Non-natural polynucleotides for modification of target nucleotide sequence

Abstract

To provide a novel non-natural polynucleotide having structural characteristics that enable improved editing efficiency in a genome editing technique using a single-stranded non-natural polynucleotide including a crosslinked nucleic acid in a part of the nucleotide sequence.SOLUTION: The present invention provides a non-natural polynucleotide that can specifically bind to a target nucleotide sequence in double-stranded DNA in a cell to modify one or more nucleotides contained in the sequence, the non-natural polynucleotide containing one or more mismatched nucleotides relative to the sequence, having a crosslinked nucleic acid represented by at least one of (A) and (B), and further having characteristics (C) and (D). (A) One or more nucleotides adjacent on the 5' upstream side of the mismatched nucleotide are crosslinked nucleic acids. (B) One or more nucleotides adjacent on the 3' downstream side of the mismatched nucleotide are crosslinked nucleic acids. (C) The 5'-terminal nucleotide is a crosslinked nucleic acid. (D) The chain length is 22 to 95 nt.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an unnatural polynucleotide that can specifically bind to a target nucleotide sequence and is used for modifying one or more nucleotides included in the target nucleotide sequence in double-stranded DNA in a cell.

Background Art

[0002] CRISPR-Cas is a genome editing technology that applies the acquired immune mechanism of eubacteria and archaea and is used as a tool for genetic engineering. CRISPR-Cas9 (Patent Document 1) using a CRISPR (Clustered Regularly interspaced short palindromic repeats) sequence, which is a DNA sequence, and Cas9, which is a DNA cleavage enzyme of Streptococcus pyogenes, is characterized by inducing cleavage of target double-stranded DNA by recognizing the target DNA sequence with RNA, and is the most popular genome editing technology due to its simplicity, speed, and high efficiency. On the other hand, CRISPR-Cas9 has problems with off-target effects such as the guide RNA sequence misrecognizing the genome sequence and unexpected mutations being introduced at locations other than the target DNA sequence due to cleavage of double-stranded DNA.

[0003] Regarding the off-target effect of CRISPR-Cas, various solutions have been proposed. For example, CRISPR-Cas3, which utilizes the fact that the recognition sequence of Cas9 is 20 bases while the recognition sequence of Cas3 derived from Escherichia coli is 27 bases, enables more specific mutagenesis (Patent Document 2). A method using a complex in which a guide RNA capable of recognizing a DNA sequence is linked to a deaminase that performs nucleic acid base conversion and a mutant Cas nuclease in which the cleavage activity of one strand of double-stranded DNA is inactivated realizes safer and more specific genome editing compared to CRISPR-Cas9 without inducing double-stranded DNA cleavage (Patent Document 3).

[0004] However, even if the problem of off-target effects can be improved, since CRISPR-Cas is a technology for introducing a Cas nuclease derived from bacteria or a gene encoding the Cas nuclease into cells, the problem of unexpected risks due to foreign gene introduction still remains. As a genome editing technology that does not use the Cas nuclease protein, a genome editing technology using single-stranded synthetic DNA containing a modified nucleic acid is known.

[0005] As an example of a method for modifying a target nucleotide sequence in double-stranded DNA using single-stranded synthetic DNA in which a part of the nucleotide sequence is replaced with a locked nucleic acid (LNA), in a cell-free system experiment, at least one mismatched nucleotide and at least two LNAs are included, and each LNA is a method using an oligonucleotide arranged at a distance of at least one nucleotide from the at least one mismatched nucleotide is known (Patent Document 4).

[0006] As another example of a genome editing technology that does not use the Cas nuclease protein, it has been reported that by introducing single-stranded synthetic DNA in which a part of the nucleotide sequence is LNA into mouse ES cells, mutations of 1 to 3 bases can be introduced into the target nucleotide sequence (Non-Patent Document 1). It is suggested that the technology of Non-Patent Document 1 can improve the problem of off-target effects as compared with the genome editing technology using the Cas nuclease protein. The authors of Non-Patent Document 1 decoded the nucleotide sequences of the regions of 335 bp around the target nucleotide sequence for 33 cells in which the modification of the target nucleotide sequence was confirmed to have been performed as intended by nucleotide sequence decoding, and confirmed that no unintended modification occurred at locations other than the target nucleotide sequence. From this result, it is explained that very accurate genome editing was achieved in mouse-derived ES cells in which the mismatch repair mechanism was functioning by the single-stranded synthetic DNA used by the authors of Non-Patent Document 1. The authors of Non-Patent Document 1 have concluded that it is important for the mismatched nucleotide to be LNA in order to avoid the mismatch repair mechanism within cells and modify the target nucleotide sequence, and have conducted experiments with a focus on this. In fact, Non-Patent Document 1 has conducted experiments using more than 60 types of single-stranded synthetic DNAs, and at least the mismatched nucleotide of 41 types of single-stranded synthetic DNAs was LNA.

[0007] Non-Patent Document 2 is a follow-up report by the same authors as Non-Patent Document 1, which is based on the discovery in Non-Patent Document 1 that the mismatch repair mechanism within cells can be avoided when the mismatched nucleotide contained in the single-stranded synthetic DNA is LNA. Non-Patent Document 2 focuses on elucidating the mechanism of how the single-stranded synthetic DNA with a mismatched nucleotide being LNA performs genomic modification of mammalian cells.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0009]

Non-Patent Document 1

[0010] As a genome editing technique without using a Cas nuclease, several reports have been made on the modification of a target nucleotide sequence using single-stranded synthetic DNA containing at least one or more LNAs, but the editing efficiency is not sufficient.

[0011] An object of the present invention is to provide a new unnatural polynucleotide having a structural feature that enables improvement of editing efficiency in a genome editing technique using an unnatural polynucleotide composed of single-stranded synthetic DNA in which a part of the nucleotide sequence is a cross-linked nucleic acid. [Means for Solving the Problems]

[0012] As a result of repeated studies to solve the above problems, the present inventors have found that a non-natural polynucleotide that can specifically bind to a target nucleotide sequence for modifying one or more nucleotides included in the target nucleotide sequence in double-stranded DNA in a cell, which contains one or more mismatched nucleotides with respect to the target nucleotide sequence, nucleotides other than the mismatched nucleotides contain nucleotides complementary to the target nucleotide sequence, and at least one nucleotide adjacent to the mismatched nucleotide or the 5'-terminal nucleotide is a cross-linked nucleic acid, and is within a specific chain length range. By using this non-natural polynucleotide, the editing efficiency (yield) of the target nucleotide sequence is improved, and the present invention has been completed.

[0013] That is, the present invention relates to the following [1] to

[38] . [1] A non-natural polynucleotide that can specifically bind to a target nucleotide sequence for modifying one or more nucleotides included in the target nucleotide sequence in double-stranded DNA in a cell, which contains one or more mismatched nucleotides with respect to the target nucleotide sequence, and has a cross-linked nucleic acid shown in at least one of the following (A) and (B), and further has the characteristics of (C) and (D). (A) One or more nucleotides adjacent to the 5'-upstream side of the mismatched nucleotide are cross-linked nucleic acids (B) One or more nucleotides adjacent to the 3'-downstream side of the mismatched nucleotide are cross-linked nucleic acids (C) The 5'-terminal nucleotide is a cross-linked nucleic acid (D) The chain length is 22 to 95 nucleotides [2] The non-natural polynucleotide according to [1], wherein the non-natural polynucleotide further has the following characteristic (E). (E) The nucleotide at the 3'-end is a cross-linked nucleic acid 〔3〕 The unnatural polynucleotide according to 〔1〕 or 〔2〕 above, further having the following characteristic (F). (F) One or more nucleotides adjacent to the nucleotide at the 5'-end are cross-linked nucleic acids 〔4〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔3〕 above, further having the following characteristic (G). (G) One or more nucleotides adjacent to the nucleotide at the 3'-end are cross-linked nucleic acids 〔5〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔4〕 above, further having the following characteristic (J). (J) One or more phosphodiester bond portions between nucleotides are replaced with phosphate moiety-modified bonds

[0014] 〔6〕 The unnatural polynucleotide according to 〔5〕 above, wherein the phosphate moiety-modified bond contains at least one selected from the group consisting of phosphorothioate bond, methyl phosphate bond, boranophosphate bond, and mesylphosphoroamidate bond. 〔7〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔6〕 above, further having the following characteristic (M). (M) One or more nucleotides are arranged between the nucleotide adjacent to the 5'-upstream side of the mismatched nucleotide and the nucleotide at the 5'-end, and are arranged at a distance of at least one nucleotide from both the nucleotide adjacent to the 5'-upstream side of the mismatched nucleotide and the nucleotide at the 5'-end, and are cross-linked nucleic acids 〔8〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔7〕 above, further having the following characteristic (N). (N) One or more nucleotides that are arranged between the nucleotide adjacent to the 3'-downstream side of the mismatched nucleotide and the nucleotide at the 3'-end, and are arranged at a distance of at least one nucleotide from both the nucleotide adjacent to the 3'-downstream side of the mismatched nucleotide and the nucleotide at the 3'-end are cross-linked nucleic acids. 〔9〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔8〕 above, wherein the unnatural polynucleotide further has the following characteristics (O) and / or (X2). (O) The pentose sugar in one or more nucleotides adjacent to the cross-linked nucleic acid at the 3'-end is ribose. (X2) The pentose sugar in one or more nucleotides adjacent to the cross-linked nucleic acid at the 5'-end is ribose.

[0015] 〔10〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔9〕 above, wherein the unnatural polynucleotide further has the following characteristic (P). (P) The nucleotide at the 3'-end is phosphorylated. 〔11〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔10〕 above, wherein the cross-linked nucleic acid contains at least one selected from the group consisting of LNA, AmNA, BNA N-H, BNA N-Me and ENA. 〔12〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔11〕 above, wherein the unnatural polynucleotide further has the following characteristic (X1). (X1) The nucleotide at the 3'-end and / or one or more nucleotides adjacent to the nucleotide at the 3'-end are nucleic acids with a modified 2'-site. 〔13〕 The unnatural polynucleotide according to 〔12〕 above, wherein the nucleic acid with a modified 2'-site contains at least one selected from the group consisting of 2'-F, 2'-OMe, 2'-MOE and 2'-O-(2-carbamoylethyl). 〔14〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔13〕 above, wherein the unnatural polynucleotide further has the following characteristic (X4). (X4) An adapter is added to the 3'-end. 〔15〕 The unnatural polynucleotide according to 〔14〕 above, wherein the adapter has a function of inhibiting mismatch repair in a host cell. 〔16〕 The unnatural polynucleotide according to 〔14〕 or 〔15〕 above, wherein the adapter has a function of protecting the unnatural polynucleotide from nuclease digestion. 〔17〕 The unnatural polynucleotide according to any one of 〔14〕 to 〔16〕 above, wherein the adapter has a function of providing an editing efficiency higher than that of the unnatural polynucleotide defined by SEQ ID NO: 9 (designated as "GEO-8"). 〔18〕 The unnatural polynucleotide according to any one of 〔14〕 to 〔17〕 above, wherein the adapter is a nucleotide containing a mismatched nucleotide. 〔19〕 The unnatural polynucleotide according to any one of 〔14〕 to 〔18〕 above, wherein the adapter is a nucleotide that forms a stem structure which may have a loop.

[0016] 〔20〕 The unnatural polynucleotide according to any one of 〔14〕 to 〔17〕 above, wherein the adapter is a compound other than a nucleotide that modifies the end (designated as "modifying compound"). 〔21〕 The unnatural polynucleotide according to 〔20〕 above, wherein the molecular weight of the modifying compound is 2,000 or less. 〔22〕 The unnatural polynucleotide according to any one of 〔1〕 to 〔21〕 above, wherein the unnatural polynucleotide further has the following characteristic of (X5). (X5) One or more nucleotides are inserted between the mismatched nucleotide and the 3'-terminal nucleotide. 〔23〕 An unnatural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides included in the target nucleotide sequence in double-stranded DNA in a cell, which contains one or more mismatched nucleotides with respect to the target nucleotide sequence, A non-natural polynucleotide having a cross-linked nucleic acid shown in at least one of the following (A) and (B), having the characteristics of (D), and further having any one or more characteristics selected from the group consisting of (H2), (Y1), and (Y3). (A) One or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are cross-linked nucleic acids (B) One or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are cross-linked nucleic acids (D) The chain length is 22 to 95 nucleotides (H2) The phosphodiester bond portion between the 5'-terminal nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide is replaced by a phosphate moiety-modified bond (Y1) The 5'-terminal nucleotide is a mismatched nucleotide, and one or more nucleotides adjacent to the 5'-terminal nucleotide are mismatched nucleotides (Y3) An adapter is added to the 5' end

[0017]

[24] The non-natural polynucleotide according to

[23] above, wherein the adapter has a function of inhibiting mismatch repair in a host cell

[25] The non-natural polynucleotide according to

[23] or

[24] above, wherein the adapter has a function of protecting the non-natural polynucleotide from nuclease digestion

[26] The non-natural polynucleotide according to any one of

[23] to

[25] above, wherein the adapter has a function of providing a higher editing efficiency than the non-natural polynucleotide (GEO-8) defined by SEQ ID NO: 9

[27] The non-natural polynucleotide according to any one of

[23] to

[26] above, wherein the adapter is a modified compound and the molecular weight of the modified compound is 2,000 or less

[28] The non-natural polynucleotide according to any one of

[23] to

[27] above, wherein the non-natural polynucleotide further has the following characteristic (Y2). (Y2) The pentose sugar in the nucleotide at the 5'-end is ribose, and the pentose sugars in one or more nucleotides adjacent to the nucleotide at the 5'-end are ribose.

[29] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides included in the target nucleotide sequence in double-stranded DNA in a cell, including one or more mismatched nucleotides with respect to the target nucleotide sequence, and having a cross-linked nucleic acid shown in at least one of the following (A) and (B), and further having the characteristics of (D) and (X7). (A) One or more nucleotides adjacent to the 5'-upstream side of the mismatched nucleotide are cross-linked nucleic acids. (B) One or more nucleotides adjacent to the 3'-downstream side of the mismatched nucleotide are cross-linked nucleic acids. (D) The strand length is 22 to 95 nucleotides. (X7) One or more nucleotides arranged between the mismatched nucleotide and the nucleotide at the 5'-end are cross-linked nucleic acids.

[0018]

[30] The non-natural polynucleotide according to

[29] , wherein the cross-linked nucleic acid includes at least one selected from the group consisting of LNA, AmNA, BNA N-H, BNA N-Me, and ENA.

[31] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides included in the target nucleotide sequence in double-stranded DNA in a cell, including one or more mismatched nucleotides with respect to the target nucleotide sequence, and having all the characteristics of the following (C), (D), (I), and (X6). (C) The nucleotide at the 5'-end is a cross-linked nucleic acid. (D) The strand length is 22 to 95 nucleotides. (I) The phosphodiester bond portion between the nucleotide at the 3'-end and one or more nucleotides adjacent to the nucleotide at the 3'-end is replaced with a phosphorothioate bond (X6) The mismatched nucleotide is a cross-linked nucleic acid

[32] The unnatural polynucleotide according to

[31] above, wherein the unnatural polynucleotide further has the following characteristic (E). (E) The nucleotide at the 3'-end is a cross-linked nucleic acid

[33] The unnatural polynucleotide according to

[31] or

[32] above, wherein the unnatural polynucleotide further has the following characteristic (P). (P) The nucleotide at the 3'-end is phosphorylated

[34] A kit for modifying a target nucleotide sequence containing the unnatural polynucleotide according to any one of [1] to

[33] above.

[35] A pharmaceutical composition containing the unnatural polynucleotide according to any one of [1] to

[33] above.

[36] A method for modifying one or more nucleotides contained in a target nucleotide sequence in double-stranded DNA in a cell, comprising the step of introducing into the cell an unnatural polynucleotide containing one or more mismatched nucleotides with respect to the target nucleotide sequence, wherein the modification of the target nucleotide sequence includes at least one or more selected from the group consisting of deletion, insertion and substitution of one or more nucleotides of the target nucleotide sequence, and the unnatural polynucleotide is the unnatural polynucleotide according to any one of [1] to

[33] above, the method.

[37] The method according to

[36] above, wherein the cell is a prokaryotic cell or a eukaryotic cell.

[38] The method according to

[37] above, 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]

[0019] According to the present invention, a non-natural polynucleotide with excellent editing efficiency of a target nucleotide sequence is provided.

Brief Description of Drawings

[0020]

Figure 1

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Figure 3A

Figure 3B

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Figure 8A

Figure 8B

Figure 8C

Figure 9

Figure 10A

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Figure 16A

Figure 16B

Figure 17A

Figure 17B

Embodiments for Carrying Out the Invention

[0021] <Summary of the Present Invention> In the prior art using a single-stranded polynucleotide, the polynucleotide introduced into a cell specifically recognizes and binds to a target nucleotide sequence in the lagging strand of a replication fork during double-stranded DNA replication, and functions as a primer for Okazaki fragment synthesis by DNA polymerase. In this way, genome sequence editing occurs by incorporating the polynucleotide into the nascent DNA strand. On the other hand, since cells of organisms are equipped with a mismatch repair mechanism that repairs mismatches that occur during DNA replication, mismatches caused by polynucleotides introduced from the outside are also immediately repaired. One reason why the editing efficiency of the target nucleotide sequence by polynucleotides introduced from the outside into cells is lower than that of the modification of the target nucleotide sequence using nucleases is considered to be the correction of editing by this mismatch repair mechanism.

[0022] On the other hand, it is presumed that the unnatural polynucleotide of the present invention promotes editing by the presence of a crosslinked nucleic acid at a specific position in the unnatural polynucleotide, simultaneously reduces the influence of the mismatch repair mechanism, and can significantly increase the editing efficiency of the target nucleotide sequence compared to conventional reports.

[0023] In the present invention, in the unnatural polynucleotide, for example, the number and position of mismatch nucleotides, the number and position of crosslinked nucleic acids, the chain length of the polynucleotide, the type of crosslinked nucleic acid, substitution with a phosphate moiety modification bond (for example, phosphorothioate bond) of the phosphodiester bond portion between one or more nucleotides, substitution of ribose of the pentose sugar in one or more nucleotides (which may be abbreviated as "RNA substitution" in this specification), phosphorylation of the 3'-end or 5'-end, addition of an adapter, etc. are appropriately combined to further increase the efficiency of target nucleotide sequence modification.

[0024] <Nucleotide> In the present invention, "nucleotide" is a general term for substances in which a phosphate group is bonded to a nucleoside. A nucleoside is a compound in which a purine base or a pyrimidine base is glycosidically bonded to the 1-position of a pentose sugar. A chain-like biopolymer composed of nucleotides as units is a polynucleotide (also referred to as "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 commonly contains A, G, and T as in DNA, but instead of T, it contains uridine monophosphate (UMP, hereinafter referred to as "U").

[0025] <Double-stranded DNA> In the present invention, "double-stranded DNA" refers to DNA in which single-stranded DNAs having complementary base sequences form hydrogen bonds between bases in opposite directions to form a double helix. The double-stranded DNA in the present invention is not particularly limited, and examples include genomic DNA, mitochondrial DNA, and chloroplast DNA. Also, in the present invention, examples of the double-stranded DNA containing a "target nucleotide sequence" preferably include genes related to genetic diseases, and more preferably genes related to human genetic diseases.

