WO2020184700A1 - Ihh発現を調節するための核酸複合体 - Google Patents
Ihh発現を調節するための核酸複合体 Download PDFInfo
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Definitions
- the present invention relates to a nucleic acid complex containing a heteroduplex oligonucleotide (HDO) for regulating the expression of the Indian hedgehog gene (hereinafter, IHH gene).
- HDO heteroduplex oligonucleotide
- IHH gene-specific inhibitors IHH gene-specific inhibitors.
- the present invention also relates to a therapeutic agent for fibrosis, which comprises an inhibitor of a transcript of the IHH gene.
- Hedgehog is a morphogenetic signaling pathway that controls the fate and tissue construction of progenitor cells during embryogenesis, and hedgehog reactivation occurs during liver injury in adults.
- Hedgehog (Hh) is a signal transduction pathway that controls important cell fate decisions including proliferation, apoptosis, migration and differentiation, and the number of adult tissues including the liver regulates the wound healing response (non-patent literature). 1).
- ECM extracellular matrix
- HSC Hepatic Stem Cell
- ECM contains a complex mixture of proteins that promote cell proliferation, migration and differentiation.
- One ECM component having such a role is osteopontin (OPN: Osteopontin), a matrigergoline phosphoprotein also known as secretory phosphoprotein 1 (Non-Patent Document 2).
- HSC Hepatic Stem Cell plays an important role in liver fibrosis.
- the expression of the hedgehog signaling components of Ihh, Smo, Ptc, Gli2 and Gli3 in HSC was obtained by constructing a hedgehog siRNA vector targeting Ihh, Smo and Gli2 and transfecting them into HSC, respectively. Expression decreased. It has been found that HSC activation and collagen secretion can be regulated by hedgehog signaling (Non-Patent Document 3).
- Nonalcoholic steatohepatitis is a major cause of liver disease worldwide.
- the transcription factor TAZ (WWTR1) is significantly higher in human and murine NASH liver than in normal or fatty liver.
- WWTR1 transcription factor 1
- TAZ factor is significantly higher in human and murine NASH liver than in normal or fatty liver.
- TAZ factor is significantly higher in human and murine NASH liver than in normal or fatty liver.
- hepatocyte TAZ silencing in a mouse model of NASH prevented or reversed hepatitis, hepatocyte death and fibrosis, but not steatohepatitis. From these facts, it was found that the TAZ factor is a factor that contributes to an important process for the progression of steatosis and NASH (Non-Patent Document 4).
- Non-Patent Documents 2 and 3 show that activation of hepatic stellate cells (HSC) plays an important role in NASH fibrosis. Although many factors for activating HSC have been proposed in NASH, research in this field is not yet complete and has not yet reached an FDA-approved therapeutic strategy (Non-Patent Document 5).
- HSC hepatic stellate cells
- the therapeutic agents for fibrosis include therapeutic agents consisting of antibiotics such as steroids, for example, pirfenidone and Nintedanib, which are therapeutic agents for idiopathic pulmonary fibrosis (IPF: (Idiopathic Pulmonary Fibrosis)).
- Pirfenidone is an anti-fibrotic drug.
- the main mechanism of action is suppression of transforming growth factor- ⁇ (TGF- ⁇ ) production.
- TGF- ⁇ promotes fibrosis by controlling the phenomenon of "epithelial-mesenchymal transition" in which type 2 alveolar epithelial cells differentiate into fibroblasts and myofibroblasts.
- Pirfenidone exerts an anti-fibrotic effect by blocking its pathway.
- b-FGF basic-fibroblast growth factor
- SDF-1 ⁇ stroma cell derived factor-1 ⁇
- IFN- ⁇ interferon- ⁇
- Nintedanib is an antifibrotic drug.
- One of the small molecule tyrosine kinase inhibitors vascular endothelial cell growth factor receptor type 1-3 (VEGFR: vascular endothelial growth factor receptor), fibroblast growth factor receptor (FGFR: fibroblast growth factor receptor), platelets It acts on the derived growth factor receptor (PDGFR).
- VEGFR vascular endothelial cell growth factor receptor type 1-3
- FGFR fibroblast growth factor receptor
- PDGFR derived growth factor receptor
- therapeutic agents for fibrosis composed of these low molecular weight compounds, therapeutic agents having a new mechanism of action are required.
- IHH gene inhibitors have not yet been used for the treatment of fibrosis and have not even been suggested.
- IHH protein is a secretory protein belonging to the hedgehog family.
- Intron 1 of the IHH gene has a binding region of the transcription factor TAZ, and the expression of the IHH gene is positively regulated by the transcription factor TAZ through this region (Patent Document 1, Non-Patent Document 4).
- TAZ is an exacerbating factor of NASH fibrosis
- the IHH gene may mediate the exacerbating effect.
- the inventors have found that the IHH gene is secreted from hepatocytes, activates hepatocytes, and is a component in which the IHH gene is also secreted from activated stellate cells. Therefore, the IHH gene is used in the pathophysiology of fibrosis. It was thought that the pathological condition would progress further by enhancing the autokupffer and parakupffer action of the liver.
- an inhibitor of the IHH gene which is one of the hedgehog families, is useful for elucidating the function of the IHH gene. Therefore, it can be expected to suppress or delay the progression of the pathological condition of fibrosis. Furthermore, the inhibitor of the IHH gene is useful not only for the liver but also for the treatment, prevention, improvement of inflammatory diseases and fibrotic diseases in tissues and organs such as kidney, lung and skin, or suppression or delay of its progression.
- the present invention is a problem to be solved to provide an inhibitor of the IHH gene. It is an issue to be solved to provide a nucleic acid complex containing a hetero double-stranded nucleic acid (HDO) that regulates the expression of the IHH gene as an inhibitor of the IHH gene.
- HDO hetero double-stranded nucleic acid
- a nucleic acid complex for reducing the expression of mRNA and protein which are transcripts of the IHH gene that is, an inhibitor of the IHH gene and a method of inhibiting the expression of the IHH gene are disclosed as means for solving the problem. ..
- IHH gene inhibitors are useful in treating, preventing, ameliorating or delaying the progression of fibrosis, fibrotic disease in patients in need.
- a nucleic acid complex comprising an oligonucleotide consisting of 12 to 30 nucleotides and having a nucleic acid base sequence in which the oligonucleotide is complementary to an IHH gene transcript.
- the nucleic acid complex of [1] wherein the oligonucleotide is a single-strand oligonucleotide.
- the nucleic acid complex of [1] which is a hetero double-stranded nucleic acid composed of an antisense strand composed of the oligonucleotide and a nucleic acid strand complementary to the antisense strand.
- nucleic acid complex according to any one of [1] to [3], wherein the oligonucleotide contains at least one modified nucleotide.
- nucleic acid complex according to [5] wherein the oligonucleotide is a phosphorothioate oligonucleotide.
- nucleic acid complex according to any one of [1] to [7], wherein the oligonucleotide contains a modified nucleobase.
- nucleobase according to [8] wherein the modified nucleobase is 5-methylcytosine, 2'-MOE, BNA, LNA or AmNA.
- the oligonucleotide is: Gap region consisting of multiple nucleic acids; 5'wing region consisting of multiple nucleic acids; 3'wing region consisting of multiple nucleic acids; The nucleic acid complex according to any one of [1] to [10], which comprises.
- the nucleic acid base sequence of the oligonucleotide consists of a base sequence complementary to an oligonucleotide consisting of 12 to 30 consecutive nucleotides in the IHH gene sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 [1] to The nucleic acid complex of any of [11].
- the nucleic acid base sequence of the oligonucleotide is SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,
- the nucleic acid complex according to [12], which comprises the sequence of any of 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 and 112.
- nucleic acid complex according to [13], wherein the nucleic acid base sequence of the oligonucleotide consists of the sequence of SEQ ID NO: 26.
- the nucleobase sequence of the oligonucleotide is SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, It consists of any of the sequences 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186 and 188, [1]-[11].
- Nucleic acid complex of any of. [16] The nucleic acid complex according to [15], wherein the nucleic acid base sequence of the oligonucleotide consists of the sequence of SEQ ID NO: 160, 170 or 178. [17] A pharmaceutical composition comprising an IHH-specific inhibitor comprising any of the nucleic acid complexes of [1] to [16]. [18] A therapeutic agent for fibrosis comprising an IHH-specific inhibitor comprising the nucleic acid complex according to [1]. [19] A therapeutic agent for Nash containing an IHH-specific inhibitor comprising any of the nucleic acid complexes of [1] to [16].
- a therapeutic agent for liver fibrosis which comprises an IHH-specific inhibitor comprising any of the nucleic acid complexes of [1] to [16].
- a therapeutic agent for renal fibrosis which comprises an IHH-specific inhibitor comprising any of the nucleic acid complexes of [1] to [16].
- a therapeutic agent for pancreatic fibrosis which comprises an IHH-specific inhibitor comprising any of the nucleic acid complexes of [1] to [16].
- a therapeutic agent for pulmonary fibrosis which comprises an IHH-specific inhibitor comprising any of the nucleic acid complexes of [1] to [16].
- a therapeutic agent for skin fibrosis which comprises an IHH-specific inhibitor comprising any of the nucleic acid complexes of [1] to [16].
- a nucleic acid complex comprising 12 to 30 oligonucleotides and having a nucleic acid base sequence containing at least 8 consecutive nucleic acid bases of any of the nucleic acid base sequences of SEQ ID NOs: 1 to 50.
- This specification includes the disclosure of Japanese Patent Application No. 2019-047703, which is the basis of the priority of the present application.
- a nucleic acid complex comprising an oligonucleotide consisting of 12 to 30 nucleotides of the present invention and having a nucleic acid base sequence in which the oligonucleotide is complementary to the IHH gene transcript can inhibit the expression of the IHH gene.
- C indicates the central vein
- G indicates the Grisson capsule
- ⁇ indicates the inflammatory cell collection
- the arrow ( ⁇ ) indicates the lipid droplet.
- It is a liver histology (Oil red O staining) of a NASH pathological model mouse 5 weeks after administration of Ren1-12-27.
- e is the Normal Diet + Vehicle administration group
- f is the Normal Diet + HDO administration group
- g is the MCD Diet + Vehicle administration group
- h is the MCD Diet + HDO administration group.
- C indicates the central vein
- G indicates the Grisson capsule
- the arrow ( ⁇ ) indicates the lipid droplet.
- A shows the expression-suppressing effect of IHH mRNA
- B shows the expression-suppressing effect of Malat-1 mRNA (positive control). It is a figure which shows the IHH mRNA expression suppression effect by Ren-1-12-27 in the skin fibroblast (MDF) derived from a normal mouse.
- MDF skin fibroblast
- A shows the expression-suppressing effect of IHH mRNA
- B shows the expression-suppressing effect of Malat-1 mRNA (positive control).
- MRPTEC renal tubular epithelial cell
- the present invention is a nucleic acid complex comprising an oligonucleotide consisting of 12 to 30 nucleotides and having a nucleic acid base sequence in which the oligonucleotide is complementary to a transcript of the IHH (Indian hedgehog) gene.
- a nucleic acid having a nucleic acid sequence complementary to the transcript of the IHH gene acts as an antisense nucleic acid to the transcript of the IHH gene. That is, it acts as a specific inhibitor of the IHH gene, and has an activity of suppressing the expression of the target gene, the IHH gene, or the normal transcript level by an antisense effect.
- the transcript of the IHH gene is an mRNA transcribed from the genomic DNA encoding the IHH gene, and includes unmodified mRNA, unspliced mRNA precursor, and the like.
- the "transcript” can be any RNA synthesized by DNA-dependent RNA polymerase.
- the oligonucleotide of the nucleic acid complex is a single-stranded oligonucleotide. That is, it is a single-strand antisense oligonucleotide (ASO: antisense oligonucleotide).
- ASO antisense oligonucleotide
- the nucleic acid complex is a heteroduplex oligonucleotide (HDO) composed of an antisense strand composed of an oligonucleotide and a sense strand which is a nucleic acid strand complementary to the antisense strand.
