. Stir for 15 minutes. The cell lysate was centrifuged at 10,000 g for 5 minutes at room temperature, and 30 μL of the obtained supernatant was used for the luciferase test. Determination of luminous intensity A Luminoskan luminometer (Thermo Scientific) was used.

siRNA

The siRNA system was commissioned and purchased by Nippon Gene Material. Further, siNegative (Universal Negative Control siRNA) is commercially available from Nippon Gene Material.

3'-end siRelA/antisense stock; 5'-cuagccagcuuggcaacagTT-3' (sequence identification number 19)

3'-end siRelA/Justice Unit: 5’-cuguugccaagcuggcuagTT-3’ (sequence identification number 20)

siPositive/antisense stock; 5'-ugacguaaagggauagggcTT-3' (sequence identification number 21)

T is the portion of the protruding portion. The siRNA system introduces dsRNA, which is a protruding part of TT, into cells.

Gapper and mixmer

Gapmer and mixmer were commissioned by GeneDesign Co., Ltd. to synthesize and purchase.

3'-end gapmer 5'-CTagccagcttGG-3' (sequence identification number 22)

5'-end LNA 5'-TCCGGCCGCGCCTGCGCGCT-3' (sequence identification number 23)

5'-end gapmer 5'-TCCGgccgcgcctgcgCGCT-3' (sequence identification number 24)

5'-end Mixmer 1 5'-TccgGccgCgccTgcgCgct-3' (SEQ ID NO: 25)

5'-end Mixmer 2 5'-tccGgccGCGCCTgcgCgct-3' (SEQ ID NO: 26)

Uppercase indicates LNA.

<Example 10>

Combination of 5'-end LNA and 3'-end siRNA

LNA Antisense Oligo (5'-end) designed to hybridize to the 5'-end of human RelA mRNA and LNA ASO (3'-end/10-mer), siRNA designed to hybridize to the 3'-UTR terminus (3'-siR), siPositive, siNegative are used alone or in combination to measure the translation inhibition effect. The case where 5'-LNA was used alone was used at a concentration of 10 nM, and the case where siRNA was used alone was used at a concentration of 40 nM, and in the case of two combinations, 5'-LNA was used at 5 nM and siRNA was used at a concentration of 20 nM. The luciferase activity is expressed as a relative value when the value of transfection of siNegative is taken as 1.

[result]

When 5'-end LNA, 3'-end siRelA, 3'-end LNA, and siPositive were allowed to act alone, 32%, 35%, 40%, and 91% inhibition effects were obtained (Fig. 16). When used in combination, 5'-end+3'-end siRelA gets 91%, 5-end+3'-end gets 92%, 3'-end+siPositive gets 91%, 5'-end+siNegative gets 43% Translation suppression effect (Figure 16). This suggests that in combination with the 5'-end LNA, not only the 3'-end LNA, but also the 3'-end siRNA is also good. From the above results, it is understood that a combination of siRNA composed of a natural nucleic acid and an antisense LNA composed entirely of a nucleic acid derivative can achieve a translation inhibitory effect.

<Example 11>

Combination of 5'-end LNA and 3'-end gapmer

Designed to hybridize to the 5'-end of human RelA mRNA The 5'-end LNA (5'-L) and the LNA gapmer (3'-end gapmer/13mer; 3'-G) designed to hybridize with the 3'-UTR end are used alone or in combination, and measured. Translation suppression effect. The case where 5'-L was used alone was used at 10 nM or 5 nM. In the case of two combinations, the 5'-LNA system was used at a ratio of 5 nM and 3'-G between 0.1 and 10 nM. The luciferase activity was expressed as the relative value of the cell treated without Antisense Oligo as a one.

[result]

When 3'-G was transfected at 0.1, 1, 5, or 10 nM alone, no translation inhibitory effect (107%) was observed at 0.1 nM, and a translation inhibitory effect of 63%, 90%, and 94% was obtained at 1 nM or more. When 5 nM 5'-L was used in combination with the respective concentrations of 3'-G, 62% was obtained with 0.1 nM combination, 93% with 1 nM combination, and 96% with 5 nM, 10 nM combination ( Figure 17). From the above results, it is suggested that the combination with 5'-L is not only a 3'-end LNA, but also a 3'-G (gapmer type). Further, by using 3'-G as the 3'-side antisense nucleic acid, a strong translation inhibitory effect can be obtained at a low concentration (10 nM or less).

