Ligand-bound nucleic acid complex

Abstract

Provided is a PTH1 ligand-bound nucleic acid complex that is delivered to a desired organ and that is for regulating the expression or editing of a target gene, a transcript thereof or a translation product thereof.　This PTH1 ligand-bound nucleic acid complex, which has a structure wherein a PTH1 ligand is bound to a nucleic acid, said nucleic acid containing an antisense strand containing an oligonucleotide that has a nucleic acid base sequence complementary to a target transcript and that comprises 12-30 nucleotides, is delivered to an organ and regulates the expression or editing of a target gene, a transcript thereof or a translation product thereof, which makes the complex useful in treating a disease caused by the gene. Further, the ligand-bound nucleic acid complex can be delivered to organs, tissues and cells in which PTH1 receptors are expressed, for example tissues such as that of the kidney, bones, and subcutaneous fat, and cells such as vascular endothelial cells, which makes the complex useful in treating diseases in these organs.

Description

Ligand-bound nucleic acid complexes

[0001] The present invention relates to a nucleic acid complex that regulates the expression or editing of a target gene, its transcription product, or its translation product, and more specifically to a ligand-bound nucleic acid complex in which a ligand that can be delivered to a target organ, cell, etc. is bound to the nucleic acid complex.

[0002] Nucleic acid drugs differ from conventional small molecule drugs in that they act on the sequence of the transcripts of disease-causing genes themselves, thereby regulating the expression of transcripts and proteins, and are therefore being developed as next-generation medicines.

[0003] Nucleic acids used as nucleic acid medicines include small interfering nucleic acids (siNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), and short hairpin RNA (shRNA) molecules that can mediate RNA interference (RNAi) against target nucleic acid sequences, as well as single-stranded antisense oligonucleotides (ASO) and antisense double-stranded DNA oligonucleotides (ADO), heteroduplex oligonucleotides (HDO) consisting of an antisense strand of DNA and a complementary strand of RNA, and single-stranded heteroduplex oligonucleotides (SDO). Chemically modified low-molecular-weight nucleic acids such as ss-HDO (ss-heteroduplex oligonucleotide) have been known for some time.

[0004] HDO consists of an antisense strand having a nucleic acid sequence capable of hybridizing to a target gene or a target transcription product, and a complementary strand (also called a sense strand), which is a nucleic acid strand complementary to the antisense strand. The structure in which the antisense strand is annealed to the complementary strand is called a heteroduplex oligonucleotide (HDO) (Patent Document 1).

[0005] ss-HDO is a single-stranded oligonucleotide at the time of production, and has a structure comprising an antisense strand composed of DNA nucleotides or DNA nucleotide analogs, a linker portion composed of 3 to 10 nucleotides, and a sense strand composed of RNA nucleotides or RNA nucleotide analogs that is complementary to the antisense strand. This single-stranded oligonucleotide has an X-L-Y structure (Patent Document 2). X is the antisense strand, Y is the strand complementary to the antisense strand, and L is composed of nucleotides that function as a linker. When this single-stranded oligonucleotide is used as a pharmaceutical composition, the antisense strand and the strand complementary to the antisense strand anneal monomolecularly at the linker in solvents used in physiological saline, aqueous injections, non-aqueous injections, suspension injections, solid injections, etc., or in blood or plasma, to form a double-stranded structure. Since such a nucleic acid complex anneals monomolecularly to form a double-stranded structure when acting as a pharmaceutical composition, this single-stranded oligonucleotide is a type of HDO.

[0006] Meanwhile, a known technique for delivering a nucleic acid complex to a target organ involves adding a ligand or the like that can be delivered to the target organ to the nucleic acid complex. The ligand or the like can be a molecule selected from lipids, peptides, and proteins. Lipids can be selected from cholesterol, fatty acids, fat-soluble vitamins, glycolipids, and glycerides. Furthermore, a ligand for a receptor expressed on the surface of a target cell can also be selected as the ligand or the like (Patent Document 1).

[0007] For example, when cholesterol is used as a ligand, it is taken up into cells via LDL receptors on the cell surface, and therefore can be delivered to the liver, which is an organ containing cells with LDL receptors. However, since many cells with LDL receptors exist in organs other than the liver, when targeting organs other than the liver, cholesterol will also be delivered to other organs, and therefore safety and side effects in other organs must be considered.

[0008] Ligand-conjugated nucleic acid complexes used as nucleic acid drugs are required not only to be delivered to organs, but also to be taken up by organ tissues or cells within the organs to exert an antisense effect. When a nucleic acid molecule with a larger molecular weight than the ligand is bound, it is difficult to predict whether the ligand-conjugated nucleic acid complex will be taken up into cells, making this a high hurdle. There are still not enough ligand-conjugated nucleic acid complexes available that can be delivered to each organ. Currently, bioventures and pharmaceutical companies are continuously researching and developing ligands that can be delivered more specifically to target organs.

[0009] PTH receptors are widely distributed throughout the body, but are most abundant in the kidneys and bones, and types 1 to 3 have been identified to date. The PTH1 receptor is a G protein-coupled receptor. Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) are known to be endogenous ligands for the PTH1 receptor.

[0010] PTH is a hormone secreted by the parathyroid gland. It is a peptide hormone consisting of 84 amino acids. It is involved in regulating blood calcium levels, and most of this occurs via the PTH1 receptor in the kidney. PTHrP is a 139-173 amino acid protein with N-terminal homology to PTH. The N-terminus of PTHrP is highly homologous to PTH, and both bind to and exert their effects on the common PTH receptor. PTHrP is secreted from various cells in various organs as needed, and physiologically acts primarily in a paracrine / autocrine manner. The biological activity of PTH can be reproduced by the N-terminal fragment of amino acids 1-34, i.e., hPTH(1-34) (Patent Document 3). PTH(1-34) and PTHrP(1-34) are known to have two amphopilic alpha helical domains. The biological activities of PTH(1-34) and PTHrP(1-34) are known to be the promotion of differentiation of osteoblast precursor cells and pre-osteoblasts, and the suppression of apoptosis of osteoblasts, thereby increasing the number of osteoblasts and promoting bone formation.

[0011] However, no attempt has been made to bind a ligand for the PTH1 receptor (hereinafter referred to as a PTH1 ligand) to a nucleic acid molecule that regulates the expression or editing of a target gene, its transcription product, or its translation product, and then use the PTH1 ligand to incorporate a nucleic acid molecule containing an antisense strand to the target gene or its transcription product into a tissue or cell for the purpose of delivering it to an organ, tissue, or cell, thereby regulating the expression or editing of the target gene, its transcription product, or its translation product with the nucleic acid molecule.

[0012] International Publication No. WO2013 / 089283 International Publication No. WO2017 / 131124 Japanese Patent Publication No. 62-28799

[0013] The PTH1 receptor is a G-protein-coupled receptor (GPCR) with seven transmembrane helices. PTH1 receptors are widely distributed throughout the body, but tend to be highly expressed in organs and tissues such as the kidney, bone, and subcutaneous fat, and in cells such as vascular endothelial cells. Among human and mouse organs, the PTH1 receptor is particularly highly expressed in the kidney.

[0014] The problem to be solved by the present invention is to deliver a ligand-binding nucleic acid complex to an organ of a subject where the PTH1 receptor is expressed via the PTH1 receptor, thereby regulating the expression or editing of a target gene, its transcription product, or its translation product.Another object of the present invention is to discover and complete a ligand-binding nucleic acid complex that can be delivered to an organ of a subject where the PTH1 receptor is expressed via the PTH1 receptor by intravenous or subcutaneous administration, which is an administration method that imposes little physical strain on the subject, and that can regulate the expression or editing of a target gene, its transcription product, or its translation product.

[0015] The inventors have confirmed that a nucleic acid complex to which a ligand for the PTH1 receptor, which is expressed in the kidney and bone tissues of subjects such as humans and mice, is delivered to organs expressing the PTH1 receptor and regulates the expression or editing of a target gene, its transcription product, or its translation product, thereby completing the invention.

[0016] As a result of further research into ligands for the PTH1 receptor, the inventors have found that the PTH1 ligand-binding nucleic acid complex of the present invention can be delivered to a target organ and regulates the expression or editing of a target gene, its transcription product, or its translation product. The ligand-binding nucleic acid complex of the present invention can be used to treat diseases of organs containing cells expressing the PTH1 receptor.

[0017] That is, the present invention is as follows: [1] A ligand-binding nucleic acid complex in which a nucleic acid molecule that regulates the expression or editing of a target gene, its transcription product, or its translation product is bound to a PTH1 ligand with or without a linker. [2] The ligand-binding nucleic acid complex of [1], in which the PTH1 ligand is a peptide consisting of an amino acid sequence represented by the following formula: [Chemical Formula 1] A １ -A ２ -A ３ -A ４ -A ５ -A ６ -A ７ -A ８ -A ９ -A １０ -A １１ -A １２ -A １３ -A １４ -A １５ -A １６ -A １７ -A １８ -A １９ -A ２０ -A ２１ -A ２２ -A ２３ -A ２４ -A ２５ -A ２６ -A ２７ -A ２８ -A ２９ -A ３０ -A ３１ -A ３２ -A ３３ -A ３４[wherein the amino acid sequence is written from the N-terminus to the C-terminus.] A4 is Glu or absent; A5 is Leu, His, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe p-X-Phe or absent, where X is OH, halogen, or CH3; A6 is Gln; A7 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe p-X-Phe or absent, where X is OH, halogen, or CH3; A8 is Met, Nva, Leu, Val, Ile, Cha, Acc, Nle or absent; A9 is His or absent; A10 is Asp or Asn; A11 is Leu, Lys, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe or p-X-Phe, where X is OH, halogen, or CH3; A12 is Gly, Acc, or Aib; A13 is Lys; A14 is Ser or His; A15 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe or p-X-Phe, where X is OH, halogen, or CH3; A16 is Ser, Gln, Asn, Ala, or Aib; A17 is Ser, Asp, Thr, or Aib; A18 is Met, Nva, Leu, Val, Ile, Nle, Acc, Cha, or Aib; A19 is Arg, Glu, or Aib; A20 is Arg; A21 is Arg, Val, Acc, Cha, or Met; A22 is Phe, Glu, Aib, Acc, or Cha; A23 is Phe, Trp, Leu, Lys, Acc, or Cha; A24 is Leu, Lys, Acc, or Cha; A25 is His, Arg, Lys, Aib, Acc, or Glu; A26 is His, Aib, Acc, or Lys; A27 is Lys, Aib, Leu, hArg, Gln, Acc, or Cha; A28 is Ile, Leu, Lys, Acc, or Cha;[3] The ligand-binding nucleic acid complex of [1], wherein the PTH1 ligand is a peptide consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 1 to 168. [4] The ligand-binding nucleic acid complex of [2], wherein the PTH1 ligand is a peptide consisting of any one of the sequences selected from the group consisting of SEQ ID NOs: 1 to 164. [5] The ligand-binding nucleic acid complex of [4], wherein the nucleic acid molecule and the PTH1 ligand are linked via a linker. [6] The ligand-binding nucleic acid complex of [5], wherein the linker is a cleavable linker having a cleavable structure. [7] The ligand-binding nucleic acid complex of [5], wherein the linker is any of the linkers having the following structure:

[0018] [8] The ligand-binding nucleic acid complex of [1], wherein the nucleic acid molecule is selected from the group consisting of a nucleic acid molecule having an antisense strand composed of an oligonucleotide having a nucleic acid base sequence complementary to a target gene or its transcription product, an aptamer having a nucleic acid base sequence that specifically binds to a target protein, and a decoy composed of an oligonucleotide having a nucleic acid sequence complementary to a target transcription factor. [9] The ligand-binding nucleic acid complex of [1], wherein the nucleic acid molecule is a nucleic acid molecule selected from ADO, ASO, HDO, and RNAi.

[10] The ligand-binding nucleic acid complex of [9], wherein the antisense strand of the nucleic acid molecule consists of 12 to 30 consecutive nucleotides.

[11] The ligand-binding nucleic acid complex of [8], wherein the nucleic acid molecule is a decoy composed of 8 to 30 nucleotides and having a nucleic acid sequence complementary to a target transcription factor.

[12] The ligand-binding nucleic acid complex of [9], wherein the oligonucleotide is an siRNA consisting of an antisense strand composed of RNA and a nucleic acid strand complementary to the antisense strand.

[13] The ligand-bound nucleic acid complex of [9], wherein the nucleic acid strand of the nucleic acid molecule contains nucleotides, modified nucleotides, and / or nucleotide analogs.

[14] The ligand-bound nucleic acid complex of

[13] , wherein the total number of nucleotides, modified nucleotides, and nucleotide analogs in the antisense strand of the nucleic acid molecule is 12 to 30 nucleotides.

[15] The ligand-bound nucleic acid complex of

[13] , wherein the nucleic acid molecule is HDO, and the total number of nucleotides, modified nucleotides, and nucleotide analogs in the antisense strand and complementary strand of the HDO each is 12 to 30 nucleotides.

[16] The ligand-bound nucleic acid complex of

[15] , wherein the antisense strand is a gapmer and contains a gap region containing nucleotides and / or modified nucleotides, and wing regions containing one or more nucleotide analogs and / or modified nucleotides located on the 5'-end and / or 3'-end of the gap region.