- the antisense strand is annealing to the sense strand nucleic acid strand.
- the antisense strand may be referred to as the first nucleic acid strand and the sense strand may be referred to as the second nucleic acid strand.
- Such a nucleic acid complex is called a double-stranded nucleic acid complex.
- the nucleic acid complex is a single-stranded oligonucleotide at the time of production, which comprises an antisense strand consisting of a DNA nucleotide or a DNA nucleotide analog, a linker moiety consisting of 3 to 10 nucleotides, and the above.
- the structure may include a sense strand consisting of an RNA nucleotide or an RNA nucleotide analog complementary to the antisense strand.
- the nucleic acid complex having this structure is called a single-strand heteroduplex oligonucleotide (ss-HDO), and has, for example, the structure of X-LY described in WO2017 / 131124A1.
- oligonucleotide It is an oligonucleotide.
- X is the antisense strand
- Y is the complementary strand to the antisense strand
- L consists of nucleotides that act as linkers.
- this single-stranded oligonucleotide is used as a pharmaceutical composition, it is antisense in a solvent used for physiological saline, aqueous injection, non-aqueous injection, suspension injection, solid injection, etc., or in blood or plasma.
- the strand and the complementary strand to the antisense strand anneal one molecule with the linker as a fulcrum to form a double chain structure.
- Such a nucleic acid complex is one of the double-stranded nucleic acid complexes because it has a double-stranded structure by annealing one molecule when acting as a pharmaceutical composition.
- HDO hetero double-stranded nucleic acid
- active strand composed of DNA, which is the active body
- sense strand Carrier strand
- a pharmaceutical composition containing an IHH gene-specific inhibitor is highly stable in human blood and is efficiently delivered to a target tissue according to the performance of the ligand.
- RNA strands are rapidly removed by RNase H after HDO is delivered intracellularly. There, a new double-stranded structure is formed between the freed DNA strand and the mRNA, and the mRNA is degraded by the action of intracellular RNase H, thereby exerting a knockdown effect.
- the nucleic acid complex comprises 12-30 oligonucleotides having a nucleic acid sequence that is complementary to the transcript of the IHH gene.
- the oligonucleotide which is the antisense strand of the nucleic acid complex of the present invention, targets mRNA, which is a transcript of the IHH gene.
- the base sequence of the antisense strand is complementary to the partial sequence in the base sequence of the human IHH gene or the partial sequence in the base sequence of the mouse IHH gene, preferably complementary to the partial sequence in the base sequence of the human IHH gene. Is the target.
- the nucleotide sequence of the human IHH gene is shown in SEQ ID NO: 1
- the nucleotide sequence of the mouse IHH gene is shown in SEQ ID NO: 2.
- the nucleic acid base sequence containing the oligonucleotide consisting of 12 to 30 nucleotides of the present invention and in which the oligonucleotide is complementary to the IHH transcript is a partial sequence in the base sequence of the human IHH gene or the mouse IHH gene. It is a sequence complementary to a partial sequence in the base sequence of.
- the nucleic acid base sequence of the oligonucleotide is SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, It may consist of any of the sequences 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 and 112.
- nucleobase sequence consisting of the sequences of SEQ ID NOs: 19, 24, 26, 28, 76, 78, 84 or 86 is preferable, and further, the nucleobase sequence consisting of the sequence of SEQ ID NO: 26 is preferable.
- the sequence of the sense strand (SEQ ID NO: 25) with respect to the sequence of SEQ ID NO: 26 is the 598th to 611th base (14 base length) of the base sequence of SEQ ID NO: 1.
- the starting site of the sequence is shifted to the 603rd and 596th bases of the base sequence of SEQ ID NO: 1
- the sequence complementary to the sense strand having a base length of 13 to 20 is the nucleic acid base sequence of the oligonucleotide. It can also be.
- the nucleic acid base sequence of the oligonucleotide is SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148. It may consist of any of the sequences 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186 and 188. .. Among these, a nucleobase sequence consisting of the sequence of SEQ ID NO: 160, 170 or 178 is preferable.
- the DHO will be described in detail below.
- the single-strand oligonucleotide can be prepared and used based on the description regarding the antisense strand described below.
- the first nucleic acid chain is (I) Containing nucleotides and optionally nucleotide analogs, the total number of the nucleotides and optionally contained nucleotide analogs in the nucleic acid chain is 8-100. (Ii) Containing at least four contiguous nucleotides recognized by RNase H when hybridized to a transcript. (Iii) Containing at least one unnatural nucleotide, (Iv) Hybridizes to the transcript.
- the second nucleic acid chain is (I) Contains RNA nucleotides, optionally nucleotide analogs, and optionally DNA nucleotides. (Ii) Contains DNA nucleotides and / or nucleotide analogs, or (Iii) Contains PNA nucleotides.
- the "antisense effect” means that the target transcript (RNA sense strand) hybridizes with, for example, a DNA strand complementary to its partial sequence, or a strand usually designed to produce an antisense effect.
- translational inhibition or splicing function conversion effects such as exon skipping that may occur by coating the transcript with a hybridization product, and / or the transcript that may occur by recognizing hybridized moieties. It means the suppression caused by the decomposition of.
- complementarity here refers to the 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. Means that. If a sufficient number of nucleobases in the antisense strand can hydrogen bond to the corresponding nucleobases in the target nucleic acid, the antisense strand and the target nucleobase are complementary to each other and thus produce the desired effect (eg, the IHH gene). Antisense inhibition of target nucleic acids such as).
- a non-complementary nucleobase between the antisense strand and the IHH gene can be tolerated provided that the antisense strand can specifically hybridize to the target nucleic acid.
- the antisense compound can hybridize to one or more segments of the tau nucleic acid so that the intervening or adjacent segments do not participate in hybridization events (eg, loop structures, mismatches or hairpin structures).
- the antisense strand is complementary to the sequence of mRNA encoding the IHH gene.
- Complementary means that the antisense strand is complementary to the extent that it can bind to the mRNA encoding the IHH gene, for example, 80% or more, 90% or more, or 95% or more, 96% or more, 97%. As mentioned above, it may be 98% or more, or 99% or more complementary. It may be 100% complementary. There may be about 0 to 4 mismatches.
- the antisense strand or particular portion thereof provided herein is 80-100%, preferably 90-100%, relative to the tau nucleic acid, target region, target segment or particular portion thereof. %, More preferably 95-100%, or 100% complementary.
- the ratio of antisense strand complementarity to the target nucleic acid can be determined using conventional methods.
- 16 of the 20 nucleobases of the antisense strand are complementary to the target region, so the specifically hybridizing antisense strand represents 80% complementarity.
- the remaining non-complementary nucleobases can collect or intersperse complementary nucleobases, and these non-complementary nucleobases need not be adjacent to each other or adjacent to the complementary nucleobases.
- an antisense strand of 18 nucleobase length with four non-complementary nucleic acid bases next to two regions that are fully complementary to the target nucleic acid has 14 complementary to the target region. Therefore, it has 77.8% overall complementarity and is therefore within the scope of the present invention.
- the ratio of the complementarity of the antisense strand to the region of the target nucleic acid can be determined by a BLAST program or the like known in the art.
- the first nucleic acid strand is an antisense nucleic acid that is complementary to the target transcript, such as a transcript of the target gene, when the first nucleic acid strand hybridizes to the transcript.
- a nucleic acid having a region containing at least four contiguous nucleotides.
- nucleic acid may mean a monomer nucleotide, or may mean an oligonucleotide composed of a plurality of monomers.
- nucleic acid chain is also used here to refer to oligonucleotides. Nucleic acid chains may be prepared in whole or in part by chemical synthesis methods such as the use of an automatic synthesizer, and are not limited to polymerase, ligase or restriction enzyme reactions, but are prepared by enzymatic treatment. You may.
- the chain length of the first nucleic acid chain is not particularly limited, but is 12 to 30 bases, 12 to 25 bases, or 13 to 20 bases. In some cases, the strand length is usually selected according to other factors such as the strength of the antisense effect of the nucleic acid strand on the target, cost, synthesis yield and the like.
- the chain length of the second nucleic acid chain may be the same as that of the first nucleic acid chain. In that case, it is 12 to 30 bases, 12 to 25 bases, or 13 to 20 bases. Further, it may be several bases to a dozen bases longer or shorter than the chain length of the first nucleic acid chain.
- At least 4 contiguous nucleotides recognized by RNase H are usually regions containing 4 to 20 contiguous nucleotides, 5 to 16 contiguous nucleotides, or 6 to 12 A region containing contiguous nucleotides of bases.
- a nucleotide recognized by RNase H which cleaves an RNA strand when hybridized to an RNA nucleotide, such as natural DNA, can be used in this region.
- Suitable nucleotides such as modified DNA nucleotides and other bases, are known in the art. It is also known that nucleotides having a hydroxy group at the 2'position, such as RNA nucleotides, are unsuitable. Those skilled in the art can readily determine the suitability of nucleotides for use in this region containing "at least four contiguous nucleotides”.
- the first nucleic acid strand comprises "nucleotides and optionally nucleotide analogs". This wording means that the first nucleic acid strand has a DNA nucleotide, an RNA nucleotide, and may optionally further have a nucleotide analog in the nucleic acid strand.
- DNA nucleotide means a naturally occurring DNA nucleotide or a DNA nucleotide in which a subunit of a base, sugar or phosphate bond thereof is modified.
- RNA nucleotide means a naturally occurring RNA nucleotide or an RNA nucleotide in which a subunit of its base, sugar or phosphate binding is modified. Modification of a subunit of a base, sugar or phosphate bond is the addition of one substituent or the substitution of one within the subunit, not the substitution of the entire subunit with a different chemical group. ..
- DNA may be a modified nucleotide from the viewpoint of high resistance to DNA degrading enzymes and the like in a part or all of the region containing a nucleotide.
- modifications include, for example, 5-methylation, 5-fluorolation, 5-bromolation, 5-iodolation, N4-methylation, 5-demethylation of thymidine, 5-fluorolation, 5-methylation of thytocin.
- phosphorothioatetization methylphosphonateization, methylthiophosphonateization, chiral-methylphosphonateization, phospho Logithioate, phosphoromidate, 2'-O-methylation, 2'-methoxyethyl (MOE), 2'-aminopropyl (AP), 2'-fluoro, but in the body From the viewpoint of excellent kinetics, phosphorothioationation is preferable. Further, such modification may be applied to the same DNA in combination of a plurality of types. Further, as described later, RNA nucleotides may be modified in order to obtain the same effect.
- the modified DNA may affect the antisense effect of the double-stranded nucleic acid disclosed here. Since these modes differ depending on the sequence of the target gene and the like, it cannot be said unconditionally, but those skilled in the art can determine them while taking into consideration the description of the literature on the antisense method described later.
- the antisense effect of the modified double-stranded nucleic acid complex is measured, and the obtained measured value is not significantly lower than that of the unmodified double-stranded nucleic acid complex (for example,). If the measured value of the double-stranded nucleic acid complex after modification 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, in cells or the like in which a test nucleic acid compound is introduced into a cell or the like and suppressed by the antisense effect exerted by the test nucleic acid compound, as shown in Examples described later.
- the expression level of the target gene (mRNA amount, cDNA amount, protein amount, etc.) can be determined by appropriately using known methods such as northern blotting, quantitative PCR, and Western blotting.
- nucleotide analog means a nucleotide that does not exist in nature, and two or more substituents are added to the nucleotide, sugar, or phosphate-binding subunit of the nucleotide, or two in the subunit. It means that it has been substituted as described above, or that the entire subunit has been substituted with a different chemical group.
- Examples of analogs with two or more substitutions include crosslinked nucleic acids.
- a crosslinked nucleic acid is a nucleotide analog to which a crosslinked unit is added based on two substitutions in the sugar ring, typically a nucleotide analog in which a carbon at the 2'position and a carbon at the 4'position are bonded. Can be mentioned.
- the first nucleic acid strand further comprises a nucleotide analog in view of increasing the affinity of the target gene for the transcript subsequence and / or resistance to the nucleolytic enzyme.
