Double-stranded nucleotide complexes for modifying target nucleotide sequences

Abstract

To provide novel polynucleotides that can be applied to genome editing technologies with improved efficiency in editing target nucleotide sequences. [Solution] A double-stranded nucleotide complex for modifying one or more nucleotides contained in a target nucleotide sequence in double-stranded DNA within a cell, wherein the double-stranded nucleotide complex comprises a first and a second polynucleotide, the first polynucleotide being capable of specifically binding to the target nucleotide sequence and containing at least one or more mismatched nucleotides and a first complementary site with respect to the target nucleotide sequence, and the second polynucleotide containing at least a second complementary site comprising a complementary base sequence that specifically binds to the first complementary site.

Description

[Technical Field]

[0001] The present invention relates to a double-stranded nucleotide complex that can specifically bind to a target nucleotide sequence for modifying one or more nucleotides contained in a target nucleotide sequence in double-stranded DNA within a cell. [Background technology]

[0002] CRISPR-Cas is a genome editing technology that applies the adaptive immune mechanisms of bacteria and archaea, and is used as a tool in genetic engineering. CRISPR-Cas9 (Patent Document 1), which uses the DNA sequence CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the DNA-cutting enzyme Cas9 from Streptococcus pyogenes, is characterized by its ability to induce cleavage of target double-stranded DNA by having RNA recognize the target DNA sequence. Due to its simplicity, speed, and high efficiency, it is the most widely used genome editing technology. On the other hand, CRISPR-Cas9 has problems with off-target effects, such as the guide RNA sequence misrecognizing the genome sequence and the introduction of unexpected mutations in locations other than the target DNA sequence due to the cleavage of double-stranded DNA.

[0003] Various solutions have been proposed to address the off-target effects of CRISPR-Cas. For example, CRISPR-Cas3, which utilizes the fact that the recognition sequence of Cas3 derived from E. coli is 27 bases compared to the recognition sequence of Cas9 (20 bases), enables more specific mutation introduction (Patent Document 2). Methods using a complex linking a guide RNA capable of DNA sequence recognition with a deaminase that performs nucleic acid base conversion, and mutant Cas nucleases in which the cleavage activity of one of the double-stranded DNA strands has been inactivated, have achieved safer and more specific genome editing compared to CRISPR-Cas9 without inducing double-stranded DNA breaks (Patent Document 3).

[0004] However, even if the problem of off-target effects can be improved, CRISPR-Cas is a technology that introduces bacterial Cas nuclease or a gene encoding Cas nuclease into cells, so the problem of unexpected risks due to the introduction of foreign genes still remains. As a genome editing technology that does not use Cas nuclease proteins, genome editing technology using single-stranded synthetic DNA containing modified nucleic acids is known.

[0005] As an example of a method for modifying the target nucleotide sequence in double-stranded DNA using single-stranded synthetic DNA in which part of the nucleotide sequence is replaced with lock nucleic acid (LNA), a method is known in cell-free experiments that uses oligonucleotides containing at least one mismatched nucleotide and at least two LNAs, where each LNA is positioned at a distance of at least one nucleotide from the at least one mismatched nucleotide (Patent Document 4).

[0006] However, the technology described in Patent Document 4 does not have sufficient efficiency in editing the target nucleotide sequence. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 6343605 [Patent Document 2] Patent No. 6480647 [Patent Document 3] Patent No. 6206893 [Patent Document 4] Patent No. 5405121 [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide a novel polynucleotide that can be applied to genome editing technology with improved efficiency in editing target nucleotide sequences without introducing foreign nucleases or genes encoding exogenous nucleases into cells. [Means for solving the problem]

[0009] As a result of extensive research to solve the above problems, the present inventors have discovered that by using a double-stranded nucleotide complex that combines a single-stranded polynucleotide that can specifically bind to a target nucleotide sequence and contains one or more mismatched nucleotides with respect to the target nucleotide sequence, with another single-stranded polynucleotide having at least one polynucleotide having a complementary sequence that specifically binds to the single-stranded polynucleotide, the target nucleotide sequence can be edited more effectively, thus completing the present invention.

[0010] In other words, the present invention relates to the following [1] to

[17] . [1] A double-stranded nucleotide complex for modifying one or more nucleotides contained in a target nucleotide sequence in double-stranded DNA within a cell, The double-stranded nucleotide complex comprises a first polynucleotide and a second polynucleotide, The first polynucleotide is capable of specifically binding to the target nucleotide sequence and contains one or more mismatched nucleotides with respect to the target nucleotide sequence. The first polynucleotide has at least one feature selected from the group consisting of (A), (B), (C), and (D) below, (A) The nucleotide at the 5' end is a cross-linked nucleic acid. (B) The nucleotide adjacent to the 5' side of the mismatched nucleotide is a cross-linked nucleic acid. (C) The nucleotide adjacent to the 3' side of the mismatched nucleotide is a cross-linked nucleic acid. (D) The mismatched nucleotide is a cross-linked nucleic acid. The first polynucleotide comprises at least a first complementary site containing a complementary base sequence that specifically binds to the second polynucleotide, and also comprises a first 3' overhang site located at the 3' end of the first complementary site and / or a first 5' overhang site located at the 5' end of the first complementary site. The second polynucleotide includes at least a second complementary site comprising a complementary base sequence that specifically binds to the first complementary site of the first polynucleotide. Double-stranded nucleotide complex.

[0011] [2] The first polynucleotide consists of a first complementary site and a first 3' overhang site, The double-stranded nucleotide complex according to [1], wherein the second polynucleotide further includes a second 3' overhang region located on the 3' end of the second complementary region, and the chain length of the second 3' overhang region is 4 nucleotides or more. [3] The double-stranded nucleotide complex according to [1] or [2], wherein the second polynucleotide further comprises a phosphorylated nucleotide at its 3' end. [4] The double-stranded nucleotide complex according to any one of [1] to [3], wherein the second polynucleotide further comprises a nucleotide in which the pentose sugar is ribose. [5] The double-stranded nucleotide complex according to any one of [1] to [4] above, wherein the second complementary site contains a mismatched nucleotide for the first complementary site. [6] The double-stranded nucleotide complex according to any one of [2] to [5], wherein the second 3' overhang region contains a mismatched nucleotide for the target nucleotide sequence. [7] The double-stranded nucleotide complex according to any one of [1] to [6], wherein the second polynucleotide further comprises a phosphate-modified bond. 〔8〕 The double-stranded nucleotide complex according to 〔7〕 above, wherein the second polynucleotide further comprises a phosphoric acid moiety-modified bond between the nucleotide at its 5'-end and one or more nucleotides adjacent to the nucleotide at the 5'-end. 〔9〕 The double-stranded nucleotide complex according to 〔7〕 or 〔8〕 above, wherein the phosphoric acid moiety-modified bond comprises at least one selected from the group consisting of phosphorothioate bond, methyl phosphate bond, boranophosphate bond and mesylphosphoramidate bond. 〔10〕 The double-stranded nucleotide complex according to any one of 〔1〕 to 〔9〕 above, wherein the second polynucleotide further comprises a bridged nucleic acid at its 3'-end. 〔11〕 The double-stranded nucleotide complex according to any one of 〔1〕 to 〔10〕 above, wherein the bridged nucleic acid comprises at least one selected from the group consisting of LNA, AmNA, BNA N-H, BNA N-Me and ENA. 〔12〕 The first polynucleotide consists of a first complementary site and a first 5'-overhang site, The second polynucleotide further comprises a second 5'-overhang site located on the 5'-end side of the second complementary site, The double-stranded nucleotide complex according to any one of 〔1〕 to 〔11〕 above. A kit for modifying a target nucleotide sequence, containing the double-stranded nucleotide complex according to any one of 〔1〕 to 〔12〕 above. 〔14〕 A pharmaceutical composition containing the double-stranded nucleotide complex according to any one of 〔1〕 to 〔12〕 above. 〔15〕 A method for modifying one or more nucleotides contained in a target nucleotide sequence in double-stranded DNA in a cell, comprising: introducing a double-stranded nucleotide complex containing a first polynucleotide and a second polynucleotide into the cell. The first polynucleotide can specifically bind to the target nucleotide sequence and contains one or more mismatched nucleotides with respect to the target nucleotide sequence, and at least includes a first complementary site containing a complementary base sequence that specifically binds to the second polynucleotide, and a first 3'-overhang site located on the 3'-terminal side of the first complementary site and / or a first 5'-overhang site located on the 5'-terminal side of the first complementary site. The second polynucleotide at least includes a second complementary site containing a complementary base sequence that specifically binds to the first complementary site of the first polynucleotide. A method for modifying one or more nucleotides contained in the target nucleotide sequence. 〔16〕 The method according to 〔15〕, wherein the cell is a prokaryotic cell or a eukaryotic cell. 〔17〕 The method according to 〔16〕, wherein the eukaryotic cell is at least one selected from the group consisting of a plant cell, an insect cell, and an animal cell.

Advantages of the Invention

[0012] According to the present invention, a double-stranded nucleotide complex, which is a polynucleotide with excellent editing efficiency of a target nucleotide sequence, is provided.

Brief Description of the Drawings

[0013] [Figure A] FIG. A is a diagram schematically showing nine modes of the double-stranded nucleotide complex in the present invention. [Figure B] FIG. B is a schematic diagram showing an experimental system for editing a mutation of an inactive luciferase gene in the genome of 293-nLD1 cells using the double-stranded nucleotide complex of the present invention and detecting the editing efficiency as the luminescence of luciferase. [Figure 1-1]Figure 1-1 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 1-1. Double-stranded nucleotide complex DS-1 consists of the first polynucleotide FS-8 and the second polynucleotide SS-53. Similarly, double-stranded nucleotide complex DS-2 consists of the first polynucleotide FS-8 and the second polynucleotide SS-52, and double-stranded nucleotide complex DS-10 consists of the first polynucleotide FS-8 and the second polynucleotide SS-58. In the figure, T indicates the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G indicates the guanine base, which is a mismatch nucleotide; and L indicates that the pentose sugar in the nucleotide has been replaced with LNA. [Figure 1-2] Figure 1-2 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 1-2. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents the guanine, which is the mismatch nucleotide; and L represents the substitution of the pentose sugar in the nucleotide with LNA. [Figure 1-3] Figure 1-3 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 1-3. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents the guanine, which is the mismatch nucleotide; and L represents the substitution of the pentose sugar in the nucleotide with LNA. [Figure 1-4] Figure 1-4 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 1-4. In the figure, T represents thymine, the base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatched nucleotide; and L represents the substitution of the pentose sugar in the nucleotide with LNA. In SSS-111, C represents cytosine, the mismatched nucleotide for the first polynucleotide; and in SSS-112 and SSS-113, A represents adenine, the mismatched nucleotide for the first polynucleotide. [Figure 1-5] Figure 1-5 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 1-5. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents the guanine mismatch nucleotide; L represents the substitution of the pentose sugar in the nucleotide with LNA; and S represents the substitution of the phosphate diester bond with a phosphorothioate bond. [Figure 1-6] Figure 1-6 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 1-6. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents the guanine mismatch nucleotide; L represents the substitution of the pentose sugar in the nucleotide with LNA; and S represents the substitution of the phosphate diester bond with a phosphorothioate bond. [Figure 1-7] Figure 1-7 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 1-7. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents the mismatched nucleotide, guanine; L represents the substitution of the pentose sugar in the nucleotide with LNA; and S represents the substitution of the phosphate diester bond with a phosphorothioate bond. Furthermore, the entire second 3' overhang region was non-complementary to the corresponding nucleotide in the target nucleotide sequence. [Figure 1-8] Figure 1-8 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 1-8. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents the mismatched nucleotide, guanine; L represents the substitution of the pentose sugar in the nucleotide with LNA; and the vertical lines indicate the substitution with RNA. [Figure 2-1]Figure 2-1 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 2-1. In the figure, T represents thymine, the base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; and L indicates that the pentose sugar in that nucleotide has been replaced with LNA. In the second polynucleotide, C represents cytosine, and T represents thymine, the mismatch nucleotide for the first polynucleotide. [Figure 2-2] Figure 2-2 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 2-2. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatched nucleotide in the first polynucleotide; and L represents the substitution of the pentose sugar in that nucleotide with LNA. [Figure 3-1] Figure 3-1 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 3-1. In the figure, T represents thymine, the base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; L represents the substitution of the pentose sugar in that nucleotide with LNA; and C represents cytosine. In addition, T in the second polynucleotide represents thymine, the mismatch nucleotide for the first polynucleotide. [Figure 3-2] Figure 3-2 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 3-2. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatched nucleotide in the first polynucleotide; and L represents the substitution of the pentose sugar in that nucleotide with LNA. [Figure 3-3]Figure 3-3 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 3-3. In the figure, T represents thymine, the base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; L represents the substitution of the pentose sugar in that nucleotide with LNA; and C represents cytosine. In addition, T in the second polynucleotide represents thymine, the mismatch nucleotide for the first polynucleotide. [Figure 3-4] Figure 3-4 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 3-4. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatched nucleotide in the first polynucleotide; and L represents the substitution of the pentose sugar in that nucleotide with LNA. [Figure 3-5] Figure 3-5 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 3-5. In the figure, T represents thymine, the base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; and L indicates that the pentose sugar in that nucleotide has been replaced with LNA. In addition, T in the second polynucleotide represents thymine, the mismatch nucleotide for the first polynucleotide. [Figure 3-6] Figure 3-6 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 3-6. In the figure, T represents thymine, the base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; L represents the substitution of the pentose sugar in that nucleotide with LNA; vertical lines indicate substitution with RNA; and shaded areas indicate mismatch nucleotides for the first polynucleotide. In addition, T in the second polynucleotide represents thymine, the mismatch nucleotide for the first polynucleotide. [Figure 3-7] Figure 3-7 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 3-7. In the figure, T represents thymine, the base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; and L indicates that the pentose sugar in that nucleotide has been replaced with LNA. In addition, T in the second polynucleotide represents thymine, the mismatch nucleotide for the first polynucleotide. [Figure 4-1] Figure 4-1 is a schematic diagram showing the structure of the single-stranded polynucleotide shown in Example 4-1. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, which is a mismatch nucleotide in the single-stranded polynucleotide; and L represents the substitution of the pentose sugar in the nucleotide with LNA. [Figure 4-2-1] Figure 4-2-1 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 4-2. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; L represents the substitution of the pentose sugar in the nucleotide with LNA; and the vertical lines indicate the substitution with RNA. [Figure 4-2-2] Figure 4-2-2 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 4-2. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; L represents the substitution of the pentose sugar in the nucleotide with LNA; and the vertical lines indicate the substitution with RNA. [Figure 4-3-1]Figure 4-3-1 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 4-3. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; L represents the substitution of the pentose sugar in the nucleotide with LNA; and the vertical line indicates the substitution with RNA. [Figure 4-3-2] Figure 4-3-2 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 4-3. In the figure, T represents the thymine base after point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide; G represents guanine, the mismatch nucleotide in the first polynucleotide; L represents the substitution of the pentose sugar in the nucleotide with LNA; and the vertical lines indicate the substitution with RNA. [Figure 5] Figure 5 is a schematic diagram showing the structure of the double-stranded nucleotide complex shown in Example 5. In the figure, T represents thymine, the base after a point mutation in the inactive luciferase gene (sense strand), which is the target polynucleotide, and G represents guanine, the mismatched nucleotide in the first polynucleotide. [Modes for carrying out the invention]

[0014] <Summary of the Invention> In conventional techniques using single-stranded polynucleotides, the polynucleotides introduced into cells specifically recognize and bind to target nucleotide sequences in the lagging strand of the replication fork during double-stranded DNA replication, acting as primers for Okazaki fragment synthesis by DNA polymerase. This integration of polynucleotides into the newly generated DNA strands leads to genome sequence editing. On the other hand, living cells have a mismatch repair mechanism that repairs mismatches that occur during DNA replication, so mismatches caused by externally introduced polynucleotides are immediately repaired. One reason why modification of target nucleotide sequences using externally introduced polynucleotides is less efficient than modification of target nucleotide sequences using nucleases is thought to be the correction of edits by this mismatch repair mechanism.

