JP7128517B2 - Antisense nucleic acid with reduced side effects - Google Patents

Description

The present invention relates to structures that reduce the side effects of antisense nucleic acids that have the activity of inhibiting or regulating the expression of target genes through antisense effects. It relates to an antisense nucleic acid capable of maintaining both the maintenance of and the reduction of side effects.

In recent years, oligonucleotides have been developed as medicines called nucleic acid medicines, and especially from the viewpoint of high selectivity of target genes, the development of nucleic acid medicines using the antisense method is vigorously progressing. It is The antisense method selectively inhibits the expression of a protein encoded by a target gene by introducing into cells an oligonucleotide (antisense nucleic acid (ASO)) complementary to a partial sequence of the mRNA (sense strand) of the target gene. is a method of inhibiting or controlling

PS-type ASOs account for the majority of ASOs in clinical trials in the pipeline. PS-type ASO is obtained by substituting one of the oxygen atoms of the phosphate group of the phosphodiester bond (Fig. 1.A), which is a natural internucleic acid bond, with a sulfur atom and phosphorothioate (PS) (Fig. 1 .B). PS conversion is seen to improve nuclease resistance (Non-Patent Document 1), uptake into cells (Non-Patent Document 2), etc., but it is known to cause various side effects such as hepatotoxicity. (Non-Patent Document 3).

One representative example of PS-type ASO is mipomersen, which was developed by Ionis Pharmaceuticals as an anticholesterolemia drug having a gapmer structure called a second-generation nucleic acid structure. In 2013, it was approved in the United States as the world's first systemically administered nucleic acid drug for homozygous familial hypercholesterolemia, but various side effects such as hepatotoxicity of PS type ASO were observed (non-patent Reference 4). In addition, it has not been approved in Europe due to side effects (Non-Patent Document 5).

Nucleic Acids Res. , 16, pp. 3209-3221, (1998) Antisense Res. Dev. , 3, pp. 53-66, (2009), doi: 10.1089/ard. 1993.3.53. Trends Biotechnol. 1996 Oct;14(10):376-87. U.S.A. S. Food and Drug Administration News Release, https://www. fda. gov/NewsEvents/Newsroom/Press Announcements/ucm337195. htm? source=govdelivery. European Medicines Agency, EMA/792736/2012, EMEA/H/C/002429, https://www. buenavistainv. com/dbimages/Isis%20Presentation. pdf.

It is an object of the present invention to provide antisense nucleic acids (ASO) with reduced side effects.

A further object of the present invention is to provide ASO with reduced side effects while maintaining the antisense effect of ASO by optimally using the PS modification that is considered to be the cause of the side effects of ASO.

Yet another object of the present invention is to provide a double-stranded nucleic acid complex comprising the ASO and a polynucleotide at least partially complementary thereto.

Other objects of the present invention will become clear from the description below.

The present inventors have found that ASO has a structure (Fig. 1.C) was found to have the effect of reducing side effects while maintaining the antisense effect by using it in the antisense active site.

The present inventors also found that a double-stranded nucleic acid comprising an ASO having the above structure and a polynucleotide at least partially complementary thereto similarly maintains the antisense effect based on the above ASO, It has been found that its side effects are reduced.

Accordingly, the present invention relates to:
1. An antisense nucleic acid comprising a PSPO unit represented by the following formula (XX)

[In the formula,
A is independently selected from optionally chemically modified nucleosides,
n represents an integer of 2 or more].
2. 1. The antisense nucleic acid according to 1 above, which has a structure represented by the following formula (XX'):

[In the formula,
A is independently selected from optionally chemically modified nucleosides,
n represents an integer of 2 or more,
Y is a 5′ wing region containing one or more chemically modified nucleotides, comprising a building block located 5′ to the PSPO unit and located at the 5′ end of the antisense nucleic acid; formula (XX′- a) or formula (XX'-b)

or Y-PSPO unit -NL- (where NL represents a nucleotide linker), and
Z is a 3' wing region containing one or more chemically modified nucleotides, comprising a building block located 3' to the PSPO unit and located at the 3' end of the antisense nucleic acid; good nucleoside; formula (XX'-c) or formula (XX'-d)

or -NL-PSPO unit-Z, where NL represents a nucleotide linker].
3. An antisense nucleic acid comprising at least four consecutive nucleotides that may be chemically modified as constituent units and comprising one or more motifs having alternating phosphodiester bonds and phosphorothioate bonds as internucleic acid bonds,
2. The antisense nucleic acid according to 1 above, wherein the motif is represented by the following formula (I) or (II).

[In the formula,
A ₁ to A _i are each independently selected from optionally chemically modified nucleosides,
R is independently selected from S ^- or O ^- such that formula (I) has alternating phosphodiester and phosphorothioated linkages as internucleic acid linkages;
Here, partial structure (Ia)

may or may not exist,
i = an integer of 6 to 100 when the partial structure (Ia) is present, and i = 5 when the partial structure (Ia) is not present],
or,

[In the formula,
A ₁ -A _i and R are as defined with respect to Formula (I);
Here, partial structure (II-a)

may or may not exist,
i=an integer of 6 to 100 when the partial structure (II-a) is present, and i=5 when the partial structure (II-a) is not present].
4. 3. The antisense nucleic acid according to 3 above, wherein at least 4 consecutive ones of A ₁ to A _i are optionally chemically modified deoxyribonucleosides.
5. (a) a 5' wing region comprising one or more chemically modified nucleotides located 5' to the motif and comprising building blocks located at the 5' end of the antisense nucleic acid, and/or
(b) a 3' wing region comprising one or more chemically modified nucleotides located 3' to the motif and comprising building blocks located at the 3' end of the antisense nucleic acid;
and optionally,
(c) at least one nucleotide linker interposed between said 5' wing region and said motif, between said motif and said 3' wing region and/or between said motif and having a length of 1-20 bases;
further comprising
the 5′ wing region and the 3′ wing region each independently have a length of 1 to 10 bases;
4. The antisense nucleic acid according to 4 above.
6. 6. The antisense nucleic acid of any one of 4 or 5 above, wherein said deoxyribonucleoside is not chemically modified.
7. comprising said 5′ wing region and said 3′ wing region;
The 5′ wing region is represented by formula (III)

[In the formula,
X is independently a chemically modified nucleoside;
c represents an integer of 1 to 9]
is represented by
The 3′ wing region is represented by formula (IV)

[In the formula,
X is independently a chemically modified nucleoside;
d represents an integer of 1 to 9]
is represented by;
or,
The 5′ wing region is represented by formula (III′) or formula (III″):

[In the formula,
X is independently a chemically modified nucleoside;
c' represents an integer of 0 to 4],
or

[In the formula,
X is independently a chemically modified nucleoside;
c' represents an integer of 0 to 4]
is represented by
wherein the 3' wing region has formula (IV') or formula (IV''):

[In the formula,
X is independently a chemically modified nucleoside;
d' represents an integer of 0 to 4]
or

[In the formula,
X is independently a chemically modified nucleoside;
d' represents an integer of 0 to 4]
represented by
The antisense nucleic acid according to 5 or 6 above.
8. 8. The antisense nucleic acid of 7 above, wherein X is independently a nucleic acid analogue.
9. 9. The antisense nucleic acid of 8 above, wherein the nucleic acid analogue is a crosslinked nucleic acid.
10. 10. The antisense nucleic acid of 9 above, wherein the cross-linked nucleic acid is selected from the group consisting of LNA, cEt-BNA, amide BNA and cMOE-BNA.
11. 11. The antisense nucleic acid of any one of 1 to 10 above, which is 8 to 100 bases in length.
12. Formula (V)

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^- or O ^- such that the region between A ₁ and A _i of formula (V) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, the partial structure (Va)

may or may not exist,
i = an integer of 6 to 96 when the partial structure (Va) exists, and i = 5 when the partial structure (Va) does not exist]
or a structure represented by
Formula (VI)

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^- or O ^- such that the region between A ₁ and A _i of formula (VI) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, partial structure (VI-a)

may or may not exist,
i = an integer of 6 to 96 when the partial structure (VI-a) exists, and i = 5 when the partial structure (VI-a) does not exist]
or a structure represented by
Formula (V')

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^— or O ^— such that the region between A ₁ and A _i of formula (V′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4 and g' represents an integer from 0 to 4,
Here, the partial structure (V'-a)

is always present, and i = an integer from 6 to 96]
or a structure represented by
Formula (VI')

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^— or O ^— such that the region between A ₁ and A _i of formula (VI′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4 and g' represents an integer from 0 to 4,
Here, the partial structure (VI'-a)

is always present, and i = an integer from 6 to 96]
12. The antisense nucleic acid according to any one of 3 to 11 above, which has a structure represented by
13. The motif is represented by formula (VII)

[wherein X is independently a chemically modified nucleoside;
Q is S ^- or O ^- , and f is an integer from 1 to 10]
13. The antisense nucleic acid according to any one of 3 to 12 above, which is interrupted at one or more positions by one or more structures represented by:
14. 4. The antisense nucleic acid of 3 above, wherein A ₁ to A _i are each independently selected from chemically modified nucleosides.
15. 15. The antisense nucleic acid of 14 above, wherein said chemically modified nucleoside is a nucleic acid analogue.
16. Formula (VIII)

[In the formula,
X and X ₁ -X _i are independently nucleic acid analogues;
R is independently selected from S ^- or O ^- such that the region between X ₁ and X _i of formula (VIII) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, partial structure (VIII-a)

may or may not exist,
i = an integer of 6 to 96 when the partial structure (VIII-a) is present, and i = 5 when the partial structure (VIII-a) is not present]
or a structure represented by
Formula (IX)

[In the formula,
X and X ₁ -X _i are independently nucleic acid analogues;
R is independently selected from S ^- or O ^- such that the region between X ₁ and X _i of formula (IX) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, partial structure (IX-a)

may or may not exist,
i = an integer of 6 to 96 when the partial structure (IX-a) exists, and i = 5 when the partial structure (IX-a) does not exist]
or a structure represented by
Formula (VIII')

[In the formula,
X and X ₁ -X _i are independently nucleic acid analogues;
R is independently selected from S ^— or O ^— such that the region between X ₁ and X _i of formula (VIII′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
e' represents an integer from 0 to 4 and g' represents an integer from 0 to 4,
Here, the partial structure (VIII'-a)

is always present, and i = an integer from 6 to 96]
or a structure represented by
Formula (IX')

[In the formula,
X and X ₁ -X _i are independently nucleic acid analogues;
R is independently selected from S ^— or O ^— such that the region between X ₁ and X _i of formula (IX′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
e' represents an integer from 0 to 4 and g' represents an integer from 0 to 4,
Here, partial structure (IX'-a)

is always present, and i = an integer from 6 to 96]
16. The antisense nucleic acid according to any one of 3, 14 and 15 above, which has a structure represented by
17. (i) the antisense nucleic acid according to any one of 1 to 16 above; ) a complementary strand comprising a polynucleotide complementary to the antisense nucleic acid;
A double-stranded nucleic acid complex comprising
18. (ii) The double-stranded nucleic acid complex according to 17 above, wherein the polynucleotide in the complementary strand comprises at least optionally chemically modified deoxyribonucleotides and/or optionally chemically modified ribonucleotides as structural units.
19. (ii) The double-stranded nucleic acid complex according to 18 above, wherein the polynucleotide in the complementary strand contains only optionally chemically modified deoxyribonucleotides as constituent units.
20. (ii) The double-stranded nucleic acid complex according to 19 above, wherein the polynucleotide in the complementary strand contains only chemically unmodified deoxyribonucleotides as constituent units.
21. (ii) The double-stranded nucleic acid complex according to 18 above, wherein the polynucleotide in the complementary strand contains only chemically unmodified ribonucleotides as constituent units.
22. (ii) The double-stranded nucleic acid complex according to 18 above, wherein the polynucleotide in the complementary strand comprises only optionally chemically modified deoxyribonucleotides and optionally chemically modified ribonucleotides as constituent units.
23. 23. The double-stranded nucleic acid complex according to 22 above, wherein the polynucleotide comprises alternating deoxyribonucleotides and ribonucleotides.
24. 24. The double-stranded nucleic acid complex according to 22 or 23 above, wherein the deoxyribonucleotides and ribonucleotides are not chemically modified.
25. (ii) The double-stranded nucleic acid complex according to any one of 17 to 24 above, wherein the polynucleotide in the complementary strand comprises at least one bulge.
26. (ii) The double-stranded nucleic acid complex according to any one of 17 to 25 above, wherein the polynucleotide in the complementary strand contains at least one mismatch to the (i) antisense nucleic acid.
27. 27. The double-stranded nucleic acid complex according to any one of 17 to 26 above, wherein the length of the polynucleotide in the (ii) complementary strand is 8 to 100 bases.
28. 28. The double-stranded nucleic acid complex according to any one of 17 to 27 above, wherein the (i) antisense nucleic acid has the same length as the polynucleotide in the (ii) complementary strand.
29. 28. The double-stranded nucleic acid complex according to any one of 17 to 27 above, wherein the length of the (i) antisense nucleic acid is different from the length of the polynucleotide in the (ii) complementary strand.
30. 30. The double-stranded nucleic acid complex according to any one of 17-29 above, comprising at least one ligand.
31. 31. The double-stranded nucleic acid complex according to 30 above, wherein at least one ligand is bound to one or more constituent units of the polynucleotide of the (ii) complementary strand.
32. 32. The two of 30 or 31 above, wherein said at least one ligand is selected from the group consisting of protein ligands, peptide ligands, aptamer ligands, carbohydrate ligands, lipid ligands, small molecule ligands and biomolecule/bioactive molecule ligands. Stranded nucleic acid complex.
33. the at least one ligand is a cyclic arginine-glycine-aspartic acid (RGD) sequence-containing peptide, N-acetylgalactosamine, cholesterol, vitamin E (tocopherol), stearic acid, docosanoic acid, anisamide, folic acid, anandamide or spermine; 32. The double-stranded nucleic acid complex according to 32 above.
34. 17. The antisense nucleic acid of any one of 1-16 above, for decreasing expression of a target gene in a mammal.
35. 35. The antisense nucleic acid of 34 above, which is an antisense strand complementary to any region of the target gene mRNA.
36. 17. The antisense nucleic acid according to any one of 1 to 16 above, for suppressing proliferation of cells in mammals in which target gene expression is enhanced.
37. 37. The antisense nucleic acid of 36 above, wherein the target gene is an oncogene and the cell is a cancer cell.
38. 34. The double-stranded nucleic acid complex according to any one of 17-33 above, for decreasing the expression of a target gene in a mammal.
39. 39. The double-stranded nucleic acid complex according to 38 above, wherein the (i) antisense nucleic acid is an antisense strand complementary to any region of the target gene mRNA.
40. 34. The double-stranded nucleic acid complex according to any one of 17 to 33 above, which is for suppressing proliferation of cells in which target gene expression is enhanced in mammals.
41. 41. The double-stranded nucleic acid complex according to 40 above, wherein the target gene is an oncogene and the cell is a cancer cell.
42. comprising the antisense nucleic acid of any one of 1 to 16 above or the double-stranded nucleic acid complex of any one of 17 to 33 above, and optionally a pharmacologically acceptable carrier, pharmaceutical composition.
43. 43. The pharmaceutical composition according to 42 above, for treating and/or preventing cancer.
44. A step of contacting a cell with the antisense nucleic acid according to any one of 1 to 16 above or the double-stranded nucleic acid complex according to any one of 17 to 33 above, and measuring the intracellular transcript level A method of reducing
A method wherein the antisense nucleic acid is an antisense strand complementary to any region of said transcript.
45. 45. The method of 44 above, wherein said transcript is an mRNA transcript encoding a protein.
46. 45. The method of 44 above, wherein said transcript is a non-protein-encoding transcript.
47. Use of the antisense nucleic acid according to any one of 1 to 16 above or the double-stranded nucleic acid complex according to any one of 17 to 33 above for reducing the expression of a target gene in a mammal.
48. The antisense nucleic acid according to any one of 1 to 16 above or the double strand according to any one of 17 to 33 for producing a drug for reducing the expression of a target gene in a mammal Use of Nucleic Acid Complexes.
49. of a target gene in a mammal, comprising administering to the mammal the antisense nucleic acid of any one of 1 to 16 or the double-stranded nucleic acid complex of any one of 17 to 33. A method of reducing expression levels comprising:
A method wherein the antisense nucleic acid is an antisense strand complementary to any region of the target gene mRNA.
50. 50. The method of 49 above, wherein said administration is enteral administration.
51. 50. The method of 49 above, wherein said administration is parenteral administration.
52. 52. The method according to any one of 49 to 51 above, wherein the dose of said nucleic acid complex is 0.001 mg/kg/day to 50 mg/kg/day.
53. 53. The method of any one of 49-52, wherein said mammal is a human.
54. 33. 33 above, wherein said at least one ligand is a cyclic peptide, N-acetylgalactosamine, cholesterol, vitamin E (tocopherol) or analogues thereof, stearic acid, docosanoic acid, anisamide, folic acid or analogues thereof, anandamide or spermine. A double-stranded nucleic acid complex as described.

In ASO, a structure with alternating phosphodiester bonds, which are natural internucleic acid bonds, and phosphorothioate (PS) bonds, in which one of the oxygen atoms of the phosphate group is substituted with a sulfur atom (Fig. 1.C), is called an anti- By using it in the sense active site, it is possible to obtain the effect of reducing side effects while maintaining the antisense effect.

Moreover, by using the ASO, it is possible to achieve both reduction of side effects and specific and efficient suppression of proliferation of target cells, preferably target cancer cells.

Furthermore, by using a double-stranded nucleic acid containing the ASO and a polynucleotide at least partially complementary thereto, it is possible to obtain the effect of reducing side effects while maintaining the antisense effect based on the ASO. is.

In the backbone structure of the DNA molecule, A. PO-type linkages of native phosphodiester linkages, B. PS-type linkages of phosphorothioate linkages, and C.I. A PS-PO type bond is shown with PS and PO types arranged in the structure. 1. This figure shows an aspect of adopting the PS-PO structure in the so-called first-generation antisense nucleic acid. The solid black line is the PS-PO structure (here, the structure composed of deoxyribonucleotides is shown). 2. FIG. 1 shows an aspect of adopting a PS-PO structure in an antisense nucleic acid having a gapmer structure, which is said to be the second generation. The central gap region (solid black line) is a substrate for RNaseH that plays a major role in antisense activity and has a PS-PO structure. On the other hand, chemically modified nucleotides, such as LNA and RNA derivatives, are used in both ends of the wing region (indicated by white frames) in order to increase nuclease resistance and affinity to the target mRNA. 3. Example with wing regions only at the 5' end. 4. Examples where the wing region is only at the 3' end. 5 and 6. It is a structural example in which the antisense active portion functions independently of RNaseH. Here, 5 indicates an aspect in which the gap region is interrupted at multiple locations by a structure similar to the wing region, and 6 indicates an aspect in which the entire antisense nucleic acid is composed of chemically modified nucleotides such as LNA and RNA derivatives. An example of the structure of a double-stranded nucleic acid in which a complementary nucleic acid strand is added to an antisense nucleic acid is shown. The antisense nucleic acids in 1-6 correspond to structures 1-6 in FIG. 2, respectively. 1 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using A549 cell line. 2 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using the A431 cell line. 2 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using an AsPC-1 cell line. 1 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using an HPAC cell line. 2 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using a HepG2 cell line. 2 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using MCF-7 cell line. 1 shows the results of a cytostatic activity test of a double-stranded nucleic acid targeting the human BCL2 gene using an OVCAR-3 cell line. 2 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using an MCAS cell line. 1 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using a Caki-1 cell line. Fig. 2 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using a DU145 cell line. 2 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using a PC-3 cell line. 2 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using a T24 cell line. 1 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using HCT116 cell line. 2 shows the results of a cytostatic activity test of a double-stranded nucleic acid targeting the human BCL2 gene using HGC-27 cell line. Fig. 2 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using the MKN45 cell line. 1 shows the results of a cell growth inhibitory activity test of a double-stranded nucleic acid targeting the human BCL2 gene using a PANC-1 cell line. 1 shows the results of an mRNA expression-suppressing activity test using a double-stranded nucleic acid targeting the human BCL2 gene using an OVCAR-3 cell line. Fig. 2 shows the results of an mRNA expression-suppressing activity test using a double-stranded nucleic acid targeting the human BCL2 gene using the A431 cell line. Fig. 2 shows the measurement results of aspartate aminotransferase (AST) levels in pancreatic cancer orthotopic transplantation mouse models. Fig. 2 shows the measurement results of alanine aminotransferase (ALT) levels when using a pancreatic cancer orthotopic transplantation mouse model. Fig. 3 shows the results of calculating the liver weight ratio using a pancreatic cancer orthotopic transplantation mouse model. Fig. 3 shows the evaluation results of the antitumor effect by comparing the pancreas weight ratio when using a pancreatic cancer orthotopic transplantation mouse model. 2 shows the results of an mRNA expression-suppressing activity test of a double-stranded nucleic acid targeting the human BCL2 gene using a PANC-1 cell line. 2 shows the results of an mRNA expression-suppressing activity test of a double-stranded nucleic acid targeting the human BCL2 gene using an ASPC-1 cell line. 2 shows the results of an mRNA expression-suppressing activity test of a double-stranded nucleic acid targeting the human BCL2 gene using the A549 cell line. Fig. 2 shows the results of an mRNA expression-suppressing activity test of a double-stranded nucleic acid targeting the human BCL2 gene with cRGD bound to the center of the complementary strand, using the PANC-1 cell line. Fig. 2 shows the results of an mRNA expression-suppressing activity test of a double-stranded nucleic acid targeting the human BCL2 gene with cRGD bound to the center of the complementary strand using the K562 cell line. 2 shows the results of an mRNA expression-suppressing activity test of a double-stranded nucleic acid targeting the human BCR-ABL gene with folic acid bound to the center of the complementary strand. The results of mRNA expression-suppressing activity tests of double-stranded nucleic acids targeting the human STAT3 gene are shown. Fig. 3 shows the results of a verification test of the interferon-α (IFN-α) expression-inducing action in PBMC (human peripheral blood mononuclear cells) of PS-PO type double-stranded nucleic acid. An example of a polynucleotide with a ligand suspended from the 2' position of the nucleotide via a linker is shown.