[0026] For example, genes related to adrenoleukodystrophy (ABCD1), genes related to medium-chain acyl-CoA dehydrogenase deficiency (ACADM), genes related to Wilson's disease (ATP7B), genes related to hereditary pulmonary hypertension (BMPR2), genes related to hereditary pulmonary hypertension (BMPR2), genes related to X-linked agammaglobulinemia (BTK), genes related to cystinuria (CTNS), genes related to Duchenne muscular dystrophy (DMD), genes related to hemophilia A (F8), genes related to hemophilia B (F9), genes related to tyrosinemia (FAH), genes related to hepatic glycogenosis type Ia (G6PC), genes related to frontotemporal lobar degeneration (GRN), genes related to mucopolysaccharidosis type II (IDS), genes related to mucopolysaccharidosis type I (IDUA), genes related to primary immunodeficiency syndrome (IKBKB), genes related to familial hypertrophic cardiomyopathy (MYH7), genes related to peroxisome biogenesis disorder (PEX2), genes related to hepatic glycogenosis type IX (PHKA2), genes related to protein C deficiency (PROC), genes related to hepatic glycogenosis type V (PYGM), genes related to familial dilated cardiomyopathy (RBM20), genes related to α1-antitrypsin deficiency (SERPINA1), genes related to citrin deficiency (SLC25A13), genes related to cystinuria (SLC7A9), genes related to Niemann-Pick disease (SMPD1), and genes related to amyotrophic lateral sclerosis (SOD1), etc. can be mentioned. Note that all of these diseases and genes are human diseases and genes.

[0027] <Complementary nucleotide and mismatched nucleotide> In double-stranded DNA, G and C, and A and T are bonded by hydrogen bonds so that it usually becomes a combination of purine bases and pyrimidine bases. The nucleotide paired with a specific nucleotide is called a "complementary nucleotide".

[0028] In the present invention, the "mismatch nucleotide" refers to a nucleotide that cannot form a Watson-Crick type hydrogen bond between two bases, or a nucleotide for which there is no corresponding nucleotide, so that a Watson-Crick type hydrogen bond between two bases cannot be formed. That is, the "nucleotide that cannot form a Watson-Crick type hydrogen bond between two bases" refers to a nucleotide other than C for G, other than T for A, other than A for T, and other than G for C. The "fact that a Watson-Crick type hydrogen bond between two bases cannot be formed because there is no corresponding nucleotide" means that there is no corresponding nucleotide among all of A, T, G, and C for G, there is no corresponding nucleotide among all of A, T, G, and C for A, there is no corresponding nucleotide among all of A, T, G, and C for T, and there is no corresponding nucleotide among all of A, T, G, and C for G.

[0029] In the non-natural type polynucleotide of the present invention, there may be one or more "mismatch nucleotides" for modifying the base to be edited, and further, there may be one or more "mismatch nucleotides" that do not participate in the modification. The latter "mismatch nucleotides" are mismatch nucleotides for the purpose of improving the editing efficiency. For example, mismatch nucleotides inserted between the mismatch nucleotide for the modification purpose and the nucleotide at the 3'-end, or mismatch nucleotides in the nucleotide when the adapter is a nucleotide are applicable.

[0030] <Modification of the target nucleotide sequence> In the present invention, the "target nucleotide sequence" is a nucleotide sequence to which a non-natural type polynucleotide described later specifically binds. Here, "specifically binds" includes the following aspects 1 and 2. Aspect 1: When the non-natural type polynucleotide has only a mismatch with the base to be edited, it means binding to a sequence complementary to the non-natural type polynucleotide except for the mismatch with the base to be edited. Embodiment 2: When the non-natural polynucleotide has mismatches other than the mismatch with the base to be edited, it means binding to a sequence complementary to the non-natural polynucleotide excluding those mismatch portions.

[0031] The "modification" of the target nucleotide sequence refers to substituting a specific nucleotide (for example, G) with any one of the other three nucleotides (A, T, or C) for one or more nucleotides of the target nucleotide sequence (hereinafter referred to as "substitution"), deletion of one or more nucleotides of the target nucleotide sequence (hereinafter referred to as "deletion"), or insertion of another nucleotide or nucleotide sequence between two specific nucleotides on the target nucleotide sequence (hereinafter referred to as "insertion"). The "modification" of the target nucleotide sequence includes substitution, deletion, and insertion occurring individually and combinations of these occurring.

[0032] <Non-natural polynucleotide> In the present invention, the "non-natural polynucleotide" refers to a polynucleotide containing nucleotides other than the naturally occurring nucleotides (A, T, G, C, or U) (hereinafter referred to as "non-natural nucleotides") that constitute the polynucleotide. The origin of the non-natural nucleotides is not particularly limited and includes those artificially synthesized and those extracted and purified from substances containing non-natural nucleotides.

[0033] The unnatural nucleotides are not particularly limited, and examples include nucleic acids with a modified phosphate moiety, nucleic acids with a modified sugar moiety, nucleic acids with a modified base moiety, nucleic acids with an adapter added to the 3'-end and / or 5'-end, and nucleic acids with one or more nucleotides inserted between a mismatched nucleotide and the nucleotide at the 3'-end. Examples of nucleic acids with a modified phosphate moiety are not particularly limited, and include nucleic acids in which the phosphodiester bond portion 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, a nucleotide having a phosphorothioate bond is referred to as a "phosphorothioated" or "S-modified" nucleotide.

[0034] Other examples of nucleic acids with a modified phosphate moiety include nucleic acids having a methylphosphate bond, nucleic acids having a boranophosphate bond in which one of the non-bridging oxygen atoms of the phosphodiester bond is replaced with a borano group (BH3), nucleic acids having a mesylphosphoramidate bond in which one of the non-bridging oxygen atoms of the phosphodiester bond is replaced with -NSO2CH3, and the like.

[0035] Examples of nucleic acids with a modified sugar moiety are not particularly limited, and include nucleic acids with a modified 2'-site, crosslinked nucleic acids in which the 2'-site and 4'-site are crosslinked, and the like. Examples of nucleic acids with a modified 2'-site are not particularly limited, and include 2'-F (fluorinated), 2'-O-Methyl (2'-OMe), 2'-O-Methoxyethyl (2'-MOE), 2'-O-(2-carbamoylethyl), and the like.

[0036] Examples of crosslinked nucleic acids are not particularly limited, and include 2'-O,4'-C-methylene-Bridged Nucleic Acid (2',4'-BNA), BNA NC , bicyclic or tricyclic crosslinked nucleic acids, and other crosslinked nucleic acids, and the like. 2',4'-BNA is also called Locked Nucleic Acid (LNA) and has the structure shown below.

[0037] [Chem.]

[0038] Examples of BNA / LNA analogs include, but are not particularly limited to, 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, xylo-LNA, 2'-O,4'-C constrained ethyl (cEt) LNA, 2'-O,4'-C constrained 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), 3,4-dihydro-2H-pyran nucleic acid (DpNA), and the like.

[0039] BNA NC is not particularly limited, and examples include BNA N-H, BNA N-Me, and BNA N-Bn.

[0040] Examples of bicyclic or tricyclic bridged nucleic acids include, but are not particularly limited to, TriNA, α-L-TriNA, F-bcDNA, tricyclic DNA (tcDNA), F-tcDNA, bicyclic carbocyclic nucleotides, bicyclic DNA (bcDNA), 2'-C-bridged bicyclic nucleotides (CBBN), and the like.

[0041] Examples of other bridged nucleic acids include, but are not particularly limited to, oxetane nucleotides, locked PMO derived from 2'-amino-LNA, cyclohexenyl nucleic acid (CeNA), altritol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA (p-DNA), and the like.

[0042] In the unnatural polynucleotide of the present invention, if there are too many cross-linked nucleic acids, the editing efficiency tends to decrease. For example, as the ratio of cross-linked nucleic acids in the unnatural polynucleotide of the present invention, assuming that the total number of all nucleotides constituting the unnatural polynucleotide is 100%, the ratio of cross-linked nucleic acids is preferably 2 to 30%, more preferably 2 to 20%, and even more preferably 2 to 15%.

[0043] In the unnatural polynucleotide of the present invention, it is preferable to add an adapter to the 3'-end and / or 5'-end because higher editing efficiency is shown. The adapter in the present specification refers to, for example, those having a function of inhibiting mismatch repair of host cells, those having a function of protecting the unnatural polynucleotide of the present invention from nuclease digestion, and those having a function of providing higher editing efficiency than the unnatural polynucleotide (GEO-8) defined by SEQ ID NO: 9 by adding to the 3'-end and / or 5'-end. The adapter in the present invention has one or more of these functions, and more specific examples of such an adapter include (1) nucleotides as an adapter, and (2) compounds other than nucleotides that modify the ends (referred to as "modifying compounds" in the present specification).

[0044] The putative mechanism for inhibiting the mismatch repair of host cells by adding an adapter to the 3'-end and / or 5'-end of the unnatural polynucleotide of the present invention is as follows. Mismatch repair starts when one of the DNA strands forming a double strand has a 3'-end structure, because the intracellular mismatch repair enzyme recognizes the strand with the 3'-end as the target for repair, and then the mismatched nucleotides on the strand with the 3'-end are removed and repaired. When the unnatural polynucleotide of the present invention specifically binds to the target sequence, since the 3'-end structure exists in the unnatural polynucleotide, the mismatched nucleotides of the unnatural polynucleotide will be repaired. When a nucleotide as an adapter is added to the 3'-end of the unnatural polynucleotide, the 3'-end of the unnatural polynucleotide may be dissociated without specifically binding to the target sequence, so it is considered possible to avoid recognition by the mismatch repair enzyme. Similarly, when a modifying compound is added to the 3'-end of the unnatural polynucleotide, it is also considered possible that the 3'-end of the unnatural polynucleotide is avoided from recognition by the mismatch repair enzyme. For these reasons, it is presumed that the editing efficiency of the unnatural polynucleotide with a nucleotide or a modifying compound as an adapter added to the 3'-end is increased by inhibiting the mismatch repair of host cells.

[0045] The putative mechanism for protecting the non-natural polynucleotide of the present invention from nuclease digestion in the host cell by adding an adapter to the 3'-end and / or 5'-end of the non-natural polynucleotide of the present invention is as follows. A single-stranded non-natural polynucleotide introduced into a host cell is a target for digestion by nucleases in the host cell. Merely adding nucleotides as an adapter to the 5'-end or 3'-end of the non-natural polynucleotide cannot completely prevent the digestion of the non-natural polynucleotide by nucleases, but it is presumed that the nucleotides as an adapter act in a buffering manner and exhibit a function of suppressing the progression of digestion by exonucleases to mismatched nucleotides located in the center of the non-natural polynucleotide. On the other hand, by adding a modifying compound to the 3'-end, it is highly likely that digestion by exonucleases from the 3'-end is suppressed. For example, IdT is utilized to enhance the nuclease resistance of antisense nucleic acids by adding it to the 3'-end of the antisense nucleic acid.

[0046] As described above, since the function of inhibiting mismatch repair in the host cell and the function of protecting the non-natural polynucleotide of the present invention from nuclease digestion are based on different mechanisms, the non-natural polynucleotide of the present invention may have both of these functions.

[0047] Examples of the structure of the nucleotides as the adapter include linear nucleotides and nucleotides that form a stem structure that may have a loop. Also, the chain length of the nucleotides as the adapter is preferably 1 nt (nucleotide) or more, more preferably 3 nt or more, still more preferably 6 or more, while preferably 50 nt or less, more preferably 40 nt or less, still more preferably 30 or less. Since the non-natural polynucleotide of the present invention does not contain nucleotides as an adapter, the chain length of the nucleotides as an adapter does not affect the chain length of the non-natural polynucleotide of the present invention. The nucleotide as an adapter is preferably a nucleotide containing a mismatched nucleotide, and the nucleotide sequence of the nucleotide as an adapter is more preferably all mismatches with the 5' upstream sequence adjacent to the target nucleotide sequence or the 3' downstream sequence adjacent to the target nucleotide sequence, but one or more may be complementary to the target nucleotide sequence.

[0048] The modified compound as the adapter is preferably a compound that can bind to the 3' end and / or 5' end of the unnatural polynucleotide of the present invention. The modified compound is preferably a compound of a certain size. For example, the molecular weight of the modified compound is preferably 50 or more, more preferably 100 or more, while preferably 2,000 or less, more preferably 1,500 or less, still more preferably 1,000 or less, still more preferably 800 or less, still more preferably 500 or less. Preferred specific examples of the modified compound include at least one selected from the group consisting of fluorescein (FAM), biotin, puromycin, cholesterol, digoxigenin (DIG), and Inverted dT. The binding of the adapter to the unnatural polynucleotide of the present invention can be carried out by a known method.

[0049] These unnatural nucleotides having such modifications have a high affinity for natural DNA and RNA, so their application to nucleic acid pharmaceuticals and the like is being promoted.

[0050] <Basic composition of unnatural polynucleotide> The unnatural polynucleotide of the present invention is a single-stranded polynucleotide, which contains one or more mismatched nucleotides with respect to the target nucleotide sequence, and the nucleotides other than the mismatched nucleotides contain nucleotides complementary to the target nucleotide sequence. Therefore, by introducing the unnatural polynucleotide of the present invention into cells, it can specifically bind to the target nucleotide sequence. Thereby, one or more nucleotides in the target nucleotide sequence can be modified to desired nucleotides. When there are a plurality of the mismatched nucleotides, two or more mismatched nucleotides may be adjacent to each other or may be present at separated positions. In the present invention, "a plurality" with respect to the number of 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 ranges with these as the upper and lower limits are also included. For example, 2 to 3, 2 to 4, 2 to 5, ··· 2 to 20, 3 to 4, 3 to 5, 3 to 6, ··· 3 to 20, 4 to 5, 4 to 6, 4 to 7, ··· 4 to 20, 5 to 6, 5 to 7, 5 to 8, ··· 5 to 20, ··· 18 to 19, 18 to 20, 19 to 20.

[0051] Examples of the basic configuration of such an unnatural polynucleotide of the present invention include those described in the following aspects (i) to (vi).

[0052] (1) Aspect (i) One aspect (aspect (i)) of the unnatural polynucleotide of the present invention is (A) One or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are cross-linked nucleic acids, (B) One or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are cross-linked nucleic acids, (C) The nucleotide at the 5' end is a cross-linked nucleic acid, and (D) The strand length is 22 to 95 nucleotides and has such a configuration. Such a configuration can exhibit the effects of the present invention.

[0053] The cross-linked nucleic acids contained in the unnatural polynucleotides of aspect (i) are not particularly limited, and examples include at least one selected from the group consisting of LNA, AmNA, BNA N-H, BNA N-Me, and ENA. In the present invention, as the cross-linked nucleic acid of (A) and / or (B), LNA or BNA N-H is preferable, and LNA is more preferable. In the present invention, as the cross-linked nucleic acid of (C), LNA, AmNA, BNA N-H, BNA N-Me, and ENA are preferable.

[0054] Here, the mismatched nucleotide may or may not be a cross-linked nucleic acid. However, in aspect (i), it is more preferable that the mismatched nucleotide is not a cross-linked nucleic acid. Among the total three nucleotides including the mismatched nucleotide and adjacent thereto, the number of cross-linked nucleic acids is preferably two. Further, the "plurality" in "a plurality of nucleotides are cross-linked nucleic acids" defined by (A) and (B) is not particularly limited, but the case where there are two consecutive cross-linked nucleic acids is preferable rather than three or more consecutive cross-linked nucleic acids, and one is more preferable.

[0055] (2) Aspect (ii) Another aspect (aspect (ii)) of the unnatural polynucleotides of the present invention is (A) one or more nucleotides adjacent to the 5'-upstream side of the mismatched nucleotide are cross-linked nucleic acids, (C) the 5'-terminal nucleotide is a cross-linked nucleic acid, and (D) the strand length is 22 to 95 nucleotides and has such a configuration (however, it does not have the feature of (B)). Even with such a configuration, the effects of the present invention can be exhibited.

[0056] The cross-linked nucleic acids contained in the unnatural polynucleotides of aspect (ii) are not particularly limited, and examples include at least one selected from the group consisting of LNA, AmNA, BNA N-H, BNA N-Me, and ENA. In the present invention, the cross-linked nucleic acid contained in the unnatural polynucleotide is preferably LNA.

[0057] Here, the mismatched nucleotide may or may not be a cross-linked nucleic acid. However, in embodiment (ii), it is more preferable that the mismatched nucleotide is not a cross-linked nucleic acid. Among the total three nucleotides including the mismatched nucleotide and adjacent thereto, the number of cross-linked nucleic acids is preferably two. Further, the "plurality" in "a plurality of nucleotides are cross-linked nucleic acids" defined in (A) above is not particularly limited, but the case where there are two consecutive cross-linked nucleic acids is preferable to the case where there are three or more consecutive cross-linked nucleic acids, and one is more preferable.

[0058] (3) Embodiment (iii) A further embodiment (embodiment (iii)) of the unnatural polynucleotide of the present invention is (B) one or more nucleotides adjacent to the 3'-downstream side of the mismatched nucleotide are cross-linked nucleic acids, (C) the 5'-terminal nucleotide is a cross-linked nucleic acid, and (D) the strand length is 22 to 95 nucleotides (however, it does not have the feature of (A) above). Even with such a configuration, the effects of the present invention can be exhibited.

[0059] The cross-linked nucleic acid contained in the unnatural polynucleotide of embodiment (iii) is not particularly limited, but examples thereof include at least one selected from the group consisting of LNA, AmNA, BNA N-H, BNA N-Me, and ENA. In the present invention, LNA is preferable as the cross-linked nucleic acid contained in the unnatural polynucleotide.

[0060] Here, the mismatched nucleotide may or may not be a cross-linked nucleic acid. However, in embodiment (iii), it is more preferable that the mismatched nucleotide is not a cross-linked nucleic acid. Among the total three nucleotides including the mismatched nucleotide and adjacent thereto, the number of cross-linked nucleic acids is preferably two. Further, the "plurality" in "a plurality of nucleotides are cross-linked nucleic acids" defined in (B) above is not particularly limited, but the case where there are two consecutive cross-linked nucleic acids is preferable to the case where there are three or more consecutive cross-linked nucleic acids, and one is more preferable.

[0061] [Chain length of unnatural polynucleotide] As described above, the chain length of the unnatural polynucleotide of the present invention is 22 to 95 nt (nucleotide), preferably 23 to 95 nt, more preferably 24 to 95 nt, still more preferably 25 to 95 nt, still more preferably 27 to 62 nt, and still more preferably 35 to 53 nt. When the chain length of the unnatural polynucleotide is within such a numerical range, it is preferable because the target polynucleotide sequence can be modified with high editing efficiency.

[0062] [Relationship between mismatched nucleotide and crosslinked nucleic acid adjacent thereto] In any of the above aspects (i) to (iii), the unnatural polynucleotide of the present invention can exhibit sufficient editing efficiency with respect to the target nucleotide sequence even when, for example, the mismatched nucleotide is a crosslinked nucleic acid. In this case, it is more preferable that the crosslinked nucleic acid is LNA.

