WO2025170027A1 - Oligonucleotide - Google Patents

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

The present invention relates to an oligonucleotide that is administered intranasally into the brain of a mammal using an administration device.

Central nervous system (CNS) diseases are often caused by pathogenic proteins, so approaches to the pathogenic proteins themselves or the genes involved in the expression of those proteins are thought to be effective in treating, preventing, and ameliorating these conditions. For example, nucleic acid medicines can control protein expression by regulating the expression of specific genes, and have long been expected to be used in the treatment of CNS diseases.

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.

In order to administer drugs into the brain more effectively than oral or intravenous administration, research is underway into administration methods that bypass the blood-brain barrier and allow direct access to the central nervous system. Currently, methods include the nasal spray method, in which drugs are sprayed into the nasal cavity; intraventricular administration, in which drugs are administered directly into the ventricles of the brain; and intrathecal administration, in which drugs are injected into the spinal cavity (Biopharm. Drug Dispos. 44(1), 26-47 (2023)).

However, although the nasal spray method is minimally invasive, it has the problem of an extremely low drug transfer rate. Furthermore, intraventricular administration involves drilling the skull and administering directly 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 faces many challenges, including insufficient drug delivery to deep brain regions such as the striatum, high invasiveness, and difficulty in administering to patients with scoliosis, a common intractable neurological disease.

The present invention was 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 high efficacy in the treatment of central nervous system diseases using nucleic acid medicines.

The inventors conducted extensive research to solve the above problems. As a result, they discovered that the above problems could be solved by administering oligonucleotides intranasally using a specific administration device, leading to the completion of the present invention.

In other words, the above-mentioned objectives can be achieved by the present invention having the following configuration, and the present invention encompasses the following aspects and configurations.

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.

2. The oligonucleotide described in 1 above is administered intranasally into the brain of a mammal using an administration device equipped with a needle having a puncture portion;
It is preferable that the injection is performed through the opening of the puncture part while the puncture part is placed in the sieve holes of the sieve plate.

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.

4. The oligonucleotide described in any of 1. to 3. above is preferably an siRNA or an antisense oligonucleotide.

5. The oligonucleotide described in any of 1. to 4. above is preferably siRNA.

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.

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

8. It is preferable that the oligonucleotide described in any of items 5 to 7 above has one or more lipophilic moieties.

9. In the oligonucleotide described in 8 above, the lipid-soluble portion 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.

10. In the oligonucleotide described in any of 5. to 9. above, the target gene of the siRNA is preferably SNCA or HTT.

11. In the oligonucleotide described in any of 5. to 10. above, it is preferable that 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. In the oligonucleotides described in 5 to 11 above, the siRNA preferably comprises any one set 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)

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.

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.

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.

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 table, "^" 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.

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.

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

One aspect of the present invention is
19. A therapeutic agent for central nervous system diseases, comprising the oligonucleotide according to any one of 1. to 17. above.

20. The oligonucleotide according to 2. above, wherein the administration device has a cannula portion formed of a tubular member that is disposed over the needle portion so that the puncture portion is exposed;
the opening is provided on the distal end side of the puncture part,
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;
The opening is preferably disposed within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.

21. The oligonucleotide according to 20. above, wherein the administration device has a hub portion to which a container containing the oligonucleotide can be attached,
The hub portion preferably 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. In the oligonucleotide described in 20. or 21. above, the cross-sectional shape of the contact portion of the stopper portion is preferably circular or elliptical.

One aspect of the present invention is
23. An administration system comprising a storage section that stores the oligonucleotide according to any one of 1. to 17. above, and an administration device equipped with a needle section having a puncture section, wherein the oligonucleotide stored in the storage section can be discharged through an opening in the puncture section.
24. The administration system according to claim 23, wherein the administration device has a cannula portion formed of a tubular member that is disposed over the needle portion so that the puncture portion is exposed;
the opening is provided on the distal end side of the puncture part,
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;
The opening is preferably disposed within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.
25. The administration system according to 24. above, wherein the administration device has a hub portion to which a container portion containing the oligonucleotide can be attached,
The hub portion preferably 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. In the dispensing system described in 24. or 25. above, it is preferable that the cross-sectional shape of the abutting portion of the stopper portion is circular or elliptical.

Furthermore, the above-mentioned objects can also be achieved by the present invention having the following configuration, and the present invention also encompasses the following aspects and configurations.

Another aspect of the present invention is
1. An oligonucleotide for use in treating a central nervous system disorder, which is administered intranasally into the mammalian brain by injection into the cribrosa of the cribrosa.

2. The oligonucleotide of 1 above is administered intranasally into the brain of a mammal using an administration device equipped with a needle having a puncture portion;
It is preferable that the injection is performed through the opening of the puncture part while the puncture part is placed in the sieve holes of the sieve plate.

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.

4. The oligonucleotide described in any of 1. to 3. above is preferably an siRNA or an antisense oligonucleotide.

5. The oligonucleotide described in any of 1. to 4. above is preferably siRNA.

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.

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

8. It is preferable that the oligonucleotide described in any of 5. to 7. above has one or more lipophilic moieties.

9. In the oligonucleotide described in 8 above, the lipid-soluble portion 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.

10. In the oligonucleotide described in any of 5. to 9. above, the target gene of the siRNA is preferably SNCA or HTT.

11. In the oligonucleotide described in any of 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.

12. In the oligonucleotide according to any one of items 5 to 11 above, the siRNA preferably comprises any one set 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).

13. In the oligonucleotide described in 5. above, the siRNA preferably comprises the sequence of any one set of oligonucleotides 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. 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.

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.

16. In the oligonucleotide described in 5. above, the siRNA preferably comprises the sequence 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.

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.

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

One aspect of the present invention is
19. A therapeutic agent for central nervous system diseases, comprising the oligonucleotide according to any one of 1. to 17. above.

20. The oligonucleotide according to 2. above, wherein the administration device has a cannula portion formed of a tubular member that is disposed over the needle portion so that the puncture portion is exposed;
the opening is provided on the distal end side of the puncture part,
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;
The opening is preferably disposed within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.

21. The oligonucleotide according to 20. above, wherein the administration device has a hub portion to which a container containing the oligonucleotide can be attached,
The hub portion preferably 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. In the oligonucleotide described in 20. or 21. above, the cross-sectional shape of the contact portion of the stopper portion is preferably circular or elliptical.

One aspect of the present invention is
23. An administration system comprising a storage section that stores the oligonucleotide according to any one of 1. to 17. above, and an administration device equipped with a needle section having a puncture section, wherein the oligonucleotide stored in the storage section can be discharged through an opening in the puncture section.
24. The administration system according to claim 23, wherein the administration device has a cannula portion formed of a tubular member that is disposed over the needle portion so that the puncture portion is exposed;
the opening is provided on the distal end side of the puncture part,
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;
The opening is preferably disposed within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.
25. The administration system according to 24. above, wherein the administration device has a hub portion to which a container portion containing the oligonucleotide can be attached,
The hub portion preferably 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. In the dispensing system described in 24. or 25. above, it is preferable that the cross-sectional shape of the abutting portion of the stopper portion is circular or elliptical.

Furthermore, the above-mentioned objects can also be achieved by the present invention having the following configuration, and the present invention also encompasses the following aspects and configurations.

Another aspect of the present invention is
1. Use of an oligonucleotide administered intranasally into the mammalian brain by infusion through the cribriform foramina of the cribriform plate for the treatment of a central nervous system disorder.

2. In the use of 1. above, the oligonucleotide is administered intranasally into the brain of a mammal using an administration device equipped with a needle having a puncture portion;
It is preferable that the injection is performed through the opening of the puncture part while the puncture part is placed in the sieve holes of the sieve plate.

3. In the use described in 1 or 2 above, it is preferable that 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. In the use described in any of 1. to 3. above, the oligonucleotide is preferably an siRNA or an antisense oligonucleotide.

5. In the use described in any of 1. to 4. above, it is preferable that the oligonucleotide is siRNA.

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.

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

8. It is preferable that the oligonucleotide described in any of 5. to 7. above has one or more lipophilic moieties.

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.

10. In the use described in any of 5. to 9. above, it is preferable that the target gene of the siRNA is SNCA or HTT.

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

12. In the use according to any one of the above items 5 to 11, the siRNA preferably comprises any one set 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).

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.

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 one of Tables 1-1 to 1-6.

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.

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.

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 one of Tables 2-1 to 2-4.

18. In the use described in 2. above, 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 distal end side of the puncture part,
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;
The opening is preferably disposed within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium.

19. In the use according to the above 18., the administration device has a hub portion to which a container portion containing the oligonucleotide can be attached,
The hub portion preferably 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. In the use described in 18. or 19. above, it is preferable that the cross-sectional shape of the abutting portion of the stopper portion be circular or elliptical.