[0015] On the other hand, the double-stranded nucleotide complex of the present invention comprises a first polynucleotide and a second polynucleotide, wherein the editing of the target nucleotide sequence is facilitated by the presence of one or more mismatched nucleotides and a cross-linked nucleic acid at a specific position within the first polynucleotide. Furthermore, it is presumed that by using a second polynucleotide containing a sequence complementary to the first polynucleotide, the influence of the mismatch repair mechanism is reduced, and the editing efficiency of the target nucleotide sequence can be significantly increased compared to previously reported methods.

[0016] In the present invention, the efficiency of target nucleotide sequence modification can be further increased by appropriately combining, for example, the number and position of mismatched nucleotides, the number and position of crosslinked nucleic acids, the chain length of the polynucleotide, the type of crosslinked nucleic acid, substitution of the phosphate diester bond portion between one or more nucleotides with a phosphate modification bond (e.g., a phosphorothioate bond), substitution of a pentose sugar with ribose in one or more nucleotides (sometimes abbreviated as "RNA substitution" herein), phosphorylation of the 3' or 5' end, or the addition of an overhang site, etc., in the first polynucleotide of the double-stranded nucleotide complex. Therefore, mismatched nucleotides for modifying the target nucleotide sequence may be located within the first complementary site or within the first 3' overhang site. If there are multiple mismatched nucleotides, two or more mismatched nucleotides may be located adjacent to each other or at different locations.

[0017] In the present invention, "multiple" with respect to the number of nucleotides or mismatched nucleotides includes integers of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, and also includes ranges with these as upper and lower limits. For example, 2-3, 2-4, 2-5, ...2-20, 3-4, 3-5, 3-6, ...3-20, 4-5, 4-6, 4-7, ...4-20, 5-6, 5-7, 5-8, ...5-20, ...18-19, 18-20, 19-20.

[0018] <Nucleotides> In this invention, "nucleotide" is a general term for substances in which a phosphate group is bonded to a nucleoside. A nucleoside is a pentose sugar to which a purine base or pyrimidine base is glycosidically bonded at position 1. A chain-like biopolymer in which nucleotides are the units is called a polynucleotide (also called "nucleic acid"). There are two types of polynucleotides: deoxyribonucleotides (DNA) and ribonucleotides (RNA). DNA is composed of four types of nucleotides: adenosine monophosphate (AMP, hereinafter referred to as "A"), guanosine monophosphate (GMP, hereinafter referred to as "G"), cytidine monophosphate (CMP, hereinafter referred to as "C"), and thymidine monophosphate (dTMP, hereinafter referred to as "T"). RNA contains A, G, and T, which are common to DNA, but uridine monophosphate (UMP, hereinafter referred to as "U") is included instead of T.

[0019] <Double-stranded DNA> In this invention, "double-stranded DNA" refers to DNA in which single-stranded DNA molecules, each having complementary base sequences, form hydrogen bonds between bases in opposite directions to form a double helix. While not particularly limited, double-stranded DNA in this invention includes genomic DNA, mitochondrial DNA, chloroplast DNA, and the like.

[0020] <Complementary nucleotides and mismatched nucleotides> Double-stranded DNA typically consists of purine and pyrimidine bases, with G and C, and A and T, linked by hydrogen bonds. A nucleotide that pairs with a specific nucleotide is called a "complementary nucleotide."

[0021] In the present invention, "mismatched nucleotide" means a nucleotide that cannot form a Watson-Crick type hydrogen bond between two bases, or a nucleotide that cannot form a Watson-Crick type hydrogen bond between two bases because there is no corresponding nucleotide. That is, "nucleotides that cannot form a Watson-Crick type hydrogen bond between two bases" refers to nucleotides other than C for G, other than T for A, other than A for T, and other than G for C. "A nucleotide that cannot form a Watson-Crick type hydrogen bond between two bases because there is no corresponding nucleotide" means that for G, there are no corresponding nucleotides for A, T, G, and C; for A, there are no corresponding nucleotides for A, T, G, and C; for T, there are no corresponding nucleotides for A, T, G, and C; and for G, there are no corresponding nucleotides for A, T, G, and C.

[0022] <Modification of target nucleotide sequence> In the present invention, the "target nucleotide sequence" is an oligonucleotide sequence containing the base to be edited, to which the first polynucleotide specifically binds. The base to be edited may be present on the sense strand or the antisense strand of the double-stranded DNA. Therefore, the specific binding of the first polynucleotide is not limited to the sense strand.

[0023] Here, "specific binding" between the first polynucleotide and the target nucleotide sequence refers to embodiments 1 and 2 described below. Embodiment 1: When the first polynucleotide has only a mismatch with the base to be edited, this means binding to a sequence complementary to the first polynucleotide, excluding the mismatch with the base to be edited. Embodiment 2: If the first polynucleotide has mismatches other than the mismatch with the base to be edited, this means binding to a sequence complementary to the first polynucleotide, excluding those mismatched portions.

[0024] In the present invention, the double-stranded DNA containing the "target nucleotide sequence" is preferably a gene involved in a genetic disorder, and more preferably a gene involved in a human genetic disorder.

[0025] For example, genes related to adrenoleukodystrophy (ABCD1), medium-chain acyl-CoA dehydrogenase deficiency (ACADM), Wilson's disease (ATP7B), hereditary pulmonary hypertension (BMPR2), X-linked agammaglobulinemia (BTK), cystinuria (CTNS), Duchenne muscular dystrophy (DMD), hemophilia A (F8), hemophilia B (F9), tyrosinemia (FAH), hepatic glycogen storage disease type Ia (G6PC), familial frontotemporal lobar degeneration (GRN), mucopolysaccharidosis type II (IDS), mucopolysaccharidosis Specific examples of double-stranded DNA containing "target nucleotide sequences" include genes related to type I (IDUA), primary immunodeficiency syndrome (IKBKB), familial hypertrophic cardiomyopathy (MYH7), peroxisome dysplasia (PEX2), hepatic glycogen storage disease type IX (PHKA2), protein C deficiency (PROC), hepatic glycogen storage disease type V (PYGM), familial dilated cardiomyopathy (RBM20), alpha-1 antitrypsin deficiency (SERPINA1), citrin deficiency (SLC25A13), cystinuria (SLC7A9), Niemann-Pick disease (SMPD1), and amyotrophic lateral sclerosis (SOD1). It should be noted that all of these diseases and genes are human diseases and genes.

[0026] "Modification" of a target nucleotide sequence refers to the substitution of one or more nucleotides in the target nucleotide sequence, i.e., the base to be edited, with a specific nucleotide (e.g., G) with one of three other nucleotides (A, T, or C) (hereinafter referred to as "substitution"), the deletion of one or more nucleotides in the target nucleotide sequence (hereinafter referred to as "deletion"), or the insertion of another nucleotide or nucleotide sequence between two specific nucleotides in the target nucleotide sequence (hereinafter referred to as "insertion"). "Modification" of a target nucleotide sequence includes substitution, deletion, and insertion occurring individually, as well as combinations thereof. The "target nucleotide sequence" to be modified may be either the sense strand or the antisense strand of a double-stranded DNA. In other words, the double-stranded nucleotide complex and modification method of the present invention can target both the sense strand and the antisense strand for modification.

[0027] <Double-stranded nucleotide complex> In the present invention, "double-stranded nucleotide complex" refers to a complex formed by the specific binding of a first complementary site in a first polynucleotide to a second complementary site in a second polynucleotide. The double-stranded nucleotide complex can be configured in nine ways, as shown in Figure A, depending on the presence or absence of a first 3' overhang site, a first 5' overhang site, a second 3' overhang site, and a second 5' overhang site. In Figure A, the first polynucleotide 1 is required to have a first complementary site and may also have a first 5' overhang site and / or a first 3' overhang site. Similarly, the second polynucleotide 2 is required to have a second complementary site and may also have a second 5' overhang site and / or a second 3' overhang site.

[0028] [First aspect] The first embodiment of the double-stranded nucleotide complex, as shown in Figure A(A), is a double-stranded nucleotide complex in which the first polynucleotide consists of a first complementary site and a first 3' overhang site, and the second polynucleotide further includes a second 3' overhang site located on the 3' end side of the second complementary site. In the examples of the present invention, many combinations of the first and second polynucleotides that can modify the target polynucleotide sequence with high editing efficiency have been identified as double-stranded nucleotide complexes, and this is a representative embodiment of the double-stranded nucleotide complex.

[0029] [Second aspect] A second aspect of the double-stranded nucleotide complex, as shown in Figure A(B), is a double-stranded nucleotide complex in which the first polynucleotide consists of a first complementary site and a first 5' overhang site, and the second polynucleotide consists of a second complementary site and a second 5' overhang site. In the examples of the present invention, multiple combinations of the first and second polynucleotides that can modify the target polynucleotide sequence with high editing efficiency have been identified as double-stranded nucleotide complexes.

[0030] [Third aspect] A third aspect of the double-stranded nucleotide complex, as shown in Figure A(C), is a double-stranded nucleotide complex in which the first polynucleotide consists of a first complementary site and a first 3' overhang site, and the second polynucleotide consists only of the second complementary site. In the examples of the present invention, multiple combinations of the first and second polynucleotides have been identified as double-stranded nucleotide complexes that can modify the target polynucleotide sequence with high editing efficiency.

[0031] [Fourth aspect] A fourth aspect of the double-stranded nucleotide complex, as shown in Figure A(D), is a double-stranded nucleotide complex in which the first polynucleotide consists of a first complementary site, a first 3' overhang site, and a first 5' overhang site, and the second polynucleotide consists only of the second complementary site. In the examples of the present invention, multiple combinations of the first and second polynucleotides that can modify the target polynucleotide sequence with high editing efficiency have been identified as double-stranded nucleotide complexes.

[0032] [Fifth aspect] A fifth aspect of the double-stranded nucleotide complex, as shown in Figure A(E), is a double-stranded nucleotide complex in which the first polynucleotide consists of a first complementary site and a first 5' overhang site, and the second polynucleotide consists only of the second complementary site. In the examples of the present invention, multiple combinations of the first and second polynucleotides have been identified as double-stranded nucleotide complexes that can modify the target polynucleotide sequence with high editing efficiency.

[0033] [Sixth aspect] A sixth aspect of the double-stranded nucleotide complex, as shown in Figure A(F), is a double-stranded nucleotide complex in which the first polynucleotide consists only of the first complementary site and the second polynucleotide consists only of the second complementary site. In the examples of the present invention, no combination of the first and second polynucleotides capable of modifying the target polynucleotide sequence as a double-stranded nucleotide complex could be identified.

[0034] [Seventh aspect] A seventh aspect of the double-stranded nucleotide complex, as shown in Figure A(G), is a double-stranded nucleotide complex in which the first polynucleotide consists only of the first complementary site, and the second polynucleotide consists of the second complementary site, the second 3' overhang site, and the second 5' overhang site. In the examples of the present invention, no combination of the first and second polynucleotides capable of modifying the target polynucleotide sequence as a double-stranded nucleotide complex could be identified.

[0035] [Eighth aspect] The eighth aspect of the double-stranded nucleotide complex is a double-stranded nucleotide complex in which the first polynucleotide consists only of the first complementary site, and the second polynucleotide consists of the second complementary site and the second 3' overhang site, as shown in Figure A(H).

[0036] [The ninth aspect] The ninth aspect of the double-stranded nucleotide complex is a double-stranded nucleotide complex in which the first polynucleotide consists only of the first complementary site, and the second polynucleotide consists of the second complementary site and the second 5' overhang site, as shown in Figure A(I).

[0037] <First polynucleotide> In the present invention, "first polynucleotide" means a polynucleotide that can specifically bind to a target nucleotide sequence and contains one or more mismatched nucleotides with respect to the target nucleotide sequence, and includes at least a first complementary site containing a complementary base sequence that specifically binds to a second polynucleotide. The first polynucleotide may also include a first 3' overhang site located at the 3' end of the first complementary site, in addition to the first complementary site. Furthermore, the first polynucleotide may also include a first 5' overhang site located at the 5' end of the first complementary site, in addition to the first complementary site. The first polynucleotide may also include both the first 3' overhang site and the first 5' overhang site, in addition to the first complementary site. The first polynucleotide is a single-stranded polynucleotide.

[0038] The first polynucleotide may be an antisense strand that can specifically bind to the sense strand of the target nucleotide sequence, or a sense strand that can specifically bind to the antisense strand of the target nucleotide sequence, but it is preferable that the first polynucleotide is an antisense strand.

[0039] The first polynucleotide may contain modified nucleic acids. Modified nucleic acids are not particularly limited, but include nucleic acids with modified phosphate groups, modified sugar groups, and modified base groups. Nucleic acids with modified phosphate groups are not particularly limited, but include nucleic acids in which the phosphate diester bond between one or more nucleotides is replaced with a phosphorothioate bond. While the bond between normal nucleotides is called a phosphodiester bond, this sulfurized bond is called a phosphorothioate bond. In the present invention, nucleotides having a phosphorothioate bond are called "phosphorothioate-modified" or "S-modified" nucleotides.

[0040] Other examples of nucleic acids with modified phosphate groups include nucleic acids having methyl phosphate bonds, nucleic acids having borano phosphate bonds in which one of the non-crosslinked oxygen atoms of the phosphate diester bond is replaced with a borano group (BH3), and nucleic acids having mesyl phosphoramidate bonds in which one of the non-crosslinked oxygen atoms of the phosphate diester bond is replaced with -NSO2CH3.

[0041] Nucleic acids with modified sugar moieties are not particularly limited, but include nucleic acids with modified 2' sites, and cross-linked nucleic acids with cross-linking of the 2' and 4' sites. Nucleic acids with modified 2' sites are not particularly limited, but include 2'-F (fluorinated), 2'-O-Methyl (2'-OMe), 2'-O-Methoxyethyl (2'-MOE), and 2'-O-(2-carbamoylethyl). Cross-linked nucleic acids are not particularly limited, but include 2'-O,4'-C-methylene-Bridged Nucleic Acid (2',4'-BNA) and BNA NC Examples include bicyclic or tricyclic cross-linked nucleic acids and other cross-linked nucleic acids. 2',4'-BNA is also called Locked Nucleic Acid (LNA) and has the structure shown below.

