Double-stranded oligonucleotide

Abstract

Provided is a double-stranded oligonucleotide which can more selectively suppress a gene including an extended CAG repeat sequence, can exhibit the suppression effect in a living body without relying on lipid nanoparticles, has a wider distribution range in the central nervous system, and / or can exhibit the suppression effect for a longer period of time in a living body. This double-stranded oligonucleotide includes a sense strand including a CAG repeat sequence and an antisense strand including a complementary sequence to the CAG repeat sequence, wherein at least one of nucleoside constituent units located in a center part of the antisense strand and / or a nucleoside constituent unit of at least one terminal part of the sense strand and / or the antisense strand is a non-cyclic nucleoside constituent unit represented by general formula (1).

Description

double-stranded oligonucleotide

[0001] The present invention relates to a double-stranded oligonucleotide and the like.

[0002] Polyglutamine diseases are a collective term for hereditary neurodegenerative disorders caused by abnormally extended CAG repeats, including nine diseases such as spinal-bulbar muscular atrophy (SBMA), Huntington's disease, and spinocerebellar ataxia (SCA). All of these diseases are intractable, resulting in irreversible progression of motor and cognitive impairment, and no curative treatments that can prevent neurodegeneration have been developed. Clinical trials targeting the causative gene for Huntington's disease have been conducted, but to date, sufficient clinical efficacy has not been observed, and practical application has not yet been achieved. One of the reasons for this is concern about neurotoxicity due to the simultaneous knockdown of normal genes in addition to the mutated disease-causing gene.

[0003] Recent studies have demonstrated that mismatches or chemical modifications of small interfering RNA (siRNA) targeting CAG repeats can selectively suppress abnormal polyglutamine proteins resulting from mutant genes. Previous studies have also reported that siRNA modified with unlocked nucleic acid (UNA), an acyclic artificial nucleic acid, can selectively suppress abnormal polyglutamine proteins in the central nervous system of SBMA model mice (Non-Patent Document 1). However, because siRNA is easily degraded in vivo, lipid nanoparticles are required as a delivery system for delivery to the central nervous system, which raises concerns about side effects such as inflammation. Furthermore, the distribution of lipid nanoparticles in the central nervous system is limited to the periventricular area, and further improvement of their distribution range is necessary for clinical application.

[0004] Hirunagi T, et al. Selective suppression of polyglutamine-expanded protein by lipid nanoparticle-delivered siRNA targeting CAG expansions in the mouse CNS. Mol Ther Nucleic Acids, 2021;24:e135633:1-10.

[0005] An objective of the present invention is to provide a double-stranded oligonucleotide that can more selectively suppress genes containing extended CAG repeat sequences, that can exert an inhibitory effect in vivo without relying on lipid nanoparticles, that has a wider distribution range in the central nervous system, and / or that can exert an inhibitory effect in vivo for a longer period of time.

[0006] In light of the above-mentioned problems, the present inventors have conducted extensive research and have found that the above-mentioned problems can be solved by a double-stranded oligonucleotide comprising a sense strand containing a CAG repeat sequence and an antisense strand containing a complementary sequence to the CAG repeat sequence, wherein at least one of the nucleoside building blocks in the central portion of the antisense strand and / or at least one terminal nucleoside building block of the sense strand and / or the antisense strand is an acyclic nucleoside building block represented by general formula (1). Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. A method for producing a nucleic acid molecule comprising a sense strand comprising a CAG repeat sequence and an antisense strand comprising a complementary sequence to the CAG repeat sequence, wherein at least one of the nucleoside building blocks in the center of the antisense strand and / or at least one terminal nucleoside building block of the sense strand and / or the antisense strand is represented by the general formula (1):

[0008] [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2(Except when both are organic groups.) Base represents a nucleic acid base.] A double-stranded oligonucleotide in which the acyclic nucleoside building block is represented by the following formula:

[0009] Item 2. The double-stranded oligonucleotide according to Item 1, wherein at least one of the nucleoside building blocks in the central portion of the antisense strand is the acyclic nucleoside building block.

[0010] Item 3. The double-stranded oligonucleotide according to Item 2, wherein the nucleoside building block at at least one terminal of the sense strand and / or the antisense strand is the acyclic nucleoside building block.

[0011] Item 4. The double-stranded oligonucleotide according to any one of Items 1 to 3, wherein the central portion is a region consisting of one nucleoside building block in the center of the complementary sequence or 1 to 5 nucleoside building blocks on each side of one internucleoside bond.

[0012] Item 5. The double-stranded oligonucleotide according to any one of Items 1 to 4, wherein the terminal portion is a region consisting of 1, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleoside constitutional units from the terminal.

[0013] Item 6. The double-stranded oligonucleotide according to any one of Items 1 to 5, wherein the terminal ends are the 5' terminal end of the sense strand, the 3' terminal end of the sense strand, and the 3' terminal end of the antisense strand.

[0014] Item 7. The double-stranded oligonucleotide according to any one of Items 1 to 6, wherein the 3'-end of the sense strand and / or the 3'-end of the antisense strand is an overhanging type.

[0015] Item 8. The double-stranded oligonucleotide according to any one of Items 1 to 7, wherein the sense strand is 18 to 30 bases long and the antisense strand is 18 to 30 bases long.

[0016] Item 9. The double-stranded oligonucleotide according to any one of Items 1 to 8, wherein the complementary sequence in the antisense strand has 0 to 5 mismatch mutations relative to the CAG repeat sequence in the sense strand.

[0017] Item 10. The double-stranded oligonucleotide according to any one of Items 1 to 9, wherein at least one of the nucleoside building blocks in the central portion of the antisense strand is the acyclic nucleoside building block, at least one terminal nucleoside building block of the sense strand and / or the antisense strand is the acyclic nucleoside building block, the central portion is a region consisting of 1 to 5 nucleoside building blocks on either side of a central nucleoside building block or an internucleoside bond in the complementary sequence, the terminal portions are regions consisting of 1, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleoside building blocks from the terminal, the 3'-end of the sense strand and / or the 3'-end of the antisense strand is an overhanging type, the sense strand is 18 to 30 bases long, and the antisense strand is 18 to 30 bases long.

[0018] Item 11. An expression inhibitor for a gene containing an extended CAG repeat sequence, comprising the double-stranded oligonucleotide according to any one of Items 1 to 10.

[0019] Item 12. A pharmaceutical comprising the double-stranded oligonucleotide according to any one of Items 1 to 10 or the expression-inhibiting agent according to Item 11.

[0020] Item 13. The pharmaceutical agent according to Item 12, which is used for the prevention or treatment of a polyglutamine disease.

[0021] Item 13A. A method for preventing or treating a polyglutamine disease, comprising administering the double-stranded oligonucleotide according to any one of Items 1 to 10 or the expression-inhibiting agent according to Item 11 to a subject in need thereof (preferably a subject with a polyglutamine disease or a subject with a gene containing an expanded CAG repeat sequence).

[0022] Item 13B: The double-stranded oligonucleotide according to any one of Items 1 to 10 or the expression-inhibiting agent according to Item 11 for use in the prevention or treatment of a polyglutamine disease.

[0023] Item 13C: Use of the double-stranded oligonucleotide according to any one of Items 1 to 10 or the expression-inhibiting agent according to Item 11 for the manufacture of a medicament for the prevention or treatment of a polyglutamine disease.