FIG. 1 shows the structures of phosphate-modified nucleotides contained in the oligonucleotides used in the Examples. FIG. 2 shows the structure of the lipophilic moiety contained in the oligonucleotide used in the Examples. FIG. 3 shows an example of an administration device and administration system used for intranasal administration of an oligonucleotide according to one embodiment of the present invention. FIG. 4-1 is a diagram showing the state in which the puncturing part of the administration device has punctured the sieve holes. FIG. 4-2A is the first diagram illustrating how to use the administration device. FIG. 4-2B is a second diagram illustrating how to use the administration device. FIG. 4-2C is a third diagram illustrating how to use the administration device. FIG. 4-2D is a fourth diagram illustrating how to use the administration device. FIG. 4-2E is a fifth diagram illustrating how to use the administration device. FIG. 5 is a diagram showing the measurement results of Reference Example 1-1. FIG. 6 is a diagram showing the measurement results of Reference Example 1-2. FIG. 7 is a diagram showing the measurement results of Reference Example 1-3. FIG. 8 is a diagram showing the measurement results of Reference Example 2. FIG. 9 is a diagram showing the configurations of administration devices 1A and 1B and a guide catheter 100A used in the examples. FIG. 10 shows the measurement results of knockdown activity in Example 1. FIG. 11 shows the measurement results of knockdown activity in Example 2. FIG. 12 shows the measurement results of the amount of nucleic acid transferred in Example 2. FIG. 13 is a diagram showing the configuration of an administration device 1C and a guide catheter 100B used in the example. FIG. 14-1 shows the measurement results of knockdown activity (each brain region) in Example 3. FIG. 14-2 shows the measurement results of knockdown activity in Example 3 (cervical spinal cord, thoracic spinal cord, and lumbar spinal cord). FIG. 15-1 shows the measurement results of the amount of nucleic acid transferred (each brain region) in Example 3. FIG. 15-2 shows the measurement results of the amount of nucleic acid transferred (cervical spinal cord, thoracic spinal cord, and lumbar spinal cord) in Example 3. FIG. 16 shows the measurement results of knockdown activity in Example 4. FIG. 17 shows the measurement results of the amount of nucleic acid transferred in Example 4. FIG. 18 shows the measurement results of knockdown activity in Example 7. FIG. 19 is a diagram showing the configuration of the administration device 1D and the guide catheter 100A used in Example 8. FIG. 20 shows the measurement results of knockdown activity in Example 8. FIG. 21 shows the measurement results of the amount of nucleic acid transferred in Example 8. FIG. 22 shows the measurement results of knockdown activity in Example 9. FIG. 23-1 shows the results of measuring knockdown activity by quantitative PCR (for 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 measurement results of the amount of nucleic acid transferred (each brain region) in Example 10. FIG. 24-2 shows the measurement results (spinal cord) of the amount of nucleic acid transferred in Example 10. FIG. 25-1 shows the results of measuring knockdown activity (for each brain region) by protein quantification in Example 10. FIG. 25-2 shows the results of measuring knockdown activity (spinal cord) by protein quantification in Example 10. FIG. 26 shows the results of measuring the time-dependent change in knockdown activity (CSF) by protein quantification in Example 10. FIG. 27 shows the measurement results of knockdown activity in Example 11.

The following describes an embodiment of the present invention. However, the present invention is not limited to the following embodiment. Furthermore, unless otherwise specified in this specification, operations and measurements of physical properties are performed at room temperature (20°C to 25°C) and a relative humidity of 40% RH to 50% RH.

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 relevant field, 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) written before and after it as the lower and upper limits, respectively, and means "greater than or equal to X and less than or equal to Y." Furthermore, unless otherwise specified, concentration "%" represents mass concentration "% by mass," and ratios are mass ratios unless otherwise specified.

An oligonucleotide according to one embodiment of the present invention is administered intranasally into the brain of a mammal by injecting it 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. An 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.

The inventors speculate that the mechanism by which the present invention can solve the above problems is as follows.

As a result of extensive research, the 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 by the epithelial barrier. The oligonucleotides of the present invention take this hypothesis into consideration, and are administered intranasally to the mammalian brain by injection through the cribriform foramina of the cribriform plate.

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. Injection can be performed using a medical device, such as a syringe needle, that has a structure that allows the oligonucleotide to be injected 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, etc., making it possible to administer the oligonucleotide intranasally into the brain. For example, an oligonucleotide according to one embodiment of the present invention can be administered intranasally into the brain by placing the puncture part of an administration device within the sieve pores of the cribriform plate and injecting the oligonucleotide (drug) through the opening of the puncture part.

In this way, 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 transfer into the brain. Furthermore, because a sufficient amount of oligonucleotide transfers into the brain, a sufficient amount of oligonucleotide is also taken up into tissue in each region of the brain, which is thought to improve the efficacy of the oligonucleotide. For example, because the amount of oligonucleotide required to reduce (knockdown) the expression of a protein that causes a central nervous system disorder (hereinafter also referred to as a central disorder) reaches each region of the brain, the knockdown efficiency in 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 in no way limited to this mechanism.

[Oligonucleotides]
The oligonucleotide according to one embodiment of the present invention may be any molecule formed by polymerizing nucleotides or molecules having a function equivalent 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 substituted with a molecule having a function equivalent to the nucleotide. Uracil (U) in RNA is unambiguously interpreted as thymine (T) in DNA.

Examples of molecules with functions equivalent to nucleotides include nucleotide derivatives, which are modified nucleotides. Although not particularly limited, the use of nucleotide derivatives offers the following advantages compared to RNA or DNA: increased translocation to various sites in the brain, improved or stabilized nuclease resistance, improved affinity with complementary nucleic acid strands, improved cell permeability, and/or visualization.

The oligonucleotide according to the present invention may be any oligonucleotide known to be used as a nucleic acid drug. In particular, 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, siRNA, miRNA (microRNA), and mRNA (messenger RNA), and it is more preferable that the oligonucleotide according to one embodiment of the present invention is an siRNA or antisense oligonucleotide. By using an oligonucleotide selected from these, it becomes possible to more effectively bind to target molecules, and more effectively control gene transcription and translation processes, inhibit protein function, and the like.

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

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 can 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 heteronucleotides (HDOs) that form a double strand with the complementary strand.

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

Decoy nucleic acids are double-stranded oligonucleotides that target nucleic acid recognition proteins such as transcription factors. These decoy nucleic acids mimic the nucleic acid sequence recognized by the nucleic acid recognition protein, and inhibit the protein from binding to the target nucleic acid.

siRNA stands for small interfering RNA and is a double-stranded oligonucleotide involved in RNA interference (RNAi). The oligonucleotides that make up siRNA may be composed of RNA or 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. Thus, from the perspective of more efficiently suppressing the expression of a target protein and more effectively treating central nervous system disorders, it is even more preferable that the oligonucleotide according to one embodiment of the present invention is siRNA.

When the oligonucleotide of one embodiment is an siRNA, it is preferable that all of the phosphodiester bonds linking the first and second nucleotides counting from both ends of the sense and antisense strands constituting the siRNA are substituted with phosphorothioate bonds. It is even more preferable that all of the phosphodiester bonds linking the first and second nucleotides and the second and third nucleotides counting from both ends of the sense and antisense strands are substituted with phosphorothioate bonds. The above configuration can improve the knockdown effect of the siRNA.

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

miRNA (microRNA) is a small RNA molecule of approximately 22 bases encoded in the genome that controls gene expression by suppressing translation from target molecules, mRNAs, to which it has a partially complementary sequence.

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.

When the oligonucleotide of 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, 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.

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.

Furthermore, when the oligonucleotide according to one embodiment of the present invention is double-stranded, each single strand forming the double strand preferably has 10 to 30 nucleotides, and more preferably 15 to 25 nucleotides. That is, the number of nucleotides constituting the sense strand is preferably 10 to 30 nucleotides, and more preferably 15 to 25 nucleotides. Similarly, the number of nucleotides constituting the antisense strand is preferably 10 to 30 nucleotides, and more preferably 15 to 25 nucleotides. 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 nucleotides, and more preferably 15 to 25 nucleotides. Similarly, the number of nucleotides constituting the antisense strand is preferably 10 to 30 nucleotides, and more preferably 15 to 25 nucleotides. When the number of nucleotides constituting the oligonucleotide is within the above range, the oligonucleotide is more efficiently taken up into cells and has stronger binding strength with the target molecule, thereby further improving its efficacy as an oligonucleotide.

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 or more and 30,000 or less, 3,000 or more and 21,000 or less, 3,000 or more and 18,000 or less, 3,000 or more and 13,500 or less, 3,000 or more and 9,000 or less, 3,000 or more and 6,000 or less, 3,600 or more and 30,000 or less, 3,600 or more and 21,000 or less, 3,600 or more and 18,000 or less, 3,600 or more and 13,500 or less, 3,600 or more and 9,000 or less, 3,600 or more and It may be 6,000 or less, 4,500 or more and 30,000 or less, 4,500 or more and 21,000 or less, 4,500 or more and 18,000 or less, 4,500 or more and 13,500 or less, 4,500 or more and 9,000 or less, 4,500 or more and 6,000 or less, 9,000 or more and 30,000 or less, 9,000 or more and 21,000 or less, 9,000 or more and 18,000 or less, 9,000 or more and 13,500 or less. 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, 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, and 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, and more preferably 4,500 to 13,500.

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.

Furthermore, when the oligonucleotide according to one embodiment of the present invention is double-stranded, the molecular weight is preferably between 6,000 and 20,000, more preferably between 9,000 and 18,000, and even more preferably between 13,000 and 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 between 6,000 and 20,000, more preferably between 9,000 and 18,000, and even more preferably between 13,000 and 16,000. When the molecular weight of the oligonucleotide is within the above range, it is more efficiently taken up into cells and its binding strength with the target molecule is stronger, thereby further improving its efficacy as an oligonucleotide.

(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, knockdown of the target gene can provide therapeutic, preventive, or ameliorative benefits for central nervous system diseases, particularly cranial nervous system diseases.

As used herein, 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 storage disease, peroxisomal disease, mitochondrial disease, glioblastoma, and cerebral infarction.

Examples of target molecules include SOD1 (Cu/Zn superoxide dismutase), FUS (fused in sarcoma), C9ORF72, and 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. Other examples of target molecules include SMN2, which is involved in spinal muscular atrophy (SMA).