As described above, the antisense nucleic acid of the present invention has a phosphodiester bond (PO-type bond), which is a natural internucleic acid bond, and an oxygen atom of a phosphate group, in at least a part of the backbone of the polynucleotide that constitutes it. It is a nucleic acid using a structure (PS-PO structure) having alternating phosphorothioated (PS) bonds (PS-type bonds) in which one of is substituted with a sulfur atom. By adopting such a structure, the antisense nucleic acid of the present invention can reduce side effects while maintaining its antisense effect. This PS-PO structure halves the lipidity due to substitution of S atoms in nucleic acid molecules, for example DNA molecules, in the antisense active site compared to the full PS-type structure, and additionally, PS-type and PO-type bonds are alternately ordered. This is intended to increase the effect of a backbone structure similar to that of nature due to the effect of a continuously continuous sequence. By greatly varying the skeleton of ASO in this way, it was possible to ensure the same degree of safety (reduction of side effects) as that of natural nucleic acids.

This PS-PO structure is described in Nucleic Acids Research, 2015, Vol. 43(9), 4569-4578, Cell, 2012, Vol. 150, 883-894, PCT/US2012/052874, etc., the mechanism of action of nucleic acids in these documents is due to RNA interference, and antisense effects based on the antisense effect of the present invention are known. Not a sense nucleic acid. In addition, there is no reported example of the effect of having a PS-PO structure in the backbone of DNA and reducing its side effects.

Here, "antisense nucleic acid" (herein also referred to as "ASO") refers to a nucleic acid composed of a polynucleotide and having an antisense effect. Antisense nucleic acids typically have a single-stranded structure.

As used herein, the term "antisense effect" refers to a target transcript (RNA sense strand) and, for example, a DNA strand complementary to a partial sequence thereof, or a strand designed to normally produce an antisense effect. hybridizing to target gene expression or target transcript levels. In some cases, inhibition of translation or splicing function conversion effects such as exon skipping, which may result from covering the transcript with a hybridization product, and/or the transcription, which may result from recognition of the hybridized portion. Said inhibition caused by product degradation may also be included.

As noted above, antisense nucleic acids of the invention include polynucleotides. In one embodiment of the invention, the antisense nucleic acid is a polynucleotide, ie consists exclusively of polynucleotides.

In one embodiment of the present invention, the present invention relates to an antisense nucleic acid having a PS-PO structure in at least part of the backbone of the polynucleotide that constitutes it. The antisense nucleic acid can be single stranded.

In one embodiment of the present invention, the present invention relates to an antisense nucleic acid comprising a PSPO unit represented by formula (XX) below

[In the formula,
A is independently selected from optionally chemically modified nucleosides,
n represents an integer of 2 or more, preferably an integer of 2 to 50, for example 5 to 20].

In a further embodiment of the present invention, the present invention also relates to an antisense nucleic acid having a structure represented by the following formula (XX')

[In the formula,
A is independently selected from optionally chemically modified nucleosides,
n is an integer of 2 or more, preferably an integer of 2 to 50, for example 5 to 20;
Y is a 5′ wing region containing one or more chemically modified nucleotides, comprising a building block located 5′ to the PSPO unit and located at the 5′ end of the antisense nucleic acid; formula (XX′- a) or formula (XX'-b)

or Y-PSPO unit -NL- (where NL represents a nucleotide linker), and
Z is a 3' wing region containing one or more chemically modified nucleotides, comprising a building block located 3' to the PSPO unit and located at the 3' end of the antisense nucleic acid; good nucleoside; formula (XX'-c) or formula (XX'-d)

or -NL-PSPO unit-Z, where NL represents a nucleotide linker].

In one embodiment of the invention, the invention relates to antisense nucleic acids comprising one or more motifs having alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. The motif can include at least four consecutive nucleotides that may be chemically modified as constituent units.

The above motifs are represented by formula (I) or (II) below:

[In the formula,
A ₁ to A _i are each independently selected from optionally chemically modified nucleosides,
R is independently selected from S ^- or O ^- such that formula (I) has alternating phosphodiester and phosphorothioated linkages as internucleic acid linkages;
Here, partial structure (Ia)

may or may not exist,
i = an integer of 6 to 100 when the partial structure (Ia) is present, and i = 5 when the partial structure (Ia) is not present],
or,

[In the formula,
A ₁ -A _i and R are as defined with respect to Formula (I);
Here, partial structure (II-a)

may or may not exist,
i=an integer of 6 to 100 when the partial structure (II-a) is present, and i=5 when the partial structure (II-a) is not present].

As shown in formula (I) or (II), the motif is alternately linked via a phosphodiester bond (hereinafter also referred to as "PO bond") and a phosphorothioate bond (hereinafter also referred to as "PS bond"). It is composed of linked nucleosides, which may be chemically modified.

The above motif has at least two repeats of PS and PO bonds. As shown below, the formula (I) when the partial structure (Ia) does not exist and i = 5 (or the formula when the partial structure (II-a) does not exist and i = 5 ( II)) corresponds to the two iterations above:

In addition, the above motif can have a structure having various numbers of repeats of PS bonds and PO bonds depending on the number of i.

For example, when i=7, the above motif is represented by formula (I) or (II) below.

The above motif when i=7 has three repeats of PS and PO bonds. and, respectively, the following parts:

but the substructure

corresponds to

As is clear from the above two formulas, in the above motif, the _first bond (bond between A1 and _A2 ) may be a PS bond or a PO bond.

Further, for example, when i=8, the above motif is represented by the following formula (I) or (II):

In this case, the motif has three repeats of PS and PO bonds, and additionally has one more PO bond (Formula (I)) or PS bond (Formula (II)). Thus, the motif does not necessarily have to start with a PO bond and end with a PS bond as the first bond, or start with a PS bond and end with a PO bond. That is, if the motif has a structure in which PS bonds and PO bonds are alternately repeated, it has a structure starting with a PS bond and ending with a PS bond (or starting with a PO bond and ending with a PO bond) as described above. can also

In formula (I) or (II) above, R is independently selected from S 2 ^— or O 2 ^— , depending on the number of i, such that there are alternating PS and PO bonds as described above.

In one preferred embodiment of the present invention, i in formula (I) or (II) above is an integer from 5 to 50, more preferably from 8 to 30, even more preferably from 10 to 25, such as 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

Antisense nucleic acids of the invention contain one or more of the above motifs. In one embodiment of the invention, the antisense nucleic acid of the invention comprises 1-10, preferably 1, 2, 3, 4 or 5 of the above motifs. In one preferred embodiment of the invention, the antisense nucleic acid of the invention contains one of the above motifs.

Also, in another embodiment of the present invention, the antisense nucleic acid of the present invention consists only of the above motif.

As described above, the motif can comprise at least 4 contiguous, optionally chemically modified, nucleotides as constituent units.

Also, in one embodiment of the invention, the antisense nucleic acid does not contain ribonucleotides, or does not contain ribonucleotides or chemically modified ribonucleotides.

In one embodiment of the present invention, the at least four consecutive optionally chemically modified nucleotides do not include ribonucleotides.
In one preferred embodiment of the present invention, the at least four consecutive optionally chemically modified nucleotides are deoxyribonucleotides.

"At least 4 consecutive optionally chemically modified deoxyribonucleotides" means at least 4, 5, 6, 7, 8, 9 or 10 consecutive optionally chemically modified deoxyribonucleotides It can be a region containing nucleotides, for example, 4 to 20 consecutive deoxyribonucleotides (which may be chemically modified), preferably 5 to 16 consecutive deoxyribonucleotides (which may be chemically modified). ), more preferably 6 to 14 bases, for example, a region containing 7 to 13 consecutive deoxyribonucleotides (which may be chemically modified). In this region, nucleotides recognized by RNase H that cleaves RNA strands when hybridized to RNA strands, such as natural deoxyribonucleotides and deoxyribonucleotides in which only the internucleic acid bond is chemically modified, can be used. In such cases, the region becomes a substrate for RNaseH. Such regions are also called "gap regions".

In one embodiment of the present invention, the deoxyribonucleotides are not chemically modified, and preferably portions other than the internucleic acid linkages are not chemically modified. Furthermore, in one embodiment of the present invention, the antisense nucleic acid of the present invention contains a region that serves as a substrate for RNaseH, and therefore the antisense nucleic acid as a whole can also serve as a substrate for RNaseH. In this case, the antisense nucleic acid exerts an antisense effect in an RNaseH-dependent manner.

In another embodiment of the present invention, each nucleotide in the above deoxyribonucleotide is independently chemically modified. Modifications available for deoxyribonucleotides are known in the art.

In one embodiment of the present invention, in the antisense nucleic acid of the present invention, at least four consecutive A ₁ to A _i in the above motif are optionally chemically modified deoxyribonucleosides. In this embodiment, at least 4, for example, at least 5, 6, 7, 8, 9 or 10 consecutive ones of A ₁ to A _i are optionally chemically modified deoxyribonucleosides. For example, 4 to 20, preferably 5 to 16, more preferably 6 to 14, for example 7 to 13 consecutive deoxyribonucleotides among A ₁ to A _i may be chemically modified. can be a nucleoside. In a further embodiment of the present invention, in the above motif, all of A ₁ to A _i are optionally chemically modified deoxyribonucleosides. In these embodiments, preferably the deoxyribonucleosides are not chemically modified.

In one embodiment of the present invention, at least four contiguous A ₁ to A _i bound via internucleic acid bonds are substrates for RNaseH. In this case, the entire antisense nucleic acid can also serve as a substrate for RNaseH.

As used herein, the term "nucleic acid" refers to natural nucleic acids, non-natural nucleic acids and/or nucleic acid analogues. It may also refer to nucleotides or polynucleotides. The term "nucleic acid strand" is also used herein to refer to oligonucleotides and polynucleotides. The nucleic acid strand may be prepared in whole or in part by chemical synthesis methods such as the use of an automatic synthesizer, and may be prepared by enzymatic treatment, although not limited to polymerase, ligase or restriction enzyme reactions. good. In one embodiment of the invention, the polynucleotides in the antisense nucleic acids of the invention (and the polynucleotides in the complementary strand) are artificially prepared by chemical synthesis or enzymatic reactions.

As used herein, the term "native nucleic acid" refers to a naturally occurring nucleic acid in which nucleotides are the basic unit and phosphoric acid is linked between nucleotides by forming a diester bond bridge between the 3' and 5' carbons of the sugar and polymerized. A polynucleotide present in a Generally, this includes RNA linked with ribonucleotides having any of adenine, guanine, cytosine and uracil bases and/or DNA linked with deoxyribonucleotides with any of adenine, guanine, cytosine and thymine bases. In the present specification, the binding between each nucleotide is also referred to as "nucleic acid binding".

As used herein, the term "non-natural nucleic acid" refers to a nucleic acid containing or consisting of non-natural nucleotides. Here, "non-natural nucleotide" is an artificially constructed or artificially chemically modified nucleotide that does not exist in nature, and has similar properties and / or structures to the naturally occurring nucleotide A nucleotide or a nucleotide containing nucleosides or bases that have similar properties and/or structures to naturally occurring nucleosides or bases. Examples include abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, α-deoxyribonucleosides, β-L-deoxyribonucleosides, and nucleotides with other sugar modifications. Furthermore, substituted pentasaccharides (2′-O-methylribose, 2′-deoxy-2′-fluororibose, 3′-O-methylribose, 1′,2′-deoxyribose), arabinose, substituted arabinose sugars; Nucleotides with substituted hexoses and alpha-anomeric sugar modifications are included. Non-natural nucleotides also include nucleotides containing artificially constructed base analogues or artificially chemically modified bases (modified bases). "Base analogs" include, for example, 2-oxo(1H)-pyridin-3-yl group, 5-substituted-2-oxo(1H)-pyridin-3-yl group, 2-amino-6-(2 -thiazolyl)purin-9-yl group, 2-amino-6-(2-thiazolyl)purin-9-yl group, 2-amino-6-(2-oxazolyl)purin-9-yl group and the like. "Modified bases" include, for example, modified pyrimidines (eg 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil), modified purines (eg 6-methyladenine, 6-thioguanosine) and other heterocyclic A base etc. are mentioned. In addition, chemical modifications such as methylphosphonate-type DNA/RNA, phosphorothioate-type DNA/RNA (that is, DNA/RNA having a thiophosphate bond), phosphoramidate-type DNA/RNA, 2′-O-methyl-type DNA/RNA, etc. Nucleic acids and nucleic acid analogues can also be included. These are also collectively referred to herein as "chemically modified nucleotides".

As used herein, the term "nucleic acid analogue" refers to an artificially constructed compound having a structure and/or properties similar to those of naturally occurring nucleic acids. For example, peptide nucleic acid (PNA: Peptide Nucleic Acid), peptide nucleic acid having a phosphate group (PHONA), crosslinked nucleic acid (BNA/LNA: Bridged Nucleic Acid/Locked Nucleic Acid), morpholino nucleic acid, basic polyamino acid (Poly L- Lys, Poly L-Arg) and the like. These nucleic acid analogues may be further chemically modified as described above.

In one embodiment of the present invention, the antisense nucleic acid or the double-stranded nucleic acid complex of the present invention comprises a nucleic acid (polynucleotide) containing a suitable combination of various nucleic acids as described above, or a combination of such nucleic acids (polynucleotide) as an antisense nucleic acid. and/or on complementary nucleic acid strands.

As used herein, "nucleotide" means a compound in which the sugar moiety of a nucleoside forms an ester with a phosphoric acid, where "nucleoside" refers to nitrogen-containing organic bases such as purine bases or pyrimidine bases. It means a glycoside compound in which a reducing group of sugar is linked via a glycosidic bond.

A "deoxyribonucleotide" is a nucleotide whose sugar moiety is D-2-deoxyribose, and a "ribonucleotide" is a nucleotide whose sugar moiety is D-ribose. A "deoxyribonucleoside" is a nucleoside whose sugar moiety is D-2-deoxyribose, and a "ribonucleoside" is a nucleoside whose sugar moiety is D-ribose.

A “polynucleotide” is a polymerized chain of multiple nucleotides in which nucleotides are the basic unit and phosphoric acid is linked between each nucleotide by forming a diester bond bridge between the 3′ and 5′ carbons of the sugar. means a substance in the form of This also includes so-called oligonucleotides. In the present specification, nucleotides (in a polymerized state) contained as constituent units in such polynucleotides are also sometimes referred to as "nucleotides".

Here, the above "deoxyribonucleotide" means a naturally occurring deoxyribonucleotide. The deoxyribonucleotides may be chemically modified at their base, sugar or phosphate linkages. Similarly, "ribonucleotide" means a naturally occurring ribonucleotide. The ribonucleotide may be chemically modified at its base, sugar or phosphate linkage. Examples of chemical modification include those described above.

In one embodiment of the present invention, chemically modified nucleotides may have modified base sites. Examples of base site modifications include cytosine 5-methylation, 5-fluorination, 5-bromination, 5-iodination, N4-methylation, thymidine 5-demethylation, 5-fluorination, 5- Bromination, 5-iodination, N6-methylation of adenine, 8-bromination, N2-methylation of guanine, 8-bromination can be mentioned. Furthermore, in chemically modified nucleotides in certain embodiments, the phosphodiester binding site may be modified. Modifications of the phosphodiester binding site include, for example, phosphorothioate, methylphosphonate, methylthiophosphonate, chiral-methylphosphonate, phosphorodithioate, and phosphoramidate, which are excellent in pharmacokinetics. phosphorothioate (thiophosphoric acid linkage) is preferred from the viewpoint of In addition, a plurality of types of such chemical modification of the base site and chemical modification of the phosphodiester binding site may be combined for the same nucleotide.

A person skilled in the art can appropriately select and apply possible chemical modifications to each type of nucleotide (eg, deoxyribonucleotide, ribonucleotide).

A plurality of chemical modifications as described above may be combined for the same nucleotide.

In one embodiment of the present invention, the chemically modified nucleotide may be modified at the sugar moiety, for example, the chemical modification is 2'-O-methylation, 2'-O-methoxyethyl (MOE), 2 selected from the group consisting of '-O-aminopropyl (AP), 2'-fluorination;

In addition, in the present invention, nucleosides may be similarly chemically modified. Specifically, each nucleoside may be chemically modified at its base or sugar moiety as described above, and the same nucleoside may be subjected to a combination of multiple chemical modifications. . "Nucleic acid analogues" as described above are also examples of chemically modified nucleosides. Nucleic acid analogues may be further chemically modified as described above. In the present specification, a nucleoside incorporated into a polynucleotide as its structural unit also refers to a nucleoside that constitutes the end of a polynucleotide (a nucleoside having an —OH group at the 5′ end or a nucleoside having an —OH group at the 3′ end). ) are also sometimes referred to as "nucleosides".

Generally, chemical modifications such as those described above do not affect base pairing, and therefore are not expected to affect complementarity between nucleotides or complementarity between polynucleotides. That is, two normally complementary nucleotides can maintain the ability to form base pairs after being chemically modified, and thus can still be complementary.

However, in some cases, depending on the number and position of chemical modifications, the antisense effects and the like exhibited by the antisense nucleic acids and double-stranded nucleic acids disclosed herein may be affected. Since these aspects differ depending on the sequence of the target gene, etc., they cannot be generalized, but a person skilled in the art can determine them by referring to the descriptions in the literature on the antisense method. In addition, if the antisense effect of the antisense nucleic acid after chemical modification is measured and the obtained measured value is not significantly lower than that of the antisense nucleic acid before chemical modification (for example, If the measured value of the antisense nucleic acid is 30% or more of the measured value of the antisense nucleic acid before chemical modification), the chemical modification can be evaluated. The antisense effect can be measured, for example, by introducing a test nucleic acid compound into a cell or the like, and measuring the expression level of a target gene (mRNA amount, cDNA amount, protein amount, etc.) can be carried out by appropriately using known techniques such as Northern blotting, quantitative PCR, and Western blotting. Alternatively, for example, a nucleic acid compound to be tested is introduced into a cell, and cell growth suppression caused by suppression of target gene expression performed by the nucleic acid compound to be tested is treated by adding WST (or similar dyes MTT, XTT, MTS, etc.) to a formazan dye. It can be measured using a known method such as a colorimetric method for measuring reducing enzyme activity, measurement of the amount of BrdU incorporated, flow cytometry, or the like.

As noted above, the polynucleotides of the antisense nucleic acids of the invention can optionally include nucleic acid analogs. Examples of nucleic acid analogues for use in the present invention include, for example, RNA derivatives. Examples of RNA derivatives include crosslinked nucleobases (also referred to as “crosslinked nucleic acids”), chemically modified sugar moieties, and the like, such as BNA/LNA, hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), Glycol nucleic acid (GNA), threose nucleic acid (TNA), tricyclo-DNA (tcDNA), 2'-OMe, 2'-F, 2'-O-MOE, 4'-O-aminoalkyl and the like.

In one embodiment of the invention, the nucleic acid analogue is BNA/LNA, preferably BNA represented by formula (1) below.

[In the formula (1), Base is an optionally substituted aromatic heterocyclic group or aromatic hydrocarbon ring group, for example, a base portion of a natural nucleoside (purine base, pyrimidine base) or a non-natural Type (chemically modified) nucleoside base sites are indicated. Examples of chemical modification of base sites are as described above. R ₁ and R ₂ may be the same or different and may be a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a silyl group. , a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or —P(R ³ )R ⁴ (wherein R ³ and R ⁴ may be the same or different, hydroxyl group, nucleic acid A hydroxyl group protected with a protective group for synthesis, a mercapto group, a mercapto group protected with a protective group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, and 1 to 1 carbon atoms. 6 cyanoalkoxy group or an amino group substituted with an alkyl group having 1 to 5 carbon atoms)].

The compounds shown in the above chemical formulas are nucleosides, but in the present specification, "LNA" and "BNA" also include forms (nucleotides) in which a phosphate group is bound to the nucleoside. That is, when the terms "LNA" and "BNA" are used in this specification, they also mean LNA/BNA incorporated as nucleotides into a polynucleotide. This is also the case when using the phrase "nucleic acid analogue".