[0024] Item 13D: Use of the double-stranded oligonucleotide according to any one of Items 1 to 10 or the expression-inhibiting agent according to Item 11 for the prevention or treatment of polyglutamine disease.

[0025] Item X1: A pharmaceutical composition comprising the double-stranded oligonucleotide according to any one of Items 1 to 10.

[0026] Item X2: The pharmaceutical composition according to Item X1, which is used for the prevention or treatment of a polyglutamine disease.

[0027] Item X3. The medicament or pharmaceutical composition according to Item 13 or X2, the method according to Item 13A, the double-stranded oligonucleotide or expression-inhibiting agent for use according to Item 13B, the use for manufacturing according to Item 13C, or the use according to Item 13D, wherein the polyglutamine disease is at least one selected from the group consisting of Huntington's disease (HD), spinal-bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3 or Machado-Joseph disease), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), and spinocerebellar ataxia type 17 (SCA17).

[0028] According to the present invention, it is possible to provide a double-stranded oligonucleotide that can more selectively suppress genes containing extended CAG repeat sequences, can exert an inhibitory effect in vivo without relying on lipid nanoparticles, has a wider distribution range in the central nervous system, and / or can exert an inhibitory effect in vivo for a longer period of time.

[0029] 1 shows the structures of DNA, RNA, and SNA and L-aTNA, which are examples of acyclic nucleoside building blocks represented by general formula (1). B or Base indicates a nucleic acid base. The results of the luciferase assay in Test Example 2 are shown. The vertical axis indicates the relative luminescence intensity, the horizontal axis indicates the siRNA used, and the legend indicates the androgen receptor (AR) gene used as the reporter. The results of the luciferase assay in Test Example 3 are shown. The vertical axis indicates the relative luminescence intensity, the horizontal axis indicates the siRNA used, and the legend indicates the androgen receptor (AR) gene used as the reporter. The results of Western blot in Test Example 4 are shown. The detected protein is shown on the left side of the photograph, and the siRNA used is shown on the top. The upper row shows the case where healthy control (AR-18Q) fibroblasts were used, and the lower row shows the case where spinal and bulbar muscular atrophy (SBMA) (AR-52Q) fibroblasts were used. The results of Western blot in Test Example 5 are shown. The detected protein is shown on the left side of the photograph, and the siRNA used is shown on the top. The upper and lower panels show the results of independent studies using SBMA model mice. The graphs on the left and right show the quantification results of the bands in Figure 5-1. The graph on the left shows the quantification results of the bands in the upper panel of Figure 5-1, and the graph on the right shows the quantification results of the bands in the lower panel of Figure 5-1. The results of Western blot for Test Example 6 are shown. The detected proteins are shown on the left side of the photograph, and the siRNA used is shown on the top side. The graph on the right shows the quantification results of the bands. Fluorescent images of each organ in Test Example 7 are shown. The siRNA used is shown on the top side of the photograph. The organs observed are shown on the left side of the photograph. The results of Western blot for Test Example 8 are shown. The detected proteins are shown on the left side of the photograph, and the siRNA used is shown on the top side. The graph on the right shows the quantification results of the bands. The results of Western blot for Test Example 9 are shown. The detected proteins are shown on the left side of the photograph, and the siRNA used is shown on the top side. The graph on the right shows the quantification results of the bands. The results of survival, grip strength, and rotarod task for Test Example 10 are shown. The horizontal axis indicates the age of the mice in weeks. The legend indicates the siRNA used. This shows the results of Western blotting in Test Example 11. The detected proteins are shown on the left side of the photograph, and the sample tissues are shown on the top side of the photograph.Of the two bands for each tissue, the one on the left is the control and the one on the right is S9-SNA-siRNA. The graph at the bottom shows the quantification results of the bands. Western blot results (when n was increased) for Test Example 8 are shown. The detected protein is shown on the left side of the photograph, and the siRNA used is shown on the top side of the photograph. The graph on the right shows the quantification results of the bands. Western blot results (when n was increased) for Test Example 9 are shown. The detected protein is shown on the left side of the photograph, and the siRNA used is shown on the top side of the photograph. The graph on the right shows the quantification results of the bands. Evaluation results for the polyglutamine aggregate positivity rate of motor neurons for Test Example 12 are shown. The siRNA used is shown on the top side of the photograph and the bottom side of the graph. Evaluation results for motor neuron atrophy for Test Example 12 are shown. The siRNA used is shown on the top side of the photograph and the bottom side of the graph. Evaluation results for quadriceps atrophy for Test Example 12 are shown. The siRNA used is shown on the top side of the photograph and the bottom side of the graph. Western blot results for Test Example 13 are shown. The detected protein is shown on the left side of the photograph, and the siRNA used is shown on the top side of the graph. The graph shows the quantification results of the bands in the photograph. Graphs show the results of Western blot for Test Example 14. The detected protein is shown on the left side of the photograph, and siRNA is shown above. The graph shows the quantification results of the bands in the photograph. Graphs show the results of staining for nuclear ATXN3 in the deep cerebellar nuclei and pontine nuclei in Test Example 15. The graph shows the percentage of nuclear ATXN3-positive cells. Graphs show the results of Western blot and immunohistochemistry for GFAP and IBA-1 in Test Example 16. The detected protein is shown on the left side of the photograph, and siRNA is shown above. The graph shows the quantification results of the GFAP and IBA-1 bands in the blot. Graphs show the results of Western blot for Test Example 17. The detected protein is shown on the left side of the photograph, and siRNA is shown above. The graph shows the results of ATXN3 in the blot. Graphs show the results of Western blot for Test Example 18. The detected protein is shown on the left side of the photograph, and siRNA is shown above. The graph shows the quantification results of the bands.

[0030] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0031] When upper and lower limits for a numerical range are separately disclosed herein, ranges consisting of any combination of any upper and any lower limits are also disclosed herein.

[0032] As used herein, the "identity" of a base sequence refers to the degree of match between the base sequences of two or more comparable base sequences. Therefore, the greater the match between two base sequences, the greater the identity or similarity between those sequences. The level of identity between base sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known, and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0033] As used herein, "complementary" refers not only to a perfect base complementarity (e.g., A and T or U, and G and C), but also to a degree of complementarity that allows hybridization under stringent conditions. Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA). For example, typical post-hybridization washing conditions include 1×SSC, 0.1% SDS, and 37°C. It is preferred that the hybridization state be maintained even after washing under such conditions. While not particularly limited, more stringent hybridization conditions include washing conditions of approximately 0.5×SSC, 0.1% SDS, and 42°C, and even more stringent hybridization conditions include washing conditions of approximately 0.1×SSC, 0.1% SDS, and 65°C.

[0034] As used herein, an oligonucleotide comprises nucleosides (nucleoside building blocks) and internucleoside linkages connecting adjacent nucleosides.

[0035] Nucleosides include natural nucleosides such as ribonucleosides and deoxyribonucleosides as well as modified nucleosides.