[0063] In the present invention, at least one of the nucleotides adjacent to the mismatched nucleotide is a crosslinked nucleic acid. In the relationship between the mismatched nucleotide and the crosslinked nucleic acid adjacent thereto, the number of consecutive crosslinked nucleic acids is not particularly limited, but when there are a plurality of consecutive crosslinked nucleic acids including the case where the mismatched nucleotide is a crosslinked nucleic acid, the number of consecutive crosslinked nucleic acids is preferably 2.

[0064] [3'-end is a crosslinked nucleic acid] In any of the above aspects (i) to (iii), the unnatural polynucleotide of the present invention can further enhance the editing efficiency of the target nucleotide sequence, for example, when the nucleotide at the 3'-end is a crosslinked nucleic acid.

[0065] [Nucleotide adjacent to 5'-end is a crosslinked nucleic acid] In any of the above aspects (i) to (iii), or in addition to the above (E), for example, when one or more nucleotides adjacent to the 5'-terminal nucleotide are cross-linked nucleic acids, the editing efficiency of the target nucleotide sequence can be further enhanced. In this case, it is preferable that one nucleotide adjacent to the 5'-terminal nucleotide is a cross-linked nucleic acid. Further, it is more preferable that the cross-linked nucleic acid is LNA. In this case, the number of consecutive cross-linked nucleic acids at the 5'-terminal nucleotide is not particularly limited. For example, when the number from the mismatched nucleotide to the 5'-terminal is 12 nt, 2 is preferable; when the number at the 5'-terminal is 25 nt, 3 is preferable.

[0066] [The nucleotide adjacent to the 3'-terminal is a cross-linked nucleic acid] In any of the above aspects (i) to (iii), or in addition to the above (E) and / or (F), for example, when one or more nucleotides adjacent to the 3'-terminal nucleotide are cross-linked nucleic acids, the editing efficiency of the target nucleotide sequence can be further enhanced. In this case, it is more preferable that the cross-linked nucleic acid is LNA. In this case, the number of consecutive cross-linked nucleic acids at the 3'-terminal nucleotide is not particularly limited. For example, when the number from the mismatched nucleotide to the 3'-terminal is 27 nt, 3 or less is preferable, more preferably 2 or less, and even more preferably 1. Further, for example, when the number from the mismatched nucleotide to the 3'-terminal is 12 nt, the number of consecutive cross-linked nucleic acids at the 3'-terminal nucleotide is preferably 3 or less, more preferably 2 or less, and even more preferably 1.

[0067] [Substitution with a phosphate moiety-modified bond] In any of the above aspects (i) to (iii), or in addition to the above (E), (F) and / or (G), for example, when one or more phosphodiester bond portions between nucleotides are replaced with phosphate moiety-modified bonds, the editing efficiency of the present invention can be further enhanced. Specific configurations are not particularly limited, but the following (J) can be mentioned.

[0068] (J) One or more phosphodiester bond moieties between nucleotides are replaced with phosphate moiety-modified bonds. In the present invention, the phosphate moiety-modified bond is a bond in which some atoms of the phosphodiester bond are replaced with another atom or substituent, for example, a sulfur atom, a boron atom, a nitrogen atom, a methyl group, or an ester group. Specific examples of the phosphate moiety-modified bond include a phosphorothioate bond, a methylphosphate bond, a boranophosphate bond, and a mesylphosphoramidate bond. In the unnatural polynucleotide of the present invention, the number and position of the phosphate moiety-modified bonds are not particularly limited. Further, the types of phosphate moiety-modified bonds in the unnatural polynucleotide of the present invention may be one type, or two or more types may be mixed. The number of phosphate moiety-modified bonds may be 1, or the ratio of the phosphate moiety-modified bonds in the phosphodiester bond may be 60% or less. However, from the viewpoint of editing efficiency, it is preferable that not all of the phosphodiester bonds are replaced.

[0069] One embodiment of (J) is as follows. (X3) The phosphodiester bond moiety between the nucleotide at the 3'-end and one or more nucleotides adjacent to the nucleotide at the 3'-end is replaced with a phosphate moiety-modified bond.

[0070] In (J), from the viewpoint of editing efficiency, a phosphorothioate bond is preferable as the phosphate moiety-modified bond. Embodiments in which the phosphate moiety-modified bond is a phosphorothioate bond include any one or a combination of the following (H1), (I), (K), and (L). (H1) The phosphodiester bond moiety between the nucleotide at the 5'-end and one or more nucleotides adjacent to the nucleotide at the 5'-end is replaced with a phosphorothioate bond. (I) The phosphodiester bond moiety between the nucleotide at the 3'-end and one or more nucleotides adjacent to the nucleotide at the 3'-end is replaced with a phosphorothioate bond. (K) The phosphodiester bond portion between the cross-linked nucleic acid adjacent to the 5'-upstream side of the mismatched nucleotide and one or more nucleotides further adjacent to the cross-linked nucleic acid is replaced with a phosphorothioate bond. (L) The phosphodiester bond portion between the cross-linked nucleic acid adjacent to the 3'-downstream side of the mismatched nucleotide and one or more nucleotides further adjacent to the cross-linked nucleic acid is replaced with a phosphorothioate bond.

[0071] In the above features (K) and (L), the phosphodiester bond portion between the cross-linked nucleic acid and one nucleotide directly adjacent to the cross-linked nucleic acid may or may not be replaced with a phosphorothioate bond.

[0072] The unnatural polynucleotide of the present invention may have both of the above features (H1) and (I). In this case, the unnatural polynucleotide of the present invention preferably has the feature that "the phosphodiester bond portions between the nucleotide at the 5'-end, the consecutive one or more nucleotides adjacent to the nucleotide at the 5'-end, the nucleotide at the 3'-end, and the consecutive one or more nucleotides adjacent to the nucleotide at the 3'-end are replaced with phosphorothioate bonds".

[0073] [There is a cross-linked nucleic acid between the cross-linked nucleic acid adjacent to the 5'-upstream side of the mismatched nucleotide and the cross-linked nucleic acid at the 5'-end] The unnatural polynucleotide of the present invention, in any of the above aspects (i) to (iii), or further in addition to the above (E), (F), (G) and / or (J), for example, (M) is arranged between the nucleotide adjacent to the 5'-upstream side of the mismatched nucleotide and the nucleotide at the 5'-end, and is arranged at a distance of at least one nucleotide from both the nucleotide adjacent to the 5'-upstream side of the mismatched nucleotide and the nucleotide at the 5'-end. When one or more nucleotides are cross-linked nucleic acids, the editing efficiency of the target nucleotide sequence can be further enhanced.

[0074] [There is a crosslinked nucleic acid between the crosslinked nucleic acid adjacent to the 3'-downstream side of the mismatched nucleotide and the crosslinked nucleic acid at the 3'-end] In any of the above aspects (i) to (iii), or further in addition to the above (E), (F), (G), (J) and / or (M), for example, (N) when one or more nucleotides arranged between the nucleotide adjacent to the 3'-downstream side of the mismatched nucleotide and the nucleotide at the 3'-end and spaced at least one nucleotide distance from either the nucleotide adjacent to the 3'-downstream side of the mismatched nucleotide or the nucleotide at the 3'-end are crosslinked nucleic acids, the editing efficiency of the target nucleotide sequence can be further enhanced.

[0075] [RNA substitution] Although the unnatural polynucleotide of the present invention is a single-stranded polynucleotide, the deoxyribose in one or more nucleotides may be replaced by ribose. Specifically, in any of the above aspects (i) to (iii), or further in addition to the above (E), (F), (G), (J), (M) and / or (N), for example, (O) when the pentose sugar in one or more nucleotides adjacent to the crosslinked nucleic acid at the 3'-end is ribose, and / or (X2) when the pentose sugar in one or more nucleotides adjacent to the crosslinked nucleic acid at the 5'-end is ribose, the editing efficiency of the target nucleotide sequence can be further enhanced.

[0076] [Phosphorylation at the 3'-end] In any of the above aspects (i) to (iii), or further in addition to the above (E), (F), (G), (J), (M), (N), (O) and / or (X2), for example, (P) when the nucleotide at the 3'-end is phosphorylated, the editing efficiency of the target nucleotide sequence can be further enhanced.

[0077] [Modification to the sugar moiety of nucleic acid] In any of the above aspects (i) to (iii), or further in addition to the above (E), (F), (G), (J), (M), (N), (O), (X2) and / or (P), the unnatural polynucleotide of the present invention, when a modification to the sugar moiety of the nucleic acid, for example, (X1) the nucleotide at the 3'-end and / or one or more nucleotides adjacent to the nucleotide at the 3'-end are replaced with a nucleic acid having a modified 2'-site, can further enhance the editing efficiency of the target nucleotide sequence. Examples of the nucleic acid having a modified 2'-site include at least one selected from the group consisting of 2'-F, 2'-OMe, 2'-MOE and 2'-O-(2-carbamoylethyl).

[0078] [Addition of an adapter to the 3'-end] In any of the above aspects (i) to (iii), or further in addition to the above (E), (F), (G), (J), (M), (N), (O), (X2), (P) and / or (X1), the unnatural polynucleotide of the present invention, when an adapter is added to the 3'-end (X4), can further enhance the editing efficiency of the target nucleotide sequence.

[0079] [Insertion of a nucleotide between the mismatched nucleotide and the nucleotide at the 3'-end] In any of the above aspects (i) to (iii), or further in addition to the above (E), (F), (G), (J), (M), (N), (O), (X2), (P), (X1) and / or (X4), when (X5) one or more nucleotides are inserted between the mismatched nucleotide and the 3'-terminal nucleotide, the editing efficiency of the target nucleotide sequence can be further enhanced. The "mismatched nucleotide" in (X5) refers to the mismatched nucleotide for the purpose of modification. Further, in (X5), the inserted nucleotide becomes a mismatched nucleotide, but the mismatched nucleotide here is for the purpose of improving the editing efficiency and does not correspond to the mismatched nucleotide for the purpose of modification. Further, the inserted nucleotide may be a nucleic acid in which the above-mentioned phosphate moiety is modified, a nucleic acid in which the above-mentioned sugar moiety is modified, or a nucleic acid in which the above-mentioned base moiety is modified. In (X5), the number of inserted nucleotides may be 1, or in the case of a plurality, preferably 2 or more, while preferably 6 or less, more preferably 3 or less.

[0080] (4) Aspect (iv) A further aspect (aspect (iv)) of the non-natural type polynucleotide of the present invention is (A) one or more nucleotides adjacent to the 5'-upstream side of the mismatched nucleotide are cross-linked nucleic acids, and / or (B) one or more nucleotides adjacent to the 3'-downstream side of the mismatched nucleotide are cross-linked nucleic acids, (D) The strand length is 22 to 95 nucleotides, and further (H2) The phosphodiester bond portion between the 5'-terminal nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide is replaced by a phosphate moiety-modified bond, (Y1) the 5'-terminal nucleotide is a mismatched nucleotide, and one or more nucleotides adjacent to the 5'-terminal nucleotide are mismatched nucleotides, and (Y3) an adapter is added to the 5'-terminal, and is one or more selected from the group consisting of It is such a configuration. Such a configuration can exhibit the effects of the present invention. (A), (B), and (D) in embodiment (iv) are the same as those in embodiment (i).

[0081] Here, the mismatched nucleotide may or may not be a cross-linked nucleic acid, but in embodiment (iv), it is more preferable that the mismatched nucleotide is not a cross-linked nucleic acid.

[0082] [Substitution to phosphate group-modified bond] In the unnatural polynucleotide of the present invention, from the viewpoint of editing efficiency, in the above-mentioned embodiment (iv), it is preferable that the phosphate diester bond portion between the nucleotide at the 5'-end and one or more nucleotides adjacent to the nucleotide at the 5'-end is substituted with a phosphate group-modified bond.

[0083] In the present invention, the phosphate group-modified bond is a bond in which some atoms of the phosphate diester bond are substituted with another atom or substituent, for example, a sulfur atom, a boron atom, a nitrogen atom, a methyl group, or an ester group. Specific examples of the phosphate group-modified bond include a phosphorothioate bond, a methyl phosphate bond, a boranophosphate bond, and a mesylphosphoramidate bond. In the unnatural polynucleotide of the present invention, the number and position of the phosphate group-modified bonds are not particularly limited. Furthermore, the type of the phosphate group-modified bond in the unnatural polynucleotide of the present invention may be one type, or two or more types may be mixed. The number of the phosphate group-modified bonds may be 1, or the ratio of the phosphate group-modified bonds in the phosphate diester bond may be 60% or less, but from the viewpoint of editing efficiency, it is preferable that not all of the phosphate diester bonds are substituted.

[0084] [Introduction of mismatched nucleotide to 5'-end side] In the non-natural polynucleotide of the present invention, from the viewpoint of editing efficiency in the above aspect (iv), it is preferable that (Y1) the nucleotide at the 5'-end is a mismatched nucleotide and one or more nucleotides adjacent to the nucleotide at the 5'-end are mismatched nucleotides. The mismatched nucleotides in (Y1) are "mismatched nucleotides for the purpose of improving editing efficiency" and do not correspond to "mismatched nucleotides for modifying the target nucleotide sequence". When there are a plurality of mismatched nucleotides in (Y1), the mismatched nucleotides may or may not be adjacent to each other. Further, the number thereof is preferably 2 or more, while preferably 10 or less, and more preferably 7 or less.

[0085] [Addition of adapter to 5'-end] In the non-natural polynucleotide of the present invention, from the viewpoint of editing efficiency in the above aspect (iv), it is preferable that (Y3) an adapter is added to the 5'-end. Details of the adapter are as described above.

[0086] [RNA substitution] The non-natural polynucleotide of the present invention is a single-stranded polynucleotide, but the deoxyribose in one or more nucleotides may be substituted with ribose. Specifically, in aspect (iv), for example, when (Y2) the pentose sugar in the nucleotide at the 5'-end is ribose and the pentose sugar in one or more nucleotides adjacent to the nucleotide at the 5'-end is ribose, the editing efficiency of the target nucleotide sequence can be further enhanced.

[0087] (5) Aspect (v) A further aspect (aspect (v)) of the non-natural polynucleotide of the present invention is (A) one or more nucleotides adjacent to the 5'-upstream side of the mismatched nucleotide are cross-linked nucleic acids, and / or (B) one or more nucleotides adjacent to the 3'-downstream side of the mismatched nucleotide are cross-linked nucleic acids, (D) the strand length is 22 to 95 nucleotides, (X7) One or more nucleotides arranged between the mismatched nucleotide and the 5'-terminal nucleotide are cross-linked nucleic acids. It is such a configuration. Such a configuration can exhibit the effects of the present invention. (A), (B) and (D) in embodiment (v) are the same as those in embodiment (i).

[0088] Here, the mismatched nucleotide may or may not be a cross-linked nucleic acid, but in embodiment (v), it is more preferable that the mismatched nucleotide is not a cross-linked nucleic acid.

[0089] In the unnatural polynucleotide of the present invention, in the above-mentioned embodiment (v), from the viewpoint of editing efficiency, (X7) one or more nucleotides arranged between the mismatched nucleotide and the 5'-terminal nucleotide are cross-linked nucleic acids. The cross-linked nucleic acid defined by (X7) is not particularly limited, but at least one selected from the group consisting of LNA, AmNA, BNA N-H, BNA N-Me and ENA can be mentioned, and LNA is preferable.

[0090] (6) Embodiment (vi) A further embodiment (embodiment (vi)) of the unnatural polynucleotide of the present invention is (C) The 5'-terminal nucleotide is a cross-linked nucleic acid, (D) The chain length is 22 to 95 nucleotides, (I) The phosphodiester bond portion between the 3'-terminal nucleotide and one or more nucleotides adjacent to the 3'-terminal nucleotide is replaced by a phosphorothioate bond, (X6) The mismatched nucleotide is a cross-linked nucleic acid It is such a configuration. Such a configuration can exhibit the effects of the present invention. (C), (D) and (I) in embodiment (vi) are the same as those in embodiment (i).

[0091] In aspect (vi) of the unnatural polynucleotide of the present invention, from the viewpoint of editing efficiency, (X6) the mismatched nucleotide is a crosslinked nucleic acid. The crosslinked nucleic acid defined by (X6) is not particularly limited, and examples thereof include at least one selected from the group consisting of LNA, AmNA, BNA N-H, BNA N-Me, and ENA, and LNA is preferred.

[0092] [The 3'-end is a crosslinked nucleic acid] In the aspect (vi) of the unnatural polynucleotide of the present invention, for example, when (E) the nucleotide at the 3'-end is a crosslinked nucleic acid, the editing efficiency of the target nucleotide sequence can be further enhanced. The crosslinked nucleic acid defined by (E) is not particularly limited, and examples thereof include at least one selected from the group consisting of LNA, AmNA, BNA N-H, BNA N-Me, and ENA, and LNA is preferred.

[0093] [Phosphorylation at the 3'-end] In the aspect (vi) of the unnatural polynucleotide of the present invention, or in addition to the above (E), for example, when (P) the nucleotide at the 3'-end is phosphorylated, the editing efficiency of the target nucleotide sequence can be further enhanced.

[0094] <Editing efficiency> The efficiency of modifying the target nucleotide sequence by the unnatural polynucleotide of the present invention (referred to as "editing efficiency" herein) is relatively expressed with the editing efficiency in the case of using GEO-8 in the following examples, which is the unnatural polynucleotide represented by SEQ ID NO: 9, as 1.0. For example, it is in the range of 0.4 or more, preferably 0.5 or more, more preferably 0.6 or more.

[0095] <Kit> In the present invention, a "kit" is a genetic engineering tool used for modifying a target nucleotide sequence containing a non-natural polynucleotide. The kit of the present invention may include, within the scope not impairing the effects of the present invention, a buffer, a stabilizer, a preservative, other reagents, an instruction manual describing a protocol for modifying a target nucleotide sequence with a non-natural polynucleotide, and the like.

[0096] <Pharmaceutical composition> By using a pharmaceutical composition containing the non-natural polynucleotide of the present invention (hereinafter referred to as the "pharmaceutical composition" of the present invention), a pharmaceutical composition capable of treating a target disease can be provided by modifying a gene having a mutation that does not function normally into a gene that functions normally by the action of the non-natural polynucleotide.

[0097] When using the pharmaceutical composition of the present invention, for example, it can be administered orally, intravenously, intramuscularly, transmucosally, rectally, vaginally, transdermally, via the nasal cavity, or via inhalation. Alternatively, an ex vivo treatment method can be used in which the pharmaceutical composition of the present invention is used for cells taken out from a patient, the treated cells are cultured as needed, and then returned to the patient's body. The non-natural polynucleotide of the pharmaceutical composition of the present invention may be formulated alone or in combination with other components, but it can also be provided in the form of a preparation by formulating it with a pharmaceutically acceptable carrier or additive for preparation. Pharmaceutically acceptable carriers or additives are not particularly limited, and examples include lipid nanoparticles, excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, pigments, diluents, solubilizers, solubilization aids, isotonic agents, pH adjusters, stabilizers, and the like.

[0098] The administration of the pharmaceutical composition of the present invention depends on the severity and responsiveness of the condition to be treated and continues for several days to several months, or until cure is achieved, or until remission of the condition is achieved. A person skilled in the art can determine the optimal dosage, administration method, and frequency of repetition.

