Oligonucleotide

Abstract

Provided is an oligonucleotide that is transnasally administered into the brain of a mammal.　The oligonucleotide is transnasally administered into the brain of a mammal by being injected into a cribriform foramen in the cribriform plate. The oligonucleotide may be transnasally administered into the brain of a mammal by using an administration device provided with a needle having a puncturing part, and may be administered through an opening of the puncturing part in a state where the puncturing part is disposed in a cribriform foramen in the cribriform plate.

Description

Oligonucleotides

[0001] The present invention relates to oligonucleotides that are administered intranasally into the mammalian brain using an administration device.

[0002] Since central nervous system (CNS) diseases are often caused by pathogenic proteins, approaches to the pathogenic proteins themselves or the genes involved in the expression of those proteins are considered effective for their treatment, prevention, and amelioration. For example, nucleic acid drugs can control protein expression by regulating the expression of specific genes, and therefore have long been expected to be applied to the treatment of CNS diseases.

[0003] On the other hand, the blood-brain barrier (BBB) ​​strictly controls the transport of substances between the circulating blood and brain tissue, and when drugs (nucleic acids, proteins, antibodies, etc.) are administered orally or intravenously, the amount of drug delivered to the brain is extremely limited.

[0004] Therefore, in order to administer drugs into the brain more effectively than oral or intravenous administration, research is being conducted on administration methods that can directly access the central nervous system by bypassing the blood-brain barrier. Currently, methods that are known include the nasal spray method, in which a drug is sprayed into the nasal cavity, the intraventricular administration method, in which a drug is administered directly into the ventricles, and the intrathecal administration method, in which a drug is injected into the spinal cavity (Biopharm. Drug Dispos. 44(1), 26-47 (2023)).

[0005] However, while the nasal spray method is minimally invasive, it has the problem of extremely low drug penetration. Furthermore, intraventricular administration involves drilling the skull and directly administering the drug into the ventricles, which carries the risk of injury from cerebral puncture. Intrathecal administration is a common method for administering nucleic acid drugs to treat central nervous system disorders, but it has many issues, including insufficient drug delivery to deep brain regions such as the striatum, high invasiveness, and difficulty in administering the drug to patients with scoliosis, a common neurological disorder.

[0006] The present invention has been made to solve the above-mentioned problems, and aims to provide an oligonucleotide that can be administered efficiently into the brain with minimal invasiveness and provides high efficacy in the treatment of central nervous system diseases using nucleic acid drugs.

[0007] The present inventors have conducted extensive research to solve the above problems, and as a result have found that the above problems can be solved by nasally administering an oligonucleotide using a specific administration device, which has led to the completion of the present invention.

[0008] That is, the above objects can be achieved by the present invention having the following configuration, and the present invention includes the following aspects and configurations.

[0009] One aspect of the present invention is: 1. An oligonucleotide administered intranasally into the mammalian brain by injection into the cribriform foramina of the cribriform plate.

[0010] 2. The oligonucleotide described in 1 above is preferably administered transnasally into the mammalian brain using an administration device equipped with a needle having a puncture portion, and injected through the opening of the puncture portion while the puncture portion is positioned within the cribriform foramina of the cribriform plate.

[0011] 3. The oligonucleotide described in 1 or 2 above is preferably any one selected from the group consisting of an aptamer, an antisense oligonucleotide, a decoy nucleic acid, a ribozyme, an siRNA, an miRNA, and an mRNA.

[0012] 4. The oligonucleotide according to any one of 1. to 3. above is preferably an siRNA or an antisense oligonucleotide.

[0013] 5. The oligonucleotide according to any one of 1. to 4. above is preferably an siRNA.

[0014] 6. In the oligonucleotide described in 5 above, the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is preferably 80% or more.

[0015] 7. In the oligonucleotide described in 5. or 6. above, all nucleotides constituting the siRNA are preferably 2'-modified nucleotides.

[0016] 8. The oligonucleotide according to any one of the above items 5 to 7 preferably has one or more lipophilic moieties.

[0017] 9. In the oligonucleotide described in 8 above, the lipophilic moiety is preferably at least one selected from the group consisting of a substituted or unsubstituted alkyl chain having 14 to 24 carbon atoms, eicosapentaenoic acid (EPA), arachidonic acid (ARA), and cholic acid.

[0018] 10. In the oligonucleotide according to any one of 5. to 9. above, the target gene of the siRNA is preferably SNCA or HTT.

[0019] 11. In the oligonucleotide according to any one of items 5 to 10 above, it is preferable that all of the phosphodiester bonds linking the first and second nucleotides counting from the ends of the sense and antisense strands of the siRNA are substituted with phosphorothioate bonds.

[0020] 12. See 5. to 11. above. In the oligonucleotide described in , the siRNA preferably comprises any one pair of nucleic acid sequences selected from the group consisting of the following (S1) to (S11): (S1) SEQ ID NO: 46 (sense strand) and SEQ ID NO: 213 (antisense strand) (S2) SEQ ID NO: 83 (sense strand) and SEQ ID NO: 250 (antisense strand) (S3) SEQ ID NO: 93 (sense strand) and SEQ ID NO: 260 (antisense strand) (S4) SEQ ID NO: 169 (sense strand) and SEQ ID NO: 336 (antisense strand) (S5) SEQ ID NO: 180 (sense strand) and SEQ ID NO: 347 (antisense strand) (S6) SEQ ID NO: 1360 (sense strand) and SEQ ID NO: 1389 (antisense strand) (S7) SEQ ID NO: 1366 (sense strand) and SEQ ID NO: 1395 (antisense strand) (S8) SEQ ID NO: 1368 (sense strand) and SEQ ID NO: 1397 (antisense strand) (S9) SEQ ID NO: 1372 (sense strand) and SEQ ID NO: 1401 (antisense strand) (S10) SEQ ID NO: 1374 (sense strand) and SEQ ID NO: 1403 (antisense strand) (S11) SEQ ID NO: 1756 (sense strand) and SEQ ID NO: 1759 (antisense strand)

[0021] 13. In the oligonucleotide described in 5. above, the siRNA preferably comprises any one set of oligonucleotide sequences selected from the group consisting of ds2, ds9, ds17 to ds249, and ds254 to ds273 listed in any one of Tables 1-1 to 1-6. In the table, "[L1]" represents a compound represented by the following formula (1), "^" represents a phosphorothioate bond, "mA", "mU", "mC", and "mG" represent 2'-O-methyl-RNA (i.e., "mA" represents 2'-O-methyladenosine, "mU" represents 2'-O-methyluridine, "mC" represents 2'-O-methylcytidine, and "mG" represents 2'-O-methylguanosine), "fA", "fU", "fC", and "fG" represent 2'-fluoro-DNA (i.e., "fA" represents 2'-Fluoro-2'-deoxyadenosine, "fU" represents 2'-Fluoro-2'-deoxyuridine, and "fC" represents 2'-Fluoro-2'-deoxycytidine). "fG" represents 2'-Fluoro-2'-deoxyguanosine; "dA," "dT," "dC," and "dG" represent DNA (i.e., "dA" represents deoxyadenosine, "dT" represents deoxythymidine, "dC" represents deoxycytidine, and "dG" represents deoxyguanosine); "rA," "rU," "rC," and "rG" represent RNA (i.e., "rA" represents adenosine, "rU" represents uridine, "rC" represents cytidine, and "rG" represents guanosine); "(vmU)" represents 5'-vinylphosphate-2'-O-methyl-uridine; and "p" represents 5'-phosphate.

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029] 14. In the oligonucleotide described in 13 above, the siRNA preferably comprises any one set of oligonucleotide sequences selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246 listed in any one of Tables 1-1 to 1-6.

[0030] 15. In the oligonucleotide described in 13 above, the siRNA is preferably any one set of oligonucleotides selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268, and ds273 described in any one of Tables 1-1 to 1-6.

[0031] 16. In the oligonucleotide described in 5. above, the siRNA preferably comprises any one set of oligonucleotide sequences selected from the group consisting of ds274 to ds461 described in any one of Tables 2-1 to 2-4. In the tables, "^" indicates a phosphorothioate bond, "mA", "mU", "mC", and "mG" indicate 2'-O-methyl-RNA (i.e., "mA" indicates 2'-O-methyladenosine, "mU" indicates 2'-O-methyluridine, "mC" indicates 2'-O-methylcytidine, and "mG" indicates 2'-O-methylguanosine), "fA", "fU", "fC", and "fG" indicate 2'-fluoro-DNA (i.e., "fA" indicates 2'-Fluoro-2'-deoxyadenosine). "fU" represents 2'-Fluoro-2'-deoxyuridine, "fC" represents 2'-Fluoro-2'-deoxycytidine, and "fG" represents 2'-Fluoro-2'-deoxyguanosine; "dA," "dT," "dC," and "dG" represent DNA (i.e., "dA" represents deoxyadenosine, "dT" represents deoxythymidine, "dC" represents deoxycytidine, and "dG" represents deoxyguanosine); and "p" represents 5'-phosphate.

[0032]

[0033]

[0034]

[0035]

[0036] 17. In the oligonucleotide described in 16 above, the siRNA preferably comprises any one set of oligonucleotide sequences selected from the group consisting of ds278, ds280, ds299, ds308, ds347, ds405, ds406, ds411, ds414, ds422, and ds432 to ds461 listed in any one of Tables 2-1 to 2-4.

[0037] One aspect of the present invention is: 18. A pharmaceutical composition comprising the oligonucleotide according to any one of 1. to 17. above.

[0038] One aspect of the present invention is: 19. A therapeutic agent for a central nervous system disease, comprising the oligonucleotide according to any one of 1. to 17. above.

[0039] 20. In the oligonucleotide described in 2. above, it is preferable that the administration device has a cannula portion formed of a tubular member that is arranged to cover the needle portion so that the puncture portion is exposed, the opening is provided on the tip side of the puncture portion, the tip of the cannula portion is formed with a stopper portion that has an abutment portion that abuts against the olfactory epithelium of the olfactory mucosa, and the opening is arranged within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.

[0040] 21. In the oligonucleotide described in 20. above, it is preferable that the administration device has a hub portion to which a container containing the oligonucleotide can be attached, and that the hub portion holds the base end of the cannula portion and / or the base end of the needle shaft portion of the needle portion inserted through the lumen of the cannula portion.

[0041] 22. In the oligonucleotide according to 20. or 21. above, the cross-sectional shape of the abutting portion of the stopper portion is preferably circular or elliptical.

[0042] One aspect of the present invention is an administration system comprising: 23. a storage section containing the oligonucleotide described in any one of 1. to 17. above; and an administration device equipped with a needle section having a puncture section, wherein the oligonucleotide contained in the storage section can be discharged through an opening in the puncture section. 24. In the administration system described in 23. above, the administration device has a cannula section formed of a tubular member that is arranged to cover the needle section so that the puncture section is exposed, the opening being provided on the tip side of the puncture section, and the tip of the cannula section is formed with a stopper section having an abutment section that abuts against the olfactory epithelium of the olfactory mucosa, and the opening is preferably arranged within the cribriform plate with the abutment section of the stopper section in contact with the olfactory epithelium. 25. In the administration system described in 24. above, the administration device has a hub section to which a storage section containing the oligonucleotide can be attached, and the hub section preferably holds the base end of the cannula section and / or the base end of the needle shaft section of the needle section inserted through the lumen of the cannula section. 26. In the dispensing system described in 24. or 25. above, it is preferable that the cross-sectional shape of the contact portion of the stopper portion is circular or elliptical.

[0043] The above-mentioned objects can also be achieved by the present invention having the following configuration, and the present invention also includes the following aspects and configurations.

[0044] Another aspect of the present invention is: 1. An oligonucleotide for use in treating a central nervous system disease, which is administered intranasally into the brain of a mammal by injection into the cribrosa of the cribrosa.

[0045] 2. Preferably, the oligonucleotide described in 1 above is administered transnasally into the mammalian brain using an administration device equipped with a needle having a puncture portion, and is injected through the opening of the puncture portion while the puncture portion is positioned within the cribriform foramina of the cribriform plate.

[0046] 3. The oligonucleotide described in 1 or 2 above is preferably any one selected from the group consisting of an aptamer, an antisense oligonucleotide, a decoy nucleic acid, a ribozyme, an siRNA, an miRNA, and an mRNA.

[0047] 4. The oligonucleotide according to any one of 1. to 3. above is preferably an siRNA or an antisense oligonucleotide.

[0048] 5. The oligonucleotide according to any one of 1. to 4. above is preferably an siRNA.

[0049] 6. In the oligonucleotide described in 5 above, the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is preferably 80% or more.

[0050] 7. In the oligonucleotide described in 5. or 6. above, all nucleotides constituting the siRNA are preferably 2'-modified nucleotides.

[0051] 8. The oligonucleotide according to any one of the above items 5 to 7 preferably has one or more lipophilic moieties.

[0052] 9. In the oligonucleotide described in 8 above, the lipophilic moiety is preferably at least one selected from the group consisting of a substituted or unsubstituted alkyl chain having 14 to 24 carbon atoms, eicosapentaenoic acid (EPA), arachidonic acid (ARA), and cholic acid.

[0053] 10. In the oligonucleotide according to any one of 5. to 9. above, the target gene of the siRNA is preferably SNCA or HTT.

[0054] 11. In the oligonucleotide according to any one of items 5 to 10 above, it is preferable that all of the phosphodiester bonds linking the first and second nucleotides counting from the ends of the sense and antisense strands of the siRNA are substituted with phosphorothioate bonds.

[0055] 12. In the oligonucleotide according to any one of 5. to 11. above, the siRNA preferably comprises any one pair of nucleic acid sequences selected from the group consisting of the following (S1) to (S11): (S1) SEQ ID NO: 46 (sense strand) and SEQ ID NO: 213 (antisense strand) (S2) SEQ ID NO: 83 (sense strand) and SEQ ID NO: 250 (antisense strand) (S3) SEQ ID NO: 93 (sense strand) and SEQ ID NO: 260 (antisense strand) (S4) SEQ ID NO: 169 (sense strand) and SEQ ID NO: 336 (antisense strand) (S5) SEQ ID NO: 180 (sense strand) and SEQ ID NO: 347 (antisense strand) (S6) SEQ ID NO: 1360 (sense strand) and SEQ ID NO: 1389 (antisense strand) (S7) SEQ ID NO: 1366 (sense strand) and SEQ ID NO: 1395 (antisense strand) (S8) SEQ ID NO: 1368 (sense strand) and SEQ ID NO: 1397 (antisense strand) (S9) SEQ ID NO: 1372 (sense strand) and SEQ ID NO: 1401 (antisense strand) (S10) SEQ ID NO: 1374 (sense strand) and SEQ ID NO: 1403 (antisense strand) (S11) SEQ ID NO: 1756 (sense strand) and SEQ ID NO: 1759 (antisense strand).

[0056] 13. In the oligonucleotide described in 5. above, the siRNA preferably comprises any one set of oligonucleotide sequences selected from the group consisting of ds2, ds9, ds17 to ds249, and ds254 to ds273 described in any one of Tables 1-1 to 1-6.

[0057] 14. In the oligonucleotide described in 13 above, the siRNA preferably comprises any one set of oligonucleotide sequences selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246 listed in any one of Tables 1-1 to 1-6.

[0058] 15. In the oligonucleotide described in 13 above, the siRNA is preferably any one set of oligonucleotides selected from the group consisting of ds2, ds254, ds257, ds259, ds261, ds263, ds267, and ds268 described in any one of Tables 1-1 to 1-6.

[0059] 16. In the oligonucleotide described in 5 above, the siRNA preferably comprises any one set of oligonucleotide sequences selected from the group consisting of ds274 to ds461 described in any one of Tables 2-1 to 2-4.

[0060] 17. In the oligonucleotide described in 16 above, the siRNA preferably comprises any one set of oligonucleotide sequences selected from the group consisting of ds278, ds280, ds299, ds308, ds347, ds405, ds406, ds411, ds414, ds422, and ds432 to ds461 listed in any one of Tables 2-1 to 2-4.

[0061] One aspect of the present invention is: 18. A pharmaceutical composition comprising the oligonucleotide according to any one of 1. to 17. above.

[0062] One aspect of the present invention is: 19. A therapeutic agent for a central nervous system disease, comprising the oligonucleotide according to any one of 1. to 17. above.

[0063] 20. In the oligonucleotide described in 2. above, it is preferable that the administration device has a cannula portion formed of a tubular member that is arranged to cover the needle portion so that the puncture portion is exposed, the opening is provided on the tip side of the puncture portion, the tip of the cannula portion is formed with a stopper portion that has an abutment portion that abuts against the olfactory epithelium of the olfactory mucosa, and the opening is arranged within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.

[0064] 21. In the oligonucleotide described in 20. above, it is preferable that the administration device has a hub portion to which a container containing the oligonucleotide can be attached, and that the hub portion holds the base end of the cannula portion and / or the base end of the needle shaft portion of the needle portion inserted through the lumen of the cannula portion.

[0065] 22. In the oligonucleotide according to 20. or 21. above, the cross-sectional shape of the abutting portion of the stopper portion is preferably circular or elliptical.

[0066] One aspect of the present invention is an administration system comprising: 23. a storage section containing the oligonucleotide described in any one of 1. to 17. above; and an administration device equipped with a needle section having a puncture section, wherein the oligonucleotide contained in the storage section can be discharged through an opening in the puncture section. 24. In the administration system described in 23. above, the administration device has a cannula section formed of a tubular member that is arranged to cover the needle section so that the puncture section is exposed, the opening being provided on the tip side of the puncture section, and the tip of the cannula section is formed with a stopper section having an abutment section that abuts against the olfactory epithelium of the olfactory mucosa, and the opening is preferably arranged within the cribriform plate with the abutment section of the stopper section in contact with the olfactory epithelium. 25. In the administration system described in 24. above, the administration device has a hub section to which a storage section containing the oligonucleotide can be attached, and the hub section preferably holds the base end of the cannula section and / or the base end of the needle shaft section of the needle section inserted through the lumen of the cannula section. 26. In the dispensing system described in 24. or 25. above, it is preferable that the cross-sectional shape of the contact portion of the stopper portion is circular or elliptical.

[0067] The above-mentioned objects can also be achieved by the present invention having the following configuration, and the present invention also includes the following aspects and configurations.

[0068] Another aspect of the present invention is: 1. Use of an oligonucleotide administered intranasally into the brain of a mammal by injecting it through the cribriform foramina of the cribriform plate for treating a central nervous system disease.

[0069] 2. In the use of 1 above, the oligonucleotide is preferably administered transnasally into the mammalian brain using an administration device equipped with a needle having a puncture portion, and injected through the opening of the puncture portion with the puncture portion positioned within the cribriform foramina of the cribriform plate.

[0070] 3. In the use according to 1 or 2 above, the oligonucleotide is preferably any one selected from the group consisting of an aptamer, an antisense oligonucleotide, a decoy nucleic acid, a ribozyme, an siRNA, an miRNA, and an mRNA.

[0071] 4. In the use according to any one of the above items 1 to 3, the oligonucleotide is preferably an siRNA or an antisense oligonucleotide.

[0072] 5. In the use according to any one of the above items 1 to 4, the oligonucleotide is preferably siRNA.

[0073] 6. In the use described in 5 above, it is preferable that the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is 80% or more.

[0074] 7. In the use described in 5. or 6. above, it is preferable that all nucleotides constituting the siRNA are 2'-modified nucleotides.

[0075] 8. The oligonucleotide according to any one of the above items 5 to 7 preferably has one or more lipophilic moieties.

[0076] 9. In the use described in 8 above, the lipid-soluble portion of the oligonucleotide is preferably at least one selected from the group consisting of a substituted or unsubstituted alkyl chain having 14 to 24 carbon atoms, eicosapentaenoic acid (EPA), arachidonic acid (ARA), and cholic acid.

[0077] 10. In the use according to any one of the above items 5 to 9, the target gene of the siRNA is preferably SNCA or HTT.

[0078] 11. In the use according to any one of the above items 5 to 10, it is preferable that the phosphodiester bonds linking the first and second nucleotides counting from both ends of the sense strand and the antisense strand of the siRNA in the oligonucleotide are all substituted with phosphorothioate bonds.

[0079] 12. In the use according to any one of 5. to 11. above, it is preferable that the siRNA comprises any one pair of nucleic acid sequences selected from the group consisting of the following (S1) to (S11): (S1) SEQ ID NO: 46 (sense strand) and SEQ ID NO: 213 (antisense strand) (S2) SEQ ID NO: 83 (sense strand) and SEQ ID NO: 250 (antisense strand) (S3) SEQ ID NO: 93 (sense strand) and SEQ ID NO: 260 (antisense strand) (S4) SEQ ID NO: 169 (sense strand) and SEQ ID NO: 336 (antisense strand) (S5) SEQ ID NO: 180 (sense strand) and SEQ ID NO: 347 (antisense strand) (S6) SEQ ID NO: 1360 (sense strand) and SEQ ID NO: 1389 (antisense strand) (S7) SEQ ID NO: 1366 (sense strand) and SEQ ID NO: 1395 (antisense strand) (S8) SEQ ID NO: 1368 (sense strand) and SEQ ID NO: 1397 (antisense strand) (S9) SEQ ID NO: 1372 (sense strand) and SEQ ID NO: 1401 (antisense strand) (S10) SEQ ID NO: 1374 (sense strand) and SEQ ID NO: 1403 (antisense strand) (S11) SEQ ID NO: 1756 (sense strand) and SEQ ID NO: 1759 (antisense strand).

[0080] 13. In the use described in 5. above, it is preferable that the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds2, ds9, ds17 to ds249, and ds254 to ds273 listed in any one of Tables 1-1 to 1-6.

[0081] 14. In the use described in 13 above, it is preferable that the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246 listed in any of Tables 1-1 to 1-6.

[0082] 15. In the use described in 13 above, the siRNA is preferably any one set of oligonucleotides selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268, and ds273 listed in any one of Tables 1-1 to 1-6.

[0083] 16. In the use described in 5. above, it is preferable that the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds274 to ds461 listed in any one of Tables 2-1 to 2-4.

[0084] 17. In the use described in 16 above, it is preferable that the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds278, ds280, ds299, ds308, ds347, ds405, ds406, ds411, ds414, ds422, and ds432 to ds461 listed in any of Tables 2-1 to 2-4.

[0085] 18. In the use described in 2 above, it is preferable that the administration device has a cannula portion formed of a tubular member that is arranged to cover the needle portion so that the puncture portion is exposed, the opening is provided on the tip side of the puncture portion, the tip of the cannula portion is formed with a stopper portion that has an abutment portion that abuts against the olfactory epithelium of the olfactory mucosa, and the opening is arranged within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.

[0086] 19. In the use described in 18 above, it is preferable that the administration device has a hub portion to which a container portion containing the oligonucleotide can be attached, and that the hub portion holds the base end of the cannula portion and / or the base end of the needle shaft portion of the needle portion inserted through the lumen of the cannula portion.

[0087] 20. In the use described in 18. or 19. above, it is preferable that the cross-sectional shape of the contact portion of the stopper portion is circular or elliptical.