- the "nucleotide analog” may be any nucleotide whose affinity for the partial sequence of the transcript of the target gene and / or resistance to the nucleolytic enzyme is increased by modification (crosslinking, substitution, etc.). 10-304889, International Publication 2005/021570, Japanese Patent Laid-Open No. 10-195098, Japanese Patent Laid-Open No. 2002-521310, International Publication No. 2007/143315, International Publication No. 2008/043753, International Publication No.
- nucleic acids disclosed in / 029619 and International Publication No. 2008/049085 are disclosed as being suitably used for the antisense method. That is, the nucleic acids disclosed in the above literature: hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), peptide nucleic acid (PNA), glycol nucleic acid (GNA), treose nucleic acid (TNA), morpholinonucleic acid, tricyclo-DNA (tcDNA).
- HNA hexitol nucleic acid
- CeNA cyclohexene nucleic acid
- PNA peptide nucleic acid
- GAA glycol nucleic acid
- TAA treose nucleic acid
- morpholinonucleic acid tricyclo-DNA (tcDNA).
- the BNA in one embodiment may be a ribonucleotide or deoxyribonucleotide in which the carbon at the 2'position and the carbon at the 4'position are crosslinked by two or more atoms.
- crosslinked nucleic acids are known to those of skill in the art.
- the carbon at the 2'position and the carbon at the 4'position are 4'-(CH 2 ) p -O-2' and 4'-(CH 2 ) p -S.
- R 3 is a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, respectively.
- R 1 and R 2 of substituents at the 3'position carbon: OR 2 and substituents at the 5'position: OR 1 are typically hydrogen atoms, although It may be the same or different, and it may be the same or different.
- a phosphate group protected by, or -P (R 4 ) R 5 (in the formula, R 4 and R 5 may be the same or different, a hydroxyl group, a hydroxyl group protected by a protective group for nucleic acid synthesis, A mercapto group, a mercapto group protected by a protective 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 a cyanoalkoxy group having 1 to 6 carbon atoms. It may be an amino group substituted with 1 to 5 alkyl groups).
- Such BNA includes, for example, ⁇ -L-methyleneoxy (4'-CH 2 -O-), which is also called LNA (Locked Nucleic Acid (registered trademark), 2', 4'-BNA). 2') BNA or ⁇ -D-methyleneoxy (4'-CH 2 -O-2') BNA, ethyleneoxy (4'-(CH 2 ) 2 -O-2') BNA, ⁇ -D-Chio (4'-CH 2 -S-2') BNA, Aminooxy (4'-CH 2 -ON (R 3 ) -2') BNA, 2', 4'-BNA Also called NC Oxyamino (4'-CH 2 -N (R 3 ) -O-2') BNA, 2', 4'-BNA COC , 3'amino-2', 4'-BNA, 5'-methyl BNA, cEt Also called -BNA (4'-CH (CH 3 ) -O-2') BNA, also called
- the base site may be modified.
- Modifications of the base site include, for example, 5-methylation, 5-fluorolation, 5-bromolation, 5-iodolation, N4-methylation of cytosine, 5-demethylation of thymidine, 5-fluorolation, 5-. Includes bromination, 5-iodolation, N6-methylation of adenine, 8-bromolation, N2-methylation of guanine, and 8-bromolation.
- the phosphodiester bond site may be modified.
- Modifications of the phosphate diester bond site include, for example, phosphorothioation, methylphosphonate, methylthiophosphonate, chiral-methylphosphonate, phosphorodithioate, and phosphoramidate, which are excellent in pharmacokinetics. From the perspective of being present, phosphorothioation is used. Further, such modification of the base site and the modification of the phosphoric acid diester bond site may be applied to the same nucleic acid in combination of a plurality of types.
- modified nucleotides and modified nucleotide analogs are not limited to those exemplified here.
- a large number of modified nucleotides and modified nucleotide analogs are known in the art, for example, the description of U.S. Pat. No. 8299039 of Tachas et al., Especially the description in columns 17-22, as embodiments of the present application. It can also be used.
- nucleotide analog is an LNA represented by the following formula (1).
- Base is an aromatic heterocyclic group or an aromatic hydrocarbon ring group which may have a substituent, for example, a base moiety (purine base, pyrimidine base) of a natural nucleoside or a non-natural type.
- a base moiety purine base, pyrimidine base
- Modification Shows the base site of the nucleoside. An example of modification of the base site is as described above.
- R 1 and R 2 may be the same or different, and may be the same or different, and may be the same or different, and may be the same or different, and may be the same or different. , Phosphate group, phosphate group protected by a protective group for nucleic acid synthesis, or -P (R 4 ) R 5 [Here, R 4 and R 5 may be the same or different, hydroxyl group, nucleic acid.
- Hydroxyl group protected by a synthetic protective group mercapto group, mercapto group protected by a nucleic acid synthesis protective group, amino group, alkoxy group having 1 to 5 carbon atoms, alkylthio group having 1 to 5 carbon atoms, 1 to 5 carbon atoms
- the cyanoalkoxy group of 6 or the amino group substituted with an alkyl group having 1 to 5 carbon atoms is shown. ] Is shown.
- the compound represented by the chemical formula is a nucleoside
- the "LNA" and the usual BNA in a certain embodiment also include a form (nucleotide) in which a phosphate group is bonded to the nucleoside. That is, BNA such as LNA is incorporated as a nucleotide into a nucleic acid chain containing a double-stranded nucleic acid complex.
- the "wing region containing nucleotide analogs consisting of a plurality of nucleic acids” is 5'of the region consisting of a plurality of nucleic acids (hereinafter, also referred to as "DNA gap region”) containing at least four or more consecutive DNA nucleotides. It is located on the distal side and / or on the 3'end side.
- a region containing a nucleotide analog located at the 5'end of the DNA gap region (hereinafter, also referred to as "5'wing region”) and a region containing a nucleotide analog located at the 3'end of the DNA gap region (hereinafter, ""
- the 3'wing regions are independent of each other, and may contain at least one nucleotide analog listed in the literature on the antisense method, and other than the nucleotide analog, a natural type. Nucleic acid (DNA or RNA) may also be included.
- the chain lengths of the 5'wing region and the 3'wing region are independently usually 1 to 10 bases, 1 to 7 bases, 1 to 5 bases, or 2 to 5 bases.
- the type, number and position of nucleotide analog and natural nucleotides may affect the antisense effect exerted by the double-stranded nucleic acid complex in a certain embodiment. Therefore, the preferred embodiment may change depending on the arrangement or the like. Although it cannot be said unconditionally, a person skilled in the art can determine a preferable mode while taking into consideration the description of the literature relating to the antisense method. Further, the antisense effect of the modified double-stranded nucleic acid was measured in the same manner as in the region containing "at least four or more consecutive DNA nucleotides", and the obtained measured value was the double-stranded nucleic acid before modification. If it is not significantly lower than that of, the modification can be evaluated as a preferred embodiment.
- the antisense strand of the IHH gene consisting of modified oligonucleotides against mRNA consists of a 5'wing site consisting of 1 to 10 bases, a gap region consisting of 8 to 25 bases, and 1 to 10 bases. It can consist of a 3'wing site consisting of the bases of.
- the antisense strand can have a motif represented by 2-10-2, a motif represented by 3-10-3, and the like.
- the first number of the motif represents the number of bases in the 5'wing region
- the second number represents the number of bases in the gap region
- the third number represents the number of bases in the 3'wing region. Represents the number of bases in.
- the antisense method consisting only of RNA and LNA which has been tried in the past, suppressed translation by binding to the target mRNA, but the effect was generally insufficient.
- the antisense method consisting only of DNA when it binds to the target gene, it has a double-stranded structure consisting of DNA and RNA. Although it was possible, the actual effect was still insufficient due to the weak binding to the target gene.
- a DNA having a strand length of at least 4 bases or more is arranged in the center, and an LNA (or other BNA) having a strong binding ability to RNA (that is, a target transcript) is arranged at both ends.
- LNA or other BNA
- RNA that is, a target transcript
- DNA having a strand length of 4 bases is not limited to DNA nucleotides, but includes at least four consecutive nucleotides recognized by RNase H when the first nucleic acid strand hybridizes to a transcript.
- the first nucleic acid chain is also intended to be included.
- At least four RNase Hs are recognized when the first nucleic acid strand hybridizes to the transcript, in view of the extremely high antisense effect produced by heteroduplex formation with the target transcript. It is desirable that the wing region containing the modified nucleic acid arranged on the 5'end side and the 3'end side of the region containing contiguous nucleotides optionally contains a nucleotide analog.
- the nucleotide analog may be BNA, for example LNA.
- the second nucleic acid strand in a certain embodiment is a nucleic acid complementary to the first nucleic acid strand described above.
- the base sequence of the second nucleic acid chain and the base sequence of the first nucleic acid chain do not have to be completely complementary, and are at least 70% or more, preferably 80% or more, and more preferably 90% or more (for example, 90% or more). It suffices to have complementarity of 95%, 96%, 97%, 98%, 99% or more).
- the second nucleic acid strand is an oligobucreotide composed of at least one nucleic acid selected from the group consisting of RNA, DNA, PNA (peptide nucleic acid) and BNA (for example, LNA). More specifically, the second nucleic acid strand comprises (i) RNA nucleotides, optionally nucleotide analogs, optionally DNA nucleotides, (ii) DNA nucleotides and / or nucleotide analogs, or ( iii) Contains PNA nucleotides.
- RNA nucleotides states that the second nucleic acid strand comprises RNA nucleotides and may further optionally contain nucleotide analogs, and further optionally DNA nucleotides. Means that it may contain.
- DNA nucleotides and / or nucleotide analogs means that the second nucleic acid strand may contain either DNA nucleotides or nucleotide analogs, or may contain both DNA nucleotides and nucleotide analogs.
- Constaining PNA nucleotides means that the second nucleic acid strand may be composed of PNA nucleotides.
- the second nucleic acid strand contains RNA.
- a functional molecule such as a peptide can be easily bound to the double-stranded nucleic acid complex in a certain embodiment, the second nucleic acid strand may be PNA.
- RNA nucleotide means a naturally occurring RNA nucleotide or an RNA nucleotide in which a subunit of its base, sugar or phosphate bond is modified. Modification of a subunit of a base, sugar or phosphate bond is the addition of one substituent or the substitution of one within the subunit, not the substitution of the entire subunit with a different chemical group. ..
- a part or all of the nucleic acid may be a modified nucleotide from the viewpoint of high resistance to a nucleic acid degrading enzyme such as an RNA degrading enzyme.
- modifications include, for example, 5-methylation, 5-fluorolation, 5-bromolation, 5-iodolation, N4-methylation of cytosine, 5-demethylation of thymidine, 5-fluorolation, 5-.
- RNA nucleotides in which the uracil base is replaced with a thymidine base, but phosphorothioatetization is used from the viewpoint of excellent pharmacokinetics.
- modification may be applied to the same nucleic acid in combination of a plurality of types, and for example, as used in Examples described later, the same modification is provided in order to impart resistance to cleavage by an enzyme.
- RNA may be phosphorothioated and 2'-O-methylated. However, if you expect or wish to cleave RNA nucleotides by RNase H, you can only perform either phosphorothioatetation or 2'-O-methylation.
- the number and position of modifications may affect the antisense effect exerted by the double-stranded nucleic acid complex in a certain embodiment
- the number and position of nucleotide analogs in the second nucleic acid strand may be a preferred embodiment. Exists. Since this preferred embodiment differs depending on the type, sequence, etc. of the nucleic acid to be modified, it cannot be said unconditionally, but like the first nucleic acid strand described above, the antisense effect of the modified double-stranded nucleic acid can be obtained. It can be identified by measuring.
- RNaseH suppresses degradation by RNA-degrading enzymes such as RNaseA until the second nucleic acid strand is delivered into the nucleus of a specific cell, and the nucleic acid strand is produced by RNaseH in the specific cell.
- the second nucleic acid strand is RNA, and the region containing the nucleotide analog of the first nucleic acid strand (that is, the 5'wing region and / or the region) from the viewpoint that the antisense effect is easily exerted by being decomposed.