[17] The ligand-bound nucleic acid complex of

[15] , wherein the antisense strand is a non-gapmer and contains nucleotides, modified nucleotides, and / or nucleotide analogs.

[18] The ligand-binding nucleic acid complex of

[17] , wherein the complementary strand comprises nucleotides, modified nucleotides and / or nucleotide analogs.

[19] The ligand-bound nucleic acid complex of

[16] , wherein the complementary strand comprises a center region containing nucleotides and / or modified nucleotides, and wing regions containing one or more nucleotide analogs and / or modified nucleotides located on the 5'-end and / or 3'-end thereof.

[20] The ligand-bound nucleic acid complex of

[13] , wherein the modified nucleotide is a nucleotide containing a 2'-O-CH3 group or a 2'-O-CH2CHOCH3 (MOE) group.

[21] The ligand-bound nucleic acid complex of

[13] , wherein the nucleotide analog comprises a bridged nucleotide independently selected from the group consisting of LNA, cEt-BNA, amide BNA (AmNA), and cMOE-BNA.

[22] The ligand-bound nucleic acid complex according to

[13] , wherein the nucleotide analogs are independently selected from the group consisting of PNA, GNA, TNA, cEt, tcDNA, morpholino nucleic acid, BNA, Guanidine bridged nucleic acid (GuNA), and 2'-O,4'-C-Spirocyclopropylene bridged nucleic acid (scpBNA).

[23] The ligand-bound nucleic acid complex according to

[13] , wherein at least one nucleotide or modified nucleotide in the nucleic acid molecule is phosphorothioated or boranophosphated.

[24] A ligand-bound nucleic acid complex having an antisense strand for reducing the expression level of a target transcript in an organ of a subject that expresses a PTH1 receptor, and a PTH1 ligand for delivering the antisense strand to the organ of the subject, wherein the antisense strand comprises a base sequence capable of hybridizing to at least a portion of the target transcript and has an antisense effect on the target transcript.

[25] The ligand-bound nucleic acid complex of

[24] , wherein the nucleic acid complex is a double-stranded nucleic acid agent comprising a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand comprises a base sequence capable of hybridizing to at least a portion of the target transcript and has an antisense effect on the target transcript, the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and is bound to a PTH1 ligand, and the first nucleic acid strand is annealed to the second nucleic acid strand.

[26] A ligand-binding nucleic acid complex according to

[25] , which is for intravenous administration. This specification includes the disclosure of Japanese Patent Application No. 2021-179771, from which the present application claims priority.

[0019] The ligand-binding nucleic acid complex of the present invention can be used to treat diseases of organs containing cells expressing the PTH1 receptor.

[0020] The ligand-binding nucleic acid complex of the present invention can be delivered in an organ- or cell-specific manner via the PTH1 ligand, and can regulate the expression or editing of a target gene, its transcription product, or its translation product via the nucleic acid molecule. By using a nucleic acid complex that targets a disease-causing gene, its transcription product, or its translation product, it can be used as a therapeutic agent for a disease of a specific organ. Because it is most easily delivered to the kidney, which is the organ where the PTH1 receptor is most expressed, the PTH1 ligand-binding nucleic acid complex can be used as a therapeutic agent for kidney disease.

[0021] Figure 1 shows changes in animal weight before and after administration of L001-HDO6. Figure 2A shows changes in blood ALT activity 3 days after administration of L001-HDO6 to mice. Figure 2B shows changes in blood AST activity 3 days after administration of L001-HDO6 to mice. Figure 3 shows changes in mMalat1 ncRNA expression levels in the liver of mice after administration of L001-HDO6. Figure 4 shows changes in mMalat1 ncRNA expression levels in the kidney of mice after administration of L001-HDO6. Figure 5 is a schematic diagram of fluorescently labeled ASO and HDO used in the kidney distribution test. Figure 6 shows immunostained images of the kidney 10 minutes, 6 hours, 24 hours, and 72 hours after a single intravenous administration of L001-HDO6-Alexa488 to mice. Figure 7 shows changes in mMalat1 ncRNA expression levels in mouse kidneys after intravenous administration of HDO, L001-HDO6, L003-HDO6, L005-HDO6, and L010-HDO6. Figure 8 shows changes in mMalat1 ncRNA expression levels in mouse kidneys after subcutaneous administration of L031-HDO6 and L021-HDO6. Figure 9 shows changes in mMalat1 ncRNA expression levels in L021-HDO6, L003-HDO6, L005-HDO6, and L010-HDO6 in an in vitro assay using a cell line stably expressing mPth1R. Figure 10 shows changes in mMalat1 ncRNA expression levels in L021-HDO6 and L011-HDO6 in an in vitro assay using a cell line stably expressing mPth1R. Figure 11 shows changes in mMalat1 ncRNA expression levels by L031-HDO6 and L040-HDO6 in an in vitro assay using a cell line stably expressing mPth1R. Figure 12 shows changes in mMalat1 ncRNA expression levels by L089-HDO6 and L098-HDO6 in an in vitro assay using a cell line stably expressing mPth1R. Figure 13 shows changes in mMalat1 ncRNA expression levels by L165-HDO6 and L168-HDO6 in an in vitro assay using a cell line stably expressing mPth1R. Figure 14 shows changes in mMalat1 ncRNA expression levels by L001-ASO in an in vitro assay using a cell line stably expressing mPth1R.FIG. 15 shows changes in the expression level of Gapdh mRNA caused by L010-siRNA, as determined by an in vitro assay using a cell line stably expressing mPth1R.

[0022] The present invention relates to a ligand-bound nucleic acid complex in which a ligand is bound to a nucleic acid complex, and the ligand and the nucleic acid complex are bound indirectly via a linker or directly without a linker.

[0023] 1. Nucleic Acid Complex In the present invention, a "nucleic acid complex" refers to a nucleic acid molecule that regulates the expression or editing of a target gene, its transcription product, or its translation product. Examples of such nucleic acid molecules include nucleic acid molecules that have a nucleic acid sequence complementary to the target gene or its transcription product and have antisense activity. More specific examples of the nucleic acid molecule include single-stranded antisense strands (single stranded antisense oligonucleotides, ASO), miRNA, anti-miR, RNA interference (RNAi), short interference RNA (siRNA), short hairpin RNA (shRNA), antisense double-stranded nucleic acid (antisense double-stranded DNA oligonucleotides, ADO), and heteroduplex oligonucleotides (heteroduplex oligonucleotides, HDO).

[0024] Further examples of such nucleic acid molecules include aptamers, which have high specificity and high binding affinity for target molecules such as proteins. Further examples of such nucleic acid molecules include decoys. The function of a decoy is to suppress the function of the original transcription factor by hybridizing to the transcription factor, whereas a transcription factor normally binds to a binding site of the transcription factor, such as a specific promoter of a gene, to activate the gene or regulate the on / off of gene function. Further examples of such nucleic acid molecules include baits, which are nucleic acid molecules that specifically bind to a specific target molecule in a cell and modify the function of the target molecule.

[0025] The term "target gene" refers to a gene to which the antisense strand of the nucleic acid complex of the present invention can bind.

[0026] The type of target gene is not particularly limited as long as it is expressed in vivo. Examples of target genes include genes derived from an organism into which the nucleic acid complex of the present invention is introduced, such as genes whose expression increases in various diseases. Examples include the Indian hedgehog gene, interferon gene, apolipoprotein B gene, huntingtin gene, dystrophin gene, and DMPK (dystrophia myotonica protein kinase) gene.

[0027] Indian hedgehog (IHH) is a secreted protein belonging to the hedgehog family. It is known to be downstream of the transcription factor TAZ and to exacerbate fibrosis in NASH.

[0028] The cytokine interleukin-1 (IL-1) gene is known to cause chronic inflammatory diseases. It has been reported that skipping exon 9, which encodes the transmembrane domain of the pre-messenger RNA (mRNA) IL-1RAcP, results in substantial inhibition of IL-1 signaling (Mol Ther Nucleic Acids. 2013 Jan 22(1):e66. doi:10.1038 / mtna.2012.58.).

[0029] The apolipoprotein B gene, ApoB-100, is known to cause familial hypercholesterolemia, a hereditary metabolic disease, and the nucleic acid drug mipomersen was launched in the United States in 2013.

[0030] Huntington's disease is a chronic, progressive neurodegenerative disease that is inherited in an autosomal dominant manner and is characterized by involuntary movements, primarily chorea, as well as psychiatric symptoms and dementia. The Huntington gene, located on the short arm of chromosome 4, 4p16.3, is known to be the causative gene for Huntington's disease.

[0031] Duchenne muscular dystrophy is caused by a mutation in the dystrophin gene, is inherited as an X-linked recessive disorder, and generally occurs in boys. Exon skipping, which skips over abnormal exons in the dystrophin gene, is an effective treatment.

[0032] The DMPK gene encodes myotonin protein kinase and is known to be the causative gene for myotonic dystrophy, which is the muscular dystrophy with the highest incidence in adults.

[0033] The COL4A3, COL4A4, and COL4A5 genes encode the α3, α4, or α5 chain of type 4 collagen, which constitutes the basement membrane. Abnormalities in any of the COL4A3, COL4A4, and COL4A5 genes can lead to Alport syndrome, a syndrome characterized by renal damage, hearing loss, and eye complications, which often progresses to end-stage renal failure.

[0034] The term "target transcript" refers to any RNA that is a direct target of the nucleic acid complex of the present invention and is synthesized by RNA polymerase. Furthermore, the term "transcript of a target gene" also refers to "target transcript." Specifically, this term includes mRNA transcribed from a target gene (including mature mRNA, pre-mRNA, and mRNA without base modifications), non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA. Examples of target transcripts include pre-mRNA, which is a transcript of the IL-1RAcP gene, mRNA, which is a transcript of the ApoB-100 gene, mRNA, which is a transcript of the IHH gene, pre-mRNA, which is a transcript of the dystrophin gene, DMPK mRNA, which is a transcript of the DMPK gene, and metastasis associated lung adenocarcinoma transcript 1 (Malat1) non-coding RNA (ncRNA).

[0035] Malat1 is a long non-coding RNA (lncRNA) that is highly expressed in malignant tumors, including lung cancer, and is known to reside in the nuclei of muscle cells.

[0036] "Target translation product" refers to a protein synthesized by catalyzing a reaction in which ribosomes link amino acids carried by transfer RNA according to codons to form a peptide chain using mRNA, excluding non-coding RNA, as a template. In addition, "translation product of target gene" and "translation product of target transcript" are also included in "target translation product."

[0037] "Aptamer" refers to a nucleic acid molecule that specifically binds to a target translation product, for example, a specific target molecule inside a cell, on a cell membrane, or outside a cell, for example, on a cell membrane or outside a cell. Aptamers include DNA-type and RNA-type aptamers, and can be prepared by methods known in the art, for example, by in vitro selection using the SELEX (systematic evolution of ligands by exponential enrichment) method. The nucleic acid base length of the aptamer is not particularly limited, but is 10 to 70 bases long, preferably 20 to 50 bases long.

[0038] The term "decoy" refers to a nucleic acid having a sequence of the binding site of a transcription factor (e.g., NF-kB) or a similar sequence, which is introduced into cells as a "decoy" to suppress the action of the transcription factor (if it is a transcription activator, it suppresses transcription, and if it is a transcription repressor, it promotes transcription). Decoy nucleic acids can be easily designed based on information about the binding sequence of the target transcription factor. There are no particular limitations on the base length of the decoy nucleic acid, but it is 8 to 30 bases long, preferably 10 to 25 bases long.

[0039] "Nucleic acid" or "nucleic acid molecule" refers to a monomer, such as a nucleoside or nucleotide, an oligomer, such as an oligonucleotide, or a polymer, such as a polynucleotide. The term "nucleic acid strand" is also used herein to refer to an oligonucleotide. A nucleic acid strand may be prepared in whole or in part by chemical synthesis, such as by use of an automated synthesizer, or by enzymatic treatment, including but not limited to, polymerase, ligase, or restriction enzyme reactions.

[0040] "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of a nucleoside is typically, but not limited to, a pentofuranosyl sugar, specific examples of which include ribose and deoxyribose. The base portion (nucleobase) of a nucleoside is typically a heterocyclic base moiety. Examples include, but are not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).

[0041] A "nucleotide" refers to a molecule in which a phosphate group is covalently linked to the sugar portion of a nucleoside. In the case of nucleotides containing a pentofuranosyl sugar, the phosphate group is typically linked to the 2', 3', or 5' hydroxyl group of the sugar.

[0042] An "oligonucleotide" refers to a linear oligomer formed by covalently linking several to several dozen hydroxyl groups in the sugar moieties and phosphate groups of adjacent nucleotides. A "polynucleotide" refers to a polymer formed by linking a larger number of nucleotides than an oligonucleotide, several dozens or more, preferably several hundred or more, by such covalent bonds. Within an oligonucleotide or polynucleotide structure, the phosphate groups are generally considered to form internucleoside bonds.