[0088] FIG. 1 shows the structure of a phosphate-modified nucleotide contained in an oligonucleotide used in the examples. FIG. 2 shows the structure of a lipophilic moiety contained in an oligonucleotide used in the examples. FIG. 3 shows an example of an administration device and administration system used for nasally administering an oligonucleotide according to one embodiment of the present invention. FIG. 4-1 shows the state in which the puncture portion of the administration device has been inserted into a sieve hole. FIG. 4-2A is a first diagram illustrating a method for using the administration device. FIG. 4-2B is a second diagram illustrating a method for using the administration device. FIG. 4-2C is a third diagram illustrating a method for using the administration device. FIG. 4-2D is a fourth diagram illustrating a method for using the administration device. FIG. 4-2E is a fifth diagram illustrating a method for using the administration device. FIG. 5 shows the measurement results of Reference Example 1-1. FIG. 6 shows the measurement results of Reference Example 1-2. FIG. 7 shows the measurement results of Reference Example 1-3. FIG. 8 shows the measurement results of Reference Example 2. FIG. 9 shows the configurations of administration devices 1A and 1B and guide catheter 100A used in the examples. FIG. 10 is a diagram showing the measurement results of knockdown activity in Example 1. FIG. 11 is a diagram showing the measurement results of knockdown activity in Example 2. FIG. 12 is a diagram showing the measurement results of the amount of nucleic acid translocation in Example 2. FIG. 13 is a diagram showing the configurations of the administration device 1C and guide catheter 100B used in the Examples. FIG. 14-1 is a diagram showing the measurement results of knockdown activity (each brain region) in Example 3. FIG. 14-2 is a diagram showing the measurement results of knockdown activity (cervical spinal cord, thoracic spinal cord, and lumbar spinal cord) in Example 3. FIG. 15-1 is a diagram showing the measurement results of the amount of nucleic acid translocation (each brain region) in Example 3. FIG. 15-2 is a diagram showing the measurement results of the amount of nucleic acid translocation (cervical spinal cord, thoracic spinal cord, and lumbar spinal cord) in Example 3. FIG. 16 is a diagram showing the measurement results of knockdown activity in Example 4. FIG. 17 is a diagram showing the measurement results of the amount of nucleic acid translocation in Example 4. FIG. 18 is a diagram showing the measurement results of knockdown activity in Example 7. FIG. 19 is a diagram showing the configurations of the administration device 1D and guide catheter 100A used in Example 8.FIG. 20 shows the results of measuring knockdown activity in Example 8. FIG. 21 shows the results of measuring the amount of nucleic acid translocation in Example 8. FIG. 22 shows the results of measuring knockdown activity in Example 9. FIG. 23-1 shows the results of measuring knockdown activity by quantitative PCR (each brain region) in Example 10. FIG. 23-2 shows the results of measuring knockdown activity by quantitative PCR (spinal cord) in Example 10. FIG. 24-1 shows the results of measuring the amount of nucleic acid translocation (each brain region) in Example 10. FIG. 24-2 shows the results of measuring the amount of nucleic acid translocation (spinal cord) in Example 10. FIG. 25-1 shows the results of measuring knockdown activity by protein quantification (each brain region) in Example 10. FIG. 25-2 shows the results of measuring knockdown activity by protein quantification (spinal cord) in Example 10. FIG. 26 shows the results of measuring the change in knockdown activity over time (CSF) by protein quantification in Example 10. FIG. 27 shows the measurement results of knockdown activity in Example 11.

[0089] Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to only the following embodiments. Furthermore, in this specification, unless otherwise specified, operations and measurements of physical properties are performed under conditions of room temperature (20°C or higher and 25°C or lower) and relative humidity of 40% RH or higher and 50% RH or lower.

[0090] Furthermore, throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to be used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of a conflict, this specification (including definitions) shall prevail. The present invention is not limited to the following embodiments and can be modified in various ways within the scope of the claims. Furthermore, in this specification, "X to Y" means a range including the numerical values ​​(X and Y) described before and after it as the lower and upper limits, respectively, and means "X or more and Y or less." Furthermore, unless otherwise specified, concentration "%" represents mass concentration "% by mass," and ratios represent mass ratios unless otherwise specified.

[0091] An oligonucleotide according to one embodiment of the present invention is administered intranasally into the brain of a mammal by injection through the cribriform foraminae of the cribriform plate. Furthermore, an oligonucleotide according to one embodiment is administered intranasally into the brain of a mammal using an administration device equipped with a needle having a puncture portion, and is administered through the opening of the puncture portion while the puncture portion is positioned within the cribriform plate. The oligonucleotide according to one embodiment of the present invention can be administered (delivered) efficiently into the brain while being minimally invasive, and furthermore, can achieve high efficacy as a nucleic acid drug.

[0092] The present inventors speculate that the mechanism by which the above problems can be solved by the present invention is as follows.

[0093] As a result of extensive research, the present inventors have hypothesized that in known, minimally invasive nasal administration methods, such as conventional nasal spray methods, drug delivery to the brain is inhibited by clearance through the epithelial barrier. The oligonucleotide of the present invention takes this hypothesis into consideration and is administered intranasally to the mammalian brain by injection through the cribriform foramina of the cribriform plate.

[0094] Here, "injecting" an oligonucleotide through the sieve pores of the cribriform plate means that the oligonucleotide is directly injected into the sieve pores of the cribriform plate. The injection can be performed using a medical device, such as a syringe needle, that has a structure capable of injecting an oligonucleotide into the sieve pores of the cribriform plate. The oligonucleotide injected into the sieve pores of the cribriform plate flows into the brain through the sieve pores of the cribriform plate due to injection pressure or the like, thereby enabling transnasal administration of the oligonucleotide into the brain. For example, an oligonucleotide according to one embodiment of the present invention can be administered transnasally into the brain by placing the puncture portion of an administration device within the sieve pores of the cribriform plate and injecting the oligonucleotide (drug) through the opening of the puncture portion.

[0095] Thus, despite being administered intranasally, the oligonucleotides of the present application can be administered from within the cribriform foramina of the cribriform plate, a location close to the brain. This suggests that the oligonucleotides of the present invention are less affected by clearance by the epithelial barrier and achieve a high rate of delivery into the brain. Furthermore, because a sufficient amount of oligonucleotide is delivered to the brain, a sufficient amount of oligonucleotide is also taken up into tissues in each region of the brain, which is thought to improve the efficacy of the oligonucleotide. For example, because the amount of oligonucleotide required for reducing (knockdown) the expression of proteins that cause central nervous system disorders (hereinafter also referred to as central disorders) reaches each region of the brain, the knockdown efficiency at each region is improved, which is thought to improve the efficacy of the oligonucleotide as a nucleic acid drug. Note that the above mechanism is speculative, and the present invention is not limited to this mechanism in any way.

[0096] [Oligonucleotide] The oligonucleotide according to one embodiment of the present invention may be any molecule formed by polymerizing nucleotides or molecules having equivalent functions to the nucleotides, such as RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; chimeric nucleic acids, which are polymers of ribonucleotides and deoxyribonucleotides; and nucleotide polymers in which at least one nucleotide of these nucleic acids (RNA, DNA, and chimeric nucleic acids) has been replaced with a molecule having equivalent functions to the nucleotide. Uracil (U) in RNA is unambiguously interpreted as thymine (T) in DNA.

[0097] Examples of molecules having functions equivalent to nucleotides include, but are not limited to, nucleotide derivatives obtained by modifying nucleotides. Use of nucleotide derivatives has the advantages of, for example, being able to improve the amount of transfer to each site in the brain, improving or stabilizing nuclease resistance, improving affinity with complementary nucleic acid strands, improving cell permeability, and / or enabling visualization, compared to RNA or DNA.

[0098] The oligonucleotide according to the present invention may be an oligonucleotide known to be used as a nucleic acid drug. Among these, the oligonucleotide according to one embodiment of the present invention is preferably selected from the group consisting of aptamers, antisense oligonucleotides (ASOs), decoy nucleic acids, ribozymes, siRNAs, miRNAs (microRNAs), and mRNAs (messenger RNAs), and more preferably, the oligonucleotide according to one embodiment of the present invention is an siRNA or an antisense oligonucleotide. By using an oligonucleotide selected from these, it is possible to more effectively bind to target molecules, and more effectively control gene transcription and translation processes, inhibit protein function, and the like.

[0099] Here, an aptamer is a single-stranded nucleic acid molecule that binds to a specific target molecule (e.g., a protein) and can bind to a disease-related target protein and inhibit its function.

[0100] Antisense oligonucleotides are DNA, RNA, or chimeric molecules of DNA and RNA that are at least partially complementary to the base sequence of a target molecule (e.g., mRNA or miRNA), and may be single-stranded. For example, antisense oligonucleotides inhibit the translation of a complementary RNA strand by binding to the complementary RNA strand. They can also control splicing by binding to a specific complementary pre-mRNA. Antisense oligonucleotides may also be heterodimers (HDOs) that form a double strand with a complementary strand.

[0101] Ribozymes, also known as RNA enzymes, are a general term for catalytic RNAs. Ribozymes can be designed to cleave their own or other target RNA molecules, making them useful as inhibitors of gene expression.

[0102] A decoy nucleic acid is a double-stranded oligonucleotide that targets a nucleic acid-recognition protein such as a transcription factor. The decoy nucleic acid mimics the nucleic acid sequence recognized by the nucleic acid-recognition protein and inhibits the protein from binding to the target nucleic acid.

[0103] siRNA stands for small interfering RNA and is a double-stranded oligonucleotide involved in RNA interference (hereinafter referred to as RNAi). The oligonucleotide constituting the siRNA may be composed of RNA or may be composed of RNA and DNA. More specifically, siRNA functions as a guide for suppressing the expression of a target gene, which is a target molecule, and can selectively suppress (knock down) the expression of a protein whose expression is controlled by messenger RNA (mRNA) through cleavage of that mRNA. Thus, from the viewpoint of more efficiently suppressing the expression of a target protein and more effectively treating central nervous system disorders, it is more preferable that the oligonucleotide according to one embodiment of the present invention is an siRNA.

[0104] When the oligonucleotide according to one embodiment is an siRNA, it is preferred that the phosphodiester bonds linking the first and second nucleotides counting from both ends of the sense strand and antisense strand constituting the siRNA are all replaced with phosphorothioate bonds.More preferably, the phosphodiester bonds linking the first and second nucleotides and the second and third nucleotides counting from both ends of the sense strand and antisense strand are all replaced with phosphorothioate bonds.By having the above-mentioned configuration, the knockdown effect of siRNA can be improved.

[0105] In one embodiment of the present invention, a hairpin siRNA can be used, which contains a sense strand and an antisense strand in a single-stranded oligonucleotide and forms complementary base pairs within the molecule.

[0106] miRNAs (microRNAs) are small RNA molecules of approximately 22 bases encoded in the genome that regulate gene expression by suppressing the translation of target mRNAs that have partially complementary sequences to themselves.

[0107] mRNA (messenger RNA) is an oligonucleotide that encodes information for a target protein, and can be used to treat or prevent disease by producing the target protein within cells.

[0108] When the oligonucleotide according to one embodiment of the present invention is single-stranded and is other than mRNA, the number of nucleotides (i.e., the number of bases) forming the single strand may be 10 to 100, 10 to 70, 10 to 60, 10 to 45, 10 to 30, 10 to 20, 12 to 100, 12 to 70, 12 to 60, 12 to 45, 12 to 30, 12 to 20, 15 to 100, 15 to 70, 15 to 60, 15 to 45, 15 to 30, 15 to 20, 30 to 100, 30 to 70, 30 to 60, 30 to 45, 40 to 100, 40 to 70, 40 to 60, or 40 to 45. For example, when the oligonucleotide according to one embodiment of the present invention is a single-stranded antisense oligonucleotide, the number of nucleotides (i.e., the number of bases) forming the single strand is preferably 10 to 30, more preferably 11 to 25, and even more preferably 12 to 20. Furthermore, for example, when the oligonucleotide according to one embodiment of the present invention is a single-stranded siRNA having a loop structure such as a hairpin, the number of nucleotides forming the single strand is preferably 30 to 70, and more preferably 40 to 60. Furthermore, when the oligonucleotide according to one embodiment of the present invention is a single-stranded aptamer, the number of nucleotides (i.e., the number of bases) forming the single strand is preferably 10 to 100, and more preferably 15 to 45.

[0109] Furthermore, when the oligonucleotide according to one embodiment of the present invention is a single-stranded mRNA, the number of nucleotides (i.e., the number of bases) forming the single strand is preferably 50 to 15,000, and more preferably 100 to 10,000.

[0110] Furthermore, when the oligonucleotide according to one embodiment of the present invention is double-stranded, each single strand forming the double strand preferably has a number of 10 to 30, more preferably 15 to 25. That is, the number of nucleotides constituting the sense strand is preferably 10 to 30, more preferably 15 to 25. Similarly, the number of nucleotides constituting the antisense strand is preferably 10 to 30, more preferably 15 to 25. For example, when the oligonucleotide according to one embodiment of the present invention is a double-stranded siRNA, the number of nucleotides constituting the sense strand is preferably 10 to 30, more preferably 15 to 25. Similarly, the number of nucleotides constituting the antisense strand is preferably 10 to 30, more preferably 15 to 25. By having the number of nucleotides constituting the oligonucleotide within the above range, it is easier to be taken up into cells more efficiently and the binding strength with the target molecule is stronger, thereby further improving the efficacy of the oligonucleotide.

[0111] Furthermore, when the oligonucleotide according to one embodiment of the present invention is single-stranded and is other than mRNA, the molecular weight (Da) may be 3,000 to 30,000, 3,000 to 21,000, 3,000 to 18,000, 3,000 to 13,500, 3,000 to 9,000, 3,000 to 6,000, 3,600 to 30,000, 3,600 to 21,000, 3,600 to 18,000, 3,600 to 13,500, 3,600 to 9,000, 3,600 to 6,000, 000 or less, 4500 to 30,000, 4500 to 21,000, 4500 to 18,000, 4500 to 13,500, 4500 to 9,000, 4500 to 6,000, 9,000 to 30,000, 9,000 to 21,000, 9,000 to 18,000, 9,000 to 13,500, 12,000 to 30,000, 12,000 to 21,000, 12,000 to 18,000, or 12,000 to 13,500. For example, when the oligonucleotide according to one embodiment of the present invention is a single-stranded antisense oligonucleotide, the molecular weight is preferably 3,000 to 9,000, more preferably 3,200 to 8,000, even more preferably 3,400 to 7,000, and particularly preferably 3,600 to 6,000. Furthermore, for example, when the oligonucleotide according to one embodiment of the present invention is a single-stranded siRNA having a loop structure such as a hairpin, the molecular weight is preferably 9,000 to 21,000, more preferably 12,000 to 18,000. Furthermore, when the oligonucleotide according to one embodiment of the present invention is a single-stranded aptamer, the molecular weight is preferably 3,000 to 30,000, more preferably 4,500 to 13,500.

[0112] Furthermore, when the oligonucleotide according to one embodiment of the present invention is a single-stranded mRNA, the molecular weight is preferably 15,000 or more and 4,500,000 or less, and more preferably 30,000 or more and 3,000,000 or less.

[0113] Furthermore, when the oligonucleotide according to one embodiment of the present invention is double-stranded, the molecular weight is preferably 6,000 to 20,000, more preferably 9,000 to 18,000, and even more preferably 13,000 to 16,000. For example, when the oligonucleotide according to one embodiment of the present invention is a double-stranded siRNA, the molecular weight is preferably 6,000 to 20,000, more preferably 9,000 to 18,000, and even more preferably 13,000 to 16,000. When the molecular weight of the oligonucleotide is within the above range, it is more easily taken up into cells and has a stronger binding strength with the target molecule, thereby further improving the efficacy of the oligonucleotide.

[0114] (Target Molecule) The target molecule targeted by the oligonucleotide according to one embodiment of the present invention is not particularly limited, and may be a molecule that can provide therapeutic, preventive, or ameliorative benefits for central nervous system diseases, particularly cranial nervous system diseases. For example, when the target molecule is a target gene, it may be a gene that can provide therapeutic, preventive, or ameliorative benefits for central nervous system diseases, particularly cranial nervous system diseases, upon knockdown of the target gene.

[0115] In this specification, central nervous system disease (also referred to as central disease) refers to a disease of the central nervous system. Examples of central nervous system diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), dementia with Lewy bodies, multiple system atrophy (MSA), spinal muscular atrophy (SMA), Dravet syndrome, Angelman syndrome, spinocerebellar ataxia, Alexander disease, progressive supranuclear palsy (PSP), multiple sclerosis, prion disease, lysosomal disease, peroxisomal disease, mitochondrial disease, glioblastoma, and cerebral infarction.

[0116] Examples of target molecules include SOD1 (Cu / Zn superoxide dismutase), FUS (fused in sarcoma), C9ORF72, or ATXN2 (ataxin 2), which are involved in amyotrophic lateral sclerosis (ALS). Other examples of target molecules include APP (amyloid precursor protein) and tau, which are involved in Alzheimer's disease. Other examples of target molecules include LRRK2 (leucine-rich repeat kinase 2) and SNCA (α-synuclein), which are involved in Parkinson's disease. Other examples of target molecules include HTT (huntingtin), which is involved in Huntington's disease. Other examples of target molecules include ATXN3, which is involved in spinocerebellar ataxia. Other examples of target molecules include UBE3A antisense transcripts, which are involved in Angelman syndrome. Other examples of target molecules include GFAP, which is involved in Alexander disease, and SMN2, which is involved in spinal muscular atrophy (SMA).

[0117] That is, the oligonucleotide according to one embodiment of the present invention may target SOD1 (Cu / Zn superoxide dismutase), FUS (fused in sarcoma), C9ORF72, ATXN2 (ataxin 2), APP (amyloid precursor protein), tau, LRRK2 (leucine-rich repeat kinase 2), SNCA (α-synuclein), HTT (huntingtin), ATXN3, UBE3A antisense transcript, GFAP, or SMN2.

[0118] Furthermore, when the oligonucleotide according to one embodiment of the present invention is an siRNA, the target gene can be any of the target molecules described above, and the target gene is preferably SNCA (α-synuclein) or HTT (huntingtin). Because the siRNA according to one embodiment of the present invention can more effectively knock down these target genes, it is expected to be highly effective in the treatment, prevention, and amelioration of synucleinopathies such as MSA and Parkinson's disease, and Huntington's disease.

[0119] (Nucleotide Derivatives) Examples of nucleotide derivatives include sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and phosphate group-modified nucleotides. Various modified nucleotides are described in detail below, but one nucleotide derivative may have the characteristics of two or more types of modified nucleotides. For example, a sugar-modified nucleotide may have the characteristics of a base-modified nucleotide, or may have the characteristics of a phosphate group-modified nucleotide.

[0120] In an oligonucleotide according to one embodiment of the present invention, the proportion of nucleotide derivatives relative to the total nucleotides constituting the oligonucleotide may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50%, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or even 100%. When the proportion of nucleotide derivatives relative to the total nucleotides constituting the oligonucleotide is within the above range, the efficiency of translocation of the oligonucleotide to each site in the brain is improved.

[0121] <Sugar-Modified Nucleotides> The sugar-modified nucleotide may be any nucleotide in which part or all of the chemical structure of the sugar of the nucleotide has been modified or substituted with any substituent, or substituted with any atom, but 2'-modified nucleotides are preferably used. That is, in an oligonucleotide according to one embodiment of the present invention, at least one of the nucleotides constituting the oligonucleotide is preferably a 2'-modified nucleotide.

[0122] Furthermore, in an oligonucleotide according to one embodiment of the present invention, the proportion of 2'-modified nucleotides relative to all nucleotides constituting the oligonucleotide is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, and particularly preferably 99% or more (upper limit: 100%). When the proportion of 2'-modified nucleotides relative to all nucleotides constituting the oligonucleotide is within the above range, the efficiency of translocation of the oligonucleotide to various sites in the brain is further improved.

[0123] Furthermore, in one embodiment of the present invention, the oligonucleotide most preferably comprises 100% 2'-modified nucleotides out of all nucleotides constituting the oligonucleotide. That is, in one embodiment of the present invention, all nucleotides constituting the oligonucleotide most preferably comprise 2'-modified nucleotides (claim 7). This configuration more sufficiently improves the amount of the oligonucleotide translocated to each site in the brain.

[0124] Examples of 2'-modified nucleotides include those in which the 2'-OH group of ribose is -H, -OR, -R, -R'OR, -SH, -SR, or -NH ２ , -NHR, -NR ２ , -N ３ , —CN, —F, —Cl, —Br, and —I (wherein R is an alkyl group or an aryl group, preferably an alkyl group having 1 to 6 carbon atoms; R′ is an alkylene group, preferably an alkylene group having 1 to 6 carbon atoms; and —NR ２(wherein the two R's may be the same or different). Among these, substitution with -H, -F, a methoxy group, a methoxyethoxy group, or an ethoxy group is preferred as the 2'-modification. That is, in one embodiment of the present invention, the 2'-modified nucleotide may be a 2'-modified nucleotide in which the 2'-OH group of ribose is substituted with a substituent selected from the group consisting of -F, a methoxy group, a methoxyethoxy group, and an ethoxy group. Examples of such 2'-modified nucleotides include 2'-O-methyl-RNA, 2'-fluoro-DNA, and 2'-O-methoxyethyl-RNA.

[0125] The 2'-modified nucleotide may also be a nucleotide having two or more substituents at the 2' position, such as -F and a methoxy group (J. Am. Chem. Soc. 2022, 144, 14517-14534). The 2'-modified nucleotide may also be a combination of a 4'-modified nucleotide or a 5'-modified nucleotide (see, for example, Nucleic Acids Research, 2018, Vol. 46, No. 16, 8090-8104, or Nucleic Acids Research, 2020, Vol. 48, No. 1810101-10124).

[0126] The 2'-modified nucleotide may be, for example, a 2'-OH group of ribose substituted with a substituent selected from the group consisting of a 2-(methoxy)ethoxy group, a 3-aminopropoxy group, a 2-[(N,N-dimethylamino)oxy]ethoxy group, a 3-(N,N-dimethylamino)propoxy group, a 2-[2-(N,N-dimethylamino)ethoxy]ethoxy group, a 2-(methylamino)-2-oxoethoxy group, a 2-(N-methylcarbamoyl)ethoxy group, and a 2-cyanoethoxy group.

[0127] Furthermore, as the sugar-modified nucleotide, a bridged structure-type artificial nucleic acid (BNA) having two cyclic structures by introducing a bridged structure into the sugar moiety is also preferably used. Specifically, there are Locked Nucleic Acids (LNAs) in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via a methylene [Tetrahedron Letters, 38, 8735 (1997) and Tetrahedron, 54, 3607 (1998)], Ethylene-bridged Nucleic Acids (ENAs) [Nucleic Acid Research, 32, e175 (2004)], Constrained Ethyl (cEt) [The Journal of Organic Chemistry, 75, 1569 (2010)], Amido-Bridged Nucleic Acid (AmNA) [Chem Bio Chem 13, 2513 (2012)], 2'-O,4'-C-Spirocyclopropylene bridged nucleic acid (scpBNA) [Chem. Commun. , 51, 9737 (2015)], tricycloDNA (tcDNA) [Nat. Biotechnol., 35, 238 (2017)], and the like.

[0128] Other sugar-modified nucleotides include Unlocked Nucleic Acid (UNA) [Mol. Ther. Nucleic Acids 2, e103 (2013)] and GNA having a glycerol backbone [RNA. 2023 Apr; 29 (4): 402-414. ], ANA or FANA having an arabinose backbone [Nucleic Acids Res. 2006; 34(2): 451-461.], TNA having a threose backbone [J. Am. Chem. Soc. 2023, 145, 19691-19706], ANA having an altritol backbone [4028-4040, Nucleic Acids Research, 2020, Vol. 48, No. 8], HNA having an anhydrohexitol backbone or cyclohexene backbone, or CeNA [Chem. Commun., 2016, 52, 13467-13470] are also preferably used. Among these, bridged artificial nucleic acids (BNAs) are preferably used from the viewpoint of increasing the thermal stability of the double-stranded structure.

[0129] Further examples of sugar-modified nucleotides 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)].