[0099] <Diseases targeted by the pharmaceutical composition> Examples of diseases targeted by the pharmaceutical composition of the present invention include diseases caused by substitution of a single nucleotide in the genome in humans, 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 glycogenosis type Ia, familial frontotemporal dementia, mucopolysaccharidosis type II, mucopolysaccharidosis type I, primary immunodeficiency syndrome, familial hypertrophic cardiomyopathy, peroxisome biogenesis disorder, hepatic glycogenosis type IX, protein C deficiency, hepatic glycogenosis type V, familial dilated cardiomyopathy, α1-antitrypsin deficiency, citrin deficiency, cystinuria, Niemann-Pick disease, and amyotrophic lateral sclerosis.

[0100] <Preparation of unnatural polynucleotides> The unnatural polynucleotides in the present invention can be easily prepared by methods known in the art of the present invention.

[0101] <Introduction of unnatural polynucleotides into cells> In the present invention, "introducing a non-natural polynucleotide into a cell" can be carried out according to known methods depending on the cell to be introduced. Known methods for introducing polynucleotides (transfection methods) are roughly classified into two types: viral vector systems and non-viral vector systems. The viral vector system is a method of introducing a gene into a cell by utilizing the cell entry mechanism originally possessed by a virus. Although not particularly limited, it is a method using adenovirus, retrovirus, lentivirus, etc. as vectors. Examples of the non-viral vector system include, but are not particularly limited to, the lipofection method, the electroporation method, the microinjection method, the particle gun method, etc. The lipofection method is a method that utilizes the phenomenon in which a cationic liposome having a positive charge binds around a polynucleotide having a negative charge to form a complex, and the polynucleotide is taken up from the cell surface into the cell by the endocytosis phenomenon. The electroporation method is a method in which a high-voltage pulse is directly applied to a cell using a dedicated machine, and the polynucleotide is taken up through small pores formed on the cell surface. The microinjection method is a method in which a substance to be introduced is placed in a glass needle having a tip diameter of about 1 μm and directly introduced into the cell. The particle gun method is a method in which DNA is coated on the surface of gold particles by co-precipitating gold particles and DNA, and the gold particles are ejected toward target cells by a helium gas pressure or the like. The present invention is a method for modifying a target nucleotide sequence by introducing only a non-natural polynucleotide without introducing a Cas nuclease into a cell, and from the viewpoint of safety, a non-viral vector system method is more preferable. The method of the present invention includes, for example, a method carried out in the human body or a method including a step carried out in the human body.

[0102] The unnatural polynucleotide introduced into the cell in this way (also referred to as "editing nucleic acid" in this specification) selectively binds to the target nucleotide sequence in genomic DNA, as schematically shown in FIG. 1. The unnatural polynucleotide of the present invention is mismatched with the base to be edited in the target nucleotide sequence in double-stranded DNA in the cell such as genomic DNA, and it is preferable to design so that all of the 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 conflict with the object of the present invention.

[0103] When the base to be edited in the target nucleotide sequence is, for example, T and it is desired to modify it to C, the mismatched nucleotide in the unnatural polynucleotide of the present invention is set to G. A person skilled in the art can set the mismatched nucleotide in the unnatural polynucleotide in order to modify the base of the base to be edited to a desired base based on common general knowledge.

[0104] <Method for modifying one or more nucleotides included in a target nucleotide sequence in double-stranded DNA in a cell> The "method for modifying one or more nucleotides included in a target nucleotide sequence in double-stranded DNA in a cell" of the present invention includes the step of introducing the above-described unnatural polynucleotide of the present invention into a cell. Here, the modification of the target nucleotide sequence includes at least one or more selected from the group consisting of deletion, insertion, and substitution of one or more nucleotides of the target nucleotide sequence. As the unnatural polynucleotide used in the modification method of the present invention, those described in the above aspects (i) to (vi) can be preferably used.

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

[0106] <Cell> The method for modifying a target nucleotide sequence using the non-natural polynucleotide of the present invention can be applied to all cells having double-stranded DNA. Furthermore, as long as the DNA replication mechanism in the cell can be utilized, not only the double-stranded DNA inherent in the cell, but also the single-stranded DNA derived from the virus in the virus-infected cell can be modified as the target nucleotide sequence. That is, the "cell" of the present invention includes prokaryotic cells and eukaryotic cells.

[0107] <Prokaryotic cell> In the present invention, the "prokaryotic cell" is not particularly limited, and examples include bacterial cells and archaeal cells.

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

[0109] <Animal cell> In the present invention, the "animal cell" is not particularly limited, and includes 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, and examples include fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells, brain cells, kidney cells, and the like. Stem cells are not particularly limited, and examples include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).

[0110] <Mammalian cell> In the present invention, "mammal" refers to a group of vertebrates belonging to the class Mammalia, and humans are also included in mammals. "Mammalian cells" refer to cells that make up a mammal or cells derived from a mammal. Examples of mammals other than humans include, but are not particularly limited to, deer, elk, hamsters, dogs, mice, wolves, whales, zebras, donkeys, weasels, bats, dolphins, armadillos, seals, cows, wild boars, horses, squirrels, bears, cats, moles, monkeys, raccoons, kangaroos, pigs, foxes, sheep, etc.

[0111] Regarding human cells, as described above, cells derived from tissues, germ cells, somatic cells, and stem cells are included. In the examples of this specification, HEK293 cells were used to confirm the modification of the target nucleotide sequence, but it can also be confirmed using other cells, for example, HeLa cells.

[0112] <Insect cells> In the present invention, "insect" refers to a general term for arthropods classified in the class Insecta. Examples of insects include, but are not particularly limited to, silkworm larvae, Drosophila, crickets, etc. In the present invention, "insect cells" include, but are not particularly limited to, cells that make up the body tissues of insects and cells derived from insect tissues, etc.

[0113] <Plant cells> In the present invention, the "plant" is not particularly limited, and examples include seed plants, fern plants, moss plants, algae, etc. The "plant cell" refers to a cell that constitutes a plant or a cell derived from a plant. Seed plants include angiosperms and gymnosperms. Angiosperms include dicots and monocots. Examples of dicots include, but are not particularly limited to, morning glory, dandelion, azalea, camellia, eggplant, rape, pea, etc. Examples of monocots include, but are not particularly limited to, rice, lily, tulip, pampas grass, corn, etc. Examples of gymnosperms include, but are not particularly limited to, pine, cedar, ginkgo, cypress, etc. Examples of fern plants include, but are not particularly limited to, bracken, horsetail, polypodium, shepherd's purse, etc. Examples of moss plants include, but are not particularly limited to, haircap moss, acrocarpous moss, cedar moss, water moss, etc. Algae include multicellular algae and unicellular algae. Examples of multicellular algae include, but are not particularly limited to, kelp, wakame, sea lettuce, green laver, etc. Examples of unicellular algae include, but are not particularly limited to, chlorella, euglena, spirulina, chlamydomonas, coccomyxa, botryococcus, micractinium, diatom, etc.

Example

[0114] Hereinafter, the present invention will be specifically described based on specific examples, but the present invention is not limited thereto.

[0115] A. Genome editing experiments using 293-nLD1 cells (1) 1. Preparation of mutant NanoLuc (registered trademark) plasmid Based on the wild-type nucleotide sequence (GenBank JQ513379) of the NanoLuc gene, which is a luciferase gene, DNA in the region from base number 847 to 1,380 was synthesized (Gene Universal Inc.), and XhoI and ApaI linkers were ligated, and then incorporated into the corresponding restriction enzyme sites of the pcDNA TM 5 / FRT / TO plasmid (Thermo Fisher Scientific Inc.) (pcDNA5-nLW1). Here, the wild-type nucleotide sequence of the NanoLuc gene is designated as SEQ ID NO: 1. On the one hand, a mutant nucleotide sequence in which cytosine (C) at base number 922 of the NanoLuc gene was changed to thymine (T) was synthesized, and it was incorporated into the pcDNA TM 5 / FRT / TO plasmid in the same manner as above to prepare a mutant (pcDNA5-nLD1). The luciferase gene possessed by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above point mutation.

[0116] 2. Preparation of a stable transformant cell line carrying the mutant NanoLuc gene Flp-In TM -293 cell line (Thermo Fisher Scientific Inc.) was seeded in a 6 cm dish at a density of 1x10 6 cells and cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc.) at 37°C in a 5% CO2 environment. After 24 hours, pcDNA5-nLD1 or pcDNA5-nLW1 plasmid (1 μg) and pOG44 plasmid (3 μg, Thermo Fisher Scientific Inc.) were transfected using Lipofectamine 3000 (Thermo Fisher Scientific Inc.) according to the normal 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, suspended in DMEM + 10% FBS medium containing hygromycin (Thermo Fisher Scientific Inc.) at a concentration of 50 μg / ml, and cultured in two 10 cm dishes. Thereafter, the culture was continued while changing the medium with the same medium every 3 days. After culturing for about 20 days, the formation of colonies of sufficient number and size was confirmed, and all the cells were TrypLE TMIt was detached from the petri dish using Express Enzyme 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 is 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. Introduction of unnatural polynucleotide into 293-nLD1 cells 293-nLD1 cells were seeded at 1x10 4 cells into a 96-well plate (Nunc (registered trademark) MicroWell TM 96, Nunclon Delta-Treated, Flat-Bottom Microplate, Thermo Fisher Scientific Inc.) and cultured in DMEM + 10% FBS medium at 37 °C in a 5% CO2 environment. After 24 hours, 0.05 or 0.1 μg of the unnatural polynucleotide was transfected with Lipofectamine 3000 according to the normal protocol.

[0119] 4. Measurement of editing efficiency · NanoLuc Luciferase Assay After continuing the culture for 72 hours from the transfection, the NanoLuc Luciferase activity was measured. The NanoLuc Luciferase activity was measured using the Nano-Glo (registered trademark) Luciferase Assay System (Promega Corporation) according to the normal protocol. For the measurement of the luciferase luminescence, an EnSpire multimode plate reader (PerkinElmer Co., Ltd.) was used.

[0120] · Counting of viable cell numbers The viable cell numbers were measured according to the normal protocol using CellTiter - Blue (registered trademark) Cell Viability Assay (Promega Corporation). An EnSpire multimode plate reader (PerkinElmer Co., Ltd.) was used to measure the fluorescence value of CellTiter - Blue. The viable cell numbers of the edited cells were from 5 x 10 3 to 2 x 10 5 A calibration curve of the measured values of CellTiter - Blue and the cell numbers was created using a two - fold dilution series of the 293 - nLD1 cell numbers, and the viable cell numbers were calculated based on this calibration curve.

[0121] · Calculation of editing efficiency When measuring the NanoLuc Luciferase activity of 293 - nLW1 cells with wild - type luciferase integrated into the genome, the measured value was approximately 20,000 counts / cell. Therefore, if the mutation of the inactive luciferase gene is repaired and wild - type NanoLuc is synthesized, luciferase activity of 20,000 counts per cell will be detected. Based on this value, the editing efficiency (%) is NanoLuc Luciferase activity ÷ 20,000 ÷ cell number x 100 calculated using the above formula.

[0122] 5. Measurement of editing efficiency by Next Generation Sequencing (NGS) Genomic DNA was prepared from 293-nLD1 cells transfected with non-natural polynucleotides according to the normal protocol using NucleoSpin Tissue (Takara Inc,). For the prepared genomic DNA, a region of approximately 200 bases upstream to downstream of the base to be edited was amplified by PCR and library-prepared, and amplicon sequencing was performed on this library using MiSeq / MiSeq Reagent Kit v3 (Illumin) (Bioengineering Lab. Co., Ltd.). For each sample, 30,000 to 50,000 amplicon sequences were analyzed, and the editing efficiency was calculated from the ratio of the number of edited sequence counts to the number of wild-type sequence counts.

[0123] 6. Synthesis of non-natural polynucleotides All non-natural polynucleotides including nucleic acid modifications were synthesized by Gene Design (Japan) and those purified by simple column or HPLC were used.

[0124] Example 1-1 Modification of target nucleotide sequences with unnatural polynucleotides An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing non-natural polynucleotides into 293-nLD1 cells. As described above, the codon (CAA) encoding the 22nd glutamine (Gln-22) of the luciferase gene introduced into 293-nLD1 cells has a point mutation to the stop codon (TAA) and does not show luciferase activity as it is. If the T of the stop codon (TAA) in the inactive luciferase gene can be replaced with C, the luciferase activity of 293-nLD1 cells will be restored, and the modification of the target nucleotide sequence can be detected by the luminescence by luciferase (Figure 2).

[0125] In Example 1-1, 11 types of unnatural polynucleotides, namely GEO-1 to GEO-9, GEO-242, and GEO-11, and as a negative control, an unnatural polynucleotide GEO-94 that cannot repair inactivating mutations because it does not have mismatched nucleotides, were each introduced into 293-nLD1 cells by transfection (Figure 3A).

[0126] In GEO-1 to GEO-9, GEO-242, and GEO-11, the nucleotide at the position corresponding to T of the above stop codon TAA in the target polynucleotide sequence is G, that is, a mismatch, and the nucleotides other than the mismatched nucleotide are complementary to the target polynucleotide sequence. GEO-1 to GEO-4 are 21 nt, and GEO-5 to GEO-9, GEO-242, GEO-11, and GEO-94 are 25 nt. In GEO-1 and GEO-5, the mismatched nucleotide G is LNA. In GEO-2 and GEO-6, one nucleotide on the 5' upstream side and the 3' downstream side adjacent to the mismatched nucleotide G is LNA. In GEO-3 and GEO-7, the mismatched nucleotide G and the nucleotide at the 5' end are LNA. In GEO-4 and GEO-8, one nucleotide on the 5' upstream side and the 3' downstream side adjacent to the mismatched nucleotide G is LNA, and the nucleotide at the 5' end is LNA. In GEO-11, the nucleotide at the 5' end is LNA. In GEO-242, the nucleotide at the 3' end is LNA. GEO-9 does not have LNA.

[0127] In GEO-94, the nucleotide at the position corresponding to the base to be edited is A, that is, it is complementary. Furthermore, in GEO-94, one nucleotide on the 5' upstream side and the 3' downstream side adjacent to this nucleotide A is LNA, and the nucleotide at the 5' end is LNA.

[0128] The base sequences, strand lengths, and editing efficiencies (%) of the above editing nucleic acids GEO-1 to GEO-9, GEO-242, GEO-11, and the negative control (GEO-94) are as shown in Table 1. Also, the editing efficiencies (%) of each editing nucleic acid are shown in a bar graph (Figure 3B).

[0129]

Table 1

[0130] In Table 1, guanosine monophosphate, which is a mismatched nucleotide, was underlined, and the nucleotides substituted with crosslinked nucleic acids were shown in capital letters. All of the crosslinked nucleic acids in Table 1 were LNA.

[0131] As shown in Table 1, the absolute value of the editing efficiency was highest for GEO-8 at 0.25%, and second highest for GEO-7 at 0.05%. Furthermore, the editing efficiency of GEO-94, which was the negative control, was almost 0%. When the editing efficiency of GEO-8 was set to 1.0, the relative editing efficiencies of GEO-1 to GEO-6, GEO-9, GEO-242, and GEO-11 were all less than 0.2. Note that GEO-1, GEO-2, GEO-3, GEO-5, GEO-6, and GEO-7 are the same as the configurations disclosed in Non-Patent Document 1. Thus, GEO-8, which is a non-natural polynucleotide of the present invention, showed an editing efficiency more than five times higher than that of the previously reported GEO-7.

[0132] In Examples 1-2 to 1-13 hereinafter, the editing efficiency of GEO-8 was used as a reference, but if the relative editing efficiency with the editing efficiency of GEO-8 set to 1.0 is 0.4 or more, it shows a value sufficiently higher than the editing efficiency reported in the past.

[0133] Example 1-2 Chain length of unnatural polynucleotides Although GEO-4 and GEO-8 in Example 1-1 had the same LNA arrangement, a large difference in editing efficiency occurred due to a 4-nt difference. In Example 1-2, in order to examine the preferred chain length of the unnatural polynucleotide, the same experiment as in Example 1-1 was conducted using nine types of unnatural polynucleotides with the same arrangement of GEO-8 and LNA but different chain lengths (Figure 4). The nine types of unnatural polynucleotides used in Example 1-2 were GEO-23 (29 nt), GEO-79 (31 nt), GEO-24 (35 nt), GEO-25 (41 nt), GEO-174 (45 nt), GEO-175 (49 nt), GEO-176 (53 nt), GEO-281 (75 nt), and GEO-282 (95 nt). The base sequences, chain lengths, and relative editing efficiencies of the above-mentioned respective edited nucleic acids are as shown in Table 2.

[0134]

Table 2

[0135] In Table 2, guanosine monophosphate, which is a mismatched nucleotide, is underlined, and the nucleotides substituted with the crosslinked nucleic acid are shown in capital letters. All of the crosslinked nucleic acids in Table 2 were LNA.

[0136] As shown in Table 2, including GEO-8 as a control, the relative editing efficiencies of the edited nucleic acids having chain lengths of 25 nt or more and 95 nt or less showed 0.4 or more, and that of GEO-174 with 45 nt showed the highest relative editing efficiency of 3.74. GEO-281 and GEO-282 with longer chain lengths tended to have a decrease in relative editing efficiency, but it was found that they still showed high values of 0.58 or more.

[0137] Example 1-3 LNA modification at the 3'-end of unnatural polynucleotides In Example 1-3, the editing efficiency of the structure in which the pentose sugar in the nucleotide at the 3'-end was replaced with LNA was examined. Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-16 in which the nucleotide at the 3'-end of GEO-6 was LNA, and GEO-17 in which the nucleotide at the 3'-end of GEO-8 was further LNA (Figure 5). The base sequences, strand lengths, and relative editing efficiencies of the above-described edited nucleic acids are as shown in Table 3.

[0138]

Table 3

[0139] In Table 3, guanosine monophosphate, which is a mismatched nucleotide, is underlined, and the nucleotides substituted with crosslinked nucleic acids are shown in capital letters. All of the crosslinked nucleic acids in Table 3 were LNAs.

[0140] As shown in Table 3, the relative editing efficiency of GEO-16 was 0.16, and that of GEO-17 was 2.00. From these results, it was found that the configuration (GEO-16) in which one nucleotide on the 5'-upstream side and one nucleotide on the 3'-downstream side adjacent to the mismatched nucleotide are LNAs and the 3'-end is an LNA has a low relative editing efficiency. In the configuration in which one nucleotide on the 5'-upstream side and one nucleotide on the 3'-downstream side adjacent to the mismatched nucleotide are LNAs and the 5'-end is an LNA (hereinafter referred to as the "configuration of GEO-8"), it was shown that the relative editing efficiency is further improved when the 3'-end is also an LNA. On the other hand, it was found that even when the 3'-end is an LNA, in the case of the configuration (GEO-16) where the 5'-end is unmodified, the editing efficiency is at the same level as GEO-6 and does not show sufficient editing efficiency. From this, it was shown that it is important for the nucleotide at the 5'-end to be a crosslinked nucleic acid such as LNA in order to exhibit sufficient editing efficiency.