[0043] "Antisense technology" refers to technology that uses nucleic acid molecules with antisense strands to regulate the amount, activity, and / or function of a target nucleic acid. The principle of antisense technology using antisense strands is that nucleic acid molecules with antisense strands hybridize to a target nucleic acid and regulate the amount, activity, and / or function of the target nucleic acid. For example, in some cases, nucleic acid molecules with antisense strands cause changes in the transcription or translation of the target. Such expression regulation can be achieved, for example, by degradation or occupancy-based inhibition of the target mRNA. One example of regulating RNA target function by degradation is the degradation of the target RNA by ribonuclease (RNase) upon hybridization with a nucleic acid molecule with a DNA-like antisense strand. An antisense strand (ASO) composed of DNA configured to hybridize with the target RNA hybridizes to the target RNA to form a double strand and is then degraded by RNase. This cycle is repeated to reduce the target RNA in cells, thereby inhibiting the expression of the target RNA or suppressing the action of the target RNA. This antisense effect is called an RNase-dependent antisense effect. Furthermore, nucleic acid molecules having an antisense strand may use splicing function conversion effects such as inhibition of transcription or translation of target RNA or exon skipping as an RNase-independent antisense effect. In this case, ASO hybridizes to the target RNA to suppress or enhance expression of the target gene. Another example of an RNase-independent antisense effect is RNA interference (RNAi). RNAi is antisense-mediated gene silencing that utilizes an RNA-induced silencing complex (RISC). Examples of RNAi include siRNA, shRNA, miRNA, and the like. This antisense effect is also called an RNAi-dependent antisense effect.

[0044] The term "antisense effect" refers to the effect of regulating the expression or editing of a target gene or its transcription product by hybridizing the antisense strand of a nucleic acid molecule to the target gene or its transcription product (RNA sense strand).

[0045] "Modulating the expression or editing of a target gene, its transcription product, or its translation product" means suppressing, decreasing, or enhancing the expression of a target gene or the expression level of a target transcript (herein, "the expression level of a target transcript" is often referred to as "the level of a target transcript"), inhibiting translation, inhibiting the function of a translation product, controlling RNA splicing (e.g., splicing switch, exon inclusion, exon skipping, etc.), degradation of a transcript, or inhibiting the binding of a target gene to a protein.

[0046] In one embodiment, the antisense effect refers to the inhibition of translation or splicing function alteration effects, such as exon skipping, that can occur by coating the transcript by hybridization, and / or the suppression that can occur by degradation of the transcript that can occur by recognition of the hybridized portion. In another embodiment, the antisense effect refers to the enhancement of normal mRNA expression that occurs in exon inclusion of a target gene or transcript, which is the opposite of exon skipping, in that an exon that would otherwise be excluded from mRNA due to a genetic abnormality is incorporated into mRNA by the action of an antisense molecule.

[0047] For example, in post-transcriptional inhibition of a target gene, when an RNA oligonucleotide is introduced into a cell as an ASO, the ASO anneals to the mRNA, the transcription product of the target gene, to form a partial duplex. This partial duplex acts as a cover to prevent ribosomal translation, thereby inhibiting the expression of the target protein encoded by the target gene at the translational level (steric blocking). On the other hand, when an oligonucleotide containing DNA is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed. This heteroduplex structure is recognized by RNase, resulting in degradation of the target gene's mRNA and inhibition of the expression of the protein encoded by the target gene at the expression level. Furthermore, antisense effects can also be achieved by targeting introns in pre-mRNA. Furthermore, antisense effects can also be achieved by targeting miRNA. In this case, inhibition of the miRNA function can increase the expression of the gene whose expression is normally controlled by the miRNA. In one embodiment, modulation of the expression of a target transcript can be achieved by reducing the amount of the target transcript.

[0048] For example, when ADO is used as a nucleic acid molecule having an antisense strand, the DNA double strand is cleaved in cells by DNA nuclease (DNase), the DNA antisense strand hybridizes to the target RNA to form a double strand, and then the target RNA is degraded by RNase, and this cycle is repeated to inhibit the expression of the target RNA, suppress the action of the target RNA, etc. In another embodiment, the DNA double strand of ADO is cleaved by DNA nuclease (DNase), and then the DNA antisense strand hybridizes to the target RNA, and then the transcription or translation of the target RNA is inhibited or RNA splicing such as exon skipping is controlled to suppress or enhance the expression of the target gene.

[0049] For example, when HDO is used as a nucleic acid molecule having an antisense strand, the complementary strand consisting of the RNA of HDO is cleaved by RNase in a cell, and then the DNA antisense strand hybridizes to the target RNA to form a double strand, and the target RNA is then degraded by RNase, and this cycle is repeated to inhibit the expression of the target RNA, suppress the action of the target RNA, etc. Furthermore, after the complementary strand of the RNA of HDO is cleaved by RNase in a cell, the DNA antisense strand hybridizes to the target RNA, and suppresses or enhances the expression of the target gene by inhibiting the transcription or translation of the target RNA or by controlling RNA splicing such as exon skipping.

[0050] For example, when RNA interference (RNAi) is used as the antisense nucleic acid molecule, RNAi refers to antisense-mediated gene silencing via a mechanism that utilizes the RNA-induced silencing complex (RISC). Examples of RNAi include siRNA and shRNA. In another example, regulation of RNA target function is via an occupancy-based mechanism, such as that naturally utilized by microRNAs. MicroRNAs are small, non-coding RNAs that regulate the expression of protein-encoding RNAs. Binding of an antisense nucleic acid molecule to a microRNA inhibits the binding of the microRNA to its mRNA target, thereby disrupting microRNA function. MicroRNA mimics can enhance native microRNA function. Nucleic acid molecules with specific antisense strands alter pre-mRNA splicing. Regardless of the specific mechanism, sequence specificity allows antisense nucleic acid molecules to be used as tools for target validation and gene functioning, and as therapeutic agents to selectively regulate the expression of genes involved in disease pathogenesis.

[0051] The length of the antisense strand is not particularly limited, but is at least 8 bases, for example, 8 to 40 bases, preferably 12 to 30 bases, more preferably 12 to 25 bases, or 13 to 20 bases. In some cases, the length is typically selected depending on other factors such as the strength of the antisense effect of the nucleic acid strand on the target, cost, and synthesis yield. In the case of a double-stranded nucleic acid, the length may be selected from lengths that provide a Tm value of the double strand of 50°C or higher, more preferably 60°C or higher.

[0052] In the case of a double-stranded nucleic acid, the length of the complementary strand may be the same as that of the antisense strand. In this case, the length is at least 8 bases, for example, 8 to 40 bases, preferably 12 to 30 bases, more preferably 12 to 25 bases, or 13 to 20 bases. In addition, the length may be several to several dozen bases longer or shorter than the length of the antisense nucleic acid strand.

[0053] "Complementary" or "complementarity" refers to a relationship in which so-called Watson-Crick base pairs (natural base pairs) and non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) can be formed via hydrogen bonds. When a sufficient number of nucleobases in the antisense strand can hydrogen bond with corresponding nucleobases in the target gene or target transcript, the antisense strand and the target gene or target transcript are complementary to each other, resulting in the desired antisense effect. Non-complementary nucleobases between the antisense strand and the target gene or target transcript are acceptable, provided that the antisense strand can specifically hybridize to the target nucleic acid. Furthermore, the antisense strand can hybridize to one or more segments of the target gene or target transcript, such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop structure, mismatch, or hairpin structure). The antisense strand is said to be complementary to the sequence of the target gene or target transcript. "Complementary" means that the antisense strand is sufficiently complementary to be able to bind to the target gene or target transcription product, for example, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more complementary. 100% complementary is also acceptable. 0 to 4 mismatches are also acceptable.

[0054] In certain embodiments, the "nucleic acid complex" of the present invention may be a single-stranded oligonucleotide at the time of production, comprising an antisense strand composed of DNA nucleotides or DNA nucleotide analogs, a linker portion composed of 3 to 10 nucleotides, and a sense strand composed of RNA nucleotides or RNA nucleotide analogs that is complementary to the antisense strand. A nucleic acid complex of this structure is known as a single-stranded heteroduplex oligonucleotide (ss-HDO), and is an oligonucleotide having an X-L-Y structure (Patent Document 4). X is the antisense strand, Y is the strand complementary to the antisense strand, and L is composed of nucleotides that function as a linker. When this single-stranded oligonucleotide is used as a pharmaceutical composition, the antisense strand and the strand complementary to the antisense strand anneal to each other via the linker in a solvent such as physiological saline, aqueous injections, non-aqueous injections, suspension injections, or solid injections, or in blood or plasma, to form a duplex structure. Such a nucleic acid complex is a type of double-stranded nucleic acid complex because it forms a double-stranded structure by annealing one molecule when acting as a pharmaceutical composition.

[0055] In the present invention, the above-mentioned nucleic acid complex may be referred to as a nucleic acid molecule.

[0056] Above, in some embodiments, preferred typical examples of single-stranded ASOs and double-stranded nucleic acid complexes have been described, but the single-stranded ASOs and double-stranded nucleic acid complexes in some embodiments are not limited to the above typical examples.

[0057] In some embodiments, the antisense strand comprises nucleotides, modified nucleotides, and / or nucleotide analogs, meaning that the antisense strand comprises DNA nucleotides, RNA nucleotides, and optionally further comprises modified nucleotides and nucleotide analogs in the nucleic acid strand.

[0058] In certain embodiments, the complementary strand comprises nucleotides, modified nucleotides and / or nucleotide analogs.

[0059] The complementary strand includes DNA nucleotides, RNA nucleotides, and may further include optionally modified nucleotides and nucleotide analogs in the nucleic acid strand.

[0060] In one embodiment, the complementary strand has a center region comprising nucleotides and / or modified nucleotides and wing regions comprising one or more nucleotide analogs and / or modified nucleotides located on the 5'-terminus and / or 3'-terminus thereof.

[0061] As used herein, "DNA nucleotide" refers to a naturally occurring DNA nucleotide or a DNA nucleotide in which the base, sugar or phosphate linkage subunit has been modified.

[0062] Similarly, an "RNA nucleotide" refers to a naturally occurring RNA nucleotide or an RNA nucleotide in which the base, sugar or phosphate linkage subunit has been modified.

[0063] A "modified nucleotide" refers to the addition of a substituent to, or substitution within, the base, sugar, or phosphate linkage subunit of a nucleotide, but not the replacement of the entire subunit with a different chemical group. DNA may contain modified nucleotides, with the aim of making some or all of the region containing the nucleotide more resistant to DNases and the like. Examples of such modifications include 5-methylation, 5-fluorolation, 5-bromination, 5-iodination, and N4-methylation of cytosine, 5-demethylation, 5-fluorolation, 5-bromination, and 5-iodination of thymidine, N6-methylation and 8-bromination of adenine, N2-methylation and 8-bromination of guanine, phosphorothioation, boranophosphate, methylphosphonate, methylthiophosphonate, chiral-methylphosphonate, phosphorodithioate, phosphoramidate, 2'-O-methylation, 2'-methoxyethyl (MOE), 2'-aminopropyl (AP), and 2'-fluorolation. From the viewpoint of superior pharmacokinetics, phosphorothioate is preferred. Furthermore, multiple types of such modifications may be combined on the same DNA. Furthermore, as described below, RNA nucleotides may also be modified to achieve the same effect.

[0064] In some cases, the number and position of modified nucleotides may affect the antisense effect, etc., of the double-stranded nucleic acid disclosed herein. These aspects vary depending on the sequence of the target gene, etc., and cannot be generalized; however, those skilled in the art can determine them by referring to the literature on antisense methods described below. Furthermore, the antisense effect of the modified double-stranded nucleic acid complex can be measured, and if the measured value is not significantly lower than that of the double-stranded nucleic acid complex before modification (e.g., if the measured value of the modified double-stranded nucleic acid complex is 30% or more of the measured value of the double-stranded nucleic acid complex before modification), the modification can be evaluated. The antisense effect can be measured, for example, by introducing a test nucleic acid compound into cells, etc., as shown in the Examples below, and measuring the expression level (mRNA amount, cDNA amount, protein amount, etc.) of the target gene in the cells, etc., suppressed by the antisense effect of the test nucleic acid compound, using known techniques such as Northern blotting, quantitative PCR, and Western blotting, as appropriate.

[0065] "Nucleotide analog" refers to a nucleotide that does not occur in nature, in which two or more substituents are added to the base, sugar, or phosphate bond subunit of the nucleotide, or two or more substitutions are made within the subunit, or the entire subunit is replaced with a different chemical group. An example of an analog with two or more substitutions is a bridged nucleic acid. A bridged nucleic acid is a nucleotide analog in which a bridge unit is added based on two substitutions in the sugar ring, and typically includes a nucleotide analog in which the 2' carbon and the 4' carbon are bonded. In one embodiment, the first nucleic acid strand further comprises a nucleotide analog in order to increase affinity for a partial sequence of a transcription product of a target gene and / or resistance to nuclease degradation. The nucleotide analog may be any nucleotide that has been modified (crosslinked, substituted, etc.) to increase its affinity for a partial sequence of a transcription product of a target gene and / or its resistance to nuclease degradation, and examples thereof include nucleic acids disclosed as being suitable for use in antisense methods in JP-A-10-304889, WO 2005 / 021570, JP-A-10-195098, JP-T-2002-521310, WO 2007 / 143315, WO 2008 / 043753, WO 2008 / 029619, and WO 2008 / 049085 (hereinafter, these documents may also be referred to as "documents related to antisense methods"). That is, examples of the nucleic acids disclosed in the above documents include hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), morpholino nucleic acid, tricyclo-DNA (tcDNA), 2'-O-methylated nucleic acid (2'-OMe), 2'-O-methoxyethylated nucleic acid (2'-MOE), 2'-O-ethylated nucleic acid (cEt), 2'-O-aminopropylated nucleic acid (2'-AP), 2'-fluorolated nucleic acid, 2'F-arabinonucleic acid (2'-F-ANA), and bridged nucleic acid (BNA).