BNAs include, for example, α-L-methyleneoxy (4'-CH ₂ -O-2 ') BNA), β-D-methyleneoxy (4'-CH ₂ -O-2') BNA, ethyleneoxy (4'-(CH ₂ ) ₂ -O-2') also called ENA BNA), β-D-thio(4′-CH ₂ -S-2′)BNA, aminooxy(4′-CH ₂ -ON(R ³ )-2′)BNA, 2′,4′- Oxyamino(4′-CH ₂ —N(R ³ )-O-2′)BNA, also called BNANC, 2′,4′-BNACOC, 3′amino-2′,4′-BNA, 5′- (4′-CH(CH ₃ )-O-2′)BNA, also called methyl BNA, cEt-BNA, (4′-CH(CH ₂ OCH ₃ )-O-2′, also called cMOE-BNA ) BNA, amide BNA (4′-C(O)—N(R)-2′) BNA (R═H, Me), also called AmNA, and other BNAs known to those skilled in the art.

In one embodiment of the invention, the nucleic acid analogues (eg LNA/BNA) may further undergo chemical modifications (or combinations of chemical modifications) as described above. In one embodiment of the invention, the nucleic acid analogues (eg LNA/BNA) are incorporated into the polynucleotide as phosphorothioated nucleotides.

Those skilled in the art can appropriately select possible chemical modifications according to the types of nucleotides (eg, deoxyribonucleotides, ribonucleotides) and nucleic acid analogs.

In one embodiment of the invention, an antisense nucleic acid of the invention can comprise wing regions comprising one or more chemically modified nucleotides. The wing region is a region in which chemically modified nucleotides are arranged mainly to increase the nuclease resistance of the antisense nucleic acid and the affinity of the nucleic acid for target mRNA. A "wing region containing one or more chemically modified nucleotides" is a region of the PSPO unit or of the motif, preferably containing the at least four contiguous deoxyribonucleotides that may be chemically modified (hereinafter "DNA (also referred to as "gap region"), preferably at the 5' and/or 3' end of the antisense nucleic acid. Thus, in one preferred embodiment of the invention, the wing region comprises a building block located at the 5' end of the antisense nucleic acid and/or a building block located at the 3' end of the antisense nucleic acid.

In one embodiment of the present invention, a region comprising a chemically modified nucleotide, such as a nucleic acid analogue, placed at the 5' end of said PSPO unit or of said motif, for example of said DNA gap region (hereinafter "5' wing region ), and a region containing chemically modified nucleotides, such as nucleic acid analogues, placed at the 3′ end of the PSPO unit or of the motif, such as the DNA gap region (hereinafter also referred to as the “3′ wing region”). ) are each independent and may contain at least one chemically modified nucleotide, and may also contain natural nucleotides (deoxyribonucleotides or ribonucleotides) in addition to such chemically modified nucleotides. . In addition, the chain length of the 5' wing region and the 3' wing region can be independently usually 1 to 10 bases, preferably 1 to 7 bases, or 2 to 5 bases, for example 2 to 4 bases. .

Furthermore, the type, number, and position of chemically modified nucleotides and natural nucleotides in the 5' wing region and 3' wing region may affect the antisense effect of the antisense nucleic acid in certain embodiments. , preferred aspects may vary depending on the sequence and the like. Although it cannot be said unconditionally, a person skilled in the art can determine a preferred embodiment while taking into consideration the description of the antisense method literature such as the description of Tachas et al., US Pat. No. 8,299,039. . In addition, similarly to the region containing "at least four or more contiguous deoxyribonucleotides that may be chemically modified", a nucleic acid (or a double-stranded nucleic acid containing the nucleic acid) after chemical modification, for example, after introduction of a nucleic acid analogue The chemical modification, e.g., nucleic acid Analogs can be evaluated as a preferred embodiment.

Note that the PS-PO structure tends to have weaker enzyme resistance than the conventional PS-type antisense strand structure. Therefore, an antisense nucleic acid having a PS-PO structure must be endowed with optimal enzyme resistance. In the present invention, in the antisense nucleic acid having a PS-PO structure as used in the present invention, DNA having a chain length of at least 4 bases (preferably chemically unmodified DNA) is arranged in the center, and RNA ( That is, the antisense strand is endowed with optimal enzyme resistance by flanking modified nucleotides, preferably nucleic acid analogues, such as LNA (or other BNA), which have strong binding capacity to target transcripts). As a result, it was found to have an effect of suppressing the expression of target transcripts and an effect of suppressing target cell growth equivalent to antisense nucleic acids having a PS structure.

Thus, in one embodiment of the invention, an antisense nucleic acid of the invention comprises the 5' wing region and the 3' wing region described above. The 5' wing region and 3' wing region preferably each independently contain a nucleic acid analogue. In one embodiment of the invention, the 5' and 3' wing regions each consist of nucleic acid analogues. In this case, the nucleic acid analog may or may not be chemically modified. The 5' and 3' wing regions may each contain BNA/LNA, in which case the LNA/BNA may be further chemically modified. In one embodiment of the invention the LNA/BNA is phosphorothioated.

Also, in one embodiment of the invention, the antisense nucleic acid does not contain ribonucleotides, or does not contain ribonucleotides or chemically modified ribonucleotides.

In one embodiment of the present invention, the nucleic acid bond in the wing region may have a PS-type structure (that is, a structure in which PS bonds are continuous), or a PO-type structure (that is, a structure in which PO bonds are continuous). or may have a PS-PO structure. When the length of the wing region is short, for example, 4 bases or less, the internucleic acid bond in the wing region may preferably have a PS-type structure.

Also, in one embodiment of the present invention, the 5' wing region has formula (III)

[In the formula,
X is independently a chemically modified nucleoside;
c represents an integer of 1 to 9]
is represented by

Also, in one aspect of the present invention, the 3' wing region has formula (IV)

[In the formula,
X is independently a chemically modified nucleoside;
d represents an integer of 1 to 9]
is represented by

Here, in formula (III) or (IV), preferably X is a nucleic acid analogue, more preferably a cross-linked nucleic acid. In one preferred embodiment of the invention, said cross-linking nucleic acid is selected from the group consisting of LNA, cEt-BNA, amide BNA and cMOE-BNA. Preferably X is an LNA. Also, c and d preferably each independently represent an integer from 1 to 6, i.e. 1, 2, 3, 4, 5 or 6, most preferably both c and d are 1 .

Further, in one embodiment of the present invention, the 5' wing region is represented by Formula (III') or Formula (III''):

[In the formula,
X is independently a chemically modified nucleoside;
c' represents an integer of 0 to 4],
or

[In the formula,
X is independently a chemically modified nucleoside;
c′ represents an integer from 0 to 4].

Also, in one embodiment of the present invention, the 3' wing region is represented by formula (IV') or formula (IV''):

[In the formula,
X is independently a chemically modified nucleoside;
d' represents an integer of 0 to 4],
or

[In the formula,
X is independently a chemically modified nucleoside;
d' represents an integer of 0 to 4].

wherein in formula (III'), (III''), (IV') or (IV''), preferably X is a nucleic acid analogue, more preferably a cross-linked nucleic acid. In one preferred embodiment of the invention, said cross-linking nucleic acid is selected from the group consisting of LNA, cEt-BNA, amide BNA and cMOE-BNA. Preferably X is an LNA. Also, c' and d' preferably each independently represent an integer of 0 to 3, i.e., 0, 1, 2 or 3, most preferably both c' and d' are 0 .

In one embodiment of the present invention, the antisense nucleic acid of the present invention comprises a nucleotide linker intervening between said 5′ wing region and said motif, between said motif and said 3′ wing region and/or between said motifs. can contain.

Here, the "nucleotide linker" is a linker that connects between the 5' wing region and the motif, between the motif and the 3' wing region, or between the motifs (when there are multiple motifs), (or chemically modified nucleotides). A "nucleotide linker" can also be a linker that connects between the 5' wing region and the PSPO unit, between the PSPO unit and the 3' wing region, or between a plurality of the PSPO units. The nucleotide linker preferably has a length of 1-20, more preferably 1-10, more preferably 1-5, especially 1-3 bases.

In one embodiment of the present invention, the nucleotide linker has a structure represented by formula (X) or formula (XI) below, or is a homopolymer consisting of units of formula (X) or formula (XI) is a random, alternating, block or gradient copolymer consisting of units of formula (X) and formula (XI), or a combination thereof:

[wherein A is independently selected from optionally chemically modified nucleosides, such as chemically unmodified deoxyribonucleosides or ribonucleosides]. In this embodiment, said homopolymer, copolymer or combination thereof comprises from 1 to 20, preferably from 1 to 10, more preferably from 1 to 5, such as 1, 2, 3, 4 or 5 of said units. can be configured.

In yet a further embodiment of the invention, said nucleotide linker is represented by formula (XII) below:

[In the formula,
A is independently selected from optionally chemically modified nucleosides, such as chemically unmodified deoxyribonucleosides or ribonucleosides;
a and b are independently of each other integers from 0 to 20, preferably from 1 to 10, more preferably from 1 to 5, especially from 1 to 3;
However, when a = 0, b is an integer of 1 to 20, preferably 1 to 10, such as 1 to 5 or 1 to 3, and when b = 0, a is 1 to 20 is an integer of preferably 1 to 10, such as 1 to 5 or 1 to 3,
When both a and b are 1 or greater, the arrangement of -AP(=O)O-- units ^{and -AP} (=O)S-- units is random, alternating, block, gradient or combinations thereof ].

For example, the nucleotide linker can have the following arrangement with respect to PS-type linkage and/or PO-type linkage (po for unit represented by formula (X), unit represented by formula (XI) in ps):

In one embodiment of the present invention, the nucleotide linker is not a structure consisting of a PS-PO structure repeated twice (-ps-po-ps-po- or -po-ps-po-ps-) , preferably free of such structures.

In one embodiment of the invention, the antisense nucleic acids of the invention do not contain a nucleotide linker as described above.

The antisense nucleic acid of the present invention has one or more selected from the above motif (M), 5' wing region (5W), 3' wing region (3W) and nucleotide linker (NL) in any combination. (provided that it contains at least one motif). Antisense nucleic acids of the present invention can have, for example, the following configurations:

In a further embodiment of the invention, the antisense nucleic acid of the invention consists of said motif, said 5' wing region and 3' wing region.

In one preferred embodiment of the invention, the antisense nucleic acids of the invention are represented by formula (V) or (VI) below:
Formula (V)

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^- or O ^- such that the region between A ₁ and A _i of formula (V) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
X is independently a chemically modified nucleoside;
e represents an integer from 1 to 9, preferably an integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6, and g represents an integer from 1 to 9, preferably an integer from 1 to 6, such as , 1, 2, 3, 4, 5 or 6;
Here, the partial structure (Va)

may or may not exist,
When the partial structure (Va) is present, i = an integer of 6 to 96, preferably 8 to 30, more preferably 9 to 25, still more preferably an integer of 10 to 20, such as 11, 12 , 13, 14, 15, 16, 17, 18, 19, and i=5 if the substructure (Va) is absent],
or,
Formula (VI)

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^- or O ^- such that the region between A ₁ and A _i of formula (VI) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
X is independently a chemically modified nucleoside;
e represents an integer from 1 to 9, preferably an integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6, and g represents an integer from 1 to 9, preferably an integer from 1 to 6, such as , 1, 2, 3, 4, 5 or 6;
Here, partial structure (VI-a)

may or may not exist,
When the partial structure (VI-a) is present, i = an integer of 6 to 96, preferably 8 to 30, more preferably 9 to 25, still more preferably 10 to 20, such as 11, 12, 13 , 14, 15, 16, 17, 18, 19, and i=5 if the substructure (VI-a) is not present].

wherein in formula (V) or (VI) preferably X is a nucleic acid analogue, more preferably a crosslinked nucleic acid, more preferably from LNA, cEt-BNA, amide BNA and cMOE-BNA selected from the group consisting of Preferably X is an LNA. Also, e and g preferably each independently represent an integer from 1 to 6, i.e. 1, 2, 3, 4, 5 or 6, most preferably both e and g are 1 .

In another preferred embodiment of the invention, the antisense nucleic acid of the invention is represented by formula (V') or (VI') below:
Formula (V')

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^— or O ^— such that the region between A ₁ and A _i of formula (V′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
X is independently a chemically modified nucleoside;
e′ represents an integer from 0 to 4, preferably an integer from 0 to 3, ie 0, 1, 2 or 3, and g′ represents an integer from 0 to 4, preferably an integer from 0 to 3, ie 0 , representing 1, 2 or 3,
Here, the partial structure (V'-a)

is always present, and i = an integer from 6 to 96, preferably an even number in the range from 6 to 96, more preferably an even number in the range from 8 to 30, more preferably an even number in the range from 8 to 26 , even more preferably an even number in the range 10 to 20, such as 10, 12, 14, 16, 18 or 20], or
Formula (VI')

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^— or O ^— such that the region between A ₁ and A _i of formula (VI′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
X is independently a chemically modified nucleoside;
e′ represents an integer from 0 to 4, preferably an integer from 0 to 3, ie 0, 1, 2 or 3, and g′ represents an integer from 0 to 4, preferably an integer from 0 to 3, ie 0 , representing 1, 2 or 3,
Here, the partial structure (VI'-a)

is always present, and i = an integer from 6 to 96, preferably an even number in the range from 6 to 96, more preferably an even number in the range from 8 to 30, more preferably an even number in the range from 8 to 26 , more preferably an even number in the range 10 to 20, such as 10, 12, 14, 16, 18 or 20].

Here, in formula (V′) or (VI′), preferably X is a nucleic acid analogue, more preferably a crosslinked nucleic acid, more preferably LNA, cEt-BNA, amide BNA and cMOE- selected from the group consisting of BNA; Preferably X is an LNA. Also, e' and g' preferably each independently represent 0, 1, 2 or 3, and most preferably both e and g are 0.

In another embodiment of the invention, in the antisense nucleic acid of the invention, said motif is represented by formula (VII)

[wherein X is independently a chemically modified nucleoside;
Q is S ^- or O ^- , and f is an integer from 1 to 10]
is interrupted at one or more places by one or more structures represented by For example, the interrupted motif in formula (VII) above has the structure:

[wherein A ₁ -A _i , X, Q and f are as defined above, preferably A ₁ -A _i are chemically unmodified deoxyribonucleotides].

Thus, the motif may be interrupted by formula (VII) above in such a way that a polynucleotide chain with appropriate internucleic acid linkages is formed.

Accordingly, in one aspect of the invention, the invention provides an antisense nucleic acid comprising one or more motifs having alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages, wherein the motifs have the formula Antisense nucleic acids represented by (I) or (II) and wherein the motif is interrupted at one or more locations by one or more structures represented by formula (VII) . This aspect corresponds to the aspect indicated by 5 in FIG.

Here, in formula (VII) above, X is preferably independently a nucleic acid analogue, more preferably a crosslinked nucleic acid, more preferably from LNA, cEt-BNA, amide BNA and cMOE-BNA. selected from the group consisting of Most preferably X is an LNA. Also, f preferably each independently represents an integer from 1 to 6, eg 1, 2, 3, 4, 5 or 6, most preferably f is 1.

Formula (VII) above is a structure similar to (or the same structure as) a portion of formula (III) or (IV), comprising a chemically modified nucleoside, preferably a nucleic acid analog, and an internucleic acid bond Become. When the motif is interrupted at one or more locations by one or more structures represented by formula (VII) above, for example, the number of interrupted structures is large and/or the number of interrupted structures is large. In this case, the antisense nucleic acid contains at least 4 consecutive nucleotides that may be chemically modified, particularly at least 4 consecutive deoxyribonucleotides that are recognized by RNaseH (only the internucleic acid bond may be chemically modified ) does not exist (see 5 in FIG. 2). In such cases, the antisense nucleic acid does not contain a substrate for RNaseH, and thus exerts an antisense effect in an RNaseH-independent manner (e.g., inhibition of translation or inhibition of translation, which may occur by covering the target transcript with a hybridization product). due to splicing function conversion effects such as exon skipping).

Therefore, in one embodiment of the present invention, the antisense nucleic acid of the present invention does not contain a region that serves as a substrate for RNaseH, and therefore the antisense nucleic acid as a whole does not serve as a substrate for RNaseH.

In a further embodiment of the invention, in the antisense nucleic acid of the invention, in said motif, A ₁ -A _i are each independently selected from chemically modified nucleosides. In one embodiment of the invention, the chemically modified nucleosides are nucleic acid analogues, more preferably crosslinked nucleic acids. The cross-linking nucleic acids are preferably selected from the group consisting of LNA, cEt-BNA, amide BNA and cMOE-BNA.

In one embodiment of the invention, the antisense nucleic acid of the invention is represented by formula (VIII) or (IX) below:

[In the formula,
X and X ₁ -X _i are independently nucleic acid analogues;
R is independently selected from S ^- or O ^- such that the region between X ₁ and X _i of formula (VIII) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
e represents an integer from 1 to 9, preferably from 1 to 6, such as 1, 2, 3, 4, 5 or 6, and g represents an integer from 1 to 9, preferably from 1 to 6, such as 1 , represents an integer of 2, 3, 4, 5 or 6,
Here, partial structure (VIII-a)

may or may not exist,
When the partial structure (VIII-a) is present, i = an integer of 6 to 96, preferably 8 to 30, more preferably 9 to 25, still more preferably an integer of 10 to 20, such as 11, 12 , 13, 14, 15, 16, 17, 18, 19, and i=5 if the substructure (VIII-a) is absent],
or,
Formula (IX)

[In the formula,
X and X ₁ -X _i are independently nucleic acid analogues;
R is independently selected from S ^- or O ^- such that the region between X ₁ and X _i of formula (IX) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
e represents an integer from 1 to 9, preferably an integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6, and g represents an integer from 1 to 9, preferably an integer from 1 to 6, such as , 1, 2, 3, 4, 5 or 6;
Here, partial structure (IX-a)

may or may not exist,
When the partial structure (IX-a) is present, i = an integer of 6 to 96, preferably 8 to 30, more preferably 9 to 25, still more preferably an integer of 10 to 20, such as 11, 12 , 13, 14, 15, 16, 17, 18, 19, and i=5 if the substructure (IX-a) is not present].

In one preferred embodiment of the present invention, in formula (VIII) or (IX) above, X and X ₁ -X _i are independently a cross-linking nucleic acid, preferably LNA, cEt-BNA, amide BNA and cMOE-BNA.

In another embodiment of the invention, the antisense nucleic acid of the invention is represented by Formula (VIII') or (IX') below:
(VIII')

[In the formula,
X and X ₁ -X _i are independently nucleic acid analogues;
R is independently selected from S ^— or O ^— such that the region between X ₁ and X _i of formula (VIII′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
e′ represents an integer from 0 to 4, preferably an integer from 0 to 3, ie 0, 1, 2 or 3, and g′ represents an integer from 0 to 4, preferably an integer from 0 to 3, ie 0 , representing 1, 2 or 3,
Here, the partial structure (VIII'-a)

is always present, and i = an integer from 6 to 96, preferably an even number in the range from 6 to 96, more preferably an even number in the range from 8 to 30, more preferably an even number in the range from 8 to 26 , even more preferably an even number in the range 10 to 20, such as 10, 12, 14, 16, 18 or 20], or
Formula (IX')

[In the formula,
X and X ₁ -X _i are independently nucleic acid analogues;
R is independently selected from S ^— or O ^— such that the region between X ₁ and X _i of formula (IX′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
e′ represents an integer from 0 to 4, preferably an integer from 0 to 3, ie 0, 1, 2 or 3, and g′ represents an integer from 0 to 4, preferably an integer from 0 to 3, ie 0 , representing 1, 2 or 3,
Here, partial structure (IX'-a)

is always present, and i = an integer from 6 to 96, preferably an even number in the range from 6 to 96, more preferably an even number in the range from 8 to 30, more preferably an even number in the range from 8 to 26 , more preferably an even number in the range 10 to 20, such as 10, 12, 14, 16, 18 or 20].

In one preferred embodiment of the present invention, in formula (VIII') or (IX') above, X and X ₁ -X _i are independently cross-linked nucleic acids, preferably LNA, cEt-BNA, A cross-linked nucleic acid selected from the group consisting of amide-BNA and cMOE-BNA.

The length of the polynucleotide in the antisense nucleic acid is not particularly limited, but is preferably at least 8 bases, at least 10 bases, at least 12 bases, at least 13 bases, at least 14 bases, or At least 15 bases. Said length may preferably be 100 bases or less, 35 bases or less, 25 bases or less, 20 bases or less, 19 bases or less, 18 bases or less or 17 bases or less. The length range is preferably 10 to 35 bases, more preferably 12 to 25 bases, even more preferably 13 to 20 bases. The length is generally selected according to the strength of the antisense effect of the nucleic acid strand on the target and other factors such as cost and synthesis yield. The polynucleotide (antisense nucleic acid, complementary strand) in the present invention may contain an abasic nucleoside or the like. Expressed as the length of

Also, in one embodiment of the invention, the antisense nucleic acid is a synthetic, purified and/or isolated antisense nucleic acid. The antisense nucleic acid has the activity of suppressing the expression of a target gene and the level of transcription products (mRNA transcripts encoding proteins or transcripts that do not encode proteins) by antisense effects.