[0036] Modified nucleosides include ribonucleosides, deoxyribonucleosides, and RNA or DNA molecules modified to improve or stabilize nuclease resistance, increase affinity with complementary nucleic acids, increase cell permeability, or facilitate visualization, compared to RNA or DNA. Examples include 2'-OMe-modified nucleosides, 2'-MOE-modified nucleosides, and nucleosides with modified sugar moieties, such as LNA (registered trademark), ENA (registered trademark), AmNA (registered trademark), GuNA, and scpBNA (hereinafter sometimes referred to as sugar-modified nucleosides). Oligonucleotides may contain, for example, modified nucleic acid molecules disclosed in Khvorova & Watts (Nature Biotechnology 35, 238-248 (2017) doi:10.1038 / nbt.3765).

[0037] Sugar-modified nucleosides refer to modified nucleosides containing modified sugars, where modified sugar refers to sugars that have substitutions and / or any changes from the natural sugar moiety (i.e., the sugar moiety found in DNA (2'-H) or RNA (2'-OH)). The sugar-modified nucleoside may be any nucleoside in which any chemical structural substance has been added or substituted to a part or all of the chemical structure of the sugar of the nucleoside, and examples thereof include modified nucleosides substituted with 2'-O-methyl in the sugar moiety, modified nucleosides substituted with 2'-O-propyl, modified nucleosides substituted with 2'-methoxyethoxy, modified nucleosides substituted with 2'-O-methoxyethyl, modified nucleosides substituted with 2'-O-[2-(guanidium)ethyl], modified nucleosides substituted with 2'-O-fluoro, bridged artificial nucleic acids having two cyclic structures by introducing a bridged structure into the sugar moiety, more specifically, locked artificial nucleic acids (LNA) in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via a methylene, and ethylene-bridged artificial nucleic acids (ENA) [Nucleic Acid Research, 32, e175 (2004)], and further examples include peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)], and peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].

[0038] 2'-O-methyl (2'-OMe) modification of the sugar moiety of RNA (2'-OMe-RNA) is a naturally occurring modification that improves the binding affinity and nuclease resistance of modified oligonucleotides while reducing their immunostimulatory potential. 2'-O-methoxyethyl (2'-MOE) modification of RNA further increases nuclease resistance compared to 2'-OMe modification, significantly increasing the binding affinity (ΔTm) of the modified nucleotide. 2'-Fluoro (2'-F) modification of RNA (2'-F-RNA) can also be used to increase the affinity of oligonucleotides. Other 2'-modified nucleic acids include 2'-F-ANA (F-arabinonucleic acid) and the 2'-modified derivatives proposed by Sekine et al. (Patent No. 5194256, JP 2015-020994).

[0039] Locked nucleic acid (LNA), which links the 2' oxygen and 4' carbon of ribose, significantly increases binding affinity. In LNA, the 2' oxygen and 4' carbon of the ribose sugar of RNA are fixed in a ring structure. This modification increases specificity, affinity, and half-life, allowing for effective delivery to target tissues with reduced toxicity. However, fully modified LNA oligomers longer than approximately eight nucleotides tend to aggregate, and are generally used in combination with DNA or other sugar-modified nucleic acids. A methylated analog of LNA, cEt (constrained ethyl bridged nucleic acid), is also useful. Tricyclo-DNA (tcDNA) is a constrained nucleotide based on a three-ring backbone.

[0040] Other examples of modified nucleosides include those in which an atom (e.g., hydrogen atom, oxygen atom) or functional group (e.g., hydroxyl group, amino group) in the base portion of a nucleic acid is substituted with another atom (e.g., hydrogen atom, sulfur atom), functional group (e.g., amino group), or alkyl group having 1 to 6 carbon atoms, or those protected with a protecting group (e.g., methyl group or acyl group), and molecules in which another chemical substance such as a lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, or dye is attached to a nucleoside.

[0041] Nucleic acid bases include not only typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C), etc.), but also other bases such as hypoxanthine (I), modified bases, etc. Modified bases include, for example, 2-thiouracil, 2,6-diaminopurine, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, Examples include 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, and cyanuric acid.

[0042] The internucleoside bond is not particularly limited as long as it is capable of forming a double-stranded oligonucleotide, and examples thereof include a phosphodiester bond, a phosphorothioate bond (also called a thiophosphate bond), a phosphorodithioate bond, a phosphotriester bond, a methylphosphonate bond, a methylthiophosphonate bond, a boranophosphate bond, and a phosphoramidate bond.

[0043] 1. Double-Stranded Oligonucleotides In one aspect, the present invention relates to a double-stranded oligonucleotide (sometimes referred to herein as the "double-stranded oligonucleotide of the present invention") comprising a sense strand containing a CAG repeat sequence and an antisense strand containing a complementary sequence to the CAG repeat sequence, wherein at least one nucleoside building block in the center of the antisense strand and / or at least one terminal nucleoside building block of the sense strand and / or the antisense strand is an acyclic nucleoside building block represented by general formula (1):

[0044] The sense strand contains a CAG repeat sequence.

[0045] The CAG repeat sequence is a sequence having a structure in which the base sequence consisting of CAG is repeated as a backbone, and is not particularly limited thereto. The CAG repeat sequence is not limited to those starting with C and terminating with G (e.g., CAGCAGCAG, etc.), but also includes those starting with a base (A or G) other than the end of the base sequence consisting of CAG and terminating with a base (C or A) other than the end of the base sequence consisting of CAG (e.g., AGCAGCAG, GCAGCAG, AGCAGC, GCAGCAGCA, etc.).

[0046] The base length of the CAG repeat sequence is not particularly limited, as long as the double-stranded oligonucleotide of the present invention can exert an inhibitory effect on a gene containing an extended CAG repeat sequence. The gene is described below. The inhibitory effect can be measured according to the method described in Test Example 2 below. The base length of the CAG repeat sequence in the sense strand is, for example, 10 to 30, preferably 12 to 28, more preferably 14 to 26, even more preferably 16 to 25, even more preferably 18 to 24, and particularly preferably 20 to 24.

[0047] The CAG repeat sequence may have a base mutation (preferably a base substitution) as long as the double-stranded oligonucleotide of the present invention can exert an inhibitory effect on genes containing the extended CAG repeat sequence. The CAG repeat sequence is a base sequence that has, for example, 85% or more identity, preferably 90% or more identity, more preferably 95% or more identity, even more preferably 98% or more identity, even more preferably 99% or more identity, and particularly preferably 100% identity to a CAG repeat sequence of the same base length without base mutations. The number of base mutations is, for example, 0 to 5, preferably 0 to 3, more preferably 0 to 2, even more preferably 0 to 1, and particularly preferably 0.

[0048] The sense strand may contain a sequence other than the CAG repeat sequence, so long as the double-stranded oligonucleotide of the present invention can exert an inhibitory effect on genes containing extended CAG repeat sequences. Examples of such sequences include sequences (e.g., overhang sequences) added to the ends (particularly the 3' ends) of the CAG repeat sequence. The base length of such sequences is, for example, 0 to 5, preferably 0 to 3, and more preferably 0 to 2. The base length of the CAG repeat sequence relative to the total base length of the sense strand (100%) is preferably 75 to 100%, more preferably 80 to 100%, even more preferably 85 to 100%, and even more preferably 90 to 100%.