[0042] [ka]

[0043] Examples of BNA / LNA analogues, though not particularly limited, include ethylene-bridged BNA (ENA), amide-bridged BNA (AmNA), 2'-(alkylamino)-LNA, 2'-(acylamino)-LNA, 2'-N-substituted-2'-amino-LNA, α-LNA, α-L-LNA, β-D-LNA, 2'-amino-LNA, 2'-thio-LNA, xyl-LNA, 2'-O,4'-C-restricted ethyl (cEt)LNA, 2'-O,4'-C-restricted methoxyethyl (cMOEt)LNA, carba(cLNA), BNACOC, spirocyclopropylene-bridged nucleic acid (scpBNA), heterocyclic-bridged BNA, urea-bridged BNA, sulfonamide-bridged BNA, 5'-methyl-substituted BNA, guanidine-bridged nucleic acid (GuNA), and 3,4-dihydro-2H-pyran nucleic acid (DpNA).

[0044] BNA NC Examples include, but are not limited to, BNA NH, BNA N-Me, and BNA N-Bn.

[0045] Examples of bicyclic or tricyclic crosslinked nucleic acids include, but are not limited to, TriNA, α-L-TriNA, F-bcDNA, tricycloDNA (tcDNA), F-tcDNA, bicyclic carbocyclic nucleotides, bicycloDNA (bcDNA), and 2'-C-crosslinked bicyclic nucleotides (CBBN).

[0046] Other cross-linked nucleic acids include, but are not limited to, oxetane nucleotides, locked PMO derived from 2'-amino-LNA, cyclohexenyl nucleic acid (CeNA), alitriol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl RNA (pRNA), and 3'-deoxypyranosyl DNA (pDNA).

[0047] The first polynucleotide is preferable if it has at least one feature selected from the group consisting of (A), (B), (C), and (D) below, because it can modify the target polynucleotide sequence with high editing efficiency. (A) The nucleotide at the 5' end is a cross-linked nucleic acid. (B) The nucleotide adjacent to the 5' side of the mismatched nucleotide is a cross-linked nucleic acid. (C) The nucleotide adjacent to the 3' side of the mismatched nucleotide is a cross-linked nucleic acid. (D) The mismatched nucleotide is a cross-linked nucleic acid.

[0048] The first polynucleotide is more preferable if it has the characteristics of (A) above and at least one characteristic selected from the group consisting of (B), (C), and (D) above, because it can modify the target polynucleotide sequence with high editing efficiency.

[0049] <First complementary site> In the present invention, the "first complementary site" refers to a polynucleotide contained in the first polynucleotide that includes a nucleotide complementary to the target nucleotide sequence. The presence of the first complementary site allows the double-stranded nucleotide complex to specifically bind to the target nucleotide sequence when introduced into a cell. The first complementary site is included in all forms of the double-stranded nucleotide complex.

[0050] In the first embodiment of the double-stranded nucleotide complex, the chain length of the first complementary site is 7 to 17 nt, preferably 7 to 15 nt. When the chain length of the first complementary site is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0051] In a second embodiment of the double-stranded nucleotide complex, the chain length of the first complementary site is 8 to 16 nt. When the chain length of the first complementary site is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0052] In a third embodiment of the double-stranded nucleotide complex, the chain length of the first complementary site is 11 to 12 nt. When the chain length of the first complementary site is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0053] In a fourth embodiment of the double-stranded nucleotide complex, the chain length of the first complementary site is 9 to 11 nt. When the chain length of the first complementary site is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0054] In a fifth embodiment of the double-stranded nucleotide complex, the chain length of the first complementary site is 10 to 12 nt. When the chain length of the first complementary site is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0055] In the first embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is (chain length of the first complementary site) / (chain length of the first polynucleotide) = 28-68%, preferably (chain length of the first complementary site) / (chain length of the first polynucleotide) = 28-60%. When the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0056] In a second embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is (chain length of the first complementary site) / (chain length of the first polynucleotide) = 32-64%. When the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0057] In a third embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is (chain length of the first complementary site) / (chain length of the first polynucleotide) = 44-48%. When the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0058] In a fourth embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is (chain length of the first complementary site) / (chain length of the first polynucleotide) = 36-44%. When the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0059] In a fifth embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is (chain length of the first complementary site) / (chain length of the first polynucleotide) = 40-48%. When the ratio of the chain length of the first complementary site to the chain length of the first polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0060] <First 3' overhang area> In the present invention, the "first 3' overhang region" refers to a polynucleotide located on the 3' side of the first complementary region among the polynucleotides included in the first polynucleotide, which contains a nucleotide complementary to the target nucleotide sequence, and which does not have a nucleotide to pair with the second polynucleotide when the first polynucleotide and the second polynucleotide form a double-stranded nucleotide complex. The first 3' overhang region may or may not be included in the first polynucleotide depending on the embodiment, and is included in the first, third, and fourth embodiments of the double-stranded nucleotide complex.

[0061] In the first embodiment of the double-stranded nucleotide complex, the chain length of the first 3' overhang region is 8 to 18 nt. When the chain length of the first 3' overhang region is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0062] In a third embodiment of the double-stranded nucleotide complex, the chain length of the first 3' overhang region is 13-14 nt. When the chain length of the first 3' overhang region is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0063] In a fourth embodiment of the double-stranded nucleotide complex, the chain length of the first 3' overhang region is 1 to 13 nt. When the chain length of the first 3' overhang region is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0064] In the first embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first 3' overhang region to the chain length of the first polynucleotide is (chain length of the first 3' overhang region) / (chain length of the first polynucleotide) = 32 to 72%. When the ratio of the chain length of the first 3' overhang region to the chain length of the first polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0065] In a third embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first 3' overhang region to the chain length of the first polynucleotide is (chain length of the first 3' overhang region) / (chain length of the first polynucleotide) = 52-56%. When the ratio of the chain length of the first 3' overhang region to the chain length of the first polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0066] In a fourth embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first 3' overhang region to the chain length of the first polynucleotide is (chain length of the first 3' overhang region) / (chain length of the first polynucleotide) = 4-52%. When the ratio of the chain length of the first 3' overhang region to the chain length of the first polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0067] <First 5' overhang section> In the present invention, the "first 5' overhang region" refers to a polynucleotide located on the 5' side of the first complementary region among the polynucleotides included in the first polynucleotide, which does not have a nucleotide to pair with the second polynucleotide when the first polynucleotide and the second polynucleotide form a double-stranded nucleotide complex. The first 5' overhang region may or may not be included in the first polynucleotide depending on the embodiment, and is included in the second, fourth, and fifth embodiments of the double-stranded nucleotide complex.

[0068] In a second embodiment of the double-stranded nucleotide complex, the chain length of the first 5' overhang region is 9 to 17 nt. When the chain length of the first 5' overhang region is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0069] In a fourth embodiment of the double-stranded nucleotide complex, the chain length of the first 5' overhang region is 1 to 13 nt. When the chain length of the first 5' overhang region is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0070] In a fifth embodiment of the double-stranded nucleotide complex, the chain length of the first 5' overhang region is 8 to 16 nt. When the chain length of the first 5' overhang region is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0071] In a second embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first 5' overhang region to the chain length of the first polynucleotide is (chain length of the first 5' overhang region) / (chain length of the first polynucleotide) = 36-68%. When the ratio of the chain length of the first 5' overhang region to the chain length of the first polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0072] In a fourth embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first 5' overhang region to the chain length of the first polynucleotide is (chain length of the first 5' overhang region) / (chain length of the first polynucleotide) = 4 to 52%. When the ratio of the chain length of the first 5' overhang region to the chain length of the first polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0073] In a fifth embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the first 5' overhang region to the chain length of the first polynucleotide is (chain length of the first 5' overhang region) / (chain length of the first polynucleotide) = 32-64%. When the ratio of the chain length of the first 5' overhang region to the chain length of the first polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0074] <Second Polynucleotide> In the present invention, "second polynucleotide" means a polynucleotide that includes at least a second complementary site containing a complementary base sequence that specifically binds to the first complementary site of the first polynucleotide. The second polynucleotide may also include a second 3' overhang site located at the 3' end of the second complementary site. Furthermore, the second polynucleotide may also include a second 5' overhang site located at the 5' end of the second complementary site. The second polynucleotide is a single-stranded polynucleotide.

[0075] The chain length of the second overhang region is 16 to 70 nt (nucleotide), preferably 16 to 60 nt, and more preferably 16 to 50 nt. A chain length of the second polynucleotide within this range is preferable because it allows for high-efficiency editing of the target polynucleotide sequence.

[0076] The second polynucleotide may contain a modified nucleic acid. The modified nucleic acid is not particularly limited, but examples include nucleic acids with modified phosphate groups, modified sugar groups, and modified base groups. The nucleic acid with modified phosphate groups is not particularly limited, but examples include nucleic acids in which the phosphate diester bond between one or more nucleotides is replaced with a phosphorothioate bond.

[0077] Other examples of nucleic acids with modified phosphate groups include nucleic acids having methyl phosphate bonds, nucleic acids having borano phosphate bonds in which one of the non-crosslinked oxygen atoms of the phosphate diester bond is replaced with a borano group (BH3), and nucleic acids having mesyl phosphoramidate bonds in which one of the non-crosslinked oxygen atoms of the phosphate diester bond is replaced with -NSO2CH3.

[0078] Nucleic acids with modified sugar moieties are not particularly limited, but, similar to the first polynucleotide, include nucleic acids with modified 2' sites and cross-linked nucleic acids with cross-linked 2' and 4' sites. Cross-linked nucleic acids are not particularly limited, but, similar to the first polynucleotide, include LNA.

[0079] The second polynucleotide preferably contains a phosphate modification bond, and more preferably contains a phosphate modification bond between its 5' terminal nucleotide and one or more nucleotides adjacent to the 5' terminal nucleotide. Such a second polynucleotide can modify the target polynucleotide sequence with high editing efficiency.

[0080] If the second polynucleotide further contains a cross-linked nucleic acid at its 3' end, it can modify the target polynucleotide sequence with high editing efficiency.

[0081] The second polynucleotide may contain a phosphorylated nucleotide at its 3' end. The presence of a phosphorylated nucleotide at the 3' end of the second polynucleotide allows for highly efficient editing of the target polynucleotide sequence.

[0082] The second polynucleotide may contain a nucleotide in which the pentose is ribose. When the pentose of the polynucleotide contained in the second 3' overhang or the second complementary site is ribose, the target polynucleotide sequence can be modified with higher editing efficiency than when the second polynucleotide consists entirely of DNA. Furthermore, when the pentose of the second polynucleotide consists entirely of ribose, the target polynucleotide sequence can be modified with higher editing efficiency than when the second polynucleotide consists entirely of DNA.

[0083] <Second complementary site> In the present invention, the "second complementary site" refers to a polynucleotide included in the second polynucleotide that contains a complementary base sequence that specifically binds to the first polynucleotide, and which has a paired nucleotide regardless of whether it specifically binds to the first polynucleotide when the first polynucleotide and the second polynucleotide form a double-stranded nucleotide complex. The second complementary site is included in all embodiments of the double-stranded nucleotide complex.

[0084] The second complementary site may contain a mismatched nucleotide relative to the first complementary site. If the second complementary site contains at least one mismatched nucleotide relative to the first complementary site, the target polynucleotide sequence can be modified with high editing efficiency.

[0085] Since the chain length of the second complementary region is the same as that of the first complementary region, the preferred numerical range for chain length is the same as that of the first complementary region.

[0086] Since the chain length of the second complementary site is the same as that of the first complementary site, the numerical range for the ratio of the chain length of the second complementary site to the chain length of the preferred second polynucleotide is the same as that of the first complementary site.

[0087] <Second 3' overhang section> In the present invention, the "second 3' overhang region" refers to a polynucleotide located on the 3' side of the second complementary region among the polynucleotides included in the second polynucleotide, which does not have a corresponding nucleotide when the first polynucleotide and the second polynucleotide form a double-stranded nucleotide complex. The second 3' overhang region may or may not be included in the second polynucleotide depending on the embodiment, and is included in the first, seventh, and eighth embodiments of the double-stranded nucleotide complex.

[0088] In the first embodiment of the double-stranded nucleotide complex, the chain length of the second 3' overhang region is preferably 4 nt or more, more preferably 4 to 38 nt. When the chain length of the second 3' overhang region is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0089] In the first embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the second 3' overhang region to the chain length of the second polynucleotide is (chain length of the first 3' overhang region) / (chain length of the first polynucleotide) = 25-76%. When the ratio of the chain length of the second 3' overhang region to the chain length of the second polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0090] The second 3' overhang region may contain one or more mismatched nucleotides relative to the target nucleotide sequence. These mismatched nucleotides may be relative to the sense strand or the antisense strand of the target nucleotide sequence.

[0091] <Second 5' overhang section> In the present invention, the "second 5' overhang region" refers to a polynucleotide located on the 5' side of the second complementary region among the polynucleotides included in the second polynucleotide, which does not have a corresponding nucleotide when the first polynucleotide and the second polynucleotide form a double-stranded nucleotide complex. The second 5' overhang region may or may not be included in the second polynucleotide depending on the embodiment, and is included in the second, seventh, and ninth embodiments of the double-stranded nucleotide complex.

[0092] In a second embodiment of the double-stranded nucleotide complex, the chain length of the second 5' overhang region is preferably 2 nt or more, more preferably 2 to 8 nt. When the chain length of the second 5' overhang region is within this numerical range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0093] In a second embodiment of the double-stranded nucleotide complex, the ratio of the chain length of the second 5' overhang region to the chain length of the second polynucleotide is (chain length of the second 5' overhang region) / (chain length of the second polynucleotide) = 14-50%. When the ratio of the chain length of the second 5' overhang region to the chain length of the second polynucleotide is within this range, it is preferable because the double-stranded nucleotide complex can modify the target polynucleotide sequence with high editing efficiency.

[0094] The second 5' overhang region may contain one or more mismatched nucleotides relative to the target nucleotide sequence. These mismatched nucleotides may be relative to the sense strand or the antisense strand of the target nucleotide sequence.

[0095] <Kit> In the present invention, "kit" refers to a genetic engineering tool containing a double-stranded nucleotide complex used for modifying a target nucleotide sequence. The kit of the present invention may include buffers, stabilizers, preservatives, other reagents, instructions describing a protocol for modifying the target nucleotide sequence using the double-stranded nucleotide complex, etc., to the extent that it does not impair the effects of the present invention.

[0096] <Pharmaceutical composition> By utilizing the pharmaceutical composition containing the double-stranded nucleotide complex of the present invention (hereinafter referred to as the "pharmaceutical composition of the present invention"), it is possible to provide a pharmaceutical composition that can treat a target disease by modifying a gene with a mutation that does not function normally into a gene that functions normally through the action of the double-stranded nucleotide complex.