In other words, the oligonucleotide according to one embodiment of the present invention can 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.

Furthermore, when the oligonucleotide according to one embodiment of the present invention is siRNA, the target gene can be any of the target molecules described above, with SNCA (α-synuclein) or HTT (huntingtin) being particularly preferred. The siRNA according to one embodiment of the present invention is capable of more effectively knocking down these target genes, and is therefore expected to be highly effective in the treatment, prevention, and amelioration of synucleinopathies such as MSA and Parkinson's disease, as well as Huntington's disease.

(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 characteristics of two or more types of modified nucleotides. For example, a sugar-modified nucleotide may have characteristics of a base-modified nucleotide or a phosphate group-modified nucleotide.

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%. Having the proportion of nucleotide derivatives relative to the total nucleotides constituting the oligonucleotide within the above range improves the efficiency of the oligonucleotide's transfer to various sites in the brain.

<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 is 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.

Furthermore, in an oligonucleotide according to one embodiment of the present invention, the proportion of 2'-modified nucleotides among 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 among all nucleotides constituting the oligonucleotide is within the above range, the efficiency of delivery of the oligonucleotide to various sites in the brain is further improved.

Furthermore, in one embodiment of the present invention, the oligonucleotide most preferably comprises 100% 2'-modified nucleotides out of all nucleotides constituting the oligonucleotide. In other words, in one embodiment of the present invention, the oligonucleotide most preferably comprises all nucleotides constituting the oligonucleotide that are 2'-modified nucleotides (Claim 7). This configuration more sufficiently improves the amount of oligonucleotide translocated to each site in the brain.

Examples of 2'-modified nucleotides include those in which the 2'-OH group of ribose is substituted with a substituent selected from the group consisting of -H, -OR, -R, _-R'OR , -SH, -SR, _-NH2 , -NHR, _-NR2 , -N3, -CN, -F, -Cl, -Br, and _-I (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 the two Rs in -NR2 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.

Furthermore, the 2'-modified nucleotide may 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). Furthermore, the 2'-modified nucleotide may be combined with 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).

The 2'-modified nucleotide may, for example, be one in which the 2'-OH group of ribose is 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.

Furthermore, bridged nucleic acids (BNAs) that have two cyclic structures due to the introduction of a bridged structure into the sugar moiety are also suitable for use as sugar-modified nucleotides. Specifically, 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)], and ethylene-bridged nucleic acids (ENAs) [Nucleic Acid Research, 32, e 175 (2004)], Constrained Ethyl (cEt) [The Journal of Or Ganic Chemistry 75, 1569 (2010)], Amido-Bridged Nucleic Acid (AmNA) [Chem Bio Chem 13, 2513 (2012)], 2'-O, 4'-C-S Pirocyclopropylene bridged nuclear acid (scpBNA) [Chem. Commun. , 51, 9737 (2015)], tricycloDNA (tcDNA) [Nat. Biotechnol., 35, 238 (2017)], etc.

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

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)].

<Phosphodiester-linked modified nucleotides>
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.

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%. Having the proportion of phosphodiester-linked nucleotides relative to the total nucleotides constituting the oligonucleotide within the above ranges further improves 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).

Examples of nucleotides modified with a phosphodiester bond include nucleotides in which a phosphodiester bond has been replaced with a phosphorothioate bond, nucleotides in which a phosphodiester bond has been replaced with a phosphorodithioate bond, nucleotides in which a phosphodiester bond has been replaced with an alkylphosphonate bond, nucleotides in which a phosphodiester bond has been replaced with a boranophosphate bond, nucleotides in which a phosphodiester bond has been replaced with an amide bond, nucleotides in which a phosphodiester bond has been replaced with a sulfonamide bond, nucleotides in which a phosphodiester bond has been replaced with a phosphonoacetate bond (PACE), nucleotides in which a phosphodiester bond has been replaced with a 2'-5' bond [Bioorg. Med. Chem. Lett. 16, 3238-3240], and nucleotides in which a phosphodiester bond has been 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. Replacing the phosphodiester bond with such a bond improves the efficiency of oligonucleotide delivery to various sites in the brain and the efficacy of the oligonucleotide (e.g., the efficiency of knocking down target genes).

Phosphodiester bond-modified nucleotides may be optical isomers (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).

<Base-modified nucleotides>
The base-modified nucleotide may be any nucleotide in which part or all of the chemical structure of the nucleotide base has been modified or substituted with any substituent or substituted with any atom.

Base-modified nucleotides include, for example, 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 5th position of cytosine is substituted with a methyl group.

<Phosphate-modified nucleotides>
The phosphate-modified nucleotide may be any nucleotide in which the phosphate group at the 5' position of the nucleotide has been modified or substituted with any substituent, or substituted with any atom. However, it is more preferable that the nucleotide be substituted with a vinylphosphonate group, (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 FIG. 1. Other phosphate-modified nucleotides are disclosed in WO 2011/39699 and WO 2011/39702.

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.

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 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.

The cyclic oligonucleotide may also be represented by formula 2.

(fat-soluble part)
The oligonucleotide according to one embodiment of the present invention preferably has one or more lipophilic moieties (also referred to as lipophilic groups).In other words, the oligonucleotide according to one embodiment of the present invention preferably has at least one nucleotide constituting the oligonucleotide being a nucleotide or nucleotide derivative having a lipophilic moiety.By having one or more lipophilic moieties in the oligonucleotide, it is expected that the efficiency of the oligonucleotide's transfer to each site in the brain and the knockdown efficiency of the target gene will be further improved.

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

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. Having a lipophilic portion in the oligonucleotide can, for example, more effectively knock down a target gene.

The term "lipid-soluble moiety" broadly refers to any compound or chemical substructure that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is, for example, by the octanol-water partition coefficient, log _Kow . Here, _Kow 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 property of a substance measured in a laboratory. However, it can also be predicted by using coefficients attributable to the chemical's components calculated using first-principles or empirical methods. In principle, a chemical is lipophilic if its log _Kow is greater than 0. The log _Kow of the lipophilic moiety of an oligonucleotide according to one embodiment of the present invention is not particularly limited as long as it is greater than 0, but may be greater than 1, 2, or 3. That is, an oligonucleotide according to one embodiment of the present invention has one or more lipophilic moieties, and the log _Kow of the lipophilic moieties may be greater than 0, greater than 1, greater than 2, or greater than 3.

The fat-soluble portion in one embodiment of the present invention is, 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, etc. , cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, 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 cellular uptake, 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.

In an oligonucleotide according to one embodiment of the present invention, the lipophilic moiety may be directly bonded (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 Figure 2 (C16U). 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.

Furthermore, in an oligonucleotide according to one embodiment of the present invention, the lipophilic moiety may be attached 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 ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, click reaction product (e.g., triazole from azide-alkyne cycloaddition), or 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 lipophilic moiety and a nucleotide are bonded using 5'-Amino-Modifier C6-PDA, and [L2] to [L5] are examples in which a lipophilic moiety and a nucleotide are bonded using 3'-Amino-Modifier C7 CPG 1000.

(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.

(SNCA-siRNA or HTT-siRNA)
The oligonucleotide according to one embodiment of the present invention is preferably an siRNA targeting 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.

Examples of siRNA targeting SNCA (SNCA-siRNA) that can be used 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.

Furthermore, examples of siRNA targeting HTT (HTT-siRNA) that can be used 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.

Among these, the siRNA base sequences (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 are therefore more suitable for use as oligonucleotides (siRNA) according to one embodiment of the present invention.

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).

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.
(S1) Modified siRNA: ds79, ds187
(S2) Modified siRNA: ds105, ds198, ds249, ds9
(S3) Modified siRNA: ds113, ds201, ds216, ds221, ds226, ds231, ds236, ds241, ds246
(S4) Modified siRNA: ds156, ds211, ds217, ds222, ds227, ds232, ds237, ds242
(S5) Modified siRNA: ds164, ds213, ds218, ds223, ds228, ds233, ds238, 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.

Furthermore, from the viewpoint of achieving 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.

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.

Furthermore, from the viewpoint of having a superior SNCA knockdown activity, it is more preferable that the oligonucleotide (siRNA) according to one embodiment of the present invention is 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 having an even superior SNCA knockdown activity, it is more preferable that the oligonucleotide according to one embodiment is 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 it is more preferable that the oligonucleotide according to one embodiment is 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 a set selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268, and ds273.

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 with 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.

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 2'-modified nucleotides have been added to the base sequences (S1) to (S11) listed above.

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.

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 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.

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 with any one set of sequences selected from the group consisting of ds2, ds254, ds257, ds258, ds259, ds261, ds263, ds267, ds268, and ds273.

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 being compared. Sequence identity is generally calculated using the following formula 1.

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 for each. N is A, U, C, or G for RNA, and A, T, C, or G for DNA.

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 this 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.

For example, if the oligonucleotide described herein has 21 bases, the shortened base sequence may have 20, 19, 18, 17, 16, or 15 bases. Therefore, the oligonucleotide according to one embodiment of the present invention may have 20, 19, 18, 17, 16, or 15 bases, and the base sequences of these oligonucleotides may have 85% or more sequence identity, 90% or more sequence identity, 95% or more sequence identity, or may be identical to the shortened base sequence.

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 bases. 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 bases, and the base sequences of these oligonucleotides may have 85% or more sequence identity, 90% or more sequence identity, 95% or more sequence identity, or may be identical to the truncated base sequence.

Here, the shortened base sequence may be an oligonucleotide described herein in which the 5' end and/or 3' end has been shortened, or an oligonucleotide sequence in which a portion of the sequence has been omitted (deleted), or a combination thereof.