[0066] In some embodiments, BNAs are ribonucleotides or deoxyribonucleotides in which the 2' carbon and the 4' carbon are bridged by two or more atoms. Examples of bridged nucleic acids are known to those of skill in the art. One subgroup of such BNAs includes those in which the 2' carbon and the 4' carbon are bridged by a 4'-(CH ２ )p-O-2',4'-(CH ２ )p-S-2',4'-(CH ２ )p-OCO-2',4'-(CH ２ )n-N(R ３ )—O—(CH ２ ) m-2′ (where p, m, and n are integers of 1 to 4, 0 to 2, and 1 to 3, respectively. R ３ represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, and a unit substituent (such as a fluorescent or chemiluminescent labeling molecule, a nucleic acid cleavage active functional group, or an intracellular or nuclear transport signal peptide). Furthermore, in one embodiment, in the BNA, the substituent at the 3' carbon: OR ２ and a substituent at the 5' carbon: OR １ R １ and R ２ are typically hydrogen atoms, but may be the same or different, and are a protecting group for a hydroxyl group in nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group in nucleic acid synthesis, or -P(R ４ ) R ５ (In the formula, R ４ and R ５may be the same or different and may represent a hydroxyl group, a hydroxyl group protected with a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with an alkyl group having 1 to 5 carbon atoms. Examples of such BNAs include α-L-methyleneoxy (4'-CH ２ -O-2')BNA) or β-D-methyleneoxy (4'-CH ２ -O-2')BNA, also known as ENA, ２ )2-O-2')BNA, β-D-thio(4'-CH ２ -S-2')BNA, aminooxy (4'-CH ２ -O-N(R ３ )-2')BNA, also known as 2',4'-BNANC, ２ -N(R ３ )-O-2')BNA, 2',4'-BNACOC, 3'amino-2',4'-BNA, 5'-methylBNA, cEt-BNA (4'-CH(CH ３ )-O-2')BNA, also called cMOE-BNA (4'-CH(CH ２ OCH ３ )-O-2') BNA, amide BNA (also known as AmNA) (4'-C(O)-N(R)-2') BNA (R = H, Me), guanidine bridged nucleic acid (GuNA), 4'-C-spirocyclopropylene bridged nucleic acid (scpBNA), and other BNAs known to those skilled in the art.

[0067] Furthermore, in certain embodiments, modified nucleic acids may have their base sites modified. Examples of modifications of base sites include 5-methylation, 5-fluorolation, 5-bromination, 5-iodination, and N4-methylation of cytosine, 5-demethylation, 5-fluorolation, 5-bromination, and 5-iodination of thymidine, N6-methylation and 8-bromination of adenine, and N2-methylation and 8-bromination of guanine. Furthermore, in certain embodiments, modified nucleic acids may have their phosphodiester bond sites modified. Examples of modifications of phosphodiester bond sites include phosphorothioation, boranophosphate, methylphosphonate, methylthiophosphonate, chiral-methylphosphonate, phosphorodithioate, and phosphoramidate, with phosphorothioation being preferred from the viewpoint of superior pharmacokinetics. Furthermore, a combination of multiple types of modifications at the base site or at the phosphodiester bond site may be performed on the same nucleic acid.

[0068] Generally, modified nucleotides and nucleotide analogs are not limited to those exemplified herein. Many modified nucleotides and nucleotide analogs are known in the art, and the descriptions in, for example, U.S. Patent No. 8,299,039 to Tachas et al., particularly columns 17-22, can be used as embodiments of the present application.

[0069] A person skilled in the art can select and use an appropriate nucleotide analog from such modified nucleic acids as a nucleic acid constituting a nucleic acid complex, taking into consideration aspects such as antisense effect, affinity for a partial sequence of a transcription product of a target gene, and resistance to nucleases. In one embodiment, the nucleotide analog is an LNA.

[0070] In one embodiment, the antisense strand comprises a region containing multiple DNA nucleotides (hereinafter also referred to as a "DNA gap region") and wing regions containing one or more nucleotide analogs located on the 5'- and / or 3'-end of the region. This antisense strand is also referred to as a gapmer. The length of the gapmer is at least 8 bases, for example, 8 to 40 bases, preferably 12 to 30 bases, and more preferably 12 to 25 bases, or 13 to 20 bases.

[0071] Additionally, in some embodiments, the plurality of DNA nucleotides may be modified nucleotides.

[0072] Additionally, in some embodiments, the plurality of DNA nucleotides may be nucleotide analogs.

[0073] The region containing a nucleotide analogue positioned at the 5'-end of the DNA gap region (hereinafter also referred to as the "5' wing region") and the region containing a nucleotide analogue positioned at the 3'-end of the DNA gap region (hereinafter also referred to as the "3' wing region") are independent of each other and may contain at least one nucleotide analogue listed in the literature on the antisense method, and may further contain natural nucleic acids (DNA or RNA) or modified nucleotides in addition to such nucleotide analogues. The chain lengths of the 5' wing region and the 3' wing region are independently usually 1 to 10 bases, 1 to 7 bases, or 2 to 5 bases.

[0074] In some embodiments, the antisense strand is not a Gapmer but is composed of multiple DNA nucleotides, modified nucleotides, or nucleotide analogs, or a combination thereof. This antisense strand is also referred to as a non-Gapmer. The length of the non-Gapmer is at least 8 bases, for example, 8 to 40 bases, preferably 12 to 30 bases, more preferably 12 to 25 bases, or 13 to 20 bases.

[0075] In some embodiments, the complementary strand in the double-stranded nucleic acid complex may be a gapmer comprising a region containing multiple DNA or RNA nucleotides and wing regions containing one or more modified nucleotides and / or nucleotide analogs located on the 5'- and / or 3'-ends of the region. By using modified nucleotides and / or nucleotide analogs in the 5'- or 3'-wing regions, binding to the antisense strand can be further strengthened.

[0076] In some embodiments, the complementary strand in a double-stranded nucleic acid complex may be a plurality of DNA nucleotides and / or modified nucleotides or nucleotide analogs, or RNA nucleotides and / or modified nucleotides or nucleotide analogs. Furthermore, the complementary strand in a double-stranded nucleic acid complex may have a center region containing nucleotides and / or modified nucleotides and wing regions containing one or more nucleotide analogs and / or modified nucleotides located on the 5'-end and / or 3'-end of the center region.

[0077] In the present invention, examples of nucleic acid molecules that regulate the expression or editing of a target gene, its transcription product, or its translation product include nucleic acid molecules selected from the group consisting of the above-mentioned nucleic acid molecules comprising an antisense strand consisting of an oligonucleotide having a nucleic acid base sequence that is complementary to the target gene or its transcription product, an aptamer having a nucleic acid base sequence that specifically binds to a target protein, and a decoy consisting of an oligonucleotide having a nucleic acid sequence that is complementary to a target transcription factor.

[0078] Furthermore, in the present invention, examples of nucleic acid molecules that regulate the expression or editing of a target gene, its transcription product, or its translation product include nucleic acid molecules selected from the group consisting of ADO, ASO, HDO, and RNAi.

[0079] 2. Ligands The term "ligand" refers to a substance that binds to a biomolecule to form a complex and fulfill a biological purpose. In some cases, the ligand has a target delivery function. For example, lipids are preferred ligands from the viewpoint of highly specific and efficient delivery of a nucleic acid complex to the liver, etc. Examples of such lipids include cholesterol, fatty acids, and other lipids (e.g., vitamin E (tocopherols, tocotrienols), vitamin A, vitamin D), fat-soluble vitamins such as vitamin K (e.g., acylcarnitine), intermediate metabolites such as acyl-CoA, glycolipids, glycerides, and derivatives thereof. Among these, cholesterol and vitamin E (tocopherols, tocotrienols) are preferred ligands from the viewpoint of higher safety. Furthermore, sugars (e.g., glucose, sucrose) are preferred ligands from the viewpoint of highly specific and efficient delivery of nucleic acid molecules to the brain. Furthermore, from the viewpoint that the nucleic acid complex can be delivered to each organ with high specificity and efficiency by binding to various proteins present on the cell surface of the organ, preferred ligands include receptor ligands, antibodies, and / or peptides or proteins such as fragments thereof.

[0080] A "PTH1 ligand" is a ligand capable of binding to a PTH1 receptor. Representative examples of ligands capable of binding to a PTH1 receptor include human parathyroid hormone (hPTH), human parathyroid hormone-related protein (hPTHrP), and human tuberoinfundibular peptide (hTIP).

[0081] In one embodiment, the PTH1 ligand is a peptide consisting of the amino acid sequence represented by the following formula: １ -A ２ -A ３ -A ４ -A ５ -A ６ -A ７ -A ８ -A ９ -A １０ -A １１ -A１２ -A １３ -A １４ -A １５ -A １６ -A １７ -A １８ -A １９ -A ２０ -A ２１ -A ２２ -A ２３ -A ２４ -A ２５ -A ２６ -A ２７ -A ２８ -A ２９ -A ３０ -A ３１ -A ３２ -A ３３ -A ３４[wherein the amino acid sequence is written from the N-terminus to the C-terminus.] A4 is Glu or absent; A5 is Leu, His, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe p-X-Phe or absent, where X is OH, halogen, or CH3; A6 is Gln; A7 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe p-X-Phe or absent, where X is OH, halogen, or CH3; A8 is Met, Nva, Leu, Val, Ile, Cha, Acc, Nle or absent; A9 is His or absent; A10 is Asp or Asn; A11 is Leu, Lys, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe or p-X-Phe, where X is OH, halogen, or CH3; A12 is Gly, Acc, or Aib; A13 is Lys; A14 is Ser or His; A15 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe or p-X-Phe, where X is OH, halogen, or CH3; A16 is Ser, Gln, Asn, Ala, or Aib; A17 is Ser, Asp, Thr, or Aib; A18 is Met, Nva, Leu, Val, Ile, Nle, Acc, Cha, or Aib; A19 is Arg, Glu, or Aib; A20 is Arg; A21 is Arg, Val, Acc, Cha, or Met; A22 is Phe, Glu, Aib, Acc, or Cha; A23 is Phe, Trp, Leu, Lys, Acc, or Cha; A24 is Leu, Lys, Acc, or Cha; A25 is His, Arg, Lys, Aib, Acc, or Glu; A26 is His, Aib, Acc, or Lys; A27 is Lys, Aib, Leu, hArg, Gln, Acc, or Cha; A28 is Ile, Leu, Lys, Acc, or Cha;A29 is Ala, Glu, Acc, or Aib; A30 is Glu, Asp, Leu, Nle, Cha, Aib, Acc, or Lys; A31 is Ile, Val, Leu, Nle, Cha, Lys, or Acc; A32 is His; A33 is Thr, Asn, Lys, or Cys; A34 is Phe, Ala, Tyr, Amp, or Aib.

[0082] In the present specification, when amino acids and the like are represented by abbreviations, they are based on the abbreviations established by the IUPAC-IUB Commission on Biochemical Nomenclature or on the abbreviations commonly used in the art, examples of which are shown below. Furthermore, when an amino acid can have optical isomers, the L-isomer is indicated unless otherwise specified. Gly or G: Glycine Ala or A: Alanine Val or V: Valine Leu or L: Leucine Ile or I: Isoleucine Ser or S: Serine Thr or T: Threonine Cys or C: Cysteine ​​Met or M: Methionine Glu or E: Glutamic acid Asp or D: Aspartic acid Lys or K: Lysine Arg or R: Arginine His or H: Histidine Phe or F: Phenylalanine Tyr or Y: Tyrosine Trp or W: Tryptophan Pro or P: Proline Asn or N: Asparagine Gln or Q: Glutamine Aib: Aminoisobutyric acid Nle: Norleucine β-Ala: β-alanine hPTH: human PTH Boc: t-butoxycarbonyl Fmoc: 9-fluorenylmethoxycarbonyl Nva: norvaline Abu: α-aminobutyric acid Ahc: 1-aminocyclohexylcarboxylic acid hArg: homoarginine Cha: 2-amino-3-cyclohexylpropionic acid Npa: 3-(2-naphthyl)-alanine Dap: 2,3-aminopropionic acid D-Ser: (D)-Ser D-Leu: (D)-Leu D-Trp: (D)-Trp

[0083] The peptides represented by the above formula include hPTH(1-34), hPTH(1-34) derivatives, for example, hPTH(1-34) in which an amino acid residue at a specific position is substituted with another amino acid residue, hPTHrP(1-34), hPTHrP(1-34) derivatives, and these peptides in which n amino acid residues (n = an integer from 1 to 9) have been deleted from the N-terminus.

[0084] In one embodiment, the PTH1 ligand includes hTIP(1-39), hTIP(1-39) derivatives, and peptides thereof lacking n amino acid residues (n=an integer from 1 to 9) from the N-terminus.

[0085] In one embodiment, the PTH1 ligand includes an hPTH(1-34) peptide, an hPTHrP(1-34) peptide, an hTIP(1-39) peptide, or a derivative thereof selected from the group of peptides represented by SEQ ID NOs: 1 to 168 in Table 2 below, and a partial peptide thereof, which is a peptide lacking n amino acid residues (n is an integer from 1 to 9) from the N-terminus of the peptide.