[0097] The pharmaceutical composition of the present invention can be administered, for example, orally, intravenously, intramuscularly, via the oral mucosa, rectally, vaginally, transdermally, nasally, or by inhalation. Alternatively, an ex vivo therapy can be used in which the pharmaceutical composition of the present invention is used on cells extracted from a patient, the treated cells are cultured as needed, and then returned to the patient's body. The double-stranded nucleotide complex of the pharmaceutical composition of the present invention may be formulated alone or in combination with other components, or it may be provided in the form of a pharmaceutical formulation by incorporating a pharmaceutically acceptable carrier or formulation additive. Examples of pharmaceutically acceptable carriers or additives are not particularly limited, but include lipid nanoparticles, excipients, disintegrants, disintegration aids, binders, lubricants, coatings, dyes, diluents, solubilizers, solubilizers, isotonic agents, pH adjusters, stabilizers, and the like.

[0098] The administration of the pharmaceutical composition of the present invention lasts for several days to several months, or until a cure is achieved or the symptoms are reduced, depending on the severity and response of the condition being treated. Those skilled in the art can determine the optimal dose, administration method, and frequency of recurrence.

[0099] <Target diseases of pharmaceutical compositions> Diseases targeted by the pharmaceutical composition of the present invention include, for example, diseases resulting from single-nucleotide substitutions in the human genome, such as adrenoleukodystrophy, medium-chain acyl-CoA dehydrogenase deficiency, Wilson's disease, hereditary pulmonary hypertension, X-linked agammaglobulinemia, cystinuria, Duchenne muscular dystrophy, hemophilia A, hemophilia B, tyrosinemia, hepatic glycogen storage disease type Ia, familial frontotemporal lobar degeneration, mucopolysaccharidosis type II, mucopolysaccharidosis type I, primary immunodeficiency syndrome, familial hypertrophic cardiomyopathy, peroxisome dysplasia, hepatic glycogen storage disease type IX, protein C deficiency, hepatic glycogen storage disease type V, familial dilated cardiomyopathy, α1-antitrypsin deficiency, citrin deficiency, cystinuria, Niemann-Pick disease, and amyotrophic lateral sclerosis.

[0100] <Preparation of double-stranded nucleotide complexes> The double-stranded nucleotide complex in the present invention can be readily prepared by methods known in the field of the present invention.

[0101] <Introduction of double-stranded nucleotide complexes into cells> In the present invention, "introducing a double-stranded nucleotide complex into cells" can be carried out according to known methods depending on the cells to be introduced. Known methods for introducing polynucleotides into cells (transfection methods) are broadly classified into two types: viral vector systems and non-viral vector systems. Viral vector systems are methods that introduce genes into cells by utilizing the cell entry mechanism inherent in viruses, and are not particularly limited, but include methods that use adenoviruses, retroviruses, lentiviruses, etc. as vectors. Non-viral vector systems are not particularly limited, but include lipofection, electroporation, microinjection, particle gun, etc. Lipofection is a method that utilizes the phenomenon in which positively charged cationic liposomes bind to negatively charged polynucleotides to form a complex, and the polynucleotides are taken up from the cell surface into the cell by endocytosis. Electroporation is a method that uses a dedicated machine to apply high-voltage pulses directly to cells, causing polynucleotides to be taken up through small pores on the cell surface. Microinjection is a method of introducing a substance into cells directly by placing it in a glass needle with a tip diameter of approximately 1 μm. Particle gun is a method of coating the surface of gold particles with DNA by co-precipitating gold particles with DNA, and then ejecting the gold particles towards target cells using helium gas pressure or the like. The present invention is a method for modifying target nucleotide sequences by introducing only non-natural polynucleotides without introducing Cas nucleases into cells, and from a safety standpoint, non-viral vector-based methods are more preferable. Examples of the methods of the present invention include methods performed in the human body and methods that include steps performed in the human body.

[0102] The double-stranded nucleotide complex (also referred to herein as "edited nucleic acid") introduced into the cell in this manner dissociates into a first polynucleotide and a second polynucleotide within the cell, and the first complementary site of the first polynucleotide selectively binds to the target nucleotide sequence in the genomic DNA. The base sequence of the first complementary site in the double-stranded nucleotide complex of the present invention is a mismatch with the base to be edited in the target nucleotide sequence of the double-stranded DNA in the cell, such as genomic DNA, and it is preferable to design the complex so that all base sequences other than the base to be edited are complementary to the target nucleotide sequence. However, there may be mismatched nucleotides other than the base to be edited, as long as it does not contradict the purpose of the present invention.

[0103] If the target nucleotide sequence is to be edited, for example, T, and it is to be changed to C, the mismatched nucleotide at the first complementary site in the double-stranded nucleotide complex of the present invention is set to G. Those skilled in the art can, based on common technical knowledge, set the mismatched nucleotide at the first complementary site in order to change the base of the target nucleotide to the desired base.

[0104] <Method for modifying one or more nucleotides in a target nucleotide sequence in double-stranded DNA within a cell> The present invention's "method for modifying one or more nucleotides contained in a target nucleotide sequence in intracellular double-stranded DNA" includes the step of introducing the above-described double-stranded nucleotide complex of the present invention into a cell. Here, the modification of the target nucleotide sequence includes at least one selected from the group consisting of deletion, insertion, and substitution of one or more nucleotides in the target nucleotide sequence. As the double-stranded nucleotide complex used in the modification method of the present invention, those described in the first to fifth embodiments above can be preferably used.

[0105] <Method for confirming modification of target nucleotide sequence> Methods for confirming the modification of the target nucleotide sequence in the present invention include measuring the activity of a gene containing the target nucleotide sequence, and directly measuring the target nucleotide sequence using a next-generation sequencer (NGS) and digital PCR.

[0106] <cell> The method for modifying a target nucleotide sequence using a double-stranded nucleotide complex according to the present invention is applicable to all cells that have double-stranded DNA. Furthermore, if the intracellular DNA replication mechanism can be utilized, the method is not limited to double-stranded DNA inherent in cells, but can also modify single-stranded DNA derived from viruses in virus-infected cells as the target nucleotide sequence. In other words, the "cells" of the present invention include prokaryotic cells and eukaryotic cells.

[0107] <Prokaryotic cells> In this invention, "prokaryotic cells" are not particularly limited, but include bacterial cells and archaeal cells.

[0108] <Eukaryotic cells> In the present invention, "eukaryotic cells" are not particularly limited, but include animal cells, insect cells, plant cells, algal cells, and fungal cells.

[0109] <Animal cells> In this invention, "animal cells" are not particularly limited but include cells of vertebrates, cells of invertebrates, cells derived from animal tissues, germ cells, somatic cells, and stem cells. Germ cells include oocytes and sperm cells. Somatic cells are not particularly limited but include fibroblasts, hematopoietic cells, neurons, muscle cells, osteocytes, hepatocytes, pancreatic cells, brain cells, kidney cells, etc. Stem cells are not particularly limited but include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).

[0110] <Mammalian cells> In this invention, "mammal" refers to a group of vertebrates belonging to the order Mammalia, and humans are included in the category of mammals. "Mammalian cell" refers to a cell that constitutes a mammal or a cell derived from a mammal. Examples of mammals other than humans are not particularly limited, but include deer, sea lions, hamsters, dogs, mice, wolves, whales, zebras, donkeys, weasels, bats, dolphins, anteaters, seals, cows, wild boars, horses, squirrels, bears, cats, moles, monkeys, raccoons, kangaroos, pigs, foxes, sheep, and the like.

[0111] Regarding human cells, as mentioned above, this includes tissue-derived cells, germ cells, somatic cells, and stem cells. In the examples described herein, HEK293 cells were used to confirm the modification of the target nucleotide sequence, but it can also be confirmed using other cells, such as HeLa cells.

[0112] <Insect cells> In this invention, "insect" refers to all arthropods classified under the class Insecta. While not particularly limited, insects such as silkworm larvae, fruit flies, and crickets can be used. In this invention, "insect cells" are not particularly limited, but include cells that constitute the body tissue of insects and cells derived from insect tissue.

[0113] <Plant cells> In this invention, "plant" is not particularly limited, but includes seed plants, ferns, mosses, algae, etc. "Plant cell" refers to a cell that makes up a plant or a cell derived from a plant. Seed plants include angiosperms and gymnosperms. Angiosperms include dicotyledons and monocotyledons. Dicotyledons are not particularly limited, but include morning glories, dandelions, azaleas, rhododendrons, eggplants, rapeseed, peas, etc. Monocotyledons are not particularly limited, but include rice, lilies, tulips, Japanese pampas grass, corn, etc. Gymnosperms are not particularly limited, but include pine, cedar, ginkgo, cypress, etc. Ferns are not particularly limited, but include bracken, fiddlehead ferns, leucocephala, horsetail, etc. Mosses are not particularly limited, but include liverworts, hornworts, sphagnum mosses, and sphagnum mosses. Algae include multicellular algae and unicellular algae. Examples of multicellular algae, though not particularly limited, include kelp, wakame seaweed, sea lettuce, and green algae. Examples of unicellular algae, though not particularly limited, include Chlorella, Euglena, Spirulina, Chlamydomonas, Coccomyxa, Botryococcus, Crepiformis, and diatoms. [Examples]

[0114] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

[0115] A. Genome editing experiments using 293-nLD1 cells 1. Production of mutant NanoLuc® plasmid Based on the wild-type base sequence of the luciferase gene NanoLuc (GenBank JQ 513379), DNA from base number 847 to 1,380 was synthesized (by Gene Universal Inc.), resulting in pcDNA. TM The gene was seamlessly integrated into the XbaI-ApaI restriction enzyme region of the 5 / FRT / TO plasmid (Thermo Fisher Scientific Inc.) by seamless cloning (pcDNA5-nLW1). Here, the wild-type sequence of the NanoLuc gene is denoted as sequence number 106. On the other hand, a mutant nucleotide sequence was synthesized in the region from nucleotide number 847 to 1,380, in which the cytosine (C) at nucleotide number 922 of the NanoLuc gene was changed to thymine (T), and a mutant was created by incorporating this into the pcDNATM5 / FRT / TO plasmid in the same manner as above (pcDNA5-nLD1). The luciferase gene in this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation described above.

[0116] 2. Creation of a stable transformed cell line containing the mutated NanoLuc gene. Flp-In, a human-derived cell line TM -293 cell line (Thermo Fisher Scientific Inc.) 1x10 6 Cells were seeded in 6 cm dishes and cultured at 37°C under 5% CO2 conditions in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc.). After 24 hours, transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific Inc.) with pcDNA5-nLD1 or pcDNA5-nLW1 plasmid (1 μg) and pOG44 plasmid (3 μg, Thermo Fisher Scientific Inc.) according to the standard protocol. After 48 hours, TrypLE TM Express Enzyme (Thermo Fisher Scientific Inc.) was added, and the cells were incubated at 37°C for 3 minutes. The detached cells were collected and suspended in DMEM + 10% FBS medium containing 50 μg / ml of hygromycin (Thermo Fisher Scientific Inc.). The cells were divided into two 10 cm dishes and cultured. Culture was then continued, with the medium being changed every 3 days. After approximately 20 days of culture, the formation of a sufficient number and size of colonies was confirmed, and all cells were subjected to TrypLE. TMUsing Express Enzyme, it was detached from the petri dish and recovered.

[0117] The cells in which the mutant NanoLuc was integrated into the genome were named 293-nLD1 cells. The mutant NanoLuc gene possessed by the 293-nLD1 cells was the inactive luciferase gene as described above. These 293-nLD1 cells were used in subsequent genome editing experiments. On the other hand, the cells in which the wild-type NanoLuc was integrated into the genome were named 293-nLW1 and used as a positive control in subsequent genome editing experiments.

[0118] 3. Synthesis of Polynucleotide All polynucleotides including nucleic acid modifications were synthesized by Gene Design Co., Ltd. (Japan) and those purified by a simple column or HPLC were used.

[0119] 4. Preparation of Double-Stranded Nucleotide Complex 0.025 μg or 0.05 μg of the first polynucleotide and the second polynucleotide with a weight such that the molar ratio to the first polynucleotide is equal (1.625 pmol for 0.025 μg and 3.25 pmol for 0.05 μg) were mixed in TE buffer, then heated at 95 °C for 5 minutes, and then gradually cooled to room temperature (25 °C) to prepare a double-stranded nucleotide complex. In this example, the first polynucleotide was named FS-XX (XX is a number), the second polynucleotide was named SS-XX (XX is a number), and the double-stranded nucleotide complex was named DS-XX (XX is a number).

[0120] 5. Introduction of Polynucleotide into 293-nLD1 Cells 293-nLD1 cells were adjusted to 1x10 4 cells and seeded in a 96-well plate (Nunc (registered trademark) MicroWell TMCells were seeded on a Nunclon Delta-Treated Flat-Bottom Microplate (96, Thermo Fisher Scientific Inc.) and cultured in DMEM + 10% FBS medium at 37°C under 5% CO2 conditions. After 24 hours, double-stranded nucleotide complexes or single-stranded polynucleotides were transfected into 293-nLD1 cells with Lipofectamine 3000 according to a standard protocol.

[0121] 6. Measuring editing efficiency • NanoLuc Luciferase Assay NanoLuc Luciferase activity was measured after 72 hours of incubation following transfection. NanoLuc Luciferase activity was measured using the Nano-Glo® Lucif erase Assay System (Promega Corporation) according to a standard protocol. Luciferase luminescence was measured using the EnSpire multimode plate reader (PerkinElmer Co., Ltd.).

[0122] • Counting the number of living cells The number of viable cells was measured using the CellTiter-Blue® Cell Viability Assay (Promega Corporation) according to a standard protocol. The fluorescence values ​​of CellTiter-Blue® were measured using the EnSpire multimode plate reader (PerkinElmer Co., Ltd.). The number of viable cells in the edited cells is 5 x 10⁻⁶ 3 From 2 x 10 5 A calibration curve was created using a 2x dilution series of 293-nLD1 cell counts between these two points to obtain CellTiter-Blue® measurement values ​​and cell counts. Based on this calibration curve, the number of viable cells was calculated.

[0123] • Calculation of editing efficiency When the NanoLuc Luciferase activity of 293-nLW1 cells with wild-type luciferase incorporated into their genome was measured, the measured value was approximately 20,000 counts / cell. Therefore, if the mutation in the inactive luciferase gene is repaired and wild-type NanoLuc is synthesized, a luciferase activity of 20,000 counts per cell will be detected. Based on this value, the editing efficiency (%) is: The calculation was performed using the formula: NanoLuc Luciferase activity ÷ 20,000 ÷ number of cells x 100.

[0124] Example 1-1 Investigation of chain length of the second 3' overhang portion In this example, the chain length of the second 3' overhang portion was investigated. Specifically, we prepared double-stranded nucleotide complexes with second 3' overhang sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences. For example, in the case of the double-stranded nucleotide complex DS-1, as shown in Table 1-1-1, the double-stranded nucleotide complex DS-1 was prepared using the method described in "4. Preparation of Double-Stranded Nucleotide Complexes" above, with the first polynucleotide FS-8 and the second polynucleotide SS-53, and introduced into 293-nLD1 cells.