[0141] Example 1-4 LNA modification of one or more nucleotides adjacent to LNA at the 5'-end of unnatural polynucleotides In Examples 1-4, the editing efficiencies of the configurations in which one or more nucleotides adjacent to the 5'-end were replaced with LNAs were examined for the configuration of GEO-8 in Example 1-1 and the configuration of GEO-17 in Example 1-3. Specifically, in GEO-8, GEO-18 in which the 1 nucleotide adjacent to the 5'-end is further LNA; in GEO-8, GEO-19 in which the 2 consecutive nucleotides adjacent to the 5'-end are further LNA; and in GEO-17, GEO-68 in which the 1 nucleotide adjacent to the 5'-end is further LNA were each used to measure the editing efficiency in the same manner as in Example 1-1 (Figure 6). The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as shown in Table 4.

[0142]

Table 4

[0143] In Table 4, guanosine monophosphate, which is a mismatched nucleotide, is underlined, and the nucleotides substituted with crosslinked nucleic acids are shown in capital letters. All of the crosslinked nucleic acids in Table 4 were LNA.

[0144] As shown in Table 4, the relative editing efficiency of GEO-18 was 2.05 and that of GEO-19 was 0.66. From these results, it was found that in the configuration of GEO-8, even a configuration in which one or more nucleotides adjacent to the 5'-end were replaced with LNA showed sufficient editing efficiency. Furthermore, the relative editing efficiency of GEO-17 was 2.00 and that of GEO-68 was 1.64. From these results, in a configuration in which the 1 nucleotide on the 5'-upstream side and the 3'-downstream side adjacent to the mismatched nucleotide are LNA, the 5'-end is LNA, and the 3'-end is LNA (hereinafter referred to as the "configuration of GEO-17"), even a configuration in which the 1 nucleotide adjacent to the 5'-end is replaced with LNA showed sufficient editing efficiency.

[0145] In the structure of GEO-8, the relative editing efficiency of GEO-19, in which the two nucleotides adjacent to the 5'-end were further replaced with LNA, was 0.66, which was slightly lower than that of the structure of GEO-8. On the other hand, in Example 1-8 described below, when the 5'-upstream side of the mismatched nucleotide was extended, in the structure of GEO-8, the relative editing efficiency of GEO-313, in which one nucleotide adjacent to the 5'-end was further replaced with LNA, was 5.23, and the relative editing efficiency of GEO-315, in which two nucleotides adjacent to the 5'-end were further replaced with LNA, was 6.59. It was suggested that even when a plurality of nucleotides adjacent to the 5'-end were replaced with LNA in the structure of GEO-8, the editing efficiency might increase if other structures such as the chain length of the unnatural polynucleotide changed.

[0146] Example 1-5 LNA modification of one or more nucleotides adjacent to LNA at the 3'-end of unnatural polynucleotides In Examples 1-5, the editing efficiency of the structure in which one or more nucleotides adjacent to the nucleotide at the 3'-end were replaced with LNA was examined for the structure of GEO-17 in Examples 1-3. Specifically, in GEO-17, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-69 in which one nucleotide adjacent to the nucleotide at the 3'-end was LNA, and GEO-71 in which two consecutive nucleotides adjacent to the nucleotide at the 3'-end were LNA (Figure 7). The base sequences, chain lengths, and relative editing efficiencies of the above-described editing nucleic acids are as shown in Table 5.

[0147]

Table 5

[0148] In Table 5, guanosine monophosphate, which is a mismatched nucleotide, was underlined, and the nucleotides replaced with the cross-linked nucleic acid were shown in capital letters. All of the cross-linked nucleic acids in Table 5 were LNA.

[0149] As shown in Table 5, the relative editing efficiency of GEO-69 was 1.01, and that of GEO-71 was 0.51. From these results, in the configuration of GEO-17, when one nucleotide adjacent to the 3'-terminal nucleotide was replaced with LNA, it showed an editing efficiency comparable to that of GEO-8, and when two nucleotides adjacent to the 3'-terminal nucleotide were replaced with LNA, it showed an editing efficiency about half that of GEO-8.

[0150] On the other hand, in Examples 1-8 described later, when the 3'-downstream side of the mismatched nucleotide was extended, in the configuration of GEO-17, the relative editing efficiency of GEO-319 in which one nucleotide adjacent to the 3'-terminal was further replaced with LNA was 2.18, and the relative editing efficiency of GEO-321 in which two nucleotides adjacent to the 3'-terminal were further replaced with LNA was 1.40. It was suggested that even when a plurality of nucleotides adjacent to the 3'-terminal were replaced with LNA in the configuration of GEO-17, the editing efficiency might increase if other configurations such as the chain length of the unnatural polynucleotide changed.

[0151] Example 1-6 Substitution of the phosphodiester bond portion between one or more nucleotides contained in unnatural polynucleotides with phosphorothioate bonds (1) In Examples 1-6 of the present invention, the editing efficiency of a configuration in which the phosphodiester bond portion between one or more nucleotides was replaced with a phosphorothioate bond was examined with respect to the configuration of GEO-8 in Example 1-1.

[0152] Specifically, in GEO-8, GEO-158, GEO-159, GEO-160, GEO-29, and GEO-161 in which the phosphodiester bond between the nucleotide at the 5'-end and the 1-5 consecutive nucleotides adjacent to the nucleotide at the 5'-end is replaced with a phosphorothioate bond (Figure 8A); in GEO-8, GEO-162, GEO-163, GEO-164, GEO-30, GEO-165, GEO-272, and GEO-196 in which the phosphodiester bond between the nucleotide at the 3'-end and the 1-7 consecutive nucleotides adjacent to the nucleotide at the 3'-end is replaced with a phosphorothioate bond (Figure 8B); in GEO-8, GEO-31 in which the phosphodiester bond between the nucleotide at the 5'-end and the 4 consecutive nucleotides adjacent to the nucleotide at the 5'-end is replaced with a phosphorothioate bond, and the phosphodiester bond between the nucleotide at the 3'-end and the 4 consecutive nucleotides adjacent to the nucleotide at the 3'-end is replaced with a phosphorothioate bond (Figure 8C); in GEO-8, GEO-126 and GEO-127 in which the phosphodiester bond between the LNA on the 5' upstream side adjacent to the mismatched nucleotide G and the 2 or 4 consecutive nucleotides adjacent to the LNA is replaced with a phosphorothioate bond (Figure 8C); and in GEO-8, GEO-128 and GEO-129 in which the phosphodiester bond between the nucleotide adjacent to the LNA on the 3' downstream side adjacent to the mismatched nucleotide G and the 2 or 4 consecutive nucleotides adjacent to the adjacent nucleotide is replaced with a phosphorothioate bond (Figure 8C) were each used to measure the editing efficiency in the same manner as in Example 1-1. The base sequences, strand lengths, and relative editing efficiencies of the above-described edited nucleic acids are as shown in Table 6.

[0153]

Table 6

[0154] In Table 6, guanosine monophosphate, which is a mismatched nucleotide, was underlined, and the nucleotides substituted with cross-linked nucleic acids were shown in capital letters. All of the cross-linked nucleic acids in Table 6 were LNA. An asterisk (*) was inserted between the corresponding two or more nucleotides at the position where the phosphodiester bond portion between two or more nucleotides was substituted with a phosphorothioate bond.

[0155] As shown in Table 6, the relative editing efficiency of GEO-158 was 2.40, that of GEO-159 was 3.19, that of GEO-160 was 3.02, that of GEO-29 was 2.86, and that of GEO-161 was 2.27. From this result, it was shown that in the composition of GEO-8, when the phosphodiester bond portion between the 5'-terminal nucleotide and the 1 to 5 nucleotides adjacent to the 5'-terminal nucleotide was substituted with a phosphorothioate bond, the editing efficiency was improved.

[0156] Furthermore, the relative editing efficiency of GEO-162 was 3.64, that of GEO-163 was 2.04, that of GEO-164 was 3.08, that of GEO-30 was 4.17, that of GEO-165 was 4.24, that of GEO-272 was 3.40, and that of GEO-196 was 3.97. From this result, it was shown that in the composition of GEO-8, when the phosphodiester bond portion between the 3'-terminal nucleotide and the 1 to 7 nucleotides adjacent to the 3'-terminal nucleotide was substituted with a phosphorothioate bond, the editing efficiency was improved.

[0157] Furthermore, the relative editing efficiency of GEO-31 was 5.68. From this result, it was shown that in the composition of GEO-8, when the phosphodiester bond portions between the 5'-terminal nucleotide and the consecutive 4 nucleotides adjacent to the 5'-terminal nucleotide and between the 3'-terminal nucleotide and the consecutive 4 nucleotides adjacent to the 3'-terminal nucleotide were substituted with phosphorothioate bonds, the editing efficiency was improved.

[0158] Furthermore, from Table 6, the relative editing efficiency of GEO-126 was 0.99, and that of GEO-127 was 0.70. From these results, in the structure of GEO-8, when the phosphodiester bond portion between the LNA adjacent to the 5' upstream side of the mismatched nucleotide and the consecutive 2 or 4 nucleotides further adjacent to the LNA was replaced with a phosphorothioate bond, it was found that the editing efficiency was comparable to that of GEO-8.

[0159] Furthermore, from Table 6, the relative editing efficiency of GEO-128 was 0.55, and that of GEO-129 was 2.15. From these results, in the structure of GEO-8, when the phosphodiester bond portion between the consecutive 2 nucleotides further adjacent to the 3' downstream side of the LNA adjacent to the 3' downstream side of the mismatched nucleotide was replaced with a phosphorothioate bond, it showed an editing efficiency half that of GEO-8, and when the phosphodiester bond portion between the consecutive 4 nucleotides further adjacent to the 3' downstream side of the LNA adjacent to the 3' downstream side of the mismatched nucleotide was replaced with a phosphorothioate bond, it was shown that the editing efficiency was improved. As shown in Table 6 and FIG. 8C, in GEO-128 and GEO-129, the phosphodiester bond portion between the LNA adjacent to the 3' downstream side of the mismatched nucleotide and the nucleotide directly adjacent to the LNA was not replaced with a phosphorothioate bond.

[0160] Example 1-7 Substitution of the phosphodiester bond portion between one or more nucleotides contained in unnatural polynucleotides with phosphorothioate bonds (2) In Examples 1-7, for the structure of GEO-17 in Examples 1-3, the editing efficiency of a structure in which the phosphodiester bond portion between one or more nucleotides was replaced with a phosphorothioate bond was examined. Specifically, in GEO-17, using GEO-155, GEO-167, GEO-168, and GEO-169 in which the phosphodiester bond portion between the 3' terminal nucleotide and the consecutive 1 to 4 nucleotides adjacent to the 3' terminal nucleotide was replaced with a phosphorothioate bond, the editing efficiency was measured in the same manner as in Example 1-1 (FIG. 9). The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as shown in Table 7.

[0161]

Table 7

[0162] In Table 7, guanosine monophosphate, which is a mismatched nucleotide, was underlined, and the nucleotides substituted with crosslinked nucleic acids were shown in capital letters. All of the crosslinked nucleic acids in Table 7 were LNA. At the positions where the phosphodiester bond portions between two or more nucleotides were substituted with phosphorothioate bonds, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0163] As shown in Table 7, the relative editing efficiency of GEO-155 was 3.63, that of GEO-167 was 6.05, that of GEO-168 was 5.81, and that of GEO-169 was 2.90. From this result, in the structure of GEO-17, it was shown that when the phosphodiester bond portion between the 3'-terminal nucleotide and 1 to 4 nucleotides adjacent to the 3'-terminal nucleotide was substituted with a phosphorothioate bond, the editing efficiency was improved.

[0164] Example 1-8 Extension of the chain lengths on the 5'-upstream side and 3'-downstream side of mismatched nucleotides and the position of LNA In Examples 1-8 of the present invention, when there was one mismatched nucleotide contained in the unnatural polynucleotide, the influence on the editing efficiency when changing the chain length of the 5'-upstream region and the 3'-downstream region of the mismatched nucleotide was examined. Furthermore, the editing efficiency when appropriately changing the number and position of LNAs near the 5'-end and the number and position of LNAs near the 3'-end was examined.

[0165] Specifically, GEO-25 with a chain length of 41 nt, a 5' upstream region of 20 nt and a 3' downstream region of 20 nt for the mismatched nucleotide G, 1 nucleotide on each of the 5' upstream side and 3' downstream side adjacent to the mismatched nucleotide G being LNA, and the 5'-terminal nucleotide being LNA; GEO-311 with a chain length of 38 nt, a 5' upstream region of 25 nt and a 3' downstream region of 12 nt for the mismatched nucleotide G, 1 nucleotide on each of the 5' upstream side and 3' downstream side adjacent to the mismatched nucleotide G being LNA, and the 5'-terminal nucleotide being LNA; GEO-312 in GEO-311, where the 12th nucleotide counting from the nucleotide on the 5' upstream side adjacent to the mismatched nucleotide G towards the 5' upstream side is LNA; GEO-313 in GEO-311, where 1 nucleotide adjacent to the 5'-terminal LNA is LNA; GEO-314 in GEO-311, where 1 nucleotide adjacent to the 5'-terminal LNA is LNA and the 12th nucleotide counting from the nucleotide on the 5' upstream side adjacent to the mismatched nucleotide G towards the 5' upstream side is LNA; GEO-315 in GEO-311, where 2 consecutive nucleotides adjacent to the 5'-terminal LNA are LNA; and GEO-316 in GEO-311, where 2 consecutive nucleotides adjacent to the 5'-terminal LNA are LNA and the 12th nucleotide counting from the nucleotide on the 5' upstream side adjacent to the mismatched nucleotide G towards the 5' upstream side is LNA. The editing efficiency was measured in the same manner as in Example 1-1 using each of them (Figure 10A).

[0166] More specifically, GEO-317 with a chain length of 40 nt, a 12-nt 5'-upstream region and a 27-nt 3'-downstream region of the mismatched nucleotide G, 1 nucleotide on the 5'-upstream side and 3'-downstream side adjacent to the mismatched nucleotide G being LNA, and the nucleotides at the 5'- and 3'-ends being LNA; GEO-318 in which the 12th nucleotide counting from the nucleotide on the 3'-downstream side adjacent to the mismatched nucleotide G towards the 3'-downstream side is LNA in GEO-317; GEO-319 in which 1 nucleotide adjacent to the LNA at the 3'-end is LNA in GEO-317; GEO-320 in which 1 nucleotide adjacent to the LNA at the 3'-end is LNA and the 12th nucleotide counting from the nucleotide on the 3'-downstream side adjacent to the mismatched nucleotide G towards the 3'-downstream side is LNA in GEO-317; GEO-321 in which 2 consecutive nucleotides adjacent to the LNA at the 3'-end are LNA in GEO-317; and GEO-322 in which 2 consecutive nucleotides adjacent to the LNA at the 3'-end are LNA and the 12th nucleotide counting from the nucleotide on the 3'-downstream side adjacent to the mismatched nucleotide G towards the 3'-downstream side is LNA in GEO-317. Using each of these, the editing efficiency was measured in the same manner as in Example 1-1 (Figure 10B). The base sequences, chain lengths, and relative editing efficiencies of the above-described editing nucleic acids are as shown in Table 8.

[0167]

Table 8

[0168] In Table 8, guanosine monophosphate, which is the mismatched nucleotide, is underlined, and the nucleotides substituted with the crosslinked nucleic acid are shown in capital letters. All of the crosslinked nucleic acids in Table 8 were LNA.

[0169] As shown in Table 8, the relative editing efficiency of GEO-25 was 3.47, that of GEO-311 was 3.31, that of GEO-312 was 4.16, that of GEO-313 was 5.23, that of GEO-314 was 6.57, that of GEO-315 was 6.59, that of GEO-316 was 7.02, that of GEO-317 was 2.55, that of GEO-318 was 2.66, that of GEO-319 was 2.18, that of GEO-320 was 2.51, that of GEO-321 was 1.40, and that of GEO-322 was 0.77.

[0170] Comparing GEO-8 and GEO-311, it was shown that extending the chain length of the 5'-upstream region of the mismatched nucleotide increased the editing efficiency. Comparing GEO-313 with GEO-18 in Example 1-4 and GEO-315 with GEO-19 in Example 1-4 respectively, it was shown that even when a plurality of nucleotides adjacent to the nucleotide at the 5'-end were LNAs, extending the chain length of the 5'-upstream region of the mismatched nucleotide increased the editing efficiency.

[0171] Comparing GEO-311 and GEO-312, GEO-313 and GEO-314, GEO-315 and GEO-316 respectively, it was shown that the editing efficiency further increased when the 12th nucleotide counting from the nucleotide on the 5'-upstream side adjacent to the mismatched nucleotide was LNA towards the 5'-upstream side.

[0172] Compared with GEO-311, GEO-312, GEO-313, GEO-314, GEO-315, GEO-316 in which the chain length of the 5'-upstream region of the mismatched nucleotide was extended, GEO-317, GEO-318, GEO-319, GEO-320, GEO-321, GEO-322 in which the chain length of the 3'-downstream region of the mismatched nucleotide was extended had relatively low editing efficiency. Therefore, it was suggested that extending the chain length of the 5'-upstream region of the mismatched nucleotide was effective.

[0173] As a result of comparing GEO-317 with GEO-318 and GEO-319 with GEO-320 respectively, since there is no significant difference in the editing efficiency, when the chain length of the 3'-downstream region of the smatch nucleotide is extended, it is suggested that even if the 12th nucleotide counting from the nucleotide on the 3'-downstream side adjacent to the mismatched nucleotide G is replaced with LNA toward the 3'-downstream side, the influence on the editing efficiency is small.

[0174] Example 1-9 RNA substitution of nucleotides near the 3'-end In Examples 1-9, the editing efficiency of a configuration in which one or more nucleotides adjacent to the nucleotide at the 3'-end were replaced with RNA was examined with respect to the configuration of GEO-17 in Examples 1-3. Specifically, in GEO-17, GEO-150 in which one nucleotide adjacent to the nucleotide at the 3'-end was replaced with RNA; GEO-73 in which two consecutive nucleotides adjacent to the nucleotide at the 3'-end were replaced with RNA; GEO-151 in which three consecutive nucleotides adjacent to the nucleotide at the 3'-end were replaced with RNA; and GEO-74 in which four consecutive nucleotides adjacent to the nucleotide at the 3'-end were replaced with RNA were each used, and the editing efficiency was measured in the same manner as in Example 1-1 (Figure 11). The base sequences, chain lengths, and relative editing efficiencies of the above-described editing nucleic acids are as shown in Table 9.

[0175]

Table 9

[0176] In Table 9, guanosine monophosphate, which is a mismatched nucleotide, is underlined, and the nucleotides replaced with crosslinked nucleic acids are shown in capital letters. All of the crosslinked nucleic acids in Table 9 were LNA. An arrow was attached to the base symbol of the corresponding nucleotide for the nucleotide replaced with RNA.

[0177] As shown in Table 9, the relative editing efficiency of GEO-150 was 1.65, that of GEO-73 was 4.20, that of GEO-151 was 0.78, and that of GEO-74 was 1.12. From these results, in the configuration of GEO-17, when 1 or 2 nucleotides adjacent to the 3'-terminal nucleotide were RNA-substituted, the editing efficiency was improved compared to GEO-8, and when 3 or 4 nucleotides were RNA-substituted, the editing efficiency was at the same level as GEO-8.