[0049] The base length of the sense strand is not particularly limited as long as the double-stranded oligonucleotide of the present invention can exert an inhibitory effect on a gene containing an extended CAG repeat sequence, and the base length is, for example, 18 to 30, preferably 19 to 28, more preferably 20 to 26, and even more preferably 21 to 24.

[0050] The antisense strand is not particularly limited as long as it contains a complementary sequence to the CAG repeat sequence of the sense strand and can form a double-stranded oligonucleotide that can exert an inhibitory effect on genes containing an extended CAG repeat sequence.

[0051] The antisense strand has, for example, 75 to 100%, preferably 80 to 100%, more preferably 85 to 100%, even more preferably 90 to 100%, and even more preferably 93 to 100% identity to the fully complementary sequence of the CAG repeat sequence of the sense strand.

[0052] The antisense strand may have mismatch mutations in the complementary sequence of the CAG repeat sequence relative to the CAG repeat sequence of the sense strand. Mismatch mutations indicate that the two opposing bases in the duplex are not in a base complementary relationship (e.g., A and T or U, and G and C). The number of mismatch mutations is, for example, 0 to 5, preferably 0 to 3, more preferably 0 to 2, and even more preferably 0 to 1. The mismatch mutations are preferably located in the center of the antisense strand (described below).

[0053] The base length of the complementary sequence to the CAG repeat sequence is not particularly limited, as long as the double-stranded oligonucleotide of the present invention can exert an inhibitory effect on genes containing an extended CAG repeat sequence. The base length of the complementary sequence to the CAG repeat sequence is, for example, 10 to 30, preferably 12 to 28, more preferably 14 to 26, even more preferably 16 to 25, even more preferably 18 to 24, and particularly preferably 20 to 24.

[0054] The antisense strand may contain a sequence other than the complementary sequence to the CAG repeat sequence, so long as the double-stranded oligonucleotide of the present invention can exert an inhibitory effect on genes containing the extended CAG repeat sequence. Examples of such a sequence include a sequence (e.g., an overhang sequence) added to the end (particularly the 3' end) of the complementary sequence. The base length of the other sequence is, for example, 0 to 5, preferably 0 to 3, and more preferably 0 to 2. The base length of the complementary sequence relative to the total base length of the antisense strand (100%) is preferably 75 to 100%, more preferably 80 to 100%, even more preferably 85 to 100%, even more preferably 90 to 100%, particularly preferably 95 to 100%, and particularly preferably 100%.

[0055] The base length of the antisense strand is not particularly limited as long as the double-stranded oligonucleotide of the present invention can exert an inhibitory effect on genes containing an extended CAG repeat sequence, and the base length is, for example, 18 to 30, preferably 19 to 28, more preferably 20 to 26, even more preferably 21 to 24, and particularly preferably 21 to 22.

[0056] The double-stranded oligonucleotide of the present invention is characterized in that at least one of the nucleoside building blocks in the central portion of the antisense strand and / or at least one terminal nucleoside building block of the sense strand and / or antisense strand is an acyclic nucleoside building block represented by general formula (1). This allows genes containing extended CAG repeat sequences to be more selectively inhibited, the inhibitory effect can be exerted in vivo without relying on lipid nanoparticles, the distribution range in the central nervous system is wider, and / or the inhibitory effect can be exerted in vivo for a longer period of time. From the viewpoint of more effectively exerting these effects, in the double-stranded oligonucleotide of the present invention, at least one of the nucleoside building blocks in the central portion of the antisense strand and at least one terminal nucleoside building block of the sense strand and / or antisense strand is an acyclic nucleoside building block represented by general formula (1).

[0057] The general formula (1) is as follows:

[0058] In general formula (1), R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 Except when both are organic groups. Base refers to a nucleic acid base.

[0059] The organic group is not particularly limited, and examples thereof include hydrocarbon groups.

[0060] The hydrocarbon group is preferably a chain-like hydrocarbon group. Examples of the chain-like hydrocarbon group include alkyl groups, alkenyl groups, and alkynyl groups, with alkyl groups being particularly preferred. Specific examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, n-hexyl, and 3-methylpentyl groups. The number of carbon atoms in the hydrocarbon group is not particularly limited. The number of carbon atoms is preferably 1 to 8, more preferably 1 to 6, even more preferably 1 to 4, still more preferably 1 to 2, and particularly preferably 1. Furthermore, alkyl groups containing an alkynyl group (—C≡C—, —C≡CH) at the end or inside thereof, which allows for the introduction of various functional groups by click reactions and the like, are even more preferred.

[0061] In addition to the above, the organic group can also be a monovalent group formed by removing one hydrogen atom or functional group from various molecules, such as molecules used to modify oligonucleotides. Examples of such molecules include polyethylene glycol chains, dye molecules, polycations (spermine), groove binders, amino groups, hydroxyl groups, thiol groups, metal ligands, photocleavable functional groups, and sugar chains. These can be linked directly or indirectly to the backbone of the above-mentioned structural units. For example, they can be linked using a click reaction (e.g., the reaction between an alkyne and an azide, as described above).

[0062] R shown in Figure 1 1 and R 2 are both hydrogen atoms, SNA and R 1 is a methyl group and R 2 By molecular modeling of acyclic polynucleotides such as L-aTNA, where R is a hydrogen atom, 1 and R 2 Even if a relatively large organic group is used, it is thought that the double-strand forming ability and its structure will not be affected.

[0063] In a preferred embodiment of the present invention, R 1 and R 2 are preferably both hydrogen atoms (SNA shown in FIG. 1).

[0064] In general formula (1), Base represents a nucleic acid base.

[0065] In general formula (1), *1 and *2 indicate the direction in which the oligonucleotide is constructed. 1 is an organic group and R 2 When R is a hydrogen atom, *1 is the 3' side and *2 is the 1' side. 1 is a hydrogen atom and R 2 When R is an organic group, *1 is the 1' side and *2 is the 3' side. 1 is a hydrogen atom and R 2 If is a hydrogen atom, *1 is the (S) side and *2 is the (R) side.

[0066] The (S) and (R) ends (SNA in Figure 1), and the 3' and 1' ends (L-aTNA in Figure 1) correspond to the 5' and 3' ends of DNA, respectively. For convenience, the (S) and (R) ends, and the 3' and 1' ends may be referred to as the 5' and 3' ends, respectively.

[0067] When the acyclic nucleoside structural unit represented by general formula (1) is at the terminal, a hydrogen atom, a sulfur atom, or a phosphate group is added to *1 and *2 according to the above.

[0068] The central portion of the antisense strand literally refers to a region other than the ends of the antisense strand. The central portion of the antisense strand is preferably a region consisting of one nucleoside building block or 1 to 5 nucleoside building blocks on either side of one internucleoside bond in the center of the complementary sequence of the antisense strand. In this preferred embodiment, when the base length of the complementary sequence is an odd number, the center of the complementary sequence is one nucleoside building block, and when the base length of the complementary sequence is an even number, the center of the complementary sequence is one internucleoside bond. In this preferred embodiment, the central region is a region consisting of 1 to 5 (preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, and even more preferably 1) nucleoside structural units on either side of the central linkage; when the base length of the complementary sequence is an odd number, the central region is a region consisting of 3 to 11 (preferably 3 to 9, more preferably 3 to 7, even more preferably 3 to 5, and even more preferably 3) nucleoside structural units in total, consisting of the central nucleoside structural unit and the nucleoside structural units on either side of it; and when the base length of the complementary sequence is an even number, the central region is a region consisting of 2 to 10 (preferably 2 to 8, more preferably 2 to 6, even more preferably 2 to 4, and even more preferably 2) nucleoside structural units, consisting of the nucleoside structural units on either side of the central internucleoside bond.