[0125] Furthermore, as a standard for editing efficiency, DS-89 was prepared using the method described in "4. Preparation of Double-Stranded Nucleotide Complexes" above, with FS-8 as the first polynucleotide and SS-160 as a negative control for the second polynucleotide, and introduced into 293-nLD1 cells. Since SS-160 is a sequence that does not bind complementaryally to FS-8 at any site, DS-89 does not form a double-stranded nucleotide complex, but for convenience, it is referred to as the double-stranded nucleotide complex DS-89.

[0126] As mentioned above, since the cytosine (C) at base number 922 was changed to thymine (T), the codon encoding glutamine (Gln-22) at position 22 (CAA) in the luciferase gene introduced into 293-nLD1 cells was point-mutated into a stop codon (TAA), and therefore did not exhibit luciferase activity. If the T in the stop codon (TAA) in the inactive luciferase gene could be replaced with C, the luciferase activity of 293-nLD1 cells could be restored, and the modification of the target nucleotide sequence could be detected by the luminescence produced by luciferase (Figure B).

[0127] In this Example 1-1, the double-stranded nucleotide complexes DS-89, DS-1 to DS-10 shown in Table 1-1-1 and Figure 1-1 were introduced into 293-nLD1 cells by transfection. In the double-stranded nucleotide complexes shown in Table 1-1-1, the amount of the double-stranded nucleotide complex introduced was 3.25 pmol in all cases.

[0128] [Table 1-1-1]

[0129] Table 1-1-2 shows the base sequences of the first polynucleotide FS-8 contained in each of the double-stranded nucleotide complexes DS-1 to DS-10 used in this embodiment, as well as the base sequences and chain lengths of the second polynucleotides SS-53, SS-52, SS-51, SS-50, SS-36, SS-54, SS-55, SS-56, SS-57, SS-58, and SS-160. In Table 1-1-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0130] Furthermore, in each table listing nucleotide sequences in this specification, uppercase letters indicate LNA, * indicates a phosphorothioate bond, arrows indicate RNA, underlines indicate mismatched nucleotides, and -p indicates phosphorylation.

[0131] The first polynucleotide, FS-8, has a G nucleotide, i.e., a mismatch, at the position corresponding to T in the stop codon TAA in the target polynucleotide sequence, while the nucleotides other than the mismatched nucleotide in FS-8 are complementary to the target polynucleotide sequence.

[0132] [Table 1-1-2]

[0133] chain length The first polynucleotide, FS-8, has a chain length of 25 nt. The chain lengths of the second polynucleotide, SS-53, etc., are as shown in Table 1-1-2. Additionally, the chain length of SS-160 is 25 nt.

[0134] Chain length of complementary regions The chain lengths of the first and second complementary sites in each of the double-stranded nucleotide complexes DS-1 to DS-10 are as shown in Table 1-1-1.

[0135] Chain length of the overhang portion The chain lengths of the first 3' overhang region and the second 3' overhang region in each of the double-stranded nucleotide complexes DS-1 to DS-10 are as shown in Table 1-1-1.

[0136] LNA location FS-8, the first polynucleotide in DS-1 to DS-10, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends that are LNA, and the nucleotide at the 5' end is also LNA. SS-53, SS-52, SS-51, SS-50, SS-36, SS-54, SS-55, SS-56, SS-57, and SS-58, the second polynucleotides in DS-1 to DS-10, do not contain LNA.

[0137] Table 1-1-3 shows the specific sequence information for the overhang and complementary regions in DS-1 to DS-10. For convenience, in Table 1-1-3, the sequences of the first polynucleotide are written from left to right in the 5'→3' direction, and the sequences of the second polynucleotide are written from left to right in the 3'→5' direction. [Table 1-1-3]

[0138] Editing efficiency The editing efficiencies of double-stranded nucleotide complexes DS-1 to DS-10 are shown in Table 1-1-1. The editing efficiency of each double-stranded nucleotide complex is shown as a relative value, with the editing efficiency of DS-89 set to 1.00. Double-stranded nucleotide complexes exhibiting a relative editing efficiency of 1.50 or higher were judged to be double-stranded nucleotide complexes that exhibit superior editing efficiency.

[0139] Consideration As shown in Table 1-1-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then the editing efficiencies of DS-1 to DS-7 all showed high values ​​of 1.50 or higher. On the other hand, the editing efficiencies of DS-8 to DS-10 were all less than 1.50. From these results, it was found that if the chain length of the second 3' overhang region in the double-stranded nucleotide complex is 4 nt or more, it exhibits excellent editing efficiency.

[0140] Example 1-2: Investigation of the chain length of the second 3' overhang region Similar to Example 1-1, the chain length of the second 3' overhang region was investigated.

[0141] In Example 1-2, the double-stranded nucleotide complexes DS-89, DS-5, and DS-11~DS-17 shown in Table 1-2-1 and Figure 1-2 were introduced into 293-nLD1 cells by transfection. In the double-stranded nucleotide complexes shown in Table 1-2-1, the amount of the double-stranded nucleotide complex introduced was 3.25 pmol in all cases.

[0142] [Table 1-2-1]

[0143] Table 1-2-2 shows the base sequences of the first polynucleotide FS-8 and the second polynucleotides SS-36, SS-38, SS-94, SS-95, SS-96, SS-97, SS-98, and SS-99 contained in each of the double-stranded nucleotide complexes used in this example. In Table 1-2-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0144] [Table 1-2-2]

[0145] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-36, etc., are as shown in Table 1-2-2.

[0146] Chain length of complementary regions The chain lengths of the first and second complementary sites in the double-stranded nucleotide complexes DS-5 and DS-11-DS-17 are as shown in Table 1-2-1.

[0147] Chain length of the overhang portion The chain lengths of the first 3' overhang region and the second 3' overhang region in each of the double-stranded nucleotide complexes DS-5, DS-11 to DS-17 are as shown in Table 1-2-1.

[0148] LNA location FS-8, the first polynucleotide in DS-5 and DS-11-DS-17, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends that are LNA, and the nucleotide at the 5' end is also LNA. SS-36 does not have LNA. SS-38, SS-94, SS-95, SS-96, SS-97, SS-98, and SS-99 all have LNA at their 3' ends.

[0149] Table 1-2-3 shows the specific sequence information for the overhang and complementary regions in DS-5 and DS-11 to DS-17, respectively. For convenience, in Table 1-2-3, the sequences of the first polynucleotide are written from left to right in the 5'→3' direction, and the sequences of the second polynucleotide are written from left to right in the 3'→5' direction. [Table 1-2-3]

[0150] Editing efficiency The editing efficiencies of the double-stranded nucleotide complexes DS-5 and DS-11-DS-17 are shown in Table 1-2-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00. Double-stranded nucleotide complexes exhibiting a relative editing efficiency of 1.50 or higher were judged to be double-stranded nucleotide complexes that exhibit superior editing efficiency.

[0151] Consideration As shown in Table 1-2-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then the editing efficiencies of DS-5 and DS-11 to DS-17 all showed high values ​​of 1.50 or higher. On the other hand, the editing efficiencies of DS-8 to DS-10 were all less than 1.50. From these results, it was found that if the chain length of the second 3' overhang region in the double-stranded nucleotide complex is at least 38 nt or less, excellent editing efficiency is observed.

[0152] Examples 1-3: Investigation of chain length of complementary regions In this example, the chain lengths of the complementary regions, specifically the chain length of the first complementary region and the chain length of the second complementary region, were examined. The chain lengths of the first and second complementary regions are identical. Specifically, we prepared double-stranded nucleotide complexes with complementary sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0153] In this example, the double-stranded nucleotide complexes DS-89, DS-18~DS-23, DS-5, and DS-24~DS-28 shown in Table 1-3-1 and Figure 1-3 were introduced into 293-nLD1 cells by transfection. In the double-stranded nucleotide complexes shown in Table 1-3-1, the amount of the double-stranded nucleotide complex introduced was 3.25 pmol in all cases.

[0154] [Table 1-3-1]

[0155] Table 1-3-2 shows the base sequences of the first polynucleotide FS-8 and the second polynucleotides SS-140, SS-137, SS-141, SS-138, SS-142, SS-139, SS-36, SS-40 to SS-44 contained in each of the double-stranded nucleotide complexes used in this example. In Table 1-3-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0156] [Table 1-3-2]

[0157] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-140, etc., are as shown in Table 1-3-2.

[0158] Chain length of complementary regions The chain lengths of the first and second complementary sites in the double-stranded nucleotide complexes DS-18 to DS-23, DS-5, and DS-24 to DS-28 are as shown in Table 1-3-1.

[0159] Chain length of the overhang portion The chain lengths of the first 3' overhang region and the second 3' overhang region in each of the double-stranded nucleotide complexes DS-18 to DS-23, DS-5, and DS-24 to DS-28 are as shown in Table 1-3-1.

[0160] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0161] Table 1-3-3 shows the specific sequence information for the overhang and complementary regions in DS-18 to DS-23, DS-5, and DS-24 to DS-28, respectively. For convenience, in Table 1-3-3, the first polynucleotide is written from left to right in the 5'→3' direction, and the second polynucleotide is written from left to right in the 3'→5' direction. [Table 1-3-3]

[0162] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 1-3-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00. Double-stranded nucleotide complexes exhibiting a relative editing efficiency of 1.50 or higher were judged to be double-stranded nucleotide complexes that exhibit superior editing efficiency.

[0163] Consideration As shown in Table 1-3-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then the editing efficiencies of DS-18 to DS-23, DS-5, and DS-24 to DS-28 all showed high values ​​of 1.50 or higher. From these results, it was found that excellent editing efficiency is observed when the chain length of the complementary site is in the range of 7 nt to 15 nt. Furthermore, when comparing DS-18 with DS-19, and DS-20 with DS-21, DS-19 showed higher editing efficiency than DS-18, and DS-21 showed higher editing efficiency than DS-20. From these results, it was found that double-stranded nucleotide complexes containing a second polynucleotide with a mismatched nucleotide for the first polynucleotide show higher editing efficiency.

[0164] Example 1-4 Examination of mismatched nucleotides within the second complementary site In this example, we investigated whether the editing efficiency is affected by the presence or absence of mismatched nucleotides relative to the first polynucleotide within the second complementary site. Here, "mismatched nucleotides relative to the first polynucleotide" refers not to "mismatched nucleotides" contained in the first polynucleotide used to modify the target base in the target polynucleotide, but rather to nucleotides in the second polynucleotide that do not bind complementaryly to the nucleotides in the first polynucleotide. Specifically, we prepared double-stranded nucleotide complexes with single-nucleotide modifications at various positions within the second complementary site and introduced them into 293-nLD1 cells to measure the editing efficiency of the target polynucleotide sequence.

[0165] In this example, the double-stranded nucleotide complexes DS-89, DS-11, and DS-29~DS-31 shown in Table 1-4-1 and Figure 1-4 were introduced into 293-nLD1 cells by transfection. In the double-stranded nucleotide complexes shown in Table 1-4-1, the amount of the double-stranded nucleotide complex introduced was 1.625 pmol in all cases.

[0166] [Table 1-4-1]

[0167] Table 1-4-2 shows the base sequence of the first polynucleotide FS-8 and the base sequences and chain lengths of the second polynucleotides SS-38, SS-111~SS-113 contained in each of the double-stranded nucleotide complexes used in this example. In Table 1-4-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0168] [Table 1-4-2]

[0169] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-38, etc., are as shown in Table 1-4-2.

[0170] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 1-4-1.

[0171] Chain length of the overhang portion The chain lengths of the first 3' overhang region and the second 3' overhang region in each double-stranded nucleotide complex are as shown in Table 1-4-1.

[0172] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is LNA. In the second polynucleotide of the double-stranded nucleotide complex used in the example, the nucleotide at the 3' end is LNA in all cases.

[0173] Mismatched nucleotides in the second complementary site In SS-111, the nucleotide at the 5' end is a mismatch nucleotide for the first polynucleotide; in SS-112, the 11th nucleotide from the 5' end is a mismatch nucleotide for the first polynucleotide; and in SS-113, the 5th nucleotide from the 5' end is a mismatch nucleotide for the first polynucleotide. The underlined nucleotides in Table 1-4-3 are the mismatch nucleotides.

[0174] Table 1-4-3 shows the specific sequence information for the overhang and complementary regions of DS-11 and DS-29 to DS-31, respectively. For convenience, in Table 1-4-3, the sequences of the first polynucleotide are written from left to right in the 5'→3' direction, and the sequences of the second polynucleotide are written from left to right in the 3'→5' direction. [Table 1-4-3]

[0175] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 1-4-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00. Double-stranded nucleotide complexes exhibiting a relative editing efficiency of 1.50 or higher were judged to be double-stranded nucleotide complexes that exhibit superior editing efficiency.

[0176] Consideration As shown in Table 1-4-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then the editing efficiencies of DS-11, DS-29 to DS-31 all showed high values ​​of 1.50 or higher. From these results, it was found that introducing at least one mismatched nucleotide into the second complementary site significantly improves editing efficiency.

[0177] Examples 1-5: Investigation of substitution of phosphorothioate bonds with the second polynucleotide. In this example, we investigated whether the editing efficiency was affected by substituting the phosphate diester bond between nucleotides within the second 3' overhang region or the complementary region with a phosphorothioate bond. Specifically, we prepared double-stranded nucleotide complexes in which the phosphate diester bonds between nucleotides within the second 3' overhang region or complementary region were replaced with phosphorothioate bonds, and introduced these complexes into 293-nLD1 cells to measure the editing efficiency of the target polynucleotide sequence.

[0178] In this example, the double-stranded nucleotide complexes DS-89, DS-5, DS-32, and DS-33 shown in Table 1-5-1 and Figure 1-5 were introduced into 293-nLD1 cells by transfection. In the double-stranded nucleotide complexes shown in Table 1-5-1, the amount of the double-stranded nucleotide complex introduced was 3.25 pmol in all cases.

[0179] [Table 1-5-1]

[0180] Table 1-5-2 shows the base sequence of the first polynucleotide FS-8 and the base sequences and chain lengths of the second polynucleotides SS-36, SS-34, and SS-35 contained in each of the double-stranded nucleotide complexes used in this example. In Table 1-5-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0181] [Table 1-5-2]

[0182] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-36, etc., are as shown in Table 1-5-2.

[0183] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 1-5-1.

[0184] Chain length of the overhang portion The chain lengths of the first 3' overhang region and the second 3' overhang region in each double-stranded nucleotide complex are as shown in Table 1-5-1.

[0185] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0186] Substitution site for phosphorothioate bond within the second polynucleotide In SS-34, the bonds from the 3' end to the 4th bond are substituted, and in SS-35, the bonds from the 5' end to the 4th bond are substituted. Specifically, the bonds indicated by * in Table 1-5-2 are phosphorothioate bonds.