Furthermore, any one pair of oligonucleotide sequences selected from the group consisting of siRNA base sequences H-0001 to H-0258 consisting of a combination of sense and antisense strands shown in any one of Tables 4-1 to 4-6, and ds274 to ds461 listed 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.

From the viewpoint of achieving a higher HTT knockdown activity, it is preferable that the oligonucleotide (siRNA) according to one embodiment of the present invention contains any one set of base sequences selected from the group consisting of (H1) to (H5) shown below, among the base sequences (H-0001 to H-0258) of siRNAs consisting of combinations of sense strands and antisense strands 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)

Oligonucleotides containing siRNAs in which 2'-modified nucleotides have been added to the base sequences of (H1) to (H5) above can be more preferably used as oligonucleotides 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 the following modified siRNAs (H1): ds278, ds405, ds432, ds437, ds442, ds447, ds452, ds457
(H2) Modified siRNA: ds280, ds406, ds433, ds438, ds443, ds448, ds453, ds458
(H3) Modified siRNA: 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

Furthermore, from the viewpoint of achieving higher HTT knockdown activity, it is preferable that the siRNA (oligonucleotide) targeting HTT contains the sequence of any one set of oligonucleotides selected from the group consisting of ds274 to ds461 listed in any 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 contain the sequence 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.

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 with 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.

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 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 2'-modified nucleotides have been added to the base sequences of (H1) to (H5) listed 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 oligonucleotides selected from the group consisting of ds274 to ds461 listed in any one of Tables 2-1 to 2-4.

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.

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

These siRNAs can be appropriately combined with the sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, phosphate group-modified nucleotides, and lipophilic moieties described above. By combining these nucleotide derivatives and lipophilic moieties, it is possible to improve the knockdown activity of the siRNA and the amount of siRNA delivered to various sites in the brain.

(Antisense oligonucleotides)
When the oligonucleotide according to one embodiment of the present invention is an antisense oligonucleotide, the target gene may be, but is not limited to, SOD1, FUS, C9ORF72, ATXN2, APP, tau, LRRK2, SNCA, HTT, ATXN3, UBE3A antisense transcript, or SMN2, which are genes associated with diseases of the central nervous system. Of these, at least one selected from the group consisting of the SNCA gene, the SOD1 gene, and the HTT gene is preferred.

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.

[Oligonucleotide manufacturing method]
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 according to 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.

Also, oligonucleotides containing nucleotide derivatives can be produced by subjecting previously prepared desired nucleotide derivatives, such as sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and phosphate group-modified nucleotides, to an extension reaction on a solid phase using the phosphoramidite method or the like. Specific methods for producing oligonucleotides containing nucleotide derivatives include those described in the Examples.

Here, 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, 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.

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).

In addition, methods for preparing oligonucleotides having a lipophilic moiety or a ligand are not particularly limited. For example, phosphoramidite units or solid-phase supports having a linker or a lipophilic group (lipophilic moiety) can be prepared in advance, and then subjected to an extension reaction on the solid phase with other nucleotides or nucleotide derivatives. Alternatively, oligonucleotides can be isolated and then post-modified in the liquid phase to produce oligonucleotides having a lipophilic moiety or a ligand. Phosphoramidite units having a lipophilic group (lipophilic moiety) can be prepared, for example, according to the methods described in International Publication No. 2021/092371 and Nature Biotechnology (40, 1500-1508, 2022). Post-modification can be performed, for example, by coupling the desired lipophilic compound to the oligonucleotide using a coupling reagent. Specific methods for producing phosphoramidite units having a lipophilic group (lipophilic moiety) and oligonucleotides having a lipophilic moiety can be adopted from the methods described in the Examples.

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, 1 equivalent of antisense strand to 0.1 to 10 equivalents of sense strand, preferably 0.5 to 2 equivalents, more preferably 0.9 to 1.1 equivalents, and even more preferably equimolar amounts (equal amounts), and the mixture is then typically annealed. It is also possible to omit the annealing step and use the mixture directly.

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

<Pharmaceutical composition and therapeutic agent for central nervous system disorders>
One aspect of the present invention may be a pharmaceutical composition comprising the above-described oligonucleotide. In other words, a pharmaceutical composition according to one aspect 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.

The pharmaceutical composition according to one embodiment of the present invention can be used for a wide variety of 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 above-described oligonucleotide and pharmaceutical composition, 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 is administered through the opening of the puncture portion with the puncture portion positioned within the cribriform foramen of the cribriform plate.

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.

Pharmaceutical compositions and therapeutic agents for central nervous system disorders containing oligonucleotides and the above-mentioned carriers can be prepared by methods known to those skilled in the art. For example, the pharmaceutical composition or therapeutic agent for central nervous system disorders of the present invention can be prepared by mixing an oligonucleotide solution with a carrier dispersion of an appropriate concentration.

Furthermore, as pharmaceutical compositions and therapeutic agents for central nervous system diseases according to one embodiment of the present invention, for example, composite particles composed of oligonucleotides and lead particles as constituent components, and compositions composed of composite particles and, optionally, a lipid membrane covering the composite particles, are also suitable for use. 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.

The pharmaceutical composition and therapeutic agent for central nervous system disorders according to one embodiment of the present invention may contain, in addition to the above-mentioned carrier, a pharmaceutically acceptable carrier or diluent. A pharmaceutically acceptable carrier or diluent is essentially a chemically inert and harmless compound (including when it is a composition) that does 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.

The pharmaceutical composition and therapeutic agent for central nervous system disorders 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, with a liquid formulation being preferred.

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, such as an emulsifying aid, stabilizer, isotonicity agent, and/or pH adjuster. 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.

Administration Devices and Systems
In one embodiment of the present invention, an oligonucleotide 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.

The administration device 1 and the administration system 200 including the administration device will be described below using Figures 3 and 4-1, but the administration device 1 and administration system 200 are not limited to the embodiments shown in Figures 3 and 4-1.

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 foramen 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 Figure 4-1. The olfactory nerve (nerve axon) Y5 passes through the foramen, extending from the olfactory bulb Y1, which is part of brain tissue B, to olfactory cells distributed in the olfactory mucosa Y2 (composed of the olfactory epithelium Y3 and the lamina propria Y4) within the nasal cavity Z1. Numerous cribriform foramen X3 exist not only on the flat surface of the cribriform plate X2 as shown in Figure 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 this device is any of these foramen that can be punctured by the puncturing portion 12 that has penetrated the olfactory mucosa Y2.

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

As shown in Figure 4-1, the administration system 200 places the guide catheter 100 in the nasal cavity Z1 with the tip facing the cribriform plate X2, inserts the administration device 1 into the guide catheter 100, and administers 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.

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

The administration device 1 is 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.

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

<Needle>
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 the 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 exposed from the distal end of the cannula portion 30 during puncturing.

The needle shaft portion 11 corresponds to the main body of the needle portion 10 and has an inner cavity 21a that communicates with the inner cavity 31a of the hub portion 20 or the cannula portion 30. A base-end opening 15 is formed at the base end of the needle shaft portion 11. The needle shaft portion 11 is connected to the hub portion 20 via the base-end opening 15 so that it can communicate with the hub portion 20.

The needle shaft 11 is formed with a length approximately 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 and the lumen 21a of the hub 20 are in communication. However, the needle shaft 11 may be shorter than the overall length of the cannula 30, with the proximal end positioned within the lumen 31a of the cannula 30. When configured in this manner, oligonucleotide A flows through the lumen 31a of the cannula 30 to the proximal opening 15.

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 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 tip opening 14 can deliver oligonucleotide A 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.

From the viewpoint of ease of puncturing the sieve hole 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, which is the axial length of the blade surface 13 shown in FIG. 3 (corresponding to the exposed length from the tip of the cannula portion 30 of the needle portion 10), is long enough to position the tip opening 14 at least within the sieve hole 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 set appropriately depending on the thickness of the olfactory mucosa Y2 of the mammal to be punctured, the overall length of the sieve hole X3, etc.

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

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

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 in order to puncture the olfactory mucosa, etc., 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.

<Hub section>
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 so as 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.

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

The connection part 22 is provided on the base 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 connection part 22 is connected to the storage part 40 via a connecting member 50 such as a tube. However, the connection part 22 may also be configured to directly and detachably fit together with the shape of the tip side of the storage part 40 (e.g., luer taper type, luer lock type). The connection part 22 is not particularly limited as long as it is configured to at least connect the storage part 40 and the hub part 20 so that oligonucleotide A can flow between them.

<Cannula section>
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.

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. Note that 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 from a separate, detachable or fixed member that can be positioned on the tip side of the cannula portion 30, and the abutment portion 32a may be formed from a portion of this separate member that comes into contact with the olfactory epithelium Y3.

When the puncturing portion 12 of the needle part 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 abutting portion 32a into contact with the olfactory epithelium Y3 as shown in Figure 4-1.

Furthermore, when the needle 10 is punctured, the stopper portion 32 stabilizes the puncture position of the administration device 1 by bringing the abutment portion 32a into contact with the olfactory epithelium Y3. This allows the needle 10 to puncture the sieve hole X3 to be punctured without misalignment. Note that it is preferable that the stopper portion 32 be pressed slightly against the olfactory epithelium Y3 so that the puncture position of the administration device 1 is fixed.

When the contact portion 32a is the tip surface of the cannula portion 30, the length (maximum width) of the longest radial portion of the tip surface of the cannula portion 30 preferably has 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 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 that the stopper function of preventing the needle portion 10 from over-puncturing the sieve hole X3 is exerted.