[0069] In a preferred embodiment of the double-stranded oligonucleotide of the present invention, at least one (e.g., 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) of the nucleoside building blocks in the central portion of the antisense strand is an acyclic nucleoside building block represented by general formula (1).

[0070] At least one end of the sense strand and / or antisense strand refers to at least one (preferably 2 to 4, more preferably 2 to 3, and particularly preferably 3) of the four ends in total, consisting of the two ends of the sense strand and the two ends of the antisense strand. In a preferred embodiment, the at least one end is at least one end selected from the group consisting of the 5' end of the sense strand, the 3' end of the sense strand, and the 3' end of the antisense strand, and particularly preferably the 5' end of the sense strand, the 3' end of the sense strand, and the 3' end of the antisense strand.

[0071] The terminus of the sense strand / antisense strand literally means a region including the end of the sense strand / antisense strand. The terminus of the sense strand / antisense strand is preferably a region consisting of 1, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleoside units from the end.

[0072] When the acyclic nucleoside structural unit represented by general formula (1) is located at the terminal, a hydrogen atom, a sulfur atom, or a phosphate group is added to *1 and *2 according to the above. When the acyclic nucleoside structural unit represented by general formula (1) is contained in the central portion, adjacent nucleoside structural units are linked by an internucleoside bond as described above.

[0073] In a preferred embodiment of the double-stranded oligonucleotide of the present invention, the nucleoside constituent units at at least one terminal of the sense strand and / or antisense strand (i.e., all of the nucleoside constituent units constituting the terminal at least one terminal) are acyclic nucleoside constituent units represented by general formula (1).

[0074] The double-stranded oligonucleotide of the present invention may have both blunt ends, or at least one end may be a protruding end. In the latter case, it is more preferable that the 3' end of the sense strand and / or the 3' end of the antisense strand be an overhanging end (i.e., not forming a double strand). The base length of the protruding portion is, for example, 1 to 5, preferably 1 to 3, more preferably 2 to 3, and particularly preferably 2.

[0075] In the double-stranded oligonucleotide of the present invention, the nucleoside constituent units other than the acyclic nucleoside constituent units represented by general formula (1) are not particularly limited as long as they can exert an inhibitory effect on genes containing extended CAG repeat sequences, and can be, for example, ribonucleosides, deoxyribonucleosides, etc.

[0076] In a particularly preferred embodiment of the present invention, the sense strand consists of the base sequence shown in SEQ ID NO: 7, or a base sequence in which one to several (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) bases have been substituted with respect to the base sequence. In this embodiment, it is preferred that 1, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleoside constitutional units from each of the two termini are acyclic nucleoside constitutional units represented by general formula (1).

[0077] In a particularly preferred embodiment of the present invention, the antisense strand comprises a base sequence set forth in any one of SEQ ID NOS: 9 to 11, or a base sequence in which one or more bases (e.g., 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1) have been substituted with respect to the base sequence. In this embodiment, it is preferred that 1, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleoside structural units from the 3'-terminus are acyclic nucleoside structural units represented by general formula (1). It is also preferred that at least one (e.g., 1 to 5, preferably 1 to 4, more preferably 1 to 3, even more preferably 1 to 2, and especially preferably 1) of the nucleoside structural units located at positions 7 to 13 (preferably positions 8 to 12, more preferably positions 8 to 10, even more preferably positions 9 to 10, and particularly preferably positions 9) from the 5'-terminus is an acyclic nucleoside structural unit represented by general formula (1).

[0078] In the double-stranded oligonucleotide of the present invention, the internucleoside bond may contain a phosphorothioate bond, which increases nuclease resistance and further improves the inhibitory effect in vivo.

[0079] The number of phosphorothioate bonds that the double-stranded oligonucleotide of the present invention may contain is, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 6, still more preferably 1 to 4, particularly preferably 2 to 4, especially more preferably 2 to 3, and particularly preferably 2.

[0080] In one embodiment, all of the internucleoside linkages in at least one terminal region of the sense strand and / or antisense strand may be phosphorothioate linkages. The internucleoside linkages in the terminal region are terminal internucleoside linkages (linkages between the terminal nucleoside and the penultimate nucleoside) or multiple consecutive internucleoside linkages including the terminal internucleoside linkage. The number of internucleoside linkages in the terminal region is, for example, 1 to 6, preferably 1 to 4, more preferably 2 to 4, even more preferably 2 to 3, and particularly preferably 2. All of the internucleoside linkages in the 5'-terminal region of the antisense strand may be phosphorothioate linkages.

[0081] The double-stranded oligonucleotide of the present invention can be an siRNA or an miRNA.

[0082] The double-stranded oligonucleotide of the present invention can be easily prepared by known chemical nucleic acid synthesis methods or genetic engineering techniques. For example, it can be prepared using solid-phase synthesis, PCR, restriction enzyme cleavage, DNA ligation techniques, etc. The synthesis method for oligonucleotides containing acyclic nucleoside building blocks represented by general formula (1) is well known, and they can be synthesized using commercially available monomers or according to or in accordance with methods described in known literature (e.g., ChemBioChem, 2014, 15, 2549).

[0083] 2. Uses The double-stranded oligonucleotide of the present invention can be used, for example, as an active ingredient in a medicine, a reagent, etc. (sometimes referred to herein as the "drug of the present invention"). More specifically, the double-stranded oligonucleotide of the present invention can be used as an active ingredient in a pharmaceutical composition, an expression inhibitor of a gene containing an extended CAG repeat sequence, a drug for preventing or treating polyglutamine diseases, etc. The above are sometimes collectively referred to as the "drug of the present invention."

[0084] The extended CAG repeat sequence is a sequence in which the number of CAG repeats is increased relative to the normal type, and is not particularly limited thereto. The number of CAG repeats in the extended CAG repeat sequence can be, for example, 5 or more, preferably 10 or more, more preferably 20 or more, increased relative to the number of CAG repeats in the normal gene, and the upper limit of the increase in the number of CAG repeats is not particularly limited, and can be, for example, 300, 200, 150, or 100. The number of CAG repeats in the extended CAG repeat sequence is, for example, 30 or more, preferably 35 or more, more preferably 40 or more, even more preferably 45 or more, and even more preferably 50 or more, and the upper limit of the number of CAG repeats is not particularly limited, and can be, for example, 400, 300, 200, or 150.

[0085] Examples of genes containing extended CAG repeat sequences include the HTT gene (associated with Huntington's disease), the AR gene (associated with spinal-bulbar muscular atrophy), the ATN1 gene (associated with DRPLA), the ATXN1 gene (associated with spinocerebellar ataxia type 1), the ATXN2 gene (associated with spinocerebellar ataxia type 2), the ATXN3 gene (associated with spinocerebellar ataxia type 3), the CACNA1A gene (associated with spinocerebellar ataxia type 6), the ATXN7 gene (associated with spinocerebellar ataxia type 7), and the TBP gene (associated with spinocerebellar ataxia type 17).