- the region complementary to the 3'wing region) is a modified nucleic acid or nucleotide analog, and the modified or analog has an effect of suppressing degradation by an enzyme such as RNA degrading enzyme.
- the modification is 2'-O-methylation and / or phosphorothioatelation of RNA.
- all of the regions complementary to the region containing the nucleotide analog of the first nucleic acid chain may be modified, and the region complementary to the region containing the modified nucleic acid of the first nucleic acid chain may be modified. Part of it may be modified. Further, the modified region may be longer or shorter than the region containing the modified nucleic acid of the first nucleic acid chain as long as the modified region is contained.
- a functional portion may be bound to the second nucleic acid strand.
- the bond between the second nucleic acid chain and the functional moiety may be a direct bond or an indirect bond via another substance, but in certain embodiments, a covalent bond, an ion. It is preferable that the second nucleic acid chain and the functional portion are directly bonded by a bond, a hydrogen bond, or the like, and a covalent bond is more preferable from the viewpoint of obtaining a more stable bond.
- the structure of the "functional portion” is not particularly limited, and a desired function is imparted to the double-stranded nucleic acid complex and / or the nucleic acid strand that binds the "functional portion".
- Desired functions include labeling function, purification function and delivery function to the target.
- the portion that imparts the labeling function include compounds such as fluorescent protein and luciferase.
- the portion that imparts the purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide.
- the second nucleic acid strand is used as a functional part. It is preferable that a molecule having an activity of delivering the double-stranded nucleic acid complex in a certain embodiment to the target site is bound.
- Examples of the portion having the "delivery function to the target" include lipids from the viewpoint of being able to deliver the double-stranded nucleic acid complex in a certain embodiment with high specificity and efficiency to the liver and the like.
- examples of such lipids include lipids such as cholesterol and fatty acids (for example, vitamin E (tocopherols and tocotrienols), vitamin A and vitamin D), fat-soluble vitamins such as vitamin K (for example, acylcarnitine), and acyl CoA.
- examples of the "functional portion" in a certain embodiment include sugars (for example, glucose and sucrose).
- examples of the "functional portion” in a certain embodiment include sugars (for example, glucose and sucrose).
- a ligand or antibody of the receptor for example, a ligand or antibody of the receptor, And / or peptides or proteins such as fragments thereof are mentioned as "functional moieties" in certain embodiments.
- the first nucleic acid chain, the second nucleic acid chain and the third nucleic acid chain can be prepared by appropriately selecting a method known to those skilled in the art.
- a commercially available nucleic acid automatic synthesizer manufactured by Applied Biosystems
- Nucleic acid can be prepared by synthesizing using (manufactured by Beckman, etc.) and then purifying the obtained oligonucleotide using a reverse phase column or the like. Then, the nucleic acids prepared in this manner are mixed in an appropriate buffer solution, denatured at about 90 to 98 ° C. for several minutes (for example, 5 minutes), and then about 30 to 70 ° C. Annealing over 1-8 hours can prepare the double-stranded nucleic acid complex in some embodiments. In addition, the double-stranded nucleic acid complex to which the functional moiety is bound can be prepared by synthesizing, purifying, and annealing as described above using a nucleic acid species to which the functional moiety is bound in advance. .. Many methods for binding functional moieties to nucleic acids are well known in the art.
- the "second nucleic acid strand" targets the antisense nucleic acid at the target site without reducing the antisense effect. It is excellent in that it can be delivered efficiently. Therefore, the double-stranded nucleic acid in some embodiments is not limited to the above embodiment, and for example, an embodiment containing the following antisense nucleic acid can be provided instead of the above-mentioned first nucleic acid strand. ..
- a double-stranded nucleic acid complex having an activity of suppressing the expression of a target gene by an antisense effect (i) an antisense nucleic acid complementary to a transcript of the target gene, and a nucleic acid containing no DNA.
- the antisense nucleic acid has an RNase H independent antisense effect.
- the "RNase H-independent antisense effect” is a splicing function such as translation inhibition and exon skipping by hybridizing a transcript (RNA sense strand) of a target gene with a nucleic acid strand complementary to its partial sequence. It means the activity of suppressing the expression of the target gene caused by the conversion effect.
- DNA-free nucleic acid means an antisense nucleic acid that does not contain natural DNA and modified DNA, and examples thereof include nucleic acids consisting of PNA or morpholino nucleic acid. Further, in the "DNA-free nucleic acid", as with the first nucleic acid chain or the second nucleic acid chain, a part or all of the nucleic acid is a modified nucleotide from the viewpoint of high resistance to nucleic acid degrading enzyme. It may be configured. Examples of such modifications are as described above, and further, a plurality of types of modifications may be applied to the same nucleic acid in combination. In addition, a preferred embodiment regarding the number of modified nucleic acids and the position of modification can be specified by measuring the antisense effect of the modified double-stranded nucleic acid, as in the case of the first nucleic acid strand described above.
- the base sequence of the "DNA-free nucleic acid” and the base sequence of the nucleic acid complementary to the nucleic acid or the base sequence of the transcript of the target gene need not be completely complementary, and are preferably at least 70% or more. May have complementarity of 80% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more).
- the chain length of the "DNA-free nucleic acid” is not particularly limited, but is usually 10 to 35 bases, preferably 12 to 25 bases, and more preferably 13 to 20 bases.
- composition containing the double-stranded nucleic acid complex in some embodiments can be formulated by a known pharmaceutical method.
- a known pharmaceutical method for example, capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, lozenges, sublinguals, chewing agents, buccal agents, pastes, syrups, suspending agents, As elixirs, emulsions, coatings, ointments, ointments, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc., enteral (oral, etc.) or non- It can be used transenterally.
- lipoproteins such as chylomicrons and chylomicrons remnants are used in the double-stranded nucleic acid complex in some embodiments in which lipids are bound as functional portions during formulation. You may form a complex with.
- a substance having an action of enhancing the permeability of the colonic mucosal epithelium for example, medium-chain fatty acid, long-chain unsaturated fatty acid or a derivative thereof (salt, ester)
- it may be a complex (mixed micelle, emulsion) of a fatty acid ()) and a surfactant (nonionic surfactant, anionic surfactant).
- the preferred administration form of the composition in some embodiments is not particularly limited, and is intrarectally (orally or the like) or non-enteric, more specifically, intravenous, intraarterial, or intraperitoneal administration. , Subcutaneous administration, intradermal administration, respiratory tract administration, rectal administration and intramuscular administration, and infusion administration.
- compositions in some embodiments can be used for animals including humans, but are not particularly limited as non-human animals, and are intended for various livestock, poultry, pets, laboratory animals and the like. can do.
- the dose or ingestion thereof depends on the age, weight, symptom, health condition, type of composition (medicine, food and drink, etc.) of the subject, and the like.
- the effective intake of the composition according to an embodiment is preferably 0.001 mg / kg / day to 50 mg / kg / day in terms of nucleotides.
- the present invention includes a therapeutic agent for fibrosis, which comprises an IHH gene-specific inhibitor or an inhibitor of a transcript of the IHH gene.
- the expression of the COL1A1 gene, CTGF gene, and ADGRE1 is decreased, and the expression of the TGFB1 gene and CCL2 gene is increased.
- Fibrosis in the liver includes fatty liver (fatty liver), alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (ASH), non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NAFLD).
- Non-alcoholic steatohepatitis NASH
- chronic hepatitis Chronic hepatitis
- liver cirrhosis Liver cirrhosis
- viral, autoimmune, biliary stagnation metabolic, congestive, drug-induced, infectious, etc.
- the renal fibrosis means kidney fibrosis (Kidney fibrosis), renal systemic fibrosis (NSF), kidney fibroma (Kidney fibroma) and the like.
- the fibrosis of the pancreas means cystic fibrosis (CF, cystic fibrosis) and the like.
- Pulmonary fibrosis includes pulmonary fibrosis, interstitial pulmonary fibrosis, acute diffuse interstitial pulmonary fibrosis, and idiopathic pulmonary fibrosis. It means pulmonary fibrosis: IPF), etc.
- Skin fibrosis includes skin fibrosis disease, scleroderma, systemic sclerroderma, localized scleroderma, and collagen disease. , Dermatofibroma, etc.
- Example 1 Primary screening Obtained coding sequence information for each gene of human IHH and mouse IHH from the NCBI website (https://www.ncbi.nlm.nih.gov/), and obtained human IHH and mouse IHH genes as shown in FIG. IHH gene coding sequence: NCBI Reference Sequence: NM_002181.3 (SEQ ID NO: 1), mouse IHH gene coding sequence: NCBI Reference Sequence: NM_010544.3 (SEQ ID NO: 2) based on the sequence (sense strand) of 55 of them. An antisense oligonucleotide (ASO) was designed. The sequence information is shown in Table 1.
- mice IHH gene coding sequence In Table 1, the sequence in the human IHH gene coding sequence or the mouse IHH gene coding sequence is shown as a sense oligonucleotide, and the sequence complementary to the sequence is shown as an antisense oligonucleotide.
- Mouse Hepa1-6 cell lines were seeded on a commercially available 24-well plate at 1 ⁇ 10 5 cells / ml / well and cultured in a CO 2 incubator for 24 hours. The next day, ASO (20 nM each) was transfected with Lipofectamine 2000 (Thermo Fisher Scientific) and cultured in a CO 2 incubator for 48 hours. Total RNA was extracted from each well of the 24-well plate after culturing using the RNeasy Mini Kit (QIAGEN).
- the reverse transcription reaction and quantitative PCR used the Rotor Gene Probe RT-PCR Kit (QIAGEN), and the quantitative PCR device used the Rotor-Gene Q (QIAGEN).
- the primers and probes for mouse IHH and mouse GAPDH used reagents designed by TaqMan Gene Expression Assays (Thermo Fisher Scientific), and the mRNA expression level was determined by measuring the Ct values of mouse IHH and mouse GAPDH, and then ⁇ Ct. Calculated by the relative quantitative method based on the method.
- FIG. 3-1 shows the screening results of Ren-1-1 to -31
- FIG. 3-2 shows the screening results of Ren-1-32 to -55.
- PBS-1 to 8 are antisense nucleic acids used for negative control
- APOB-1 to 2 are antisense nucleic acids used for positive control, and sequence information was obtained from the literature (Nat Communi. 2015 Aug 10; 6: 7969.).
- FIGS. 3-1 and 3-2 a knockdown effect of 78% was confirmed in No. 12 (Ren-1-12) (FIGS. 3-1 and 3-2).
- All the sequences listed in the Antisense column in the table contain phosphorothioate (PS) bonds between nucleotides.
- the bases in bold are containing LNA modifications.
- 3-8-3 in the Motif column consists of 3 LNA-modified nucleic acids-8 unmodified nucleic acids-3 LNA-modified nucleic acids (including PS modification between all nucleotides), for a total of 14 mer nucleic acid bases. Shows antisense nucleic acid.
- Human / mouse in the Species specificity column indicates that it is an antisense nucleic acid that perfectly matches between human and mouse. Further, it is shown that mouse is an antisense nucleic acid that completely matches the mouse sequence but does not completely match the human sequence.
- GC content was calculated based on the website (http://www.ngrl.co.jp/tools/0217oligocalc.htm).
- the Tm value of the LNA-modified antisense nucleic acid was calculated based on the website (https://www.exiqon.com/ls/pages/exiqontmpredictiontool.aspx). Sequence information within the human and mouse coding regions was obtained from the NCBI website (https://www.ncbi.nlm.nih.gov/).
- Example 2 Calculation of IC50 value of IHH gene knockdown action of Ren-1-12 ASO
- the IC50 value of IHH gene knockdown activity was obtained for Ren-1-12 which was hit in the screening of Example 1.
- the experiment was basically performed in the same manner as in Example 1.
- the IC50 value of knockdown action in Ren-1-12 was calculated to be 1.07 nM
- the IC50 value of APOB ASO used as a control was calculated to be 2.52 nM (Fig. 4A, B).