The antisense nucleic acids of the present invention can inhibit the proliferation of target cells, preferably target cancer cells, depending on the selection of target transcripts (typically gene transcripts). Typically, target cells are cells with increased expression of the target gene, and the antisense nucleic acid of the present invention can suppress their proliferation. For example, by using the antisense nucleic acid of the present invention to suppress the expression of an oncogene (e.g., BCL2 gene having an apoptosis-suppressing function, BCR-ABL gene or STAT3 gene) in target cancer cells as a target gene, It can suppress the growth of cancer cells.

The "target gene" or "target transcript" whose expression is suppressed by the antisense effect is not particularly limited, and examples thereof include genes whose expression is enhanced in various diseases. In one embodiment of the invention, the target gene is an oncogene. The term "transcription product of a target gene" refers to mRNA transcribed from genomic DNA encoding the target gene, and includes unmodified mRNA and unspliced mRNA precursors. . Generally, a "transcript" is any RNA synthesized by a DNA-dependent RNA polymerase.

The phrase "synthetic, purified and/or isolated antisense nucleic acid" as used herein refers to an antisense nucleic acid comprising a polynucleotide chain, even if each constituent unit of the polynucleotide is a naturally occurring substance, It may be a non-naturally occurring substance, but the polynucleotide itself (i.e., the entire polynucleotide) is non-naturally occurring and/or substantially non-naturally occurring (typically artificially constructed). means that the

The term "complementary" refers to the so-called Watson-Crick base pairing (natural base pairing) or non-Watson-Crick base pairing (Hoogsteen type base pairs, etc.). For example, in DNA adenine (A) is complementary to thymidine (T) and in RNA adenine (A) is complementary to uracil (U).

For example, when a nucleotide at a certain position in a polynucleotide of an antisense nucleic acid can form a base pair with a nucleotide at a certain position in a transcription product of a target gene through hydrogen bonding, the polynucleotide and the transcription product form the hydrogen bond. are considered to be complementary at the positions of

In order for one polynucleotide to hybridize or anneal (the terms are used interchangeably herein) with another polynucleotide, the two polynucleotides are complementary at all positions in their base sequences. No need. Also in the present invention, in order for the antisense nucleic acid to exert its function, it is not necessary to be complementary at all positions of the base sequence, and mismatches at some positions are allowed.

For example, the base sequence of the target transcript and the base sequence of the polynucleotide in the antisense nucleic acid need not be completely complementary, ie, the two need not be complementary at all positions.

In the present invention, the polynucleotide in the antisense nucleic acid is at least partially complementary to the target transcript. In one embodiment of the present invention, the base sequence of the target transcript and the base sequence of the polynucleotide in the antisense nucleic acid are at least 70% or more, preferably 80% or more, more preferably 90% or more (e.g., 95% , 96%, 97%, 98%, 99% or more, or 100%) are complementary. For example, if 70% of the nucleotides are complementary, the two have 70% "complementarity." In one preferred embodiment of the invention, both are fully complementary, ie have 100% complementarity.

In addition, the complementarity of two polynucleotides (for example, the complementarity between the target transcript and the polynucleotide of the antisense nucleic acid), when both have different lengths, is can be calculated as complementarity in the entire double-strand forming region including them).

Sequence complementarity can be determined by using the BLAST program or the like. A person skilled in the art can easily design an antisense nucleic acid complementary to the target transcript, for example, based on information about the base sequence of the target gene.

In another aspect of the invention, the invention comprises:
(i) the antisense nucleic acid; and (ii) a polynucleotide comprising at least nucleotides that may be chemically modified as structural units, at least a portion of which is complementary to the (i) antisense nucleic acid. a complementary strand comprising nucleotides,
a double-stranded nucleic acid complex comprising
Regarding.

In one embodiment of the invention, the polynucleotide in the complementary strand consists of nucleotides, optionally chemically modified, as building blocks. In yet a further embodiment of the invention said complementary strand consists of said polynucleotide.

As described above, the PS-PO structure tends to have weaker enzyme resistance than the PS-type antisense strand structure of the prior art. need to have. In the present invention, the antisense nucleic acid having a PS-PO structure as used in the present invention is made into a double-stranded nucleic acid structure with a strand complementary thereto, so that the double-stranded nucleic acid can be used as an optimal enzyme. It was also found that it has resistance, and as a result, it has an effect of suppressing the expression of target transcripts and the proliferation of target cells equal to or greater than that of an antisense nucleic acid having a PS-type structure.

The complementary strand is a nucleic acid that is at least partially complementary to the antisense nucleic acid as described above. The above complementary nucleic acids are synthetic, purified and/or isolated nucleic acids. Here, the phrase "synthetic, purified and/or isolated nucleic acid" includes a polynucleotide chain and is a nucleic acid that is at least partially complementary to the antisense nucleic acid, wherein each constituent of the polynucleotide Each unit may be a naturally occurring substance or a non-naturally occurring substance, but the polynucleotide itself (i.e., the entire polynucleotide) is non-naturally occurring and/or substantially means something that does not occur (typically constructed artificially).

The polynucleotide in the complementary strand includes at least "nucleotides that may be chemically modified" as structural units. This term means that the polynucleotide has nucleotides that are chemically modified or not. Here, the above nucleotides, which are essential structural units, may be deoxyribonucleotides, ribonucleotides, or combinations thereof. In addition, each of the nucleotides may or may not be chemically modified independently. As chemical modifications, chemical modifications such as those described for antisense nucleic acids can be used. Nucleotides that have been chemically modified are, for example, nucleic acid analogs linked via internucleic acid linkages (herein, for convenience, nucleic acid analogs in the form of such nucleotides are also referred to in the form of nucleosides). A given nucleic acid analogue may also be referred to as a “nucleic acid analogue”), and the nucleic acid analogue may be chemically modified as described above. Thus, other than nucleotides, PNAs having peptide chains, morpholino nucleic acids, basic polyamino acids (Poly L-Lys, Poly L-Arg), etc., can be used as long as they have affinity for antisense nucleic acids. can be used as building blocks of complementary strands. In the present specification, structures containing such nucleic acid analogues as structural units are also referred to as polynucleotides (or nucleic acids or nucleic acid chains).

Here, the polynucleotide constituting the complementary strand may have a continuous PS-type structure, a continuous PO-type structure, or a PS-PO structure as an internucleic acid bond. or a combination thereof.

In one embodiment of the present invention, the polynucleotide in the complementary strand contains at least optionally chemically modified deoxyribonucleotides and/or optionally chemically modified ribonucleotides as constituent units.

In a further embodiment of the present invention, the polynucleotide in the complementary strand contains only optionally chemically modified deoxyribonucleotides as constituent units. In another embodiment of the present invention, the polynucleotide in the complementary strand contains only chemically unmodified deoxyribonucleotides as building blocks.

In another embodiment of the present invention, the polynucleotide in the complementary strand contains only ribonucleotides, which may be chemically modified, as constituent units. In one embodiment of the present invention, the polynucleotide in the complementary strand contains only chemically unmodified ribonucleotides as constituent units. In a further embodiment of the present invention, the polynucleotide in the complementary strand is chemically modified. It contains only deoxyribonucleotides which may be optionally chemically modified and ribonucleotides which may be chemically modified as constituent units. Preferably, said deoxyribonucleotides and ribonucleotides are not chemically modified.

In this aspect, the ratio of the number of deoxyribonucleotides to the number of ribonucleotides in the polynucleotide is not particularly limited. In one embodiment of the present invention, the polynucleotide is 1-90%, preferably 5-70%, more preferably 10-50%, for example 30-50% of the total number of nucleotides. It can contain ribonucleotides. For example, the polynucleotide can comprise, for example, 10, 20, 30, 40, 50, 60, 70, 80 or 90% of the total number of nucleotides in the polynucleotide. In these cases the remainder are deoxyribonucleotides. In yet further embodiments of the invention, the polynucleotides in the complementary strand can comprise 1-12 ribonucleotides, such as 2-10, 3-9 or 5-8 ribonucleotides. In a further embodiment of the invention the polynucleotide in the complementary strand is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ribonucleotides including. These also apply to deoxyribonucleotides. The position of the ribonucleotide and the position of the deoxyribonucleotide in the polynucleotide of the complementary strand are not particularly limited.

When the polynucleotide of the complementary strand contains only deoxyribonucleotides that may be chemically modified and ribonucleotides that may be chemically modified as constituent units, and the polynucleotide contains a plurality of ribonucleotides, each ribonucleotide Nucleotides may be present separately from each other, or may be present consecutively, or both may be present in combination. These also apply to deoxyribonucleotides.

In one aspect of the present invention, the polynucleotide of the complementary strand comprises a plurality of each of optionally chemically modified ribonucleotides and optionally chemically modified deoxyribonucleotides, and the ribonucleotides and deoxyribonucleotides are alternately or exist almost alternately. In this case, ribonucleotides and deoxyribonucleotides may be alternately (or nearly alternately) present one at a time, two at a time, three at a time, four at a time, five at a time, or six at a time. Preferably, said deoxyribonucleotides and ribonucleotides are not chemically modified.

In a further embodiment of the present invention, the polynucleotide of the complementary strand is a deoxyribonucleotide in which all structural units in the 5'-terminal region and/or 3'-terminal region of the polynucleotide may be chemically modified, and other is a ribonucleotide in which all structural units in the region may be chemically modified. Preferably, said deoxyribonucleotides and ribonucleotides are not chemically modified. Here, the "5' end region" refers to the structural unit located at the 5' end of the polynucleotide (the first structural unit counting from the 5' end) and the 2nd to nth structural units counting from the 5' end is a region containing Here n can be an integer from 3 to 20, preferably an integer from 3 to 10, eg 4, 5, 6, 7, 8 or 9. The "3' terminal region" includes a structural unit located at the 3' end of the polynucleotide (the 1st structural unit counting from the 3' end) and the 2nd to mth structural units counting from the 3' end. area. Here m can be an integer from 3 to 20, preferably an integer from 3 to 10, eg 4, 5, 6, 7, 8 or 9.

In one preferred embodiment of the present invention, the polynucleotide of the complementary strand has deoxyribonucleotides as all structural units in the 5'-terminal region and the 3'-terminal region of the polynucleotide, and the 5'-terminal and 3'-terminal All structural units in the region other than the region are ribonucleotides, wherein the 5' end region is the 2nd to nth (n = 3 to 6 ), and the 3′-terminal region is a region consisting of the structural unit located at the 3′-end and the 2nd to m-th (m=3-6) structural units counted from the 3′-end. is. For example, the complementary strand represented by SEQ ID NO: 12 described in Example 1 corresponds to this embodiment, and the structural unit located at the 5' end and the 2nd to 5th structural units counted from the 5' end All structural units in the region consisting of and all structural units in the region consisting of the structural unit located at the 3' end and the 2nd to 5th structural units counting from the 3' end are deoxyribonucleotides, and other (the region consisting of the 6th to 11th structural units counted from the 5' end) are all ribonucleotides. Preferably, said deoxyribonucleotides and ribonucleotides are not chemically modified.

In another embodiment of the present invention, the complementary strand of polynucleotide consists only of chemically unmodified deoxyribonucleotides and chemically modified sugar nucleotides, or the complementary strand of polynucleotide is chemically modified. It consists only of ribonucleotides without sugars and nucleotides with chemically modified sugars. Preferably, the chemical modification is selected from the group consisting of 2'-O-methylation, 2'-O-methoxyethyl (MOE), 2'-O-aminopropyl (AP), and 2'-fluorination. be done.

In a further embodiment of the present invention, the polynucleotide of the complementary strand is a nucleotide in which all constituent units in the 5′-terminal region and/or the 3′-terminal region of the polynucleotide are chemically modified nucleotides, preferably sugars are chemically modified ( Chemical modification is selected from the group consisting of, for example, 2'-O-methylation, 2'-O-methoxyethyl (MOE), 2'-O-aminopropyl (AP), and 2'-fluorination ), more preferably 2′-O-methyl deoxyribonucleotides/ribonucleotides, and all structural units in regions other than the 5′-terminal and 3′-terminal regions are chemically unmodified deoxyribonucleotides or ribonucleotides be. Here, the 5'-terminal region and/or the 3'-terminal region have the meanings given above. In one preferred embodiment of the present invention, the complementary strand of the polynucleotide is a nucleotide in which all structural units in the 5′-terminal region and 3′-terminal region of the polynucleotide are chemically modified sugars (chemical modification is, for example, 2′-O-methylation, 2′-O-methoxyethyl (MOE), 2′-O-aminopropyl (AP), 2′-fluorination), more preferably 2 '-O-methyl type deoxyribonucleotides/ribonucleotides, and all constituent units in the region other than the 5'-terminal and 3'-terminal regions are deoxyribonucleotides or ribonucleotides, wherein the 5'-terminal region is A region consisting of structural units located at the 5' end and structural units 2 to n (n = 3 to 6) counted from the 5' end, and the 3' end region is a structural unit located at the 3' end. and the 2nd to m-th structural units (m=3 to 6) counted from the 3' end. For example, the complementary strand represented by SEQ ID NO: 7 described in Example 1 corresponds to this embodiment, and the structural unit located at the 5' end and the second and third structural units counted from the 5' end All structural units in the region consisting of and all structural units in the region consisting of the structural unit located at the 3' end and the 2nd and 3rd structural units counting from the 3' end are 2'-O-methyl type It is a deoxyribonucleotide/ribonucleotide, and all structural units in the other region (region consisting of the 4th to 13th structural units counted from the 5' end) are chemically unmodified ribonucleotides. The 5'-terminal region and/or 3'-terminal region composed of chemically modified nucleotides may have a continuous PS-type structure or a continuous PO-type structure as an internucleic acid bond. or may have a PS-PO structure. For example, the 5' terminal region can have a continuous PS-type structure as the internucleic acid bond, and the 3' terminal region can have a continuous PO-type structure as the internucleic acid bond (eg, SEQ ID NO: 7).

In a further embodiment of the present invention, the polynucleotide in the complementary strand comprises at least four consecutive deoxyribonucleotides, which may be chemically modified, and a 5' wing region located on the 5' side thereof and a 3' located on the 3' side thereof. wing area; In this embodiment, part or all of the deoxyribonucleotides may be chemically modified, preferably part or all, more preferably all of the deoxyribonucleotides are phosphorothioated bonds as internucleic acid bonds. have. Preferably, the deoxyribonucleotides are not chemically modified except for internucleic acid linkages. Preferably, the 5' and 3' wing regions consist of LNA/BNA with phosphorothioated bonds as internucleic acid linkages.

As noted above, the polynucleotide in the complementary strand is a polynucleotide that is at least partially complementary to the antisense nucleic acid. The base sequence of the polynucleotide in the complementary strand and the base sequence of the polynucleotide in the antisense nucleic acid do not need to be completely complementary as long as they can at least partially form a double strand. , for example, at least 70% or more, preferably 80% or more, more preferably 90% or more (eg, 95%, 96%, 97%, 98%, 99% or more) of complementarity.

In one preferred embodiment of the invention, both polynucleotides have 100% complementarity. Here, complementarity is calculated as complementarity in the double-strand forming region (entire double-strand forming region) as described above. In this aspect, for example, when the length of the polynucleotide of the complementary strand is longer than the length of the polynucleotide of the antisense nucleic acid, the entire polynucleotide of the antisense nucleic acid is completely complementary to a portion of the polynucleotide of the complementary strand. and its completely complementary portion becomes the double-strand forming region. In addition, when the length of the complementary strand polynucleotide is shorter than that of the antisense nucleic acid polynucleotide, the entire complementary strand polynucleotide is completely complementary to a portion of the antisense nucleic acid polynucleotide, and the complete The complementary portion becomes the double-strand forming region. When the polynucleotide of the antisense nucleic acid and the polynucleotide of the complementary strand have the same length, both polynucleotides are completely complementary throughout.

In the present invention, the polynucleotides in the antisense nucleic acid and the polynucleotides in the complementary strand can "anneal" to form a double strand based on the complementarity described above. A person skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which two nucleic acid strands can be annealed.

The polynucleotides in the complementary strand contain nucleotides that may be chemically modified, as described above. From the viewpoint of high resistance to nucleases such as RNases, in the polynucleotide in the complementary strand, nucleotides (e.g., deoxyribonucleotides and/or ribonucleotides) are chemically modified as described for antisense nucleic acids. good too. For example, until the polynucleotide of the complementary strand is delivered to the nucleus of a specific cell, the polynucleotide is degraded by RNaseH in a specific cell while suppressing the degradation by an RNA degrading enzyme such as RNaseA. The polynucleotide in the complementary strand may contain a nucleic acid analogue, from the viewpoint that antisense effects are likely to be exerted.

Chemical modifications, such as the number and positions of nucleic acid analogues, may affect the antisense effect of a nucleic acid complex in certain embodiments. There are preferred aspects of the position. Since this preferred embodiment differs depending on the type, sequence, etc. of the nucleic acid to be chemically modified, it cannot be generalized. It can be identified by measuring the antisense effect of the nucleic acid complex.

In one embodiment of the invention, the polynucleotide in the complementary strand has chemically modified nucleotides, such as nucleic acid analogues, in the region complementary to the 5' wing region and/or 3' wing region of the antisense nucleic acid. .

The length of the polynucleotide in the complementary strand is not particularly limited, but is at least 8 bases, at least 10 bases, at least 12 bases, at least 13 bases, at least 14 bases, at least 15 bases, or at least 16 bases. Said length may preferably be 100 bases or less, 35 bases or less, 25 bases or less, 20 bases or less, 19 bases or less, 18 bases or less or 17 bases or less. The length range is preferably 10 to 35 bases, more preferably 12 to 25 bases, even more preferably 13 to 20 bases. The length is usually selected according to its effect on enzyme resistance and other factors such as cost and synthetic yield.

In one embodiment of the present invention, the length of the polynucleotide in the antisense nucleic acid and the length of the polynucleotide in the complementary strand may be the same or different.

In one embodiment of the invention, the length of the polynucleotide in the antisense nucleic acid is greater than the length of the polynucleotide in the complementary strand.

In other embodiments of the invention, the length of the polynucleotide in the antisense nucleic acid is less than the length of the polynucleotide in the complementary strand.

Furthermore, in one embodiment of the invention, the polynucleotide in the complementary strand contains at least one mismatch to the polynucleotide in the antisense nucleic acid.

A "mismatch" is a position within a double-strand forming region formed by annealing between a polynucleotide in an antisense nucleic acid and a polynucleotide in a complementary strand (hereinafter also simply referred to as a "double-strand forming region"). In the above, it means that Watson-Crick base pairs are not formed between the nucleotides of both polynucleotide chains.

However, the mismatch here means that the polynucleotide of the antisense nucleic acid and the polynucleotide of the complementary strand have the same number of nucleotides and / or nucleic acid analogues (hereinafter collectively referred to as "nucleotides etc." ), and although base pairs are formed at all positions other than the above position, base pairs are not formed only at the position. Therefore, it does not include overhangs or bulge-like structures caused by differences in length of both polynucleotides within the duplex forming region. This is intended to distinguish between mismatches and overhangs or bulges, and does not prevent the simultaneous presence of mismatches and bulges (and/or overhangs) in the nucleic acid complex of the present invention. .

Therefore, "the polynucleotide in the complementary strand contains at least one mismatch with respect to the polynucleotide in the antisense nucleic acid" means that the polynucleotide of the antisense nucleic acid and the polynucleotide of the complementary strand are identical in the double-strand forming region. and the latter contains at least one such nucleotide in the double-strand forming region that does not form a Watson-Crick base pair between both polynucleotides. Examples of mismatches include A vs. G, C vs. A, U vs. C, A vs. A, G vs. G, C vs. C, etc., and U vs. G, U vs. C, U vs. T, etc. using ribonucleotides. but also includes, for example, non-basic residues to nucleotides, etc., acyclic residues to nucleotides, etc. In a broad sense, a mismatch as used herein includes a mismatch at a particular position such that the thermodynamic stability of the duplex at that position is lower than the thermodynamic stability of the Watson-Crick base pair at that position. Also included is any transformation at that position that reduces the thermodynamic stability at or near it.

In one embodiment of the invention, the nucleic acid complex contains at least one mismatch in the polynucleotide in the complementary strand to the polynucleotide in the antisense nucleic acid. The number of mismatches is not particularly limited as long as it does not interfere with the formation of a double-stranded nucleic acid. It may exist as a combination of both. In one embodiment of the invention, the polynucleotide in the complementary strand contains one mismatch to the polynucleotide in the antisense nucleic acid.

The position of the mismatch is not particularly limited as long as it can form a mismatch structure.

For example, when the polynucleotide of the antisense nucleic acid and the polynucleotide of the complementary strand each have a length of 10 to 35 bases, for example, the 5′ end of the double strand forming region (the above region of the polynucleotide of the antisense nucleic acid Mismatches can be introduced at positions 2 to 15, eg, positions 6 to 11 (eg, 6, 7, 8, 9, 10 or 11), counting from the 5′ end of the DNA).

When the nucleic acid complex of the present invention has at least one mismatch, the portion of the mismatch, that is, the portion where the double strand is not formed, is moderately attacked by DNase or RNase. By the time it is delivered to the target mRNA, only the complementary strand is moderately cleaved (whether the polynucleotide of the complementary strand is deoxyribonucleotides, ribonucleotides, or a combination thereof) to form an antisense It is also conceivable that the polynucleotide of the antisense nucleic acid is only a polynucleotide of the nucleic acid, and that the polynucleotide of the antisense nucleic acid promotes good double-strand formation with the target mRNA.