The cannula portion 30 may be formed in a straight cylindrical shape, or may be partially curved in order to facilitate insertion of the needle portion 10 into the sieve hole X3. Furthermore, in order to facilitate insertion into the nasal cavity Z1 and insertion of the needle portion 10 into the sieve hole X3, 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.

There are no particular restrictions on the length of the cannula portion 30, 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.

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

The cannula portion 30 may be configured to be integrally arranged with the hub portion 20, or may be configured to be detachable from the hub portion 20.

<Containment Unit>
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.

The storage unit 40 is connected to the hub unit 20 so as to be able to communicate with the needle unit 10. The tip opening 42a of the liquid delivery unit 42, formed at the tip of the liquid delivery unit 42, is in communication 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 11. The storage unit 40 is preferably configured so that the dosage of oligonucleotide A stored in the storage space 41 can be adjusted; for example, a syringe with a plunger can be used. Note that the storage unit 40 is not limited to a syringe; it can be 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. Furthermore, the storage unit 40 may be attached to a medical device having an operation unit that can control the amount and timing of oligonucleotide A discharged, and the liquid delivery unit 42 and hub unit 20 can be connected so as to be able to communicate with each other.

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

As shown in Figure 3, the guide catheter 100 has a catheter body 110 made of a tubular member having an inner lumen 111 that runs longitudinally from the tip to the base end.

The guide catheter 100 can be made from materials such as metals and resins that can be used in the medical field; for example, stainless steel such as SUS304 or polyurethane can be used.

The distal end of the catheter body 110 may be provided with a curved section arranged adjacent to the distal end, curving away from the axis of the catheter body 110. The outer surface of the guide catheter 100 may also be subjected to circumferential processing such as spiral cutting or various surface treatments to improve insertability.

The catheter body 110 may be used in a straight state as shown in Figure 3, or may 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.

Here, an example of the shape of the guide catheter 100 is 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. The guide catheter 100 can also 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.

The guide catheter 100 can be combined with the administration device 1 and supplied to the market as an administration system 200. Furthermore, 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.

[How to use the device]
Next, a method of using the administration device 1 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 oligonucleotides is initiated, and a procedure for administering the oligonucleotides 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 while 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 while the puncturing portion 12 is positioned within the cribriform plate X2, and administering the oligonucleotide into the brain via the tip opening 14.

As shown in Figure 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.

Next, the user inserts the needle portion 10 into the olfactory mucosa Y2 (in the order of olfactory epithelium Y3 and lamina propria Y4) as shown in Figure 4-2B.

Next, as shown in Figure 4-2C, the user further advances the needle portion 10 into the olfactory mucosa Y2, causing the needle tip portion 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 covers 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. The tip of the cannula portion 30 is formed with a stopper portion 32 having an abutment portion 32a that abuts against the olfactory epithelium Y3 of the olfactory mucosa Y2. The opening (tip opening 14) is positioned within the cribriform plate X2 with the abutment portion 32a of the stopper portion 32 in contact with the olfactory epithelium Y3. Once the puncture portion 12 has completed puncturing the cribriform hole X3 and the tip opening 14 is positioned within the cribriform hole X3 or beyond the cribriform plate X2 into the ventricle, administration of oligonucleotides can begin. Figure 4-2D shows the tip opening 14 positioned within the cribriform hole X3.

Then, as shown in Figure 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 appropriate amount of oligonucleotide required for administration. This causes the oligonucleotide to be administered through the tip opening 14 of the puncture part 12 into the sieve holes X3. After administering the oligonucleotide, the user removes the administration device 1 from the nasal cavity Z1, completing the series of processes.

As shown in Figure 4-2E, the oligonucleotide administered into the phloem X3 flows toward the olfactory bulb 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.

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

With this configuration, the puncture section 12 is inserted into the cribriform foramen X3 of the cribriform plate X2, and the tip opening 14 provided in the puncture section 12 is positioned within the cribriform foramen X3 or beyond the cribriform plate X2 into the ventricle. Then, oligonucleotides, such as drugs, are administered through the tip opening 14. This allows the oligonucleotides 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 oligonucleotides, such as proteins or antibodies, which are currently difficult to administer to brain tissue B, the oligonucleotides can be efficiently delivered to brain tissue B by bypassing the blood-brain barrier. Furthermore, because the oligonucleotides are 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, while minimizing the amount of oligonucleotides flowing into the blood vessels and lymphatic vessels. Furthermore, compared to conventional methods of administering oligonucleotides to brain tissue B, such as intrathecal administration, nasal spray, and intraventricular administration, the present invention enables oligonucleotides to be delivered to the brain in a minimally invasive and extremely efficient manner.

The oligonucleotide of the present invention has a cannula portion 30 formed of a tubular member that is positioned over 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 mammalian brain using an administration device 1 that is placed within the cribriform plate X2 with the abutment portion 32a of the stopper portion 32 in contact with the olfactory epithelium Y3.

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 lumen of the cannula portion 30.

The oligonucleotides 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 contact portion 32a of the stopper portion 32 of the cannula portion 30 is circular or elliptical.

[Other forms]
Other embodiments of the present invention include therapeutic methods using the above-described oligonucleotides. Here, therapeutic methods include not only methods for treating diseases, but also methods for prevention and amelioration. Furthermore, 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.

1. A method for treating a brain disease, comprising administering an oligonucleotide intranasally into the brain of a mammal by injecting it through the cribriform foramen of the cribriform plate;
2. The method for treating a brain disease according to 1. above, wherein in the administration step, the oligonucleotide is transnasally injected into the mammalian brain through an opening of an administration device equipped with a needle having a puncture portion, with the puncture portion being positioned within the cribriform foramina of the cribriform plate;
3. The method of treatment 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, an siRNA, an miRNA, and an mRNA;
4. The method of any one of 1. to 3. above, wherein the oligonucleotide is an siRNA or an antisense oligonucleotide;
5. The method of any one of 1. to 4. above, wherein the oligonucleotide is siRNA;
6. The method for treatment according to 5 above, wherein the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is 80% or more;
7. The method of treatment according to 5. or 6. above, wherein all nucleotides constituting the siRNA are 2'-modified nucleotides;
8. The method for treatment according to any one of 5. to 7. above, wherein the siRNA has one or more lipophilic moieties;
9. The method of treatment according to 8. above, wherein the fat-soluble 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 method of any one of 5. to 9. above, wherein the target gene of the siRNA is SNCA or HTT;
11. The method of treatment according to any one of 5 to 10 above, wherein all of the 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 method of treatment according to any one of 5 to 11 above, wherein the siRNA comprises any one set 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);
13. The method of treatment according to 5 above, 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 of 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;
15. 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 administration device has a cannula portion formed of a tubular member that is disposed over the needle portion so that the puncture portion is exposed;
the opening is provided on the distal end side of the puncture part,
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;
2. The method of treatment according to the above 2., wherein the opening is placed in the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium;
19. The administration device has a hub portion to which a container portion containing the oligonucleotide can be attached,
19. The method of treatment according to claim 18, wherein 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. A treatment method as described in 18 or 19 above, wherein the cross-sectional shape of the contact portion of the stopper portion is circular or elliptical.
21. The method for treating a brain disease according to any one of 1. to 20. above, wherein the brain disease is a central nervous system disease;
22. The method for treatment 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.

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

That is, the present invention may also include the following embodiments.
1. A method for administering an oligonucleotide, comprising the step of administering the oligonucleotide intranasally into the brain of a recipient (mammal) by injecting it through the cribriform foramina of the cribriform plate;
2. The administration method according to 1. above, wherein the administration step comprises administering the oligonucleotide transnasally into the mammalian brain through an opening of an administration device equipped with a needle having a puncture portion, with the puncture portion being positioned within a cribriform foramen 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, an siRNA, an miRNA, and an mRNA;
4. The administration method according to any one of 1. to 3. above, wherein the oligonucleotide is an siRNA or an antisense oligonucleotide;
5. The administration method according to any one 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 one of 5. to 7. above, wherein the siRNA has one or more lipophilic moieties;
9. The administration method according to 8. above, wherein the fat-soluble 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 any one of 5 to 9 above, wherein the target gene of the siRNA is SNCA or HTT;
11. The administration method according to any one of 5 to 10 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;
12. The administration method according to any one of 5 to 11 above, wherein the siRNA comprises any one set 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);
13. The administration method according to 5. above, 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 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 administration device has a cannula portion formed of a tubular member that is disposed over the needle portion so that the puncture portion is exposed;
the opening is provided on the distal end side of the puncture part,
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;
2. The administration method according to the above 2., wherein the opening is placed in the cribriform plate with the abutting portion of the stopper portion in contact with the olfactory epithelium;
19. The administration device has a hub portion to which a container portion containing the oligonucleotide can be attached,
19. The administration method according to claim 18, wherein 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. An administration method described in 18 or 19 above, wherein the cross-sectional shape of the abutment portion of the stopper portion is circular or elliptical.

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, allowing for efficient delivery to the brain while remaining minimally invasive, and furthermore, exhibiting high efficacy as a nucleic acid drug. As such, the oligonucleotides described herein are particularly suitable for intranasal administration, but at the same time, certain efficacy as a nucleic acid drug (e.g., reduction (knockdown) of the expression of a specific protein) can also be expected when administered non-intranasally. In other words, the oligonucleotides described herein are not limited to a specific administration method, and they themselves possess certain efficacy as nucleic acid drugs. For example, the oligonucleotides described herein can also be used as siRNA targeting a specific gene, without being limited to a specific administration method, as shown below.