[0130] <Phosphodiester bond-modified nucleotide> The phosphodiester bond-modified nucleotide may be any nucleotide in which part or all of the chemical structure of the phosphodiester bond of the nucleotide has been modified or substituted with any substituent or substituted with any atom.

[0131] In an oligonucleotide according to one embodiment of the present invention, at least one of the nucleotides constituting the oligonucleotide is preferably a phosphodiester-linked modified nucleotide. Furthermore, in an oligonucleotide according to one embodiment of the present invention, the proportion of phosphodiester-linked modified nucleotides relative to the total nucleotides constituting the oligonucleotide may be 5% to 50%, 5% to 40%, 7% to 40%, 7% to 30%, 7% to 25%, 10% to 40%, 10% to 30%, 10% to 25%, 15% to 40%, 15% to 30%, or 15% to 25%. When the proportion of phosphodiester-linked nucleotides relative to the total nucleotides constituting the oligonucleotide is within the above range, the efficiency of delivery of the oligonucleotide to various sites in the brain and the efficacy of the oligonucleotide (e.g., the efficiency of knocking down a target gene) are further improved.

[0132] Examples of nucleotides modified with a phosphodiester bond include nucleotides in which a phosphodiester bond is replaced with a phosphorothioate bond, nucleotides in which a phosphodiester bond is replaced with a phosphorodithioate bond, nucleotides in which a phosphodiester bond is replaced with an alkylphosphonate bond, nucleotides in which a phosphodiester bond is replaced with a boranophosphate bond, nucleotides in which a phosphodiester bond is replaced with an amide bond, nucleotides in which a phosphodiester bond is replaced with a sulfonamide bond, nucleotides in which a phosphodiester bond is replaced with a phosphonoacetate bond (PACE), nucleotides in which a phosphodiester bond is replaced with a 2'-5' bond [Bioorg. Med. Chem. Lett. 16, 3238-3240], and nucleotides in which a phosphodiester bond is replaced with a (mesyl)phosphoramidate bond [Nucleic Acids Research, 2021, Vol. 49, No. 16, 9026-9041], and preferably nucleotides in which the phosphodiester bond is replaced with a phosphorothioate bond. By replacing the phosphodiester bond with such a bond, the efficiency of delivery of the oligonucleotide to various sites in the brain and the efficacy of the oligonucleotide (e.g., the efficiency of knocking down the target gene) are further improved.

[0133] The phosphodiester bond-modified nucleotide may be an optical isomer (Rp, Sp). Methods for selectively synthesizing optical isomers of phosphorothioate bonds are disclosed, for example, in J. Am. Chem. Soc., 124, 4962 (2002), Nucleic Acids Research, 42, 13546 (2014), and Science, 361, 1234 (2018).

[0134] <Base-Modified Nucleotide> A base-modified nucleotide may be any nucleotide in which part or all of the chemical structure of the base of the nucleotide is modified or substituted with any substituent or substituted with any atom.

[0135] Examples of base-modified nucleotides include nucleotides in which the oxygen atom in the base is replaced with a sulfur atom, nucleotides in which the hydrogen atom in the base is replaced with an alkyl group having 1 to 6 carbon atoms, a halogen, or the like, nucleotides in which the methyl group is replaced with hydrogen, hydroxymethyl, an alkyl group having 2 to 6 carbon atoms, or the like, and nucleotides in which the amino group is replaced with an alkyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms, an oxo group, a hydroxy group, or the like. Examples of base-modified nucleotides are disclosed in J. Org. Chem. 2011, 76, 7295-7300. Specific examples include 5-methylcytosine, in which the 5-position of cytosine is substituted with a methyl group.

[0136] <Phosphate group-modified nucleotide> The phosphate group-modified nucleotide may be any nucleotide in which the phosphate group at the 5' position of the nucleotide is modified or substituted with any substituent or substituted with any atom. However, vinyl phosphonate groups, (PO(OH) ２ (CH═CH—)), or (PO(OH) ２ (CH ２ CH ２ )-). Substitution with such a modification group can improve the knockdown efficiency of the target gene. Specific examples of phosphate-modified nucleotides include (vnT) and (vmU) shown in Figure 1. Other phosphate-modified nucleotides are disclosed in WO 2011 / 39699 and WO 2011 / 39702.

[0137] In an oligonucleotide according to one embodiment of the present invention, at least one of the nucleotides constituting the oligonucleotide is preferably a phosphate-modified nucleotide. When the oligonucleotide contains one or more phosphate-modified nucleotides, the efficiency of the oligonucleotide's delivery to various sites in the brain and the efficiency of target gene knockdown are further improved.

[0138] Furthermore, an oligonucleotide according to one embodiment of the present invention may include an oligonucleotide derivative described in WO 2018 / 199340. That is, an oligonucleotide according to one embodiment of the present invention may include an oligonucleotide derivative or a salt thereof comprising a cyclic oligonucleotide and a linear oligonucleotide, wherein the cyclic oligonucleotide and the linear oligonucleotide have complementary base sequences, and the cyclic oligonucleotide and the linear oligonucleotide form a complex via hydrogen bonds between the complementary base sequences. Furthermore, the cyclic oligonucleotide may have a length of 10 to 40 bases and may contain at least one phosphorothioate bond. Furthermore, the base length of the cyclic oligonucleotide may be the same as or longer than the base length of the linear oligonucleotide.

[0139] The cyclic oligonucleotide may also be represented by Formula 2:

[0140]

[0141] (Fat-soluble portion) The oligonucleotide according to one embodiment of the present invention preferably has one or more fat-soluble portions (also referred to as fat-soluble groups).In other words, the oligonucleotide according to one embodiment of the present invention preferably has at least one of the nucleotides constituting the oligonucleotide be a nucleotide or nucleotide derivative having a fat-soluble portion.By having one or more fat-soluble portions in the oligonucleotide, it is expected that the efficiency of oligonucleotide transfer to each site in the brain and the knockdown efficiency of target gene can be further improved.

[0142] The lipophilic moiety is conjugated to a nucleotide or nucleotide derivative directly or via a linker. In a nucleotide or nucleotide derivative, the site to which the lipophilic moiety is conjugated may be the sugar moiety, the base moiety, or the phosphate moiety, but is preferably the sugar moiety or the phosphate moiety. Furthermore, in a nucleotide or nucleotide derivative, when the lipophilic moiety is conjugated to the sugar moiety, the conjugation position is preferably the 2'-position, the 3'-position, or the 5'-position.

[0143] Furthermore, the position of the conjugated nucleotide or nucleotide derivative in the oligonucleotide sequence is not particularly limited, and may be the 3' end, the 5' end, or anywhere between the 3' end and the 5' end of the oligonucleotide. When the oligonucleotide has a lipophilic moiety, for example, it can more effectively knock down a target gene.

[0144] The term "lipid-soluble moiety" broadly refers to any compound or chemical substructure that has an affinity for lipids. Methods for characterizing the lipid solubility of a lipid-soluble moiety include, for example, the octanol-water partition coefficient, log K ｏｗ Here, K ｏｗ is the ratio of a chemical's concentration in the octanol phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributable to the chemical's constituent components calculated using first-principles or empirical methods. In principle, a chemical can be calculated using its log K ｏｗ The lipophilicity is determined when the log K of the lipophilic portion of the oligonucleotide according to one embodiment of the present invention is greater than 0. ｏｗ is not particularly limited as long as it exceeds 0, but may be greater than 1, greater than 2, or greater than 3. That is, an oligonucleotide according to one embodiment of the present invention has one or more lipophilic moieties, and the log K ｏｗ may be greater than 0, may be greater than 1, may be greater than 2, or may be greater than 3.

[0145] The lipophilic moiety according to one embodiment of the present invention may be, for example, a lipid (a substituted or unsubstituted alkyl chain having 7 to 30 carbon atoms, and an alkyl chain having 7 to 30 carbon atoms substituted with an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne), cholesterol, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA), retinoic acid, Examples of the lipid-soluble moiety include cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, sphingolipids, fat-soluble vitamins, and phenoxazines. Among these, from the viewpoint of efficient uptake into cells, the fat-soluble moiety according to one embodiment of the present invention is preferably at least one selected from the group consisting of a substituted or unsubstituted alkyl chain having 14 to 24 carbon atoms, eicosapentaenoic acid (EPA), arachidonic acid (ARA), and cholic acid.

[0146] In an oligonucleotide according to one embodiment of the present invention, the lipophilic moiety may be directly conjugated to the nucleotide. When directly conjugated, the lipophilic moiety can be conjugated to the nucleotide via the OH group at the 2'-position of the sugar moiety of the nucleotide, as shown in (C16U) in Figure 2. For example, a substituted or unsubstituted alkyl chain having 14 to 24 carbon atoms can be conjugated to the nucleotide via the OH group at the 2'-position of the sugar moiety.

[0147] Furthermore, in an oligonucleotide according to one embodiment of the present invention, the lipophilic moiety may be linked via a linker. The linker is not particularly limited as long as it can mediate a bond between the lipophilic moiety and the nucleotide, but examples include those containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, a click reaction product (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate. More specifically, the linker can be formed using, for example, 5'-Amino-Modifier C6-PDA, 3'-PT-Amino-Modifier C6 CPG, or 3'-Amino-Modifier C7 CPG 1000 (all manufactured by Glen Research). [L1] shown in Figure 2 is an example in which a fat-soluble moiety and a nucleotide are bonded together using 5'-Amino-Modifier C6-PDA, and [L2] to [L5] are examples in which a fat-soluble moiety and a nucleotide are bonded together using 3'-Amino-Modifier C7 CPG 1000.

[0148] (Ligand) An oligonucleotide according to one embodiment of the present invention may contain a ligand. That is, an oligonucleotide according to one embodiment of the present invention may have at least one ligand. The ligand is not particularly limited as long as it is a molecule that binds to a biomolecule such as a protein, and examples thereof include targeting ligands that target receptors that mediate delivery to specific central nervous system tissues. In this specification, the term "ligand" excludes the above-mentioned lipophilic moiety.

[0149] (SNCA-siRNA or HTT-siRNA) The oligonucleotide according to one embodiment of the present invention is preferably an siRNA that targets SNCA or HTT. The siRNA is not particularly limited as long as it targets SNCA or HTT, and those disclosed in the following International Publications can be used.

[0150] Examples of siRNA targeting SNCA (SNCA-siRNA) include those described in International Publication Nos. WO 2005 / 004794, WO 2006 / 039253, WO 2007 / 135426, WO 2008 / 086079, WO 2009 / 079399, WO 2012 / 027713, WO 2020 / 028816, and WO 2022 / 072447.

[0151] Furthermore, examples of siRNA targeting HTT (HTT-siRNA) include those described in International Publication Nos. WO 2004 / 101787, WO 2005 / 027980, WO 2005 / 105995, WO 2007 / 051045, WO 2008 / 005562, WO 2016 / 161374, and WO 2021 / 087036.

[0152] Among these, the nucleotide sequences of siRNA (S-0001 to 0196) consisting of combinations of sense and antisense strands shown in Tables 3-1 to 3-5, and the oligonucleotides ds1, ds2, ds9, ds10, and ds17 to ds273 shown in the Examples have high SNCA knockdown activity and can therefore be more preferably used as oligonucleotides (siRNA) according to one embodiment of the present invention.

[0153] From the viewpoint of achieving a higher SNCA knockdown activity, the oligonucleotide (siRNA) according to one embodiment of the present invention preferably contains any one pair of base sequences selected from the group consisting of (S1) to (S11) shown below, and more preferably any one pair of base sequences selected from the group consisting of (S6) to (S11), among the siRNA base sequences (S-0001 to S-0199) consisting of combinations of sense strands and antisense strands shown in Tables 3-1 to 3-5. Here, "set" refers to the combination of a sense strand and an antisense strand. (S1) SEQ ID NO: 46 (sense strand) and SEQ ID NO: 213 (antisense strand) (S-0020) (S2) SEQ ID NO: 83 (sense strand) and SEQ ID NO: 250 (antisense strand) (S-0057) (S3) SEQ ID NO: 93 (sense strand) and SEQ ID NO: 260 (antisense strand) (S-0067) (S4) SEQ ID NO: 169 (sense strand) and SEQ ID NO: 336 (antisense strand) (S-0143) (S5) SEQ ID NO: 180 (sense strand) and SEQ ID NO: 347 (antisense strand) (S-0154) (S6) SEQ ID NO: 1360 (sense strand) and SEQ ID NO: 1389 (antisense strand) (S-0179) (S7) SEQ ID NO: 1366 (sense strand) and SEQ ID NO: 1395 (antisense strand) (S-0186) (S8) SEQ ID NO: 1368 (sense strand) and SEQ ID NO: 1397 (antisense strand) (S-0187) (S9) SEQ ID NO: 1372 (sense strand) and SEQ ID NO: 1401 (antisense strand) (S-0191) (S10) SEQ ID NO: 1374 (sense strand) and SEQ ID NO: 1403 (antisense strand) (S-0193) (S11) SEQ ID NO: 1756 (sense strand) and SEQ ID NO: 1759 (antisense strand) (S-0197).

[0154] An oligonucleotide containing an siRNA in which 2'-modified nucleotides have been added to the base sequences of (S1) to (S11) above can be more preferably used as an oligonucleotide according to one embodiment of the present invention. Among the oligonucleotides in Tables 1-1 to 1-6, oligonucleotides containing an siRNA in which 2'-modified nucleotides have been added to the base sequences of (S1) to (S11) are shown below. Modified siRNA (S1): ds79, ds187 Modified siRNA (S2): ds105, ds198, ds249, ds9 Modified siRNA (S3): ds113, ds201, ds216, ds221, ds226, ds231, ds236, ds241, ds246 Modified siRNA (S4): ds156, ds211, ds217, ds222, ds227, d232, d237, ds242 Modified siRNA (S5): ds164, ds213, ds218, ds223, ds228, d233, d238, ds243, ds247 (S6) Modified siRNA: ds28, ds172, ds214, ds219, ds224, ds229, ds234, ds239, ds244 (S7) Modified siRNA: ds35, ds175, ds248 (S8) Modified siRNA: ds36, ds176 (S9) Modified siRNA: ds40, ds177, ds215, ds220, ds225, ds230, ds235, ds240, ds245 (S10) Modified siRNA: ds42, ds178.

[0155] Furthermore, from the viewpoint of achieving a higher SNCA knockdown activity, it is more preferable that the oligonucleotide (siRNA) according to one embodiment of the present invention comprises any one set of oligonucleotide sequences selected from the group consisting of ds2, ds9, ds17 to ds249, and ds254 to ds273 listed in any one of Tables 1-1 to 1-6.

[0156] Furthermore, from the viewpoint of achieving even better SNCA knockdown activity, it is even more preferable that the oligonucleotide (siRNA) according to one embodiment of the present invention comprises any one set of oligonucleotide sequences selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246 listed in any one of Tables 1-1 to 1-6.

[0157] Furthermore, from the viewpoint of having a superior SNCA knockdown activity, the oligonucleotide (siRNA) according to one embodiment of the present invention is more preferably any one set selected from the group consisting of ds2, ds17 to 249, and ds254 to ds273 listed in any of Tables 1-1 to 1-6. Furthermore, from the viewpoint of even superior SNCA knockdown activity, the oligonucleotide according to one embodiment is more preferably any one set selected from the group consisting of ds2 and ds250 to ds273 listed in any of Tables 1-1 to 1-6, and more preferably any one set selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172 , ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211 and ds213 to ds246, and most preferably any one set selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268 and ds273.

[0158] Furthermore, the oligonucleotide according to one embodiment of the present invention may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity to any one set of oligonucleotides selected from the group consisting of oligonucleotides S-0001 to S-0196 shown in Tables 3-1 to 3-5, and oligonucleotides ds1, ds2, ds9, ds10, and ds17 to ds273 shown in the Examples.

[0159] Furthermore, an oligonucleotide according to one embodiment of the present invention may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity with any one set of base sequences selected from the group consisting of (S1) to (S11) shown in Tables 3-1 to 3-5 above. Furthermore, an oligonucleotide according to one embodiment of the present invention may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity with any one set of base sequences selected from the group consisting of (S6) to (S11) shown in Tables 3-1 to 3-5 above. Furthermore, an oligonucleotide according to one embodiment of the present invention may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity with an siRNA in which the base sequences of (S1) to (S11) listed above have been modified with 2'-modified nucleotides.

[0160] Furthermore, the oligonucleotide according to one embodiment may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity to the sequence of any one set of oligonucleotides selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246 listed in any one of Tables 1-1 to 1-6.

[0161] Furthermore, the oligonucleotide of one embodiment may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity with any one set of sequences selected from the group consisting of ds2, ds17 to 249, and ds254 to ds273 listed in any one of Tables 1-1 to 1-6.

[0162] Furthermore, the oligonucleotide of one embodiment may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity with any one set of sequences selected from the group consisting of ds2 and ds250 to ds273 listed in any one of Tables 1-1 to 1-6. Furthermore, the oligonucleotide according to one embodiment may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity to any one set of sequences selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246. Furthermore, the oligonucleotide according to one embodiment may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity to any one set of sequences selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268, and ds273.

[0163] As used herein, "sequence identity" refers to the degree to which a specific nucleic acid sequence matches another sequence at a certain rate. Sequence identity is usually calculated using known algorithms such as BLAST (Basic Local Alignment Search Tool) or CLUSTALW. Specifically, the percentage of matching bases or amino acids is calculated taking into account gaps (insertions or deletions) between the sequences to be compared. Generally, sequence identity is calculated using the following formula 1:

[0164]

[0165] If the nucleic acid contained in the sequence is modified, the sequence identity is calculated based on the state before modification. For example, if "mN" or "fN" is present in the sequence, the sequence identity is calculated using N as the N. In the case of RNA, N is A, U, C, or G, and in the case of DNA, N is A, T, C, or G.

[0166] Furthermore, an oligonucleotide according to one embodiment of the present invention may have the same base sequence as a base sequence obtained by shortening the base sequence of an oligonucleotide shown in the present specification by 1 base, 2 bases, 3 bases, 4 bases, 5 bases, or 6 bases (hereinafter referred to as a shortened base sequence). Furthermore, an oligonucleotide according to one embodiment of the present invention may have 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity with the shortened base sequence.

[0167] For example, when the number of bases in the oligonucleotide shown herein is 21, the number of bases in the truncated base sequence is a base sequence of 20, 19, 18, 17, 16, or 15. Thus, the number of bases in the oligonucleotide according to one embodiment of the present invention may be 20, 19, 18, 17, 16, or 15, and the base sequences of these oligonucleotides may have 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity to the truncated base sequence, or may even be identical.

[0168] Furthermore, when the number of bases in the oligonucleotide described herein is 23, the number of bases in the truncated base sequence is a base sequence of 22, 21, 20, 19, 18, or 17. Therefore, the number of bases in the oligonucleotide according to one embodiment of the present invention may be 22, 21, 20, 19, 18, or 17, and the base sequences of these oligonucleotides may have 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity to the truncated base sequence, or may even be identical.

[0169] Here, the shortened base sequence may be an oligonucleotide shown in the present specification in which the 5'-end and / or 3'-end has been shortened, or an oligonucleotide in which a portion of the sequence has been omitted (deleted), or a combination thereof.

[0170] Furthermore, any one pair of oligonucleotide sequences selected from the group consisting of nucleotide sequences H-0001 to H-0258 of siRNA consisting of a combination of a sense strand and an antisense strand shown in any one of Tables 4-1 to 4-6, and ds274 to ds461 shown in any one of Tables 2-1 to 2-4, have high HTT knockdown activity and can therefore be more preferably used as an oligonucleotide according to one embodiment of the present invention.

[0171] From the viewpoint of achieving a higher HTT knockdown activity, the oligonucleotide (siRNA) according to one embodiment of the present invention preferably contains any one pair of base sequences selected from the group consisting of (H1) to (H5) shown below, among the nucleotide sequences of siRNA consisting of a combination of a sense strand and an antisense strand (H-0001 to H-0258) shown in Tables 4-1 to 4-6. (H1) SEQ ID NO: 365 (sense strand) and SEQ ID NO: 623 (antisense strand) (H-0005) (H2) SEQ ID NO: 370 (sense strand) and SEQ ID NO: 628 (antisense strand) (H-0010) (H3) SEQ ID NO: 396 (sense strand) and SEQ ID NO: 654 (antisense strand) (H-0036) (H4) SEQ ID NO: 407 (sense strand) and SEQ ID NO: 665 (antisense strand) (H-0047) (H5) SEQ ID NO: 469 (sense strand) and SEQ ID NO: 727 (antisense strand) (H-0109)

[0172] An oligonucleotide containing an siRNA in which 2'-modified nucleotides have been added to the base sequences of (H1) to (H5) above can be more preferably used as an oligonucleotide according to one embodiment of the present invention. Among the oligonucleotides in Tables 2-1 to 2-4, oligonucleotides containing siRNAs in which 2'-modified nucleotides have been added to the base sequences of (H1) to (H5) are shown below: Modified siRNA (H1): ds278, ds405, ds432, ds437, ds442, ds447, ds452, ds457 Modified siRNA (H2): ds280, ds406, ds433, ds438, ds443, ds448, ds453, ds458 Modified siRNA (H3): ds299, ds411, ds434, ds439, ds444, ds449, ds454, ds459 (H4) Modified siRNA: ds308, ds414, ds435, ds440, ds445, ds450, ds455, ds460 (H5) Modified siRNA: ds347, ds422, ds436, ds441, ds446, ds451, ds456, ds461

[0173] Furthermore, from the viewpoint of achieving higher HTT knockdown activity, it is preferable that the siRNA (oligonucleotide) targeting HTT comprises any one set of oligonucleotide sequences selected from the group consisting of ds274 to ds461 listed in any one of Tables 2-1 to 2-4. Furthermore, from the viewpoint of achieving even better HTT knockdown activity, it is even more preferable that the siRNA (oligonucleotide) targeting HTT comprises any one set of oligonucleotide sequences selected from the group consisting of ds278, ds280, ds299, ds308, ds347, ds405, ds406, ds411, ds414, ds422, and ds432 to ds461 listed in any one of Tables 2-1 to 2-4.

[0174] Furthermore, the oligonucleotide according to one embodiment of the present invention may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity to any one set of oligonucleotides selected from the group consisting of H-0001 to H-0258 shown in any of Tables 4-1 to 4-6, and ds274 to ds461 shown in any of Tables 2-1 to 2-4.

[0175] Furthermore, an oligonucleotide according to one embodiment of the present invention may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity with any one set of base sequences selected from the group consisting of (H1) to (H5) shown in Tables 4-1 to 4-6. Furthermore, an oligonucleotide according to one embodiment of the present invention may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity with an siRNA in which the base sequences of (H1) to (H5) listed above have been modified with 2'-modified nucleotides.

[0176] Furthermore, the oligonucleotide according to one embodiment of the present invention may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity to any one set of oligonucleotides selected from the group consisting of ds274 to ds461 listed in any one of Tables 2-1 to 2-4.

[0177] Furthermore, the oligonucleotide according to one embodiment of the present invention may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, or 95% or more sequence identity to any one set of oligonucleotides selected from the group consisting of ds278, ds280, ds299, ds308, ds347, ds405, ds406, ds411, ds414, ds422, and ds432 to ds461 listed in any one of Tables 2-1 to 2-4.

[0178] In this specification, the base sequences shown in the tables are written from left to right in the direction from the 5' end to the 3' end.