When there is a mismatch as described above, the polynucleotide in the antisense nucleic acid and the polynucleotide in the complementary strand are not completely complementary. good too. In one preferred embodiment of the present invention, when complementary polynucleotides contain mismatches, both polynucleotides have 100% complementarity in the double-strand forming region excluding the mismatched portion.

In another embodiment of the invention, the polynucleotides in the complementary strand do not contain mismatches.

Also, for example, from the viewpoint of moderately cleaving the polynucleotide in the complementary strand, instead of forming mismatches as described above, or in addition to mismatch formation, the polynucleotide in the complementary strand may be added with at least one It is also possible to include ribonucleotides. Since ribonucleotides generally have weaker resistance to nucleolytic enzymes than deoxyribonucleotides and chemically modified nucleotides (for example, nucleic acid analogues), cleavage of polynucleotides starting from the ribonucleotide introduction site in the complementary strand can be moderately promoted. Conceivable.

Thus, in one embodiment of the invention, the polynucleotides in the complementary strand comprise one or more ribonucleotides as building blocks. In a further embodiment of the invention, the polynucleotide in the complementary strand contains at least one mismatch to the polynucleotide in the antisense nucleic acid and contains ribonucleotides as building blocks. In a further embodiment of the present invention, the polynucleotide in the complementary strand contains at least one mismatch to the polynucleotide in the antisense nucleic acid, and contains ribonucleotides as constituent units, and at least One forms the mismatch. For example, the polynucleotide in the complementary strand can contain one G to U mismatch (G in the antisense nucleic acid and U in the complementary strand) to the polynucleotide in the antisense nucleic acid.

When the complementary strand contains ribonucleotides, the positions of the ribonucleotides are not particularly limited. When the polynucleotide has a length of 10 to 35 bases, counting from the 5' end, 1st to 35th, for example, 2nd to 30th, 3rd to 25th, 4th to 20th, 5th to 15th, Ribonucleotides at positions 6-11 (eg, 6, 7, 8, 9, 10 or 11) can be included.

Also, in one embodiment of the invention, the polynucleotide in the complementary strand comprises at least one bulge.

Here, the "bulge" is a double-stranded region formed by annealing between the polynucleotide of the antisense nucleic acid and the polynucleotide of the complementary strand (hereinafter also simply referred to as "double-stranded region"). Nucleotides in the latter polynucleotide ( and/or chemically modified nucleotides).

The number of bulges is not particularly limited as long as it allows the formation of double strands. In one embodiment of the invention, the polynucleotide in the complementary strand can contain 1-4 bulges, eg, 1-3 bulges. In a further embodiment of the invention the polynucleotide in the complementary strand comprises one bulge.

The position of the bulge is not particularly limited as long as the bulge structure can be formed. For example, when the polynucleotide of the antisense nucleic acid and the polynucleotide of the complementary strand each have a length of 10 to 35 bases, for example, the 5′ end of the double strand forming region (the above region of the polynucleotide of the antisense nucleic acid 5′ end of the complementary strand), at positions 2 to 15, for example, at positions 6 to 12 (for example, 6, 7, 8, 9, 10, 11 or 12). A bulge can be introduced at the corresponding position of the nucleotide.

The number of nucleotides constituting the bulge (the number of nucleotides not forming base pairs with the polynucleotide of the antisense nucleic acid) is not particularly limited as long as it can form a bulge structure. In one embodiment of the invention, the bulge can consist of 1-7, such as 1-5 or 2-4 nucleotides per bulge.

When a plurality of bulges are provided, each bulge may be composed of the same number of nucleotides or the like, or each bulge may be composed of independently different numbers of nucleotides or the like.

In one embodiment of the invention, the bulge is composed of nucleotides and/or modified nucleotides.

In a further embodiment of the present invention, deoxyribonucleotides and/or ribonucleotides (both of which may or may not be modified) are used as the nucleotides constituting the bulge. Preferably, said deoxyribonucleotides and ribonucleotides are not chemically modified.

A person skilled in the art can appropriately select the constituent unit of the polynucleotide in the complementary strand so as to form a bulge according to the type and base sequence of the polynucleotide in the antisense nucleic acid.

In one preferred embodiment of the present invention, part or all of the nucleotides constituting the bulge are ribonucleotides, preferably uracil (U). More preferably, part or all of the nucleotides constituting the bulge are chemically unmodified ribonucleotides, preferably chemically unmodified uracil (U).

The nucleic acid complex of the present invention, in which the polynucleotide in the complementary strand contains a bulge, is moderately attacked by DNase or RNase in the portion of the bulge structure (that is, the portion where the double strand is not formed). In vivo, only the complementary strand is moderately cleaved by the time it is delivered to the target mRNA (regardless of whether the polynucleotide of the complementary strand is deoxyribonucleotide, ribonucleotide, or a combination thereof) ), resulting in only the polynucleotide of the antisense nucleic acid, which is thought to promote good double-stranded formation of the polynucleotide of the antisense nucleic acid with the target mRNA.

When the polynucleotide in the complementary strand contains at least one bulge, the above complementarity is calculated after excluding the bulge structure portion from the double-strand forming region. That is, the bulge structure portion is not included in the double-strand forming region when calculating the above complementarity. Also, as noted above, the antisense nucleic acid and the complementary strand have 100% complementarity in one preferred embodiment of the present invention. In this aspect, for example, when the length of the complementary polynucleotide (excluding the bulge-forming portion) is longer than the length of the polynucleotide of the antisense nucleic acid, the entire polynucleotide of the antisense nucleic acid is the complementary polynucleotide (excluding the bulge-forming portion), and the fully complementary portion becomes the double-strand forming region. In addition, when the length of the complementary strand polynucleotide (excluding the bulge-forming portion) is shorter than that of the antisense nucleic acid, the entire complementary strand polynucleotide (excluding the bulge-forming portion) is the antisense nucleic acid polynucleotide. It is completely complementary to a part of the nucleotide, and the completely complementary part becomes the double-strand forming region. When the polynucleotide of the antisense nucleic acid and the polynucleotide of the complementary strand (excluding the bulge-forming portion) have the same length, both polynucleotides are completely complementary except for the bulge-structure portion.

As described above, when a double-stranded nucleic acid containing the antisense nucleic acid and its complementary strand is used, the double-stranded nucleic acid has optimal enzyme resistance, and as a result, antisense having a PS structure. It was found to have an effect of suppressing the expression of target transcripts and an effect of suppressing target cell growth equal to or greater than those of nucleic acids. Various polynucleotides can be used as complementary strands in the double-stranded nucleic acid complex.

Furthermore, in the present invention, when a specific polynucleotide is used as the complementary strand, for example, when the complementary strand contains at least one mismatch to the antisense nucleic acid, and when the complementary strand contains deoxyribonucleotides and ribonucleotides alternately, side effects of the nucleic acid complex are further reduced compared to the case where other polynucleotides are used as complementary strands, specifically hepatotoxicity (especially AST value) is further reduced It was also found to reduce

Moreover, in one embodiment of the present invention, the nucleic acid complex can have overhangs.

Here, an "overhang" is a terminal base-paired non-forming nucleotide (or chemically modified nucleotide ). Each overhang contains at least one nucleotide (or chemically modified nucleotide). Preferably each overhang is a two nucleotide overhang. The nucleotides that make up the overhang can be chosen arbitrarily. The overhanging nucleotides may or may not base pair with the target transcript. Examples of such two nucleotide overhangs include, but are not limited to, UU, TT, AA, GG, CC, AC, CA, AG, GA, GC and CG.

The position of the overhang includes the 5' end of the polynucleotide in the antisense nucleic acid, the 3' end of the polynucleotide in the antisense nucleic acid, the 5' and 3' ends of the polynucleotide in the antisense nucleic acid, and the polynucleotide in the complementary strand. the 5' end of the polynucleotide on the complementary strand, the 3' end of the polynucleotide on the complementary strand, the 5' and 3' ends of the polynucleotide on the complementary strand, the 5' end of the polynucleotide on the antisense nucleic acid and the 5' end of the polynucleotide on the complementary strand, or The 3' end of the polynucleotide of the antisense nucleic acid and the 3' end of the polynucleotide of the complementary strand are possible.

In another embodiment of the invention, the nucleic acid complex does not contain overhangs.

Also, in one embodiment of the present invention, the polynucleotide in the complementary strand comprises at least deoxyribonucleotides and optionally nucleic acid analogues as constituent units. The above deoxyribonucleotides may or may not be chemically modified. In one embodiment of the present invention, the polynucleotide in the complementary strand comprises deoxyribonucleotides, which may be chemically modified, and optionally nucleic acid analogues only as building blocks. In another embodiment of the invention, the polynucleotides in the complementary strand comprise only chemically modified deoxyribonucleotides and optionally nucleic acid analogues as building blocks.

Furthermore, in one embodiment of the invention, the polynucleotides in the complementary nucleic acid strands comprise nucleic acid analogues. In other embodiments of the invention, the polynucleotides in the complementary nucleic acid strand do not contain nucleic acid analogues. Also, in another embodiment of the invention, the polynucleotides in the complementary nucleic acid strand do not contain ribonucleotides. In yet another embodiment of the invention, the polynucleotides in the complementary nucleic acid strand do not contain deoxyribonucleotides.

In addition, as described above, in one embodiment of the present invention, the complementary strand consists only of deoxyribonucleotides. In this embodiment, the double-stranded nucleic acid complex can also successfully suppress target transcript levels, which is believed to be due to the following mechanism: a polynucleotide in the antisense nucleic acid and a polynucleotide in the complementary nucleic acid strand. The DNA-DNA duplex formed by is recognized by DNase before it is delivered to the target transcript, and the DNase degrades the polynucleotide in the complementary nucleic acid strand. Polynucleotides in the remaining antisense-active portion then form DNA-RNA duplexes with target transcripts, which are then recognized and degraded by RNaseH.

In one embodiment of the invention, said antisense nucleic acid or said double-stranded nucleic acid complex is an antisense nucleic acid or nucleic acid complex for reducing expression of a target gene in a cell or mammal. In a further embodiment of the present invention, the antisense nucleic acid or nucleic acid complex suppresses the expression of target genes (preferably oncogenes) in target cells (preferably cancer cells), antisense nucleic acid or nucleic acid complex for inhibiting the proliferation of Typically, the target cell can be a cell with increased expression of the target gene. In a further embodiment of the present invention, the antisense nucleic acid or nucleic acid complex is an antisense nucleic acid or nucleic acid complex for suppressing target gene expression in a target tissue and/or target site.

In a further embodiment of the invention, the polynucleotide in the antisense nucleic acid is the antisense strand complementary to any region of the target gene mRNA. In one embodiment of the invention, the target gene is human bcl-2, human BCR-ABL or human STAT3.

In one embodiment of the present invention, the polynucleotide in the antisense nucleic acid is a polynucleotide selected from (a) to (d) below:
(a) a polynucleotide having a nucleotide sequence represented by SEQ ID NO: 2, 17, 19 or 22;
(b) a polynucleotide having 70% or more sequence identity with the polynucleotide of (a) above;
(c) a polynucleotide in which a small number of nucleotides of the polynucleotide of (a) have been substituted, deleted, added and/or inserted;
(d) A polynucleotide comprising any of the polynucleotides (a) to (c) above as a partial sequence.

The polynucleotide (b) is 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more of the base sequence of the polynucleotide (a) , eg, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 99.5% or greater, sequence identity. Preferably, the polynucleotide of (b) above has the activity of suppressing the expression of the target transcript and/or has reduced side effects.

The polynucleotide of (c) is a small number of the polynucleotides of (a), preferably one to several, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or It can be a polynucleotide in which 10 nucleotides have been replaced, deleted, added and/or inserted. Preferably, the polynucleotide of (c) above has the activity of suppressing the expression of the target transcript and/or has reduced side effects.

The polynucleotide (d) above is a polynucleotide comprising any of the polynucleotides (a) to (c) above as a partial sequence, preferably has an activity to suppress the expression of a target transcript and/or has side effects is reduced. In one embodiment of the present invention, the length of the polynucleotide of (d) above is 8-100 bases. Preferably, said length is at least 10 bases, at least 12 bases, or at least 13 bases. Said length may preferably be 100 bases or less, 35 bases or less, 25 bases or less or 20 bases or less.

As used herein, the term "sequence identity" with respect to base sequences means that two base sequences to be compared are aligned so that as many bases as possible match each other, and the number of matched bases is divided by the total number of bases. is expressed as a percentage (%). In the above alignment, gaps are appropriately inserted in one or both of the two sequences to be compared, if necessary. Such sequence alignments can be performed using well-known programs such as BLAST, FASTA, CLUSTAL W (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1993; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997). When a gap is inserted, the above total number of bases is the number of bases with one gap being counted as one base. If the total number of bases counted in this way differs between the two sequences being compared, the percent identity is calculated by dividing the number of matched bases by the total number of bases in the longer sequence. be.

Generally, the presence of such chemical modifications (eg, nucleic acid analogs) does not affect the sequence identity.

In one embodiment of the present invention, the polynucleotide in (ii) the complementary strand is a polynucleotide selected from (a′) to (d′) below:
(a') a polynucleotide having a nucleotide sequence represented by any one of SEQ ID NOS: 7-16, 18, 20, 21 and 23;
(b') a polynucleotide having 70% or more sequence identity with the polynucleotide of (a') above;
(c') a polynucleotide in which a small number of nucleotides of the polynucleotide of (a') are substituted, deleted, added and/or inserted;
(d') A polynucleotide comprising any one of the polynucleotides (a') to (c') above as a partial sequence.

The polynucleotide (b') is 70% or more, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 95% or more of the base sequence of the polynucleotide (a'). % or greater, such as 96% or greater, 97% or greater, 98% or greater, 99% or greater or 99.5% or greater, sequence identity. Preferably, the polynucleotide of (b') above is at least partially complementary to the antisense nucleic acid, more preferably 100% complementary to the antisense nucleic acid.

The polynucleotide of (c′) is a small number of the polynucleotides of (a′), preferably one to several, for example 1, 2, 3, 4, 5, 6, 7, 8, It can be a polynucleotide in which 9 or 10 nucleotides have been replaced, deleted, added and/or inserted. Preferably, the polynucleotide of (c') above is at least partially complementary to the antisense nucleic acid, more preferably 100% complementary to the antisense nucleic acid.

The polynucleotide (d') above is a polynucleotide comprising any of the polynucleotides (a') to (c') above as a partial sequence, and is preferably at least partially complementary to the antisense nucleic acid. , more preferably 100% complementary to the antisense nucleic acid. In one embodiment of the present invention, the length of the polynucleotide of (d') is 8-100 bases. Preferably, said length is at least 10 bases, at least 12 bases, at least 13 bases, at least 14 bases, at least 15 bases, or at least 16 bases. The length is preferably 100 bases or less, 45 bases or less, 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, or 20 bases or less. can.

In one embodiment of the present invention, the antisense nucleic acid or double-stranded nucleic acid complex comprises, for example, 1 to 10, preferably 1 to 6, such as 1, 2, 3, 4 or 5 may be included. When the antisense nucleic acid or double-stranded nucleic acid complex contains multiple ligands, the ligands may be the same or different from each other.

When the double-stranded nucleic acid complex has a ligand, for example, a ligand molecule may be bound to the complementary strand.

In the present invention, the term "ligand" means a substance capable of binding to a receptor, and typically substances capable of specifically binding to a receptor can be used. The receptor is not particularly limited as long as it is a substance capable of binding a ligand, and examples thereof include various receptors present on cell membranes. That is, as used herein, the terms "ligand" and "receptor" are used to mean a partner capable of binding to each other, preferably a partner capable of specifically binding to each other, and their binding elicits some biological reaction. is not limited to For example, a substance that can specifically bind to a receptor present on the cell membrane surface of target cells can be used as a ligand.

The ligands can be, for example but not limited to, proteins, peptides, aptamers, carbohydrates, lipids, small molecules, biomolecules/bioactive molecules.

"Protein" means a linear chain of α-L-amino acids (including glycine) linked by peptide bonds. As used herein, protein is a concept that includes both simple proteins consisting of only amino acids and complex proteins containing components other than amino acids, and refers to those consisting of 100 or more amino acid residues. Examples of proteins used as ligands (hereinafter also referred to as "protein ligands") include fatty acid binding proteins FABP1-12 (fatty acid binding protein 1-12), outer membrane-specific lipoprotein molecular chaperone LolA, outer membrane-specific lipoprotein receptor LolB, lipoxygenase and cyclooxygenase.

Proteins that can be used as ligands also include antibodies (hereinafter also referred to as "antibody ligands"). For example, by selecting an appropriate receptor and using an antibody that recognizes it as an antigen as a ligand, the antisense nucleic acid of the present invention can be used as a target cell or tissue in which the receptor exists (typically, the receptor can be specifically delivered to target cells or target tissues on which the drug is present.

Peptides can also be used as ligands. A "peptide" is a substance in which two or more amino acid molecules are linked by peptide bonds, and in this specification, refers to a substance in which less than 100 amino acid residues are linked. In the present invention, preferably 60 amino acids or less, such as 50 residues or less, 40 residues or less, 30 residues or less, 20 residues or less, or 10 residues or less, such as 9 residues or less, A peptide in which 8 residues or less, 7 residues or less, 6 residues or less, or 5 residues or less are linked is used as a ligand. Peptides used as ligands (hereinafter also referred to as "peptide ligands") are not particularly limited, but examples thereof include cyclic peptides such as cyclic RGD sequence-containing peptides, insulin, glucagon-like peptide-1, vasopressin, and oxytocin. is mentioned.

Here, the "cyclic RGD sequence-containing peptide" (hereinafter also referred to as "cRGD" or "cRGD peptide") is a peptide having at least one arginine-glycine-aspartic acid (RGD) sequence and forming a cyclic structure. be. The above "cyclic peptides" are preferably peptides with identical or similar ligand properties, particularly target specificities, to the cyclic RGD sequence-containing peptides. Although the sequence length of the cyclic peptide is not particularly limited, it is preferable that the number of amino acids is usually 3 or more, for example, 4-15 or 5-10, from the viewpoint of forming a ring structure.

The RGD sequence binds to and activates integrin molecules ₍ particularly _αVβ3 and _αVβ5 ) _, which are cell adhesion molecules on the cell surface, and induces cell-side endocytosis. is known, and the use of RGD peptides as tumor-targeting ligands is also known.

In the present invention, any cRGD peptide can be used as long as it has such an RGD sequence and forms a cyclic structure.

The sequence length of the cRGD peptide is not particularly limited, but from the viewpoint of forming a ring structure, it preferably has 3 or more amino acids, for example, 4-15 or 5-10 amino acids.

In addition, the types of amino acids that constitute portions other than the RGD sequence and their sequences are arbitrary. Therefore, the cRGD peptide may contain other types of natural amino acids and synthetic amino acids in addition to the 20 types of amino acids that constitute the body. However, amino acids and sequences thereof that do not exert undesired effects on subjects to which the antisense nucleic acid of the present invention is administered, do not substantially impair the activity of the complex, and do not interfere with the function of the RGD sequence are preferred.

As noted above, cRGD peptides have at least one RGD sequence. In one embodiment of the invention, the cRGD peptide has one RGD sequence.

Specific examples of cRGD peptides include, but are not limited to, peptides having the following amino acid sequences:
cRGDfK (Arg-Gly-Asp-D-Phe-Lys) (SEQ ID NO:24).

The cRGD peptide can be synthesized by a well-known automatic synthesizer, or a commercially available product (eg, “Cyclo(-RGDfK)” manufactured by Selleck) can be used.

Furthermore, aptamers can also be used as ligands (hereinafter also referred to as "aptamer ligands"). Aptamers are nucleic acid molecules that specifically bind to a specific molecule, typically by means other than hybridizing, usually by randomly altering the sequence of the nucleic acid to specifically bind to the target molecule. can be obtained by selecting those that do using methods known to those skilled in the art (for example, the SELEX method and the one-step selection method). Therefore, by using an aptamer that specifically binds to a target molecule as a ligand, the antisense nucleic acid of the present invention can be used as a target cell or tissue in which the target molecule is present (typically, the target molecule is present on the surface thereof). specific delivery to existing target cells or target tissues).

Sugar chains can also be used as ligands. Here, the term "sugar chain" refers to a molecule in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and derivatives thereof are bound in a chain by glycosidic bonds. Point. Sugar chains used as ligands (hereinafter also referred to as "sugar chain ligands") are not particularly limited, but examples thereof include N-acetylgalactosamine, hyaluronic acid, dextrin and cellulose.

Lipids can also be used as ligands. As used herein, "lipid" includes simple lipids, complex lipids and derived lipids. Lipids used as ligands (hereinafter also referred to as “lipid ligands”) are not particularly limited, but examples thereof include phospholipids and glycolipids that are complex lipids, and fatty acids and steroids that are derivative lipids. Examples of phospholipids are sphingophospholipids, examples of glycolipids are glycosphingolipids, examples of fatty acids are linear or branched _C4 - _C30 saturated or unsaturated fatty acids such as lauric acid, myristic Acids, palmitic acid, stearic acid, oleic acid, linolenic acid, docosanoic acid and examples of steroids include cholesterol. Vitamin E (tocopherol), which is a fat-soluble vitamin, is also an example of a lipid that can be used as a ligand. Tocopherols are for example selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol. Analogues of tocopherol can also be used in the present invention.