[0187] Table 1-5-3 shows the specific sequence information for the overhang and complementary regions of DS-5, DS-32, and DS-33, respectively. For convenience, in Table 1-3-3, the sequences of the first polynucleotide are written from left to right in the 5'→3' direction, and the sequences of the second polynucleotide are written from left to right in the 3'→5' direction. [Table 1-5-3]

[0188] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 1-5-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00. Double-stranded nucleotide complexes exhibiting a relative editing efficiency of 1.50 or higher were judged to be double-stranded nucleotide complexes that exhibit superior editing efficiency.

[0189] Consideration As shown in Table 1-5-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then the editing efficiencies of DS-5, DS-32, and DS-33 all showed high values ​​of 1.50 or higher. From these results, it was found that the editing efficiency is greatly improved by substituting a phosphorothioate bond within the second 3' overhang or second 3' complementary site.

[0190] Example 1-6: Investigation of combinations of introducing LNA into the second polynucleotide and substituting it with a phosphorothioate bond. In this example, we investigated whether editing efficiency is affected by a combination of introducing LNA to the 3' end of the second polynucleotide and substituting the phosphate diester bond between nucleotides within the overhang or complementary region of the second polynucleotide with a phosphorothioate bond. Specifically, we prepared multiple types of double-stranded nucleotide complexes combining these mutations and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0191] In this example, the double-stranded nucleotide complexes DS-89, DS-5, DS-34, DS-11, DS-32, DS-35, DS-33, and DS-36-DS-38 shown in Table 1-6-1 and Figure 1-6 were introduced into 293-nLD1 cells by transfection. In the double-stranded nucleotide complexes shown in Table 1-6-1, the amount of the double-stranded nucleotide complex introduced was 3.25 pmol in all cases.

[0192] [Table 1-6-1]

[0193] The nucleotide sequences of the first polynucleotide FS-8 and the second polynucleotides SS-36, SS-77, SS-38, SS-34, SS-78, SS-35, SS-79, SS-20, and SS-80 contained in each of the double-stranded nucleotide complexes used in this example, and their chain lengths are shown in Table 1-6-2. The sequences in Table 1-6-2 are described in the 5'→3' direction from left to right for both the first polynucleotide and the second polynucleotide.

[0194]

Table 1-6-2

[0195] Chain length The chain length of FS-8, which is the first polynucleotide, is 25 nt. The chain lengths of the second polynucleotides such as SS-36 are as described in Table 1-6-2.

[0196] Chain length of complementary region The chain lengths of the first complementary region and the second complementary region are as described in Table 1-6-1.

[0197] Chain length of overhang region The chain lengths of the first 3' overhang region and the second 3' overhang region in each of the double-stranded nucleotide complexes are as described in Table 1-6-1.

[0198] Position of LNA In the double-stranded nucleotide complex used in the example, FS-8, the first polynucleotide, has one nucleotide adjacent to the mismatch nucleotide G at the 5' upstream and 3' downstream ends that are LNA, and the nucleotide at the 5' end is also LNA. Among the second polynucleotides of the double-stranded nucleotide complex used in the example, SS-38, SS-78 to SS-80 all have an LNA nucleotide at the 3' end, while the remaining second polynucleotides do not have LNA.

[0199] Substitution site for phosphorothioate bond within the second polynucleotide In SS-34 and SS-78, the bonds from the 3' end to the 4th bond are substituted; in SS-35 and SS-79, the bonds from the 5' end to the 4th bond are substituted; and in SS-20 and SS-80, the bonds from the 3' end to the 4th bond and the bonds from the 5' end to the 4th bond are substituted. Specifically, the bonds indicated by * in Table 1-6-2 are phosphorothioate bonds.

[0200] Table 1-6-3 shows the specific sequence information for the overhang and complementary regions of DS-5, DS-34, DS-11, DS-32, DS-35, DS-33, and DS-36-DS-38. For convenience, in Table 1-3-3, the sequences of the first polynucleotide are written from left to right in the 5'→3' direction, and the sequences of the second polynucleotide are written from left to right in the 3'→5' direction. [Table 1-6-3]

[0201] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 1-6-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00. Double-stranded nucleotide complexes exhibiting a relative editing efficiency of 1.50 or higher were judged to be double-stranded nucleotide complexes that exhibit superior editing efficiency.

[0202] Consideration As shown in Table 1-6-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then the editing efficiencies of DS-5, DS-34, DS-11, DS-32, DS-35, DS-33, and DS-36-DS-38 all showed high values ​​of 1.50 or higher. Furthermore, in the second polynucleotide, those with LNA at the 3' terminal showed a tendency for decreased activity, but the activity improved by introducing a phosphorothioate bond to the 5' terminal region. This suggests that the combination of LNA and phosphorothioate bond synergistically improves editing efficiency.

[0203] Example 1-7: Investigation of introducing mismatched nucleotides into the second 3' overhang region. In this example, we investigated whether the editing efficiency is affected by introducing a mismatched nucleotide into the second 3' overhang region. Specifically, we prepared a double-stranded nucleotide complex in which all nucleotides in the second 3' overhang region were replaced with mismatched nucleotides, and further replaced with phosphorothioate bonds at various positions within the second complementary region. This complex was then introduced into 293-nLD1 cells, and the editing efficiency of the target polynucleotide sequence was measured.

[0204] In this example, the double-stranded nucleotide complexes DS-89, DS-39~DS-41 shown in Table 1-7-1 and Figure 1-7 were introduced into 293-nLD1 cells by transfection. In the double-stranded nucleotide complexes shown in Table 1-7-1, the amount of the double-stranded nucleotide complex introduced was 1.625 pmol in all cases.

[0205] [Table 1-7-1]

[0206] The nucleotide sequences and chain lengths of the first polynucleotide FS-8 and the second polynucleotides SS-101, SS-104, and SS-105 included in each of the double-stranded nucleotide complexes used in this example are shown in Table 1-7-2. The sequences in Table 1-7-2 are described in the 5'→3' direction from left to right for both the first polynucleotide and the second polynucleotide.

[0207]

Table 1-7-2

[0208] Chain length The chain length of FS-8, which is the first polynucleotide, is 25 nt. The chain lengths of the second polynucleotides such as SS-36 are as described in Table 1-7-2.

[0209] Chain length of complementary region The chain lengths of the first complementary region and the second complementary region are as described in Table 1-7-1.

[0210] Chain length of overhang region The chain lengths of the first 3' overhang region and the second 3' overhang region in each of the double-stranded nucleotide complexes are as described in Table 1-7-1.

[0211] Position of LNA In FS-8, which is the first polynucleotide of the double-stranded nucleotide complexes used in the example, the 1 nucleotide on the 5' upstream side and the 3' downstream side adjacent to the mismatched nucleotide G, and the nucleotide at the 5' end are LNA. None of the second polynucleotides of the double-stranded nucleotide complexes used in this example have LNA.

[0212] Substitution site for phosphorothioate bond in the second polynucleotide In SS-104, the bonds from the 5' end to the third position are substituted, while in SS-105, the bonds from the 5' end to the fourth position and the bonds between the sixth and seventh positions are substituted. Specifically, the bonds indicated by * in Table 1-7-2 are phosphorothioate bonds.

[0213] Substitution site for mismatched nucleotides in the second 3' overhang region As shown in the underlined section of Table 1-7-3, in SS-101, SS-104, and SS-105, all nucleotides in the second 3' overhang region are replaced with mismatched nucleotides.

[0214] Table 1-7-3 shows the specific sequence information for the overhang and complementary regions of each of DS-39 to DS-41. For convenience, in Table 1-3-3, the sequences of the first polynucleotide are written from left to right in the 5'→3' direction, and the sequences of the second polynucleotide are written from left to right in the 3'→5' direction.

[0215] [Table 1-7-3]

[0216] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 1-7-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00. Double-stranded nucleotide complexes exhibiting a relative editing efficiency of 1.50 or higher were judged to be double-stranded nucleotide complexes that exhibit superior editing efficiency.

[0217] Consideration As shown in Table 1-7-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then the editing efficiencies of DS-39 to DS-41 all showed high values ​​of 1.50 or higher. Furthermore, it was found that having a mismatched nucleotide in the second 3' overhang region resulted in higher editing efficiency. In addition, it was found that having a phosphorothioate bond in the second complementary region significantly increased the editing efficiency.

[0218] Example 1-8: Investigation of RNA substitution of nucleotides constituting the second polynucleotide. In this example, we investigated whether the editing efficiency is affected by substituting one or more nucleotides constituting the second polynucleotide from DNA to RNA. Specifically, we prepared double-stranded nucleotide complexes in which one or more nucleotides in the second 3' overhang region and / or the second complementary region were replaced from DNA to RNA, and introduced these complexes into 293-nLD1 cells to measure the editing efficiency of the target polynucleotide sequence.

[0219] In this example, the double-stranded nucleotide complexes DS-89, DS-5, and DS-42 to DS-51 shown in Table 1-8-1 and Figure 1-8 were introduced into 293-nLD1 cells by transfection. In the double-stranded nucleotide complexes shown in Table 1-8-1, the amount of the double-stranded nucleotide complex introduced was 3.25 pmol in all cases.

[0220] [Table 1-8-1]

[0221] Table 1-8-2 shows the base sequences of the first polynucleotide FS-8 and the second polynucleotides SS-36, SS-81, SS-82, SS-73~SS-76, SS-24, SS-25, SS-28, and SS-31 contained in each of the double-stranded nucleotide complexes used in this example. In Table 1-8-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0222] [Table 1-8-2]

[0223] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-36, etc., are as shown in Table 1-8-2.

[0224] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 1-8-1. The second complementary site may contain a nucleotide corresponding to a mismatched nucleotide of the first polynucleotide, as in DS-48 to DS-51.

[0225] Chain length of the overhang portion The lengths of the first 3' overhang and the second 3' overhang in each double-stranded nucleotide complex are as shown in Table 1-8-1.

[0226] LNA location In the double-stranded nucleotide complex used in the example, FS-8, the first polynucleotide, has one nucleotide adjacent to the mismatch nucleotide G at the 5' upstream and 3' downstream ends that are LNA, and the nucleotide at the 5' end is LNA. Among the second polynucleotides of the double-stranded nucleotide complex used in the example, SS-81, SS-82, SS-75, and SS-25 all have LNA at the 3' end, SS-74 and SS-28 all have LNA at the 5' end, SS-76 and SS-31 have LNA at both the 5' and 3' ends, and SS-73 and SS-24 do not have LNA.

[0227] Substitution site for RNA in the second polynucleotide The nucleotides indicated by the arrows in Table 1-8-2 have been replaced with RNA.

[0228] Table 1-8-3 shows the specific sequence information for the overhang and complementary regions of DS-5 and DS-42 to DS-51, respectively. For convenience, in Table 1-3-3, the sequences of the first polynucleotide are written from left to right in the 5'→3' direction, and the sequences of the second polynucleotide are written from left to right in the 3'→5' direction.

[0229] [Table 1-8-3]

[0230] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 1-8-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00. Double-stranded nucleotide complexes exhibiting a relative editing efficiency of 1.50 or higher were judged to be double-stranded nucleotide complexes that exhibit superior editing efficiency.

[0231] Consideration As shown in Table 1-8-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then the editing efficiencies of DS-5 and DS-42 to DS-51 all showed high values ​​of 1.50 or higher. Thus, it was found that those in which one or more nucleotides of the second polynucleotide are RNA showed higher editing efficiency.

[0232] Example 2-1 Investigation of the chain length of the second complementary region In Examples 1-1 to 1-8, the activity of double-stranded nucleotide complexes with a structure in which both the first and second 3' overhang regions protruded was investigated. Examples 2-1 and 2-1 investigated the activity of a double-stranded nucleotide complex in a different configuration, specifically one in which both the first and second 5' overhang regions protrude. Specifically, we prepared double-stranded nucleotide complexes with second complementary sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0233] In this example, the double-stranded nucleotide complexes DS-89, DS-52~DS-61 shown in Table 2-1-1 and Figure 2-1 were introduced into 293-nLD1 cells by transfection. In the double-stranded nucleotide complexes shown in Table 2-1-1, the amount of the double-stranded nucleotide complex introduced was 1.625 pmol in all cases.

[0234] [Table 2-1-1]

[0235] Table 2-1-2 shows the base sequences of the first polynucleotide FS-8 contained in each of the double-stranded nucleotide complexes used in this example, as well as the base sequences and chain lengths of the second polynucleotides SS-147, SS-143, SS-148, SS-144, SS-149, SS-145, SS-150, SS-146, SS-128, and SS-129. In Table 2-1-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0236] [Table 2-1-2]

[0237] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-147, etc., are as shown in Table 2-1-2.

[0238] Chain length of complementary regions The chain lengths of the first and second complementary regions are as shown in Table 2-1-1. In this embodiment, the chain length of the second complementary region is in the range of 8 nt to 16 nt.

[0239] Chain length of the overhang portion The chain lengths of the first 5' overhang and the second 5' overhang are as shown in Table 2-1-1.

[0240] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0241] Mismatched nucleotides in the second polynucleotide SS-143, SS-144, SS-145, and SS-146 have mismatched nucleotides relative to the first polynucleotide. Specifically, the underlined thymine in Table 2-1-2 are the mismatched nucleotides.

[0242] Table 2-1-3 shows the specific sequence information of the overhang and complementary regions in each of the double-stranded nucleotide complexes used in this example. For convenience, the sequences in Table 2-1-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0243] [Table 2-1-3]

[0244] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 2-1-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00.

[0245] Consideration As shown in Table 2-1-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, it was found that the double-stranded nucleotide complex used in this example had an editing efficiency equivalent to or higher than DS-89. A tendency was observed for editing efficiency to increase as the chain length of the second complementary site shortened. Furthermore, when comparing DS-52 with DS-53, DS-54 with D-55, DS-56 with DS-57, and DS-58 with DS-59, DS-53 showed higher editing efficiency than DS-52, D-55 than DS-54, DS-57 than DS-56, and DS-59 than DS-58. From these results, it was found that double-stranded nucleotide complexes containing a second polynucleotide with a mismatched nucleotide for the first polynucleotide show higher editing efficiency.

[0246] Example 2-2: Investigation of the chain length of the second 5' overhang portion In this example, we prepared double-stranded nucleotide complexes having second 5' overhang sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0247] In this example, the double-stranded nucleotide complexes DS-89 and DS-62~DS-65 shown in Table 2-2-1 and Figure 2-2 were introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex introduced into 293-nLD1 cells, as shown in Table 2-2-1, was 1.625 pmol in all cases.

[0248] [Table 2-2-1]

[0249] Table 2-2-2 shows the base sequence of the first polynucleotide FS-8 and the base sequences and chain lengths of the second polynucleotides SS-124, SS-125, SS-126, and SS-127 contained in each of the double-stranded nucleotide complexes used in this example. In Table 2-2-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0250] [Table 2-2-2]

[0251] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-124, etc., are as shown in Table 2-2-2.

[0252] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 2-2-1.

[0253] Chain length of the overhang portion The chain lengths of the first 5' overhang and the second 5' overhang are as shown in Table 2-2-1. The chain length of the second 5' overhang of the double-stranded nucleotide complex used in this example is in the range of 2 nt to 8 nt.