1. An siRNA targeting SNCA, comprising any one set 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);
2. The siRNA according to 1. above, wherein the proportion of 2'-modified nucleotides in 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 one of 1. to 3. above, which has one or more lipophilic moieties.
5. The siRNA according to 4. above, wherein the fat-soluble 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 one 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.

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 one of Tables 1-1 to 1-6.

8. SNCA-targeting siRNA, which is any one pair 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.

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. The siRNA according to 9. above, wherein the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is 80% or more;
11. The siRNA according to 9. or 10. above, wherein all nucleotides constituting the siRNA are 2'-modified nucleotides;
12. The siRNA according to any one of 9. to 11. 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.

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

14. An siRNA targeting HTT, comprising 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.

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

The present invention will be explained below using examples. However, the present invention is not limited to these examples.

The effects of the present invention will be explained using the following examples and comparative examples. The materials, amounts used, ratios, processing details, processing 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," "% 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.

<Preparation of siRNAs (ds1 to ds6) used in the Examples and Reference Examples>
(Step 1) Synthesis of oligonucleotides (ss6 and ss9) The 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. 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.

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)). 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, as 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, as well as the calculated and measured molecular weights of the resulting oligonucleotides, are shown in Table 5.

(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).

Cleavage from the solid support and deprotection were performed using 28% aqueous ammonia or 28% aqueous ammonia with DEA added to a concentration of 3% (v/v) (3% DEA/aqueous ammonia solution) according to the protocol described in the above reference. The resulting crude product was concentrated under reduced pressure and purified by anion exchange chromatography (system: AKTA pure® 150 (Cytiva), column: TSKgel® SuperQ-5PW, 13 maikurom, 21.5 x 150 mm (Tosoh), 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 using 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 within the chain were synthesized (see Table 5).

(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.

(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.

(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.

(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.

<Preparation of siRNAs (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 the Reference Example and the calculated and measured molecular weights of the obtained oligonucleotides are shown in Tables 5 and 6.

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

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

(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.

(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.

(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.

(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.

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).

(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.

(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.

(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.

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

(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.

(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.

<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), and 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 1x PBS in the combinations shown in Tables 7-1 to 7-5. Synthesis of ds17 to ds171 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.

<Preparation of SNCA-targeting siRNA (ds250 to ds253)>
For ds250-253, single-stranded oligonucleotides were synthesized using 2'-TBDMS ribonucleoside phosphoramidite (TFS) according to the same method as in Steps 2 and 4 of the preparation of siRNA (ds1-ds6). Then, double-stranded oligonucleotides were prepared by mixing equal amounts of the antisense and sense strands in the combinations shown in Table 8 using the same method as in Step 6. 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.

<Preparation of SNCA-targeting siRNA (ds254 to ds257)>
For ds254 to 257, single-stranded oligonucleotides were synthesized using a method similar to steps 2 and 4 of the preparation of siRNA (ds1 to ds6), and then double-stranded oligonucleotides were prepared using the same method as 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.

<Preparation of SNCA-targeting siRNA (ds258 to ds273)>
For ds258 to ds273, single-stranded oligonucleotides were synthesized using a method similar to steps 2 and 4 of the preparation of siRNA (ds1 to ds6), and then double-stranded oligonucleotides were prepared using the same method as 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.

<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.

<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, prepared into aCSF solution, and used 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.

Reference Example 1-1: Knockdown test of hypoxanthine-guanine phosphoribosyltransferase 1 (hereinafter referred to as HPRT1)-targeting siRNA administered to rat cervical vertebrae
30 μL (600 μg/head) of ds4, ds3, or ds7 diluted to 20 mg/mL with D-PBS was administered intracervically (intracisternal administration) to 9-week-old male SD rats, and brain samples were collected 7 days after administration and analyzed (N = 5). Two of the five ds7 rats showed abnormalities, so the remaining three (N = 3) showed no abnormalities and were analyzed.

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 the KingFisher® Flex (TFS) and the KingFisher MagMax-96 Total RNA Isolation Kit (TFS, AM1830). cDNA was produced by reverse transcription using EvoScript Universal cDNA Master (Roche Diagnostics, 07912455001) according to the instructions provided with the kit.

This cDNA was used as a template for PCR reactions, and a QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems) was used to perform PCR reactions using the TaqMan® probe method on the HPRT1 gene and, as a control, the beta-actin (hereinafter referred to as ACTB) gene, measuring the amount of mRNA amplified. Using ACTB mRNA amplified as an internal control, a semi-quantitative value for HPRT1 mRNA was calculated. Furthermore, the amounts of HPRT1 and ACTB mRNA amplified in the negative control group were similarly measured, and a semi-quantitative value for HPRT1 mRNA was calculated.

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 was TaqMan® Gene Expression Master Mix (Applied Biosystems, 4369542), and the measurements were performed according to the attached protocols. The amount of target mRNA in nucleic acid-administered individuals was calculated as a relative proportion, with the amount of HPRT1 mRNA in the negative control (non-administered) group set at 1. Figure 5 shows the results of the relative proportion of mRNA amount, expressed as the mean ± standard error. The data for each site in Figure 5 shows, from left to right, the results for the negative control (non-administered), ds4, ds3, and ds7.

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

[Reference Example 1-2: Knockdown test using HPRT1-targeting siRNA administered to rat cervical vertebrae]
Nine-week-old male SD rats were administered 30 μL (300 μg/head) of ds4, ds3, ds5, and ds8 diluted to 10 mg/mL with D-PBS into the cervical spine. 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, with the amount of HPRT1 mRNA in the negative control group (non-administered group) set at 1. The results, expressed as the mean ± standard error of the relative 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.

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

Reference Example 1-3: Knockdown test using alpha-synuclein (hereinafter referred to as SNCA)-targeting siRNA administered to rat cervical vertebrae
30 μL (300 μg/head) of ds9, ds1, ds10, or ds2 diluted to 10 mg/mL with D-PBS was administered to 10-week-old male SD rats via the cervical spine, 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.

This cDNA was used as a template for PCR reactions, and the SNCA gene and, as a control, the ACTB gene were subjected to PCR reactions using the TaqMan (registered trademark) probe method using a QuantStudio 12K Flex real-time PCR system, to measure the amount of mRNA amplified. The amount of ACTB mRNA amplified was used as an internal control, and a semi-quantitative value for SNCA mRNA was calculated. Furthermore, the amounts of SNCA and ACTB mRNA amplified in the negative control group were similarly measured, and a semi-quantitative value for SNCA mRNA was calculated.

The SNCA gene was measured using TaqMan probe Rn01425143_m1 (Applied Biosystems), and the ACTB gene was measured using Rn00667869_m1 (Applied Biosystems). TaqMan® Gene Expression Master Mix was used as the reaction reagent, and measurements were performed according to the attached protocols. The amount of target mRNA in nucleic acid-administered individuals was calculated as a relative proportion, with the amount of SNCA mRNA in the negative control group (non-administered group) set at 1. Figure 7 shows the results of the relative proportion of mRNA amount, expressed 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.

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

[Reference Example 2: Knockdown test using HPRT1-targeting siRNA administered intracerebroventricularly to mice]
Six-week-old male ICR mice were intracerebroventricularly administered 5 μL (125 μg/head) of ds11, ds12, ds13, ds14, ds15, and ds16 diluted to 25 mg/mL in D-PBS. Seven days after administration, brain samples were collected and analyzed (N=5). One individual with ds14 and three with ds15 failed administration, so they were excluded from further analysis. RNA was extracted and purified using the 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.

This cDNA was used as a template for PCR reactions, and the HPRT1 gene and, as a control, the beta-actin (hereinafter referred to as ACTB) gene were subjected to PCR reactions using the TaqMan® probe method with a CFX Connect® Real-Time System (BIO-RAD) to measure the amount of mRNA amplified. Using ACTB mRNA amplified as an internal control, the semi-quantitative value of HPRT1 mRNA was calculated. Furthermore, the amounts of HPRT1 and ACTB mRNA amplified in the negative control group were similarly measured, and the semi-quantitative value of HPRT1 mRNA was calculated.

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 measurements were performed according to the attached protocols. The amount of target mRNA in nucleic acid-administered individuals was calculated as a relative proportion, with the amount of HPRT1 mRNA in the negative control group (D-PBS-administered group) set at 1. Figure 8 shows the results of the relative proportion of mRNA amount, expressed as the mean ± standard error. The data for each site in Figure 8 shows, from left to right, the results for the negative control (D-PBS-administered), ds11, ds12, ds13, ds14, ds15, and ds16.

All test samples (ds11, ds12, ds13, ds14, ds15, ds16) demonstrated knockdown activity compared to the negative control (D-PBS).

[Example 1: Nasal administration knockdown test using HPRT1-targeting siRNA in rats]
In Example 1, live rats 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 oligonucleotide transferred to each site in the brain and the strength of knockdown were evaluated for each rat.

The administration device 1A produced in Example 1 had the following specifications. Figure 9 shows a structural diagram of the administration device 1A produced in Example 1.

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 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 115 mm. A double-lumen catheter was created by gluing the tip of the gap with UV adhesive, leaving 1 mm of the inner catheter exposed distally from the outer 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 double-lumen catheter was then inserted into the needle tube from the tip of the cut hub, with the 1 mm exposed distal end first, and the gap was glued with UV adhesive. Next, at the end of the double-lumen catheter opposite the end glued to the hub needle, the tip of the inner catheter was cut 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-lumen 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-lumen catheter.