As used herein, the term "analog" refers to a compound having the same or similar basic skeleton and similar structures and properties as a given compound, especially compounds having the same or similar ligand properties. Analogs include, for example, biosynthetic intermediates, metabolites, compounds with substituents, and the like. One skilled in the art can determine whether a compound is an analog of another compound.
As used herein, "tocopherol analogs" include unsaturated analogs of tocopherol (e.g., tocotrienols such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol), pharmaceutical compounds of tocopherol or the unsaturated analogs described above. acceptable esters (eg, acetates, succinates, propionates), and pharmaceutically acceptable salts thereof (eg, sodium, potassium, calcium, magnesium salts). The above salts can be formed especially in the case of esters with dicarboxylic acids such as succinic acid. The tocopherol analogues described above may have the same or similar ligand properties, particularly target specificities, as tocopherol.

A low-molecular-weight compound can also be used as a ligand. Low-molecular-weight compounds that can be used as ligands (hereinafter also referred to as "low-molecular-weight ligands") are not particularly limited, but examples thereof include anisamide, tirofiban, and 2-pyrrolidin-1-yl-N-[ 4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide.

Biomolecules/bioactive molecules can also be used as ligands. Biomolecules/bioactive molecules that can be used as ligands (hereinafter also referred to as “biomolecule/bioactive molecule ligands”) are particularly limited as long as they are molecules that are relevant to living organisms or molecules that have activity that can affect living organisms. Examples include, but are not limited to, folic acid, anandamide, and spermine.

For example, "folic acid" is a vitamin that all cells require for DNA synthesis. Folate receptors have been reported to be overexpressed in many cancer cells, and folate is therefore widely used as a cancer targeting ligand. It has also been reported that complexes of folic acid and folate receptors are taken up into cells by endocytosis, and that many cancer cells take up a lot of folic acid.

In the present invention, such folic acid can also be used as the ligand, and folic acid can be easily obtained commercially (for example, "Folic acid" manufactured by Sigma-Aldrich). Analogues of folic acid can also be used in the present invention.

Here, as used herein, a "folic acid analogue" is a compound having a fused pyrimidine heterocycle attached to an amino acid moiety (preferably a glutamic acid moiety) via a benzoyl moiety, namely an amino acid moiety, a benzoyl moiety and a fused pyrimidine heterocycle. Refers to ring-based compounds (excluding folic acid itself). Examples of the above-mentioned "fused pyrimidine heterocycle" include pyrimidine further fused with a 5- or 6-membered heterocyclic ring such as pteridine or bicyclic pyrrolopyrimidine. Examples of folic acid or analogs thereof include optionally substituted folic acid, folinic acid, pteropolyglutamic acid based on the folic acid (pteroyl-glutamic acid) backbone, and folate receptor-binding pteridines such as tetrahydropterin, dihydrofolic acid. , tetrahydrofolic acid, deaza and dideaza analogues. The folic acid analogues described above may have the same or similar ligand properties, particularly target specificities, as folic acid.

In addition, those skilled in the art will also consider other biomolecules/bioactive molecule ligands, antisense nucleic acids of the present invention, while taking into consideration the information of each receptor, for example, the expression level and expression specificity in various tissues and cells. Appropriate biomolecules/bioactive molecule ligands can be selected as appropriate, depending on the desired target of .

In one preferred embodiment of the present invention, the ligand is selected from the group consisting of protein ligands, peptide ligands, aptamer ligands, carbohydrate ligands, lipid ligands, small molecule ligands and biomolecule/bioactive molecule ligands, more preferably selected from the group consisting of peptide ligands, carbohydrate ligands, lipid ligands, small molecule ligands and biomolecule/bioactive molecule ligands.

In a further preferred embodiment of the invention, the ligand is a cyclic peptide such as a cyclic arginine-glycine-aspartic acid (RGD) sequence-containing peptide, folic acid or analogues thereof, vitamin E (tocopherol) or analogues thereof, stearic acid, docosane selected from acids, anandamide, spermine, cholesterol, anisamide or N-acetylgalactosamine.

In another embodiment of the invention, the ligand is selected from peptide ligands, biomolecule/bioactive molecule ligands or lipid ligands, eg selected from cyclic RGD sequence-containing peptides, folic acid or vitamin E (tocopherol).

Preferably, the antisense nucleic acid or double-stranded nucleic acid complex of the present invention with a ligand as described above targets a cell or tissue having a receptor (e.g., on their surface) to which the ligand can specifically bind. It can be a cell or a target tissue. The types of target cells and target tissues are not particularly limited, and appropriate cells or tissues can be targeted depending on the purpose. For example, target cells may be cells that form a tissue or organ. Alternatively, the target cell may be a solitary cell, such as a leukemic cell, which is a tumor-forming cell in a tissue, such as a solid cancer cell, or invades a lymphoid tissue or other tissue. It may be a cell or the like (hereinbelow, these cells are also collectively referred to simply as "cancer cells").

In one embodiment of the invention, the ligand may be directly bound or indirectly bound via a linker. For example, the ligand may be at the 5′ or 3′ end of the polynucleotide and/or at other sites, such as within the central region defined below, having a number of m % relative to the total number of polynucleotides. (where m is an integer from 1 to 70, such as an integer from 2 to 60, an integer from 5 to 50, an integer from 10 to 40 is an integer of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 be able to). Here, when the polynucleotide consists of an odd number of structural units, the “central region” refers to one structural unit located in the center of the polynucleotide, and 5′ and 3 A region containing the same number of structural units in the ' direction, wherein the center of the polynucleotide is the same as the center of the region, and the polynucleotide consists of an even number of structural units. is a region containing two structural units located in the center of the polynucleotide and the same number of structural units in the 5′ direction and the 3′ direction from the central structural unit, and It means a region that is the same as the center of the region.

In one embodiment of the invention, the ligand is attached to the 5' end of the polynucleotide. In another embodiment of the present invention, when the polynucleotide has a length of 10-35 bases, the ligand is 1-35, such as 2-30, counted from the 5' end of the polynucleotide. th, 3rd to 25th, 4th to 20th, 5th to 15th, 6th to 11th (eg, 6th, 7th, 8th, 9th, 10th or 11th) structural unit, such as its 2′ or 4′ It is connected through the position.

Methods for attaching such ligands, optionally via linkers, to polynucleotides are well known to those skilled in the art. A person skilled in the art can achieve the above binding by appropriately selecting an appropriate known method depending _on the type of ligand and linker to be used. - A brief description of the method when the NHS ester is used to suspend the ligand from the 2' position of the nucleotide:

(a) Coupling of Ligand and Linker cRGDfK and Azido-PEG ₄ -NHS ester were coupled by a condensation reaction between the NHS ester and the amino group of the lysine of cRGDfK, and azido-PEG ₄ was linked to cRGDfK via an amide bond. to generate azide cRGDfK.

(b) Preparation of Polynucleotide Containing Alkyne-Modified Nucleotide A polynucleotide having alkyne-modified nucleotide as a structural unit at a position to which a ligand is to be bound (via a linker) is prepared. Methods for preparing such polynucleotides are known to those skilled in the art, and can be synthesized, for example, by using alkyne phosphoramidites in standard oligosynthesis. For the preparation of alkyne modified nucleotides, for example 2'-O-propargyladenosine, 2'-O-propargylcytidine, 2'-O-propargylguanosine, 2'-O-propargyluridine or 2'-O-propargyl Abasic ribose can be used.

(c) Click Reaction Azide-modified cRGDfK and alkyne-modified nucleotide are combined using an addition sensitization reaction (click reaction) with azide and alkyne.

As a result, PEG ₄ is attached via a triazole to the 2′ site of the nucleotide (or chemically modified nucleotide or nucleic acid analogue) at the desired position, and a polynucleotide having cRGDfK attached thereto by an amide bond, i.e., A polynucleotide is obtained in which the ligand is suspended from the 2' position of the nucleotide via the linker. Examples of such polynucleotides are shown in FIG.

As described above, when a ligand is bound to a complementary polynucleotide via a linker, the constituent unit responsible for binding in the polynucleotide may be chemically modified for binding to the linker. For example, by aminating, thiolating, alkylating or dibenzocyclooctylating the linker binding site of the above building blocks (deoxyribonucleotides, ribonucleotides or nucleic acid analogues), amine-reactive groups in the linker, thiol Covalent bonds can be formed between the building blocks and linkers through reaction with reactive groups, alkyne-reactive groups or dibenzocyclooctyne-reactive groups. Such chemically modified nucleotides are also referred to herein as "chemically modified nucleotides for binding to a linker", and such modifications constitute one aspect of the chemical modification described above. be. In addition, since the "nucleotide chemically modified for binding to the linker" forms a covalent bond with one end of the linker as a result of the reaction as exemplified above, it can have a different structure before and after the reaction. However, a nucleotide in a form that forms a covalent bond with a linker can also be referred to herein as a "nucleotide chemically modified for attachment to a linker." Similarly, in the case of directly binding the ligand to a polynucleotide, for example, chemical modifications and reactive groups as described above with respect to binding of the ligand via a linker are appropriately added to the polynucleotide constituting unit (nucleotide) to which the ligand is bound. or nucleic acid analogues) and/or ligands to form covalent bonds between the building blocks and the ligands using known reactions, such as those described above. In this case, the chemically modified nucleotides (deoxyribonucleotides or ribonucleotides) as described above are also referred to as "nucleotides chemically modified for binding to ligands."

In one embodiment of the present invention, the polynucleotide in the complementary strand is a nucleotide (deoxyribonucleotide or ribonucleotide) chemically modified for binding to the linker as a constituent unit to which a ligand is bound via a linker. , preferably at least one ribonucleotide chemically modified for attachment to a linker. A ligand can be attached to a ribonucleotide that has been chemically modified for attachment to this linker.

In another embodiment of the present invention, the polynucleotide in the complementary strand comprises as building blocks only chemically unmodified deoxyribonucleotides and chemically modified ribonucleotides for attachment to a linker. In a further embodiment of the present invention, the polynucleotide in the complementary strand consists only of deoxyribonucleotides that are not chemically modified, ribonucleotides that are not chemically modified, and ribonucleotides that are chemically modified for binding to a linker as constituent units. include. In these embodiments, ligands can be attached to ribonucleotides that have been chemically modified for attachment to this linker.

In one embodiment of the present invention, the polynucleotide in the complementary strand contains at least one mismatch to the polynucleotide in the antisense nucleic acid and is a nucleotide chemically modified for attachment to a linker as a building block ( preferably ribonucleotides). In a further embodiment of the invention, the polynucleotide in the complementary strand contains at least one mismatch to the polynucleotide in the antisense nucleic acid and is chemically modified for attachment to the linker (preferably ribonucleotides) as constituent units, and at least one of the nucleotides forms the mismatch, ie, in this embodiment, the constituent unit to which the ligand is bound forms the mismatch. For example, the mismatch can be a G to U mismatch (G for the antisense nucleic acid and U for the complementary strand).

In addition, when the complementary strand contains a plurality of ligands, these ligands may be bound to the same structural unit (for example, they are bound to the same position (eg, 2' position) of the same structural unit). may be used), or they may be bound to structural units different from each other. In the former case, multiple ligands are multimerized (eg 2-10-mers, preferably 2-6-mers, eg 2-, 3-, 4-, 5- or 6-mers) by using suitable branched linkers, for example. may be bound to the same structural unit as In the present application, it was also found that in the nucleic acid complex of the present invention, good activity is achieved even when multiple ligands are bound to the same structural unit as multimers.

Various branched linkers are known, and a person skilled in the art can appropriately select an appropriate branched linker according to the ligand to be used.

In the present invention, for example, a linker that connects a polynucleotide and two cRGDfKs as shown below can be used as a branched linker. In this case, two cRGDfKs can be bound to the same building block nucleotide in the form of a dimer.

また、例えば、４分岐型の分岐リンカーを使用して４つのｃＲＧＤｆＫとポリヌクレオチドとを連結することにより、以下に示すように、４つのｃＲＧＤｆＫを４量体の形態で、同一の構成単位ヌクレオチドに結合させることができる。

Alternatively, for example, by linking the four cRGDfKs to the polynucleotide using a 4-branched branched linker, the four cRGDfKs are formed in the form of a tetramer, as shown below, in the same building block nucleotide. can be combined.

In one embodiment, the antisense nucleic acid consists solely of polynucleotides. In another embodiment of the invention, the antisense nucleic acid consists only of a polynucleotide and a ligand. In a further embodiment of the invention said antisense nucleic acid consists only of a polynucleotide, a ligand and a linker.
In another embodiment of the invention, the double-stranded nucleic acid complex consists only of the antisense nucleic acid and the complementary strand. In a further embodiment of the invention the double-stranded nucleic acid complex consists only of the antisense nucleic acid, the complementary strand and the ligand. In yet another embodiment of the invention, the double-stranded nucleic acid complex consists only of the antisense nucleic acid, complementary strand, ligand and linker.

Preferred typical examples of antisense nucleic acids or double-stranded nucleic acid complexes have been described above in some embodiments, and antisense nucleic acids or double-stranded nucleic acid complexes in some embodiments are It is not limited. In some embodiments, the polynucleotide of the antisense nucleic acid and/or the polynucleotide of the complementary nucleic acid strand can be prepared by appropriately selecting methods known to those skilled in the art. For example, based on the information of the nucleotide sequence of the target transcript (typically the nucleotide sequence of the target gene), the nucleotide sequence of the nucleic acid is designed, and a commercially available automatic nucleic acid synthesizer (Applied Biosystems, Beckman) etc.), and then purifying the resulting polynucleotide using a reversed-phase column or the like, a nucleic acid can be prepared. Then, the nucleic acid thus prepared is mixed in a suitable buffer solution and denatured at about 90-98° C. for several minutes (eg, 5 minutes). The antisense nucleic acid or double-stranded nucleic acid complexes in some embodiments can be prepared by annealing for about 1-8 hours at 0°C. In the double-stranded nucleic acid complex, when a ligand is bound to the polynucleotide of the complementary strand, the double-stranded nucleic acid complex is prepared, for example, by using a polynucleotide bound with a ligand in advance, It can be prepared by annealing an antisense nucleic acid to a polynucleotide as described above.

As described above, when an oligonucleotide composed of DNA is introduced into a cell as an antisense nucleic acid (ASO), the transcription product (mRNA) of the target gene binds to the ASO to partially form a double strand. , this double strand is known to act as a lid to prevent translation by the ribosome and inhibit the expression of the protein encoded by the target gene.

In addition, when an oligonucleotide composed of DNA is introduced into cells as an ASO, a DNA-RNA heterooligonucleotide is partially formed, this portion is recognized by RNaseH, and the mRNA of the target gene is degraded. is also known to inhibit the expression of proteins encoded by It has also been clarified that, in many cases, this RNaseH-dependent pathway is more effective in suppressing gene expression than when ASO acts as a lid.

In relation to the antisense effect when DNA is used as an ASO, Japanese Patent Application Publication No. 2015-502134 discloses a double-stranded antisense nucleic acid obtained by annealing an LNA/DNA gapmer and a complementary strand consisting of an RNA to the LNA/DNA gapmer. disclosed. Here, the antisense effect of the double-stranded nucleic acid is described. A double-stranded nucleic acid as in the literature is called an HDO (heteroduplex oligonucleotide).

When an oligonucleotide consisting of modified DNA that is complementary to a specific region of the pre-mRNA and is not a substrate for RNase H is introduced into cells as an ASO, a DNA-RNA heterooligonucleotide is partially formed, skipping exons of the target gene. (exon skipping method) is known.

<Composition for Suppressing Target Gene Expression or Target Transcript>
The antisense nucleic acid or double-stranded nucleic acid complex of the present invention is efficiently incorporated into target cells with high specificity, suppresses the expression of target genes or the level of target transcripts in the cells, and inhibits the proliferation of the cells. It can be suppressed very effectively. Therefore, a composition containing the antisense nucleic acid or double-stranded nucleic acid complex as an active ingredient, for example, for suppressing the expression of a target gene and/or the proliferation of target cells (eg, cancer cells) by the antisense effect , the present invention can provide. The antisense nucleic acid or double-stranded nucleic acid complex of the present invention can be delivered to a target tissue or target site with excellent specificity and efficiency, and can suppress the proliferation of target cells with high specificity and efficiency. High drug efficacy can be obtained by administration of a high concentration. In addition, the antisense nucleic acid or double-stranded nucleic acid complex of the invention particularly has the advantage of less side effects. Therefore, it is possible to provide pharmaceutical compositions for treating and preventing diseases associated with increased expression of target genes, such as metabolic diseases, tumors, and infectious diseases.

Therefore, in one embodiment of the present invention, the present invention relates to the above antisense nucleic acid or double-stranded nucleic acid complex for reducing the expression of a target gene in a mammal, preferably in a target tissue/target site. .

In a further embodiment of the present invention, the present invention provides the antisense nucleic acid or It relates to a double-stranded nucleic acid complex. The antisense nucleic acid or double-stranded nucleic acid complex can be an antisense nucleic acid or double-stranded nucleic acid complex for inhibiting cancer cell growth, or an antisense nucleic acid for cancer treatment and/or prevention. It can be a nucleic acid or a double-stranded nucleic acid complex.

Furthermore, in one embodiment of the present invention, the present invention relates to a pharmaceutical composition comprising the above antisense nucleic acid or double-stranded nucleic acid complex and optionally a pharmacologically acceptable carrier.

Moreover, in another embodiment of the present invention, the present invention relates to the use of the above antisense nucleic acid or double-stranded nucleic acid complex for producing the above pharmaceutical composition.

In one embodiment of the present invention, the present invention relates to a method for treating or preventing a disease associated with increased gene expression, comprising the step of administering the above antisense nucleic acid or double-stranded nucleic acid complex to a mammal.
In yet another embodiment, the present invention relates to a method of treating cancer in a mammal, comprising administering the antisense nucleic acid or double-stranded nucleic acid complex described above to the mammal.

The pharmaceutical composition can be a pharmaceutical composition for suppressing the growth of cancer cells, or the pharmaceutical composition can be a pharmaceutical composition for treating and/or preventing cancer.

Cancers of said cancer cells and said cancers are, for example, brain tumors; squamous cell carcinoma of the head, neck, lung, uterus or esophagus; melanoma; adenocarcinoma of the lung or uterus; renal cancer; carcinoma; gingival sarcoma; intraoral mass; perianal adenocarcinoma; anal cyst mass; anal sac apocrine adenocarcinoma; thyroid cancer; colorectal cancer; bronchial adenocarcinoma; adenocarcinoma; ductal carcinoma; breast adenocarcinoma; chondrosarcoma; soft tissue sarcoma; histiocytic sarcoma; myxosarcoma; undifferentiated sarcoma; lung cancer; Leukemia; lymphoma; gastrointestinal lymphoma; gastrointestinal lymphoma; small-to-medium cell lymphoma; adrenal medullary tumor; glioma; glioblastoma; astrocytoma; glioblastoma multiforme; inflammatory breast cancer; Wilms tumor; cancer; giant cell tumor of bone; thyroid cancer; lymphoblastic T-cell leukemia; chronic myeloid leukemia; chronic lymphocytic leukemia; chronic neutrophilic leukemia; acute lymphoblastic T-cell leukemia; plasmacytoma; immunoblastic large cell leukemia; mantle cell leukemia; multiple myeloma; Hodgkin's Lymphoma; Non-Hodgkin's Lymphoma; Lymphoblastic T-cell Lymphoma; Burkitt's Lymphoma; Follicular Lymphoma; Cancer; Endometrial cancer; Mesothelioma; Esophageal cancer; Salivary gland cancer; Hepatocellular carcinoma; It is selected from the group consisting of epithelial adenocarcinoma and testicular cancer.

In a further embodiment of the invention, the invention relates to a method of reducing transcript levels in a cell comprising contacting said antisense nucleic acid or double-stranded nucleic acid complex with said cell.

In another embodiment of the present invention, the present invention relates to a method for suppressing proliferation of cells, preferably cancer cells, comprising the step of contacting the above antisense nucleic acid or double-stranded nucleic acid complex with said cells.

In another embodiment of the present invention, the present invention relates to the use of the above antisense nucleic acid or double-stranded nucleic acid complex for reducing expression of a target gene in a mammal, preferably in a target tissue/target site. .

In another embodiment of the present invention, the present invention provides the above antisense nucleic acid or double-stranded nucleic acid for suppressing proliferation of target cells, preferably cancer cells, preferably in target tissues/target sites in mammals. Concerning the use of complexes.

The present invention further relates to the use of the above antisense nucleic acid or double-stranded nucleic acid complex for manufacturing a drug for reducing target gene expression in a mammal, preferably in a target tissue/target site. The present invention also provides the above antisense nucleic acid or double-stranded nucleic acid complex for producing a drug for suppressing the growth of target cells, preferably cancer cells, preferably in target tissues/target sites in mammals. regarding the use of

In a further embodiment, the present invention reduces the expression level of a target gene in a mammal, preferably in a target tissue/target site, comprising the step of administering the antisense nucleic acid or double-stranded nucleic acid complex to the mammal. on how to. In another embodiment of the present invention, the present invention provides a target cell, preferably in a target tissue/target site, in a mammal, comprising the step of administering the antisense nucleic acid or double-stranded nucleic acid complex to the mammal. Preferably, it relates to a method for suppressing proliferation of cancer cells.