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190] These siRNAs can be appropriately combined with the sugar-modified nucleotides, phosphodiester-linked nucleotides, base-modified nucleotides, phosphate-modified nucleotides, and lipophilic moieties described above. By combining these nucleotide derivatives and lipophilic moieties, the knockdown activity of the siRNA and the amount of siRNA delivered to various sites in the brain can be improved.

[0191] (Antisense Oligonucleotide) When the oligonucleotide according to one embodiment of the present invention is an antisense oligonucleotide, the target gene thereof is not particularly limited, and examples thereof include genes related to central nervous system diseases, such as SOD1, FUS, C9ORF72, ATXN2, APP, tau, LRRK2, SNCA, HTT, ATXN3, UBE3A antisense transcript, and SMN2. Among these, at least one selected from the group consisting of the SNCA gene, the SOD1 gene, and the HTT gene is preferred.

[0192] In the oligonucleotide according to one embodiment of the present invention, the pattern (modification pattern) for arranging sugar-modified nucleotides in the oligonucleotide is not particularly limited, but those disclosed in WO 2004 / 015107, WO 2012 / 058210, WO 2013 / 074974, WO 2022 / 072447, WO 2021 / 087036, Mol. Ther. 2018,26 (3): 708-717, Nucleic Acids Research, 2022, Vol. 50, No. 9, 4840-4859, etc. can be suitably used.

[0193] [Method for Producing Oligonucleotides] The method for producing an oligonucleotide according to one embodiment of the present invention is not particularly limited as long as it is a method capable of producing the oligonucleotide according to the present invention, and examples thereof include known chemical synthesis methods and enzymatic transcription methods. Examples of known chemical synthesis methods include the phosphoramidite method, H-phosphonate method, phosphite method, phosphate triester method, and phosphate diester method. For example, the oligonucleotide of the present invention can be synthesized using an ABI3900 high-throughput nucleic acid synthesizer (manufactured by Applied Biosystems) or an automated nucleic acid synthesizer nS-8 (manufactured by Gene Design). After synthesis is complete, removal from the solid phase, deprotection of protecting groups, and purification of the target product are carried out. It is desirable to obtain nucleic acids with a purity of 90% or more, preferably 95% or more, by purification.

[0194] Alternatively, a desired nucleotide derivative, such as a sugar-modified nucleotide, a phosphodiester bond-modified nucleotide, a base-modified nucleotide, or a phosphate group-modified nucleotide, which has been prepared in advance, can be subjected to an extension reaction on a solid phase by the phosphoramidite method, etc. As a specific method for producing an oligonucleotide containing a nucleotide derivative, the method described in the Examples can be adopted.

[0195] The sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and phosphate group-modified nucleotides can be synthesized according to known methods, or commercially available products can be used. For example, the sugar-modified nucleotides are not particularly limited, but 2'-O-methyl-RNA and 2'-fluoro-DNA phosphoramidites commercially available from Glen Research can be used.

[0196] Furthermore, sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and phosphate group-modified nucleotides are not particularly limited, but can be synthesized according to the methods described in, for example, Bioconjugate Chemistry (2020, 31, 5, 1213-1233) or Chemical Society Reviews (2021, 50, 5126-5164).

[0197] In addition, the method for preparing oligonucleotides having a lipophilic moiety or a ligand is not particularly limited, but can be achieved by preparing a phosphoramidite unit or solid-phase support having a linker or a lipophilic group (lipophilic moiety) in advance, and then subjecting them to an extension reaction on the solid phase with other nucleotides or nucleotide derivatives, or by isolating the oligonucleotide and then post-modifying it in the liquid phase to prepare an oligonucleotide having a lipophilic moiety or a ligand. The phosphoramidite unit having a lipophilic group (lipophilic moiety) is not particularly limited, but can be prepared, for example, according to the methods described in International Publication No. 2021 / 092371 and Nature Biotechnology (40, 1500-1508, 2022). In addition, post-modification is not particularly limited, but can be performed by coupling the desired lipophilic compound to the oligonucleotide using a coupling reagent. Specific methods for preparing phosphoramidite units having a lipophilic group (lipophilic moiety) and oligonucleotides having a lipophilic moiety can be adopted from the methods described in the Examples.

[0198] When the oligonucleotide according to one embodiment of the present invention is double-stranded, the synthesized and purified sense strand and antisense strand are mixed in an appropriate ratio, for example, 0.1 to 10 equivalents, preferably 0.5 to 2 equivalents, more preferably 0.9 to 1.1 equivalents, and even more preferably equimolar amounts (equal amounts) of sense strand to 1 equivalent of antisense strand, and then the mixture is typically annealed. Alternatively, the annealing step may be omitted and the mixture may be used directly.

[0199] Annealing may be performed under any conditions that allow the sense strand and the antisense strand to form a double strand, but is usually performed by mixing the antisense strand and the sense strand in approximately equimolar amounts, heating the mixture at about 85°C for about 5 minutes, and then allowing it to cool to room temperature.

[0200] Pharmaceutical Composition and Therapeutic Agent for Central Nervous System Disease One embodiment of the present invention may be a pharmaceutical composition comprising the above-described oligonucleotide. In other words, a pharmaceutical composition according to one embodiment of the present invention may contain, as an active ingredient, the oligonucleotide according to one embodiment of the present invention. The pharmaceutical composition, like the oligonucleotide, is administered transnasally into the brain of a mammal using an administration device equipped with a needle having a puncture portion, as described below, and is administered through the opening of the puncture portion with the puncture portion positioned within the cribriform foramina of the cribriform plate.

[0201] The pharmaceutical composition according to one embodiment of the present invention can be widely used for various diseases, but is particularly suitable for the treatment, prevention, and / or amelioration of central nervous system diseases. That is, one aspect of the present invention may be a therapeutic agent for central nervous system diseases, comprising the above-described oligonucleotide. Here, the term "therapeutic agent" refers to an agent used for treatment, prevention, and / or amelioration. Like the oligonucleotide and pharmaceutical composition described above, the therapeutic agent for central nervous system diseases is administered transnasally into the brain of a mammal using an administration device equipped with a needle having a puncture portion, as described below, and administered through the opening of the puncture portion with the puncture portion positioned within the cribriform foramina of the cribriform plate.

[0202] The pharmaceutical composition and therapeutic agent for central nervous system disorders according to one embodiment of the present invention may contain various components in addition to the oligonucleotide. For example, they may further contain a carrier effective for transporting the oligonucleotide into cells. Examples of carriers effective for transporting the oligonucleotide into cells include cationic carriers. Examples of cationic carriers include cationic liposomes and cationic polymers. Furthermore, carriers utilizing viral envelopes may also be used as carriers effective for transporting the oligonucleotide into cells.

[0203] The pharmaceutical composition and therapeutic agent for central nervous system diseases containing the oligonucleotide and the carrier can be prepared by methods known to those skilled in the art. For example, the pharmaceutical composition or therapeutic agent for central nervous system diseases of the present invention can be prepared by mixing a carrier dispersion and an oligonucleotide solution of appropriate concentrations.

[0204] Furthermore, as the pharmaceutical composition and therapeutic agent for central nervous system diseases according to one embodiment of the present invention, for example, composite particles comprising an oligonucleotide and a lead particle as constituent components, and a composition comprising a composite particle and, optionally, a lipid membrane covering the composite particle, are also preferably used. Examples of lead particles include lipid aggregates, liposomes, emulsion particles, polymers, metal colloids, and microparticle preparations, with liposomes being preferred, and cationic liposomes being more preferred.

[0205] In addition to the above-mentioned carrier, the pharmaceutical composition and therapeutic agent for central nervous system disorders according to one embodiment of the present invention may contain a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents are essentially chemically inert and harmless compounds (including compositions) that do not affect the biological activity of the pharmaceutical composition or therapeutic agent of the present invention. Examples of pharmaceutically acceptable carriers or diluents include, but are not limited to, water, salt solutions, sugar solutions, glycerol solutions, and ethanol.

[0206] The pharmaceutical composition and therapeutic agent for central nervous system diseases according to one embodiment of the present invention preferably contain an amount of oligonucleotide effective for treating, preventing, or ameliorating a disease, and are provided in a form that can be appropriately administered intranasally to a patient, and a liquid formulation is preferred as such a form.

[0207] Furthermore, the pharmaceutical composition and therapeutic agent for central nervous system diseases of the present invention may contain an appropriate amount of any pharmaceutically acceptable additive, for example, an emulsifying aid, a stabilizer, an isotonicity agent, and / or a pH adjuster, etc. Any pharmaceutically acceptable additive can be added at an appropriate step either before or after preparation of the pharmaceutical composition or therapeutic agent for central nervous system diseases of the present invention.

[0208] [Administration Device and Administration System] An oligonucleotide according to one embodiment of the present invention is administered transnasally into the brain of a mammal using an administration device equipped with a needle having a puncture portion, and the oligonucleotide is administered through an opening in the puncture portion when the puncture portion is positioned within the cribriform foramina of the cribriform plate. The administration device is equipped with a needle having a puncture portion, and when the puncture portion is positioned within the cribriform foramina of the cribriform plate, the oligonucleotide is administered through the opening in the puncture portion, thereby enabling the oligonucleotide to be administered transnasally into the brain of a mammal.

[0209] Below, the administration device 1 and the administration system 200 including the administration device will be described using Figures 3 and 4-1, but the administration device 1 and the administration system 200 are not limited to the embodiments of Figures 3 and 4-1.

[0210] In this specification, the "cribriform foramen X3" punctured by the puncturing portion 12 of the needle portion 10 of the administration device 1 is a hole formed in the cribriform plate X2 of the ethmoid bone X1, which has a nasal cavity opening and an olfactory bulb opening as shown in FIG. 4-1 . The olfactory nerve (nerve axon) Y5 extends from the olfactory bulb Y1, which is part of the brain tissue B, to olfactory cells distributed in the olfactory mucosa Y2 (composed of the olfactory epithelium Y3 and the lamina propria Y4) in the nasal cavity Z1. Numerous cribriform foramen X3 exist not only on the flat surface of the cribriform plate X2 as shown in FIG. 4-1 , but also on the midline, lateral wall, posterior wall of the olfactory cavity, etc. The cribriform foramen X3 to be punctured by the needle portion 10 of the present device is any hole among all these holes that can be punctured by the puncturing portion 12 that has penetrated the olfactory mucosa Y2.

[0211] (Administration System) As shown in FIG. 3 , the administration system 200 includes the administration device 1 and a guide catheter 100 .

[0212] As shown in FIG. 4-1 , the administration system 200 involves placing the guide catheter 100 in the nasal cavity Z1 with the tip thereof facing the cribriform plate X2, inserting the administration device 1 into the guide catheter 100, and administering oligonucleotide A into the brain with the opening (tip opening 14) of the puncture portion 12 of the needle portion 10 positioned within the cribriform hole X3 formed in the cribriform plate X2. The administration system 200 can deliver oligonucleotide A transnasally into the brain of a mammal such as a human via a delivery medium such as cerebrospinal fluid C or the olfactory nerve Y5. That is, the administration system 200 includes a storage portion 40 containing the oligonucleotide of the present invention and an administration device 1, and is configured so that the oligonucleotide contained in the storage portion 40 can be discharged through the opening (tip opening 14) of the puncture portion 12.

[0213] (Administration Device) As shown in FIG. 3 or FIG. 4-1, the administration device 1 includes a needle portion 10, a hub portion 20, a cannula portion 30, and a storage portion 40.

[0214] The administration device 1 is an administration device 1 equipped with a needle portion 10 having a puncture portion 12 for transnasally delivering oligonucleotide A into the brain of a mammal, and the opening (tip opening 14) of the puncture portion 12 is positioned within the sieve hole X3, and oligonucleotide A is administered through the tip opening 14.

[0215] The administration device 1 can be used in combination with the guide catheter 100 as an administration system 200, or can be used alone.

[0216] <Needle portion> The needle portion 10 has a needle shaft portion 11, a puncturing portion 12 formed on the distal end side of the needle shaft portion 11, a distal opening portion 14 (corresponding to the "opening" in the claims) formed in a needle tip portion 12a of the puncturing portion 12, and a proximal opening portion 15 formed on the proximal end of the needle shaft portion 11. The needle portion 10 has a cylindrical shape with a lumen 10a that runs longitudinally from the distal opening portion 14 formed on the distal end side to the proximal opening portion 15 formed on the proximal end side. As shown in Figure 3, the needle shaft portion 11 of the needle portion 10 is disposed within the lumen 31a of the cannula portion 30, and the puncturing portion 12 is disposed exposed from the distal end of the cannula portion 30 during puncturing.

[0217] The needle shaft 11 corresponds to the main body of the needle 10, and has a lumen 21a that communicates with the lumen 31a of the hub 20 or the cannula 30. A proximal end opening 15 is formed at the proximal end of the needle shaft 11. The needle shaft 11 is connected to the hub 20 via the proximal end opening 15 so as to be able to communicate with it.

[0218] The needle shaft 11 is formed with a length substantially equal to the overall length of the cannula 30, and is inserted through the lumen 31a of the cannula 30 and connected to the hub 20 so that the proximal opening 15 communicates with the lumen 21a of the hub 20. However, the needle shaft 11 may be shorter than the overall length of the cannula 30, with the proximal end disposed within the lumen 31a of the cannula 30. In this configuration, oligonucleotide A flows through the lumen 31a of the cannula 30 to the proximal opening 15.

[0219] The puncturing portion 12 is formed at the tip end of the needle shaft portion 11. A needle tip portion 12a is formed at the tip end of the puncturing portion 12. The needle tip portion 12a has an open end with a blade surface 13 formed by cutting the needle shaft portion 11 at an angle relative to the longitudinal direction at the tip end. The inner edge of the blade surface 13 defines a tip opening 14 that connects the inner cavity of the needle shaft portion 11 to the outside. When administering oligonucleotide A, the tip opening 14 is positioned within the sieve hole X3 with at least a portion of the puncturing portion 12 puncturing the sieve hole X3. By being positioned within the sieve hole X3, the oligonucleotide A can be delivered to the brain side against the cerebrospinal fluid C flowing out from the sieve hole X3. Note that the needle tip portion 12a is not limited to a pointed shape with a blade surface 13 at the tip, and may be a straight cylindrical shape or a cylindrical shape with a rounded, approximately hemispherical tip.

[0220] From the viewpoints of ease of puncturing the sieve holes X3 and the ability to deliver oligonucleotide A, the puncturing portion 12 preferably has an outer diameter of 0.05 mm to 2.1 mm, and more preferably 0.075 mm to 1.2 mm. The overall length of the puncturing portion 12 (corresponding to the exposed length from the tip of the cannula portion 30 of the needle portion 10), which is the axial length of the blade surface 13 shown in FIG. 3 , is long enough to allow the tip opening 14 to be positioned within the sieve holes X3, and is preferably 0.25 mm to 5.4 mm. The overall length of the puncturing portion 12 is the longitudinal length of the portion exposed from the cannula portion 30 of the needle portion 10, and is the length from the tip of the needle tip portion 12a along the longitudinal direction of the needle portion 10. The length of the puncturing portion 12 can be appropriately set depending on the thickness of the olfactory mucosa Y2 of the mammal to be punctured, the overall length of the sieve holes X3, and the like.

[0221] The needle portion 10 can be made of metals such as stainless steel (e.g., SUS304 or SUS316L), titanium, or resin materials. However, the material of the needle portion 10 is not particularly limited as long as it is usable in the medical field and suitable for the needle portion 10, in addition to the materials mentioned above.

[0222] Furthermore, in order to improve the ease of puncturing the sieve holes X3, the needle portion 10 may be formed from a plastically deformable material or a shape-memory material, and when using the device, the orientation of the puncturing portion 12 may be changed to deform the needle shaft portion 11. In this case, the shape of the needle portion 10 may be deformed into any shape immediately before use, may be deformed in advance, or may be deformed in advance and then fine-tuned when used.

[0223] Furthermore, the needle portion 10 may use different materials for the puncture portion 12 exposed from the cannula portion 30 and the needle shaft portion 11. For example, the needle portion 10 may be made of a rigid material that does not or is difficult to deform so that the puncture portion 12 punctures the olfactory mucosa or the like, and the needle shaft portion 11 may be made of a material that can be deformed to match the shape of the guide catheter 100, etc.

[0224] <Hub portion> The hub portion 20 holds the proximal end of the needle shaft portion 11 of the needle portion 10 and / or the proximal end of the cannula portion 30 to allow the flow of oligonucleotide A. The hub portion 20 has a main body portion 21 and a connecting portion 22. The hub portion 20 is connected to the storage portion 40 in a state in which the tip opening 42a of the storage portion 40 communicates with the proximal opening 15 of the needle portion 10.

[0225] The main body portion 21 has an inner cavity 21a through which oligonucleotide A can flow, and connects the tip opening 42a of the connected storage portion 40 with the base opening 15 of the needle shaft portion 11 of the needle portion 10 or the inner cavity 31a of the cannula portion 30.

[0226] The connecting part 22 is provided on the proximal end side of the hub part 20 and connects to the storage part 40 to maintain communication between the hub part 20 and the storage part 40. In this embodiment, the connecting part 22 is connected to the storage part 40 via a connecting member 50 such as a tube. However, the connecting part 22 may also be configured to fit the shape of the distal end of the storage part 40 so that the two parts are directly and detachably fitted together (e.g., a luer taper type or luer lock type). The connecting part 22 is not particularly limited as long as it is configured to connect at least the storage part 40 and the hub part 20 so that oligonucleotide A can flow between them.

[0227] <Cannula Portion> The cannula portion 30 is a tubular member made of a flexible material, and has a tubular main body portion 31 having a lumen 31a that communicates from the distal end to the proximal end. As an example, the cannula portion 30 holds at least a part of the needle shaft portion 11 of the needle portion 10.

[0228] A stopper portion 32 having an abutment portion 32a that comes into contact with the olfactory epithelium Y3 of the olfactory mucosa Y2 is provided at the tip of the main body portion 31 of the cannula portion 30. In the embodiment shown in Fig. 3, the stopper portion 32 is the tip portion of the main body portion 31, and the tip surface of the main body portion 31 functions as the abutment portion 32a. In this embodiment, the stopper portion 32 is the tip portion of the main body portion 31, and the abutment portion 32a is the tip surface of the main body portion 31. However, the stopper portion 32 and the abutment portion 32a are not limited to these configurations. The stopper portion 32 may be formed as a separate, detachable or fixed member that can be arranged on the tip side of the cannula portion 30, and the abutment portion 32a may be formed as a portion of this separate member that comes into contact with the olfactory epithelium Y3.

[0229] When the puncturing portion 12 of the needle portion 10 punctures the sieve hole X3, the stopper portion 32 functions as a stopper that prevents the needle tip portion 12a of the puncturing portion 12 from puncturing too deeply by bringing the abutment portion 32a into contact with the olfactory epithelium Y3 as shown in Figure 4-1.

[0230] Furthermore, when the needle 10 punctures the olfactory epithelium Y3, the stopper 32 brings the abutting portion 32a into contact with the olfactory epithelium Y3, thereby stabilizing the puncture posture of the administration device 1. This allows the needle 10 to puncture the sieve hole X3 to be punctured without misalignment. Note that the stopper 32 is preferably pressed against the olfactory epithelium Y3 so as to slightly sink into it, so that the puncture posture of the administration device 1 is fixed.

[0231] When the contact portion 32a is the distal end surface of the cannula portion 30, the length (maximum width) of the longest radial portion of the distal end surface of the cannula portion 30 is preferably a minor axis of 0.2 mm to 3.0 mm and a major axis of 0.2 mm to 15 mm when the cross-sectional shape of the cannula portion 30 is approximately circular (e.g., elliptical) similar to the structure of the upper nasal cavity. When the cross-sectional shape of the cannula portion 30 is circular, the diameter of the contact portion 32a is preferably 0.2 mm to 3.0 mm, and more preferably 0.2 mm to 2.1 mm. The maximum width of the contact portion 32a can be appropriately set to be at least larger than the diameter of the needle portion 10 so as to perform the stopper function of preventing the needle portion 10 from over-puncturing the sieve hole X3.

[0232] The cannula portion 30 may be formed in a straight cylindrical shape, or may have a shape with a portion curved in advance from the viewpoint of facilitating insertion of the needle portion 10 into the sieve hole X3. Furthermore, from the viewpoint of facilitating insertion into the nasal cavity Z1 and puncturing the sieve hole X3 with the needle portion 10, the cannula portion 30 may be formed in part or entirely from a material that can be plastically deformed into any shape. This allows the cannula portion 30 to be inserted while deforming to conform to the shape of the lumen of the guide catheter 100 when an insertion aid is used to guide the administration device 1, such as the guide catheter 100, to the olfactory mucosa Y2.

[0233] The length of the cannula portion 30 is not particularly limited as long as the base end is exposed from the external nares and the tip is long enough to be inserted into the olfactory mucosa Y2 near the cribriform plate X2 and allow manipulation of the needle tip portion 12a. For example, the total length may be 25 mm or more and 2000 mm or less, 30 mm or more and 1500 mm or less, 40 mm or more and 1000 mm or less, or 55 mm or more and 410 mm or less.

[0234] The cannula portion 30 may be arranged so as to cover the entire length of the needle shaft portion 11 of the needle portion 10, or may be arranged so that the base end side of the needle shaft portion 11 is exposed.

[0235] The cannula portion 30 may be configured to be disposed integrally with the hub portion 20 or may be configured to be detachable from the hub portion 20 .

[0236] <Storage section> The storage section 40 stores oligonucleotide A to be administered to the sieve hole X3. The storage section 40 has a storage space 41 that stores oligonucleotide A, and a liquid delivery section 42 that delivers the oligonucleotide A in the storage space 41 to the hub section 20. In Figure 3, the storage section 40 is connected to the hub section 20 via a connecting member 50 such as a tube so that the oligonucleotide A can flow through it.

[0237] The storage unit 40 is connected to the hub unit 20 so as to be able to communicate with the needle unit 10. A tip opening 42a of the liquid delivery unit 42 formed at the tip of the liquid delivery unit 42 communicates with the base opening 15 of the needle unit 10 through the lumen 21a of the hub unit 20. This allows the oligonucleotide A stored in the storage unit 40 to flow to the needle shaft unit 11. The storage unit 40 is preferably configured so that the dosage of the oligonucleotide A stored in the storage space 41 can be adjusted, and for example, a syringe with a plunger or the like can be used. Note that the storage unit 40 is not limited to a syringe, and any device that can store at least oligonucleotide A and allow the oligonucleotide A to flow to the needle unit 10 via the hub unit 20 may be used. Furthermore, the storage unit 40 may be connected so as to be able to communicate with the liquid delivery unit 42 and the hub unit 20 when attached to a medical device having an operation unit that can control the amount and timing of oligonucleotide A discharged.

[0238] <Guide Catheter> The guide catheter 100 is used as an insertion aid when inserting the administration device 1 into the nasal cavity Z1.

[0239] As shown in FIG. 3, the guide catheter 100 has a catheter body 110 made of a tubular member having a lumen 111 passing through in the longitudinal direction from the distal end to the proximal end.

[0240] The guide catheter 100 can be made of a material such as metal or resin that can be used in the medical field, and examples thereof include stainless steel such as SUS304 and polyurethane.

[0241] The distal end of the catheter main body 110 may be provided with a curved portion disposed adjacent thereto that curves toward the distal end so as to move away from the axis of the catheter main body 110. In addition, the outer surface of the guide catheter 100 may be subjected to a circumferential process such as a spiral cut or various surface processes to improve insertability.

[0242] The catheter body 110 may be used in a straight state as shown in FIG. 3, or may be configured to be pre-shaped to facilitate insertion into the nasal cavity Z1, or may be configured to be plastically deformable by fine adjustment before or during use.