[0254] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0255] Table 2-2-3 shows the specific sequence information of the overhang and complementary regions for each of the double-stranded nucleotide complexes used in this example. For convenience, the sequences in Table 2-2-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0256] [Table 2-2-3]

[0257] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 2-2-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00. Double-stranded nucleotide complexes exhibiting a relative editing efficiency of 1.50 or higher were judged to be double-stranded nucleotide complexes that exhibit superior editing efficiency.

[0258] Consideration As shown in Table 2-2-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, it was found that the double-stranded nucleotide complex used in this example has high editing efficiency.

[0259] Example 3-1 Investigation of the chain length of the second complementary region In Example 3-1, the activity of a double-stranded nucleotide complex with a structure in which the first and second complementary sites are present, the second 3' overhang site is absent, and the first 3' overhang site protrudes was investigated. Specifically, we prepared double-stranded nucleotide complexes with second complementary sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0260] In this example, the double-stranded nucleotide complexes DS-89, DS-66~DS-71 shown in Table 3-1-1 and Figure 3-1 were introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex introduced into 293-nLD1 cells, as shown in Table 3-1-1, was 3.25 pmol in all cases.

[0261] [Table 3-1-1]

[0262] Table 3-1-2 shows the base sequences of the first polynucleotide FS-8 and the second polynucleotides SS-4, SS-5, SS-59, SS-121, SS-122, and SS-123 contained in each of the double-stranded nucleotide complexes used in this example. In Table 3-1-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0263] [Table 3-1-2]

[0264] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-4, etc., are as shown in Table 3-1-2.

[0265] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 3-1-1.

[0266] Chain length of the overhang portion The chain length of the first 3' overhang region is as shown in Table 3-1-1. As shown in Table 3-1-1 and Figure 3-1, because the first polynucleotide is fixed, as the chain length of the second complementary region decreases, the chain length of the first complementary region of the same length also decreases, resulting in an increase in the chain length of the first 3' overhang region.

[0267] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0268] Mismatched nucleotides in the second polynucleotide SS-5 has a mismatched nucleotide relative to the first polynucleotide. Specifically, the underlined thymine in Table 3-1-2 is the mismatched nucleotide.

[0269] Table 3-1-3 shows the specific sequence information of the overhang and complementary regions in each of the double-stranded nucleotide complexes used in this embodiment. For convenience, the sequences in Table 3-1-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0270] [Table 3-1-3]

[0271] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 3-1-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00.

[0272] Consideration As shown in Table 3-1-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then DS-68 to DS-71 exhibit some degree of editing efficiency, while DS-66 and DS-67 show no editing efficiency. From these results, it is suggested that the effect is achieved when the chain lengths of the first and second complementary regions are 9 to 12 nt, and the chain length of the first 3' overhang region is 13 to 15 nt.

[0273] Example 3-2 Investigation of the chain length of the second complementary region In Example 3-2, the activity of a double-stranded nucleotide complex was investigated in which the first polynucleotide had a 5' overhang, a complementary region, and a 3' overhang region, and the second polynucleotide had only a complementary region. Specifically, we prepared double-stranded nucleotide complexes with second complementary sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0274] In this example, the double-stranded nucleotide complexes DS-89, DS-72~DS-74 shown in Table 3-2-1 and Figure 3-2 were introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex introduced into 293-nLD1 cells, as shown in Table 3-2-1, was 1.625 pmol in all cases.

[0275] [Table 3-2-1]

[0276] Table 3-2-2 shows the base sequence of the first polynucleotide FS-8 and the base sequences and chain lengths of the second polynucleotides SS-118 to SS-120 contained in each of the double-stranded nucleotide complexes used in this example. In Table 3-2-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0277] [Table 3-2-2]

[0278] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-118, etc., are as shown in Table 3-2-2.

[0279] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 3-2-1.

[0280] Chain length of the overhang portion The chain lengths of the first 5' overhang and the 3' overhang are as shown in Table 3-2-1.

[0281] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0282] Table 3-2-3 shows the specific sequence information of the overhang and complementary regions in each of the double-stranded nucleotide complexes used in this embodiment. For convenience, the sequences in Table 3-2-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0283] [Table 3-2-3]

[0284] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 3-2-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00.

[0285] Consideration As shown in Table 3-2-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, it was found that the double-stranded nucleotide complex used in this embodiment has a higher editing efficiency.

[0286] Example 3-3 Investigation of the chain length of the second complementary region In Example 3-3, the activity of a double-stranded nucleotide complex in which the first polynucleotide has a 5' overhang and a complementary region, and the second polynucleotide has only a complementary region, was investigated. Specifically, we prepared double-stranded nucleotide complexes with second complementary sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0287] In this example, the double-stranded nucleotide complexes DS-89, DS-75~DS-80 shown in Table 3-3-1 and Figure 3-3 were introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex introduced into 293-nLD1 cells, as shown in Table 3-3-1, was 1.625 pmol in all cases.

[0288] [Table 3-3-1]

[0289] Table 3-3-2 shows the base sequence of the first polynucleotide FS-8 and the base sequences and chain lengths of the second polynucleotides SS-6, SS-7, and SS-130-SS-133 contained in each of the double-stranded nucleotide complexes used in this embodiment. In Table 3-3-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0290] [Table 3-3-2]

[0291] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-118, etc., are as shown in Table 3-3-2.

[0292] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 3-3-1.

[0293] Chain length of the overhang portion The chain length of the first 5' overhang is as shown in Table 3-3-1.

[0294] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0295] Mismatched nucleotides in the second polynucleotide SS-7 has a mismatched nucleotide relative to the first polynucleotide. Specifically, the underlined thymine in Table 3-3-2 is the mismatched nucleotide.

[0296] Table 3-3-3 shows the specific sequence information of the overhang and complementary regions for each of the double-stranded nucleotide complexes used in this embodiment. For convenience, the sequences in Table 3-3-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0297] [Table 3-3-3]

[0298] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 3-3-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00.

[0299] Consideration As shown in Table 3-3-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, it was found that all the double-stranded nucleotide complexes used in this embodiment had a certain level of editing efficiency.

[0300] Example 3-4 Investigation of the chain length of the second complementary region In Examples 3-4, the activity of double-stranded nucleotide complexes was investigated in which the first polynucleotide had a 5' overhang, a complementary region, and a 3' overhang region, and the second polynucleotide had only a complementary region. Specifically, we prepared double-stranded nucleotide complexes with second complementary sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0301] In this example, the double-stranded nucleotide complexes DS-89, DS-81 to DS-83 shown in Table 3-4-1 and Figure 3-4 were introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex introduced into 293-nLD1 cells, as shown in Table 3-4-1, was 3.25 pmol in all cases.

[0302] [Table 3-4-1]

[0303] Table 3-4-2 shows the base sequence of the first polynucleotide FS-8 and the base sequences and chain lengths of the second polynucleotides SS-134 to SS-136 contained in each of the double-stranded nucleotide complexes used in this example. In Table 3-4-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0304] [Table 3-4-2]

[0305] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-134, etc., are as shown in Table 3-4-2.

[0306] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 3-4-1.

[0307] Chain length of the overhang portion The chain lengths of the first 5' overhang and the 3' overhang are as shown in Table 3-4-1.

[0308] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0309] Table 3-4-3 shows the specific sequence information of the overhang and complementary regions for each of the double-stranded nucleotide complexes used in this embodiment. For convenience, the sequences in Table 3-4-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0310] [Table 3-4-3]

[0311] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 3-4-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00.

[0312] Consideration As shown in Table 3-4-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, it was found that the double-stranded nucleotide complex used in this embodiment has a higher editing efficiency.

[0313] Example 3-5: Investigation of the chain length of the second complementary region In Examples 3-5, the activity of double-stranded nucleotide complexes was investigated in which the first polynucleotide had a 5' overhang, a complementary region, and a 3' overhang region, and the second polynucleotide had only a complementary region. Specifically, we prepared double-stranded nucleotide complexes with second complementary sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0314] In this example, the double-stranded nucleotide complexes DS-89, DS-84 to DS-86 shown in Table 3-5-1 and Figure 3-5 were introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex introduced into 293-nLD1 cells, as shown in Table 3-5-1, was 3.25 pmol in all cases.

[0315] [Table 3-5-1]

[0316] Table 3-5-2 shows the base sequence of the first polynucleotide FS-8 and the base sequences and chain lengths of the second polynucleotides SS-154 to SS-156 contained in each of the double-stranded nucleotide complexes used in this embodiment. In Table 3-5-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0317] [Table 3-5-2]

[0318] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-154, etc., are as shown in Table 3-5-2.

[0319] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 3-5-1.

[0320] Chain length of the overhang portion The chain lengths of the first 5' overhang and the 3' overhang are as shown in Table 3-5-1.

[0321] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0322] Table 3-5-3 shows the specific sequence information of the overhang and complementary regions in each of the double-stranded nucleotide complexes used in this embodiment. For convenience, the sequences in Table 3-4-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0323] [Table 3-5-3]

[0324] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 3-5-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00.

[0325] Consideration As shown in Table 3-5-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then all the double-stranded nucleotide complexes used in this example had an editing efficiency of 0.22 or less, indicating only a small editing efficiency.

[0326] Example 3-6 Examination of the presence or absence of overhanging areas In Examples 3-6, the activity of double-stranded nucleotide complexes in which the first and second polynucleotides have only complementary sites was investigated. Specifically, we prepared double-stranded nucleotide complexes using FS-8 as the first polynucleotide and various modified second polynucleotides with the same chain length as FS-8, and introduced these complexes into 293-nLD1 cells to measure the editing efficiency of the target polynucleotide sequence.

[0327] In this example, the double-stranded nucleotide complexes DS-87 to DS-89 shown in Table 3-6-1 and Figure 3-6 were introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex introduced into 293-nLD1 cells, as shown in Table 3-6-1, was 1.625 pmol in all cases.

[0328] [Table 3-6-1]

[0329] Table 3-6-2 shows the base sequence of the first polynucleotide FS-8 and the base sequences and chain lengths of the second polynucleotides SS-1, SS-15, and SS-160 contained in each of the double-stranded nucleotide complexes used in this example. In Table 3-6-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0330] [Table 3-6-2]

[0331] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-1, etc., are as shown in Table 3-6-2.

[0332] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 3-6-1.

[0333] LNA location In the double-stranded nucleotide complex used in the example, FS-8, the first polynucleotide, has one nucleotide adjacent to the mismatch nucleotide G at the 5' upstream and 3' downstream ends that are LNA, and the nucleotide at the 5' end is also LNA. Of the second polynucleotides in the double-stranded nucleotide complex used in the example, SS-15 has four LNA bases at both the 5' end and the 3' end. Neither SS-1 nor SS-160 have LNA.

[0334] Mismatched nucleotides in the second polynucleotide SS-1 has a mismatched nucleotide relative to the first polynucleotide. Specifically, the underlined thymine in Table 3-6-2 is the mismatched nucleotide. In SS-160, all of the bases that make up the molecule are mismatched nucleotides relative to the first polynucleotide.

[0335] Table 3-6-3 shows the specific sequence information of the complementary sites in each of the double-stranded nucleotide complexes used in this embodiment. For convenience, the sequences in Table 3-6-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0336] [Table 3-6-3]

[0337] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 3-6-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of DS-89 set to 1.00.

[0338] Consideration As shown in Table 3-5-1, if the editing efficiency of DS-89, i.e., FS-8, is set to 1.00, then all the double-stranded nucleotide complexes used in this example had an editing efficiency of 0.28 or less, showing only a small editing efficiency.

[0339] Example 3-7: Examination of the presence or absence of overhang regions in the first polynucleotide. In Examples 3-7, the activity of double-stranded nucleotide complexes in which the first polynucleotide had only complementary sites and the second polynucleotide also had only complementary sites was investigated. Specifically, we prepared double-stranded nucleotide complexes with second complementary sites of various chain lengths and introduced them into 293-nLD1 cells to measure the editing efficiency of target polynucleotide sequences.

[0340] In this example, the double-stranded nucleotide complexes DS-90 to DS-92 shown in Table 3-7-1 and Figure 3-7 were introduced into 293-nLD1 cells by transfection. As a control, FS-8 shown in Table 3-7-1 was introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex or FS-8 introduced into 293-nLD1 cells, as shown in Table 3-7-1, was 3.25 pmol in all cases.

[0341] [Table 3-7-1]

[0342] Table 3-7-2 shows the base sequence of FS-8 used in this example, the base sequence of the first polynucleotide FS-8 contained in each double-stranded nucleotide complex, and the base sequences and chain lengths of the second polynucleotides SS-157 to SS-159. In Table 3-7-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0343] [Table 3-7-2]

[0344] chain length The chain length of the first polynucleotide, FS-8, is 25 nt. The chain lengths of the second polynucleotide, SS-157, etc., are as shown in Table 3-7-2.

[0345] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 3-7-1.

[0346] Chain length of the overhang portion The chain lengths of the second 5' overhang and the 3' overhang are as shown in Table 3-7-1.

[0347] LNA location In the double-stranded nucleotide complex used in the example, the first polynucleotide, FS-8, has one nucleotide adjacent to the mismatched nucleotide G at the 5' upstream and 3' downstream ends, and the nucleotide at the 5' end is also LNA. The second polynucleotide in the double-stranded nucleotide complex used in this example does not have LNA.

[0348] Mismatched nucleotides in the second polynucleotide SS-1 has a mismatched nucleotide relative to the first polynucleotide. Specifically, the underlined thymine in Table 3-6-2 is the mismatched nucleotide. In SS-160, all of the bases that make up the molecule are mismatched nucleotides relative to the first polynucleotide.

[0349] Table 3-7-3 shows the specific sequence information of the overhang and complementary regions for each of the double-stranded nucleotide complexes used in this embodiment. For convenience, the sequences in Table 3-4-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0350] [Table 3-7-3]

[0351] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 3-7-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of FS-8 set to 1.00.

[0352] Consideration As shown in Table 3-7-1, if the editing efficiency of FS-8 is set to 1.00, all of the double-stranded nucleotide complexes used in this example had an editing efficiency of 0.15 or less, indicating only a small editing efficiency.

[0353] Example 4-1: Investigation of the effect of the first polynucleotide alone In Example 4-1, the activity of only the first polynucleotide was examined. Specifically, we prepared mutants of the first polynucleotide, FS-8, as the base, by making various modifications without changing the chain length, and introduced them into 293-nLD1 cells to measure the editing efficiency of the target polynucleotide sequence.

[0354] In this example, each of the single-stranded polynucleotides shown in Table 4-1-1 and Figure 4-1 was introduced into 293-nLD1 cells by transfection. As a control, FS-8, shown in Table 4-1-1, was introduced into 293-nLD1 cells by transfection. The amount of single-stranded polynucleotides introduced into 293-nLD1 cells, as shown in Table 4-1-1, was 3.25 pmol in all cases.

[0355] [Table 4-1-1]

[0356] chain length The chain length of a single-stranded polynucleotide is 25 nt.