- the APOB sequence used this time is a sequence whose effectiveness has been confirmed in vivo in the literature information (Nat Commun. 2015 Aug 10; 6: 7969.).
- Example 3 IHH gene knockdown effect of Toc-Ren-1-12 HDO in the liver To what extent the Ren-1-12 ASO hit in the screening of Example 1 exhibits a knockdown effect in the mouse liver was investigated. ..
- HDO in which tocopherol (Toc) was added as a ligand to the sense strand was applied. Used (Table 2).
- Toc-APOB HDO (Table 2) was used as a positive control.
- the R Easy Mini Kit (QIAGEN) was used to extract Total RNA from mouse liver.
- the reverse transcription reaction and quantitative PCR used the Rotor Gene Probe RT-PCR Kit (QIAGEN), and the quantitative PCR device used the Rotor-Gene Q (QIAGEN).
- the primers and probes for mouse IHH and mouse 18S rRNA used reagents designed by TaqMan Gene Expression Assays (Thermo Fisher Scientific). The mRNA expression level was calculated by a relative quantification method based on the ⁇ Ct method after measuring each Ct value of mouse IHH and mouse 18S rRNA.
- Toc-Ren-1-12 HDO When the IHH gene knockdown effect was examined 1, 3, and 7 days after a single iv administration of Toc-Ren-1-12 HDO and Toc-APOB HDO by the above method, Toc-Ren-1-12 was examined 7 days later. HDO showed almost the same knockdown effect as Toc-APOB HDO (Fig. 5). Since it has already been shown that Toc-APOB HDO can be used for efficacy evaluation in vivo (Nat Commun. 2015 Aug 10; 6: 7969.), Toc-Ren-1-12 HDO also has efficacy in vivo. It was shown to be a nucleic acid complex that could be fully used for evaluation.
- Example 4 Secondary screening 36 new ASOs were designed based on Ren-1-12, which was a hit in the screening of Example 1.
- the sequence of the sense chain of Ren-1-12 is the 598th to 611th bases (14 bases long) of the base sequence of SEQ ID NO: 1, whereas the start of the sequence is The site was shifted to the 603rd and 596th bases of the base sequence of SEQ ID NO: 1 and the base length was set to 13 to 20.
- the results are shown in Table 3.
- FIG. 6 shows the results of knockdown screening of the IHH gene for these 36 sequences (Ren-1-12-1 to -36) by the same method as in Example 1. From the results of the secondary screening, the three sequences Ren-1-12-22, 27, and 31 showed a particularly strong knockdown effect.
- All the sequences listed in the Antisense column in the table contain phosphorothioate (PS) bonds between nucleotides.
- the bases in bold are containing LNA modifications.
- 3-8-3 in the Motif column consists of 3 LNA-modified nucleic acids-8 unmodified nucleic acids-3 LNA-modified nucleic acids (including PS modification between all nucleotides), for a total of 14 mer nucleic acid bases. Shows antisense nucleic acid.
- Human / mouse in the Species specificity column indicates that it is an antisense nucleic acid that perfectly matches between human and mouse. Further, it is shown that mouse is an antisense nucleic acid that completely matches the mouse sequence but does not completely match the human sequence.
- GC content was calculated based on the website (http://www.ngrl.co.jp/tools/0217oligocalc.htm).
- the Tm value of the LNA-modified antisense nucleic acid was calculated based on the website (https://www.exiqon.com/ls/pages/exiqontmpredictiontool.aspx). Sequence information within the human and mouse coding regions was obtained from the NCBI website (https://www.ncbi.nlm.nih.gov/).
- Example 5 Comparison of IHH gene knockdown effect in liver when each HDO of Toc-Ren-1-12-22, -27, -31 was administered once iv to normal mice Selected in Example 4.
- Toc-Ren-1-12-22, -27 in normal mice, in order to determine the sequence to be advanced to the drug efficacy evaluation in the NASH pathology model from the three sequences of Ren-1-12-22, -27, -31.
- the knockdown effects of each of the -31 HDOs (Table 4) 3 days after a single iv administration at a dose of 10 nmol / kg were compared.
- Ren-1-12-27 showed the strongest knockdown rate (48%) (Fig. 7). Based on the above results, we decided to use Ren-1-12-27 for drug efficacy evaluation in the NASH pathological model.
- Example 6 Dose-dependent IHH gene knockdown effect in the liver when Toc-Ren-1-12-27 HDO was administered to normal mice in a single dose iv Toc showed the strongest knockdown effect in Example 5.
- the IHH gene knockdown effect was investigated 3 days after a single iv administration of -Ren-1-12-27 HDO at 1, 3, 10, 30 nmol / kg.
- Ren-1-12-27 showed a dose-dependent knockdown effect (55% inhibition at 3 nmol / kg, 57% inhibition at 10 nmol / kg, inhibition, 30 nmol). 72% suppression at / kg).
- Example 7 Comparison of time-dependent changes in IHH gene knockdown action in the liver when Toc-Ren-1-12-27 HDO was administered to normal mice in a single dose. Comparison between Day 3 and Day 7 Toc-Ren in Example 5 The dose dependence of the knockdown effect of -1-12-27 HDO in the liver was evaluated 3 days after administration, and the knockdown effect of the IHH gene was further examined 7 days later. As a result, as shown in FIG. 9, it was shown that Ren-1-12-27 maintained a knockdown rate of about 60 to 70% from 3 days to 7 days after administration at a dose of 30 nmol / kg. .. Based on the above, when evaluating with the NASH pathological model, the dose of Ren-1-12-27 was set to 30 nmol / kg, and it was decided to administer once a week.
- Example 8 Calculation of IC50 value of IHH gene knockdown effect by Ren-1 ASO in mouse Hepa 1-6 cells IC50 value was calculated for the top 19 ASOs showing strong knockdown effect found in Example 4. .. The results are shown in Table 5. In the secondary screening of Example 4, Ren-1-12-22, 27, 31 showed a particularly strong knockdown effect, but when the IC50 value was actually calculated, Ren-1-12-34 had the strongest IC50. The value is shown.
- Example 9 Effect of Toc-Ren-1-12-27 HDO on IHH gene expression in NASH pathological model mice prepared with a methionine / choline deficient diet (MCD diet) 6-week-old female C57BL / 6J mice were produced by Charles Japan. I bought it from the river. The methionine / choline deficient diet (MCD diet) and control diet (normal diet) were purchased from Research Diet. First, C57BL / 6J mice were divided into the Vehicle (saline) -administered group (V group) and the Toc-Ren-1-12-27 HDO (30 nmol (0.3 mg) / kg) -administered group [I (IHH) group].
- V group Vehicle (saline) -administered group
- Toc-Ren-1-12-27 HDO 30 nmol (0.3 mg) / kg) -administered group [I (IHH) group.
- mice used in this experiment were divided into the following four groups in total [(1) Vehicle-administered / Normal feed group (VN group), (2) Vehicle-administered / MCD feed group (VM group), (3) Toc- Ren-1-12-27 HDO (30 nmol (0.3 mg) / kg) administration / Normal feed group (IN group), (4) Toc-Ren-1-12-27 HDO (30 nmol (0.3 mg) / kg) ) Administration / MCD feed group (IM group)].
- Feeding was started 1 week before the first administration (Day 0), and Vehicle and Toc-Ren-1-12-27 HDO were administered once a week from Day 0 for 5 weeks. Sampling was performed once a week, the body weight of the mice was measured, heparin blood was collected from the heart, liver tissue was collected, and then the liver weight was measured. Blood liver deviation enzyme (ALT) activity, blood triglyceride concentration, and blood cholesterol concentration were measured from serum samples.
- ALT Blood liver deviation enzyme
- the reverse transcription reaction was performed using PrimeScript (TM) RT Master Mix (TaKaRa Bio).
- the quantitative PCR reaction was performed using Luna Universal qPCR Master Mix (NEB), and the quantitative PCR device used was StepOnePlus-01 (Thermo Fisher Scientific).
- the TaqMan Gene Expression Assay reagent designed for each gene by Thermo Fisher Scientific was used.
- the mRNA expression level was calculated by a relative quantification method based on the ⁇ Ct method after measuring each Ct value of each mouse gene and mouse 18S rRNA.
- Statistical processing was performed using a three-factor analysis of variance (3-way ANOVA), and a significance level of less than 5% was considered significant.
- Example 10 Effect of Toc-Ren-1-12-27 HDO on inflammatory marker gene (TNFA and CCL2) expression in NASH pathological model mice prepared with MCD diet Vehicle or in NASH pathological model mice prepared with MCD diet
- TNFA and CCL2 inflammatory marker gene
- Example 11 Effect of Toc-Ren-1-12-27 HDO on macrophage marker (ADGRE1) gene expression in NASH pathological model mice prepared with MCD diet Vehicle or Toc- As a result of investigating the effect on ADGRE1 gene expression in mice treated with Ren-1-12-27 HDO once a week for 5 weeks, as shown in FIG. 12, the expression of ADGRE1 tends to decrease as of Day 14. Indicated.
- Example 12 Effect of Toc-Ren-1-12-27 HDO fibrosis marker (COL1A1, CTGF, TGFB1, TIMP, ACTA2) gene expression in NASH pathological model mice prepared with MCD feed. Investigate the effect on fibrosis marker (COL1A1, CTGF, TGFB1, TIMP, ACTA2) gene expression in mice treated with Vehicle or Toc-Ren-1-12-27 HDO once a week for 5 weeks in NASH pathological model mice. It was. As a result, as shown in FIG. 13-1A, the expression of the COL1A1 gene was significantly increased at the time of Day 7, and the expression of the COL1A1 gene was significantly decreased at the time of Day 35 (P ⁇ 0.05).
- CTGF CTGF
- TGFB1 TIMP and ACTA2 did not tend to decrease over the entire period.
- Example 13 Effect of Toc-Ren-1-12-27 HDO on blood liver deviation enzyme (ALT) activity in NASH pathological model mice prepared with MCD feed Vehicle or in NASH pathological model mice prepared with MCD feed
- ALT activity was significantly increased in the VM group (P ⁇ 0.0001).
- Example 14 Effect of Toc-Ren-1-12-27 HDO on body weight and liver weight in NASH pathological model mice prepared with MCD diet Vehicle or Toc-Ren-1 in NASH pathological model mice prepared with MCD diet As a result of investigating the effect of -12-27 HDO on body weight and liver weight in mice administered once a week for 5 weeks, as shown in FIGS. 15A and 15B, body weight and liver in the VM group as compared with the VN group. Weight was significantly reduced (P ⁇ 0.0001). The effects of vehicle or Toc-Ren-1-12-27 HDO on body weight and liver weight of mice administered with vehicle or Toc-Ren-1-12-27 HDO once a week for 6 weeks to NASH pathological model mice prepared with MCD diet are shown in FIG. As described above, the body weight and liver weight were significantly reduced in the VM group as compared with the VN group (both P ⁇ 0.0001). At this time, no significant change was confirmed between the VM group and the IM group.
- Example 15 Effect of Toc-Ren-1-12-27 HDO on blood triglyceride concentration and blood cholesterol concentration in NASH pathological model mice prepared with MCD diet Vehicle or in NASH pathological model mice prepared with MCD diet The effects of Toc-Ren-1-12-27 HDO on blood triglyceride concentration and blood cholesterol concentration in mice treated once a week for 5 weeks were investigated. As a result, as shown in FIGS. 16A and 16B, the blood triglyceride concentration and the blood cholesterol concentration were significantly reduced in the VM group as compared with the VN group (both P ⁇ 0.0001). At this time, no significant change in blood triglyceride concentration was confirmed between the VM group and the IM group, but the blood cholesterol concentration increased significantly (P ⁇ 0.05).
- IHH mRNA expression was expressed by administering Toc-Ren-1-12-27 HDO (30 nmol (0.3 mg) / kg) once a week for 5 weeks to NASH pathological model mice prepared with MCD diet. It was shown that the expression of COL1A1 mRNA, which is a typical fibrosis marker, was significantly reduced (P ⁇ 0.05). Therefore, it was verified that Toc-Ren-1-12-27 HDO is a compound that can be a therapeutic agent for NASH.