[0086] In Table 2, the homology score indicates the homology between the amino acid sequence of the peptide assigned the ligand number in the first column and the amino acid sequence of the comparison peptide. That is, if the peptide in the first column is L011 and the comparison peptide is L001, the second K and C from the C-terminus of the 34 amino acid residues do not match, but the remaining 33 amino acid residues are identical, so the homology score is calculated as 33 / 34 = 0.971 (97.1%). Furthermore, when the peptide has an amino acid sequence in which n amino acid residues (n = an integer from 1 to 9) are deleted from the N-terminus of a full-length peptide derived from hPTHrP(1-34), hPTH(1-34), or hTIP(1-39), for example, if the peptide in the first row is L012 (n = 1 deleted) and the comparison peptide is L001, L012 and L001 correspond to L012, and of the 33 amino acid residues, the second K and C from the C-terminus are mismatched, but the remaining 32 amino acid residues are identical in sequence, the homology score is calculated to be 32 / 33 = 0.970 (97.0%).

[0087] All of the PTH1 ligands listed in Table 2 are human PTH1 ligands.

[0088] In one embodiment, the PTH1 ligand is a peptide selected from the group of hPTHrP(1-34) peptides and partial peptides thereof, which are represented by SEQ ID NOS: 1-88 in Table 1.

[0089] In one embodiment, the PTH1 ligand is a peptide selected from the group of hPTH(1-34) peptides of SEQ ID NOS: 89-164 contained in Table 1 and subpeptides thereof.

[0090] In one embodiment, the PTH1 ligand is a peptide selected from the group consisting of hTIP(1-39) peptides of SEQ ID NOS: 165-168 contained in Table 1 and partial peptides thereof.

[0091] In some embodiments, the PTH1 ligand may be a peptide having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or more identical or homologous to L001, L011, L021, L031, L041, L051, L072 derived from hPTHrP(1-34).

[0092] In some embodiments, the PTH1 ligand may be a peptide having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or more identical or homologous to L089, L099, L109 from hPTH(1-34).

[0093] In some embodiments, the PTH1 ligand may be a peptide having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or more identical or homologous to L165 from hTIP(1-39).

[0094] In the nucleic acid complex of the present invention, the ligand is attached to the antisense strand and / or complementary strand of the nucleic acid molecule. In some embodiments, the ligand is attached to the 3'-end or 5'-end of the nucleic acid strand. In some embodiments, the ligand is attached to a site other than the end of the nucleic acid strand. Methods for attaching a ligand to the 2-position of the pentose sugar of a nucleotide are known, and can be performed, for example, by the method described in International Publication No. WO2018 / 003739.

[0095] The "PTH1 ligand" may be a salt of the above peptide, and examples of the salt of the above peptide include sodium salt, potassium salt, calcium salt, hydrochloride, sulfate, nitrate, acetate, methanesulfonate, toluenesulfonate, citrate, fumarate, maleate, hydrobromide, etc.

[0096] The above peptides can be prepared by known methods such as solid phase synthesis.

[0097] 3. Binding of Ligands to Nucleic Acid Molecules Binding of a ligand to a nucleic acid can be achieved by covalent or non-covalent bonding, such as hydrogen bonding, electrostatic interaction, or hydrophobic interaction, between the binding group of the ligand and the binding group of the nucleic acid molecule. Examples of binding groups on the ligand include, but are not limited to, amino groups, hydroxy groups, carboxylic acids, thiol groups, disulfides, and azide groups possessed by peptides that are PTH1 ligands. Examples of binding groups on nucleic acids include the carbon atom at the 2nd position, the hydroxy group at the 3rd position, or the hydroxy group at the 5th position of the sugar of a nucleoside, the phosphate group at the 5th position of a nucleotide, or the base moiety of a nucleoside. Examples of binding groups on nucleic acid molecules include phosphate groups in oligonucleotides. The nucleic acid molecule and the ligand can be bound with or without a linker.

[0098] 4. Linker In some embodiments, the ligand is bound to the nucleic acid molecule via a linker. When the nucleic acid molecule is HDO, the ligand may be bound to the antisense strand and / or complementary strand of the HDO via a linker. The linker may be a cleavable or non-cleavable linker.

[0099] The term "cleavable linker" refers to a linking group that is cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within the human body). In certain embodiments, the cleavable linker is selectively cleaved by an endogenous enzyme such as a nuclease. Examples of cleavable linkers include amide, ester, phosphodiester (or both) esters, phosphate ester, carbamate, and disulfide bonds, as well as natural DNA linkers. The term "non-cleavable linker" refers to a linker that is not cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within the human body). Examples of non-cleavable linkers include, but are not limited to, phosphorothioate bonds and linkers consisting of modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked by phosphorothioate bonds. When the linker is a nucleic acid such as DNA or an oligonucleotide, the chain length is not particularly limited, but is generally 2 to 20 bases long, preferably 3 to 10 bases long.

[0100] In some embodiments, linkers for use in the present invention include chain structures such as hydrocarbyl chains, or oligomers of repeating units such as ethylene glycol, nucleoside or amino acid units.

[0101] In certain embodiments, the linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino.

[0102] In certain embodiments, the linker comprises a group selected from alkyl, amino, oxo, amido, and ether groups.

[0103] In certain embodiments, the linker comprises a group selected from an alkyl and an amide group.

[0104] In certain embodiments, the linker comprises a group selected from alkyl and ether groups.

[0105] In some embodiments, the linker comprises at least one phosphorus moiety.

[0106] In some embodiments, the linker comprises at least one phosphate group.

[0107] In some embodiments, the linker comprises at least one neutral linking group.

[0108] In some embodiments, the linker has a length of 1-1000 Å. Particular linkers have a length of 3-500 Å. Preferably, the linker has a length of 3-200 Å (lengths estimated based on the crystal structure of the PTH1R receptor bound to a PTH analog (PDB 6F3J)).

[0109] In some embodiments, the linker and the oligonucleotide can be bonded using a bifunctional bond. Generally, a bifunctional bond is a bond that uses at least two functional groups. One functional group of the linker is selected to bind to a specific site on the oligonucleotide, and the other is selected to bind to the ligand moiety. Examples of functional groups used in the bifunctional bond of the linker include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, the bifunctional bond can utilize one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

[0110] Examples of linkers include, but are not limited to, 6-aminohexanoic acid (AHA or AHEM), (2,5-dioxypyrrolidin-1-yl) 4-(2-azatricyclo[10.4.0.04,9]hexadeca-1(16),4,6,8,12,14-hexen-10-yn-2-yl-4-oxobutanoate (DBCO-NHS), 3-mercaptopropionic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. Other linkers include, but are not limited to, substituted or unsubstituted C １ -C １０ Alkyl, substituted or unsubstituted C ２ -C １０ Alkenyl or substituted or unsubstituted C ２ -C １０alkynyl, a non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0111] In certain embodiments, the linker can be selected from compounds having the following structures in Table 3 and derivatives thereof:

[0112]

[0113] In Table 3, n of the polyethylene glycol group is 1-200, preferably 1-100, and more preferably 1-50.

[0114] In certain embodiments, a linker may be composed of 2 to 20 linker-nucleosides. In certain embodiments, such linker-nucleosides are contiguous modified nucleosides. In certain embodiments, such linker-nucleosides may include a modified sugar moiety. In certain embodiments, a linker-nucleoside is unmodified. In certain embodiments, a linker-nucleoside optionally includes a protected heterocyclic base selected from a purine, a substituted purine, a pyrimidine, or a substituted pyrimidine.

[0115] In certain embodiments, the linker can be selected from compounds having the following structure and derivatives thereof:

[0116] where X is a ligand binding site and Y is a nucleic acid molecule binding site.

[0117] In certain embodiments, a compound having an active ester of the formula: (wherein X is a ligand binding site) with a compound of the following formula: wherein Y is a nucleic acid molecule binding site, (In the formula, X is a ligand binding site and Y is a nucleic acid molecule binding site. The nucleic acid molecule is selected from natural nucleotides, modified nucleotides, and nucleotide analogs, or is an oligonucleotide using them.) A linker shown in the above formula 6 can be obtained.

[0118] In certain embodiments, a linker comprising phosphate / triethylene glycol can be used.

[0119] In certain embodiments, a linker of the following formula can be used:

[0120] wherein X is a ligand binding site, Y is a nucleic acid molecule binding site, and Bx is a modified or unmodified nucleobase.

[0121] In certain embodiments, a compound having an amidite of the formula (7) above, (wherein X is a ligand) The compound represented by the formula (8) is reacted with an oligonucleotide moiety Y on a solid support, and then cleaved from the solid support. (wherein X is a ligand and Y is a nucleic acid molecule) A linker shown in the above formula (9) can be obtained.

[0122] In certain embodiments, the linker has the structure shown in the following formula:

[0123] wherein X is a ligand binding site, Y is a nucleic acid molecule binding site, W is a phosphodiester or amino group, Z is a pyrrolidinyl group represented by the formula: j is 0 or 1; n is from about 1 to about 10; m is from about 1 to about 10; l is 0 or 1-4; and when X is an amino group, l is 1.

[0124]

[0125] In certain embodiments, the linkers are prepared using click chemistry. Additional linkers suitable for use in some embodiments can be prepared by click chemistry as described in "Click Chemistry for Biotechnology and Materials Science," Ed. Jörg Laham, Wiley 2009, which is incorporated herein by reference in its entirety.

[0126] In certain embodiments, a compound of the formula: with a compound represented by the following formula: wherein Y is a nucleic acid molecule binding site, with an oligonucleotide having a terminal amine, The compound represented by the formula 14 was obtained, which was reacted with an azide-containing ligand to give: (wherein X represents a ligand binding site and Y represents a nucleic acid molecule binding site) A linker represented by the above formula 15 was obtained.

[0127] In certain embodiments, the linker can be a maleimide linker, which has the structure shown in the following formula:

[0128] wherein X is a ligand binding site and Y is a nucleic acid molecule binding site.

[0129] In certain embodiments, a compound having the following formula: with a compound represented by the following formula: wherein Y is a nucleic acid molecule binding site, by reacting with an oligonucleotide having a terminal amine, This gave a compound of formula 19, which was reacted with a maleimide-containing ligand conjugate moiety to give: (wherein X is a ligand binding site and Y is a nucleic acid molecule binding site) A linker represented by the above formula 20 was obtained.

[0130] In certain embodiments, a compound of the formula: and a compound represented by the following formula: wherein Y is a nucleic acid molecule binding site, by reacting with an oligonucleotide having a terminal amine, The compound represented by the formula (23) above was obtained, which was reacted with a thiol-containing ligand conjugate moiety to give: (wherein X is a ligand binding site, and Y is a nucleic acid molecule binding site) A linker represented by the above formula 24 was obtained.

[0131] Specific Examples and Attachment Methods of Disulfide Linkers In certain embodiments, the linker may contain a disulfide bond. In certain embodiments, the linker contains an activated disulfide that forms a disulfide bond with the ligand-binding moiety.

[0132] In certain embodiments, a compound having the following formula: and a compound represented by the following formula: wherein Y is a nucleic acid molecule binding site, and reacting the resulting oligonucleotide with an oligonucleotide having a terminal amine, The compound represented by the formula 27 above was obtained. By cleaving the disulfide bond, The compound represented by the formula (28) above was obtained, which was reacted with a thiol-containing ligand conjugate moiety to give: (wherein X is a ligand binding site and Y is a nucleic acid molecule binding site) A linker represented by the above formula 29 was obtained.

[0133] In certain embodiments, the PTH1 ligand is chemically bound to a linker, through which the PTH1 ligand is linked to a functional group for attachment to an oligonucleotide.

[0134] In certain embodiments, including but not limited to, a compound having the following formula can be used in combination with an amino group (X-NH2; X is the ligand conjugate moiety excluding the amino group) of the ligand binding moiety: (where Y is a functional group for direct or indirect attachment to the oligo, such as azide, maleimide, etc.) (wherein X is a ligand binding site and Y is a nucleic acid molecule binding site) A linker was obtained by introducing a linker into the ligand binding site shown in the formula 31 above.

[0135] 5. Pharmaceutical Compositions Diseases targeted by the ligand-binding nucleic acid complexes of the present invention may be diseases associated with the target organs and tissues or cells within those organs. Examples of such diseases include, but are not limited to, diabetes, metabolic syndrome, heart disease, muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, oculopharyngeal muscular dystrophy, chronic kidney disease (CKD), renal fibrosis, diabetic nephropathy, chronic glomerulonephritis, IgA nephropathy, lupus nephritis, primary glomerular disease, chronic obstructive pulmonary disease (COPD), emphysema, interstitial pneumonia, pulmonary fibrosis, heart disease, and muscle disease.

[0136] Compositions comprising the ligand-binding nucleic acid complex in some embodiments can be formulated by known pharmaceutical methods, for example, as capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated formulations, pellets, troches, sublingual tablets, chewable tablets, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc., and can be used enterally (e.g., orally) or parenterally.

[0137] In preparing these formulations, the pharmaceutical composition may be appropriately combined with a carrier that is pharmacologically or food- or beverage-acceptable, specifically, sterilized water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, pH adjuster, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, solubilizing agent, or other additives.