[0243] Here, examples of the shape of the guide catheter 100 are shown. The guide catheter 100 can have a total length of 90 mm, an outer diameter of 0.82 mm, an inner diameter of 0.68 mm, and a bending angle of 45° at the curved portion 113. Alternatively, the guide catheter 100 can have a total length of 45 mm, an outer diameter of 1.35 mm, and a double lumen with inner diameters of 0.45 mm and 0.70 mm.

[0244] The guide catheter 100 can be supplied to the market in combination with the administration device 1 as an administration system 200. In addition, the administration device 1 or the guide catheter 100 can function as the administration system 200 by utilizing either the administration device 1 or the guide catheter 100 that is already supplied separately.

[0245] [Method of Using the Device] Next, a method of using the administration device 1 described above will be described. The method of use described below includes a procedure corresponding to a preparatory stage from placing the administration device 1 in a predetermined position until the administration of oligonucleotide is initiated, and a procedure for administering the oligonucleotide after the preparatory stage. The method of using the administration device 1 includes the steps of inserting the administration device 1 at least through the external nostril and puncturing the olfactory mucosa Y2 with the needle portion 10 and puncturing the cribriform hole X3 of the cribriform plate X2 to position the puncturing portion 12 within the cribriform plate X2, positioning the tip opening 14 within the cribriform hole X3 or beyond the cribriform plate X2 into a ventricle with the puncturing portion 12 positioned within the cribriform plate X2, and administering the oligonucleotide into the brain via the tip opening 14.

[0246] As shown in FIG. 4-2A , a user such as a doctor first inserts the administration device 1 from the patient's external nostril toward the olfactory mucosa Y2. When inserting the administration device 1 into the nasal cavity Z1, the patient's nose is anesthetized and the insertion position is confirmed using a rigid endoscope or the like. The administration device 1 can be inserted using a straight instrument such as a rigid endoscope, or the device alone can be inserted directly into the nasal cavity Z1, or an insertion aid such as a guide catheter 100 can be used.

[0247] Next, as shown in FIG. 4-2B, the user inserts the needle portion 10 into the olfactory mucosa Y2 (the olfactory epithelium Y3, then the lamina propria Y4).

[0248] Next, as shown in FIG. 4-2C , the user further advances the needle portion 10 into the olfactory mucosa Y2, causing the needle tip 12a of the puncturing portion 12 to puncture the sieve hole X3. At this time, the abutment portion 32a of the stopper portion 32 of the cannula portion 30 comes into contact with the olfactory epithelium Y3, thereby restricting the puncturing movement of the needle portion 10 and preventing the needle portion 10 from over-puncturing the sieve hole X3. Furthermore, when the stopper portion 32 is pressed against the olfactory epithelium Y3, the puncturing position of the administration device 1 is stabilized, allowing the puncturing portion 12 to puncture the sieve hole X3 without misalignment. That is, the administration device 1 has a cannula portion 30 formed of a tubular member that is disposed over the needle portion 10 so that the puncture portion 12 is exposed, an opening (tip opening 14) is provided at the tip of the puncture portion 12, and a stopper portion 32 having an abutment portion 32a that abuts against the olfactory epithelium Y3 of the olfactory mucosa Y2 is formed at the tip of the cannula portion 30, and the opening (tip opening 14) is disposed within the cribriform plate X2 with the abutment portion 32a of the stopper portion 32 in contact with the olfactory epithelium Y3. The administration device 1 can begin administering oligonucleotides once the puncture portion 12 has completed puncturing the sieve hole X3 and the tip opening 14 has been positioned within the sieve hole X3 or beyond the sieve plate X2 into the ventricle. Figure 4-2D shows the tip opening 14 positioned within the sieve hole X3.

[0249] Then, as shown in FIG. 4-2D, the user administers the oligonucleotide contained in the container 40. If the container 40 is a syringe, the user operates the plunger manually or using a syringe pump or the like to administer the required amount of oligonucleotide. This causes the oligonucleotide to be administered through the tip opening 14 of the puncture part 12 into the sieve hole X3. After administering the oligonucleotide, the user removes the administration device 1 from the nasal cavity Z1, completing the series of processes.

[0250] As shown in Figure 4-2E, the oligonucleotide administered into the phloem X3 flows to the olfactory bulb side opening of the phloem X3 and is delivered to brain tissue B via delivery vehicles such as cerebrospinal fluid C and olfactory nerve Y5. Note that in Figures 4-2D and 4-2E, cerebrospinal fluid C is not shown to make it easier to understand the flow of the oligonucleotide after administration.

[0251] As described above, the administration device 1 of this embodiment is an administration device 1 comprising a needle portion 10 having a puncture portion 12 for transnasally delivering an oligonucleotide into the brain of a mammal, and with the puncture portion 12 positioned within the cribriform plate X2, the oligonucleotide is administered into the brain through the opening (tip opening 14) of the puncture portion 12.

[0252] With this configuration, the puncture portion 12 is inserted into the cribriform foramen X3 of the cribriform plate X2, and the distal end opening 14 provided in the puncture portion 12 is positioned within the cribriform foramen X3 or beyond the cribriform plate X2 into the ventricle, and an oligonucleotide such as a drug is administered through the distal end opening 14. This allows the oligonucleotide to be minimally invasively delivered to brain tissue B via a delivery medium such as cerebrospinal fluid C or the olfactory nerve Y5. In particular, when administering a therapeutic drug of a high molecular weight, such as a protein or antibody, which is currently difficult to administer to brain tissue B, the oligonucleotide can be efficiently delivered to brain tissue B while avoiding the blood-brain barrier. Furthermore, because the oligonucleotide is administered within the cribriform foramen X3 formed in the cribriform plate X2 after passing through the olfactory mucosa Y2, which is rich in blood vessels and lymphatic vessels, leakage into the nasal cavity Z1 is suppressed, and the amount of oligonucleotide flowing into the blood vessels and lymphatic vessels is minimized. Furthermore, the present invention enables oligonucleotides to be delivered to the brain in a minimally invasive and extremely efficient manner, compared to the conventional methods of administering oligonucleotides to brain tissue B, such as intrathecal administration, nasal spray, and intraventricular administration.

[0253] The oligonucleotide of the present invention has a cannula portion 30 formed of a tubular member that is arranged to cover the needle portion 10 so that the puncture portion 12 is exposed, an opening (tip opening 14) is provided on the tip side of the puncture portion 12, and a stopper portion 32 having an abutment portion 32a that abuts against the olfactory epithelium Y3 of the olfactory mucosa Y2 is formed at the tip of the cannula portion 30, and the opening (tip opening 14) can be administered transnasally into the brain of a mammal using an administration device 1 that is placed in the cribriform plate X2 with the abutment portion 32a of the stopper portion 32 in contact with the olfactory epithelium Y3.

[0254] Furthermore, the oligonucleotide of the present invention has a hub portion 20 to which a storage portion 40 containing the oligonucleotide can be attached, and the hub portion 20 can be administered transnasally into the brain of a mammal using an administration device 1 that holds the base end of the cannula portion 30 and / or the base end of the needle shaft portion 11 of the needle portion 10 inserted through the inner cavity of the cannula portion 30.

[0255] The oligonucleotide of the present invention can be administered transnasally into the brain of a mammal using an administration device 1 in which the cross-sectional shape of the abutting portion 32a of the stopper portion 32 of the cannula portion 30 is circular or elliptical.

[0256] [Other embodiments] Other embodiments of the present invention include a therapeutic method using the above-mentioned oligonucleotide. Here, the therapeutic method includes not only a method for treating a disease, but also a method for prevention and a method for improvement. In addition, the disease is not particularly limited, but the therapeutic method according to one embodiment of the present invention is preferably used for central nervous system diseases. That is, the present invention may include the following embodiments.

[0257] 1. A method for treating a brain disease, comprising an administration step of administering an oligonucleotide into the brain of a mammal transnasally by injecting it through the cribriform holes of the cribriform plate; 2. A method for treating a brain disease as set forth in 1. above, wherein in the administration step, an administration device is provided with a needle part having a puncture part, and the puncture part is placed within the cribriform holes of the cribriform plate, and the oligonucleotide is injected transnasally into the brain of the mammal through an opening of the puncture part; 3. A method for treating a brain disease as set forth in 1. or 2. above, wherein the oligonucleotide is any one selected from the group consisting of an aptamer, an antisense oligonucleotide, a decoy nucleic acid, a ribozyme, an siRNA, a miRNA, and an mRNA; 4. A method for treating a brain disease as set forth in any of 1. to 3. above, wherein the oligonucleotide is an siRNA or an antisense oligonucleotide; 5. A method for treating a brain disease as set forth in any of 1. to 4. above, wherein the oligonucleotide is an siRNA; 6. A method for treating a brain disease as set forth in 5. above, wherein the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is 80% or more; 7. The therapeutic method according to 5. or 6. above, wherein all nucleotides constituting the siRNA are 2'-modified nucleotides; 8. The therapeutic method according to any one of 5. to 7. above, wherein the siRNA has one or more lipophilic moieties; 9. The therapeutic method according to 8. above, wherein the lipophilic moiety is at least one selected from the group consisting of a substituted or unsubstituted alkyl chain having 14 to 24 carbon atoms, eicosapentaenoic acid (EPA), arachidonic acid (ARA), and cholic acid; 10. The therapeutic method according to any one of 5. to 9. above, wherein the target gene of the siRNA is SNCA or HTT; 11. The therapeutic method according to 5. to 10. above, wherein all phosphodiester bonds connecting the first and second nucleotides counting from both ends of the sense and antisense strands of the siRNA are substituted with phosphorothioate bonds;12. The siRNA according to any one of items 5 to 11 above, wherein the siRNA comprises any one set of nucleic acid sequences selected from the group consisting of (S1) to (S11) below: (S1) SEQ ID NO: 46 (sense strand) and SEQ ID NO: 213 (antisense strand) (S2) SEQ ID NO: 83 (sense strand) and SEQ ID NO: 250 (antisense strand) (S3) SEQ ID NO: 93 (sense strand) and SEQ ID NO: 260 (antisense strand) (S4) SEQ ID NO: 169 (sense strand) and SEQ ID NO: 336 (antisense strand) (S5) SEQ ID NO: 180 (sense strand) and SEQ ID NO: 347 (antisense strand) (S6) SEQ ID NO: 1360 (sense strand) and SEQ ID NO: 1389 (antisense strand) (S7) SEQ ID NO: 1366 (sense strand) and SEQ ID NO: 1395 (antisense strand) (S8) SEQ ID NO: 1368 (sense strand) and SEQ ID NO: 1397 (antisense strand) (S9) SEQ ID NO: 1372 (sense strand) and SEQ ID NO: 1401 (antisense strand) (S10) SEQ ID NO: 1374 (sense strand) and SEQ ID NO: 1403 (antisense strand) (S11) SEQ ID NO: 1756 (sense strand) and SEQ ID NO: 1759 (antisense strand); 13. The above-mentioned 5., wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds2, ds9, ds17 to ds249, and ds254 to ds273 listed in any one of Tables 1-1 to 1-6. 14. The method for treatment according to 13., wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246 listed in any one of Tables 1-1 to 1-6; 14. The method for treatment according to 13 above, wherein the siRNA is any one set of oligonucleotides selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268, and ds273 listed in any one of Tables 1-1 to 1-6;16. The method of treatment according to 5. above, wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds274 to ds461 listed in any one of Tables 2-1 to 2-4; 17. The method of treatment according to 16. above, wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds278, ds280, ds299, ds308, ds347, ds405, ds406, ds411, ds414, ds422, and ds432 to ds461 listed in any one of Tables 2-1 to 2-4; 18. The treatment method according to 2. above, wherein the administration device has a cannula portion formed of a tubular member that is placed over the needle portion so that the puncture portion is exposed, the opening is provided on the tip side of the puncture portion, and a stopper portion having an abutment portion that abuts against the olfactory epithelium of the olfactory mucosa is formed at the tip of the cannula portion, and the opening is placed within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium; 19. The treatment method according to 18. above, wherein the administration device has a hub portion to which a storage portion containing the oligonucleotide can be attached, and the hub portion holds the base end of the cannula portion and / or the base end of the needle shaft portion of the needle portion that has passed through the lumen of the cannula portion; 20. The treatment method according to 18. or 19, wherein the cross-sectional shape of the abutment portion of the stopper portion is circular or elliptical; 21. The treatment method according to any of 1. to 20. above, wherein the brain disease is a central nervous system disease; 22. The method for treating a brain disease according to any one of 1. to 21. above, wherein the brain disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, or dementia with Lewy bodies.

[0258] Another embodiment of the present invention is a method for administering the above-described oligonucleotide to a recipient, wherein the recipient is not particularly limited as long as it is a mammal, but is preferably a human.

[0259] That is, the present invention may also include the following embodiments: 1. An administration method for an oligonucleotide, comprising an administration step of administering an oligonucleotide transnasally into the brain of a recipient (mammal) by injecting it through the sieve holes of the cribriform plate; 2. The administration method according to 1. above, wherein the administration step comprises administering the oligonucleotide transnasally into the brain of a mammal through an opening of an administration device having a needle part with a puncture part, with the puncture part being placed within the sieve holes of the cribriform plate; 3. The administration method according to 1. or 2. above, wherein the oligonucleotide is any one selected from the group consisting of an aptamer, an antisense oligonucleotide, a decoy nucleic acid, a ribozyme, siRNA, miRNA, and mRNA; 4. The administration method according to any of 1. to 3. above, wherein the oligonucleotide is siRNA or an antisense oligonucleotide; 5. The administration method according to any of 1. to 4. above, wherein the oligonucleotide is siRNA; 6. The administration method according to 5. above, wherein the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is 80% or more. 7. The administration method according to 5. or 6. above, wherein all nucleotides constituting the siRNA are 2'-modified nucleotides; 8. The administration method according to any of 5. to 7. above, wherein the siRNA has one or more lipophilic moieties; 9. The administration method according to 8. above, wherein the lipophilic moiety is at least one selected from the group consisting of a substituted or unsubstituted alkyl chain having 14 to 24 carbon atoms, eicosapentaenoic acid (EPA), arachidonic acid (ARA), and cholic acid; 10. The administration method according to 5. to 9. above, wherein the target gene of the siRNA is SNCA or HTT; 11. The administration method according to 5. to 10. above, wherein all phosphodiester bonds linking the first and second nucleotides counting from both ends of the sense strand and antisense strand of the siRNA are substituted with phosphorothioate bonds;12. The siRNA according to any one of items 5 to 11 above, wherein the siRNA comprises any one set of nucleic acid sequences selected from the group consisting of (S1) to (S11) below: (S1) SEQ ID NO: 46 (sense strand) and SEQ ID NO: 213 (antisense strand) (S2) SEQ ID NO: 83 (sense strand) and SEQ ID NO: 250 (antisense strand) (S3) SEQ ID NO: 93 (sense strand) and SEQ ID NO: 260 (antisense strand) (S4) SEQ ID NO: 169 (sense strand) and SEQ ID NO: 336 (antisense strand) (S5) SEQ ID NO: 180 (sense strand) and SEQ ID NO: 347 (antisense strand) (S6) SEQ ID NO: 1360 (sense strand) and SEQ ID NO: 1389 (antisense strand) (S7) SEQ ID NO: 1366 (sense strand) and SEQ ID NO: 1395 (antisense strand) (S8) SEQ ID NO: 1368 (sense strand) and SEQ ID NO: 1397 (antisense strand) (S9) SEQ ID NO: 1372 (sense strand) and SEQ ID NO: 1401 (antisense strand) (S10) SEQ ID NO: 1374 (sense strand) and SEQ ID NO: 1403 (antisense strand) (S11) SEQ ID NO: 1756 (sense strand) and SEQ ID NO: 1759 (antisense strand); 13. The above-mentioned 5., wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds2, ds9, ds17 to ds249, and ds254 to ds273 listed in any one of Tables 1-1 to 1-6. 14. The administration method according to 13., wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246 listed in any one of Tables 1-1 to 1-6;15. The administration method according to any one of 13. above, wherein the siRNA is any one set of oligonucleotides selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268, and ds273 listed in any one of Tables 1-1 to 1-6; 16. The administration method according to 5. above, wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds274 to ds461 listed in any one of Tables 2-1 to 2-4. 17. The administration method described in 16. above, wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds278, ds280, ds299, ds308, ds347, ds405, ds406, ds411, ds414, ds422, and ds432 to ds461 listed in any of Tables 2-1 to 2-4; 18. The administration method described in 2. above, wherein the administration device has a cannula portion formed of a tubular member that is placed over the needle portion so that the puncture portion is exposed, the opening is provided on the tip side of the puncture portion, and a stopper portion is formed at the tip of the cannula portion that has an abutment portion that abuts against the olfactory epithelium of the olfactory mucosa, and the opening is placed within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium; 19. The administration method according to claim 18, wherein the administration device has a hub portion to which a storage portion containing the oligonucleotide can be attached, and the hub portion holds the base end of the cannula portion and / or the base end of the needle shaft portion of the needle portion inserted through the lumen of the cannula portion; 20. The administration method according to claim 18 or 19, wherein the cross-sectional shape of the abutting portion of the stopper portion is circular or elliptical.

[0260] Furthermore, the oligonucleotides of the present invention can be administered intranasally into the mammalian brain by injection through the cribriform foramen of the cribriform plate, thereby achieving efficient delivery to the brain with minimal invasiveness and exhibiting high efficacy as a nucleic acid drug. Thus, while the oligonucleotides described herein are particularly suitable for intranasal administration, certain efficacy as nucleic acid drugs (e.g., reduction of the expression of a specific protein (knockdown)) can also be expected through non-nasal administration. In other words, the oligonucleotides described herein are not limited to a specific administration method and possess certain efficacy as nucleic acid drugs themselves. For example, the oligonucleotides described herein can also be used as siRNAs targeting specific genes, as shown below, without being limited to a specific administration method.

[0261] 1. SNCA-targeting siRNA, comprising any one pair of oligonucleotide sequences selected from the group consisting of (S6) to (S11) below: (S6) SEQ ID NO: 1360 (sense strand) and SEQ ID NO: 1389 (antisense strand) (S-0179) (S7) SEQ ID NO: 1366 (sense strand) and SEQ ID NO: 1395 (antisense strand) (S-0186) (S8) SEQ ID NO: 1368 (sense strand) and SEQ ID NO: 1397 (antisense strand) (S-0187) (S9) SEQ ID NO: 1372 (sense strand) and SEQ ID NO: 1401 (antisense strand) (S-0191) (S10) SEQ ID NO: 1374 (sense strand) and SEQ ID NO: 1403 (antisense strand) (S-0193) (S11) SEQ ID NO: 1756 (sense strand) and SEQ ID NO: 1759 (antisense strand) (S-0197); The siRNA according to 1. above, wherein the ratio of 2'-modified nucleotides to the nucleotides constituting the siRNA is 80% or more; 3. The siRNA according to 1. or 2. above, wherein all nucleotides constituting the siRNA are 2'-modified nucleotides; 4. The siRNA according to any of 1. to 3. above, which has one or more lipophilic moieties; 5. The siRNA according to 4. above, wherein the lipophilic moiety is at least one selected from the group consisting of a substituted or unsubstituted alkyl chain having 14 to 24 carbon atoms, eicosapentaenoic acid (EPA), arachidonic acid (ARA), and cholic acid; 6. The siRNA according to any of 1. to 5. above, wherein all of the phosphodiester bonds linking the first and second nucleotides counting from both ends of the sense strand and antisense strand of the siRNA are substituted with phosphorothioate bonds.

[0262] 7. An siRNA targeting SNCA, comprising the sequences of any one set of oligonucleotides selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246 listed in any of Tables 1-1 to 1-6.

[0263] 8. An siRNA targeting SNCA, which is any one set of oligonucleotides selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268, and ds273 listed in any one of Tables 1-1 to 1-6.

[0264] One aspect of the present invention is: 9. An siRNA targeting HTT, comprising any one pair of oligonucleotide sequences selected from the group consisting of (H1) to (H5) below: (H1) SEQ ID NO: 365 (sense strand) and SEQ ID NO: 623 (antisense strand) (H-0005) (H2) SEQ ID NO: 370 (sense strand) and SEQ ID NO: 628 (antisense strand) (H-0010) (H3) SEQ ID NO: 396 (sense strand) and SEQ ID NO: 654 (antisense strand) (H-0036) (H4) SEQ ID NO: 407 (sense strand) and SEQ ID NO: 665 (antisense strand) (H-0047) (H5) SEQ ID NO: 469 (sense strand) and SEQ ID NO: 727 (antisense strand) (H-0109); 10. An siRNA according to the above item 9, wherein the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is 80% or more; 11. 12. The siRNA according to any one of items 9. to 11. above, wherein all nucleotides constituting the siRNA are 2'-modified nucleotides; 13. The siRNA according to any one of items 9. to 11. above, wherein all phosphodiester bonds linking the first and second nucleotides counting from both ends of the sense strand and antisense strand of the siRNA are substituted with phosphorothioate bonds.

[0265] 13. An siRNA targeting HTT, comprising the sequences of any one set of oligonucleotides selected from the group consisting of ds274 to ds461 listed in any one of Tables 2-1 to 2-4.

[0266] 14. An siRNA targeting HTT, comprising the sequences of any one set of oligonucleotides selected from the group consisting of ds278, ds280, ds299, ds308, ds347, ds405, ds406, ds411, ds414, ds422, and ds432 to ds461 listed in any of Tables 2-1 to 2-4.

[0267] Although the embodiments of the present invention have been described in detail above, it is clear that these are for illustrative and exemplary purposes only and are not limiting, and the scope of the present invention should be interpreted by the appended claims.

[0268] The present invention will be described below with reference to examples, although the present invention is not limited to these examples.

[0269] The effects of the present invention will be explained using the following examples and comparative examples. The materials, amounts used, ratios, treatment details, treatment procedures, etc. shown in the following manufacturing examples and examples can be modified as appropriate without departing from the spirit of the present invention. Therefore, the technical scope of the present invention is not limited to the following examples. The quantitative ratios of raw materials used in solutions or dispersions (units: "parts by mass" and "% by mass") are all values ​​converted to solid content. Unless otherwise specified, each operation was performed at room temperature (hereinafter, room temperature is defined as 25°C). In the following steps, the "N" in "mN", "fN", "dN", "rN", "eN", and "lN" represents A, T, C, and G, respectively, for DNA, and A, U, C, and G, respectively, for RNA.

[0270] <Preparation of siRNA (ds1 to ds6) used in the Examples and Reference Examples> (Step 1) Synthesis of oligonucleotides (ss6 and ss9) Oligonucleotides were synthesized using ns-8 (Gene Design). The solid support was 2'-OMe-RNA CPG (Glen Research), the phosphoramidites were 2'-OMe and 2'-F ribonucleoside phosphoramidites (Glen Research), and Solid Chemical Phosphorylation Reagent II (Glen Research) was used, and the solution was adjusted to 0.12 mol / L with ultra-dehydrated MeCN (Fujifilm Wako Pure Chemical Industries, Ltd.). The activator was 0.25 mol / L BTT in MeCN (Glen Research), the oxidizing agent was 0.02 mol / L iodine in THF / pyridine / water (7:2:1, Glen Research), the sulfurizing agent was 0.1 mol / L DDTT in pyridine (ChemGenes), the deblocking reagent was 3% TCA in dichloromethane (Fujifilm Wako Pure Chemical Industries), and the capping agents were CAP A (AcO / lutidine / THF = 1:1:8, Glen Research) and CAP B (10% 1-methylimidazole in THF, Glen Research). Solid-phase synthesis of oligonucleotides was carried out using RNA1.0 or RNA10, the standard method attached to ns-8.