- Example 16 Third screening 37 ASOs (Ren-1-2, Ren-1-3, Ren-1-4, Ren-1-5, Ren-1) used in Examples 1 and 4 -6, Ren-1-7, Ren-1-9, Ren-1-11, Ren-1-12, Ren-1-12-13, Ren-1-12-27, Ren-1-14, Ren -1-15, Ren-1-16, Ren-1-17, Ren-1-18, Ren-1-19, Ren-1-23, Ren-1-24, Ren-1-25, Ren-1 -26, Ren-1-28, Ren-1-29, Ren-1-33, Ren-1-35, Ren-1-36, Ren-1-37, Ren-1-38, Ren-1-39 , Ren-1-40, Ren-1-41, Ren-1-43, Ren-1-44, Ren-1-48, Ren-1-47, Ren-1-49, Ren-1-50) and PBS as a negative control and antisense nucleic acid of APOB gene as a positive control were screened again at a nucleic acid concentration of 50 nM different from that of Examples 1 and 4.
- the screening method was the same as in Example 1 and Example 4 except that the nucleic acid concentration of
- Example 17 Knockdown activity of Ren-1-12-27, Ren-1-11, Ren-1-39, Ren-1-41 on IHH gene expression in normal mouse liver This experiment is newly performed in vitro schooling. Using four ASOs (Ren-1-12-27, Ren-1-11, Ren-1-39, Ren-1-41) that showed a strong knockdown effect on IHH gene expression, in The knockdown activity in vivo was examined. Twenty-five 6-week-old normal mice (c57BL / 6j) were divided into 5 groups (Vehicle, Ren-1-12-27, Ren-1-11, Ren-1-39, Ren-1-41) according to body weight. .. Knockdown activity was compared with positive control Ren-1-12-27 (17 mer) and negative control Vehicle administration group.
- the dose of ASO was set to 30 nmol / 10 ml / kg and administered from the tail vein of mice (the administration day was set to Day 0).
- the mice underwent intracardiac blood sampling under isoflurane anesthesia, the abdomen was opened, and the liver was removed.
- Total mRNA was extracted from liver tissue, reverse transcription and qPCR were performed, and the expression of IHH mRNA was measured. The results are shown in FIG.
- Ren-1-41-administered group Compared with the Vehicle-administered group, the IHH mRNA expression of any Ren-1 ASO-administered group was decreased. Ren-1-11 and Ren-1-39 showed the same level of knockdown activity as Ren-1-12-27 (20%). The IHH mRNA expression in the Ren-1-41-administered group was reduced by about half (53%) as compared with the Vehicle-administered group, and was significantly knocked down.
- Example 18 Effect of Toc-Ren-1-12-27 on mouse NASH / liver fibrosis by MCD diet 6-week-old mice were divided into a Normal Diet group and an MCD (Methionine and Choline Deficient) Diet group, and MCD Diet A model of NASH / liver fibrosis was created.
- MCD Methionine and Choline Deficient
- FIG. 19 shows a HE-stained diagram of liver tissue 5 weeks after administration of Ren1-12-27.
- the Vehicle-administered group as compared with the Normal Diet group (Fig. 19a), the collection of inflammatory cells showed more hepatic lipid droplets and balloon-like changes due to MCD Diet administration (Fig. 19c) (inside the black circle).
- FIG. 20 shows an Oil red O staining diagram of liver tissue 5 weeks after administration of Ren1-12-27.
- the Vehicle-administered group more fatty degeneration of hepatic tissue cells was observed in the MCD Diet administration (Fig. 20 g) than in the Normal Diet group (Fig. 20e) (stained in red by Oil red O staining).
- Fig. 20 g MCD Diet administration
- Fig. 20e Normal Diet group
- many lipid droplet vacuoles were observed.
- FIG. 21 shows a Sirius-stained diagram of liver tissue 5 weeks after administration of Ren-1-12-27.
- liver fibrosis due to MCD Diet (Fig. 21k) was not observed compared to the Normal Diet group (Fig. 21i) because the observation period was short.
- MCD Diet group no significant change was observed in the collagen staining of the Ren-1HDO administration group (Fig. 21l) as compared with the Vehicle administration group (Fig. 21k).
- Table 6 shows the judgment results of NAFLD activity score * (NAS).
- NAS NAFLD activity score *
- Ren-1 HDO administration was found to have a therapeutic effect on fatty liver formation and infiltration of inflammatory cells by MCD Diet.
- the fibrosis stage of each administration group was 0 to 1A, and no significant difference was observed.
- NAFLD nonalcoholic fatty liver disease
- NASH nonalcoholic steatohepatitis
- NAS NAFLD activity score
- Kleiner DE1 Brunt EM, Van Natta M, et al., Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005 Jun; 41 (6): 1313-21.
- Example 19 Effect of suppressing IHH mRNA expression by Ren-1-12-27 in normal mouse-derived pulmonary fibroblasts (MPF) Normal mouse pulmonary fibroblasts [Mouse pulmonary fibroblasts, MPF: Cat No. M3300-57] , Purchased from ScienCell Research Laboratories. An experiment to investigate the inhibitory effect of Ren-1-12-27 on IHH mRNA expression using MPF was carried out by the following method.
- FIG. 22 shows the results of examining the effect of Ren-1-12-27ASO on suppressing IHH mRNA expression in MPF by this method.
- Example 20 Effect of suppressing IHH mRNA expression by Ren-1-12-27 in normal mouse-derived skin fibroblasts (MDF) Normal mouse skin fibroblasts [Mouse dermal fibroblasts, MDF: Cat No. M2300-57] , Purchased from ScienCell Research Laboratories.
- MDF normal mouse-derived skin fibroblasts
- MDF normal mouse skin fibroblasts
- the experiment for investigating the inhibitory effect of Ren-1-12-27 on IHH mRNA expression using MDF was carried out in the same manner as in Example 19 as follows. That is, 1 ⁇ 10 5 cells / well on a 24-well plate for adhering cell culture in which MDF previously cultured in a special medium (Fibroblast Medium-2, Cat. # 2331) was pre-coated with PLL.
- FIG. 23 shows the results of investigating the effect of Ren-1-12-27ASO on suppressing IHH mRNA expression in MDF by such a method.
- Example 21 Effect of suppressing IHH mRNA expression by Ren-1-12-27 on TGF-beta1-stimulated normal mouse-derived renal tubular epithelial cells
- MRPTEC Cat No. M4100
- An experiment to investigate the inhibitory effect of Ren-1-12-27 on IHH mRNA expression using MRPTEC was carried out by the following method. That is, 1 ⁇ 10 4 cells / well on a 24-well plate for adhering cell culture coated with MRPTEC previously cultured in a special medium (Epithelial Cell Medium-animal, Cat. # 4131 NZ).
- FIG. 24 shows the results of investigating the inhibitory effect of Ren-1-12-27ASO on IHH mRNA expression in MRPTEC when stimulated with TGF-beta1 by the above method.
- the nucleic acid complex of the present invention is useful as a therapeutic agent for fibrosis.
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Abstract
Description
[1] 12~30のヌクレオチドからなるオリゴヌクレオチドを含み、当該オリゴヌクレオチドがIHH遺伝子転写産物に対して相補的である核酸塩基配列を有する核酸複合体。
[2] 前記オリゴヌクレオチドは一本鎖オリゴヌクレオチドである[1]の核酸複合体。