In these aspects, preferably the polynucleotide of the antisense nucleic acid is the antisense strand complementary to the region of any of the above transcripts.

In another embodiment, the present invention can be utilized using various drug delivery systems (DDS) as a means of delivering antisense nucleic acids. DDS complexes include surfactant peptide (Med. Mol. Morphol., 46, pp.86, 2013), polysaccharide schizophyllan (J. Control. Release, 151, pp.155, 2011), cyclodextrin (Mol. Pharm., 12, pp.3129, 2015), cationic liposomes (J. Control. Release., 100, pp.165, 2004), nucleic acid micelle particles (Mol. Pharm., 11, pp.904, 2014), Atelocollagen (Proc. Natl. Acad. Sci. USA, 102, pp. 12179, 2005) and the like.

In one embodiment, the transcript is a protein-encoding mRNA transcript. Preferably, said protein is human BCL2, human BCR-ABL or human STAT3. In other embodiments, the transcript is a non-protein-encoding transcript.

Also, in one embodiment of the invention, the target gene is human bcl-2, human BCR-ABL or human STAT3.

In one preferred embodiment, the mammal is human.

The above compositions and drugs containing antisense nucleic acids or double-stranded nucleic acid complexes can be formulated into desired administration forms and dosage forms by known pharmaceutical methods. For example, capsules, tablets, pills, liquids, powders, granules, fine granules, film coating agents, pellets, lozenges, sublingual agents, chewing agents, buccal agents, pastes, syrups, suspensions, Enteral (oral, etc.) or It can be used enterally.

In these formulations, pharmacologically acceptable carriers or foods and beverages, specifically sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, pH adjusters , stabilizers, flavoring agents, fragrances, excipients, vehicles, preservatives, binders, diluents, tonicity agents, soothing agents, bulking agents, disintegrants, buffers, coating agents, lubricants, Coloring agents, sweetening agents, thickening agents, flavoring agents, solubilizing agents, other additives, and the like can be appropriately combined.

Preferred administration forms of the antisense nucleic acid or double-stranded nucleic acid complex, composition, or drug are not particularly limited, and can be enteral (oral, etc.) or parenteral, more specifically, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration and intramuscular administration, and administration by infusion.

In each embodiment, the antisense nucleic acid or double-stranded nucleic acid complex or composition can be used for animals including humans, that is, for humans and/or non-human animals. Animals other than humans are not particularly limited, and various livestock, poultry, pets, experimental animals, and the like can be used.

When administering or ingesting an antisense nucleic acid (or double-stranded nucleic acid complex) or composition in some embodiments, the dosage or ingestion is determined according to the subject's age, weight, symptoms, health condition, composition Depending on the type (medicine, food and drink, etc.), etc., the effective intake of the antisense nucleic acid or composition according to an embodiment is 0.001 mg/kg/day to 50 mg/kg in terms of nucleotides. / day.

As shown in Examples below, the double-stranded nucleic acid complex of the present invention, which targets the oncogene bcl-2 (or BCR-ABL or STAT3), can proliferate target cells with very high efficiency. can be suppressed. In addition, the double-stranded antisense nucleic acid of the present invention can be delivered to target tissues and target sites with excellent specificity and efficiency. Furthermore, the double-stranded nucleic acid complex of the present invention has extremely few side effects. Accordingly, the present invention provides a method of adding or administering the antisense nucleic acid or double-stranded nucleic acid complex of the present invention to a subject to suppress the expression of a target gene or target transcript by the antisense effect. can be done. In addition, the present invention provides that administration of the antisense nucleic acid or double-stranded nucleic acid complex of the present invention to a subject results in tissue- or site-specific cell proliferation accompanied by, for example, target gene expression enhancement. It is also possible to provide a method for suppressing and thereby treating and preventing various diseases associated with such increased expression.

Hereinafter, the present invention will be described more specifically based on examples, but the embodiments are not limited to the following examples.

Example 1
[Synthesis of double-stranded nucleic acid]
Based on the sequence of the human BCL2 gene, a double-stranded nucleic acid having a PS-PO type antisense nucleic acid targeting the gene as a basic skeleton was designed. Specific examples of double-stranded nucleic acids for the BCL2 gene are shown in SEQ ID NOS: 1-6 for antisense strand nucleotide sequences (16mer) and SEQ ID NOS: 7-16 for complementary strand nucleotide sequences (Table 1). A double-stranded nucleic acid is formed by annealing the antisense strand and each complementary strand in each gene sequence. The double-stranded nucleic acids formed by SEQ ID NOs: 1-6 and 7-12, respectively, are designated as BNSP-1-36 (see Table 2). Further, the double-stranded nucleic acid formed from SEQ ID NOS: 1 and 14 is BNSP-37, the double-stranded nucleic acid formed from SEQ ID NOS: 2 and 13 is BNSP-38, and the double-stranded nucleic acid formed from SEQ ID NOS: 2 and 14 BNSP-39 for the nucleic acid, BNSP-40 for the double-stranded nucleic acid formed from SEQ ID NOS:2 and 15, BNSP-41 for the double-stranded nucleic acid formed from SEQ ID NOS:2 and 16, and BNSP-41 for the double-stranded nucleic acid formed from SEQ ID NOS:17 and 10 This double-stranded nucleic acid is referred to as BNSP-42. In addition, at the 5′ ends of SEQ ID NOs: 13 to 16,

5'-Octyl-tochopherol (manufactured by Link Technology) indicated by is bound. Among these double-stranded nucleic acids, SEQ ID NO: 17 was synthesized using a nucleic acid synthesizer (model number: M-8-MX_A8S) manufactured by Nippon Techno Service Co., Ltd., and others were synthesized by Gene Design Co., Ltd. Commissioned and synthesized. Nucleotides underlined as sequence notation are RNA;

represents LNA, otherwise represents DNA. In addition, ps indicates a thiophosphate bond, po indicates a phosphodiester bond, and N _(m) indicates a 2′-OMe modified nucleotide as the binding mode of nucleotides.

Example 2
[Cell culture]
Human cancer-derived cell lines used in the experiments described later were cell lines maintained by the following method.

Human epithelial-like cell carcinoma-derived cell line (A431 cell line: purchased from JCRB Cell Bank, cell number: JCRB0004), human liver cancer-derived cell line (HepG2 cell line: purchased from Riken Cell Bank, cell number: RBRC-RCB1886) and human pancreas Cancer-derived cell line (HPAC cell line: purchased from ATCC cell bank, cell number: CRL-2119) was grown in growth medium (DMEM: GIBCO), human pancreatic cancer-derived cell line (AsPC-1 cell line: purchased from ATCC cell bank, cell number: CRL-1682), human gastric cancer-derived cell line (MKN45 cell line: purchased from JCRB cell bank, Cell number: JCRB0254), human pancreatic cancer-derived cell line (PANC-1 cell line: purchased from Riken Cell Bank, cell number: RBRC-RCB2095), human prostate cancer-derived cell line (DU145 cell line: purchased from Riken Cell Bank, cell number : RBRC-RCB2143), human prostate cancer-derived cell line (PC-3 cell line: purchased from Riken Cell Bank, cell number: RBRC-RCB2145), and human ovarian cancer-derived cell line (OVCAR-3 cell line: purchased from Riken Cell Bank , cell number: RBRC-RCB2135) is human alveolar basal epithelial adenocarcinoma using a growth medium (RPMI1640: manufactured by GIBCO) supplemented with 10 mass% fetal bovine serum, 100 units/ml penicillin and 100 μg streptomycin. Derived cell line (A549 cell line: purchased from JCRB cell bank, cell number: JCRB0076), human kidney cancer-derived cell line (Caki-1 cell line: purchased from JCRB cell bank, cell number: JCRB0801), human gastric cancer-derived cell line (HGC-27 cell line: purchased from Riken Cell Bank, cell number: RBRC-RCB0500) and human bladder cancer-derived cell line (T24 cell line: purchased from Riken Cell Bank, cell number: RBRC-RCB2536) Fetal bovine serum, non-essential amino acids for MEM (NEAA), 100 units / ml of penicillin and 100 μg of streptomycin using a growth medium (MEM: GIBCO), human ovarian cancer-derived cell line (MCAS cell line: JCRB cell Purchased from Bank, cell number: JCRB0240) contains 20% by mass fetal bovine serum, 100 units/ml penicillin and 100 μg streptomycin. Human mammary adenocarcinoma-derived cell line (MCF-7 cell line: purchased from JCRB cell bank, cell number: JCRB0134) was added with growth medium (MEM: GIBCO), 10 mass% fetal bovine serum, 1 mM pyruvate Using a growth medium (MEM: GIBCO) supplemented with sodium, 10 μg/ml insulin, non-essential amino acids for MEM (NEAA), 100 units/ml penicillin and 100 μg streptomycin, human colon cancer-derived cell line (HCT116 cell line : purchased from ATCC cell bank, cell number: CCL-247), using a growth medium (McCoy's 5a: GIBCO) supplemented with 10 mass% fetal bovine serum, 100 units/ml penicillin and 100 μg streptomycin , 37° C., 5 wt % CO ₂ conditions.

Example 3
[Cell Proliferation Assay]
Unless otherwise specified, the cell proliferation assays performed in the experiments described later were performed by the following method.

Cells were cultured in 100 μl/well medium under humidified conditions (37° C., 5% CO ₂ ) using 96-well microplates. In the cell proliferation assay, 10 μl of cell proliferation reagent WST-1 (manufactured by Roche) was added per well, and absorbance was measured using a microplate reader (manufactured by Bio Rad).

Example 4
[Quantitative RT-PCR]
Quantitative RT-PCR performed in the experiments described later was performed by the following method unless otherwise specified. Total RNA was extracted from cultured cells using Rneasy Mini Kit (QIAGEN). Quantitative RT-PCR using the above total RNA was performed using Quant-Fast Probe RT-PCR Kit (manufactured by QIAGEN) under recommended conditions. TaqMan Gene Expression Assays Probes manufactured by Applied Biosystems were used as primers. β-Actin manufactured by the same company was used as an endogenous control primer. Amplification of the quantitative RT-PCR was performed using ROTOR-Gene Q (manufactured by QIAGEN). The mRNA expression level was calculated by the Delta Delta CT method.

example 5
[Test of cell growth inhibitory activity of double-stranded nucleic acid targeting human BCL2 gene]
In order to examine the in vitro cell growth inhibitory activity of the double-stranded nucleic acid targeting the human BCL2 gene, the following experiment was performed. First, double-stranded nucleic acids BNPS-1 to 36 (36 in total) shown in Table 2 were prepared. A total of 16 cell lines shown in Example 2 above were then transfected with 3 nM of double-stranded nucleic acid using Lipofectamine 2000 (Invitrogen). The transfected cell lines were cultured for 72 hours after transfection and the cytostatic activity of the double-stranded nucleic acid was measured by the cell proliferation assay described in Example 3 above.

Also, a similar cell proliferation assay was performed using a double-stranded nucleic acid against a non-target gene instead of the above double-stranded nucleic acid.

The results are shown in Figure 4 for A549 cell line, Figure 5 for A431 cell line, Figure 6 for AsPC-1 cell line, Figure 7 for HPAC cell line, Figure 8 for HepG2 cell line, Figure 9 for MCF-7 cell line, and Figure 9 for OVCAR. -3 cell line in Figure 10, MCAS cell line in Figure 11, Caki-1 cell line in Figure 12, DU145 cell line in Figure 13, PC-3 cell line in Figure 14, T24 cell line in Figure 15, HCT116 cell line 16, HGC-27 cell line in FIG. 17, MKN45 cell line in FIG. 18, and PANC-1 cell line in FIG. As can be seen from FIGS. 4 to 18, the double-stranded nucleic acids of BNSP-7 to BNSP-12, in which complementary strands of various structures are annealed to the antisense strand with 6 fewer PS bonds, all the antisense strands are based on PS bonds. By targeting the skeleton BNSP-1 to 6 and the human BCL2 gene, it showed similar growth inhibitory activity against various cancer cell lines. In addition, as can be seen from FIG. 19, antisense strands having a structure in which PS bonds and PO bonds are completely alternated target BNSP-1 and 4, all of which have PS bonds as a backbone, and the human BCL2 gene. showed equivalent antiproliferative activity against the PANC-1 cell line.

Example 6
[Test of mRNA expression suppression activity of double-stranded nucleic acid targeting human BCL2 gene]
In order to examine the in vitro mRNA expression-suppressing activity of the double-stranded nucleic acid targeting the human BCL2 gene, the following experiment was performed. Double-stranded nucleic acids BNSP-4, BNSP-10, BNSP-16, BNSP-22, BNSP-28 and BNSP-34 were prepared by annealing the antisense sequence numbers 1 to 6 and the complementary strand sequence number 10. OVCAR-3 and A431 cell lines were then transfected with 10 nM of the double-stranded nucleic acid using Lipofectamine 2000 (Invitrogen). The transfected cell line was cultured for 24 hours after transfection, and the mRNA expression-suppressing activity of the double-stranded nucleic acid was quantified by the quantitative RT-PCR method described in Example 4 above.

In addition, instead of the double-stranded nucleic acid described above, a double-stranded nucleic acid against a non-target gene was used to quantify similar mRNA expression-suppressing activity.

The results are shown in FIG. 20 for the OVCAR-3 cell line and in FIG. 21 for the A431 cell line. As can be seen from FIG. 20, by targeting the human BCL2 gene, BNSP-10 exhibits an mRNA expression-suppressing activity equivalent to that of BNSP-4, in which all antisense strands have a PS bond as a basic skeleton, against various cancer cell lines. rice field. Moreover, as can be seen from FIGS. 20 and 21, BNSP-10 exhibited stronger mRNA expression suppression activity than other BNSP-16, BNSP-22, BNSP-28 and BNSP-34.

Example 7
[Verification of expression of hepatotoxicity and efficacy evaluation in pancreatic cancer orthotopic transplantation mouse model of PS-PO type double-stranded nucleic acid]
The following experiment was performed in order to compare the PS-PO type double-stranded nucleic acid and the double-stranded nucleic acid in which all the antisense strands have a PS bond as a basic skeleton and evaluate whether there is a difference in the two items described later. rice field.
1. Verification of expression of hepatotoxicity 2 . Proliferation inhibitory effect on pancreatic cancer sites

BALB/cA Jcl-nu/nu mice (6-week-old: male, purchased from Clea Japan, Inc.) acclimated for several days in a breeding environment were treated with a triple anesthetic (0.3 mg/kg of domitol + 4 mg/kg of dormicum + 5 mg/kg of betorfal). kg) was administered intraperitoneally at a dose of 80 μl per 10 g of mouse body weight to give general anesthesia, and then 1×10 ⁶ human cancer cell lines per animal were injected into the pancreas to prepare pancreatic orthotopic transplantation model mice ( A mouse injected with PBS was also prepared as a sham). In addition, the body weight was measured every day (afternoon) from the day after the transplantation, and from the humane endpoint, the mouse fell into a serious state of rapid weight loss (more than 20% in several days), inability to walk on its own, and inability to eat or drink water. was to be euthanized. Four days after cancer cell transplantation (day 4), the animals were divided into groups (6 groups x 6 animals) using body weight as an index, and 3 mg/kg of Saline and BNSP-37 to 41 were administered through the tail vein. In addition, Saline was administered to the Sham group. The administration period and frequency were once a day (days 4, 6, 8, 12, 15, 18, 20, 22, 25), 9 times in total. The progress was observed by weight measurement and the like, and 26 days after cancer cell transplantation, venous blood was collected from an abdominal vein under anesthesia, and then the liver and pancreas were excised. Blood biochemical tests for hepatotoxicity were entrusted to LSI Medience Co., Ltd., and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were quantified. In addition, in order to evaluate liver hypertrophy, the weight of the excised liver was measured, and the liver weight ratio was calculated by correcting with the body weight of the mouse. The results are shown in FIG. 22 for AST values, FIG. 23 for ALT values, and FIG. 24 for liver weight ratios. Furthermore, the anti-tumor effects of BNSP-37-41 were evaluated by comparing pancreas weight ratios. The results are shown in FIG. As can be seen from FIGS. 22 to 24, in the double-stranded nucleic acid (BNSP-37) in which the antisense strands all have PS bonds as a basic skeleton, AST, ALT, and all liver weight ratios manifest hepatotoxicity more clearly than in the saline-administered group. However, BNSP-38 to 41, which reduced 6 PS bonds of the antisense strand, were comparable to the Saline-administered group, and statistically significantly reduced hepatotoxicity markers and liver hypertrophy compared to BNSP-37. was found to suppress the Furthermore, as can be seen from FIG. 25, in the antitumor effect, BNSP-39 exhibits a statistically significant cancer growth inhibitory effect comparable to that of BNSP-37, making it possible to significantly reduce hepatotoxicity while maintaining drug efficacy. structure was found.

Example 8
[Synthesis of double-stranded nucleic acid]
Based on the sequences of the human BCL2, BCR-ABL and STAT3 genes, a double-stranded nucleic acid was designed having a DNA-DNA skeleton targeting the genes and containing a base having a ligand-binding site in the sugar moiety. As a specific example of the double-stranded nucleic acid for each target gene, SEQ ID NO: 18 shows the complementary strand nucleotide sequence (16mer) for the BCL2 gene. Also, the antisense strand nucleotide sequence (20mer) for the BCR-ABL gene is shown in SEQ ID NO: 19, and the complementary strand nucleotide sequences are shown in SEQ ID NOS: 20 and 21. Furthermore, the antisense strand nucleotide sequence (20mer) for the STAT3 gene is shown in SEQ ID NO:22, and the complementary strand nucleotide sequence is shown in SEQ ID NO:23. A double-stranded nucleic acid is formed by annealing the antisense strand and each complementary strand in each gene sequence. The double-stranded nucleic acid formed from SEQ ID NOS: 2 and 18 targeting the human BCL2 gene is BNSP-43, and the double-stranded nucleic acid formed from SEQ ID NOS: 19 and 20 targeting the human BCR-ABL gene is BNSP- 44, the double-stranded nucleic acid formed from SEQ ID NOs: 19 and 21 is called BNSP-45, and the double-stranded nucleic acid formed from SEQ ID NOs: 22 and 23 targeting the human STAT3 gene is called BNSP-46. Nucleic acids were synthesized in-house using an automatic DNA/RNA synthesizer (NTSH-8: manufactured by Nippon Techno Service Co., Ltd.) according to a conventional method. The formation of double-stranded nucleic acids by annealing the antisense strand and their respective complementary strands was also performed in-house.
Nucleotides underlined as sequence notation are RNA;

はＬＮＡ、それ以外はＤＮＡを表す。また、ヌクレオチドの結合様式としてｐｓはチオリン酸結合、ｐｏはホスホジエステル結合を示し、Ｎ_１は２’－Ｏ－Ｐｒｏｐａｒｇｙｌ Ｃｙｔｉｄｉｎｅ、Ｎ_２は２’－Ｏ－Ｐｒｏｐａｒｇｙｌ Ｕｒｉｄｉｎｅを表す。

represents LNA, otherwise represents DNA. In addition, ps indicates a thiophosphate bond, po indicates a phosphodiester bond, N ₁ indicates 2′-O-Propargyl Cytidine, and N ₂ indicates 2′-O-Propargyl Uridine, as binding modes of nucleotides.

Example 9
[Addition of RGD to sugar moiety 2′ site]
Of the above complementary strands, addition of cRGD to the BNSP-43 complementary strand (SEQ ID NO: 18) having a Propargyl group was carried out as follows.
(1) Azido-PEG4-NHS ester was bound to the N-terminus of a peptide of cRGDfK (Monomeric RGD: Cyclo(-RGDfK) Selleck S7834) by a condensation reaction to prepare an azide peptide.
(2) Using the BNSP-43 complementary strand (SEQ ID NO: 18) and the azide peptide as starting materials, a click reaction was performed in the presence of a Cu(II) complex in a 30% DMSO solution. In a 30% DMSO solution, 5 equivalents of peptide-N3 and 5 equivalents of _{CuSO4.5H2O were} added and reacted at 40°C for 16 hours.
(3) Excess Cu due to the click reaction may be attached to the phosphodiester, base portion, or triazole of the oligo-DNA, and the cation exchange resin (Ag ^{(registered trademark)} MP-50 (manufactured by Bio-Rad) was substituted for Na+ by.
(4) BNSP-43-cRGD is a double-stranded nucleic acid obtained by annealing the antisense strand (SEQ ID NO: 2) and the cRGD-added BNSP-43 complementary strand (SEQ ID NO: 18).
(5) Furthermore, in the case of a double-stranded nucleic acid to which a multimeric cRGD is added, it is called by adding "DicRGD" (dimer) or "TEcRGD" (tetramer) to the name of the double-stranded nucleic acid ( For example, "BNSP-43-DEcRGD", "BNSP-43-TEcRGD").

example 10
[Addition of folic acid]
Folic acid was added to the BNSP-45 complementary strand (SEQ ID NO: 21) having a Propargyl group by click reaction with PEG3-Folate (manufactured by base click: BCFA-111-1). A double-stranded nucleic acid obtained by annealing the antisense strand (SEQ ID NO: 19) and the BNSP-45 complementary strand (SEQ ID NO: 21) added with PEG3-Folate is designated as BNSP-45-PEG-3-Folate. Furthermore, a double-stranded nucleic acid obtained by annealing the antisense strand (SEQ ID NO: 19) and the in-house synthesized PEG11-Folate and PEG23-Folate-added BNSP-45 complementary strand (SEQ ID NO: 21) to BNSP-45-PEG- 11-Folate and BNSP-45-PEG-23-Folate.