[0357] LNA location The LNAs of single-stranded polynucleotides are the bases shown in uppercase in Table 4-1-1.

[0358] Mismatched nucleotides Mismatched nucleotides for the target polynucleotide sequence are the underlined mismatched nucleotides in Table 4-1-1.

[0359] Editing efficiency The editing efficiencies of each single-stranded polynucleotide are shown in Table 4-1-1. The editing efficiencies of each single-stranded polynucleotide are shown as relative values, with the editing efficiency of FS-8 set to 1.00.

[0360] Consideration As shown in Table 4-1-1, if the editing efficiency of FS-8 is set to 1.00, it was found that the editing efficiency of all single-stranded polynucleotides used in this example was inferior to that of FS-8.

[0361] Example 4-2: Investigation of editing efficiency when the second polynucleotide is replaced with RNA. In this example, a polynucleotide in which all nucleotides were replaced with RNA (SS-28) was used as the second polynucleotide, and various modified first polynucleotides were used in combination with it to investigate the editing efficiency. Specifically, we used SS-28 as the second polynucleotide and FS-8, etc., as the first polynucleotide to prepare double-stranded nucleotide complexes as shown in Table 4-2-1, Figure 4-2-1, and Figure 4-2-2-, and introduced them into 293-nLD1 cells to measure the editing efficiency of the target polynucleotide sequence. On the other hand, as a control, the single-stranded polynucleotide FS-8 shown in Table 4-2-1 was introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex and FS-8 introduced was 3.25 pmol for both.

[0362] [Table 4-2-1]

[0363] Table 4-2-2 shows the base sequences of the first polynucleotides FS-8, FS-9, FS-11, FS-242, FS-228, FS-283, FS-284, FS-6, FS-285, FS-286, FS-37, FS-38, FS-5, FS-7, FS-230, and FS-76 contained in each of the double-stranded nucleotide complexes used in this example, as well as the base sequence and chain length of the second polynucleotide SS-28. In Table 4-2-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0364] [Table 4-2-2]

[0365] chain length The chain lengths of both the first and second polynucleotides are 25 nt.

[0366] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 4-2-1.

[0367] Chain length of the overhang portion The chain lengths of the first 3' overhang region and the second 3' overhang region in each double-stranded nucleotide complex are as shown in Table 4-2-1.

[0368] LNA location The location of the LNA is the nucleotide indicated in uppercase in Table 4-2-2.

[0369] Substitution site for RNA in the second polynucleotide The second polynucleotide, SS-28, has all nucleotides replaced with RNA, as shown in Table 4-2-2.

[0370] Table 4-2-3 shows the specific sequence information for the overhang and complementary regions of each double-stranded nucleotide complex. For convenience, the sequences in Table 4-2-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0371] [Table 4-2-3]

[0372] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 4-2-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of FS-8 set to 1.00.

[0373] Consideration From the above results, it was found that the first polynucleotide of the double-stranded nucleotide complex, in which the nucleotides constituting the second polynucleotide are converted to RNA, tends to exhibit high editing efficiency if it possesses at least one characteristic selected from the group consisting of (A), (B), (C), and (D) below. (A) The nucleotide at the 5' end is LNA. (B) The nucleotide adjacent to the 5' end of the mismatched nucleotide relative to the target nucleotide sequence is the LNA. (C) The nucleotide adjacent to the 3' end of the mismatched nucleotide relative to the target nucleotide sequence is the LNA. (D) The LNA is a mismatched nucleotide for the target nucleotide sequence. Furthermore, the above results indicate that the first polynucleotide of the double-stranded nucleotide complex, in which the nucleotides constituting the second polynucleotide are converted to RNA, tends to exhibit particularly high editing efficiency if it possesses the following characteristic (A) and at least one characteristic selected from the group consisting of (B), (C), and (D). (A) The nucleotide at the 5' end is LNA. (B) The nucleotide adjacent to the 5' end of the mismatched nucleotide relative to the target nucleotide sequence is the LNA. (C) The nucleotide adjacent to the 3' end of the mismatched nucleotide relative to the target nucleotide sequence is the LNA. (D) The LNA is a mismatched nucleotide for the target nucleotide sequence.

[0374] Example 4-3: Investigation of editing efficiency when the second polynucleotide is replaced with RNA. In this example, a polynucleotide in which all nucleotides were replaced with RNA (SS-74) was used as the second polynucleotide, and various modified first polynucleotides were used in combination with it to investigate the editing efficiency. Specifically, we used SS-74 as the second polynucleotide and FS-8 as the first polynucleotide to prepare double-stranded nucleotide complexes as shown in Table 4-3-1, Figure 4-3-1, and Figure 4-3-2. These complexes were then introduced into 293-nLD1 cells to measure the editing efficiency of the target polynucleotide sequence. As a control, single-stranded polynucleotide FS-8, shown in Table 4-2-1, was introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex and FS-8 introduced was 3.25 pmol for both.

[0375] [Table 4-3-1]

[0376] Table 4-3-2 shows the base sequences of the first polynucleotides FS-8, FS-9, FS-11, FS-242, FS-228, FS-283, FS-284, FS-6, FS-285, FS-286, FS-37, FS-38, FS-5, FS-7, FS-230, and FS-76 contained in each of the double-stranded nucleotide complexes used in this example, as well as the base sequence and chain length of the second polynucleotide SS-28. In Table 4-3-2, the sequences of both the first and second polynucleotides are written from left to right in the 5'→3' direction.

[0377] [Table 4-3-2]

[0378] chain length The first polynucleotides all have a chain length of 25 nt, while the second polynucleotide has a chain length of 20 nt.

[0379] Chain length of complementary regions The chain lengths of the first and second complementary sites are as shown in Table 4-3-1.

[0380] Chain length of the overhang portion The lengths of the first 3' overhang and the second 3' overhang in each double-stranded nucleotide complex are as shown in Table 4-3-1.

[0381] LNA location The position of LNA is the nucleotide indicated in capital letters in Table 4-3-2.

[0382] Substitution site for RNA in the second polynucleotide The second polynucleotide, SS-74, has all nucleotides replaced with RNA, as shown in Table 4-3-2.

[0383] Table 4-3-3 shows the specific sequence information for the overhang and complementary regions of each double-stranded nucleotide complex. For convenience, the sequences in Table 4-3-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0384] [Table 4-3-3]

[0385] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 4-3-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of FS-8 set to 1.00.

[0386] Consideration From the above results, it was found that the first polynucleotide of the double-stranded nucleotide complex, in which the nucleotides constituting the second polynucleotide are converted to RNA, tends to exhibit high editing efficiency if it possesses at least one characteristic selected from the group consisting of (A), (B), (C), and (D) below. (A) The nucleotide at the 5' end is LNA. (B) The nucleotide adjacent to the 5' end of the mismatched nucleotide relative to the target nucleotide sequence is the LNA. (C) The nucleotide adjacent to the 3' end of the mismatched nucleotide relative to the target nucleotide sequence is the LNA. (D) The LNA is a mismatched nucleotide for the target nucleotide sequence. Furthermore, the above results indicate that the first polynucleotide of the double-stranded nucleotide complex, in which the nucleotides constituting the second polynucleotide are converted to RNA, tends to exhibit particularly high editing efficiency if it possesses the following characteristic (A) and at least one characteristic selected from the group consisting of (B), (C), and (D). (A) The nucleotide at the 5' end is LNA. (B) The nucleotide adjacent to the 5' end of the mismatched nucleotide relative to the target nucleotide sequence is the LNA. (C) The nucleotide adjacent to the 3' end of the mismatched nucleotide relative to the target nucleotide sequence is the LNA. (D) The LNA is a mismatched nucleotide for the target nucleotide sequence.

[0387] Example 5: Investigation of double-stranded nucleotide complexes with simpler structures. In this example, we investigated the editing efficiency of double-stranded nucleotide complexes with simpler structures that do not involve substitution of LNA or phosphorothioate bonds. Specifically, we prepared double-stranded nucleotide complexes using FS-9 as the first polynucleotide and SS-53, etc., as the second polynucleotide, as shown below, and introduced them into 293-nLD1 cells to measure the editing efficiency of the target polynucleotide sequence. As a control, single-stranded polynucleotide FS-9 was introduced into 293-nLD1 cells by transfection. The amount of double-stranded nucleotide complex and FS-9 introduced was 3.25 pmol for both.

[0388] [Table 5-1]

[0389] Table 5-2 shows the base sequences of the first polynucleotide FS-9 and the base sequences and chain lengths of the second polynucleotides SS-53, SS-36, SS-54, and SS-46 contained in each of the double-stranded nucleotide complexes used in this embodiment. In Table 5-2, the sequences of both the first and second polynucleotides are written from left to right in the direction of 5'→3'.

[0390] [Table 5-2]

[0391] chain length The chain length of the first polynucleotide is 25 nt in all cases, and the chain lengths of the second polynucleotide are as shown in Table 5-2.

[0392] Chain length of complementary regions The chain lengths of the first and second complementary regions are as shown in Table 5-1.

[0393] Chain length of the overhang portion The lengths of the first 3' overhang and the second 3' overhang in each double-stranded nucleotide complex are shown in Table 5-1.

[0394] LNA location In this embodiment, neither the first polynucleotide nor the second polynucleotide constituting the double-stranded nucleotide complex contains LNA.

[0395] Table 5-3 shows the specific sequence information for the overhang and complementary regions of each double-stranded nucleotide complex. For convenience, the sequences in Table 5-3 are written from left to right in the direction of 5'→3' for the first polynucleotide and from left to right in the direction of 3'→5' for the second polynucleotide.

[0396] [Table 5-3]

[0397] Editing efficiency The editing efficiencies of each double-stranded nucleotide complex are shown in Table 4-3-1. The editing efficiencies of each double-stranded nucleotide complex are shown as relative values, with the editing efficiency of single-stranded polynucleotide FS-9 set to 1.00.

[0398] Consideration From the results above, it was found that even if the first and second polynucleotides do not contain modified nucleic acids such as cross-linked nucleic acids, preparing them as a double-stranded nucleotide complex can sometimes result in high editing efficiency. [Industrial applicability]

[0399] The method using the double-stranded nucleotide complex of the present invention enables modification of the target nucleotide sequence solely by introducing the double-stranded nucleotide complex, without introducing an exogenous nuclease or a gene encoding an exogenous nuclease into the cell. Because it can be applied to genome editing technology with extremely high safety, it has industrial applicability. [Explanation of Symbols]

[0400] 1. The first polynucleotide 2. Second polynucleotide

Claims

1. A double-stranded nucleotide complex for modifying one or more nucleotides contained in a target nucleotide sequence in double-stranded DNA within a cell, The double-stranded nucleotide complex comprises a first polynucleotide and a second polynucleotide, The first polynucleotide is capable of specifically binding to the target nucleotide sequence and contains one or more mismatched nucleotides with respect to the target nucleotide sequence. The first polynucleotide has at least one feature selected from the group consisting of (A), (B), (C), and (D) below, (A) The nucleotide at the 5' end is a cross-linked nucleic acid. (B) The nucleotide adjacent to the 5' side of the mismatched nucleotide is a cross-linked nucleic acid. (C) The nucleotide adjacent to the 3' side of the mismatched nucleotide is a cross-linked nucleic acid. (D) The mismatched nucleotide is a cross-linked nucleic acid. The first polynucleotide comprises at least a first complementary site containing a complementary base sequence that specifically binds to the second polynucleotide, and a first 3' overhang site located at the 3' end of the first complementary site and / or a first 5' overhang site located at the 5' end of the first complementary site. The second polynucleotide includes at least a second complementary site comprising a complementary base sequence that specifically binds to the first complementary site of the first polynucleotide. Double-stranded nucleotide complex.

2. The first polynucleotide consists of a first complementary site and a first 3' overhang site. The double-stranded nucleotide complex according to claim 1, wherein the second polynucleotide further comprises a second 3' overhang region located at the 3' end of the second complementary region, and the chain length of the second 3' overhang region is 4 nucleotides or more.

3. The double-stranded nucleotide complex according to claim 1 or 2, wherein the second polynucleotide further comprises a phosphorylated nucleotide at its 3' end.

4. The double-stranded nucleotide complex according to any one of claims 1 to 3, wherein the second polynucleotide further comprises a nucleotide in which the pentose sugar is ribose.

5. The double-stranded nucleotide complex according to any one of claims 1 to 4, wherein the second complementary site includes a mismatched nucleotide for the first complementary site.

6. The double-stranded nucleotide complex according to any one of claims 2 to 5, wherein the second 3' overhang region contains a mismatched nucleotide with respect to the target nucleotide sequence.

7. The double-stranded nucleotide complex according to any one of claims 1 to 6, wherein the second polynucleotide further comprises a phosphate-modified bond.

8. The double-stranded nucleotide complex according to claim 7, wherein the second polynucleotide further comprises a phosphate modification bond between its 5' terminal nucleotide and one or more nucleotides adjacent to the 5' terminal nucleotide.

9. The double-stranded nucleotide complex according to claim 7 or 8, wherein the phosphate modification bond comprises at least one selected from the group consisting of a phosphorothioate bond, a methyl phosphate bond, a boranophosphate bond, and a mesylphosphoamidate bond.

10. The double-stranded nucleotide complex according to any one of claims 1 to 9, wherein the second polynucleotide further comprises a cross-linked nucleic acid at its 3' end.

11. The double-stranded nucleotide complex according to any one of claims 1 to 10, wherein the cross-linked nucleic acid comprises at least one selected from the group consisting of LNA, AmNA, BNANH, BNAN-Me, and ENA.

12. The first polynucleotide consists of a first complementary site and a first 5' overhang site. The second polynucleotide further includes a second 5' overhang region located at the 5' end of the second complementary region. A double-stranded nucleotide complex according to any one of claims 1 to 11.

13. A kit for modifying a target nucleotide sequence, comprising a double-stranded nucleotide complex according to any one of claims 1 to 12.

14. A pharmaceutical composition containing a double-stranded nucleotide complex according to any one of claims 1 to 12.

15. A method for modifying one or more nucleotides contained in a target nucleotide sequence in double-stranded DNA within a cell, The process includes the step of introducing a double-stranded nucleotide complex containing a first polynucleotide and a second polynucleotide into a cell. The first polynucleotide is capable of specifically binding to the target nucleotide sequence and contains one or more mismatched nucleotides with respect to the target nucleotide sequence, and includes at least a first complementary site containing a complementary base sequence that specifically binds to the second polynucleotide, and includes a first 3' overhang site located at the 3' end of the first complementary site and / or a first 5' overhang site located at the 5' end of the first complementary site. The second polynucleotide includes at least a second complementary site comprising a complementary base sequence that specifically binds to the first complementary site of the first polynucleotide. A method for modifying one or more nucleotides contained in the target nucleotide sequence.

16. The method according to claim 15, wherein the cells are prokaryotic cells or eukaryotic cells.

17. The method according to claim 16, wherein the eukaryotic cell is at least one selected from the group consisting of plant cells, insect cells, and animal cells.

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