[0086] Polyglutamine diseases are diseases caused by expanded CAG repeat sequences, and include, for example, Huntington's disease (HD), spinal-bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), spinocerebellar ataxia type 1 (SCA1), spinocerebellar ataxia type 2 (SCA2), spinocerebellar ataxia type 3 (SCA3 or Machado-Joseph disease), spinocerebellar ataxia type 6 (SCA6), spinocerebellar ataxia type 7 (SCA7), and spinocerebellar ataxia type 17 (SCA17). The agents of the present invention are useful as pharmaceuticals for preventing or treating these diseases.

[0087] The pharmaceutical agent of the present invention is not particularly limited as long as it contains an active ingredient, and may further contain additives as necessary, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, and chelating agents.

[0088] The mode of use of the agent of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type of agent. Depending on the intended use, the agent of the present invention can be used, for example, in vitro (e.g., added to a culture medium for cultured cells) or in vivo (e.g., administered to an animal).

[0089] The target subjects of the agents of the present invention are not particularly limited, and examples of mammals include humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer. Target subjects of the agents of the present invention can be, for example, patients, subjects with polyglutamine diseases, subjects with genes containing expanded CAG repeat sequences, etc. Target cells include animal cells, etc. The type of cell is also not particularly limited, and examples include neurons, glial cells, blood cells, hematopoietic stem and progenitor cells, gametes (sperm, ova), fibroblasts, epithelial cells, vascular endothelial cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0090] The agents of the present invention can be in any dosage form, for example, parenteral formulations such as injectable preparations (e.g., intraventricular administration, lumbar administration, infusion injections (e.g., intravenous infusion preparations, etc.), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye drops, eye ointments, nasal drops, and ear drops, or oral formulations such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, and jelly drops), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), and jellies. Furthermore, the active ingredient can be administered in a complexed state with particles (e.g., lipid particles or exosomes) or encapsulated in the particles. In one embodiment, the double-stranded oligonucleotides of the present invention can be administered without being complexed with particles such as lipid particles.

[0091] The content of the active ingredient in the drug of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0092] The dosage of the agent of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exerts a pharmacological effect, and is generally 0.1 to 1000 mg / kg body weight, preferably 0.5 to 500 mg / kg body weight per day in terms of the weight of the active ingredient, in the case of oral administration, and 0.01 to 100 mg / kg body weight, preferably 0.05 to 50 mg / kg body weight per day in the case of parenteral administration. The dosage and administration interval can be increased or decreased as appropriate depending on the age, pathological condition, symptoms, etc.

[0093] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0094] Test Example 1. Design of CAG repeat-targeting siRNA with central base mismatch / SNA modification. 21-base-long siRNAs were designed, including a sequence complementary to the CAG repeat (REP), a sequence with a central base mismatch (P) or SNA modification (S) added to the complementary sequence, and a negative control (NTC), and synthesis was outsourced to Hokkaido System Science Co., Ltd. Table 1 shows the names of the synthesized siRNAs, the base sequences of the sense and antisense strands, the type of nucleotides constituting each base (DNA, RNA, or SNA; see Figure 1 for the structure of each nucleotide), and the presence and location of mismatches. The numbers in parentheses to the right of each sequence in Table 1 indicate the sequence number.

[0095]

[0096] Test Example 2. siRNA Screening Using NSC34 Cells A construct was prepared by linking the NanoLuc® luciferase (Promega) gene to the androgen receptor (AR) gene, and then transfected into NSC34 cells to screen for siRNA using a reporter assay. Specifically, the NanoLuc gene was linked to a normal AR (28 CAG repeats) gene or a mutant AR (54 CAG repeats) gene to generate a construct expressed under the CMV promoter. To assess transfection efficiency, pGL4.53-PGK-firefly (Promega) was used as a reporter assay control. 25 nM of candidate siRNA (Test Example 1), 25 ng of pGL4.53-PGK-firefly, and 10 ng of constructs containing normal or mutant AR genes were co-transfected, and luciferase activity was measured 24 hours later.

[0097] The results are shown in Figure 2. While REP suppressed the expression of AR-28Q (normal AR protein) and AR-54Q (mutant AR protein) to a similar extent, P9, S9, P9S9, and P9S910 were found to more selectively suppress the expression of mutant AR proteins. In particular, S9, P9S9, and P9S910, which have SNAs introduced into the central region, suppressed the expression of mutant AR proteins even more selectively.

[0098] Test Example 3: Verification of the effect of siRNA with SNA modifications added to both ends Next, in order to confer nuclease resistance, SNA modifications were added to the terminal two bases of the antisense strand of these siRNAs, excluding the 5' end, to prepare 23-base-long sense strands and 21-base-long antisense strands (NTC-SNA, REP-SNA, P9-SNA, S9-SNA, P9S10-SNA, P12-SNA, S12-SNA).

[0099] The names of the synthesized siRNAs, the base sequences of the sense and antisense strands, the types of nucleotides constituting each base, and the presence and location of mismatches are shown in Table 2. The numbers in parentheses to the right of each sequence in Table 2 indicate the sequence number.

[0100]

[0101] A reporter assay was carried out in the same manner as in Test Example 2 using the above siRNA and the constructs of the normal AR gene and / or mutant AR gene prepared in Test Example 2.

[0102] The results are shown in Figure 3. It was revealed that the selectivity of P9-SNA and S9-SNA in suppressing the expression of mutant AR proteins was maintained even when SNA modifications were added to both ends.

[0103] Test Example 4: Verification of allele selectivity of SNA-siRNA in human fibroblasts. Using Lipofectamine RNAiMAX (Thermo Fisher Scientific), fibroblasts from a genetically confirmed SBMA (AR-52Q) patient and healthy control (AR-18Q) fibroblasts were administered 25 nM of siRNAs containing SNAs at both ends (Test Example 3: NTC-SNA, REP-SNA, P9-SNA, S9-SNA, P9S10-SNA, P12-SNA, S12-SNA) and Western blot analysis was performed 72 hours later to examine the suppression of AR protein expression. Dihydrotestosterone, an AR ligand, was added to the cell culture medium at 10 nM. The antibodies used were the anti-AR antibody Androgen Receptor (D6F11) XP (Cell Signaling Technology) and the anti-GAPDH antibody ab9485 (Abcam).

[0104] The results are shown in Figure 4. REP showed strong expression suppression without allele selectivity, while the others showed allele-selective expression suppression.

[0105] Test Example 5: Verification of S9-SNA-siRNA Knockdown in the Central Nervous System Using Neonatal Mice. This was evaluated by intracerebroventricular injection into AR-97Q mice, a mouse model of SBMA that expresses human AR with an expanded CAG repeat. AR-97Q mice were generated and maintained as described previously (Katsuno et al. Neuron (2002) 35:843-54). On postnatal day 1 (P1), AR-97Q mice were intracerebroventricularly injected with 2 μl of saline, Cy5-labeled siRNA S9-siRNA-Cy5, or S9-SNA-siRNA-Cy5. Seven days later, at P8, the mice were dissected, and spinal cord samples were collected and AR protein levels were compared by Western blotting. A total of 6 μg of S9-siRNA-Cy5 and S9-SNA-siRNA-Cy5 was administered per mouse. The antibodies used were anti-AR antibody Androgen Receptor (D6F11) XP (Cell Signaling Technology) and anti-GAPDH antibody ab9485 (Abcam).