[0178] Example 1-10 Phosphorylation of nucleotides at the 3'-end and / or 5'-end In Examples 1-10, the editing efficiency of a configuration in which the nucleotides at the 3'-terminal and / or 5'-terminal were further phosphorylated was examined with respect to the configuration of GEO-8 in Example 1-1 or GEO-17 in Example 1-3. Specifically, in GEO-8, GEO-26 in which the 5'-terminal nucleotide was phosphorylated; GEO-27 in which the 3'-terminal nucleotide was phosphorylated; and GEO-28 in which both the 5'-terminal and 3'-terminal nucleotides were phosphorylated; in GEO-17, GEO-223 in which the 3'-terminal nucleotide was phosphorylated; and in GEO-17, GEO-224 in which the phosphodiester bond portion between the 3'-terminal nucleotide and the adjacent nucleotide was replaced with a phosphorothioate bond and the 3'-terminal nucleotide was phosphorylated. The editing efficiency was measured in the same manner as in Example 1-1 using each of them (Figure 12). The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as described in Table 10.

[0179]

Table 10

[0180] In Table 10, guanosine monophosphate, which is a mismatched nucleotide, was underlined, and the nucleotides substituted with crosslinked nucleic acids were shown in capital letters. All of the crosslinked nucleic acids in Table 10 were LNA. At the positions where the phosphodiester bond portion between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. Furthermore, in order to indicate that the 5'-end and / or 3'-end was phosphorylated, "p" was attached to the 5'-end and / or 3'-end.

[0181] As shown in Table 10, the relative editing efficiency of GEO-26 was 1.14, that of GEO-27 was 2.23, that of GEO-27 was 2.23, that of GEO-28 was 2.91, the relative editing efficiency of GEO-223 was 2.70, and that of GEO-224 was 2.29. From these results, in the composition of GEO-8, it was shown that phosphorylating only the 5'-end had no effect on the editing efficiency, but phosphorylating the 3'-end improved the editing efficiency. Also, when both the 5'-end and 3'-end were phosphorylated, a higher editing efficiency was shown than when only the 3'-end was phosphorylated. Similarly, in the composition of GEO-17, it was shown that phosphorylating the 3'-end improved the editing efficiency. In this case, even when the phosphodiester bond portion between the nucleotide at the 3'-end and the adjacent nucleotide was replaced with a phosphorothioate bond, no increase in the editing efficiency was observed.

[0182] Example 1-11 Types of cross-linked nucleic acids In Example 1-11, in the composition of GEO-8 of Example 1-1, the editing efficiency of the composition in which the LNA at the 5'-end was replaced with another crosslinked nucleic acid was examined. Specifically, in GEO-8, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-41 in which the LNA at the 5'-end was replaced with BNA-N-H; GEO-43 in which the LNA at the 5'-end was replaced with BNA-N-Me; and GEO-57 in which the LNA at the 5'-end was replaced with ENA (Figure 13). The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as described in Table 11.

[0183]

Table 11

[0184] In Table 11, guanosine monophosphate, which is a mismatched nucleotide, was underlined, and the nucleotides substituted with cross-linked nucleic acids were shown in capital letters. The types of cross-linked nucleic acids in each edited nucleic acid in Table 11 are as described above.

[0185] As shown in Table 11, the relative editing efficiency of GEO-41 was 1.38, that of GEO-43 was 1.51, and that of GEO-57 was 1.57. From this result, in the structure of GEO-8, it was shown that not only LNA but also other cross-linked nucleic acids such as BNA-N-H, BNA-N-Me, and ENA could be substituted at the 5'-end, and in some cases, the editing efficiency might increase by the substitution.

[0186] Example 1-12 Combinations of nucleic acid modifications (1) In Examples 1-12, in the structure of GEO-8 of Example 1-1, the editing efficiency of a structure further combined with the substitution of the phosphodiester bond part with phosphorothioate bond and the phosphorylation of the 3'-end was examined. Specifically, in GEO-8, using GEO-226 in which the phosphodiester bond part between five consecutive nucleotides adjacent to the nucleotide at the 3'-end was substituted with a phosphorothioate bond and the 3'-end was phosphorylated, the editing efficiency was measured in the same manner as in Example 1-1 (Figure 14). The base sequences, strand lengths, and relative editing efficiencies of the above-mentioned each edited nucleic acid are as described in Table 12.

[0187]

Table 12

[0188] In Table 12, guanosine monophosphate, which is a mismatched nucleotide, was underlined, and the nucleotides substituted with cross-linked nucleic acids were shown in capital letters. All of the cross-linked nucleic acids in Table 12 were LNA. At the positions where the phosphodiester bond portion between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. Furthermore, in order to indicate that the 3'-end was phosphorylated, "p" was attached to the 3'-end.

[0189] As shown in Table 12, the relative editing efficiency of GEO-226 was 8.19. From this result, it was shown that in the composition of GEO-8, when the substitution of the phosphodiester bond portion with a phosphorothioate bond and the phosphorylation of the 3'-end were further combined, the editing efficiency increased extremely greatly.

[0190] Example 1-13 Combinations of nucleic acid modifications (2) In Examples 1-13, in the composition of GEO-23 in Examples 1-3, the editing efficiency of a composition in which the substitution of the phosphodiester bond portion with a phosphorothioate bond, RNA substitution, and phosphorylation of the 3'-end were further combined was examined. Specifically, in GEO-23, the phosphodiester bond portion between four consecutive nucleotides adjacent to the 5'-terminal nucleotide was replaced with a phosphorothioate bond, two consecutive nucleotides adjacent to the 3'-terminal nucleotide were RNA-substituted, and the editing efficiency was measured in the same manner as in Example 1-1 using GEO-172 in which the 3'-end was phosphorylated (Figure 15). The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as described in Table 13.

[0191]

Table 13

[0192] In Table 13, guanosine monophosphate, which is a mismatched nucleotide, was underlined, and the nucleotides substituted with crosslinked nucleic acids were shown in capital letters. All of the crosslinked nucleic acids in Table 13 were LNA. An asterisk (*) was inserted between the corresponding two or more nucleotides at the position where the phosphodiester bond portion between two or more nucleotides was substituted with a phosphorothioate bond. An arrow was attached to the base symbol of the corresponding nucleotide for the RNA-substituted nucleotide. Further, in order to indicate that the 3'-end was phosphorylated, "p" was attached to the 3'-end.

[0193] As shown in Table 13, the relative editing efficiency of GEO-172 was 8.48. From this result, it was shown that in the composition of GEO-17, further combining the substitution of the phosphodiester bond portion with a phosphorothioate bond, RNA substitution, and phosphorylation of the 3'-end significantly increased the editing efficiency.

[0194] Discussion The above Examples 1-1 to 1-13 of the present invention are compared with the disclosures in patent documents and non-patent documents. (1) From Example 1-1, it was found that GEO-8, which is a non-natural polynucleotide of the present invention, showed an editing efficiency more than 5 times higher than that of GEO-7 (that is, the chain length, the position of the mismatched nucleotide, and the position of LNA are the same as those of GEO-8), which corresponds to the polynucleotide with the highest editing efficiency among the configurations disclosed in Non-Patent Document 1. Comparing GEO-8 and GEO-7, GEO-8 of the present invention is different in that the mismatched nucleotide itself is not LNA, but both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide are substituted with LNA.

[0195] Regarding this point, in Patent Document 4, although oligonucleotides containing LNA adjacent to mismatched nucleotides did not show improvement, it is described that oligonucleotides having LNA arranged at an interval one nucleotide further away from the mismatch showed a five-fold average increase. It can be said that Patent Document 4 actively rejects the configuration containing LNA adjacent to mismatched nucleotides.

[0196] Non-Patent Document 1 focuses on avoiding mismatch repair. Further, Non-Patent Documents 1 and 2 focus on elucidating the mechanism of how single-stranded synthetic DNA with a mismatched nucleotide being LNA performs genome modification in mammalian cells. Among the more than 60 types of single-stranded synthetic DNA tested in Non-Patent Document 1, only one has both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide replaced with LNA. All of the more than 50 types of single-stranded DNA tested in Non-Patent Document 2 have the mismatched nucleotide itself as LNA, and those with both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide replaced with LNA have not been tested. Also, in the discussion of Non-Patent Document 1, it is emphasized that making the mismatched nucleotide itself LNA is important for avoiding mismatch repair, and there is no mention of those with both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide replaced with LNA.

[0197] Therefore, the descriptions in Patent Document 4 and Non-Patent Documents 1 and 2 can be said to be inhibitory factors for adopting the motivation to adopt the feature that both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide are replaced with LNA, that is, Feature (A) and Feature (B) of the present invention, in order to improve the editing efficiency. Furthermore, the experimental results shown in Example 1-1 of this specification are results completely opposite to the suggestion of Patent Document 4, and from the perspective of the effects of the invention, it can be said that the effects of the present invention are heterogeneous effects that cannot be predicted from Patent Document 4 and Non-Patent Documents 1 and 2.

[0198] Among the more than 110 types of single-stranded synthetic DNAs tested in Non-Patent Documents 1 and 2, there was only one type of single-stranded synthetic DNA in each document where the nucleotide at the 5'-end was LNA and the 3'-end was not LNA. Although the single-stranded synthetic DNA with two nucleotides, the nucleotide at the 5'-end and the mismatched nucleotide, being LNA showed an editing efficiency approximately twice that of the single-stranded synthetic DNA with only the mismatched nucleotide being LNA, no further investigation was conducted on the single-stranded synthetic DNA with the 5'-end being LNA.

[0199] In Non-Patent Document 2, it was concluded that the region 5' to the mismatched nucleotide was degraded by an endonuclease rather than an exonuclease. Therefore, it is suggested that the effect of protecting the 5'-end by LNA modification from exonuclease is limited. In fact, since the editing efficiency only doubled due to the LNA modification at the 5'-end, it is deduced that the LNA modification at the 5'-end is not so important. Therefore, the combination of substituting both the 5'- and 3'-nucleotides adjacent to the mismatched nucleotide with LNA and the LNA modification at the 5'-end has not been tried.

[0200] Therefore, the descriptions in Non-Patent Documents 1 and 2 can be said to be inhibitory factors for the attempt to substitute the 5'-end of the unnatural polynucleotide with LNA in order to improve the editing efficiency.

[0201] (2) From Examples 1-2, it was found that if the chain length of the unnatural polynucleotide is 25 nt or more and 95 nt or less, an editing efficiency of a certain level or more can be observed. Regarding the length of the strand, conventionally, strands of about 25 bases were considered good, and there were many documents stating that if the strand length was long, the effect would be low. In contrast, in the present invention, it was different from existing research in that even when the strand length was long, the effect was high. Specifically, in Non-Patent Document 1, the optimal length of the edited nucleic acid was examined using a polynucleotide in which only mismatched nucleotides were made into LNA, and as a result, it was shown that a length of 25 bases was optimal, and for those longer than 25 bases, the editing efficiency decreased. Also, in Patent Document 4, a polynucleotide with a length of 24 bases was used. On the other hand, in the non-natural type polynucleotide of the present invention, it has been discovered that those longer than 25 bases have an even higher editing efficiency.

[0202] (3) From Examples 1-3, it was shown that in order for those in which both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide are replaced with LNA to exhibit an effect, the feature (C) that the 5'-terminal nucleotide is a crosslinked nucleic acid is important. Furthermore, from the comparison with GEO-8, GEO-16, and GEO-17, it was found that when both the 5'-terminal and 3'-terminal nucleotides are crosslinked nucleic acids, the editing efficiency increased by 2-fold. Also, it was found that the effect of the 3'-terminal nucleotide being LNA is strongly shown when the 5'-terminal nucleotide is LNA. (4) Regarding Examples 1-4 and 1-5, it was shown that even when one or more nucleotides adjacent to the 5'-terminal or 3'-terminal are replaced with LNA, sufficient editing efficiency is exhibited, and depending on the position and number of the nucleotides replaced with LNA, the editing efficiency may be improved. Such an effect was not described or suggested in any of the above-mentioned patent documents or non-patent documents.

[0203] (5) From Examples 1-6 and 1-7, it was shown that when both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide are replaced with LNA, the editing efficiency is improved when the phosphodiester bond portion between the 5'-terminal or 3'-terminal nucleotide and the 1 to 7 nucleotides adjacent thereto is replaced with a phosphorothioate bond. Since the editing efficiency does not decrease even when the number of phosphorothioate bonds is increased one by one on both the 5'-terminal side and the 3'-terminal side, it is deduced that there is a high possibility that sufficient editing efficiency can be exhibited even when the number of phosphodiester bonds is 8 or more. Such an effect was not described or suggested in any of the above patent documents or non-patent documents. Also, when the 5'-terminal or 3'-terminal is LNA, sulfurization from both terminals particularly strongly improves the editing effect, which is a completely new discovery clarified by the present inventors in this specification.

[0204] (6) From Example 1-8, it was shown that the editing efficiency increases when the chain lengths of the 5'-upstream region and the 3'-downstream region of the mismatched nucleotide are extended, and further, it was shown that the editing efficiency increases by replacing some nucleotides in the extended region with LNA. Such an effect was not described or suggested in any of the above patent documents or non-patent documents.

[0205] (7) From Examples 1-9, 1-10, 1-12, and 1-13, in addition to substitution with a crosslinked nucleic acid, it was shown that the editing efficiency is significantly increased by combining modifications such as substitution of the phosphodiester bond portion with a phosphorothioate bond, RNA substitution, phosphorylation of the 3'-terminal, or phosphorylation of the 5'-terminal. Such an effect was not described or suggested in any of the above patent documents or non-patent documents.

[0206] (8) From Example 1-11, regarding substitution of the 5'-terminal with a crosslinked nucleic acid, it was shown that not only LNA but also substitution with other crosslinked nucleic acids such as BNA-N-H, BNA-N-Me, and ENA exhibits an effect of improving the editing efficiency. Such an effect was not described or suggested in any of the above patent documents or non-patent documents.

[0207] B. Genome editing experiments using 293-nLD2 cells, 293-nLD3 cells, 293-nLD4-1 cells, 293-nLD4-2 cells, 293-nLD4-3 cells, 293-nLD5 cells, 293-nLD6 cells, 293-nLD7 cells, 293-nLD8 cells or 293-nLD9 cells 1. Preparation of various mutant NanoLuc (registered trademark) plasmids (1) nDL2 A mutant base sequence in which guanine (G) at base number 894 of the NanoLuc gene was changed to adenine (A) and cytosine (C) at base number 895 was changed to thymine (T) was synthesized, and pcDNA TM 5 / FRT / TO plasmid was incorporated to create a mutant (pcDNA5-nLD2). The luciferase gene possessed by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above mutation.

[0208] (2) nDL3 A mutant base sequence in which thymine (T) at base number 956 of the NanoLuc gene was changed to adenine (A), thymine (T) at base number 957 was changed to guanine (G), and cytosine (C) at base number 958 was changed to thymine (T) was synthesized, and pcDNA TM 5 / FRT / TO plasmid was incorporated to create a mutant (pcDNA5-nLD3). The luciferase gene possessed by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above mutation.

[0209] (3) nDL4-1 A mutant base sequence in which cytosine (C) at base number 922 of the NanoLuc gene was changed to thymine (T) and guanine (G) at base number 931 was changed to thymine (T) was synthesized, and pcDNA TM 5 / FRT / TO plasmid was incorporated to create a mutant (pcDNA5-nLD4-1). The luciferase gene possessed by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above mutation.

[0210] (4) nDL4-2 A mutant base sequence in which cytosine (C) at base number 922 of the NanoLuc gene was changed to thymine (T) and guanine (G) at base number 937 was changed to thymine (T) was synthesized, and pcDNATM Mutants incorporated into the 5 / FRT / TO plasmid (pcDNA5-nLD4-2) were created. The luciferase gene carried by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above point mutation.

[0211] (5)nDL4-3 A mutant base sequence in which guanine (G) at base number 937 and cytosine (C) at base number 958 of the NanoLuc gene were changed to thymine (T) was synthesized, and pcDNA was prepared in the same manner as above. TM Mutants incorporated into the 5 / FRT / TO plasmid (pcDNA5-nLD4-3) were created. The luciferase gene carried by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above point mutation.

[0212] (6)nDL5 A mutant base sequence in which guanine (G) at base number 976 of the NanoLuc gene was deleted was synthesized, and pcDNA was prepared in the same manner as above. TM Mutants incorporated into the 5 / FRT / TO plasmid (pcDNA5-nLD5) were created. The luciferase gene carried by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above point mutation.

[0213] (7)nDL6 A mutant base sequence in which adenine (A) at base number 911 and cytosine (C) at base number 912 of the NanoLuc gene were deleted was synthesized, and pcDNA was prepared in the same manner as above. TM Mutants incorporated into the 5 / FRT / TO plasmid (pcDNA5-nLD6) were created. The luciferase gene carried by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above point mutation.

[0214] (8)nDL7 A mutant base sequence in which thymine (T) was inserted between thymine (T) at base number 930 and guanine (G) at base number 931 of the NanoLuc gene was synthesized, and pcDNA was prepared in the same manner as above.TM A mutant incorporated into the 5 / FRT / TO plasmid (pcDNA5-nLD7) was created. The luciferase gene possessed by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above point mutation.

[0215] (9)nDL8 A mutant base sequence in which thymine (T) and adenine (A) were inserted in this order between adenine (A) at base number 978 and adenine (A) at base number 979 of the NanoLuc gene was synthesized, and pcDNA was prepared in the same manner as above. TM A mutant incorporated into the 5 / FRT / TO plasmid (pcDNA5-nLD8) was created. The luciferase gene possessed by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above point mutation.

[0216] (10)nDL9 A mutant base sequence in which thymine (T) at base number 981, cytosine (C) at base number 982, cytosine (C) at base number 983, and guanine (G) at base number 984 of the NanoLuc gene were deleted was synthesized, and pcDNA was prepared in the same manner as above. TM A mutant incorporated into the 5 / FRT / TO plasmid (pcDNA5-nLD9) was created. The luciferase gene possessed by this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above point mutation.

[0217] 2. Generation of stable transfected cell lines carrying various mutant NanoLuc genes Flp-In, a cell line derived from humans TM -293 cell line (Thermo Fisher Scientific Inc.) was used at 1x10 6Seeded in a 6-cm dish to become cells and cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific Inc.) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific Inc.) at 37°C in a 5% CO2 environment. After 24 hours, pcDNA5-nLD2, pcDNA5-nLD3, pcDNA5-nLD4-1, pcDNA5-nLD4-2, pcDNA5-nLD4-3, pcDNA5-nLD5, pcDNA5-nLD6, pcDNA5-nLD7, pcDNA5-nLD8 or pcDNA5-nLD9 plasmid (1 μg) and pOG44 plasmid (3 μg, Thermo Fisher Scientific Inc.) were transfected using Lipofectamine 3000 (Thermo Fisher Scientific Inc.) according to the normal protocol. After 48 hours, TrypLE TM Express Enzyme (Thermo Fisher Scientific Inc.) was added, incubated at 37°C for 3 minutes, the detached cells were collected, suspended in DMEM + 10% FBS medium containing hygromycin (Thermo Fisher Scientific Inc.) at a concentration of 50 μg / ml, and cultured in two 10-cm dishes. Thereafter, the culture was continued while changing the medium with the same medium every three days. After culturing for about 20 days, the formation of colonies with sufficient numbers and sizes was confirmed, and all cells were TrypLE TM Express Enzyme was used to detach from the petri dish and collected.