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) serving as the needle portion 10 was inserted 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 of the puncture portion of the needle portion 10 in the long axis direction) was 0.3 mm, and the needle tip was adhered with UV adhesive to produce an administration device. A microsyringe (product name: Gastight Syringe 1705TLL, manufactured by Hamilton) was attached to the base end of the hub portion 20 as the storage portion 40.

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.

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

First, the test subjects (male 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 conducted under continuous anesthesia (1.5-3%).

Next, a guide catheter was inserted into one of the subject's nasal cavities at an angle parallel to the rat's nasal ridge. During insertion, the tip opening of the guide catheter was oriented toward the cribriform plate.

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 section). 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 25-33 mm.

By verifying the above 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, if the length of the portion of the device inserted into the rat's body is 25 to 33 mm, it can be confirmed that the puncture portion has at least reached the cribriform plate. In addition, if the length of the puncture portion does not change when it is pushed 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 opening end toward the brain), so when the needle portion 10 is pushed in during puncture, the tip of the needle portion 10 slides along the annulus and is inserted into the cribriform foramen. However, if the pushing continues, the stopper portion at the tip of the cannula portion 30 comes into contact with the olfactory epithelium, preventing further pushing.

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

After surviving for seven days from the date of administration, the animals were euthanized by carbon dioxide inhalation. The skulls were then cut open, and the extracted brains were cooled on a cooling plate. Each section was then sampled, flash-frozen in liquid nitrogen, and stored frozen.

Then, the 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 proportion, with the amount of HPRT1 mRNA in the negative control group (non-administered group) set at 1. The results of the relative proportion of mRNA levels, 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.

The test samples (ds4, ds5, ds6) demonstrated knockdown activity compared to the negative control (untreated group).

[Example 2: Nasal administration knockdown test using HPRT1-targeting siRNA in rats (varying puncture depth)]
In Example 2, rats were used as subjects, and intranasal administration was performed into the cribriform foramina of the cribriform plate. After the rats were allowed 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.

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

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

Administration device 1B was inserted into the nasal cavity using guide catheter 100A as an insertion aid. Guide catheter 100A was made of polyurethane and had a total length of 45 mm, an outer diameter of φ1.35, and a double lumen shape with inner diameters of φ0.45 and φ0.70.

In Example 2, the following test procedure was followed to deeply puncture the sieve holes in the cribriform plate.

First, the test subjects (male 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 conducted under continuous anesthesia (1.5-3%).

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

Next, the device was inserted into the guide catheter with an inner diameter of 0.45 mm, and the needle tip was punctured into the olfactory mucosa by the exposed length of the needle tip (length of the puncture section). A 16-channel X-ray CT scanner (product name: Bright Speed Elite, manufactured by GE Healthcare) confirmed that the tip of the puncture section had reached the area deep inside the olfactory epithelium where the olfactory nerves are concentrated.

Next, the microsyringe was operated to administer 30 μL of the nucleic acid solution at 20 μL/min using a microsyringe pump (product name: IC3200, manufactured by KD Scientific) and allowed to stand for 5 minutes. For administration, a nucleic acid solution containing intrastrand C16-siHPRT1(ds3) oligonucleotide dissolved in 1x PBS to a concentration of 60 mg/mL was used.

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

Then, the mRNA expression level was 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 proportion, with the amount of HPRT1 mRNA in the negative control group (non-administered group) set at 1. The results of the relative proportion of 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).

The test sample ds3 (deeper) showed improved knockdown activity compared to ds3 (shallower).

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.

Figure 12 shows the results of expressing the nucleic acid amount in each brain region as the mean ± standard error. Note that the data for each region in Figure 12 shows, from left to right, ds3 (shallow), ds3 (deep).

This amount of nucleic acid is thought to be the amount of administered nucleic acid that migrated to each brain region (olfactory bulb, striatum, and cerebral cortex (neck side)). The test sample ds3 (deeper) showed an improved amount of nucleic acid migration compared to ds3 (shallower).

[Example 3: Knockdown test in cynomolgus monkeys using HPRT1-targeting siRNA by intranasal administration]
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 subject.

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

Administration devices 1C are available with different exposed needle tip lengths (exposed lengths 1.3 mm to 4.2 mm).

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. The inner catheter of the double-lumen catheter was then inserted into the needle tube from the tip of the cut hub, with the 1 mm exposed distal end glued in place. The gap was then glued with UV adhesive. Next, at the end of the double-lumen catheter opposite the end bonded to the needle tube of the hub, the tip of the inner catheter was cut so that 7 mm of the inner catheter was exposed from the outer catheter. The step between the catheters near the cut was then filled in with an epoxy adhesive (product name: Bondquick 30, manufactured by Konishi Co., Ltd.) to smooth the gap, thereby producing a double-lumen catheter structure. The hub portion 20 of the produced administration device 1C was the hub portion of the 18G hub-equipped needle described above, and the cannula portion 30 was composed of the double-lumen catheter.

Each administration device was produced by inserting the 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 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.

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 bending angle of 45° at the curved tip. The guide catheter was also given a spiral cut in the straight section to give it flexibility and allow it to be bent.

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

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 for animals 1% "Mylan", route of administration: intravenous administration, dosage: 0.5 to 30 mg/kg/hour (0.05 to 3 mL/kg/hour).

Next, a 16-channel X-ray CT scanner (product name: Bright Speed Elite, manufactured by GE Healthcare) was used to create an image, and 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 (length of the puncture area) that would allow for puncture of at least 60% of the measured cribriform plate thickness was calculated, and a puncture area with a length equivalent to 60% to 100% of the thickness of the cribriform plate was selected.

Next, the selected 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 section). The X-ray CT scanner described above confirmed that the needle tip had punctured the cribriform foramen. The microsyringe was operated to administer 2 mL of nucleic acid solution 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.

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

Subsequently, cDNA was prepared and quantitative PCR was performed for each brain region and spinal cord (cervical, thoracic, and lumbar) 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 the mean ± standard error of the relative proportions of mRNA amounts, 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.

The test sample (ds3) demonstrated significant knockdown activity of the HPRT1 gene in various brain regions compared to the control (aCSF).

Furthermore, the amount of nucleic acid transfer in each brain region was 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.

[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.

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

First, the test subjects (male 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 conducted under continuous anesthesia (1.5-3%) with a small animal anesthesia mask attached.

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 not inserted into the nostril, but was brought close to the nostril on the administration side, and the solution was 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 was complete, the rat was kept in a supine position in the anesthesia box for 5 minutes.

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

Subsequently, cDNA preparation and quantitative PCR were performed using the same method as in Reference Examples 1-3. The results, which show the relative proportions of mRNA amounts expressed as mean ± standard error, are shown in Figure 16. Note that the data for each site in Figure 16 shows, from left to right, the results for aCSF, ds1, and ds2 (all administered intranasally), and aCSF, ds1, and ds2 (all administered intranasally).

Nasal administration demonstrated significant knockdown activity of the SNCA gene in various parts of the brain compared to nasal administration.

Furthermore, the amount of nucleic acid migration in each brain region was measured 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 template for PCR, 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.

Figure 17 shows the results of expressing the amount of nucleic acid in each brain region as the mean ± standard error. 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 shows, from left to right, the results for ds1, ds2 (both nasal administration), and ds1, ds2 (both nasal administration).

Example 5: In vitro evaluation of siSNCA
<Design of siRNA>
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 custom Perl scripts, with reference to the base sequence described in WO 2022/072447. 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).

<Cell culture>
BE(2)-C cells (KAC) were cultured at 37°C under 5% CO2 in a medium prepared by mixing equal volumes of EMEM medium (ATCC) and Ham's F-12 medium (TFS), supplemented with 15% heat-inactivated fetal bovine serum (hereinafter also referred to as FBS). Cell subculture was carried out _twice a week by washing the cells with PBS (TFS) and then treating them with 0.25% Trypsin-EDTA (TFS). Cells that had been passaged 6 or 7 times were used for the assay.

<Addition of siRNA>
A 500 nmol/L PBS solution of 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 10 μL. Subsequently, Lipofectamine RNAiMAX (TFS) was diluted 80-fold with Opti-MEM medium, and 10 μL was added to the plate containing siRNA. 30 μL of a BE(2)-C cell suspension prepared at 1.33 x 10 ⁵ cells/mL was added to the plate to achieve 4000 cells/well. The plate was left at room temperature for approximately 15 minutes and then cultured overnight at 37°C under 5% CO ₂ .

<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. The SNCA gene and the ACTB gene were PCR-reacted using a Quant Studio 7 Flex (Applied Biosystems) to measure the Ct values. The ACTB Ct value 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.

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

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.

<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. The HTT gene and the ACTB gene (control) were subjected to PCR reactions, and Ct values were measured. The ACTB Ct value was used as an internal control to calculate the semi-quantitative value of HTT mRNA. The Ct values of HTT and ACTB in the negative control group (0 pmol of siRNA) were also measured in the same manner, and the 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.

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

[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 for each subject. Administration was performed in the same manner as in Reference Example 2.

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.

Seven days after administration, brain samples were collected from various regions and analyzed (N=4). RNA was then purified using the same method as in Reference Example 2. cDNA was created by reverse transcription using FastGene Scriptase II (NIPPON Genetics, NE-LS65) according to the instructions provided with the kit.

This cDNA was used as a template for PCR reactions, and the human SNCA gene and, as a control, the ACTB gene were subjected to PCR reactions using the TaqMan (registered trademark) probe method using a QuantStudio 12K Flex real-time PCR system, to measure the amount of mRNA amplified. Using the amount of ACTB mRNA amplified as an internal control, a semi-quantitative value for SNCA mRNA was calculated. Furthermore, the amounts of human SNCA and ACTB mRNA amplified in the negative control group were similarly measured, and a semi-quantitative value for human SNCA mRNA was calculated.