[0271] Cleavage from the solid support and deprotection were carried out according to the protocol provided by the reagent company, using an AMA solution prepared by mixing 28% aqueous ammonia and 40% aqueous methylamine in a 1:1 ratio. The resulting crude product was concentrated under reduced pressure and then purified by anion exchange chromatography (system: Prominence (Shimadzu Corporation), column: Mono Q (registered trademark) 5 / 50 GL or Mono Q (registered trademark) 10 / 100 GL (Cytiva), solution A: 30% MeCN, 10 mmol / L Tris-HCl (pH 8), solution B: 1 mol / L NaBr, 30% MeCN, 10 mM Tris-HCl (pH 8)), and the resulting fractions were analyzed by LC-MS (system: Infinity 1260 (Agilent), column: ACQUITY Premier Oligonucleotide C18 Column, 130 Å, 1.7 μm, 2.1 × 50 mm (Waters), Solution A: 8.6 mmol / L TEA / 100 mmol / L HFIP, Solution B: MeOH, Gradient: 10% B to 90% B in 9 min, Flow rate: 0.6 mL / min, Column temperature: 60°C, Detection: PDA (260 nm), ESI-MS. Fractions containing the target product were desalted with distilled water (Otsuka Pharmaceutical Factory) using an Amicon® Ultra-15, Ultracel, 3 kDa (Merck). The resulting solution was microfiltered using Ultrafree (registered trademark)-MC or -CL, PVDF, 0.22 μm pore size, sterile (Merck) to synthesize oligonucleotides ss6 and ss9 shown in Table 5. The molecular weights of the resulting oligonucleotides were calculated by deconvolution using the software provided with the LC-MS. The sequences of all oligonucleotides used in the examples, and the calculated and measured molecular weights of the resulting oligonucleotides are shown in Table 5.

[0272] (Step 2) Synthesis of oligonucleotides (ss1, ss2, ss4, and ss5) using special amidites Oligonucleotides were synthesized using AKTA Oligopilot (manufactured by Cytiva). The solid phase support was NittoPhase® HL 2′OMeU350 (Kinovate) or NittoPhase® HL 2′OMeA350 (Kinovate). The phosphoramidites were synthesized according to the methods described in Journal of Medicinal Chemistry (2018), 61(3), 734-744 and WO 2021 / 092371, including phosphoramidites corresponding to (vnT), (vmU), and [C16U] shown in FIGS. 1 and 2, as well as 2′-OMe and 2′-F ribonucleoside phosphoramidites (Thermo Fisher Scientific). The solution was prepared using a solution of 0.15 mol / L of ultra-dehydrated MeCN. The activator was 0.25 mol / L BTT in MeCN (manufactured by Glen Research), the oxidizing agent was 0.05 mol / L iodine in pyridine / water (9:1, manufactured by Sigma-Aldrich), the sulfurizing agent was 0.1 mol / L DDTT in pyridine (manufactured by ChemGenes), the deblocking reagent was 3% DCA in toluene (manufactured by Fujifilm Wako Pure Chemical Industries), and the capping agents were CAP A (MeCN:N-methylimidazole = 8:2, manufactured by Sigma-Aldrich), CAP B1 (AcO:MeCN = 4:6, manufactured by Fujifilm Wako Pure Chemical Industries), and CAP B2 (2,6-lutidine:MeCN = 6:4, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was used. Solid-phase synthesis of oligonucleotides was carried out using Column 6 and 12 ml AKTAop plus 100 Ed CA, a standard method included with AKTA Oligopilot, with some modifications to the conditions (Coupling: 4 eq. phosphoramidite, 10 min; Oxidation: 4 eq., 1 min; Sulfurization: 5 CV, 10 min).

[0273] Cleavage from the solid support and deprotection were carried out using 28% aqueous ammonia or 28% aqueous ammonia with DEA ​​added to 3% (v / v) (3% DEA / aqueous ammonia solution) according to the protocol described in the above literature. The resulting crude product was concentrated under reduced pressure and then purified by anion exchange chromatography (system: AKTA pure® 150 (manufactured by Cytiva), column: TSKgel® SuperQ-5PW, 13 maikurom, 21.5 x 150 mm (manufactured by Tosoh Corporation), solution A: 20% MeCN, 10 mmol / L Tris-HCl (pH 8), solution B: 1 mol / L NaBr, 20% MeCN, 10 mM Tris-HCl (pH 8)). The resulting fractions were analyzed by LC-MS under the same conditions as in step 1. Fractions containing the target product were desalted with distilled water (Otsuka Pharmaceutical Factory) using an LV Centramate TFF system (PALL) equipped with a Centramate® cassette, Omega PES membrane, and 1 kDa cut-off (PALL). The resulting solution was microfiltered through a Millex-GP Syringe Filter Unit, 0.22 μm, polyethersulfone, 33 mm, gamma sterilized (Merck). Oligonucleotides ss2 and ss5 bearing a vinylphosphonate group at the 5'-end and oligonucleotides ss1 and ss4 bearing a hexadecyl group at each nucleotide in the chain were synthesized (see Table 5).

[0274] (Step 3) Preparation of EPA-PFP ester Eicosapentaenoic acid (300 mg, 990 μmol, 1.2 equivalents) was dissolved in DMF (N,N-dimethylformamide) (8.3 mL), followed by the addition of triethylamine (350 μL, 2.5 Mmol, 3 equivalents) and PFTU (pentafluorophenol-tetramethyluronium hexafluorophosphate) (350 mg, 830 μmol, 1 equivalent) and stirring at room temperature for 1 hour to obtain a solution of PFP ester of eicosapentaenoic acid (pentafluorophenyl esters) (approximately 100 mM). The resulting compound was used in the subsequent conjugation reaction without isolation.

[0275] (Step 4) Synthesis of 5'-EPA-linked oligonucleotide (ss3) Using PDA-C6-aminomodifier amidite (Glen Research), the oligonucleotide having an amino group at the 5' end, synthesized according to the method of Step 1, was reacted with the PFP ester obtained in Step 3 in a 75% aqueous DMF solution containing Pierce (registered trademark) 2X boronate buffer (TFS) at pH 8.5. The reaction solution containing the reactants was then purified by ion-pair reversed-phase HPLC to obtain oligonucleotide ss3.

[0276] (Step 5) Synthesis of 3′-EPA-linked oligonucleotides (ss7 and ss8) Using 3′-PT-aminomodifier C6 CPG (manufactured by Glen Research), the oligonucleotide having an amino group at the 3′ end, synthesized according to the method of Step 1, was reacted with the PFP ester obtained in Step 3 in a 75% aqueous DMF solution containing Pierce (registered trademark) 2× boronate buffer at pH 8.5, and then purified by ion-pair reversed-phase HPLC to obtain oligonucleotides ss7 and ss8.

[0277] (Step 6) Preparation of double-stranded siRNA Equal amounts of antisense and sense strands were mixed in the combinations shown in Table 5, heated at 85°C for 5 minutes, and then allowed to cool to room temperature. After allowing to cool, approximately 200 pmol of the sample was diluted with 30% MeCN in 1x PBS and analyzed by SEC-HPLC (system: Prominence (Shimadzu Corporation), column: X-Bridge Protein BEH SEC Column, 200 Å, 3.5 μm, 7.8 mm x 300 mm (Water), buffer: 30% MeCN in 1x PBS (isocratic conditions)) to confirm the formation of double strands. The resulting double-stranded chains were lyophilized, and compounds ds1 and ds2 were dissolved in aCSF, ds3 in aCSF or 1x PBS, and ds4 to 6 in 1x PBS, for evaluation. Figure 2 shows the structures of the nucleotides and elements contained in the siRNA. In Table 5, "mN" represents 2'-O-methyl-RNA, "fN" represents 2'-fluoro-DNA, "dN" represents DNA, "(vnT)" represents 5'-vinylphosphate-2'-O-(N-methylacetamide)-thymidine, "(vmU)" represents 5'-vinylphosphate-2'-O-methyl-uridine, "(C16U)" represents 2'-O-hexadecyl-uridine, "p" represents 5'-phosphate, and "^" represents a phosphorothioate bond. The structures of "[L1]", "(C16U)", "(vnT)", and "(vmU)" in Table 5 are shown in Figures 1 and 2.

[0278]

[0279] <Preparation of siRNA (ds7 to ds16) used in Reference Examples> (Step 7) Synthesis of single-stranded oligonucleotides (ss12, ss14) Oligonucleotides ss12 and ss14 were synthesized in the same manner as in Steps 1 and 2. The sequences of the oligonucleotides used in Reference Examples, and the calculated and measured molecular weights of the obtained oligonucleotides are shown in Tables 5 and 6.

[0280] (Step 8) Synthesis of cyclic single-stranded oligonucleotide (ss10) Oligonucleotide ss10 was synthesized by the method described in WO 2018 / 199340.

[0281] (Step 9) Synthesis of 3'-EPA-linked oligonucleotide (ss13) Oligonucleotide ss13 was synthesized in the same manner as in step 5.

[0282] (Step 10) Preparation of C16-PFP ester Palmitic acid (11 mg, 43 μmol, 1 equivalent) was dissolved in DMF (850 μL), and then triethylamine (18 μL, 130 μmol, 3 equivalents) and pentafluorophenyl trifluoroacetate (13 mg, 47 μmol, 1.1 equivalents) were added and stirred at room temperature for 5 hours to obtain a PFP ester solution of palmitic acid (approximately 50 mM). The resulting compound was used in the subsequent conjugation reaction without isolation.

[0283] (Step 11) Synthesis of 3'-C7-C22 linked oligonucleotide (ss16) C22-C7-CPG was synthesized by the method described in Tetrahedron Letters (Vol. 55, pp. 94-97, 2014). Subsequently, oligonucleotide ss16 was obtained using this C22-C7-CPG in the same manner as in Step 1.

[0284] (Step 12) Preparation of ARA-PFP ester A solution of PFP ester of arachidonic acid (approximately 50 mM) was obtained using arachidonic acid in the same manner as in Step 10. The obtained compound was used in the subsequent conjugation reaction without isolation.

[0285] (Step 13) Preparation of Cholic Acid-PFP Ester Trifluoroacetylcholic acid (400 mg, 570 μmol, 1 equivalent), synthesized according to the method described in Journal of the Chemical Society, Perkin Transactions 1 (Vol. 8, pp. 2245-2250, 1990), was dissolved in dichloromethane (10 mL), and N,N-diisopropylethylamine (500 μL, 2.9 Mmol, 5 equivalents) was added under ice cooling, followed by the addition of pentafluorophenyl trifluoroacetate (0.12 mL, 700 μmol, 1.2 equivalents). The resulting reaction mixture was warmed to room temperature and stirred for 2 hours. After completion of the reaction, the reaction mixture was diluted with dichloromethane and washed with saturated aqueous sodium bicarbonate and brine. The resulting solution was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to give a crude product, which was purified by reverse-phase column chromatography eluting with 100% acetonitrile (no buffer) to give Cholic acid-PFP ester (180 mg, 210 μmol, 35%) as a white solid.

[0286] The compound isolated under the following conditions was measured using a nuclear magnetic resonance spectrometer (apparatus name: Ascend (registered trademark) 400, manufactured by Bruker). １ H NMR (400MHz, DMSO-d6): δ (ppm): 0.79 (s, 3H), 0.86 (d, J = 5.6Hz, 3H), 0.96 (s, 3H), 1.15-1.48 (m, 8H), 1.50-2.02 (m , 13H), 2.14-2.19 (m, 1H), 2.68-2.70 (m, 1H), 2.78-2.79 (m, 1H), 4.81-4.83 (m, 1H), 5.14 (brs, 1H), 5.39 (brs, 1H).

[0287] (Step 14) Synthesis of 3'-C7-C16 linked oligonucleotide (ss15) Using 3'-amino modifier C7 CPG (Glen Research), the oligonucleotide having an amino group at the 3' end, synthesized according to Step 1, was reacted with the C16-PFP ester obtained in Step 10 in a 70% DMF aqueous solution containing Pierce (registered trademark) 2X boronate buffer (TFS) at pH 8.5 to obtain a reaction solution. The reaction solution was then purified by ion-pair reversed-phase HPLC to obtain oligonucleotide ss15.

[0288] (Step 15) Synthesis of 3'-C7-ARA-linked oligonucleotide (ss18) Oligonucleotide ss18 was obtained in the same manner as in Step 14, except that the ARA-PFP ester obtained in Step 12 was used.

[0289] (Step 16) Synthesis of 3'-C7-Cholic acid-linked oligonucleotide (ss19) A reaction solution was obtained in the same manner as in Step 14, except that the Cholic acid-PFP ester obtained in Step 13 was reacted in a 70% DMF aqueous solution containing Pierce (registered trademark) 2X boronate buffer (manufactured by TFS) at pH 8.5. After confirming completion of the reaction by LC-MS analysis, 600 μL of 40% aqueous methylamine was added, and completion of the deprotection reaction was confirmed by LC-MS analysis. The resulting crude product was purified by ion-pair reversed-phase HPLC to obtain oligonucleotide ss19.

[0290] (Step 17) Synthesis of 5′-EPA-linked oligonucleotide (ss11) Oligonucleotide ss11 was synthesized in the same manner as in step 4.

[0291] (Step 18) Synthesis of 3'-C7-EPA-linked oligonucleotide (ss17) In the same manner as in Step 14, oligonucleotide ss17 was obtained using the EPA-PFP ester obtained in Step 3.

[0292] (Step 19) Preparation of double-stranded siRNA Double-stranded siRNAs were prepared by mixing equal amounts of antisense and sense strands in the combinations shown in Table 6 in the same manner as in step 6. The resulting double-stranded siRNAs were lyophilized as needed, and then reconstituted in 1x PBS for evaluation. In Table 6, "mN" represents 2'-O-methyl-RNA, "fN" represents 2'-fluoro-DNA, "dN" represents DNA, "(vnT)" represents 5'-vinylphosphate-2'-O-(N-methylacetamide)-thymidine, "(vmU)" represents 5'-vinylphosphate-2'-O-methyl-uridine, "(C16U)" represents 2'-O-hexadecyl-uridine, "p" represents 5'-phosphate, and "^" represents a phosphorothioate bond. The structures of "[L1]", "[L2]", "[L3]", "[L4]", "[L5]", "[L6]", "[CL]", "[C16U]", "[vnT]" and "[vmU]" in Table 6 are shown in Figures 1 and 2.

[0293]

[0294] <Preparation of SNCA-targeting siRNA (ds17 to ds249)> For ds17 to ds249, single-stranded oligonucleotides were synthesized using a method similar to that used in step 1 of the preparation of siRNA (ds1 to ds6). Double-stranded oligonucleotides were then prepared using the same method as in step 6, by mixing equal amounts of the antisense and sense strands in 1x PBS in the combinations shown in Tables 7-1 to 7-5. Synthesis of ds17 to 171 was outsourced to Gene Design. In Tables 7-1 to 7-5, "mN" represents 2'-O-methyl-RNA, "fN" represents 2'-fluoro-DNA, "dN" represents DNA, "(vmU)" represents 5'-vinylphosphate-2'-O-methyl-uridine, "p" represents 5'-phosphate, and "^" represents a phosphorothioate bond. The structure of "(vmU)" is shown in Figure 1.

[0295]

[0296]

[0297]

[0298]

[0299]

[0300] <Preparation of SNCA-targeting siRNA (ds250 to ds253)> For ds250 to ds253, single-stranded oligonucleotides were synthesized using 2'-TBDMS ribonucleoside phosphoramidite (manufactured by TFS) in a manner similar to steps 2 and 4 of the preparation of siRNA (ds1 to ds6). Then, double-stranded oligonucleotides were prepared in the same manner as in step 6 by mixing equal amounts of the antisense and sense strands in the combinations shown in Table 8. The resulting double-stranded oligonucleotides were lyophilized and prepared into an aCSF solution for evaluation. In Table 8, "mN" represents 2'-O-methyl-RNA, "fN" represents 2'-fluoro-DNA, "rN" represents RNA, "(vmU)" represents 5'-vinylphosphate-2'-O-methyl-uridine, and "^" represents a phosphorothioate bond. The structures of "[L1]" and "(vmU)" are shown in Figures 1 and 2.

[0301]

[0302] <Preparation of SNCA-targeting siRNA (ds254-ds257)> For ds254-257, single-stranded oligonucleotides were synthesized using a method similar to steps 2 and 4 of the preparation of siRNA (ds1-ds6). Then, double-stranded oligonucleotides were prepared using the same method as in step 6 by mixing equal amounts of the antisense and sense strands in the combinations shown in Table 9. The resulting double-stranded oligonucleotides were lyophilized and prepared into an aCSF solution for evaluation. In Table 9, "mN" represents 2'-O-methyl-RNA, "fN" represents 2'-fluoro-DNA, "(vmU)" represents 5'-vinylphosphate-2'-O-methyl-uridine, and "^" represents a phosphorothioate bond. The structures of "[L1]" and "(vmU)" are shown in Figures 1 and 2.

[0303]

[0304] <Preparation of SNCA-targeting siRNA (ds258-ds273)> For ds258-273, single-stranded oligonucleotides were synthesized using a method similar to steps 2 and 4 of the preparation of siRNA (ds1-ds6). Then, double-stranded oligonucleotides were prepared using the same method as in step 6, by mixing equal amounts of antisense and sense strands in the combinations shown in Table 10. The resulting double-stranded oligonucleotides were lyophilized and prepared into an aCSF solution for evaluation. In Table 10, "mN" represents 2'-O-methyl-RNA, "fN" represents 2'-fluoro-DNA, "(vmU)" represents 5'-vinylphosphate-2'-O-methyl-uridine, and "^" represents a phosphorothioate bond. The structures of "[L1]" and "(vmU)" are shown in Figures 1 and 2.

[0305]

[0306] <Preparation of siRNA (ds274 to ds461) targeting HTT> Single-stranded oligonucleotides were synthesized using a method similar to that used in step 1 of preparing siRNA (ds1 to ds6), and then double-stranded oligonucleotides were prepared by mixing equal amounts of antisense and sense strands in 1x PBS in the combinations shown in Tables 11-1 to 11-4 using the same method as in step 6. In Tables 11-1 to 11-4, "mN" represents 2'-O-methyl-RNA, "fN" represents 2'-fluoro-DNA, "dN" represents DNA, "p" represents 5'-phosphate, and "^" represents a phosphorothioate bond.

[0307]

[0308]

[0309]

[0310]

[0311] <Preparation of antisense oligonucleotides (sequences 1346-1348)> Sequence 1346 was synthesized using LNA phosphoramidites (Fujifilm Wako Pure Chemical Industries, Ltd.) with reference to the method described in JCI Insight (2021) 6(5), e135633. Sequence 1347 was synthesized according to the method described in The Journal of Clinical Investigation (2006) 116(8), 2290-2296, and sequence 1348 was synthesized according to the method described in Nucleic Acids Research. The resulting oligonucleotides were lyophilized and prepared into an aCSF solution for evaluation. In Table 12, "eN" represents 2'-O-methoxyethyl-RNA, "lN" represents LNA, "dN" represents DNA, "H" represents 5-methylcytosine, and "^" represents a phosphorothioate bond. Furthermore, "eH" represents 2'-O-methoxyethyl-5-methylcytosine, and "lH" represents LNA having 5-methylcytosine as the base.

[0312]

[0313] Reference Example 1-1: Knockdown test using hypoxanthine-guanine phosphoribosyltransferase 1 (hereinafter referred to as HPRT1)-targeting siRNA administered to rats via cervical administration. 30 μL (600 μg / head) of ds4, ds3, or ds7 diluted to 20 mg / mL with D-PBS was administered to 9-week-old male SD rats via cervical administration (intracisternal administration). Seven days after administration, brain samples were collected and analyzed (N=5). Two of the five ds7 rats showed abnormalities, so the remaining three (N=3) showed no abnormalities.

[0314] 500 μL of TRIzol® Reagent (TFS, 15596-018) and zirconia balls φ5 mm (Nikkato, YTZ-5) were added to each collected organ, and the organs were disrupted using a TissueLyser II (QIAGEN). After centrifugation, the supernatant was collected and mixed with 40% chloroform. After centrifugation, the supernatant was collected and RNA was purified using a KingFisher® Flex (TFS) with a KingFisher MagMax-96 Total RNA Isolation Kit (TFS, AM1830). Using EvoScript Universal cDNA Master (Roche Diagnostics, 07912455001), a reverse transcription reaction was carried out according to the instructions attached to the kit to prepare cDNA.

[0315] This cDNA was used as a template for PCR reaction, and the HPRT1 gene and the beta actin (hereinafter referred to as ACTB) gene as a control were subjected to PCR reaction using the TaqMan (registered trademark) probe method using a QuantStudio 12K Flex real-time PCR system (Applied Biosystems), and the mRNA amplification levels were measured, and the ACTB mRNA amplification level was used as an internal control to calculate the semi-quantitative value of HPRT1 mRNA. Furthermore, the mRNA amplification levels of HPRT1 and ACTB in the negative control group were similarly measured, and the semi-quantitative value of HPRT1 mRNA was calculated.

[0316] The HPRT1 gene was measured using TaqMan probe Rn01527840_m1 (Applied Biosystems), and the ACTB gene was measured using Rn00667869_m1 (Applied Biosystems). The reaction reagent used was TaqMan® Gene Expression Master Mix (Applied Biosystems, 4369542), and the assay was performed according to the attached protocol. The target mRNA amount in nucleic acid-administered individuals was calculated as a relative ratio when the amount of HPRT1 mRNA in the negative control group (non-administered group) was set to 1. The results, expressed as the mean ± standard error of the relative proportion of the mRNA amount, are shown in Figure 5. The data for each site in Figure 5 show, from left to right, the results for the negative control (non-administered), ds4, ds3, and ds7.

[0317] The test samples (ds3 and ds7) showed improved knockdown activity compared to ds4.

[0318] Reference Example 1-2: Rat Cervical Administration Knockdown Test Using HPRT1-Targeting siRNA. 30 μL (300 μg / head) of ds4, ds3, ds5, and ds8 diluted to 10 mg / mL with D-PBS was administered cervically to 9-week-old male SD rats. Seven days after administration, brain samples were collected and analyzed (N=5). The cerebral cortex of one individual in the non-administered group and one individual in the ds3 group was reduced to four individuals due to sample loss. Thereafter, mRNA expression levels were measured using the same method as in Reference Example 1-1. The target mRNA levels in the nucleic acid-administered individuals were calculated as a relative ratio when the amount of HPRT1 mRNA in the negative control group (non-administered group) was set to 1. The results, expressed as the mean ± standard error of the relative proportions of mRNA levels, are shown in Figure 6. The data for each region in Figure 6 show, from left to right, the results for the negative control (non-administered), ds4, ds3, ds5, and ds8.

[0319] The test samples (ds3, ds5, ds8) showed improved knockdown activity compared to ds4.

[0320] Reference Example 1-3: Rat Cervical Knockdown Test Using siRNA Targeting Alpha-Synuclein (hereinafter referred to as SNCA) ds9, ds1, ds10, and ds2 diluted to 10 mg / mL with D-PBS were administered to 10-week-old male SD rats at 30 μL (300 μg / head) cervically, and 7 days after administration, brain samples were collected and analyzed (N=5). cDNA was then prepared in the same manner as in Reference Example 1-1.

[0321] This cDNA was used as a template for PCR reaction, and the SNCA gene and the ACTB gene (as a control) were subjected to PCR reaction using a QuantStudio 12K Flex real-time PCR system by the TaqMan (registered trademark) probe method, and the mRNA amplification levels were measured. The mRNA amplification level of ACTB was used as an internal control, and a semi-quantitative value of SNCA mRNA was calculated. In addition, the mRNA amplification levels of SNCA and ACTB in the negative control group were similarly measured, and a semi-quantitative value of SNCA mRNA was calculated.