[0106] The results are shown in Figure 5. S9-SNA-siRNA-Cy5 suppressed the expression of mutant AR protein compared to the saline-treated group, but S9-siRNA did not (top panel of Figure 5). We also tested the effect of S9-SNA-siRNA without the Cy5 label, and found that it had an inhibitory effect on expression equal to or greater than that of S9-SNA-siRNA-Cy5 (bottom panel of Figure 5).

[0107] Test Example 6: Verification of allele selectivity of S9-SNA-siRNA in the central nervous system using neonatal mice. To confirm the allele selectivity of S9-SNA-siRNA in vivo, AR-24Q transgenic mice expressing normal human AR repeats were intracerebroventricularly administered 2 μl of saline or S9-SNA-siRNP-Cy5 at P1. AR-24Q mice were generated and maintained as described previously (Katsuno et al. Neuron (2002) 35:843-54). Each mouse was administered 6 μg of siRNA. Seven days after administration, at P8, mice were dissected, and spinal cords were harvested and AR protein levels were compared by Western blotting. The antibodies used were the anti-AR antibody Androgen Receptor (D6F11) XP (Cell Signaling Technology) and the anti-GAPDH antibody ab9485 (Abcam).

[0108] The results are shown in Figure 6. No suppression of normal AR protein expression was observed in AR-24Q mice.

[0109] Test Example 7: Examination of distribution of S9-SNA-siRNA in adult mice 60 μg of Cy5-labeled S9-SNA-siRNA-Cy5 was intracerebroventricularly administered to 13-week-old AR-97Q mice, and distribution was evaluated 4 days later.

[0110] The results are shown in Figure 7. Widespread distribution in the central nervous system, including the brain and spinal cord, was observed, but no distribution in the liver was observed.

[0111] Test Example 8: Suppression of mutant AR protein expression in adult mice by S9-SNA-siRNA To confirm the duration of effect and therapeutic efficacy of S9-SNA-siRNA, 5 μl of saline or S9-SNA-siRNA was intracerebroventricularly administered to 9-week-old AR-97Q mice. 60 μg and 200 μg of siRNA were administered per mouse for comparison. Seven days after administration, the mice were dissected, and spinal cords were collected and AR protein levels were compared by Western blotting. The antibodies used were the anti-AR antibody Androgen Receptor (D6F11) XP (Cell Signaling Technology) and the anti-GAPDH antibody ab9485 (Abcam).

[0112] The results are shown in Figure 8. Suppression of the expression of spinal mutant AR protein was also observed in adult mice in a dose-dependent manner.

[0113] The same test was performed on other mice with a larger number of mice. The results are shown in Figure 12. The dose-dependent inhibitory effect of S9-SNA-siRNA on AR-97Q protein expression was demonstrated.

[0114] Test Example 9: Duration of protein expression suppression by S9-SNA-siRNA Saline or S9-SNA-siRNA was intracerebroventricularly administered to 7-week-old AR-97Q mice. On the 28th day, the mice were dissected and their spinal cords were collected. The mutant AR expression was compared by Western blotting. Each mouse was administered 200 μg / 5 μl of siRNA. The antibodies used were the anti-AR antibody Androgen Receptor (D6F11) XP (Cell Signaling Technology) and the anti-GAPDH antibody ab9485 (Abcam).

[0115] The results are shown in Figure 9. Suppression of the expression of the spinal cord mutant AR protein was observed even 28 days after injection.

[0116] The same test was performed on other mice with a larger n number. The results are shown in Figure 13. As above, suppression of spinal cord mutant AR protein expression was observed even 28 days after injection.

[0117] Test Example 10. Phenotype-ameliorating effect of S9-SNA-siRNA in AR-97Q mice Saline or S9-SNA-siRNA was intracerebroventricularly administered to 7-week-old AR-97Q mice, and survival, grip strength, and rotarod task were evaluated. Each group consisted of 13 mice, with 200 μg / 5 μl of siRNA administered per mouse. Body weight was measured using a scale (A & D Company Limited), grip strength was measured using a grip dynamometer (Muromachi Kikai), and the rotarod task was performed using a mouse rotarod (Ugo Basile).

[0118] The results are shown in Figure 10. Improvements in survival rate, grip strength, and rotarod test were observed in the S9-SNA-siRNA group.

[0119] Test Example 11: Suppression of Mutant Protein Expression in Spinocerebellar Ataxia Type 3 Mouse Models. To verify efficacy in other polyglutamine diseases, we also evaluated ATXN3-84Q mice (SCA3-YAC-84Q mice purchased from Jackson, MI), a mouse model of spinocerebellar ataxia type 3 (SCA3). Seven-week-old adult ATXN3-84Q mice were intracerebroventricularly administered 5 μl of saline or S9-SNA-siRNA. 400 μg of siRNA was administered for comparison. Seven days after administration, the mice were dissected, and the cerebrum, cerebellum, brainstem, and spinal cord were collected and compared for mutant ATXN3 protein expression by Western blotting. The antibodies used were anti-Ataxin3 antibody ab221143 (Abcam) and anti-GAPDH antibody ab9485 (Abcam).

[0120] The results are shown in Figure 11. High levels of suppression of mutant ATXN3 protein expression were observed in the cerebral cortex, cerebellum, and spinal cord, and mild suppression was observed in the brainstem.

[0121] Test Example 12: Verification of the pathological improvement effect of siRNA in AR-97Q mice. Vehicle or S9-SNA-siRNA (200 μg / 5 μl) was intracerebroventricularly administered to 7-week-old AR-97Q mice. Spinal cords were collected at 14 weeks of age, and the polyglutamine aggregate positivity rate in motor neurons, motor neuron atrophy, and quadriceps atrophy were evaluated by immunohistochemistry. Anti-polyglutamine antibody (anti-1C2 antibody, MAB1574), anti-ChAT antibody (AB144P), and HE staining were used.

[0122] The results are shown in Figures 14 to 16. The S9-SNA-siRNA group showed a decrease in the rate of spinal motor neuron polyglutamine aggregate-positive cells, and suppression of atrophy of spinal motor neurons and quadriceps femoris muscles.

[0123] Test Example 13: Verification of the Abnormal Protein Suppression Effect of siRNA in a Spinocerebellar Ataxia Type 3 Mouse Model. Spinocerebellar ataxia type 3 (SCA3) model mice (ATXN3-84Q mice) were used to verify the efficacy of siRNA against other polyglutamine diseases. Seven-week-old ATXN3-84Q mice were intracerebroventricularly administered 5 μl each of vehicle or S9-SNA-siRNA (200 or 400 μg of siRNA). Seven days after administration, the cerebellum was harvested and ATXN3 protein expression was verified by Western blot. Anti-Ataxin3 antibody (ab221143) and anti-GAPDH antibody (ab9485) were used.

[0124] The results are shown in Figure 17. In the cerebellum, S9-SNA-siRNA was found to have an inhibitory effect on the expression of mutant ATXN3, while no inhibition of the expression of endogenous mouse ATXN3 was observed.