[0138] In the case of formulation, etc., the ligand-bound nucleic acid complex in an embodiment for enteral administration may be a complex (mixed micelle, emulsion) of a substance having the effect of enhancing colonic mucosal epithelial permeability (e.g., a medium-chain fatty acid, a long-chain unsaturated fatty acid, or a derivative thereof (salt, ester, or ether)) and a surfactant (a nonionic surfactant, an anionic surfactant) from the viewpoint of further increasing the efficiency of enteral administration.

[0139] In some embodiments, preferred modes of administration of compositions comprising ligand-bound nucleic acid complexes include, but are not limited to, enteral (e.g., oral) or parenteral, more specifically, intravenous, intraarterial, intraperitoneal, subcutaneous, intradermal, intrathecal, intratracheal, rectal, and intramuscular administration, and administration by infusion.

[0140] In some embodiments, compositions containing ligand-bound nucleic acid complexes can be used in animals, including humans, but there is no particular limitation on non-human animals, and they can include various livestock, poultry, pets, laboratory animals, etc.

[0141] When administering or ingesting a composition containing a ligand-bound nucleic acid complex in some embodiments, the dosage or intake amount is selected appropriately depending on the subject's age, weight, symptoms, health condition, type of composition (drug, food, beverage, etc.), etc., but the effective intake amount of the composition in some embodiments is preferably 0.001 mg / kg / day to 50 mg / kg / day in terms of nucleotides.

[0142] Example 1 Synthesis and Purification of PTH1 Ligand The polypeptides shown in Table 1, which are PTH1 ligands of the present invention, are introduced into oligonucleic acids according to the synthesis pattern shown in Table 4.

[0143]

[0144] In Table 4, modification pattern A is a modification pattern in which, for example, a PEG3-Azide group is substituted for the amino group in the side chain of lysine, the second amino acid from the C-terminus of a polypeptide such as L001-L009, L031-L040, or L080-L098 (K-PEG3-Azide), and the azide group of K-PEG3-Azide is linked to a 5'-dibenzocyclooctyne-succinyl-hexylamino oligonucleic acid, which has been modified at the 5' end with a dibenzocyclooctyne-succinyl-hexylamino group, thereby linking the polypeptide such as L001-L009, L031-L040, or L080-L098 to the oligonucleotide.

[0145] In Table 4, modification pattern C is a modification pattern in which the amino group of the side chain of lysine at the C-terminus of a polypeptide such as L021-L030, L051-L088, or L109-L168 is substituted with a PEG3-Azide group (K-PEG3-Azide), and the azide group of K-PEG3-Azide is linked to a 5'-dibenzocyclooctyne-succinyl-hexylamino oligonucleic acid in which the 5' end of the oligonucleotide has been modified with a dibenzocyclooctyne-succinyl-hexylamino group, thereby linking the polypeptide such as L021-L030, L051-L088, or L109-L168 to the oligonucleotide.

[0146] (K-PEG3-Azide)

[0147] When L001 is linked to an oligonucleotide using this method, the resulting linker structure is K-PEG3-Unit. Here, Unit refers to the linker portion of the linker structure on the oligo side. In the structural formula (Chemical Formula 33) below, the linking site (the structure formed by the reaction of azide with the DBCO structure) and the phosphate group located at the 5' end of the oligo side are Units.

[0148] (K-PEG3-Unit)

[0149] In Table 4, modification pattern B is a modification pattern in which the thiol of cysteine, the second amino acid from the C-terminus of a polypeptide such as L011-L020, L041-L050, or L099-L108, is linked via an SS bond to a 5'-3-pyridyldithiopropionyl-hexylamino oligonucleotide, which has been modified with a 3-pyridyldithiopropionyl group at the 5' end, and introduced into an oligonucleotide.

[0150] When L099 is linked to an oligonucleotide using this method, the linker structure is a C-S-Unit. Here, "Unit" refers to the linker portion on the oligo side of the linker structure. In the structural formula (Chemical Formula 34) below, the linking site (SS bond) and the phosphate group located at the 5' end of the oligo side are the Units.

[0151] (CS-Unit)

[0152] The ligand-linker LG001 for introducing oligonucleotides into L001 can be synthesized using the solid-phase peptide synthesis method developed by Merrifield et al. A commonly available automated peptide synthesizer may also be used. For example, the automated peptide synthesizer 430A (Applied Biosystems) can be used to synthesize polypeptide L001. The resulting peptide-containing resin is subjected to gel filtration to obtain a purified polypeptide.

[0153] Other peptides that are PTH1 ligands for use in the present invention can be prepared in a similar manner by those skilled in the art.

[0154] The peptides listed in Table 1 can be synthesized according to the following method.

[0155] A synthetic peptide is immobilized on a solid support via a cleavable spacer. Suitable protecting groups, such as Boc, Fmoc, benzyl, or t-butyl, have been previously introduced into the main chain and side chains of the amino acid corresponding to the C-terminus. A reagent capable of selectively deprotecting the amino group in the main chain of the amino acid is then introduced to selectively remove only the amino group in the main chain. The deprotected amino group in the main chain is then reacted with the amino acid to be elongated. The amino acid has been protected with a suitable protecting group containing a carboxylic acid activated by the addition of a condensing agent such as HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) and a base such as DIEA (diisopropylethylamine), forming an amide bond, thereby extending the main chain toward the N-terminus. This process is repeated to synthesize a polypeptide having the desired amino acid sequence. In the case of L-001, the protecting group of the amine side chain of the amino group of the lysine side chain at the 33rd residue from the N-terminus is selectively deprotected, and then the azide group is introduced by reaction with an activated carboxylic acid containing an azide group, such as 2,5-dioxypyrrolidin-1-yl 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetate. Thereafter, the remaining protecting groups and the spacer moiety connecting the peptide to the solid phase support are cleaved. The resulting crude peptide is purified using a reverse-phase column, and the solvent is removed by evaporation using lyophilization or the like to obtain the desired peptide chain.

[0156] In one embodiment, L001, which is the polypeptide of SEQ ID NO: 1 in Table 1, was used as the PTH1 ligand. The ligand linker LG001 for introducing L001 (the polypeptide of SEQ ID NO: 1) described in Table 1 was obtained from Peptide Institute, Inc. The purity was approximately 99% by HPLC. The molecular weight determined by mass spectrometry (ESI-MS) was 4188.8 (which agreed with the calculated molecular weight of 4188.8). L002-L168 can also be synthesized by a similar method.

[0157] [Example 2] The nucleic acid complex of the present invention may be any nucleic acid complex that has an antisense strand that can hybridize to a target gene or target transcript and exerts an antisense effect. In the case of a double-stranded nucleic acid complex, a combination of a complementary strand that can hybridize with the antisense strand can be used. For example, it is possible to select from ASO, HDO, and siRNA having the following sequences. When targeting different diseases, since the target transcripts are different, it is possible to use nucleic acid molecules having an antisense strand with a nucleic acid sequence corresponding to each target.

[0158] In one embodiment, a nucleic acid complex can be used in which a PTH1 ligand is linked to an ASO having an antisense strand consisting of the following nucleic acid sequence in Table 5 and / or an HDO and / or siRNA having a strand complementary to the antisense strand.

[0159]

[0160] In Table 5, lowercase letters represent DNA, underlined capital letters represent LNA (C stands for LNA methylcytosine), uppercase letters represent RNA, uppercase italics represent 2'-O-methyl sugar modifications, lowercase italics represent morpholino nucleic acids, underlined lowercase italics represent 2'-fluoro-sugar modifications, and an asterisk represents a phosphorothioate linkage.

[0161] In Example 2, L001-HDO6 targeting mouse Malat1 (mMalat1) ncRNA was prepared using an antisense strand (SEQ ID NO: 179), a complementary strand (SEQ ID NO: 180), and the PTH1 ligand L001.

[0162]

[0163] Lower case letters represent DNA, underlined capital letters represent LNA (C stands for LNA methylcytosine), capital letters represent RNA, double underlined capital letters represent 2'-O-methyl sugar modifications, and asterisks represent phosphorothioate linkages.

[0164] SEQ ID NO: 179 is an antisense strand consisting of 16 nucleic acids (16mer) targeting mMalat1 ncRNA, and is composed of a sequence that targets mMalat1 ncRNA and can hybridize to the target site of mouse Malat1 ncRNA.

[0165] SEQ ID NO: 180 is a complementary strand consisting of 16 nucleic acids (16mer) having a sequence complementary to the antisense strand of SEQ ID NO: 179, and is composed of a sequence that can hybridize to the antisense strand of SEQ ID NO: 179.

[0166] A conjugation reaction can be carried out as follows to attach a ligand molecule to the 5' end of the complementary strand of SEQ ID NO:180.

[0167] 5'dibenzocyclooctyne-succinyl-hexylamino oligonucleic acid (1.0 mM, 3.0 mL) in which the 5' end of the complementary strand of SEQ ID NO: 180 was modified with a dibenzocyclooctyne-succinyl-hexylamino group To the mixture (X represents a dibenzocyclooctyne-succinyl-hexylamino group, * represents a phosphorothioate bond, double-underlined capital letters represent 2'-O-methyl sugar modification, and capital letters represent RNA), the L001 solution obtained in Example 1 was added, stirred, and allowed to stand. After adding aqueous sodium chloride solution and stirring, an alcohol such as 2-isopropanol or acetonitrile solvent was added, stirred, and allowed to stand at -20°C. The mixture was centrifuged for a certain period to precipitate the solid, and the supernatant was removed, followed by purification by HPLC. After distilling off the solvent, the mixture was redissolved in water, and aqueous sodium chloride solution was added and stirred. After that, alcohol was added and centrifuged to obtain the L001-bound complementary strand as a precipitate. Next, equal amounts of the antisense strand of SEQ ID NO: 179 and the L001-bound complementary strand of SEQ ID NO: 180 were added at equimolar concentrations to a microtube, stirred, and the microtube was incubated at 95°C for 5 minutes. Thereafter, the microtube is gradually cooled to room temperature to produce L001-HDO6.

[0168] In the case of PTH1 ligands introduced using modification patterns A and C in Table 4, by using the ligands L002-L010, L021-L040, L051-L098, and L0109-L168 instead of L001 in the above method, ASO, HDO, and siRNA bound to each of the ligands L002-L010, L021-L040, L051-L098, and L0109-L168 can be prepared.

[0169] In the case of a PTH1 ligand introduced using modification pattern B, in the above method, X in formula 36 is changed to a 3-pyridyldithiopropionyl group, and further, L001 is replaced with ligands LG012-LG020, LG041-LG050, and LG099-LG108, respectively, thereby making it possible to prepare ASO, HDO, and siRNA bound to ligands LG012-LG020, LG041-LG050, and LG099-LG108.

[0170] The HPLC purity, theoretical molecular weight and detection value of the complementary strands into which L001, L003, L005, L010, L021, L040, L089, L098, L098, L165 and L168 were introduced using the above method are shown in Table 6 below.

[0171]

[0172] The antisense strand of SEQ ID NO: 179 (0.2 mM, 750 μL) and the complementary strand of SEQ ID NO: 180 bound to L001 (0.20 mM, 750 μL) were mixed to obtain L001-HDO6 (0.1 mM, 1.5 mL), which is a ligand-bound HDO.

[0173] The double-stranded complex L001-HDO6 was prepared by adding L001-HDO6 to normal saline (Otsuka Saline Injection (Otsuka Pharmaceuticals)), heating the solution at 95°C for 5 minutes, and slowly cooling the solution to room temperature to form an annealed double-stranded complex.

[0174] PTH1 ligand-binding HDO can also be produced by a similar method using an oligonucleic acid in which L002-L168 has been introduced instead of L001, or an oligonucleic acid in which the sequence has been changed to HDO, HDO2, HDO3, HDO4, or HDO5.

[0175] The Tm of HDO6 into which L001, L003, L005, L010, L021, L040, L089, L098, L098, L165, and L168 were introduced was as shown in Table 7 below.

[0176]

[0177] Example 3 Reagents The nucleic acid complex used in this example was L001-HDO6, in which L001 was conjugated as a ligand to the 16-mer HDO targeting mouse Malat1 ncRNA prepared in Example 2. The inventors prepared these oligos at 100 μM in physiological saline (Otsuka Pharmaceuticals), and denatured the double-stranded HDO at 90°C for 5 minutes in a block bath (CDB-105, AS ONE), followed by natural cooling (approximately 2 hours) to return to room temperature, thereby carrying out an annealing procedure.

[0178] Because there are few species differences between humans and mice in the PTH1 receptor, PTH, and PTHrP, c57BL / 6J mice (25 mice, Charles River Japan, female, 4 weeks old upon arrival) were used as subjects. The animals were housed in plastic cages of five mice each under an environment of room temperature (24±2°C), humidity (55±5%), and 12 hours of lighting (8:00-20:00). They were given free access to water and solid feed (MF, Oriental Yeast Co., Ltd.) and allowed to acclimate for one week.