<Example 12>

Combination of 3'-end gapmer with 5'-end gapmer, 5'-mixmer1, 5'-mixmer2

A gapmer (5'-G), a mixmer1 (5'-M1), a mixmer 2 (5'-M2) designed to hybridize to the 5'-end of human RelA mRNA, and a hybrid to the 3'-UTR terminus The LNA gapmer (3'-G) designed by the method is used alone or in combination to measure the translation inhibition effect. The case of each being used alone is used at 5 nM. Combine two situations The 5'-side Antisense Oligo was used at 1 nM, 3 nM, 10 nM, and the 3'-G was used at 5 nM. The luciferase activity was expressed as a relative value when the value of the cells not treated with Antisense Oligo was taken as 1.

[result]

The 3'-end gapmer (3'-G) was transfected at a concentration of 5 nM. 3'-G alone showed an 81% translation inhibition effect at 5 nM. 5'-end gapmer (5'-G), 5'-mixmer1 (5'-M1), 5'-mixmer2 (5'-M2) were each at a concentration of 1, 3, 10 nM with a 3'-end gapmer ( 3'-G) 5nM was transfected together. When each concentration of 5'-G, 5'-M1, 5'-M2 and 5 nM of 3'-G were combined and acted on, the translation inhibitory activity was confirmed (Fig. 18). From the above results, it is suggested that even a combination of 3'-G and 5'-end gapmer (5'-G), 5'-mixmer1 (5'-M1), or 5'-mixmer2 (5'-M2) Can inhibit translation.

<Example 13>

Antisense oligonucleic acid coating of 5' and 3' end regions of mouse RelA mRNA and inhibition of inflammation-related genes

A total of 10 μg of the following nucleic acid derivative was mixed with a transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific) and applied to both ear shell portions of mouse BALB/c 7 weeks old. One mouse was used per sample.

A nucleic acid derivative having a scramble sequence (control group, 5'-GtgtAacaCgtcTataCgccCA-3')

2.5'-RelA (5'-GgtcCcgtTcccGgccCcgC-3' (SEQ ID NO: 27)

3.3'-RelA (5'-CagcGtgaTaagAcatTtaT-3' (sequence recognition) No. 28)

4.5’-RelA+3’-RelA

The 5'-RelA and 3'-RelA lines use a sequence (antisense) that hybridizes to the 5'-end and 3'-end of mouse RelA mRNA. The uppercase of the sequence is LNA. A mixmer containing LNA and DNA was used as an antisense oligo. The nucleic acid system was synthesized by GeneDesign Co., Ltd.

Two days later, 10 μl of 0.15% DNFB was applied to both ear shells to cause inflammation. Two hours later, the mice were euthanized by cervical dislocation and the ear shells were recovered and stored at -80 °C. The ear shell was cooled with liquid nitrogen, and 300 μl of RIPA buffer was added by crushing under pressure. Next, the Physcotron was finely crushed until the tissue block disappeared, and the ultrasonic generator UR-20P (manufactured by TOMY SEIKO Co., Ltd.) was further used, and the power control was set to 7 (the sample did not fly out from the Eppendorf tube). The intensity of the degree), 10 times of ultrasonic processing was performed at intervals of 15 seconds. The solution was centrifuged, and a portion of the clear solution was recovered to about 150 μl, and the protein concentration was measured by the BCA method. 10% SDS polypropylene guanamine colloid was used for electrophoresis in a manner of 15 μg of protein per lane, and transferred to a membrane by using an anti-RelA protein antibody (Santa Cruz, NFκB p65 (c-20) The Western blotting method of sc-732), anti-tubulin protein antibody (SIGMA, T6557), and anti-JunB protein antibody (Santa Cruz, JunB (210) sc-73) was used to detect the respective proteins. Further, for the purpose of confirming whether or not there is an equivalent amount of protein migration in each of the electrophoresis channels, the film after transfer was stained with Ponceau.