Example 11
[Cell culture]
Human cancer-derived cell lines used in the experiments described later were cell lines maintained by the following method.
Human alveolar basal epithelial adenocarcinoma-derived cell line (A549 cell line: purchased from JCRB cell bank, cell number: JCRB0076) was prepared with 10 mass% fetal bovine serum, non-essential amino acids for MEM (NEAA), 100 units/ml Growth medium supplemented with penicillin and 100 μg of streptomycin (MEM: GIBCO), human pancreatic cancer-derived cell line (PANC-1 cell line: purchased from Riken Cell Bank, cell number: RBRC-RCB2095), human chronic myelogenous leukemia cell line (K562 cell line: purchased from JCRB cell bank, cell number: JCRB0019), human pancreatic cancer-derived cell line (AsPC-1 cell line: purchased from ATCC cell bank, cell number: CRL-1682) is 10% by mass of bovine Using a growth medium supplemented with fetal serum, 100 units/ml penicillin and 100 μg streptomycin (RPMI1640: manufactured by GIBCO), human peripheral blood mononuclear cells (PBMC: purchased from Kurabo Industries, product number: HC-0001) were , 5% by mass of fetal bovine serum and 10 μg/mL of DNase I (LGM-3 lymphocyte: manufactured by Lonza) was used.

Example 12
[Test of mRNA expression suppression activity of double-stranded nucleic acid targeting human BCL2 gene]
In order to examine the in vitro mRNA expression-suppressing activity of the double-stranded nucleic acid targeting the human BCL2 gene, the following experiment was conducted. Double-stranded nucleic acids BNSP-1, BNSP-4, BNSP-7, BNSP-10 and antisense strand (SEQ ID NO: 17) and complementary strand (SEQ ID NO: 10) having a structure in which PS and PO bonds are completely alternated was prepared by annealing BNSP-42. Then, using Lipofectamine 2000 (Invitrogen), PANC-1 cell line, AsPC-1 cell line and A549 cell line were transfected with the double-stranded nucleic acid at 10 nM. The transfected cell line was cultured for 24 hours after transfection, and the mRNA expression-suppressing activity of the double-stranded nucleic acid was quantified by the quantitative RT-PCR method described in Example 4 above.

In addition, instead of the double-stranded nucleic acid described above, a double-stranded nucleic acid against a non-target gene was used to quantify similar mRNA expression-suppressing activity.

The results are shown in FIG. 26 for the PANC-1 cell line, FIG. 27 for the ASPC-1 cell line, and FIG. 28 for the A549 cell line. As can be seen from FIG. 26, antisense strands with a structure in which PS bonds and PO bonds are completely alternated target BNSP-1, 4 and human BCL2 genes, all of which have PS bonds as a backbone, resulting in PANC- Equivalent mRNA expression-suppressing activity was shown for 1 cell line. As can be seen from Figures 27 and 28, BNSP-7 and BNSP-10 showed mRNA expression-suppressing activity by targeting the human BCL2 gene.

Example 13
[Test of mRNA expression-suppressing activity of double-stranded nucleic acid targeting human BCL2 gene with cRGD bound to the center of complementary strand]
In order to examine the in vitro mRNA expression-suppressing activity of the above-described cRGD-bound double-stranded nucleic acid targeting the human BCL2 gene, the following experiment was performed. Prepare double-stranded nucleic acids BNSP-43-cRGD, BNSP-43-DicRGD, BNSP-43-TEcRGD, and BNSP-10 in which monomer, dimer, and tetramer cRGD are bound to the center of the complementary strand 3 μM to the PANC-1 cell line and 300 nM to the K562 cell line. After addition of the double-stranded nucleic acid, the cells were cultured for 24 hours, and the mRNA expression-suppressing activity of the double-stranded nucleic acid was quantified by the quantitative RT-PCR method described in Example 4 above.

In addition, instead of the double-stranded nucleic acid described above, a double-stranded nucleic acid against a non-target gene was used to quantify similar mRNA expression-suppressing activity.

The results are shown in FIG. 29 for the PANC-1 cell line and in FIG. 30 for the K562 cell line. As can be seen from FIG. 29, by binding a monomeric cRGD to a double-stranded nucleic acid targeting the human BCL2 gene, mRNA expression was suppressed more in the PANC-1 cell line than in the unliganded BNSP-10. It was found that the activity was enhanced. In addition, as can be seen from FIG. 30, it was revealed that the mRNA expression-suppressing activity against the K562 cell line was enhanced depending on the number of cRGDs bound to the double-stranded nucleic acid targeting the human BCL2 gene. .

Example 14
[Test of mRNA expression suppression activity of double-stranded nucleic acid targeting human BCR-ABL gene with folic acid bound to the center of complementary strand]
In order to examine the in vitro mRNA expression-suppressing activity of the double-stranded nucleic acid bound with folic acid targeting the human BCR-ABL gene, the following experiment was conducted. Double-stranded nucleic acids BNSP-45-PEG3-Folate, BNSP-45-PEG11-Folate, BNSP-45-PEG23-Folate, and BNSP- 44 was prepared and added to the K562 cell line at 300 nM. After addition of the double-stranded nucleic acid, the cells were cultured for 24 hours, and the mRNA expression-suppressing activity of the double-stranded nucleic acid was quantified by the quantitative RT-PCR method described in Example 4 above.

In addition, instead of the double-stranded nucleic acid described above, a double-stranded nucleic acid against a non-target gene was used to quantify similar mRNA expression-suppressing activity.
The results are shown in FIG. As can be seen from FIG. 31, by binding folic acid to the double-stranded nucleic acid targeting the human BCR-ABL gene, the mRNA expression-suppressing activity against the K562 cell line was enhanced more than that of BNSP-44 to which no ligand was bound. It became clear that Moreover, a comparison of PEG3, PEG11, and PEG23 revealed that the shorter the PEG, the higher the mRNA expression-suppressing activity.

Thus, in the present application, polyalkylene glycol (eg, PEG)-based linkers, ie, polyalkylene glycol linkers (eg, PEG linkers), can be used to attach the polynucleotide to the ligand. The length of the polyalkylene glycol (eg, PEG) of the linker is an integer of 1-100, such as an integer of 1-50, an integer of 2-30 or an integer of 1-12, such as 1, 2, 3, 4, 5. , 6, 7 or 8, but in the above comparison it was found that shorter polyalkylene glycol (eg PEG) lengths are advantageous. Thus, in one embodiment of the invention, the length is preferably 1-30, more preferably 1-20, particularly preferably 2-15.

example 15
[Test of mRNA expression suppression activity of double-stranded nucleic acid targeting human STAT3 gene]
In order to examine the in vitro mRNA expression-suppressing activity of the double-stranded nucleic acid targeting the human STAT3 gene, the following experiment was performed. A double-stranded nucleic acid BNSP-46 was prepared and transfected into AsPC-1 cell line at 0.3, 1 and 3 nM using Lipofectamine 2000 (Invitrogen). The transfected cell line was cultured for 24 hours after transfection, and the mRNA expression-suppressing activity of the double-stranded nucleic acid was quantified by the quantitative RT-PCR method described in Example 4 above.

In addition, instead of the double-stranded nucleic acid described above, a double-stranded nucleic acid against a non-target gene was used to quantify similar mRNA expression-suppressing activity.
The results are shown in FIG. As can be seen from FIG. 32, targeting the human STAT3 gene showed dose-dependent mRNA expression-suppressing activity against the AsPC-1 cell line.

Example 16
[Verification of interferon-α (IFN-α) expression-inducing action in PBMC (human peripheral blood mononuclear cells) of PS-PO type double-stranded nucleic acid]
The above PS-PO type double-stranded nucleic acid, a single-stranded nucleic acid in which all antisense strands have PS bonds as a basic skeleton, and a double-stranded nucleic acid in which all antisense strands have PS bonds as a basic skeleton are compared, and IFN In order to evaluate whether there is a difference in the effect of inducing -α expression, the following experiment was performed.

A single-stranded nucleic acid (SEQ ID NO: 1), double-stranded nucleic acids BNSP-1, BNSP-4, BNSP-7 and BNSP-10 were prepared. Then, using Lipofectamine 2000 (Invitrogen), PBMC cells were transfected with the above double-stranded nucleic acid at concentrations of 30, 100 and 300 nM. The transfected cell line was cultured for 24 hours after transfection, and the expression level of IFN-α was quantified by ELISA in a conventional manner.

In addition, CpGPo (Invitrogen: ODN2216) was used as an IFN-α expression positive control in place of the double-stranded nucleic acid, and the IFN-α expression level was similarly quantified.

The results are shown in FIG. As can be seen from FIG. 33, compared to the single-stranded nucleic acid, the double-stranded nucleic acid reduces the IFN-α inducing ability to about half, and the antisense strand is PS-PO type. It was found that IFN-α was not induced at all with the main strand nucleic acid. Such results also support that the double-stranded nucleic acid complex of the present application has the effect of reducing side effects.

Claims (30)

(i) and (ii) below :
(i) Formula (V)

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^- or O ^- such that the region between A ₁ and A _i of formula (V) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, the partial structure (Va)

may or may not exist,
i = an integer of 6 to 96 when the partial structure (Va) exists, and i = 5 when the partial structure (Va) does not exist]
or a structure represented by
Formula (VI)

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^- or O ^- such that the region between A ₁ and A _i of formula (VI) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages;
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, partial structure (VI-a)

may or may not exist,
i = an integer of 6 to 96 when the partial structure (VI-a) exists, and i = 5 when the partial structure (VI-a) does not exist]
or a structure represented by
Formula (V')

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^— or O ^— such that the region between A ₁ and A _i of formula (V′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4 and g' represents an integer from 0 to 4,
Here, the partial structure (V'-a)

is always present, and i = an integer from 6 to 96]
or a structure represented by
Formula (VI')

[In the formula,
A ₁ to A _i are independently chemically unmodified deoxyribonucleosides;
R is independently selected from S ^— or O ^— such that the region between A ₁ and A _i of formula (VI′) has alternating phosphodiester and phosphorothioated bonds as internucleic acid linkages. ,
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4 and g' represents an integer from 0 to 4,
Here, the partial structure (VI'-a)

is always present, and i = an integer from 6 to 96]
and (ii) a polynucleotide comprising at least a nucleotide that may be chemically modified as a constituent unit, at least a portion of which is complementary to the (i) antisense nucleic acid a complementary strand comprising a specific polynucleotide ,
A pharmaceutical composition comprising a double-stranded nucleic acid complex comprising
The polynucleotide in the above (ii) complementary strand is
- having a continuous phosphodiester bond type structure as an internucleic acid bond,
- All structural units in the 5' terminal region and / or 3' terminal region of the polynucleotide are chemically modified nucleotides, and all structural units in regions other than the region having the chemically modified nucleotide as a structural unit are chemical an unmodified deoxyribonucleotide or ribonucleotide,
- Here, the 5' end region is a region consisting of a structural unit located at the 5' end and the 2nd to n-th (n = 3 to 6) structural units counted from the 5' end, and the 3' The terminal region is a region consisting of a structural unit located at the 3' end and the 2nd to m-th (m = 3 to 6) structural units counted from the 3' end,
- However, the region having the chemically modified nucleotide as a constituent unit has a continuous phosphorothioate bond type structure or a continuous phosphodiester bond type structure as an internucleic acid bond,
A pharmaceutical composition as described above. (i) and (ii) below:
(i) Formula (V)

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (V) ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, the partial structure (Va)

may or may not exist,
when the partial structure (Va) is present, i = an integer of 6 to 96, and
i=5 when the partial structure (Va) does not exist]
or a structure represented by
Formula (VI)

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (VI) ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, partial structure (VI-a)

may or may not exist,
when the partial structure (VI-a) is present, i = an integer from 6 to 96, and
i=5 when the partial structure (VI-a) does not exist]
or a structure represented by
Formula (V')

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (V') ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4, and g' represents an integer from 0 to 4,
Here, the partial structure (V'-a)

is always present, and
i = an integer from 6 to 96]
or a structure represented by
Formula (VI')

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (VI') ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4, and g' represents an integer from 0 to 4,
Here, the partial structure (VI'-a)

is always present, and
i = an integer from 6 to 96]
An antisense nucleic acid having a structure represented by, and
(ii) a complementary strand comprising a polynucleotide comprising at least a nucleotide that may be chemically modified as a constituent unit, at least a portion of which is complementary to the above (i) antisense nucleic acid;
A pharmaceutical composition comprising a double-stranded nucleic acid complex comprising
The polynucleotide in the above (ii) complementary strand is
- Contains only deoxyribonucleotides that may be chemically modified and ribonucleotides that may be chemically modified as structural units,
- having a continuous phosphodiester bond type structure as an internucleic acid bond,
- contains at least one mismatch to the (i) antisense nucleic acid;
- the optionally chemically modified ribonucleotide is used as the nucleotide constituting the mismatch,
A pharmaceutical composition as described above. (i) and (ii) below:
(i) Formula (V)

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (V) ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, the partial structure (Va)

may or may not exist,
when the partial structure (Va) is present, i = an integer of 6 to 96, and
i=5 when the partial structure (Va) does not exist]
or a structure represented by
Formula (VI)

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (VI) ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, partial structure (VI-a)

may or may not exist,
when the partial structure (VI-a) is present, i = an integer from 6 to 96, and
i=5 when the partial structure (VI-a) does not exist]
or a structure represented by
Formula (V')

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (V') ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4, and g' represents an integer from 0 to 4,
Here, the partial structure (V'-a)

is always present, and
i = an integer from 6 to 96]
or a structure represented by
Formula (VI')

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (VI') ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4, and g' represents an integer from 0 to 4,
Here, the partial structure (VI'-a)

is always present, and
i = an integer from 6 to 96]
An antisense nucleic acid having a structure represented by, and
(ii) a complementary strand comprising a polynucleotide comprising at least a nucleotide that may be chemically modified as a constituent unit, at least a portion of which is complementary to the above (i) antisense nucleic acid;
A pharmaceutical composition comprising a double-stranded nucleic acid complex comprising
The polynucleotide in the above (ii) complementary strand is
- Contains only deoxyribonucleotides that may be chemically modified and ribonucleotides that may be chemically modified as structural units,
- having a continuous phosphodiester bond type structure as an internucleic acid bond,
- includes at least one bulge;
- All of the nucleotides that make up the bulge are ribonucleotides that may be chemically modified,
A pharmaceutical composition as described above. (i) and (ii) below:
(i) Formula (V)

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (V) ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, the partial structure (Va)

may or may not exist,
when the partial structure (Va) is present, i = an integer of 6 to 96, and
i=5 when the partial structure (Va) does not exist]
or a structure represented by
Formula (VI)

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (VI) ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e is an integer of 1 to 9, g is an integer of 1 to 9,
Here, partial structure (VI-a)

may or may not exist,
when the partial structure (VI-a) is present, i = an integer from 6 to 96, and
i=5 when the partial structure (VI-a) does not exist]
or a structure represented by
Formula (V')

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (V') ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4, and g' represents an integer from 0 to 4,
Here, the partial structure (V'-a)

is always present, and
i = an integer from 6 to 96]
or a structure represented by
Formula (VI')

[In the formula,
A. ₁ ～A _i is independently a chemically unmodified deoxyribonucleoside,
R is independently A of formula (VI') ₁ from A _i so that the region between S ^- or O ^- is selected from
X is independently a chemically modified nucleoside;
e' represents an integer from 0 to 4, and g' represents an integer from 0 to 4,
Here, the partial structure (VI'-a)

is always present, and
i = an integer from 6 to 96]
An antisense nucleic acid having a structure represented by, and
(ii) a complementary strand comprising a polynucleotide comprising at least a nucleotide that may be chemically modified as a constituent unit, at least a portion of which is complementary to the above (i) antisense nucleic acid;
A pharmaceutical composition comprising a double-stranded nucleic acid complex comprising
The polynucleotide in the above (ii) complementary strand is
- Contains only deoxyribonucleotides that may be chemically modified and ribonucleotides that may be chemically modified as structural units,
- having a continuous phosphodiester bond type structure as an internucleic acid bond,
- comprising alternating deoxyribonucleotides and ribonucleotides as described above,
A pharmaceutical composition as described above. In the (ii) polynucleotide in the complementary strand, all structural units in the regions other than the 5'-terminal and 3'-terminal regions are chemically unmodified ribonucleotides, and the 5'-terminal region is an internucleic acid bond, 2. The pharmaceutical composition of claim 1, having a continuous phosphorothioate-linked structure, and wherein the 3' terminal region has a continuous phosphodiester-linked structure as an internucleic acid bond. 3. The pharmaceutical composition according to claim 2, wherein all of the nucleotides that do not form mismatches are optionally chemically modified deoxyribonucleotides. 4. The pharmaceutical composition according to claim 3, wherein all of the nucleotides that do not constitute the bulge are deoxyribonucleotides that may be chemically modified. The pharmaceutical composition according to any one of claims 1 to 7, wherein the length of the polynucleotide in the (ii) complementary strand is 8 to 100 bases. The (i) antisense nucleic acid polynucleotide and the (ii) complementary strand polynucleotide each have a length of 10 to 35 bases, and in the (ii) complementary strand polynucleotide, the double-strand forming region The pharmaceutical composition according to claim 2 or 6, wherein the mismatch is present at positions 2 to 15 counted from the 5' end. The (i) antisense nucleic acid polynucleotide and the (ii) complementary strand polynucleotide each have a length of 10 to 35 bases, and in the (ii) complementary strand polynucleotide, the double-strand forming region The pharmaceutical composition according to claim 3 or 7, wherein the bulge is present at the 2nd to 15th positions counting from the 5' end. 11. The pharmaceutical composition of any one of claims 1-10, wherein the (i) antisense nucleic acid has the same length as the polynucleotide in the (ii) complementary strand. The pharmaceutical composition according to any one of claims 1 to 10, wherein the length of said (i) antisense nucleic acid is different from the length of the polynucleotide in said (ii) complementary strand. The pharmaceutical composition according to any one of claims 1-12, wherein said double-stranded nucleic acid complex comprises at least one ligand. 14. The pharmaceutical composition of claim 13, wherein at least one ligand is bound to one or more constituent units of (ii) the complementary strand of polynucleotide. 15. The claim 13 or 14, wherein said at least one ligand is selected from the group consisting of protein ligands, peptide ligands, aptamer ligands, carbohydrate ligands, lipid ligands, small molecule ligands and biomolecule/bioactive molecule ligands. pharmaceutical composition. Claim 15, wherein said at least one ligand is a cyclic peptide, N-acetylgalactosamine, cholesterol, vitamin E (tocopherol) or analogues thereof, stearic acid, docosanoic acid, anisamide, folic acid or analogues thereof, anandamide or spermine. The pharmaceutical composition according to . A pharmaceutical composition according to any one of claims 1 to 16 for reducing target gene expression in a mammal. 18. The pharmaceutical composition according to claim 17, wherein the (i) antisense nucleic acid is an antisense strand complementary to any region of the target gene mRNA. The pharmaceutical composition according to any one of claims 1 to 16, which is used for suppressing proliferation of cells in which target gene expression is enhanced in mammals. 20. The pharmaceutical composition of claim 19, wherein said target gene is an oncogene and said cell is a cancer cell. A pharmaceutical composition according to any one of claims 1 to 16 for treating and/or preventing cancer. 22. The pharmaceutical composition according to claim 20 or 21, wherein said cancer is pancreatic cancer. 17. The pharmaceutical composition of any one of claims 1-16 for use in a method of reducing transcript levels in a cell, said method comprising the step of contacting said pharmaceutical composition with a cell. wherein said antisense nucleic acid is an antisense strand complementary to any region of said transcript. 24. The pharmaceutical composition of claim 23, wherein said transcript is a protein-encoding mRNA transcript. 24. The pharmaceutical composition of claim 23, wherein said transcript is a non-protein-encoding transcript. 17. The pharmaceutical composition of any one of claims 1-16 for use in a method of reducing the expression level of a target gene in a mammal, said method comprising administering said pharmaceutical composition to said mammal. wherein the antisense nucleic acid is an antisense strand complementary to any region of the target gene mRNA. 27. The pharmaceutical composition of claim 26, wherein said administration is enteral administration. 27. The pharmaceutical composition of claim 26, wherein said administration is parenteral administration. The pharmaceutical composition according to any one of claims 26-28, wherein the dosage of said nucleic acid complex is 0.001 mg/kg/day to 50 mg/kg/day. The pharmaceutical composition according to any one of claims 26-29, wherein said mammal is a human.

Images