[3] 前記オリゴヌクレオチドからなるアンチセンス鎖と、前記アンチセンス鎖に相補的である核酸鎖からなるヘテロ2重鎖核酸である[1]の核酸複合体。
[4] 前記オリゴヌクレオチドが少なくとも1つの修飾ヌクレオチドを含む、[1]~[3]のいずれかの核酸複合体。
[5] 前記オリゴヌクレオチドが少なくとも1つのホスホロチオエートオリゴヌクレオチドを含む、[1]~[4]のいずれかの核酸複合体。
[6] 前記オリゴヌクレオチドが少なくとも1つのホスホジエステルオリゴヌクレオレオチドを含む、[1]~[5]の核酸複合体。
[7] 前記オリゴヌクレオチドがホスホロチオエートオリゴヌクレオチドである、[5]に記載の核酸複合体。
[8] 前記オリゴヌクレオチドが修飾核酸塩基を含む、[1]~[7]のいずれかの核酸複合体。
[9] 前記修飾核酸塩基が5-メチルシトシン、2’-MOE、BNA、LNA若しくはAmNAである、[8]に記載の核酸複合体。
[10] 前記アンチセンス鎖に相補的である核酸鎖がRNAであることを特徴とする[3]~[9]のいずれかの核酸複合体。
[11] 前記オリゴヌクレオチドが:
複数の核酸からなるギャップ領域;
複数の核酸からなる5’ウイング領域;
複数の核酸からなる3’ウイング領域;
を含むことを特徴とする[1]~[10]のいずれかの核酸複合体。
[12] 前記オリゴヌクレオチドの核酸塩基配列が、配列番号1又は配列番号2に表すIHH遺伝子配列中の連続する12~30のヌクレオチドからなるオリゴヌクレオチドに相補的な塩基配列からなる、[1]~[11]のいずれかの核酸複合体。
[13] 前記オリゴヌクレオチドの核酸塩基配列が、配列番号4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110及び112の何れかの配列からなる、[12]に記載の核酸複合体。
[14] 前記オリゴヌクレオチドの核酸塩基配列が、配列番号26の配列からなる、[13]に記載の核酸複合体。
[15] 前記オリゴヌクレオチドの核酸塩基配列が、配列番号118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186及び188の何れかの配列からなる、[1]~[11]のいずれかの核酸複合体。
[16] 前記オリゴヌクレオチドの核酸塩基配列が、配列番号160、170又は178の配列からなる、[15]に記載の核酸複合体。
[17] [1]~[16]のいずれかの核酸複合体を含む、IHH特異的な阻害剤を含む医薬組成物。
[18] [1]に記載の核酸複合体を含む、IHH特異的な阻害剤を含む線維症の治療薬。
[19] [1]~[16]のいずれかの核酸複合体を含む、IHH特異的な阻害剤を含むNashの治療薬。
[20] [1]~[16]のいずれかの核酸複合体を含む、IHH特異的な阻害剤を含む肝臓の線維症の治療薬。
[21] [1]~[16]のいずれかの核酸複合体を含む、IHH特異的な阻害剤を含む腎臓の線維症の治療薬。
[22] [1]~[16]のいずれかの核酸複合体を含む、IHH特異的な阻害剤を含む膵臓の線維症の治療薬。
[23] [1]~[16]のいずれかの核酸複合体を含む、IHH特異的な阻害剤を含む肺の線維症の治療薬。
[24] [1]~[16]のいずれかの核酸複合体を含む、IHH特異的な阻害剤を含む皮膚の線維症の治療薬。
[25] 12~30のオリゴヌクレオチドを含み、配列番号1~50の核酸塩基配列のいずれかの、少なくとも8の連続した核酸塩基を含む核酸塩基配列を有する核酸複合体。
本明細書は本願の優先権の基礎となる日本国特許出願番号2019-047703号の開示内容を包含する。
本発明は、12~30のヌクレオチドからなるオリゴヌクレオチドを含み、当該オリゴヌクレオチドがIHH(Indian hedgehog)遺伝子の転写産物に対して相補的である核酸塩基配列を有する核酸複合体である。IHH遺伝子の転写産物に対して相補的である核酸塩基配列を有する核酸はIHH遺伝子の転写産物に対してアンチセンス核酸として作用する。すなわち、IHH遺伝子の特異的な阻害剤として作用し、標的遺伝子であるIHH遺伝子の発現、又は通常転写産物レベルをアンチセンス効果によって抑制する活性を有する。
(i)ヌクレオチドと任意にヌクレオチドアナログとを含み、該核酸鎖における該ヌクレオチド及び任意に含まれる該ヌクレオチドアナログの総数は8~100であり、
(ii)転写産物にハイブリダイズした際に、RNaseHによって認識される少なくとも4つの連続したヌクレオチドを含み、
(iii)少なくとも1つの非天然ヌクレオチドを含み、
(iv)前記転写産物にハイブリダイズする。
(i)RNAヌクレオチドと、任意にヌクレオチドアナログと、任意にDNAヌクレオチドとを含み、
(ii)DNAヌクレオチド及び/又はヌクレオチドアナログを含み、又は、
(iii)PNAヌクレオチドを含む。
NCBIのウェブサイト(https://www.ncbi.nlm.nih.gov/)よりヒトIHHとマウスIHHの各遺伝子におけるcoding配列情報を入手し、図2に示すヒトIHH遺伝子coding配列:NCBI Reference Sequence: NM_002181.3(配列番号1)、マウスIHH遺伝子coding配列:NCBI Reference Sequence: NM_010544.3(配列番号2)の配列(センス鎖)に基づいて中から55本のアンチセンスオリゴヌクレオチド(ASO)をデザインした。それらの配列情報を表1に示す。表1には、ヒトIHH遺伝子coding配列又はマウスIHH遺伝子coding配列中の配列をセンスオリゴヌクレオチドとして示し、その配列に相補的な配列をアンチセンスオリゴヌクレオチドとして示す。マウスHepa1-6細胞株を市販の24-well plateに1×105 cells/ml/wellずつ播種し、24時間CO2インキュベータ中で培養した。その翌日にLipofectamine 2000(Thermo Fisher Scientific)を用いてASO(各20 nM)のトランスフェクションを行い、CO2インキュベータ中にて48時間培養した。培養後の24-well plateの各ウェルよりRNeasy Mini Kit (QIAGEN)を用いてTotal RNAを抽出した。その後、逆転写反応と定量PCRはRotor Gene Probe RT-PCR Kit(QIAGEN)を用い、定量PCR装置はRotor-Gene Q(QIAGEN)を使用した。その際、マウスIHHとマウスGAPDHのプライマーとプローブはTaqMan Gene Expression Assays(Thermo Fisher Scientific)でデザインされた試薬を使用し、mRNA発現量はマウスIHHとマウスGAPDHの各Ct値を測定した後、ΔΔCt法に基づく相対定量法により算出した。
実施例1のスクリーニングでヒットしたRen-1-12についてIHH遺伝子ノックダウン活性のIC50値を求めた。実験は基本的に実施例1と同様の方法で行った。その結果、Ren-1-12におけるノックダウン作用のIC50値は1.07 nM、対照に用いたAPOB ASOのIC50値は2.52 nMと算出された(図4A, B)。今回用いたAPOBの配列は文献情報(Nat Commun. 2015 Aug 10;6:7969.)でもin vivoで有効性が確認済の配列である。本実験のin vitroノックダウン試験においてRen-1-12 ASOのIHH遺伝子ノックダウン活性は対照に用いたAPOB ASOよりも明らかに強かったことから、in vivoでも有効性が期待できる配列であることが示唆された。
実施例1のスクリーニングでヒットしたRen-1-12 ASOがマウス肝臓においてどの程度のノックダウン作用を示すか調べた。In vivo試験には、Ren-1-12 ASOより、該アンチセンス鎖に相補的なセンス鎖を含むヘテロ2本鎖構造をデザインした後、センス鎖にトコフェロール(Toc)をリガンドとして付与したHDOを用いた(表2)。ポジティブコントロールとしてToc-APOB HDO(表2)を使用した。マウス肝臓からのTotal RNAの抽出にはRNeasy Mini Kit (QIAGEN)を用いた。その後、逆転写反応と定量PCRはRotor Gene Probe RT-PCR Kit(QIAGEN)を用い、定量PCR装置はRotor-Gene Q(QIAGEN)を使用した。その際、マウスIHHとマウス18SrRNAのプライマーとプローブはTaqMan Gene Expression Assays(Thermo Fisher Scientific)でデザインされた試薬を使用した。mRNA発現量はマウスIHHとマウス18SrRNAの各Ct値を測定した後、ΔΔCt法に基づく相対定量法により算出した。以上の方法でToc-Ren-1-12 HDO及びToc-APOB HDOを単回i.v.投与した1、3、7日後のIHH遺伝子ノックダウン効果を調べたところ、7日後にToc-Ren-1-12 HDOはToc-APOB HDOとほぼ同等のノックダウン作用を示した(図5)。Toc-APOB HDOは既にin vivoでの薬効評価に使えることが示されている(Nat Commun. 2015 Aug 10;6:7969.)ことから、Toc-Ren-1-12 HDOもin vivoでの薬効評価に十分使える可能性のある核酸複合体であることが示された。
実施例1のスクリーニングでヒットしたRen-1-12を基に新たに36本のASOをデザインした。36本のASOの配列は、Ren-1-12のセンス鎖の配列が配列番号1の塩基配列の598番目の塩基から611番目の塩基(14塩基長)であるのに対して、配列のスタート部位を配列番号1の塩基配列の603番596番目の塩基にずらすと共に塩基長を13から20とした。その結果を表3に示す。これらの36本(Ren-1-12-1~-36)の配列について実施例1と同様の方法でIHH遺伝子のノックダウンスクリーニングを行った結果を図6に示す。2次スクリーニングの結果から、Ren-1-12-22, 27, 31の3配列が特に強いノックダウン作用を示した。
実施例4で選出されたRen-1-12-22, -27, -31の3配列の中からNASH病態モデルでの薬効評価に進める配列を決めるために、正常マウスにToc-Ren-1-12-22, -27, -31の各HDO(表4)を10 nmol/kgの用量で単回i.v.投与した3日後におけるノックダウン作用を比較した。その結果、Ren-1-12-27が最も強いノックダウン率(48%)を示した(図7)。以上の結果から、Ren-1-12-27をNASH病態モデルでの薬効評価に用いることとした。
実施例5で最も強いノックダウン作用を示したToc-Ren-1-12-27 HDOの投与量を1, 3, 10, 30 nmol/kgで単回i.v.投与したときの3日後におけるIHH遺伝子ノックダウン作用を調べた。その結果、図8に示すようにRen-1-12-27 は用量依存的なノックダウン作用を示した(3 nmol/kgで55%,抑制、10 nmol/kgで57%,抑制、30 nmol/kgで72%抑制)。
実施例5でToc-Ren-1-12-27 HDOの肝臓におけるノックダウン作用の用量依存性を投与3日後で評価したが、さらに7日後におけるIHH遺伝子ノックダウン作用を調べた。その結果、図9に示すように、Ren-1-12-27 は30 nmol/kgの投与量で投与3日後から7日後にかけて60~70%程度のノックダウン率を維持することが示された。以上より、NASH病態モデルでの評価の際にはRen-1-12-27の投与量を30 nmol/kgとし、週1回の投与で行うこととした。
実施例4で見出した強いノックダウン作用を示すASOのうち上位19検体についてIC50値を算出した。結果を表5に示す。実施例4の2次スクリーニングではRen-1-12-22, 27, 31が特に強いノックダウン作用を示したが、実際にIC50値を算出したところ、Ren-1-12-34が最も強いIC50値を示した。
6週齢雌性C57BL/6Jマウスを日本チャールズリバーから購入した。メチオニン・コリン欠乏飼料(MCD飼料)及びコントロール飼料(通常食)はリサーチダイエット社より購入した。まずC57BL/6JマウスをVehicle(生理食塩水)投与群(V群)とToc-Ren-1-12-27 HDO(30 nmol(0.3mg)/kg)投与群[I(IHH)群]とに分け、さらにそれぞれの群についてMCD飼料群(M群)とNormal飼料群(N群)とに分けた。すなわち、本実験に用いるマウスは以下の計4群に分けた[(1)Vehicle投与/Normal 飼料群(VN群)、(2)Vehicle投与/MCD飼料群(VM群)、(3)Toc-Ren-1-12-27 HDO(30 nmol(0.3mg)/kg)投与/Normal飼料群(IN群)、(4)Toc-Ren-1-12-27 HDO(30 nmol(0.3mg)/kg)投与/MCD飼料群(IM群)]。給餌は初回投与(Day 0)の1週間前から開始し、Vehicle及びToc-Ren-1-12-27 HDOの投与はDay 0より週1回、5週間実施した。サンプリングは週1回行い、マウスの体重を測定後、心臓よりヘパリン採血し、肝臓組織を採取した後、肝重量を測定した。血清サンプルより血中肝臓逸脱酵素(ALT)活性、血中トリグリセライド濃度及び血中コレステロール濃度を測定した。これらの測定には、トランスアミナーゼCII-テストワコー(富士フイルム和光純薬株式会社)、ラボアッセイ(TM)トリグリセライド(富士フイルム和光純薬株式会社)、ラボアッセイ(TM)コレステロール(富士フイルム和光純薬株式会社)を使用した。各種肝臓遺伝子発現(IHH, COL1A1, CTGF, ADGRE1, ACTA2, TGFB1, CCL2, TIMP1, TNF)は、以下の方法により測定した。マウス肝臓からのTotal RNAの抽出にはReliaPrep(商標) RNA Tissue Miniprep System (Promega)を用いた。逆転写反応はPrimeScript(TM) RT Master Mix (TaKaRa Bio)を用いて行った。定量PCR反応はLuna Universal qPCR Master Mix(NEB)を用いて行い、定量PCR装置はStepOnePlus-01(Thermo Fisher Scientific)を使用した。各遺伝子毎のプライマーとプローブはThermo Fisher Scientific にて遺伝子毎にデザイン済のTaqMan Gene Expression Assay試薬を使用した。mRNA発現量はマウス各遺伝子とマウス18SrRNAの各Ct値を測定した後、ΔΔCt法に基づく相対定量法により算出した。統計処理は3要因の分散分析(3-way ANOVA)を用いて行い、危険率は5%未満を有意とした。
MCD飼料により作製したNASH病態モデルマウスにVehicle又はToc-Ren-1-12-27 HDO を週1回、5週間投与したマウスでの炎症マーカー遺伝子(TNFA及びCCL2)発現に及ぼす影響を調べた結果、図11A、Bに示すように、VN群と比較してVM群においてTNFAとCCL2の発現が顕著に上昇し、Day14でVM群と比較してIM群においてTNFAとCCL2遺伝子の発現が顕著に低下した。