[0322] The SNCA gene was measured using TaqMan probe Rn01425143_m1 (Applied Biosystems), and the ACTB gene was measured using Rn00667869_m1 (Applied Biosystems). The reaction reagent was TaqMan® Gene Expression Master Mix, and the assay was performed according to the attached protocol. The target mRNA amount in nucleic acid-administered individuals was calculated as a relative ratio when the SNCA mRNA amount in the negative control group (non-administered group) was set to 1. The relative ratio of the mRNA amount is shown in Figure 7 as the mean ± standard error. The data for each site in Figure 7 shows, from left to right, the results for the negative control (non-administered), ds9, ds1, ds10, and ds2.

[0323] The test samples (ds1, ds10, ds2) showed improved knockdown activity compared to ds9.

[0324] Reference Example 2: Mouse intracerebroventricular knockdown test using HPRT1-targeting siRNA. 5 μL (125 μg / head) of ds11, ds12, ds13, ds14, ds15, and ds16 diluted to 25 mg / mL with D-PBS were administered intracerebroventricularly to 6-week-old male ICR mice. Seven days after administration, brain samples were collected and analyzed (N=5). One individual with ds14 and three with ds15 were excluded from subsequent analysis due to poor administration. RNA was extracted and purified using an RNeasy mini kit (QIAGEN, 74104). Using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, 04897030001), a reverse transcription reaction was carried out according to the instructions attached to the kit to prepare cDNA.

[0325] This cDNA was used as a template for PCR reaction, and the HPRT1 gene and the beta actin (hereinafter referred to as ACTB) gene as a control were subjected to PCR reaction using the TaqMan probe method with a CFX Connect® Real-Time System (BIO-RAD), and the mRNA amplification levels were measured, and the mRNA amplification level of ACTB was used as an internal control to calculate a semi-quantitative value for HPRT1 mRNA. Furthermore, the mRNA amplification levels of HPRT1 and ACTB in the negative control group were similarly measured, and a semi-quantitative value for HPRT1 mRNA was calculated.

[0326] The HPRT1 gene was measured using TaqMan probe Mm01545399_m1 (Applied Biosystems), and the ACTB gene was measured using Mm00607939_s1 (Applied Biosystems). The reaction reagent was TaqMan® Gene Expression Master Mix, and the assay was performed according to the attached protocol. The amount of target mRNA in nucleic acid-administered individuals was calculated as a relative ratio when the amount of HPRT1 mRNA in the negative control group (D-PBS-administered group) was set to 1. The results, expressed as the mean ± standard error of the relative proportion of the mRNA amount, are shown in Figure 8. The data for each site in Figure 8 show, from left to right, the results for the negative control (D-PBS-administered), ds11, ds12, ds13, ds14, ds15, and ds16.

[0327] All of the test samples (ds11, ds12, ds13, ds14, ds15, and ds16) showed knockdown activity compared to the negative control (D-PBS).

[0328] [Example 1: Nasal administration knockdown test in rats using HPRT1-targeting siRNA] In Example 1, rats were used as subjects, and nasal administration was performed into the cribriform foramina of the cribriform plate. After allowing the rats to survive for 7 days after administration, the amount of oligonucleotide transferred to each site in the brain and the strength of knockdown were evaluated for each rat.

[0329] The administration device 1A produced in Example 1 had the following specifications: Figure 9 shows a configuration diagram of the administration device 1A produced in Example 1.

[0330] A double-lumen catheter was fabricated by inserting an inner catheter (made of PEEK) with an outer diameter of 0.47 mm, an inner diameter of 0.37 mm, and a total length of 125 mm into an outer catheter (made of PEEK) with an outer diameter of 0.6 mm, an inner diameter of 0.52 mm, and a total length of 115 mm. The inner catheter was exposed 1 mm distally from the outer catheter, and the tip of the gap was glued with UV adhesive to create a double-lumen catheter. Next, an 18G hub-equipped needle (product name: Terumon Bevel Needle 18G1 1 / 2, manufactured by Terumo Corporation) was cut 10 mm from the proximal end of the exposed needle tube. The inner catheter of the fabricated double-lumen catheter was then inserted into the needle tube from the distal end of the cut hub, with the 1 mm exposed distal end first, and the gap was glued with UV adhesive. Next, the tip of the inner catheter was cut at the end of the double-lumen catheter opposite the end glued to the hub needle, so that 7 mm of the inner catheter was exposed from the outer catheter. Next, the step between the catheters near the cut portion was filled in with an epoxy adhesive (product name: Bondquick 30, manufactured by Konishi Co., Ltd.) to smooth out the gap, thereby producing a double-tube catheter structure. The hub portion 20 of the produced administration device 1A was the hub portion of the 18G hub-equipped needle described above, and the cannula portion 30 was composed of a double-tube catheter.

[0331] An administration device was produced by inserting the needle tip (outer diameter 0.1 mm, inner diameter 0.06 mm, total length 15 mm, blade length 0.1 mm, made of SUS304) as the needle portion 10 into the tip of the inner catheter in the cannula portion 30 of the above structure so that the exposed length of the needle tip from the tip of the inner catheter (the length in the longitudinal direction of the puncture portion of the needle portion 10) was 0.3 mm and bonding it with a UV adhesive. A microsyringe (product name: Gastight Syringe 1705TLL, manufactured by Hamilton) was attached to the proximal end side of the hub portion 20 as the storage portion 40.

[0332] The prepared administration device 1A was inserted into the nasal cavity using a guide catheter 100A as an insertion aid. The guide catheter 100A was made of polyurethane and had a double lumen shape with a total length of 45 mm, an outer diameter of 1.35 mm, and inner diameters of 0.45 mm and 0.70 mm.

[0333] Example 1 was carried out according to the test procedures shown below.

[0334] First, male rats (slc: SD rats (16 weeks old), manufactured by Japan SLC) were given induction anesthesia (2-4%) using isoflurane inhalation anesthetic solution (manufactured by Pfizer), and then the experiment was carried out under continuous anesthesia (1.5-3%).

[0335] Next, a guide catheter was inserted into one of the nasal cavities of the subject at an angle parallel to the nasal ridge of the rat, with the tip opening of the guide catheter facing the cribriform plate.

[0336] Next, the device was inserted into the guide catheter and punctured into the olfactory mucosa by the exposed length of the needle tip (length of the puncture part). At this time, it was confirmed that the insertion length remained constant when the device was pushed in with a force of approximately 0.8 N, and that the length of the part of the device inserted into the rat's body was 25 to 33 mm.

[0337] By verifying these two points, it can be confirmed that the puncture portion of the device is positioned within the cribriform foramen of the cribriform plate. More specifically, first, the length of the portion of the device inserted into the rat's body is 25 to 33 mm, which confirms that the puncture portion has at least reached the cribriform plate. Additionally, if the length of the puncture portion does not change when pressed with a force of approximately 0.8 N, it can be confirmed that the puncture portion is positioned within the cribriform foramen. This is because the annulus of the nasal cavity opening of the cribriform foramen has a concave shape (i.e., the nasal cavity opening of the cribriform foramen has a tapered shape that gradually narrows from the open end toward the brain). Therefore, when the needle portion 10 is pressed during puncture, the tip of the needle portion 10 slides along the annulus and is inserted into the cribriform foramen. However, continued pressing causes the stopper portion at the tip of the cannula portion 30 to come into contact with the olfactory epithelium, preventing further pressing.

[0338] Next, 30 μL of each nucleic acid solution was administered at 20 μL / min using a microsyringe pump (product name: IC3200, manufactured by KD Scientific) and allowed to stand for 5 minutes. For each administration, nucleic acid solutions containing three types of oligonucleotides, ds4, ds5, and ds6, dissolved in 1×PBS at a concentration of 60 mg / mL were used.

[0339] After surviving for 7 days from the day of administration, the animals were euthanized by carbon dioxide inhalation. Then, the skull was cut, and the extracted brain was cooled on a cooling plate. Each part was collected, flash-frozen in liquid nitrogen, and then cryopreserved.

[0340] Thereafter, mRNA expression levels were measured using the same method as in Reference Example 1-1. The amount of target mRNA in nucleic acid-administered individuals was calculated as a relative ratio when the amount of HPRT1 mRNA in the negative control group (non-administered group) was set to 1. The results of the relative ratio of the mRNA amount expressed as mean ± standard error are shown in Figure 10. The data for each site in Figure 10 shows, from left to right, the results for the negative control (non-administered), ds4, ds5, and ds6.

[0341] The test samples (ds4, ds5, ds6) showed knockdown activity compared to the negative control (non-administered group).

[0342] [Example 2: Nasal administration knockdown test in rats using HPRT1-targeting siRNA (varying puncture depth)] In Example 2, live rats were used as subjects, and nasal administration was performed into the cribriform foramina of the cribriform plate. After allowing the rats to survive for 7 days after administration, the amount of oligonucleotide transferred to each site in the brain of each rat and the strength of knockdown were evaluated.

[0343] In Example 2, nasal administration into the cribriform pores of the cribriform plate was carried out at two different puncture depths. For shallower punctures, nasal administration to live rats was carried out using the same method as in Example 1. On the other hand, for deeper punctures, experiments were carried out using administration device 1B using the method described below.

[0344] In order to deeply puncture the sieve holes of the sieve plate, the administration device 1B prepared in Example 2 had the same specifications as the administration device 1A prepared in Example 1, except that the exposed length (0.3 mm) was adjusted to 0.85 mm. Figure 9 shows a structural diagram of the administration device 1B prepared in Example 2.

[0345] The administration device 1B was inserted into the nasal cavity using a guide catheter 100A as an insertion aid. The guide catheter 100A was made of polyurethane and had a total length of 45 mm, an outer diameter of 1.35 mm, and a double lumen shape with inner diameters of 0.45 and 0.70 mm.

[0346] In Example 2, in order to deeply puncture the sieve holes of the cribriform plate, the test procedure shown below was carried out.

[0347] First, male rats (slc: SD rats (16 weeks old), manufactured by Japan SLC) were given induction anesthesia (2-4%) using isoflurane inhalation anesthetic solution (manufactured by Pfizer), and then the experiment was carried out under continuous anesthesia (1.5-3%).

[0348] Next, a guide catheter was inserted into one of the nasal cavities of the subject at an angle of approximately 45° relative to the nasal ridge of the rat. During insertion, the tip opening of the guide catheter was adjusted to face the cribriform plate and to be positioned at the cribriform foramen (total length approximately 2 mm) near the skull, where the olfactory nerves are concentrated deep inside the olfactory epithelium of the rat.

[0349] Next, the device was inserted into the guide catheter on the inner diameter φ0.45 side, and the olfactory mucosa was punctured by the exposed length of the needle tip (length of the puncture part). A 16Ch X-ray CT scanner (product name: Bright Speed ​​Elite, manufactured by GE Healthcare) confirmed that the tip of the puncture part had reached the area deep inside the olfactory epithelium where the olfactory nerves are concentrated.

[0350] Next, 30 μL of the nucleic acid solution was administered at 20 μL / min using a microsyringe pump (product name: IC3200, manufactured by KD Scientific) by operating the microsyringe, and the mixture was allowed to stand for 5 minutes. For administration, a nucleic acid solution prepared by dissolving intrastrand C16-siHPRT1(ds3) oligonucleotide in 1×PBS to a concentration of 60 mg / mL was used.

[0351] After surviving for 7 days from the day of administration, the animals were euthanized by exsanguination under isoflurane anesthesia. The skull was then cut, and the extracted brain was cooled on a cooling plate. Each part was then sampled, flash-frozen in liquid nitrogen, and cryopreserved.

[0352] Thereafter, the mRNA expression level was measured using the same method as in Reference Example 1-1. The amount of target mRNA in the nucleic acid-administered individuals was calculated as a relative ratio when the amount of HPRT1 mRNA in the negative control group (non-administered group) was set to 1. The results of the relative ratio of the mRNA amount expressed as mean ± standard error are shown in Figure 11. Note that the data for each site in Figure 11 shows, from left to right, the negative control (non-administered), ds3 (shallow), and ds3 (deep).

[0353] Test sample ds3 (deeper) showed improved knockdown activity compared to ds3 (shallower).

[0354] A nine-fold volume of Cell Lysis Buffer 2 (TFS, FNN0021) and zirconia beads were added to the collected organs, which were then disrupted using a TissueLyser II (QIAGEN). The organs were then centrifuged and the supernatant was collected. The supernatant and standard siRNA were converted to cDNA using TaqMan® MicroRNA RT Kit (TFS, 4369016) and Primer 1. This cDNA was used as a PCR template, and quantitative PCR was performed using a QuantStudio 12K Flex Real-Time PCR System (TFS). Primer 2, Primer 3, and TaqMan® MGB Probe 1 (TFS, 4316033) were used in the reaction. The base sequences of each primer and probe are shown in Table 13.

[0355]

[0356] The results, expressed as mean ± standard error, of the amount of nucleic acid in each brain region are shown in Figure 12. Note that the data for each region in Figure 12 show, from left to right, ds3 (shallow) and ds3 (deep).

[0357] The amount of nucleic acid is considered to be the amount of the administered nucleic acid transferred to each brain region (olfactory bulb, striatum, and cerebral cortex (neck side)). The test sample ds3 (deeper) showed an improved amount of nucleic acid transfer compared to ds3 (shallower).

[0358] Example 3: Intranasal administration knockdown test in cynomolgus monkeys using HPRT1-targeting siRNA In Example 3, living cynomolgus monkeys were used as subjects, and a nucleic acid solution (ds3) was administered intranasally into the cribriform foramina of the cribriform plate. After allowing the monkeys to survive for 7 days from the day of the final administration, the amount of oligonucleotide transferred to each site in the brain and the strength of knockdown were evaluated for each monkey.

[0359] The administration device used in Example 3 had the following specifications: Figure 13 shows a structural diagram of the administration device 1C ((a) in Figure 13) and guide catheter 100B ((b) in Figure 13) produced in Example 3.

[0360] The administration device 1C was prepared with different exposed needle tip lengths (exposed length 1.3 mm to 4.2 mm).

[0361] First, an inner catheter (made of PEEK) with an outer diameter of 0.47 mm, an inner diameter of 0.37 mm, and a total length of 165 mm was inserted into an outer catheter (made of PEEK) with an outer diameter of 0.6 mm, an inner diameter of 0.52 mm, and a total length of 155 mm. The inner catheter was exposed 1 mm distally from the outer catheter, and the tip of the gap was glued with UV adhesive to create a double-lumen catheter. Next, an 18G hub-equipped needle (product name: Terumon Bevel Needle 18G1 1 / 2, manufactured by Terumo Corporation) was cut 10 mm from the proximal end of the exposed needle tube. Next, the inner catheter of the double-lumen catheter was inserted into the needle tube from the distal end of the cut hub, with the 1 mm exposed distal end first, and the gap was glued with UV adhesive. Next, the tip of the inner catheter was cut at the end of the double-lumen catheter opposite the end glued to the hub needle, so that 7 mm of the inner catheter was exposed from the outer catheter. Next, the step between the catheters near the cut portion was filled in with an epoxy adhesive (product name: Bondquick 30, manufactured by Konishi Co., Ltd.) to smooth out the gap, thereby producing a double-tube catheter structure. The hub portion 20 of the produced administration device 1C is the hub portion of the 18G hub-equipped needle described above, and the cannula portion 30 is composed of a double-tube catheter.

[0362] Each administration device was produced by inserting a needle tip (outer diameter 0.2 mm, inner diameter 0.12 mm, total length 15 mm, blade length 0.2 mm, made of SUS304) as the needle portion 10 into the tip of the inner catheter and bonding it with a UV adhesive so that the exposed length from the tip of the inner catheter in the cannula portion 30 of the above structure to the needle tip (the length in the longitudinal direction of the puncture portion 12 of the needle portion 10) was 1.3 mm to 4.2 mm, as shown in Table 14. A microsyringe (product name: Gastight Syringe 1002TLL, manufactured by Hamilton) was attached as the storage portion 40 to the proximal end of the hub portion 20 of each device.

[0363]

[0364] The prepared administration device 1C was inserted into the nasal cavity using a guide catheter 100B as an insertion aid. The guide catheter 100B was made of SUS304, had a total length of 90 mm, an outer diameter of 0.82 mm, an inner diameter of 0.68 mm, and a bend angle of 45° at the curved tip. The guide catheter used had a spiral cut on the straight portion to give it flexibility and make it bendable.

[0365] Example 3 was carried out according to the test procedures shown below.

[0366] First, the subject cynomolgus monkeys (4 years, 0 months to 4 years, 9 months, male) were anesthetized. The anesthetic and administration method were as follows. Anesthesia and administration were performed four times for each subject, and each administration was performed using a device with the puncture length shown in Table 14. - Induction anesthesia: Ketalar intramuscular injection 500 mg, administration route: intramuscular administration, dosage: 10 mg / kg (0.2 mL / kg) - Maintenance anesthesia: Propofol injection 1% for veterinary use "Mylan", administration route: intravenous administration, dosage: 0.5 to 30 mg / kg / hour (0.05 to 3 mL / kg / hour).

[0367] Next, a 16-channel X-ray CT scanner (product name: Bright Speed ​​Elite, manufactured by GE Healthcare) was used to perform contrast imaging. A guide catheter was inserted into one of the subject's nasal cavities while checking the contrast image. During insertion, the tip opening of the guide catheter was adjusted and fixed so that it was facing the cribriform plate and the tip was positioned directly below the cribriform plate. Next, the thickness of the cribriform plate located on a straight line extending from the opening of the guide catheter was measured. The exposed length of the needle tip (the length of the puncture portion) that could puncture 60% or more of the measured cribriform plate thickness was calculated, and a puncture portion with a length equivalent to 60% to 100% of the cribriform plate thickness was selected.

[0368] Next, the selected device was inserted into the guide catheter, and the exposed length of the needle tip (the length of the puncture portion) was inserted into the olfactory mucosa. The needle tip was confirmed to have penetrated the cribriform pores using the X-ray CT scanner. Using a microsyringe, 2 mL of the nucleic acid solution was administered at 100 μL / min using a microsyringe pump (product name: IC3200, manufactured by KD Scientific). For administration, a nucleic acid solution containing ds3 oligonucleotide dissolved in aCSF at a concentration of 60 mg / mL was used.

[0369] The same method was used to administer 2 mL of the solution to the other nostril on the side of the first administration every week at a rate of 100 μL / min. A total of four administrations, totaling 8 mL, were performed. After 7 days of survival from the final fourth administration, the rats were euthanized by exsanguination, with the chest opened under isoflurane anesthesia, the descending aorta blocked, a catheter inserted into the left ventricle, and perfused with heparin-containing saline. The skull was then cut, and the brain was cut into 6 mm sections in the coronal plane using a brain matrix. Each brain region was sampled, flash-frozen in liquid nitrogen, and cryopreserved. The spinal cord (cervical, thoracic, and lumbar spinal cord) was also sampled and cryopreserved in the same manner. A control group received aCSF and underwent the same procedure.

[0370] Subsequently, cDNA preparation and quantitative PCR were performed for each brain region and spinal cord (cervical, thoracic, and lumbar spinal cord) using the same method as in Reference Example 1-1. TaqMan probe Hs99999909_m1 (Applied Biosystems) was used to measure the HPRT1 gene, and Hs01060665_g1 (Applied Biosystems) was used to measure the ACTB gene. The results, expressed as mean ± standard error of the relative proportion of mRNA levels, are shown in Figures 14-1 and 14-2. Note that the data for each region in Figures 14-1 and 14-2 show, from left to right, the negative control (aCSF), ds3.

[0371] The test sample (ds3) showed significant knockdown activity of the HPRT1 gene in each region of the brain compared to the control (aCSF).

[0372] The amount of nucleic acid transfer in each brain region was also measured using the same method as in Example 2. The results, expressed as the mean ± standard error, of the amount of nucleic acid in each brain region and spinal cord (cervical, thoracic, and lumbar spinal cord) are shown in Figures 15-1 and 15-2. The test sample (ds3) showed significant nucleic acid transfer in each brain region. Note that the data for each region in Figure 15-1 shows, from left to right, the results for the left brain and the right brain.

[0373] [Example 4] In Example 4, live rats were used as subjects, and intranasal administration was performed into the cribriform foramina of the cribriform plate. After survival for 7 days after administration, the amount of the administered substance transferred to each site in the brain of each subject and the strength of knockdown were evaluated.

[0374] In Example 4, administration was performed using two methods: nasal administration and nasal instillation. For nasal administration, nasal administration to live rats was performed using the administration device 1B (puncture length: 0.85 mm) prepared in Example 2 in the same manner as in Example 2 (see Figure 5). For nasal instillation, a Pipetman (manufactured by GILSON) was used as the nasal administration device, and the experiment was performed using the method described below. For nasal administration and nasal instillation, two types of nucleic acid solutions were used: ds1 and ds2 oligonucleotides dissolved in aCSF to a concentration of 60 mg / mL. Administration of aCSF alone was also performed in the same manner as the administration of the nucleic acid solution described below.

[0375] First, male rats (slc: SD rats (16 weeks old), manufactured by Japan SLC) were given induction anesthesia (2-4%) using isoflurane inhalation anesthetic solution (manufactured by Pfizer), and then the experiment was carried out under continuous anesthesia (1.5-3%) with a small animal anesthesia mask attached.

[0376] Next, the nasal administration device was operated to fill the tip with 5 μL of nucleic acid solution. Next, the small animal anesthesia mask attached to the rat's nose was removed, and the rat was placed on its back. The tip of the pipette was placed close to the nostril on the administration side without being inserted into the nostril, and slowly pushed out over 2-3 minutes. After administering 5 μL, the rat was kept in a supine position in the anesthesia box for 2 minutes. The above method was repeated six times in the same nostril as the first administration, for a total of 30 μL. After administration, the rat was kept in a supine position in the anesthesia box for 5 minutes.

[0377] After surviving for 7 days from the day of administration, the animals were euthanized by exsanguination under isoflurane anesthesia. The skull was then cut, and the extracted brain was cooled on a cooling plate. Each part was then sampled, flash-frozen in liquid nitrogen, and cryopreserved.

[0378] Thereafter, cDNA preparation and quantitative PCR were performed in the same manner as in Reference Examples 1-3. The relative proportions of mRNA amounts are shown as mean ± standard error in Figure 16. The data for each site in Figure 16 show, from left to right, the results for aCSF, ds1, and ds2 (all administered intranasally), and aCSF, ds1, and ds2 (all administered intranasally).

[0379] Nasal administration showed a significant knockdown activity of the SNCA gene in each region of the brain compared to nasal administration.

[0380] The amount of nucleic acid transfer in each brain region was also analyzed using the same method as in Example 2. cDNA was converted using TaqMan® MicroRNA RT Kit (TFS, 4369016) and Primer 4. This cDNA was used as a PCR template, and quantitative PCR was performed using a QuantStudio 12K Flex Real-Time PCR System. Primer 5, Primer 3, and TaqMan® MGB Probe 2 (TFS, 4316033) were used in the reaction. The base sequences of each primer and probe are shown in Table 13.

[0381] The results, expressed as the mean ± standard error of the nucleic acid amount in each brain region, are shown in Figure 17. Nasal administration showed improved nucleic acid transfer in each brain region compared to nasal administration. Note that the data for each region in Figure 17 show, from left to right, the results for ds1, ds2 (both nasal administration), and ds1, ds2 (both nasal administration).