[result]

As a result of Western blotting, antisense oligonucleic acid 5'-RelA and In the case of 3'-RelA, or a mixture thereof, the amount of RelA protein was significantly lowered as compared with scrambling (Fig. 19). The JunB gene is activated by binding to the NFκB binding site of the promoter region of the JunB gene in the subunit with the transcription factor NFκB line of RelA. As a result, the JunB protein line which is expressed and induced acts as a transcription factor AP-1, and contributes to the deterioration of the inflammatory response. This time, in the experiment using the mouse ear shell portion, the amount of JunB protein was also decreased by mixing the antisense oligonucleic acid having the 5' and 3' ends of RelA mRNA as a target (Fig. 19). This suggests that antisense oligonucleic acids against RelA mRNA can be therapeutic agents for inflammation. That is, it was confirmed that the anti-inflammatory antisense oligonucleic acid of the present invention has an effect not only in vitro but also at the cell level, but also in vivo and at the individual level. Further, since it can suppress the expression of the JunB protein, it is considered to have an effect as an anticancer agent. After the Western blotting method, the color of the electrophoresis was stained at almost the same concentration, and the amount of protein in each of the electrophoresis channels was confirmed to be equal (Fig. 20).

[Industrial availability]

The target mRNA translation inhibitor and translation inhibition method of the present invention can be used for manufacturing, agriculture, and the like of reagents and/or medicines.

<110> Osaka University Yangjindo Co., Ltd. (Yoshindo Inc.)

<120> Translation inhibitors and translation suppression methods

<130> P2-15007091

<150> JP 2014-263578

<151> 2014-12-25

<150> JP 2015-078049

<151> 2015-04-06

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> RelA5’-end20mer

<400> 1

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> RelA ATG

<400> 2

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> RelA 3’end 19

<400> 3

<210> 4

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> Rel3’end10

<400> 4

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> junB 5’-end

<400> 5

<210> 6

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> 10mer 3MM

<400> 6

<210> 7

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> 10mer 4MM

<400> 7

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> RelA Forward Primer 5

<400> 8

<210> 9

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> RelA reverse introduction 5'

<400> 9

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> β-actin forward primer

<400> 10

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> β-actin reverse primer

<400> 11

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> RelA5’-20 Antisense Oligo

<400> 12

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Antisense Oligo with RelA ATG area

<400> 13

<210> 14

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> RelA 3’-19mer Antisense Oligo

<400> 14

<210> 15

<211> 10

<212> DNA

<213> Artificial sequence

<220>

<223> RelA 3’-end 10mer Antisense Oligo

<400> 15

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> junB 5’-20mer aso

<400> 16

<210> 17

<211> 1869

<212> DNA

<213> Artificial sequence

<220>

<223> RelA 5'-UTR-Fluorescent Enzyme ORF-RelA 3'-UTR

<400> 17

<210> 18

<211> 2025

<212> DNA

<213> Artificial sequence

<220>

<223> junB 5’-UTR-LUC-3’UTR

<400> 18

<210> 19

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> 3’-end siRelA/antisense stock

<400> 19

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> 3’-end siRelA/Justice Unit

<400> 20

<210> 21

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> siPositive/anti-sense stock

<400> 21

<210> 22

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223> 3'-end gapmer (spacer)

<400> 22

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> 5’-end LNA

<400> 23

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> 5’-end gapmer

<400> 24

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> 5'-(Mixed) 1

<400> 25

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> 5'-(Mixed) 2

<400> 26

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> mouse 5’-RelA mixmer

<400> 27

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse 3'-RelA mixmer

<400> 28

Claims (31)