MCD飼料により作製したNASH病態モデルマウスにVehicle又はToc-Ren-1-12-27 HDO を週1回、5週間投与したマウスでのADGRE1遺伝子発現に及ぼす影響を調べた結果、図12に示すように、Day14の時点でADGRE1の発現が低下する傾向を示した。
MCD飼料により作製したNASH病態モデルマウスにVehicle又はToc-Ren-1-12-27 HDO を週1回、5週間投与したマウスでの線維化マーカー(COL1A1, CTGF, TGFB1, TIMP, ACTA2)遺伝子発現に及ぼす影響を調べた。その結果、図13-1Aに示すように、Day7の時点でCOL1A1遺伝子の発現が顕著に上昇し、Day35の時点でCOL1A1遺伝子の発現が有意に低下した(P<0.05)。CTGFの発現は図13-1Bに示すようにDay14とDay28で低下傾向は示したものの、有意ではなかった。TGFB1の発現は図13-2Aに示すようにDay14とDay35で低下傾向は示したものの、有意ではなかった。TIMPとACTA2の発現については図13-2B及び図13-3に示すように全期間において低下傾向はみられなかった。
MCD飼料により作製したNASH病態モデルマウスにVehicle又はToc-Ren-1-12-27 HDO を週1回、5週間投与したマウスでの血中肝臓逸脱酵素(ALT)活性に及ぼす影響を調べた結果、図14に示すように、VN群と比較してVM群においてALT活性が有意に上昇した(P<0.0001)。
MCD飼料により作製したNASH病態モデルマウスにVehicle又はToc-Ren-1-12-27 HDO を週1回、5週間投与したマウスでの体重及び肝重量に及ぼす影響を調べた結果、図15A、Bに示すように、VN群と比較してVM群において体重と肝重量が有意に低下した(P<0.0001)。
MCD飼料により作製したNASH病態モデルマウスにビークル又はToc-Ren-1-12-27 HDOを週1回、6週間投与したマウスでの体重及び肝重量に及ぼす影響を調べた結果、図16に示すように、VN群と比較してVM群において体重及び肝重量が有意に低下(いずれもP<0.0001)した。このときVM群とIM群との間に有意な変化は確認されなかった。
MCD飼料により作製したNASH病態モデルマウスにVehicle又はToc-Ren-1-12-27 HDO を週1回、5週間投与したマウスでの血中トリグリセライド濃度及び血中コレステロール濃度に及ぼす影響を調べた。その結果、図16A、Bに示すように、VN群と比較してVM群において血中トリグリセライド濃度及び血中コレステロール濃度が有意に低下した(いずれもP<0.0001)。このとき、VM群とIM群との間に血中トリグリセライド濃度で有意な変化は確認されなかったが、血中コレステロール濃度は有意に上昇した(P<0.05)。
したがって、Toc-Ren-1-12-27 HDOはNASH治療薬となりうる化合物であることが検証された。
実施例1、実施例4で使用した37本のASO(Ren-1-2、Ren-1-3、Ren-1-4、Ren-1-5、Ren-1-6、Ren-1-7、Ren-1-9、Ren-1-11、Ren-1-12、Ren-1-12-13、Ren-1-12-27、Ren-1-14、Ren-1-15、Ren-1-16、Ren-1-17、Ren-1-18、Ren-1-19、Ren-1-23、Ren-1-24、Ren-1-25、Ren-1-26、Ren-1-28、Ren-1-29、Ren-1-33、Ren-1-35、Ren-1-36、Ren-1-37、Ren-1-38、Ren-1-39、Ren-1-40、Ren-1-41、Ren-1-43、Ren-1-44、Ren-1-48、Ren-1-47、Ren-1-49、Ren-1-50)及びネガティブコントロールとしてPBS、ポジティブコントロールとしてAPOB遺伝子のアンチセンス核酸について、実施例1、実施例4と異なる核酸濃度50nMにて再度スクリーニングを実施した。スクリーニングの方法は、核酸濃度50nMが異なる以外は、実施例1、実施例4と同様の方法で行った。結果を図17に示す。50nMの濃度では、Ren-1-11とRen-1-41がIHHのmRNAの発現を阻害することが示された。
本実験は新たにin vitroスクーリングにより、IHH遺伝子発現に対し、強いノックダウン作用を示した4つのASO(Ren-1-12-27、Ren-1-11、Ren-1-39、Ren-1-41)を用いて、in vivoでのノックダウン活性を調べた。
25匹6週齢の正常マウス(c57BL/6j)を体重により5群(Vehicle、Ren-1-12-27、Ren-1-11、Ren-1-39、Ren-1-41)に分けた。ノックダウン活性はポジティブコントロールのRen-1-12-27(17 mer)とネガティブコントールのVehicle投与群と比較した。ASOの投与量は30 nmol/10ml/kgに設定し、マウスの尾静脈より投与を行った(投与日をDay 0とした)。投与三日後(Day 3)に、マウスはイソフルラン麻酔下にて心内採血を行い、開腹し、肝臓を摘出した。肝臓組織からトータルmRNAを抽出し、逆転写、qPCRし、IHH mRNAの発現を計測した。結果を図18に示す。
6週齢のマウスをNormal Diet群とMCD(Methionine and Choline Deficient)Diet群に分け、MCD DietによるNASH・肝線維化モデルを作成した。MCD投与1週間後、Toc-Ren1-12-27を30 nmol/kgの用量で週1回、5週間、iv投与を行った。
Vehicle投与群では、Normal Diet群(図19a)に比べ、炎症性細胞の集まりはMCD Diet投与(図19c)により(黒丸内)、肝臓内脂肪滴、風船様変化が多く見られる。
* Kleiner DE1, Brunt EM, Van Natta M, et al., Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005 Jun;41(6):1313-21.
正常マウス肺線維芽細胞 [Mouse pulmonary fibroblasts, MPF: Cat No.M3300-57]は、ScienCell Research Laboratories社より購入した。MPFを用いたRen-1-12-27によるIHH mRNA発現抑制効果を調べる実験は以下の方法で実施した。市販の接着細胞培養用24ウェルプレートに、滅菌水で700倍に希釈したPoly-L-Lysine (PLL)溶液(ScienCell Research Laboratories)を0.5 ml/wellずつ分注し、CO2インキュベータ中にて2時間インキュベートすることによりPLLコーティングを行った。次に、事前に専用培地(Fibroblast Medium, Cat. #2301)を用いて培養させたMPFをPLLコーティングした接着細胞培養用24ウェルプレートに1×105 cells/wellになるように播種した。その翌日にリポフェクトアミン2000(Thermo Fisher Scientific)を用いてRen-1-12-27ASOをトランスフェクションした。その際のASOの用量は0、0.3、1、3、10、30 (nM)を用い、ポジティブコントロールとしてのMalat-1ASOの用量は0、10 (nM)を用いた。次にトランスフェクションしてから2日後にSV96 Total RNA Isolation System(Promega)を用いてTotal RNAを精製し、Total RNAからのcDNA合成はPrimeScriptTMRT Master Mix(TAKARA BIO INC.)を用いて実施した。qPCR反応はStepOnePlus-01(Thermo Fisher Scientific)を用いて実施し、マウスRen-1とマウス18SrRNAのプライマー/プローブはTaqMan Gene Expression Assays(Thermo Fisher Scientific)でデザインされた試薬を使用し、mRNA発現量はマウスRen-1とマウス18SrRNAの各Ct値を測定した後、ΔΔCt法に基づく相対定量法により算出した。この方法でMPFにおけるRen-1-12-27ASOによるIHH mRNA発現抑制効果を調べた結果を図22に示す。
正常マウス皮膚線維芽細胞 [Mouse dermal fibroblasts, MDF: Cat No.M2300-57]は、ScienCell Research Laboratories社より購入した。MDFを用いたRen-1-12-27によるIHH mRNA発現抑制効果を調べる実験は実施例19と同様の方法で以下の様に実施した。すなわち、あらかじめ専用培地(Fibroblast Medium-2, Cat. #2331)を用いて培養させておいたMDFを事前にPLLコーティングした接着細胞培養用24ウェルプレートに1×105 cells/wellになるように播種し、実施例22と同様の方法でトランスフェクション、RNA抽出、cDNA合成、qPCRを行った。このような方法でMDFにおけるRen-1-12-27ASOによるIHH mRNA発現抑制効果を調べた結果を図23に示す。
正常マウス由来腎尿細管上皮細胞 [Mouse renal proximal tubular epithelial cells, MRPTEC: Cat No.M4100]は、ScienCell Research Laboratories社より購入した。MRPTECを用いたRen-1-12-27によるIHH mRNA発現抑制効果を調べる実験は以下の方法で実施した。すなわち、あらかじめ専用培地(Epithelial Cell Medium-animal, Cat. #4131 NZ)中にて培養させたMRPTECを事前にPLLコーティングした接着細胞培養用24ウェルプレートに1×104cells/wellになるように播種し、その翌日に実施例19と同様の方法でトランスフェクションを行った。次にその翌日に1mlのPBSで2回細胞を洗浄した後、RPMI1640、0.2%FBS、ペニシリン/ストレプトマイシンを含む培地(0.5 ml)に交換した。さらにその翌日にRen-1-12-27無添加(ネガティブコントロール)のウェルを除くすべてのウェルにTGF-beta1(10 ng/ml)を添加し、さらに24時間培養した後、RNA抽出、cDNA合成、qPCRを行った。以上の方法でTGF-beta1刺激したときのMRPTECにおけるRen-1-12-27ASOによるIHH mRNA発現抑制効果を調べた結果を図24に示す。
本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。
Claims (25)
- 12~30のヌクレオチドからなるオリゴヌクレオチドを含み、当該オリゴヌクレオチドがIHH遺伝子転写産物に対して相補的である核酸塩基配列を有する核酸複合体。
- 前記オリゴヌクレオチドは一本鎖オリゴヌクレオチドである請求項1に記載の核酸複合体。
- 前記オリゴヌクレオチドからなるアンチセンス鎖と、前記アンチセンス鎖に相補的である核酸鎖からなるヘテロ2重鎖核酸である請求項1に記載の核酸複合体。
- 前記オリゴヌクレオチドが少なくとも1つの修飾ヌクレオチドを含む、請求項1~3のいずれか1項に記載の核酸複合体。
- 前記オリゴヌクレオチドが少なくとも1つのホスホロチオエートオリゴヌクレオチドを含む、請求項1~4のいずれか1項に記載の核酸複合体。
- 前記オリゴヌクレオチドが少なくとも1つのホスホジエステルオリゴヌクレオレオチドを含む、請求項1~5のいずれか1項に記載の核酸複合体。
- 前記オリゴヌクレオチドがホスホロチオエートオリゴヌクレオチドである、請求項5に記載の核酸複合体。
- 前記オリゴヌクレオチドが修飾核酸塩基を含む、請求項1~7のいずれか1項に記載の核酸複合体。
- 前記修飾核酸塩基が5-メチルシトシン、2’-MOE、BNA、LNA若しくはAmNAである、請求項8に記載の核酸複合体。
- 前記アンチセンス鎖に相補的である核酸鎖がRNAであることを特徴とする請求項3~9のいずれか1項に記載の核酸複合体。
- 前記オリゴヌクレオチドが:
複数の核酸からなるギャップ領域;
複数の核酸からなる5’ウイング領域;
複数の核酸からなる3’ウイング領域;
を含むことを特徴とする請求項1~10のいずれか1項に記載の核酸複合体。 - 前記オリゴヌクレオチドの核酸塩基配列が、配列番号1又は配列番号2に表すIHH遺伝子配列中の連続する12~30のヌクレオチドからなるオリゴヌクレオチドに相補的な塩基配列からなる、請求項1~11のいずれか1項に記載の核酸複合体。
- 前記オリゴヌクレオチドの核酸塩基配列が、配列番号4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110及び112の何れかの配列からなる、請求項12に記載の核酸複合体。
- 前記オリゴヌクレオチドの核酸塩基配列が、配列番号26の配列からなる、請求項13に記載の核酸複合体。
- 前記オリゴヌクレオチドの核酸塩基配列が、配列番号118、120、122、124、126、128、130、132、134、136、138、140、142、144、146、148、150、152、154、156、158、160、162、164、166、168、170、172、174、176、178、180、182、184、186及び188の何れかの配列からなる、請求項1~11のいずれか1項に記載の核酸複合体。
- 前記オリゴヌクレオチドの核酸塩基配列が、配列番号160、170又は178の配列からなる、請求項15に記載の核酸複合体。
- 請求項1~16のいずれか1項に記載の核酸複合体を含む、IHH特異的な阻害剤を含む医薬組成物。
- 請求項1に記載の核酸複合体を含む、IHH特異的な阻害剤を含む線維症の治療薬。
- 請求項1~16のいずれか1項に記載の核酸複合体を含む、IHH特異的な阻害剤を含むNashの治療薬。
- 請求項1~16のいずれか1項に記載の核酸複合体を含む、IHH特異的な阻害剤を含む肝臓の線維症の治療薬。
- 請求項1~16のいずれか1項に記載の核酸複合体を含む、IHH特異的な阻害剤を含む腎臓の線維症の治療薬。
- 請求項1~16のいずれか1項に記載の核酸複合体を含む、IHH特異的な阻害剤を含む膵臓の線維症の治療薬。
- 請求項1~16のいずれか1項に記載の核酸複合体を含む、IHH特異的な阻害剤を含む肺の線維症の治療薬。
- 請求項1~16のいずれか1項に記載の核酸複合体を含む、IHH特異的な阻害剤を含む皮膚の線維症の治療薬。
- 12~30のオリゴヌクレオチドを含み、配列番号1~50の核酸塩基配列のいずれかの、少なくとも8の連続した核酸塩基を含む、核酸塩基配列を有する核酸複合体。
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