[0218] Each cell line obtained using the above various plasmids was named as follows. The cells in which pcDNA5-nLD2 was integrated into the genome were named 293-nLD2 cells. The cells in which pcDNA5-nLD3 was integrated into the genome were named 293-nLD3 cells. The cells in which pcDNA5-nLD4-1 was integrated into the genome were named 293-nLD4-1 cells. The cells in which pcDNA5-nLD4-2 was integrated into the genome were named 293-nLD4-2 cells. The cells in which pcDNA5-nLD4-3 was integrated into the genome were named 293-nLD4-3 cells. The cells in which pcDNA5-nLD5 was integrated into the genome were named 293-nLD5 cells. The cells in which pcDNA5-nLD6 was integrated into the genome were named 293-nLD6 cells. The cells in which pcDNA5-nLD7 was integrated into the genome were named 293-nLD7 cells. The cells in which pcDNA5-nLD8 was integrated into the genome were named 293-nLD8 cells. The cells in which pcDNA5-nLD9 was integrated into the genome were named 293-nLD9 cells. The mutant NanoLuc gene possessed by each of the above cells is an inactive luciferase gene as described above. Each of the above cells was used in subsequent genome editing experiments.

[0219] 3. Introduction of unnatural polynucleotide into 293-nLD2~9 cells 293-nLD2 cells were seeded at 1x10 4 cells into a 96-well plate (Nunc (registered trademark) MicroWell TM 96, Nunclon Delta-Treated, Flat-Bottom Microplate, Thermo Fisher Scientific Inc.) and cultured in DMEM + 10% FBS medium at 37 °C in a 5% CO2 environment. After 24 hours, unnatural polynucleotides at each concentration of 0.0125 μg, 0.025 μg, 0.050 μg, 0.100 μg, and 0.200 μg were transfected with Lipofectamine 3000 according to the normal protocol. The 293-nLD3 cells, 293-nLD4-1 cells, 293-nLD4-2 cells, 293-nLD4-3 cells, 293-nLD5 cells, 293-nLD6 cells, 293-nLD7 cells, 293-nLD8 cells, and 293-nLD9 cells were also transfected in the same manner as the 293-nLD2 cells.

[0220] 4. Measurement of Editing Efficiency · NanoLuc Luciferase Assay After culturing for 72 hours continuously from transfection, the NanoLuc Luciferase activity was measured. The NanoLuc Luciferase activity was measured according to the normal protocol using the Nano-Glo® Luciferase Assay System (Promega Corporation). For the measurement of the luminescence of luciferase, an EnSpire multimode plate reader (PerkinElmer Co., Ltd.) was used.

[0221] · Counting of Viable Cell Number The viable cell number was measured according to the normal protocol using the CellTiter-Blue® Cell Viability Assay (Promega Corporation). For the measurement of the fluorescence value of CellTiter-Blue, an EnSpire multimode plate reader (PerkinElmer Co., Ltd.) was used. The viable cell number of the edited cells was determined by creating a calibration curve of the measured values of CellTiter-Blue and the cell number using a two-fold dilution series of the 293-nLD2 cell number between 5 x 10 3 and 2 x 10 5 , and calculating the viable cell number based on this calibration curve. The viable cell numbers of the 293-nLD3 cells, 293-nLD4-1 cells, 293-nLD4-2 cells, 293-nLD4-3 cells, 293-nLD5 cells, 293-nLD6 cells, 293-nLD7 cells, 293-nLD8 cells, and 293-nLD9 cells were also calculated in the same manner as the 293-nLD2 cells.

[0222] · Calculation of editing efficiency When measuring the NanoLuc Luciferase activity of 293-nLW1 cells with wild-type luciferase integrated into the genome, the measured value was approximately 20,000 counts / cell. Therefore, if the mutation of the inactive luciferase gene is repaired and wild-type NanoLuc is synthesized, luciferase activity of 20,000 counts per cell will be detected. Based on this value, the editing efficiency (%) is NanoLuc Luciferase activity ÷ 20,000 ÷ number of cells x 100 calculated using the following formula.

[0223] 5. Evaluation of editing efficiency of unnatural polynucleotides by AUC log The editing efficiency of each unnatural polynucleotide was evaluated as follows. The NanoLuc Luciferase activity was measured at 5 concentrations (0.0125 μg, 0.025 μg, 0.050 μg, 0.100 μg, 0.200 μg) of the unnatural polynucleotide, and a dose-response curve was created with the axis (X-axis) indicating the concentration of the unnatural polynucleotide on a logarithmic scale. From the created dose-response curve, the value with the X-axis of the area under the curve (hereinafter referred to as "AUC") on a logarithmic scale (hereinafter referred to as "AUC log") was obtained. Prism (GraphPad) was used for the calculation of AUC log.

[0224] 6. Synthesis of unnatural polynucleotides All unnatural polynucleotides including nucleic acid modifications were synthesized by Gene Design (Japan) and those purified by simple column or HPLC were used.

[0225] Example 2-1 Examination of two-base substitutions (1) An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing the unnatural polynucleotide into 293-nLD2 cells.

[0226] In this example, each of five types of non-natural polynucleotides, D2-1 to D2-6, was introduced into 293-nLD2 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described respective edited nucleic acids are as shown in Table 14.

[0227]

Table 14

[0228] In Table 14, mismatched nucleotides are underlined, and nucleotides substituted with crosslinked nucleic acids are shown in capital letters. All of the crosslinked nucleic acids in Table 14 were LNA. An asterisk (*) was inserted between the corresponding two or more nucleotides at the position where the phosphodiester bond portion between two or more nucleotides was substituted with a phosphorothioate bond. The structure of D2-2 is the same as the structure reported in Non-Patent Document 1. The relative editing efficiency was calculated with the value of the AUC of D2-2 as a reference (1.00) and shown in Table 14. Incidentally, the AUC when Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 7.4, hereinafter referred to as "TE") was used as a negative control was 123, and the AUC of D2-2 was 64,642. As shown in Table 14, the relative editing efficiency of D2-1 was 0.13, that of D2-3 was 4.94, that of D2-4 was 3.13, that of D2-5 was 1.71, and that of D2-6 was 1.64. From this result, it was found that according to the non-natural polynucleotide of the present invention, two nucleotides contained in the target nucleotide sequence can be modified.

[0229] Example 2-2 Examination of three-base substitutions An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD3 cells.

[0230] In this example, each of the four types of non-natural polynucleotides D3-1 to D3-5 was introduced into 293-nLD3 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as shown in Table 15.

[0231]

Table 15

[0232] In Table 15, mismatched nucleotides are underlined, and nucleotides substituted with cross-linked nucleic acids are shown in capital letters. All of the cross-linked nucleic acids in Table 15 were LNA. An asterisk (*) was inserted between the corresponding two or more nucleotides at the position where the phosphodiester bond portion between two or more nucleotides was substituted with a phosphorothioate bond.

[0233] All of the structures shown in Table 15 are three-base substitutions, and such examples have not been reported in the past. The relative editing efficiencies were calculated with the AUC value of D3-2 as the reference (1.00) and are shown in Table 15. The AUC when TE was used as the negative control was 119, and the AUC of D3-2 was 14,436. As shown in Table 15, the relative editing efficiency of D3-1 was 0.23, that of D3-3 was 3.44, that of D3-4 was 2.47, and that of D3-5 was 7.89. From this result, it was found that according to the non-natural polynucleotide of the present invention, three nucleotides contained in the target nucleotide sequence can be modified.

[0234] Example 2-3 Examination of two-base substitutions (2) An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing the non-natural polynucleotide into 293-nLD4-1 cells, 293-nLD4-2 cells, or 293-nLD4-3 cells.

[0235] In the experiment using 293-nLD4-1 cells, each of the three types of unnatural polynucleotides, D4-1-1 to D4-1-4, was introduced into 293-nLD4-1 cells by the transfection method. In the experiment using 293-nLD4-2 cells, each of the three types of unnatural polynucleotides, D4-2-1 to D4-2-4, was introduced into 293-nLD4-2 cells by the transfection method. In the experiment using 293-nLD4-3 cells, each of the three types of unnatural polynucleotides, D4-3-1 to D4-3-4, was introduced into 293-nLD4-3 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-mentioned edited nucleic acids are as shown in Table 16.

[0236]

Table 16

[0237] In Table 16, mismatched nucleotides are underlined, and nucleotides substituted with crosslinked nucleic acids are shown in capital letters. All of the crosslinked nucleic acids in Table 16 were LNA. An asterisk (*) was inserted between the corresponding two or more nucleotides at the position where the phosphodiester bond part between two or more nucleotides was substituted with a phosphorothioate bond.

[0238] All of the structures shown in Table 16 are two-base substitutions at distant positions, and such examples have not been reported in the past. In D4-1-1 to D4-1-4, the two mismatched nucleotides with respect to the target nucleotide sequence have an interval of 8 bases. The relative editing efficiency was calculated with the AUC value of D4-1-2 as a reference (1.00) and shown in Table 16. When TE was used as a negative control, the AUC was 47.16, and the AUC of D4-1-2 was 14904. As shown in Table 16, the relative editing efficiency of D4-1-1 was 0.67, that of D4-1-3 was 3.19, and that of D4-1-4 was 9.45. In D4-2-1 to D4-2-4, the two mismatched nucleotides with respect to the target nucleotide sequence are separated by 14 bases. The relative editing efficiencies were calculated with the AUC value of D4-2-2 as the reference (1.00) and are shown in Table 16. The AUC when TE was used as the negative control was 47.16, and the AUC of D4-2-2 was 63770. As shown in Table 16, the relative editing efficiency of D4-2-1 was 0.04, that of D4-2-3 was 2.83, and that of D4-2-4 was 3.87. In D4-3-1 to D4-3-4, the two mismatched nucleotides with respect to the target nucleotide sequence are separated by 20 bases. The relative editing efficiencies were calculated with the AUC value of D4-3-2 as the reference (1.00) and are shown in Table 16. The AUC when TE was used as the negative control was 47.16, and the AUC of D4-3-2 was 104690. As shown in Table 16, the relative editing efficiency of D4-3-1 was 0.00, that of D4-3-3 was 1.76, and that of D4-3-4 was 4.70.

[0239] From these results, it was found that when there are multiple modifications of nucleotides contained in the target nucleotide sequence, it is not necessary for the modification sites to be adjacent, and even when they are at least 20 bases apart, multiple nucleotides contained in the target nucleotide sequence can be modified simultaneously.

[0240] Example 2-4 Examination of one-base insertions An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing an unnatural polynucleotide into 293-nLD5 cells.

[0241] In this example, each of four types of unnatural polynucleotides, D5-2, D5-4, D5-5, and D5-6, and the unnatural polynucleotide D5-1 as a control were introduced into 293-nLD5 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as described in Table 17.

[0242]

Table 17

[0243] In Table 17, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were shown in capital letters. All of the cross-linked nucleic acids in Table 17 were LNA. An asterisk (*) was inserted between the corresponding two or more nucleotides at the position where the phosphodiester bond portion between two or more nucleotides was substituted with a phosphorothioate bond. Furthermore, the nucleotides within [ ] indicate the nucleotides for inserting nucleotides complementary to the nucleotides within [ ] into the target nucleotide sequence.

[0244] The structure of D5-2 was the same as the structure reported in Non-Patent Document 1. The relative editing efficiency based on the AUC value of D5-2 (1.00) was calculated and shown in Table 17. The AUC when using Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 7.4, hereinafter referred to as "TE") as a negative control was 672.3, and the AUC of D5-2 was 75293.

[0245] As shown in Table 17, the relative editing efficiency of the control D5-1 was 0.33, that of D5-4 was 10.44, that of D5-5 was 16.19, and that of D5-6 was 2.11. From this result, it was found that according to the non-natural polynucleotide of the present invention, a desired base can be inserted into the deletion site contained in the target nucleotide sequence.

[0246] Example 2-5 Examination of two-base insertions An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing the non-natural polynucleotide into 293-nLD6 cells.

[0247] In this example, each of nine types of non-natural polynucleotides of D6-1 to D6-10 was introduced into 293-nLD6 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of each of the above-described edited nucleic acids are as shown in Table 18.

[0248]

Table 18

[0249] In Table 18, mismatched nucleotides are underlined, and nucleotides substituted with crosslinked nucleic acids are shown in capital letters. All of the crosslinked nucleic acids in Table 18 were LNAs. An asterisk (*) was inserted between the corresponding two or more nucleotides at the position where the phosphodiester bond portion between two or more nucleotides was substituted with a phosphorothioate bond. Furthermore, the nucleotides within [ ] indicate the nucleotides for inserting nucleotides complementary to the nucleotides within [ ] into the target nucleotide sequence.

[0250] All of the structures shown in Table 18 were two-base insertions, and such examples have not been reported in the past. Relative editing efficiencies were calculated with the value of the AUC of D6-2 as the reference (1.00) and shown in Table 18. The AUC when TE was used as a negative control was 158, and the AUC of D3-2 was 4924. As shown in Table 18, the relative editing efficiency of D6-1 was 0.60, that of D6-3 was 12.62, that of D6-4 was 17.70, that of D6-5 was 2.29, that of D6-6 was 7.52, that of D6-7 was 40.12, that of D6-8 was 24.11, that of D6-9 was 7.66, and that of D6-10 was 17.37. From this result, it was found that according to the non-natural type polynucleotide of the present invention, even if there are two deletion sites contained in the target nucleotide sequence, a desired base can be inserted.

[0251] Example 2-6 Examination of one-base deletions An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing the non-natural type polynucleotide into 293-nLD7 cells.

[0252] In this example, each of the three types of non-natural polynucleotides D7-1 to D7-4 was introduced into 293-nLD7 cells by the transfection method. The nucleotide sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as shown in Table 19.

[0253]

Table 19

[0254] In Table 19, mismatched nucleotides are underlined, and nucleotides substituted with crosslinked nucleic acids are shown in capital letters. The mismatched nucleotide x in Table 19 means that one nucleotide complementary to the nucleotide present in the target nucleotide sequence is missing in the editing nucleic acid. All of the crosslinked nucleic acids in Table 19 were LNA. An asterisk (*) was inserted between the corresponding two or more nucleotides at the position where the phosphodiester bond portion between two or more nucleotides was replaced with a phosphorothioate bond.

[0255] All of the structures shown in Table 19 were single-base deletions, and such examples have not been reported in the past. The relative editing efficiency was calculated with the AUC value of D7-2 as the reference (1.00) and shown in Table 19. When TE was used as a negative control, the AUC was 9964, and the AUC of D7-2 was 12715. As shown in Table 19, the relative editing efficiency of D7-1 was 0.72, that of D7-3 was 2.10, and that of D7-4 was 11.31. From this result, it was found that according to the non-natural polynucleotide of the present invention, a desired base can be removed from the target nucleotide sequence.

[0256] Example 2-7 Examination of two-base deletions An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing the non-natural polynucleotide into 293-nLD8 cells.

[0257] In this example, each of the three types of non-natural polynucleotides D8-1 to D8-4 was introduced into 293-nLD8 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described edited nucleic acids are as shown in Table 20.

[0258]

Table 20

[0259] In Table 20, mismatched nucleotides are underlined, and nucleotides substituted with cross-linked nucleic acids are shown in capital letters. The mismatched nucleotide xx in Table 20 means that two nucleotides complementary to the nucleotide present in the target nucleotide sequence are missing in the edited nucleic acid. All of the cross-linked nucleic acids in Table 20 were LNA. An asterisk (*) was inserted between the corresponding two or more nucleotides at the position where the phosphodiester bond portion between two or more nucleotides was replaced with a phosphorothioate bond.

[0260] All of the structures shown in Table 20 were two-base deletions, and such examples have not been reported in the past. The relative editing efficiencies were calculated with the AUC value of D7-2 as the reference (1.00) and are shown in Table 19. When TE was used as a negative control, the AUC was 1243, and the AUC of D8-2 was 1303. As shown in Table 20, the relative editing efficiency of D8-1 was 1.03, that of D8-3 was 4.84, and that of D8-4 was 22.95. From this result, it was found that according to the non-natural polynucleotide of the present invention, a plurality of desired bases can be removed from the target nucleotide sequence.

[0261] Example 2-8 Examination of four-base insertions An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD9 cells.

[0262] In this example, each of four types of non-natural polynucleotides, D9-1, D9-2, D9-3, D9-5, and D9-6, was introduced into 293-nLD9 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as shown in Table 21.

[0263]

Table 21

[0264] In Table 21, mismatched nucleotides are underlined, and nucleotides substituted with cross-linked nucleic acids are shown in capital letters. All of the cross-linked nucleic acids in Table 21 were LNA. An asterisk (*) was inserted between the two or more nucleotides corresponding to the location where the phosphodiester bond portion between two or more nucleotides was replaced with a phosphorothioate bond. Further, the nucleotides within [ ] indicate the nucleotides for inserting nucleotides complementary to the nucleotides within [ ] into the target nucleotide sequence.

[0265] The structure of D9-2 is the same as the structure reported in Non-Patent Document 1. The relative editing efficiency based on the AUC value of D9-2 as a reference (1.00) was calculated and shown in Table 21. The AUC when using Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 7.4, hereinafter referred to as "TE") as a negative control was 383.8, and the AUC of D9-2 was 18543. As shown in Table 21, the relative editing efficiency of D9-1 was 0.37, that of D9-3 was 5.12, that of D9-5 was 7.02, and that of D9-6 was 3.14. From this result, it was found that according to the non-natural polynucleotide of the present invention, even when there are four deletion sites contained in the target nucleotide sequence, a desired base can be inserted.

[0266] From the above Examples 2-1 to 2-8, by using the non-natural type polynucleotide of the present invention, it was found that a plurality of two or more bases on the target polynucleotide can be substituted, one base or a plurality of two or more bases can be inserted into the target polynucleotide, and one base or a plurality of two or more bases can be removed from the target polynucleotide.

[0267] C. Genome editing experiments using 293-nLD1 cells (2) Using the 293-nLD1 cells prepared in the above "A. Genome editing experiment (1) using 293-nLD1 cells", the following genome editing experiment was conducted.

[0268] 1. Introduction of non-natural type 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 TM 96, Nunclon Delta-Treated, Flat-Bottom Microplate, Thermo Fisher Scientific Inc.), and cultured in DMEM + 10% FBS medium at 37 °C in a 5% CO2 environment. After 24 hours, 0.05 or 0.1 μg of non-natural type polynucleotide was transfected with Lipofectamine 3000 according to the normal protocol.

[0269] 2. Measurement of editing efficiency and evaluation of editing efficiency of non-natural type polynucleotide by AUC log For the measurement of editing efficiency and the evaluation of editing efficiency, the methods described in the above "B. Genome editing experiment using 293-nLD2 cells, 293-nLD3 cells, 293-nLD4-1 cells, 293-nLD4-2 cells, 293-nLD4-3 cells, 293-nLD5 cells, 293-nLD6 cells, 293-nLD7 cells, 293-nLD8 cells or 293-nLD9 cells" were used.