A target mRNA translation inhibitor comprising a first polynucleotide that hybridizes to a 5'-non-translated region and/or a translation region of a target mRNA, and a vector that hybridizes to a 3'-non-translated region of the mRNA Two polynucleotides. A translational inhibitor of mRNA as claimed in claim 1, wherein the first polynucleotide is a polynucleotide which hybridizes to a region of the target mRNA comprising a 5'-non-translated region. A translational inhibitor of mRNA as claimed in claim 1 or 2, wherein the first polynucleotide hybridizes to a region of the target mRNA comprising a 5'-end or a translation initiation codon. The translational inhibitor of the target mRNA of any one of claims 1 to 3, wherein the second polynucleotide is hybridized immediately prior to the poly-A sequence of the target mRNA. The translation inhibitor of the target mRNA according to any one of claims 1 to 4, wherein the first polynucleotide is a polynucleotide comprising 18 to 30 bases. The translational inhibitor of the target mRNA according to any one of claims 1 to 5, wherein the second polynucleotide is a polynucleotide comprising 6 to 30 bases. The translational inhibitor of the target mRNA according to any one of claims 1 to 6, wherein the polynucleotide is DNA, RNA, a polynucleotide comprising LNA or Alkranyl oligonucleic acid. The translation inhibitor of the subject mRNA of any one of claims 1 to 7, wherein the polynucleotide comprises at least one nucleotide analog. The translation inhibitor of the target mRNA according to any one of claims 1 to 8, wherein at least one spacer is interposed to link the first plurality The nucleotides are made with the polynucleotide of the second polynucleotide, each of which can hybridize to the 5'-non-translated region and/or the translation region and the 3'-non-translated region of the target mRNA. The translational inhibitor of the target mRNA according to any one of claims 1 to 9, wherein the target mRNA is an mRNA of a protein that causes an inflammatory response. The translational inhibitor of the target mRNA according to any one of claims 1 to 10, wherein the target mRNA is an mRNA of NFκB. An anti-inflammatory agent and/or anticancer agent comprising a translational inhibitor of mRNA as claimed in claim 10 or 11. A cell into which a translational inhibitor of the target mRNA of any one of claims 1 to 11 or an anti-inflammatory agent and/or an anticancer agent according to claim 12 is introduced. A kit comprising a translational inhibitor of the target mRNA according to any one of claims 1 to 11 and/or an anti-inflammatory agent and/or an anticancer agent according to claim 12. A method of inhibiting translation of a target mRNA by first hybridizing a first polynucleotide that hybridizes to a 5'-non-translated region and/or a translation region of a target mRNA, and hybridizing to a 3'-non-translated region of the mRNA The second polynucleotide hybridizes to the target mRNA. A method of translating an inhibitory target mRNA of claim 15, wherein the first polynucleotide is a polynucleotide which hybridizes to a region of the target mRNA comprising a 5'-non-translated region. A method of translating an inhibitory target mRNA of claim 15 or 16, wherein the first polynucleotide hybridizes to a region of the target mRNA comprising a 5'-end or a translation initiation codon. A method of translating a suppressor mRNA of any one of clauses 15 to 17, wherein the second polynucleotide is hybridized immediately prior to the poly-A sequence of the target mRNA. The method of claim 15, wherein the first polynucleotide is a polynucleotide comprising 18 to 30 bases. The method of claim 15, wherein the second polynucleotide is a polynucleotide comprising 6 to 30 bases. A method of translating an inhibitory target mRNA according to any one of claims 15 to 20, wherein the polynucleotide is DNA, RNA, a polynucleotide comprising LNA or Alkranyl oligonucleic acid. The method of claim 15, wherein the polynucleotide is a polynucleotide comprising at least one nucleotide analog. A method of translating an inhibitory target mRNA according to any one of claims 15 to 22, wherein: using at least one spacer to link the first polynucleotide to the multinuclear of the second polynucleotide The glucuronide can be made to hybridize to each of the 5'-non-translated region and/or translation region and the 3'-untranslated region of the target mRNA. A method of translating an inhibitory target mRNA according to any one of items 15 to 23, wherein the target mRNA is an mRNA of a protein causing an inflammatory reaction. The method of claim 15, wherein the target mRNA is NFκB mRNA. An inflammatory therapeutic method and/or a method of treating cancer, which is a method of using a translation of a suppressor mRNA as claimed in claim 24 or 25. A method of screening for a substance having a translational inhibitory activity by using an in vitro translation system to screen for a substance having a translational inhibitory activity. The method of claim 27, wherein the substance having the translation inhibitory activity is a polynucleotide. The method of claim 28, wherein the polynucleotide is the first polynucleotide and/or the second polynucleotide of claim 1. The translational inhibitor of the target mRNA of any one of claims 1 to 11, the anti-inflammatory agent and/or the anticancer agent of claim 12, the cell of claim 13, and/or the anti-inflammatory agent of claim 14 and / or a set of anticancer agents, wherein the second polynucleotide is siRNA. A method of inhibiting translation of claims 15 to 25, and/or an inflammatory therapeutic method and/or a method of treating cancer of claim 26, wherein the second polynucleotide is siRNA.