[0382] Example 5: In vitro evaluation of siSNCA siRNA design A set of 405 siRNAs targeting the human synuclein alpha gene (SNCA; human NCBI refseq ID NM_000345.4; NCBI GeneID: 6622) was designed using a custom Perl script. The nucleotide sequence described in WO 2022 / 072447 was also used as a reference for siRNA design. Scoring of the designed siRNA was performed with reference to the s-Biopredsi method (Nucleic Acids Research (2007) 35(18), e123) and the siDirect method (Nucleic Acids Research (2004) 32 (Web Server Issue), W124-W129), and off-target evaluation was performed using GGGenome (Retriever).

[0383] <Cell Culture> BE(2)-C cells (KAC) were cultured in a medium prepared by mixing equal parts of EMEM medium (ATCC) and Ham's F-12 medium (TFS) with 15% inactivated fetal bovine serum (FBS) at 37°C under 5% CO ２The cells were cultured under 5% CO₂ at 25°C for 1 hour. The cells were subcultured twice a week by washing with PBS (TFS) and then treating with 0.25% Trypsin-EDTA (TFS). Cells that had been passaged 6 or 7 times were used for the assay.

[0384] <Addition of siRNA> A PBS solution of 500 nmol / L siRNA was diluted with Opti-MEM medium (TFS) to 50 pmol / L, 500 pmol / L, and 5 nmol / L in a 384-well black bottom clear plate (Greiner) to make 10 μL. Next, Lipofectamine RNAiMAX (TFS) was diluted 80-fold with Opti-MEM medium, and 10 μL was added to the plate to which 1.33 x 10 ５ 30 μL of a suspension of BE(2)-C cells prepared to a concentration of 4000 cells / mL was added to the plate to give a concentration of 4000 cells / well. The plate was left to stand at room temperature for approximately 15 minutes, and then incubated at 37°C in 5% CO ２ The mixture was incubated overnight under

[0385] <Evaluation of knockdown activity> Cell lysis, reverse transcription, and qPCR were performed according to the standard protocol of the Taqman Fast Advanced Cells-to-CT kit (TFS). cDNA prepared from cells lysed using the kit was used with Taqman probes Hs00240906_m1 SNCA (Applied Biosystems) to measure the SNCA gene and Hs01060665_g1 (Applied Biosystems) to measure the ACTB gene. PCR reactions were performed using Quant Studio 7 Flex (Applied Biosystems) to measure the SNCA gene and the ACTB gene as a control, and the Ct values ​​were measured. The Ct value of ACTB was used as an internal control to calculate the quasi-quantitative value of SNCA mRNA. In addition, the Ct values ​​of SNCA and ACTB in the negative control group (0 pmol of siRNA) were measured in the same manner, and the semi-quantitative value of SNCA mRNA was calculated. The results of siRNAs (ds17-171) that showed 70% or more knockdown activity at 100 pmol / L or 1 nmol / L as the average of duplicate measurements are shown in Tables 15-1 to 15-4.

[0386]

[0387]

[0388]

[0389]

[0390] In vitro evaluation of ds9 and ds172 to 249 was carried out according to the procedure described above in [In vitro evaluation of siSNCA]. The results are shown in Tables 16-1 to 16-2.

[0391]

[0392]

[0393] [Example 6: In vitro evaluation of siHTT] <Design of siRNA> A set of 236 siRNAs was designed with reference to the base sequences in WO 2022 / 072447.

[0394] <Evaluation of knockdown activity> Using a method similar to that described in Example 5, TaqMan probe Hs00918174_m1 (Applied Biosystems) was used to measure the HTT gene, and Hs01060665_g1 (Applied Biosystems) was used to measure the ACTB gene. PCR reactions were performed on the HTT gene and the ACTB gene as a control, and Ct values ​​were measured. Using the ACTB Ct value as an internal control, a semi-quantitative value of HTT mRNA was calculated. Furthermore, the Ct values ​​of HTT and ACTB in the negative control group (0 pmol of siRNA) were similarly measured, and a semi-quantitative value of HTT mRNA was calculated. The results for siRNAs (ds274-402) that showed an average of 50% or more knockdown activity at 100 pmol / L or 60% or more at 1 nmol / L, based on duplicate measurements, are shown in Tables 17-1 to 17-3.

[0395]

[0396]

[0397]

[0398] Similarly, in vitro evaluation of ds403 to 461 was carried out according to the method described in the above [In vitro evaluation of siHTT]. The results are shown in Tables 18-1 to 18-2.

[0399]

[0400]

[0401] Example 7 In Example 7, human SNCA transgenic mice (Neuroscience Research (2012) 73(2), 173-177) were used as subjects. The mice were administered intracerebroventricularly and allowed to live for 7 days after administration. The amount of the administered substance transferred to each site in the brain and the strength of knockdown were then evaluated. Administration was performed in the same manner as in Reference Example 2.

[0402] For intraventricular administration, 5 μL of a nucleic acid solution was administered, in which each of the oligonucleotides ds2, ds254, and ds257 to ds273 was dissolved in aCSF to a concentration of 20 mg / mL. Administration of aCSF alone was also performed in the same manner as administration of the nucleic acid solution.

[0403] Seven days after administration, brain samples were collected from each site and analyzed (N=4). RNA was then purified in the same manner as in Reference Example 2. cDNA was prepared by reverse transcription using FastGene Scriptase II (NIPPON Genetics, NE-LS65) according to the instructions attached to the kit.

[0404] This cDNA was used as a template for PCR reaction, and the human SNCA gene and the ACTB gene (as a control) were subjected to PCR reaction using a QuantStudio 12K Flex real-time PCR system by the TaqMan (registered trademark) probe method, and the mRNA amplification levels were measured, and the ACTB mRNA amplification level was used as an internal control to calculate a semi-quantitative value for SNCA mRNA. Furthermore, the mRNA amplification levels of human SNCA and ACTB in the negative control group were similarly measured, and a semi-quantitative value for human SNCA mRNA was calculated.

[0405] Measurement of the human SNCA gene was performed using TaqMan probe Hs00240907_m1 (Applied Biosystems), and measurement of the ACTB gene was performed using Mm00607939_s1 (Applied Biosystems). TaqMan® Gene Expression Master Mix was used as the reaction reagent, and the measurements were performed according to the attached protocol. The target mRNA levels in nucleic acid-administered individuals were calculated as a relative proportion to the human SNCA mRNA level in the negative control group (aCSF group). The relative proportions of mRNA levels are shown as mean ± standard error in Figure 18. The data for each site in Figure 18 show, from left to right, the results for the negative control (aCSF-administered), ds2, ds254, and ds257 to ds273.

[0406] All test samples showed knockdown activity compared to the aCSF-administered group, and ds2, ds254, ds257, ds259, ds261, ds263, ds267, and ds268 showed particularly strong knockdown activity.

[0407] [Example 8] In Example 8, male rats (slc: SD rats (13 weeks old), manufactured by Nippon SLC) were used as subjects, and intranasal administration was performed into the cribriform foramina of the cribriform plate. After allowing the rats to survive for 7 days after administration, the amount of the administered substance transferred to each site in the brain and the strength of knockdown were evaluated for each rat.

[0408] In Example 8, in order to accommodate use with a 3D micro X-ray CT device described below, an administration device 1D was used in which the total length (115 mm) of the outer catheter of the administration device 1A produced in Example 1 was changed to 915 mm, and the total length (125 mm) of the inner catheter was changed to 930 mm. The configuration other than the total lengths of the outer catheter and inner catheter was the same as that of the administration device 1A produced in Example 1. Figure 19 shows a configuration diagram of the administration device 1D produced in Example 8.

[0409] Using the administration device 1D, live rats were imaged under triple anesthesia using a 3D micro X-ray CT scanner R_mCT2 (Rigaku Corporation). A guide catheter was inserted into one of the subject's nasal cavities while observing the contrast image. The administration device 1D (puncture length: 0.85 mm) was then inserted into the guide catheter. After confirming that the needle tip had passed through the cribriform foramen without penetrating the brain, intranasal administration was performed. For intranasal administration, ds2 and ds250-253 oligonucleotides were each dissolved in aCSF at a concentration of 60 mg / mL and 30 μL was administered.

[0410] Seven days after administration, brain samples were collected from each site, and then cDNA was prepared in the same manner as in Example 7, followed by quantitative PCR in the same manner as in Example 4. The results, which show the relative proportions of mRNA amounts as mean ± standard error, are shown in Figure 20. Note that the data for each site in Figure 20 shows, from left to right, the results for non-administration, ds2, and ds250-253.

[0411] ds2 (5'EPA-siSNCA) showed significant knockdown activity of the SNCA gene in various brain regions compared with ds250 and 251, which have low phosphate modification rates, and ds252 and ds253, which have low sugar modification rates. However, ds253, which has a low sugar modification rate on the sense strand, showed higher knockdown activity than ds2, although not as high as ds2.

[0412] The amount of nucleic acid transfer in each brain region was also measured using the same method as in Example 4. The results, expressed as mean ± standard error, of the amount of nucleic acid in each brain region are shown in Figure 21. Note that the data for each region in Figure 21 show, from left to right, the results for ds2, ds250 to 253. The test sample ds2 (5'EPA-siSNCA) showed significant nucleic acid transfer in each brain region.

[0413] [Example 9] In Example 9, male rats (slc: SD rats (13 weeks old), manufactured by Nippon SLC) were used as subjects, and intranasal administration was performed into the cribriform foramina of the cribriform plate. After allowing the rats to survive for 7 days after administration, the amount of the administered substance transferred to each site in the brain and the strength of knockdown were evaluated for each subject.

[0414] In Example 9, nasal administration was carried out in the same manner as in Example 8. For nasal administration, each of the oligonucleotides ds2 and ds254 to 257 was dissolved in aCSF to a concentration of 60 mg / mL, and 30 μL was administered.

[0415] Seven days after administration, brain samples were collected from each site, and then cDNA was prepared and quantitative PCR was performed using the same method as in Example 8. The results, which show the relative proportions of mRNA amounts as mean ± standard error, are shown in Figure 22. Note that the data for each site in Figure 22 show, from left to right, the results for non-administration, ds2, and ds254 to 257.

[0416] The test samples (ds2, ds254-257) showed significant knockdown activity of the SNCA gene in each region of the brain compared to the non-administration group.

[0417] Example 10 In Example 10, living cynomolgus monkeys were used as subjects, and a nucleic acid solution (ds2) was administered intranasally into the cribriform foramina of the cribriform plate. After allowing the monkeys to survive for 56 days from the date of the first administration, the amount of oligonucleotide translocated to each site in the brain and the strength of knockdown were evaluated for each subject (N=3).

[0418] The administration was carried out in a volume of 2 mL in the same manner as in Example 3. For administration, a nucleic acid solution prepared by dissolving ds2 oligonucleotide in aCSF to a concentration of 60 mg / mL was used.

[0419] For the two-dose group, 2 mL was administered at a rate of 100 μL / min into the same nostril as the first administration one week later, using the same method. After 56 days of survival from the date of the first administration, brain samples were collected in the same manner as in Example 3, flash-frozen in liquid nitrogen, and then cryopreserved. CSF samples were also collected and cryopreserved in the same manner. Spinal cord samples (cervical, thoracic, and lumbar spinal cord) were also collected and cryopreserved in the same manner. A control group was administered aCSF, and the same procedures were followed.

[0420] Subsequently, cDNA preparation and quantitative PCR were performed for each brain region using the same method as in Example 7. TaqMan probe Mf02793033_m1 (Applied Biosystems) was used to measure the SNCA gene, and Mf04354341_g1 (Applied Biosystems) was used to measure the ACTB gene. Figure 23-1 shows the relative proportions of mRNA levels, expressed as mean ± standard error. The data for each region in Figure 23-1, from left to right, represent the negative control (aCSF), one dose of ds2, and two doses of ds2. The left graph in Figure 23-1 shows the results for the treated side, and the right graph shows the results for the untreated side. Here, the treated side refers to the brain administered with oligonucleotide, either the right or left brain, and the untreated side refers to the other brain not administered with oligonucleotide. A similar analysis was also performed on the spinal cord (cervical, thoracic, and lumbar). The results are shown in Figure 23-2.

[0421] The test samples (ds2 single administration and ds2 double administration) showed significant knockdown activity of the SNCA gene in each region of the brain and the spinal cord compared to the control (aCSF).

[0422] The same method as in Example 4 was used to analyze the amount of nucleic acid transfer in each brain region. The results, expressed as mean ± standard error, of the amount of nucleic acid in each brain region are shown in Figure 24-1. The test samples (one administration of ds2 and two administrations of ds2) showed significant nucleic acid transfer in each brain region. Note that the data for each region in Figure 24-1, from left to right, show the results for one administration of ds2 and two administrations of ds2. The graph on the left in Figure 24-1 shows the results for the administration side, and the graph on the right shows the results for the non-administration side. A similar analysis was also performed on the spinal cord (cervical, thoracic, and lumbar spinal cord). The results are shown in Figure 24-2.

[0423] Furthermore, the amount of α-synuclein protein contained in the brain extract was quantified. Quantification was performed using the LEGEND MAX Human α-Synuclein (Colorimetric) ELISA Kit (Biolegend, 448607) according to the instructions attached to the kit. The results, expressed as mean ± standard error, of the α-synuclein protein levels in each brain region and CSF are shown in Figure 25-1. Note that the data for each region in Figure 25-1 represent, from left to right, the negative control (aCSF), ds2 administered once, and ds2 administered twice. The graph on the left in Figure 25-1 shows the results for the administered group, and the graph on the right shows the results for the unadministered group. A similar analysis was also performed on spinal cord extracts (cervical, thoracic, and lumbar spinal cord). The results are shown in Figure 25-2. Furthermore, CSF was analyzed in the same manner. The percentages of α-synuclein protein levels 14, 28, 42, and 56 days after the start of administration, relative to the α-synuclein protein level in the CSF before the start of administration, are shown in Figure 26. The test samples (ds2 single administration and ds2 double administration) showed a significant decrease in α-synuclein protein levels in each region of the brain, the spinal cord, and the CSF.

[0424] [Example 11] In Example 11, male rats (slc: SD rats (13 weeks old), manufactured by Nippon SLC) were used as subjects, and intranasal administration was performed into the cribriform foramina of the cribriform plate. After allowing the rats to survive for 7 days after administration, the amount of the administered substance transferred to each site in the brain and the strength of knockdown were evaluated for each rat.

[0425] In Example 11, nasal administration was carried out in the same manner as in Example 8. For nasal administration, each of the antisense oligonucleotides (sequences 1346 to 1348) was dissolved in aCSF to a concentration of 60 mg / mL, and 30 μL was administered.

[0426] Seven days after administration, brain samples were collected from various parts of the brain, and then cDNA was prepared in the same manner as in Example 8.

[0427] This cDNA was used as a template for PCR reaction, and the SNCA gene, SOD1 gene, MALAT1 gene, and ACTB gene (as a control) were subjected to PCR reaction using a QuantStudio 12K Flex real-time PCR system by the TaqMan (registered trademark) probe method, and the mRNA amplification amount was measured for each gene. The mRNA amplification amount of ACTB was used as an internal control, and the mRNA amplification amount of each target gene was calculated. In addition, the mRNA amplification amount of each target gene and ACTB in the negative control group was similarly measured, and the mRNA amplification amount of each target gene was calculated.

[0428] The SNCA gene was measured using TaqMan probe Rn01425143_m1 (Applied Biosystems), the SOD1 gene was measured using TaqMan probe Rn00566938_m1 (Applied Biosystems), the MALAT gene was measured using TaqMan MGB probe 3 (SEQ ID NO: 1755) (Applied Biosystems), primer 6 (SEQ ID NO: 1753) and primer 7 (SEQ ID NO: 1754), and the ACTB gene was measured using Rn00667869_m1 (Applied Biosystems), and the reaction reagent was TaqMan (registered trademark) Gene Expression Master Mix, and the reaction was carried out according to the attached protocol. The base sequences of each primer and probe are shown in Table 13.

[0429] The amount of target mRNA in nucleic acid-administered individuals was calculated as a relative proportion when the amount of mRNA for each target gene in the negative control group (non-administered group) was set to 1. The relative proportions of the mRNA amounts are shown as mean ± standard error in Figure 27. The data for each site in Figure 27 shows, from left to right, the results for the negative control (non-administered) and the antisense oligonucleotide.

[0430] The test samples (SEQ ID NOs: 1346 to 1348) showed significantly improved knockdown activity for each target gene compared to the non-administration group.

[0431] This application is based on Japanese Patent Application No. 2024-017851, filed on February 8, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0432] DESCRIPTION OF SYMBOLS 1 Administration device, 10 Needle portion (10a lumen), 11 Needle shaft portion, 12 Puncture portion (12a needle tip portion), 13 Blade surface, 14 Tip opening, 15 Base opening, 20 Hub portion 21 Main body portion (21a lumen), 22 Connection portion, 30 Cannula portion, 31 Main body portion (31a lumen), 32 Stopper portion (32a abutment portion), 40 Storage portion, 41 Storage space, 42 Liquid delivery portion (tip opening 42a), 50 Connection member, 100 Guide catheter, 110 Catheter main body, 111 Lumen, 200 Administration system, A Oligonucleotide, B Brain tissue, C Cerebrospinal fluid (CSF), X1 Ethmoid bone, X2 Cribrosa plate, X3 Cribrosa foramen, Y1 olfactory bulb, Y2 olfactory mucosa, Y3 olfactory epithelium, Y4 lamina propria, Y5 olfactory nerve, Z1 nasal cavity.

Claims

1. Oligonucleotides administered intranasally into the mammalian brain by injection into the cribriform foramina of the cribriform plate.

2. The oligonucleotide according to claim 1, which is administered intranasally into the brain of a mammal using an administration device equipped with a needle having a puncture portion, and which is injected through the opening of the puncture portion with the puncture portion positioned within the cribriform foramina of the cribriform plate.

3. The oligonucleotide according to claim 1, wherein the oligonucleotide is any one selected from the group consisting of an aptamer, an antisense oligonucleotide, a decoy nucleic acid, a ribozyme, an siRNA, an miRNA, and an mRNA.

4. The oligonucleotide of claim 1, wherein the oligonucleotide is an siRNA or an antisense oligonucleotide.

5. The oligonucleotide of claim 1, wherein the oligonucleotide is an siRNA.

6. The oligonucleotide according to claim 5, wherein the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is 80% or more.

7. The oligonucleotide according to claim 5, wherein all nucleotides constituting the siRNA are 2'-modified nucleotides.

8. The oligonucleotide of claim 5, wherein the siRNA comprises one or more lipophilic moieties.

9. The oligonucleotide of claim 8, wherein the lipid-soluble portion is at least one selected from the group consisting of a substituted or unsubstituted alkyl chain having 14 to 24 carbon atoms, eicosapentaenoic acid (EPA), arachidonic acid (ARA), and cholic acid.

10. The oligonucleotide of claim 5, wherein the target gene of the siRNA is SNCA or HTT.

11. The oligonucleotide of claim 5, wherein all of the phosphodiester bonds linking the first and second nucleotides counting from both ends of the sense and antisense strands of the siRNA are substituted with phosphorothioate bonds.

12. The oligonucleotide according to claim 5, wherein the siRNA comprises any one pair of nucleic acid sequences selected from the group consisting of the following (S1) to (S11): (S1) SEQ ID NO:46 and SEQ ID NO:213 (S2) SEQ ID NO:83 and SEQ ID NO:250 (S3) SEQ ID NO:93 and SEQ ID NO:260 (S4) SEQ ID NO:169 and SEQ ID NO:336 (S5) SEQ ID NO:180 and SEQ ID NO:347 (S6) SEQ ID NO:1360 and SEQ ID NO:1389 (S7) SEQ ID NO:1366 and SEQ ID NO:1395 (S8) SEQ ID NO:1368 and SEQ ID NO:1397 (S9) SEQ ID NO:1372 and SEQ ID NO:1401 (S10) SEQ ID NO:1374 and SEQ ID NO:1403 (S11) SEQ ID NO:1756 and SEQ ID NO:1759 13. The oligonucleotide according to claim 5, wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds2, ds9, ds17 to ds249, and ds254 to ds273 listed in any of Tables 1-1 to 1-6. In the tables, "[L1]" represents a compound represented by formula (1) below, "^" represents a phosphorothioate bond, "mA," "mU," "mC," and "mG" represent 2'-O-methyl-RNA, "fA," "fU," "fC," and "fG" represent 2'-fluoro-DNA, "dA," "dT," "dC," and "dG" represent DNA, "(vmU)" represents 5'-vinylphosphate-2'-O-methyl-uridine, and "p" represents 5'-phosphate.

14. The oligonucleotide of claim 13, wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds9, ds28, ds35, ds36, ds40, ds42, ds79, ds105, ds113, ds156, ds164, ds172, ds175, ds176, ds177, ds178, ds187, ds198, ds201, ds211, and ds213 to ds246 listed in any one of Tables 1-1 to 1-6.

15. The oligonucleotide of claim 13, wherein the siRNA is any one set of oligonucleotides selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268, and ds273 listed in any one of Tables 1-1 to 1-6.

16. The oligonucleotide of claim 5, wherein the siRNA comprises any one set of oligonucleotide sequences selected from the group consisting of ds274 to ds461 listed in any one of Tables 2-1 to 2-4. In the tables, "^" represents a phosphorothioate bond, "mA," "mU," "mC," and "mG" represent 2'-O-methyl-RNA, "fA," "fU," "fC," and "fG" represent 2'-fluoro-DNA, "dA," "dT," "dC," and "dG" represent DNA, and "p" represents 5'-phosphate.

17. The oligonucleotide of claim 16, wherein the oligonucleotide comprises any one set of oligonucleotide sequences selected from the group consisting of ds278, ds280, ds299, ds308, ds347, ds405, ds406, ds411, ds414, ds422, and ds432 to ds461 listed in any one of Tables 2-1 to 2-4.

18. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 17.

19. A therapeutic agent for central nervous system disorders, comprising the oligonucleotide according to any one of claims 1 to 17.

20. The oligonucleotide described in claim 2, wherein the administration device has a cannula portion formed of a tubular member that is positioned over the needle portion so that the puncture portion is exposed, the opening is provided on the tip side of the puncture portion, the tip of the cannula portion is formed with a stopper portion having an abutment portion that abuts against the olfactory epithelium of the olfactory mucosa, and the opening is positioned within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.

21. The oligonucleotide described in claim 20, wherein the administration device has a hub portion to which a container containing the oligonucleotide can be attached, and the hub portion holds the base end of the cannula portion and / or the base end of the needle shaft portion of the needle portion inserted through the lumen of the cannula portion.

22. The oligonucleotide according to claim 20, wherein the cross-sectional shape of the abutting portion of the stopper portion is circular or elliptical.

23. An administration system comprising a storage section containing the oligonucleotide according to any one of claims 1 to 17, and an administration device equipped with a needle section having a puncture section, wherein the oligonucleotide contained in the storage section can be discharged through the opening of the puncture section.

24. The administration device according to claim 23, wherein the administration device has a cannula portion formed of a tubular member that is positioned over the needle portion so that the puncture portion is exposed, the opening is provided on the tip side of the puncture portion, the tip of the cannula portion is formed with a stopper portion having an abutment portion that abuts against the olfactory epithelium of the olfactory mucosa, and the opening is positioned within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.

25. The administration system according to claim 24, wherein the administration device has a hub portion to which a container containing the oligonucleotide can be attached, and the hub portion holds the base end of the cannula portion and / or the base end of the needle shaft portion of the needle portion inserted through the lumen of the cannula portion.

26. The dispensing system according to claim 24, wherein the cross-sectional shape of the abutting portion of the stopper portion is circular or elliptical.

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