[0179] Methods: Mice were divided into three groups based on their body weight: Vehicle (Saline, Otsuka Pharmaceutical), L001 complementary chain (1 μmol / kg), and L001-HDO6 (1 μmol / kg) groups (n = 5 animals per group). On the day of reagent administration, mice were restrained in a restrainer while awake, and the reagent was slowly administered via the tail vein (10 ml / kg, Day 0). The L001 complementary chain (1 μmol / kg) group served as a negative control. After administration, hemostasis at the administration site was confirmed, and the mice were returned to their cages. Necropsies were performed three days after administration (Day 3). On the day of necropsy, mice were weighed, and under isoflurane anesthesia (induction at 3-5%, maintenance at 1-3%), intracardiac blood was collected (Terumo syringe SS-01P2525, containing the anticoagulant heparin) and euthanized by exsanguination. The abdomen was opened, and the liver and kidneys were removed. The removed organs were immediately immersed in ISOGEN and subjected to mRNA extraction. The blood sample was placed in a 1.5 ml Eppendorf tube and centrifuged at 10,000 rpm for 5 minutes. The supernatant was frozen (-30°C) and used to measure the blood levels of the liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

[0180] Tissues were disrupted using a biomasher, GentleMACS Dissociators (Miltenyi Biotec), or FastPrep-24 5G (MP Biomedicals). Total RNA was extracted from the tissue homogenate using ReliaPrep. ＴＭ RNA Tissue Miniprep System (Z6112, Promega) was used. cDNA was prepared from total RNA using PrimeScript. ＴＭ Reverse transcription was performed using RT Master Mix (TaKaRa, RR036A). mRNA expression of each gene was measured using the Step One Plus real-time PCR system (Applied Biosystems). TaqMan probe sets for mouse Malat1 (Mm 01227912-s1, Thermo Fisher Scientific) and 18S rRNA (Mm 03928990-g1, Thermo Fisher Scientific) mRNA were used.

[0181] Blood ALT / AST activity was measured using a Test Wako ALT / AST measurement kit (Fujifilm Wako Pure Chemical Industries, Ltd., 431-3090) and an enzyme calibrator (Fujifilm Wako Pure Chemical Industries, Ltd., 416-57191).

[0182] All measured values ​​were expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 7.04 with one-way ANOVA, and comparisons between groups were performed using Dunnett's multiple comparisons. A significance level of less than 5% was considered significant.

[0183] Results 1. General condition No clear changes were observed before and after administration in the coat, behavior, feces, etc. of the mice. The reagent was administered via the tail vein at a dose of 10 ml / kg, and no abnormal behavior was observed immediately after administration.

[0184] 2. Body Weight The average body weight of animals in each group before administration was 17 g, and no significant change was observed between before and after administration. Furthermore, there was no significant change in the average body weight of the oligo-administered group compared to the vehicle group (Figure 1).

[0185] 3. Blood ALT / AST Activity Figure 2A shows the change in blood ALT activity 3 days after administration of mMalat1 oligo to mice. Figure 2B shows the change in blood AST activity 3 days after administration of mMalat1 oligo to mice. No significant changes were observed in ALT and AST activity in the mMalat1 oligo administration group compared to the vehicle administration group.

[0186] 4. Changes in mMalat1 ncRNA Expression in Tissues 4.1 Liver The expression of mMalat1 ncRNA in the liver was significantly reduced in the L001-HDO6-administered group compared to the vehicle-administered group and the L001 complementary strand-administered group (FIG. 3).

[0187] 4.2 Kidneys mMalat1 ncRNA expression in the kidney was significantly reduced in the L001-HDO6-administered group compared to the vehicle-administered group and the L001 complementary strand-administered group (FIG. 4).

[0188] In this example, L001-HDO6 was administered intravenously to a subject (intravenous administration), resulting in delivery of L001-HDO6 to organs expressing the PTH1 receptor, such as the liver, and suppressing the expression of the target transcription product, mMalat1 ncRNA.

[0189] Example 4 Histological Examination of Kidneys by Immunostaining Images After Administration of L001-HDO6-Alexa488 Fluorescently labeled HDO used for histological examination by immunostaining images is shown in FIG.

[0190] Fluorescently labeled ASO, in which Alexa488 was bound to the 5' end of the antisense strand (SEQ ID NO: 179) prepared in Example 2, was annealed to the L001 complementary strand (SEQ ID NO: 180) by the method described in Example 2 to form a double-stranded complex, thereby producing fluorescently labeled HDO (L001-HDO6-Alexa488).

[0191] Immunohistochemistry was performed using 12 c57BL / 6J mice. A single intravenous dose of 1 μmol / kg of fluorescently labeled HDO (L001-HDO6-Alexa488) was administered to the mice. Ten minutes, six hours, 24 hours, and 72 hours later, blood was collected intracardially under isoflurane anesthesia. The kidneys were then perfused with 10 ml of saline via left ventricular intubation, followed by 10 ml of 4% paraformaldehyde. The kidneys were then excised and immersed in 4% paraformaldehyde to prepare frozen sections. For each frozen section, kidney glomeruli were stained with nephrin (primary antibody Mouse Nephrin Affinity Purified Polyclonal Antibody, Goat IgG (R&D Systems: AF3459)), secondary antibody Alexa Flour 594 chicken anti-goat IgG (H+L) (A-21468, Invitrogen), and cell nuclei containing DNA were stained with DAPI (4',6-diamidino-2-phenylindole) solution. Fluorescent images of the pathological sections were captured using an Olympus VS120 Slide Scanning System.

[0192] Figure 6 shows immunohistochemical staining images of kidneys 10 minutes, 6 hours, 24 hours, and 72 hours after a single i.v. administration of 1 μmol / kg of fluorescently labeled HDO (L001-HDO6-Alexa488) to c57BL / 6J mice. Each time point after administration consists of four photographs. The upper left photograph is an ultraviolet (UV) fluorescent image showing the results of DAPI staining. The lower left photograph is a red fluorescent image (red-Alexa594) showing the results of staining with an anti-nephrin antibody. The upper right photograph is a green fluorescent image showing the results of staining with L001-HDO6-Alexa488 (green-Alexa488). The lower right photograph is a merged image of the above three photographs.

[0193] Results 1. Glomeruli Alexa-488 fluorescence was faintly observed within the glomeruli 10 minutes after administration of the L001-HDO6 reagent, but was not observed at any of the observation time points of 6, 24, and 72 hours.

[0194] 2. Renal Tubule In renal tubule cells, Alexa-488 fluorescence was observed 10 minutes after administration of the L001-HDO6 reagent, became stronger at 6 hours, was strongest at 24 hours, and was still observed in many renal tubules at 72 hours.

[0195] In this example, the results showed that by intravenously administering L001-HDO6 to a subject, L001-HDO6 was delivered to an organ expressing the PTH1 receptor, such as the liver, and accumulated in the renal tubules in the liver for 72 hours.

[0196] Example 5 The nucleic acid complexes used in this example were prepared by attaching a PTH1 ligand to the 16-mer HDO or ASO targeting mouse Malat1 ncRNA prepared in the Examples. The inventors prepared these oligos in saline (Otsuka Pharmaceutical Factory). The oligos for animal evaluation were denatured in a block bath (CDB-105, AS ONE) and the double-stranded HDOs for cellular evaluation were denatured in a T100 thermal cycler (Bio-Rad) at 95°C for 10 minutes, followed by cooling (approximately 2 hours) and annealing to room temperature. The oligos for animal evaluation were adjusted to a final concentration of 100 nmol / mL in saline, and the oligos for cellular evaluation were adjusted to various concentrations in DMEM (Invitrogen) with 0.1% FCS.

[0197] Animals: C57BL / 6J mice (Japan SLC, female, 4 weeks old at time of arrival) were used. Animals were housed in plastic cages of five mice each under an environment of room temperature (24 ± 2°C), humidity (55 ± 5%), and 12-hour lighting (7:00-19:00). They were given free access to water and solid feed (MF, Oriental Yeast Co., Ltd.) and allowed to acclimate for one week.

[0198] In Vivo Evaluation Method: Mice were divided into vehicle (Saline, Otsuka Pharmaceutical Factory) and various PTH1 ligand-HDO (1 μmol / kg) groups based on their body weight (n=5 animals per group). On the day of reagent administration, mice were restrained in a restrainer while awake, and the test agent was slowly administered (10 mL / kg, Day 0) via the tail vein or subcutaneously in the dorsal neck. After administration, hemostasis at the administration site was confirmed, and the mice were returned to their cages. Autopsies were performed three days after administration (Day 3). On the day of autopsy, mice were euthanized by exsanguination under isoflurane anesthesia (induction at 3-5%, maintenance at 1-3%). Laparotomy was performed, and the liver, kidneys, lungs, heart, and thigh muscles were removed. The removed organs were immediately immersed in ISOGEN and subjected to mRNA extraction.

[0199] For mRNA extraction, tissue was disrupted by bead disruption using TissueLyser II (QIAGEN). Total RNA was extracted from the tissue lysate using ReliaPrep. ＴＭThe RNA Tissue Miniprep System (Promega) was used. cDNA was prepared from total RNA using PrimeScript. ＴＭ Reverse transcription was performed using RT Master Mix (TaKaRa). mRNA expression of each gene was measured using the real-time PCR system Step One Plus (Applied Biosystems). TaqMan probe sets for mouse Malat1 (Mm 01227912-s1, Applied Biosystems) and mouse Gapdh (Mm99999915_g1, Applied Biosystems) mRNA were used.

[0200] All measured values ​​were expressed as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 9.4.0. Comparisons between groups were performed using one-way analysis of variance followed by Dunnett's multiple comparison test, and comparisons between two independent samples were performed using Student's t-test. A risk rate of less than 5% was considered statistically significant in all cases.

[0201] Results 1. Study of PHT1 Ligand Peptide Amino Acid Sequence Length Modifications Renal Malat1 ncRNA expression was not statistically significantly suppressed in the HDO intravenous administration group (HDO) without PTH1 ligand administration, but was statistically significantly reduced in all HDO intravenous administration groups with PTH1 ligand administration (L001-HDO6, L003-HDO6, L005-HDO6, and L010-HDO6) compared to the saline administration group. Furthermore, compared to HDO, renal Malat1 ncRNA expression was statistically significantly reduced in all HDO intravenous administration groups with PTH1 ligand administration (L001-HDO6, L003-HDO6, L005-HDO6, and L010-HDO6). The results are shown in Figure 7.

[0202] L001 is a peptide consisting of 34 amino acid residues, L003 is a peptide consisting of 32 amino acid residues with 2 amino acid residues deleted from the N-terminus of L001, L005 is a peptide consisting of 30 amino acid residues with 4 amino acid residues deleted from the N-terminus of L001, and L010 is a peptide consisting of 25 amino acid residues with 9 amino acid residues deleted from the N-terminus of L001.

[0203] HDO bound to a peptide consisting of 34 amino acid residues of L001 inhibited target gene expression. Furthermore, HDO bound to peptides consisting of 32, 30, and 25 amino acid residues, which were obtained by deleting 2, 4, and 9 amino acid residues from the N-terminus of the 34 amino acid peptide, also inhibited target gene expression, demonstrating that peptides with at least 9 amino acid residues deleted from the N-terminus have antisense effects.

[0204] In Figure 7 and other figures, the symbols * and *** represent P<0.05 or 0.001 (Dunnett) vs. Saline, and the symbols #, ##, and ### represent P<0.05, 0.01, or 0.001 (Dunnett) vs. HDO.

[0205] 2. Subcutaneous Administration: Renal Malat1 ncRNA expression was not statistically significantly suppressed in the unliganded HDO subcutaneous administration group (HDO). On the other hand, in the liganded HDO subcutaneous administration groups (L031-HDO6 and L021-HDO6), both groups showed a statistically significant decrease compared to the saline administration group (Figure 8). Furthermore, L021-HDO6 showed significant inhibitory activity against the target gene in both the subcutaneous and intravenous administration groups.

[0206] L031 is a peptide consisting of 35 amino acid residues, and L021 is a peptide consisting of 34 amino acid residues. The difference in amino acid sequence between these peptides is the presence or absence of T at the C-terminus and the position of K (Lys), but both peptides significantly inhibited the expression of the target gene.

[0207] 3. In Vitro Assay Cells Mouse Pth1R (mPth1R) was stably expressed in NIH / 3T3 cells using the PiggyBac transposon method. The mPth1R stably expressing clone cell line used to evaluate the effects of PTH1 ligands has a cAMP induction ability with an IC50 of approximately 100 pM and 33K agonist activity.

[0208] In vitro evaluation method: mPth1R stable expressing cell line was placed in a 96-well plate at 1.5 x 10 ４ Cells were seeded at 100 μL / well. 24 hours after seeding, the medium in each well was aspirated, and 100 μL of various concentrations of PTH1 ligand-HDO prepared in DMEM containing 0.1% FBS was added to each well. Total RNA extraction from the cells was performed according to the protocol of the SV 96 Total RNA Isolation System (Promega) RNA purification kit. Specifically, 24 hours after the addition of PTH1 ligand-HDO, the medium in each well was aspirated, and then each well was washed with PBS. The PBS was aspirated, and then 100 μL of the RNA lysis buffer included with the RNA purification kit was added to each well to lyse the cells. The subsequent procedures were carried out according to the SV 96 Total RNA Isolation System (Promega) to obtain total RNA. Reverse transcription from total RNA to cDNA was carried out using PrimeScript. ＴＭ RT Master Mix (TaKaRa) was used. mRNA expression of each gene was measured using the real-time PCR system Step One Plus (Applied Biosystems). Malat1 (Mm 01227912-s1, Applied Biosystems) was used to analyze the target gene, and a TaqMan probe for Gapdh (Mm99999915_g1, Applied Biosystems) was used as an internal standard gene.