The TaqMan probe Hs00240907_m1 (Applied Biosystems) was used to measure the human SNCA gene, and Mm00607939_s1 (Applied Biosystems) was used to measure the ACTB gene. TaqMan® Gene Expression Master Mix was used as the reaction reagent, and measurements were performed according to the attached protocols. The target mRNA level in nucleic acid-administered individuals was calculated as a relative percentage to the human SNCA mRNA level in the negative control group (aCSF group). Figure 18 shows the results, expressed as the mean ± standard error, of the relative mRNA levels. The data for each site in Figure 18, from left to right, shows the results for the negative control (aCSF administration), ds2, ds254, and ds257 to ds273.

All test samples demonstrated knockdown activity compared to the aCSF-treated group, with ds2, ds254, ds257, ds259, ds261, ds263, ds267, and ds268 demonstrating particularly strong knockdown activity.

[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 of each rat and the strength of knockdown were evaluated.

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

Using the administration device 1D, a live rat was imaged under triple anesthesia using a 3D micro X-ray CT scanner R_mCT2 (Rigaku Corporation). While checking the image, a guide catheter was inserted into one of the subject's nasal cavities. Next, the administration device 1D (puncture length: 0.85 mm) was inserted into the guide catheter, and 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 to a concentration of 60 mg/mL, and 30 μL was administered.

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

ds2 (5'EPA-siSNCA) demonstrated significant knockdown activity of the SNCA gene in various brain regions compared to 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, demonstrated higher knockdown activity than ds2, although this was not as effective as ds2.

Furthermore, the same method as in Example 4 was used to measure the amount of nucleic acid transfer in each brain region. The results, expressed as the 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 shows, from left to right, the results for ds2, ds250-253. The test sample ds2 (5'EPA-siSNCA) showed significant nucleic acid transfer in each brain region.

[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 of each rat and the strength of knockdown were evaluated.

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 ds254-257 was dissolved in aCSF to a concentration of 60 mg/mL, and 30 μL was administered.

Seven days after administration, brain samples were collected from each area, 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 expressed as mean ± standard error, are shown in Figure 22. Note that the data for each area in Figure 22 shows, from left to right, the results for non-administration, ds2, and ds254-257.

The test samples (ds2, ds254-257) showed significant knockdown activity of the SNCA gene in various parts of the brain compared to the untreated group.

[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 transferred to each site in the brain and the strength of knockdown were evaluated for each subject (N=3).

Administration was performed in the same manner as in Example 3, with 2 mL administered. A nucleic acid solution prepared by dissolving ds2 oligonucleotide in aCSF to a concentration of 60 mg/mL was used for administration.

For the two-dose group, 2 mL was administered at a rate of 100 μL/min in the same manner one week later into the nostril on the side where the first administration was given. 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 similarly cryopreserved. Spinal cord samples (cervical, thoracic, and lumbar spinal cord) were also collected and similarly cryopreserved. The control group was administered aCSF, and the same procedures were followed.

cDNA was then prepared and quantitative PCR was performed for each brain region using the same method as in Example 7. The 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 hemisphere, 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.

The test samples (ds2 single dose and ds2 double dose) demonstrated significant knockdown activity of the SNCA gene in various parts of the brain and spinal cord compared to the control (aCSF).

Furthermore, the amount of nucleic acid transfer in each brain region was analyzed using the same method as in Example 4. The results, expressed as the mean ± standard error of the amount of nucleic acid in each brain region, are shown in Figure 24-1. The test samples (one dose of ds2 and two doses 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, shows one dose of ds2 and two doses of ds2. The graph on the left in Figure 24-1 shows the results for the administered side, and the graph on the right shows the results for the unadministered 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.

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 provided with 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, from left to right, represent 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, a similar analysis was performed on CSF. Figure 26 shows the percentage of α-synuclein protein 14, 28, 42, and 56 days after the start of administration, with the amount of α-synuclein protein in the CSF before administration set at 100. The test samples (ds2 single administration and ds2 double administration) showed a significant decrease in α-synuclein protein levels in various parts of the brain, spinal cord, and CSF.

[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 of each rat and the strength of knockdown were evaluated.

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 1346-1348) was dissolved in aCSF to a concentration of 60 mg/mL, and 30 μL was administered.

Seven days after administration, brain samples were collected from various regions, and cDNA was then prepared using the same method as in Example 8.

This cDNA was used as a template for PCR reactions, and the SNCA gene, SOD1 gene, MALAT1 gene, and the ACTB gene (as a control) were subjected to PCR reactions using the TaqMan (registered trademark) probe method using a QuantStudio 12K Flex real-time PCR system, and the mRNA amplification levels were measured for each gene. Using the ACTB mRNA amplification level as an internal control, the mRNA quasi-quantitative values for each target gene were calculated. Furthermore, the mRNA amplification levels for each target gene and ACTB in the negative control group were similarly measured, and the mRNA quasi-quantitative values for each target gene were calculated.

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 (sequence number 1755) (Applied Biosystems), primer 6 (sequence number 1753) and primer 7 (sequence number 1754), and the ACTB gene was measured using Rn00667869_m1 (Applied Biosystems). The reaction reagent used was TaqMan® Gene Expression Master Mix, and the assay was carried out according to the attached protocol. The base sequences of each primer and probe are shown in Table 13.

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 mRNA amount are shown in Figure 27, expressed as mean ± standard error. The data for each site in Figure 27 shows, from left to right, the results for the negative control (non-administered) and antisense oligonucleotide.

The test samples (sequence numbers 1346-1348) showed significantly improved knockdown activity for each target gene compared to the untreated group.

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

1 administration device,
10 needle part (10a inner cavity),
11 needle shaft section,
12 Puncture part (12a needle tip part),
13 Blade surface,
14 tip opening;
15 proximal opening;
20 Hub portion 21 Main body portion (21a inner cavity),
22 connection part,
30 cannula part,
31 main body (31a inner cavity),
32 stopper portion (32a abutment portion),
40 storage section,
41 storage space,
42 liquid delivery section (tip opening 42a),
50 connecting member,
100 guide catheter,
110 catheter body,
111 lumen,
200 administration system,
A oligonucleotide,
B. Brain tissue;
C. Cerebrospinal fluid (CSF),
X1 ethmoid bone,
X2 sieve plate,
X3 sieve hole,
Y1 olfactory bulb,
Y2 olfactory mucosa,
Y3 olfactory epithelium,
Y4 lamina propria,
Y5 olfactory nerve,
Z1 Nasal cavity.

Claims (26)

An oligonucleotide administered intranasally into the mammalian brain by injection through the cribriform foramina of the cribriform plate. The compound is administered intranasally into the brain of a mammal using an administration device having a needle portion with a puncture portion,
The oligonucleotide according to claim 1 , which is injected through the opening of the puncture portion while the puncture portion is positioned within the sieve pores of the cribriform plate. The oligonucleotide of 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. The oligonucleotide of claim 1, wherein the oligonucleotide is an siRNA or an antisense oligonucleotide. The oligonucleotide of claim 1, wherein the oligonucleotide is siRNA. The oligonucleotide of claim 5, wherein the proportion of 2'-modified nucleotides in the nucleotides constituting the siRNA is 80% or more. The oligonucleotide of claim 5, wherein all nucleotides constituting the siRNA are 2'-modified nucleotides. The oligonucleotide of claim 5, wherein the siRNA has one or more lipophilic moieties. 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. The oligonucleotide of claim 5, wherein the target gene of the siRNA is SNCA or HTT. The oligonucleotide of claim 5, wherein all 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. The oligonucleotide according to claim 5, wherein the siRNA comprises any one set 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 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 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, "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.






The oligonucleotide according to 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. The oligonucleotide according to 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. The oligonucleotide according to 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 table, "^" 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.



The oligonucleotide according to claim 16, wherein the oligonucleotide comprises the sequence 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 one of Tables 2-1 to 2-4. A pharmaceutical composition comprising the oligonucleotide described in any one of claims 1 to 17. A therapeutic agent for central nervous system disorders, comprising the oligonucleotide described in any one of claims 1 to 17. the administration device has a cannula portion formed of a tubular member that is disposed over the needle portion so that the puncture portion is exposed;
the opening is provided on the distal end side of the puncture part,
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;
The oligonucleotide according to claim 2 , wherein the opening is positioned within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium. the administration device has a hub portion to which a container portion containing the oligonucleotide can be attached,
The oligonucleotide according to claim 20 , wherein the hub portion holds the proximal end of the cannula portion and/or the proximal end of the needle shaft portion of the needle portion inserted through the lumen of the cannula portion. The oligonucleotide described in claim 20, wherein the cross-sectional shape of the abutting portion of the stopper portion is circular or elliptical. An administration system comprising a storage section containing the oligonucleotide described in 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. the administration device has a cannula portion formed of a tubular member that is disposed over the needle portion so that the puncture portion is exposed;
the opening is provided on the distal end side of the puncture part,
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;
24. The administration system of claim 23, wherein the opening is positioned within the cribriform plate with the abutment portion of the stopper portion in contact with the olfactory epithelium. the administration device has a hub portion to which a container portion containing the oligonucleotide can be attached,
25. The administration system according to claim 24, wherein the hub portion holds the proximal end of the cannula portion and/or the proximal end of the needle shaft portion of the needle portion inserted through the lumen of the cannula portion. The dispensing system described in claim 24, wherein the cross-sectional shape of the abutment portion of the stopper portion is circular or elliptical.