[0125] Test Example 14: Verification of the durability of siRNA protein expression suppression in ATXN3-84Q mice To verify the durability of protein expression suppression, 5 μl of vehicle or S9-SNA-siRNA (400 μg of siRNA) was administered intracerebroventricularly as a bolus injection into 7-week-old ATXN3-84Q mice. Two months after administration, the cerebellum was harvested and mutant ATXN3 protein expression was verified by Western blot analysis. Anti-Ataxin3 antibody (ab221143) and anti-GAPDH antibody (ab9485) were used.

[0126] The results are shown in Figure 18. In the cerebellum, the effect of S9-SNA-siRNA in suppressing the expression of mutant ATXN3 was observed, but suppression of the expression of endogenous mouse ATXN3 was not observed.

[0127] Test Example 15: Verification of the pathology-ameliorating effect of siRNA in ATXN3-84Q mice. Seven-week-old ATXN3-84Q mice were administered 5 μl of vehicle or S9-SNA-siRNA (400 μg of siRNA) via intracerebroventricular bolus injection. Two months after administration, the cerebellum and brainstem were collected and the intranuclear ATXN3 positivity rates in the deep cerebellar nuclei and pontine nuclei were compared. Antibodies used were anti-Ataxin3 antibody (MAB5360), anti-NeuN antibody (ABN78A4A), and anti-Hoechst antibody (33342).

[0128] The results are shown in Figure 19. A decrease in the rate of nuclear ATXN3-positive cells was observed in the S9-SNA-siRNA administration group.

[0129] Test Example 16: Investigation of neuroinflammation induction by siRNA Seven-week-old ATXN3-84Q mice were administered 5 μl of vehicle or S9-SNA-siRNA (400 μg siRNA) via a lateral cerebroventricular bolus injection. Two months later, the cerebellum was harvested and the expression of the neuroinflammatory markers GFAP and IBA-1 was examined by Western blot and immunohistochemistry. Anti-GFAP antibody (GA5) and anti-IBA-1 / AIF-1 antibody (E4O4W) were used for Western blot, and anti-GFAP antibody (GA5) and anti-IBA-1 antibody (013-27691) were used for immunohistochemistry.

[0130] The results are shown in Figure 20. S9-SNA-siRNA did not show an increase in the expression of GFAP or IBA-1. Administration of the siRNA of the present invention did not induce inflammation in the nerves.

[0131] Test Example 17: Verification of siRNA allele selectivity using human fibroblasts. Fibroblasts derived from a patient with spinocerebellar ataxia type 3 (SCA3) (ATXN3-71 / 23Q) were treated with 25 nM NTC-SNA-siRNA, 25 nM REP-SNA-siRNA, and 2.5 or 25 nM S9-SNA-siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific). The suppression of ATXN3 protein expression after 48 hours was examined by Western blotting. Antibodies used were anti-ATXN3 antibody (EPR22418-147, Abcam) and anti-GAPDH antibody (ab9485, Abcam).

[0132] The results are shown in Figure 21. REP exhibited allele-nonselective expression suppression, while S9-SNA-siRNA exhibited mutant allele-selective expression suppression.

[0133] Test Example 18: Effect of Phosphorothioate-Modified S9-SNA-siRNA To achieve higher nuclease resistance, two internucleoside bonds in the 5'-terminal region of the antisense strand of S9-SNA-siRNA (the bond between the 5'-terminal nucleoside (G) and the penultimate nucleoside (C), and the bond between the penultimate nucleoside (C) and the third penultimate nucleoside (U)) were modified with phosphorothioate bonds (S9-SNA-siRNA-PS). 5 μl of vehicle or S9-SNA-siRNA-PS (200 μg of siRNA) was administered intracerebroventricularly as a bolus injection into 6-week-old AR-97Q mice. One month after administration, spinal cord samples were collected and mutant ATXN3 protein expression was examined by Western blot analysis. The antibodies used were an anti-AR antibody (Androgen Receptor (D6F11) XP (registered trademark): Cell signaling technology) and an anti-GAPDH antibody (ab9485: Abcam).

[0134] The results are shown in Figure 22. In the spinal cord, greater suppression of AR-97Q protein expression was observed compared to non-PS-modified siRNA under the same conditions.

Claims

1. A method for producing a nucleic acid molecule comprising a sense strand comprising a CAG repeat sequence and an antisense strand comprising a complementary sequence to the CAG repeat sequence, wherein at least one of the nucleoside building blocks in the center of the antisense strand and / or at least one terminal nucleoside building block of the sense strand and / or the antisense strand is represented by the general formula (1): [In the formula: R 1 and R 2 are the same or different and represent a hydrogen atom or an organic group (provided that R 1 and R 2 (Except when both are organic groups.) Base represents a nucleic acid base.] A double-stranded oligonucleotide in which the acyclic nucleoside building block is represented by the following formula:

2. The double-stranded oligonucleotide according to claim 1, wherein at least one of the nucleoside building blocks in the central portion of the antisense strand is an acyclic nucleoside building block.

3. The double-stranded oligonucleotide according to claim 2, wherein the nucleoside building block at at least one terminal of the sense strand and / or the antisense strand is the acyclic nucleoside building block.

4. The double-stranded oligonucleotide according to claim 1, wherein the central portion is a region consisting of one nucleoside building block or one to five nucleoside building blocks on either side of one internucleoside bond in the center of the complementary sequence.

5. The double-stranded oligonucleotide according to claim 1, wherein the terminal portion is a region consisting of 1, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleoside structural units from the end.

6. The double-stranded oligonucleotide of claim 5, wherein the termini are the 5' terminus of the sense strand, the 3' terminus of the sense strand, and the 3' terminus of the antisense strand.

7. The double-stranded oligonucleotide according to claim 1, wherein the 3'-end of the sense strand and / or the 3'-end of the antisense strand is an overhanging type.

8. The double-stranded oligonucleotide of claim 1, wherein the sense strand is 18 to 30 bases long and the antisense strand is 18 to 30 bases long.

9. The double-stranded oligonucleotide of claim 1, wherein the complementary sequence in the antisense strand has 0 to 5 mismatch mutations relative to the CAG repeat sequence in the sense strand.

10. The double-stranded oligonucleotide according to claim 1, wherein at least one of the nucleoside building blocks in the central portion of the antisense strand is the acyclic nucleoside building block; at least one terminal nucleoside building block of the sense strand and / or the antisense strand is the acyclic nucleoside building block; the central portion is a region consisting of 1 to 5 nucleoside building blocks on either side of a central nucleoside building block or an internucleoside bond in the complementary sequence; the terminal portions are regions consisting of 1, 1 to 2, 1 to 3, 1 to 4, or 1 to 5 nucleoside building blocks from the terminal; the 3'-end of the sense strand and / or the 3'-end of the antisense strand is an overhanging type; the sense strand is 18 to 30 bases long; and the antisense strand is 18 to 30 bases long.

11. An agent for inhibiting the expression of a gene containing an extended CAG repeat sequence, comprising the double-stranded oligonucleotide according to any one of claims 1 to 10.

12. A medicine comprising the double-stranded oligonucleotide according to any one of claims 1 to 10 or the expression inhibitor according to claim 11.

13. The pharmaceutical composition according to claim 12, which is used for the prevention or treatment of polyglutamine diseases.

Images