[0209] All measured values ​​are expressed as mean ± standard deviation of N = 3. IC50 was calculated using GraphPad Prism 9.4.0.

[0210] 3.1. L021-HDO6, L003-HDO6, L005-HDO6, L010-HDO6 L021-HDO6, L003-HDO6, L005-HDO6, and L010-HDO6 all reduced Malat1 ncRNA expression in a concentration-dependent manner, with IC50 values ​​of 46.8 nM, 88.9 nM, 121.3 nM, and 166.7 nM, respectively (Figure 9). These results indicate that HDOs bound to PTH1 ligands consisting of peptides represented by SEQ ID NOs: 21, 3, 5, 6, and 10 have a concentration-dependent antisense effect on the target gene.

[0211] Both L021-HDO6 and L011-HDO6 reduced Malat1 ncRNA expression in a concentration-dependent manner, with IC50 values ​​of 6.94-10.2 nM and 11.8-14.1 nM, respectively (Figure 10). These results indicate that HDO bound to PTH1 ligands consisting of peptides of SEQ ID NO:21 and SEQ ID NO:11 has a concentration-dependent antisense effect on the target gene.

[0212] 3.3 L031-HDO6 and L040-HDO6 Both L031-HDO6 and L040-HDO6 reduced Malat1 ncRNA expression in a concentration-dependent manner, with IC50 values ​​of 41.5-71 nM and 92.2-111 nM, respectively (Figure 11). These results indicate that HDO bound to PTH1 ligands consisting of peptides represented by SEQ ID NO:31 and SEQ ID NO:40 has a concentration-dependent antisense effect on the target gene.

[0213] 3.4 L089-HDO6 and L098-HDO6 Both L089-HDO6 and L098-HDO6 reduced Malat1 ncRNA expression in a concentration-dependent manner, with IC50 values ​​of 1.14-1.61 nM and 47.7-82.7 nM, respectively (Figure 12). These results indicate that HDO bound to PTH1 ligands consisting of peptides represented by SEQ ID NOs: 89 and 98 has a concentration-dependent antisense effect on the target gene.

[0214] 3.5. L165-HDO6 and L168-HDO6 Both L165-HDO6 and L168-HDO6 reduced Malat1 ncRNA expression in a concentration-dependent manner, with IC50 values ​​of 27.8-185 nM and 11.4-96.5 nM, respectively (Figure 13). These results indicate that HDO bound to PTH1 ligands consisting of peptides represented by SEQ ID NOs: 165 and 168 has a concentration-dependent antisense effect on the target gene.

[0215] 3.6. L001-ASO Both L001-ASOs, which are ASOs bound to the PTH1 ligand L001, reduced Malat1 ncRNA expression in a concentration-dependent manner, with an IC50 of 1.25 nM (Figure 14). These results indicate that ASOs bound to the PTH1 ligand consisting of the peptide of SEQ ID NO: 1 have a concentration-dependent antisense effect on the target gene. The PTH1 ligand of the present invention bound to a single-stranded nucleic acid molecule and exhibited a concentration-dependent antisense effect on the target gene.

[0216] [Example 6] The nucleic acid complex of the present invention may be any complex that has an antisense strand that can hybridize to a target gene or target transcript and exerts an antisense effect. In the case of a double-stranded complex, a combination of a complementary strand that can hybridize with the antisense strand can be used. For example, siRNAs having the following sequences can be selected. When targeting different diseases, since the target transcripts are different, nucleic acid molecules having antisense strands with nucleic acid sequences corresponding to the respective targets can be used.

[0217] In one embodiment, the nucleic acid complex can use an siRNA consisting of the following antisense strand (SEQ ID NO: 181) and guide strand (SEQ ID NO: 182).

[0218] SEQ ID NO:181:

[0219] SEQ ID NO:182:

[0220] In the above SEQ ID NOs: 181 and 182, capital italic letters represent 2'-O-methyl sugar modifications, underlined lowercase italic letters represent 2'-fluoro-sugar modifications, and an asterisk represents a phosphorothioate bond.

[0221] SEQ ID NO: 181 is an antisense strand consisting of 23 nucleic acids (23mer) targeting mGapdh mRNA, and is composed of a sequence capable of hybridizing to the target site of mouse Gapdh mRNA. SEQ ID NO: 182 is a complementary strand consisting of 23 nucleic acids (21mer) having a sequence complementary to the antisense strand of SEQ ID NO: 181, and is composed of a sequence capable of hybridizing to the antisense strand of SEQ ID NO: 181.

[0222] A conjugation reaction can be carried out in the same manner as in HDO, and a ligand molecule can be bound to the 5' end of the complementary strand of SEQ ID NO:182.

[0223] The PTH1 ligand-binding nucleic acid complex used in this example was L010-siRNA, in which the PTH1 ligand L010 was conjugated to the 23-mer siRNA targeting mouse Gapdh mRNA described above, according to the method of Example 2. The inventors prepared these oligos in physiological saline (Otsuka Pharmaceutical). The siRNA for cell evaluation was denatured at 95°C for 10 minutes in a T100 thermal cycler (Bio-Rad), followed by cooling (approximately 2 hours) and returning to room temperature for annealing. The oligos for animal evaluation were finally prepared at 100 nmol / mL in physiological saline, and the oligos for cell evaluation were prepared at various concentrations in Opti-MEM (Invitrogen).

[0224] Cells A cell line in which mouse Pth1R (mPth1R) was stably expressed in an NIH / 3T3 cell line by the PiggyBac transposon method was used.

[0225] In vitro evaluation method: mPth1R stable expressing cell line was placed in a 96-well plate at 3.75 x 10 ３Cells were seeded at 100 μL / well. Four hours after seeding, the medium in each well was aspirated, and 100 μL of various concentrations of L010-siRNA prepared in Opti-MEM was added to each well. Total RNA extraction from the cells was performed according to the protocol of the SV96 Total RNA Isolation System (Promega) RNA purification kit. 72 hours after the addition of L010-siRNA, the medium in each well was aspirated, and then each well was washed with PBS. The PBS was aspirated, and then 100 μL of the RNA Lysis Buffer included with the RNA purification kit was added to each well to lyse the cells. Subsequent procedures for obtaining total RNA followed the SV96 Total RNA Isolation System. PrimeScript RT Master Mix (TaKaRa) was used for reverse transcription from total RNA to cDNA. mRNA expression of each gene was measured using the real-time PCR system Step One Plus (Applied Biosystems). Gapdh (Mm99999915_g1, Applied Biosystems) was used for target gene analysis, and a TaqMan probe for Actb (Mm01205647_g1, Applied Biosystems) was used as an internal control gene. All measurements were expressed as mean ± standard deviation (N = 4).

[0226] The test results are shown in Figure 15. 10 μM of siRNA bound to the PTH1 ligand L010 reduced mGapdh mRNA expression by 14% compared to the control (0 μM) administration group. The PTH1 ligand of the present invention also exhibited an antisense effect on the target gene when bound to siRNA.

[0227] The ligand-bound nucleic acid complex of the present invention can be used as a nucleic acid drug that is delivered to an organ having a PTH1 receptor and regulates the expression or editing of a target gene or its transcription product.

[0228] 1-182 Synthesis

[0229] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A ligand-bound nucleic acid complex in which a nucleic acid molecule that regulates the expression or editing of a target gene, its transcription product, or its translation product is bound to a PTH1 ligand with or without a linker, wherein the PTH1 ligand is a peptide having an amino acid sequence represented by the following formula: [Chemical formula 1] A １ -A ２ -A ３ -A ４ -A ５ -A ６ -A ７ -A ８ -A ９ -A １０ -A １１ -A １２ -A １３ -A １４ -A １５ -A １６ -A １７ -A １８ -A １９ -A ２０ -A ２１ -A ２２ -A ２３ -A ２４ -A ２５ -A ２６ -A ２７ -A ２８ -A ２９ -A ３０ -A ３１ -A ３２ -A ３３ -A ３４ In the formula, the amino acid sequence is written from the N-terminus to the C-terminus. During the ceremony, A １ is Ala, Ser, Dap or deleted; A ２ is Val; A ３ is Ser, Thr, Aib; A ４ is Glu; A ５ is Leu, His, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe, or P-X-Phe, where X is OH, halogen, or CH ３ and A ６ is Gln; A ７ is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe or P-X-Phe, where X is OH, halogen, or CH ３ and A ８ are Met, Nva, Leu, Val, Ile, Cha, Acc, Nle; A ９ is His; A １０ is Asp or Asn; A １１ is Leu, Lys, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe or P-X-Phe, where X is OH, halogen, or CH ３ and A １２ is Gly, Acc, or Aib; A １３ is Lys; A １４ is Ser or His; A １５ is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Acc, Phe or P-X-Phe, where X is OH, halogen, or CH ３ and A １６ is Ser, Gln, Asn, Ala, or AiB; A １７ is Ser, Asp, Thr, or AiB; A １８ is Met, Nva, Leu, Val, He, Nle, Acc, Cha, or Aib; A １９ is Arg, Glu or AiB; A ２０ is Arg; A ２１ is Arg, Val, Acc, Cha, or Met; A ２２ is Phe, Glu, Aib, Acc, or Cha; A ２３ is Phe, Trp, Leu, Lys, Acc, or Cha; A ２４ is Leu, Lys, Acc, or Cha; A ２５ is His, Arg, Lys, AiB, Acc, or Glu; A ２６ is His, Aib, Acc, or Lys; A ２７ is Lys, Aib, Leu, hArg, Gln, Acc, or Cha; A ２８ is Ile, Leu, Lys, Acc, or Cha; A ２９ is Ala, Glu, Acc, or Aib; A ３０ is Glu, Asp, Leu, Nle, Cha, Aib, Acc, or Lys; A ３１ is Ile, Val, Leu, Nle, Cha, Lys, or Acc; A ３２ is His; A ３３ is Thr, Asn, Lys, or Cys; A ３４ is Phe, Ala, Tyr, Amp or Aib.

2. A ligand-binding nucleic acid complex in which a nucleic acid molecule that regulates the expression or editing of a target gene, its transcription product, or its translation product is bound to a PTH1 ligand with or without a linker, wherein the PTH1 ligand is a peptide consisting of any one of the sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61 to SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 89, and SEQ ID NO: 165, and the second amino acid residue from the C-terminus of the PTH1 ligand peptide is either Thr, Asn, Lys, or Cys.

3. 3. The ligand-bound nucleic acid complex according to claim 1, wherein the nucleic acid molecule and the PTH1 ligand are linked via a linker.

4. The ligand-bound nucleic acid complex of claim 3 , wherein the linker is a cleavable linker having a cleavable structure.

5. The ligand-bound nucleic acid complex of claim 4, wherein the linker is any of the linkers having the following structures: 【Table 1】

6. 3. The ligand-binding nucleic acid complex of claim 1, wherein the nucleic acid molecule is a nucleic acid molecule selected from the group consisting of a nucleic acid molecule comprising an antisense strand consisting of an oligonucleotide having a nucleic acid base sequence complementary to a target gene or its transcription product, an aptamer having a nucleic acid base sequence that specifically binds to a target protein, and a decoy consisting of an oligonucleotide having a nucleic acid sequence complementary to a target transcription factor.

7. The ligand-binding nucleic acid complex according to claim 1 or claim 2, wherein the nucleic acid molecule is selected from the group consisting of ADO, ASO, HDO and siRNA, and the antisense strand of the nucleic acid molecule consists of 12 to 30 consecutive nucleotides.

8. The ligand-bound nucleic acid complex of claim 7 , wherein the nucleic acid strand of the nucleic acid molecule comprises nucleotides, modified nucleotides and / or nucleotide analogs.

9. 9. The ligand-bound nucleic acid complex according to claim 8, wherein the nucleic acid molecule is HDO, and the total number of nucleotides, modified nucleotides, and nucleotide analogs in the antisense strand and complementary strand of HDO each consists of 12 to 30 nucleotides.

10. The ligand-bound nucleic acid complex of claim 9, wherein the antisense strand is a gapmer and comprises a gap region containing nucleotides and / or modified nucleotides and wing regions containing one or more nucleotide analogs and / or modified nucleotides located on the 5'-end and / or 3'-end thereof.

11. The ligand-bound nucleic acid complex of claim 9, wherein the complementary strand comprises a center region containing nucleotides and / or modified nucleotides and wing regions containing one or more nucleotide analogs and / or modified nucleotides located on the 5'-end and / or 3'-end thereof.

12. The modified nucleotide is 2'-O-CH ３ group or 2'-O-CH ２ CH ２ OCH ３ 10. The ligand-bound nucleic acid complex of claim 9, wherein the nucleotide comprises a (MOE) group.

13. 10. The ligand-bound nucleic acid complex of claim 9, wherein the nucleotide analog comprises a bridged nucleotide independently selected from the group consisting of LNA, cEt-BNA, amide BNA (AmNA), and cMOE-BNA.

14. 10. The ligand-binding nucleic acid complex of claim 9, wherein the nucleotide analogs are independently selected from the group consisting of PNA, GNA, TNA, cEt, tcDNA, morpholino nucleic acid, BNA, GuNA, and scpBNA.

15. 10. The ligand-bound nucleic acid complex of claim 9, wherein at least one nucleotide or modified nucleotide in the nucleic acid molecule is phosphorothioated or boranophosphated.

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