[0270] 3. Synthesis of non-natural type polynucleotide All unnatural polynucleotides, including modified nucleic acids, were synthesized by Gene Design (Japan) and used after purification by simple column or HPLC.

[0271] Example 3-1 Modification of target nucleotide sequences with unnatural polynucleotides having various characteristics An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing unnatural polynucleotides into 293-nLD1 cells.

[0272] In this example, each of the unnatural polynucleotides shown in Table 31 was introduced into 293-nLD1 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-mentioned editing nucleic acids are as described in Table 31.

[0273]

Table 31-1

[0274]

Table 31-2

[0275] In Table 31, mismatched nucleotides are underlined, and nucleotides substituted with crosslinked nucleic acids are shown in capital letters. All crosslinked nucleic acids in Table 31 were LNA. For nucleotides substituted with RNA, an arrow was attached to the base symbol of the corresponding nucleotide. An asterisk (*) was inserted between the corresponding nucleotides at the position where the phosphodiester bond between nucleotides was substituted with a phosphorothioate bond. For unnatural polynucleotides with phosphorylated 5'-end and / or 3'-end, "p" was attached to the 5'-end and / or 3'-end. Furthermore, G(F) and T(F) in GEO-204 indicate that each is 2'-fluoro modified, A(MT) in GEO-213 indicates that it is 2'-O-methyl modified, and t*(MP)a in GEO-651 indicates that the bond between t and a is a methyl phosphate bond.

[0276] As shown in Table 31, it was found that the non-natural polynucleotide of the present invention has an editing efficiency 4 times or more higher than that of the reference GEO-8.

[0277] Example 3-2 Modification of Target Nucleotide Sequence with Unnatural Polynucleotide with Mismatched Nucleotide at 3' End An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing the non-natural polynucleotide into 293-nLD1 cells.

[0278] In this example, each of the non-natural polynucleotides shown in Table 32 was introduced into 293-nLD1 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as described in Table 32. In Table 32, the strand lengths of the editing nucleic acid main body and the adapter are described separately, but each sequence defined as a sequence number is the combination of the editing nucleic acid main body and the adapter.

[0279]

Table 32-1-1

[0280]

Table 32-1-2

[0281]

Table 32-2-1

[0282]

Table 32-2-2

[0283]

Table 32-3

[0284]

Table 32-4

[0285]

Table 32-5

[0286]

Table 32-6

[0287] In Table 32, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were shown in capital letters. All of the cross-linked nucleic acids in Table 32 were LNA. For nucleotides substituted with RNA, an arrow was attached to the base symbol of the corresponding nucleotide. An asterisk (*) was inserted between the corresponding nucleotides at the position where the phosphodiester bond portion between nucleotides was substituted with a phosphorothioate bond. For unnatural polynucleotides phosphorylated at the 5'-end and / or 3'-end, "p" was attached to the 5'-end and / or 3'-end. Furthermore, [t], [tt], and [ttt] indicate that 1, 2, and 3 thymidine monophosphates were inserted, respectively, and [a], [aa], and [aaa] indicate that 1, 2, and 3 adenosine monophosphates were inserted, respectively.

[0288] As shown in Table 32, it was found that the editing efficiency of the unnatural polynucleotide of the present invention was higher than that of the reference GEO-8.

[0289] Example 3-3 Modification of Target Nucleotide Sequence with Unnatural Polynucleotide with Adapter at 3' End An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing the unnatural polynucleotide into 293-nLD1 cells.

[0290] In this example, each of the unnatural polynucleotides shown in Table 33 was introduced into 293-nLD1 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described edited nucleic acids are as shown in Table 33. In Table 33, the strand lengths of the edited nucleic acid main body and the adapter are described separately, but each sequence defined as a sequence number is the combination of the edited nucleic acid main body and the adapter.

[0291]

Table 33-1

[0292]

Table 33-2

[0293] In Table 33, mismatched nucleotides are underlined, and nucleotides substituted with crosslinked nucleic acids are shown in capital letters. All crosslinked nucleic acids in Table 33 were LNA. Arrows were attached to the base symbols of the corresponding nucleotides for nucleotides substituted with RNA. An asterisk (*) was inserted between the corresponding nucleotides at the positions where the phosphodiester bond portion between nucleotides was substituted with a phosphorothioate bond. For unnatural polynucleotides phosphorylated at the 5'-end and / or 3'-end, "p" was attached to the 5'-end and / or 3'-end. Furthermore, among the unnatural polynucleotides shown in Table 33, the nucleotides in the second row are adapters, and each adapter forms a stem structure that may have a loop. Schematic diagrams of the stem structures of each unnatural polynucleotide are shown in FIGS. 16A, 16B, 17A, and 17B.

[0294] As shown in Table 33, it was found that the unnatural polynucleotides of the present invention have an editing efficiency equal to or higher than that of the reference GEO-8.

[0295] Example 3-4 Modification of Target Nucleotide Sequence with Unnatural Polynucleotide of Aspect (vi) An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing the unnatural polynucleotide into 293-nLD1 cells.

[0296] In this example, each of the unnatural polynucleotides shown in Table 34 was introduced into 293-nLD1 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as described in Table 34.

[0297]

Table 34

[0298] In Table 34, mismatched nucleotides were underlined, and the nucleotides substituted with crosslinked nucleic acids were shown in capital letters. All of the crosslinked nucleic acids in Table 34 were LNA. An asterisk (*) was inserted between the corresponding nucleotides at the positions where the phosphodiester bond portion between nucleotides was substituted with a phosphorothioate bond. For unnatural polynucleotides with phosphorylated 5'-ends and / or 3'-ends, "p" was attached to the 5'-end and / or 3'-end.

[0299] As shown in Table 34, it was found that the unnatural polynucleotides of the present invention had higher editing efficiency than the reference GEO-8.

[0300] Example 3-5 Modification of Target Nucleotide Sequence with Unnatural Polynucleotide Where Nucleotide at 5' End Is Not LNA An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing an unnatural polynucleotide into 293-nLD1 cells.

[0301] In this example, each of the unnatural polynucleotides shown in Table 41 was introduced into 293-nLD1 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as described in Table 41.

[0302]

Table 41-1

[0303]

Table 41-2

[0304] In Table 41, mismatched nucleotides were underlined, and nucleotides substituted with crosslinked nucleic acids were shown in capital letters. All of the crosslinked nucleic acids here were LNA. For nucleotides substituted with RNA, an arrow was attached to the base symbol of the corresponding nucleotide. An asterisk (*) was inserted between the corresponding nucleotides at the position where the phosphodiester bond portion between nucleotides was substituted with a phosphorothioate bond. The crosslinked nucleic acids in GEO-827 and GEO-831 were both LNA. Furthermore, the mismatched nucleotides at the 5'-end or in the vicinity thereof in GEO-649, GEO-650, GEO-653, GEO-654, GEO-656, and GEO-657 were mismatched nucleotides for the purpose of improving the editing efficiency, and not mismatched nucleotides for the purpose of modification.

[0305] As shown in Table 41, it was found that the non-natural polynucleotide of the present invention had a higher editing efficiency than the reference GEO-8.

[0306] Example 3-6 Modification of Target Nucleotide Sequence with Unnatural Polynucleotide with Modifying Compound at 5' End or 3' End An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing the non-natural polynucleotide into 293-nLD1 cells.

[0307] In this example, each of the non-natural polynucleotides shown in Tables 42 and 43 was introduced into 293-nLD1 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described editing nucleic acids are as described in Tables 42 and 43.

[0308] [Table 42]

[0309] [Table 43]

[0310] In Tables 42 to 43, mismatched nucleotides were underlined, and nucleotides substituted with crosslinked nucleic acids were shown in capital letters. All of the crosslinked nucleic acids in Tables 42 to 43 were LNA. An asterisk (*) was inserted between the corresponding nucleotides at positions where the phosphodiester bond portion between nucleotides was substituted with a phosphorothioate bond. Note that in Tables 42 to 43, Biotin is biotin as a modifying compound, FAM is fluorescein (FAM) as a modifying compound, Pur is puromycin as a modifying compound, Chol is cholesterol as a modifying compound, DIG is digoxigenin as a modifying compound, and IdT is Inverted dT as a modifying compound. Here, the molecular weight of biotin (Biotin) is 244.31, the molecular weight of FAM is 376.32, the molecular weight of puromycin (Pur) is 471.51, the molecular weight of cholesterol (Chol) is 386.65, the molecular weight of digoxigenin (DIG) is 414.31, and the molecular weight of Inverted dT (IdT) is 304.2. Furthermore, the mismatched nucleotides at the 5'-end or in the vicinity thereof in GEO-649, GEO-650, GEO-653, GEO-654, GEO-656, and GEO-657 are mismatched nucleotides for the purpose of improving the editing efficiency and are not mismatched nucleotides for modification purposes.

[0311] As shown in Tables 42 to 43, it was found that the non-natural polynucleotide of the present invention has a higher editing efficiency than GEO-8 used as a reference.

[0312] Example 3-7 Examination of Introduction of Phosphoric Acid Moiety Modifying Bond to 5' Side or 3' Side An experiment was conducted to measure the editing efficiency of the target polynucleotide sequence by introducing a non-natural polynucleotide having a phosphate group-modified bond on the 5'-side or 3'-side from a mismatched nucleotide for modification purposes into 293-nLD1 cells.

[0313] In this example, each of the non-natural type polynucleotides shown in Tables 44 to 45 was introduced into 293-nLD1 cells by the transfection method. The base sequences, strand lengths, and relative editing efficiencies of the above-described edited nucleic acids are as described in Tables 44 to 45.

[0314]

Table 44

[0315]

Table 45

[0316] In Tables 44 to 45, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were shown in capital letters. All of the cross-linked nucleic acids in Tables 44 to 45 were LNA. An asterisk (*) was inserted between the corresponding nucleotides at the positions where the phosphodiester bond portion between nucleotides was substituted with a phosphorothioate bond.

[0317] As shown in Tables 44 to 45, it was found that the non-natural type polynucleotide of the present invention has a higher editing efficiency than the reference GEO-8.

Industrial Applicability

[0318] The method using the non-natural type polynucleotide of the present invention enables modification of a target nucleotide sequence only by introducing the polynucleotide without introducing an exogenous nuclease or a gene encoding an exogenous nuclease into the cell, and can be applied to a highly safe genome editing technology, and thus has industrial applicability.

Claims

1. A non-naturally occurring polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides contained in the target nucleotide sequence in a double-stranded DNA in a cell, comprising: containing one or more mismatched nucleotides relative to the target nucleotide sequence, A non-natural polynucleotide having at least one of the following crosslinked nucleic acids (A) and (B) and further having the characteristics (C) and (D). (A) one or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are bridged nucleic acids (B) one or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are bridged nucleic acids. (C) The 5'-terminal nucleotide is a bridged nucleic acid. (D) The length is between 22 and 95 nucleotides.

2. The non-naturally occurring polynucleotide of claim 1, further comprising the following characteristic (E): (E) The 3'-terminal nucleotide is a bridged nucleic acid.

3. The non-naturally occurring polynucleotide according to claim 1 or 2, further comprising the following characteristic (F): (F) one or more nucleotides adjacent to the 5'-terminal nucleotide are bridged nucleic acids

4. The non-naturally occurring polynucleotide according to any one of claims 1 to 3, further comprising the following characteristic (G): (G) one or more nucleotides adjacent to the 3'-terminal nucleotide are bridged nucleic acids

5. The non-naturally occurring polynucleotide according to any one of claims 1 to 4, further comprising the following characteristic (J): (J) one or more internucleotide phosphodiester bonds are replaced with phosphate-modified bonds

6. 6. The non-natural polynucleotide according to claim 5, wherein the phosphate moiety modified bond comprises at least one bond selected from the group consisting of a phosphorothioate bond, a methyl phosphate bond, a boranophosphate bond and a mesyl phosphoramidate bond.

7. The non-naturally occurring polynucleotide according to any one of claims 1 to 6, further comprising the following characteristic (M): (M) one or more nucleotides located between the nucleotide adjacent to the 5' upstream side of the mismatched nucleotide and the 5' terminal nucleotide and located at a distance of at least one nucleotide from both the nucleotide adjacent to the 5' upstream side of the mismatched nucleotide and the 5' terminal nucleotide are bridged nucleic acids.

8. The non-naturally occurring polynucleotide according to any one of claims 1 to 7, further comprising the following characteristic (N): (N) one or more nucleotides arranged between the nucleotide adjacent to the 3' downstream side of the mismatched nucleotide and the 3' terminal nucleotide and arranged at a distance of at least one nucleotide from both the nucleotide adjacent to the 3' downstream side of the mismatched nucleotide and the 3' terminal nucleotide are bridged nucleic acids.

9. The non-natural polynucleotide according to any one of claims 1 to 8, further comprising the following characteristic (O) and / or (X2): (O) The pentose sugar in one or more nucleotides adjacent to the 3'-terminal bridged nucleic acid is ribose. (X2) The pentose in one or more nucleotides adjacent to the 5'-terminal bridged nucleic acid is ribose.

10. The non-naturally occurring polynucleotide according to any one of claims 1 to 9, further comprising the following characteristic (P): (P) The 3'-terminal nucleotide is phosphorylated

11. The non-natural polynucleotide according to any one of claims 1 to 10, wherein the crosslinked nucleic acid comprises at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me and ENA.

12. The non-natural polynucleotide according to any one of claims 1 to 11, further comprising the following characteristic (X1): (X1) The 3'-terminal nucleotide and / or one or more nucleotides adjacent to the 3'-terminal nucleotide are nucleic acids modified at the 2' site.

13. The non-natural polynucleotide according to claim 12, wherein the nucleic acid modified at the 2' site comprises at least one selected from the group consisting of 2'-F, 2'-OMe, 2'-MOE and 2'-O-(2-carbamoylethyl).

14. The non-natural polynucleotide according to any one of claims 1 to 13, further comprising the following characteristic (X4): (X4) An adapter is added to the 3' end

15. The non-natural polynucleotide according to claim 14 , wherein the adaptor has a function of inhibiting mismatch repair in a host cell.

16. The non-natural polynucleotide according to claim 14 or 15, wherein the adaptor has a function of protecting the non-natural polynucleotide from nuclease digestion.

17. The non-natural polynucleotide according to any one of claims 14 to 16, wherein the adapter has a function of imparting a higher editing efficiency than the non-natural polynucleotide defined by SEQ ID NO: 9 (designated "GEO-8").

18. The non-natural polynucleotide according to any one of claims 14 to 17, wherein the adaptor is a nucleotide containing a mismatched nucleotide.

19. The non-natural polynucleotide according to any one of claims 14 to 18, wherein the adaptor is a nucleotide that forms a stem structure which may have a loop.

20. The non-natural polynucleotide according to any one of claims 14 to 17, wherein the adaptor is a compound other than a nucleotide that modifies the end (referred to as a "modifying compound").

21. The non-natural polynucleotide according to claim 20, wherein the molecular weight of the modified compound is 2,000 or less.

22. The non-natural polynucleotide according to any one of claims 1 to 21, further comprising the following characteristic (X5): (X5) one or more nucleotides are inserted between the mismatched nucleotide and the 3'-terminal nucleotide

23. A non-naturally occurring polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides contained in the target nucleotide sequence in a double-stranded DNA in a cell, comprising: containing one or more mismatched nucleotides relative to the target nucleotide sequence, A non-naturally occurring polynucleotide having at least one of the following crosslinked nucleic acids (A) and (B), having characteristic (D), and further having one or more characteristics selected from the group consisting of (H2), (Y1), and (Y3). (A) one or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are bridged nucleic acids (B) one or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are bridged nucleic acids. (D) The length is between 22 and 95 nucleotides. (H2) The phosphodiester bond between the 5'-terminal nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide is replaced with a phosphate moiety-modified bond. (Y1) the 5'-terminal nucleotide is a mismatched nucleotide, and one or more nucleotides adjacent to the 5'-terminal nucleotide are mismatched nucleotides; (Y3) An adapter is added to the 5' end

24. The non-naturally occurring polynucleotide according to claim 23 , wherein the adaptor has the function of inhibiting mismatch repair in a host cell.

25. The non-natural polynucleotide according to claim 23 or 24, wherein the adaptor has a function of protecting the non-natural polynucleotide from nuclease digestion.

26. The non-natural polynucleotide according to any one of claims 23 to 25, wherein the adaptor has a function of imparting higher editing efficiency than the non-natural polynucleotide (GEO-8) defined by SEQ ID NO:

9.

27. The non-natural polynucleotide according to any one of claims 23 to 26, wherein the adaptor is a modified compound, and the molecular weight of the modified compound is 2,000 or less.

28. The non-natural polynucleotide according to any one of claims 23 to 27, wherein the non-natural polynucleotide further has the following characteristic (Y2): (Y2) The pentose in the 5'-terminal nucleotide is ribose, and the pentose in one or more nucleotides adjacent to the 5'-terminal nucleotide is ribose.

29. A non-naturally occurring polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides contained in the target nucleotide sequence in a double-stranded DNA in a cell, comprising: containing one or more mismatched nucleotides relative to the target nucleotide sequence, A non-natural polynucleotide having at least one of the following crosslinked nucleic acids (A) and (B) and further having the characteristics (D) and (X7): (A) one or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are bridged nucleic acids (B) one or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are bridged nucleic acids. (D) The length is between 22 and 95 nucleotides. (X7) one or more nucleotides located between the mismatched nucleotide and the 5'-terminal nucleotide are bridged nucleic acids

30. 30. The non-natural polynucleotide of claim 29, wherein the bridged nucleic acid comprises at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me and ENA.

31. A non-naturally occurring polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides contained in the target nucleotide sequence in a double-stranded DNA in a cell, comprising: containing one or more mismatched nucleotides relative to the target nucleotide sequence, A non-naturally occurring polynucleotide having all of the following characteristics (C), (D), (I) and (X6). (C) The 5'-terminal nucleotide is a bridged nucleic acid. (D) The length is between 22 and 95 nucleotides. (I) The phosphodiester bond between the 3'-terminal nucleotide and one or more nucleotides adjacent to the 3'-terminal nucleotide is replaced with a phosphorothioate bond. (X6) The mismatched nucleotide is a bridged nucleic acid.

32. The non-naturally occurring polynucleotide of claim 31, further comprising the following characteristic (E): (E) The 3'-terminal nucleotide is a bridged nucleic acid.

33. 33. The non-naturally occurring polynucleotide of claim 31 or 32, further comprising the following characteristic (P): (P) The 3'-terminal nucleotide is phosphorylated

34. A kit for modifying a target nucleotide sequence, comprising the non-natural polynucleotide according to any one of claims 1 to 33.

35. A pharmaceutical composition comprising the non-naturally occurring polynucleotide according to any one of claims 1 to 33.

36. A method for modifying one or more nucleotides contained in a target nucleotide sequence in double-stranded DNA in a cell, comprising: introducing into a cell a non-naturally occurring polynucleotide that contains one or more mismatch nucleotides relative to the target nucleotide sequence; 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 of the target nucleotide sequence; The method, wherein the non-naturally occurring polynucleotide is a non-naturally occurring polynucleotide according to any one of claims 1 to 33.

37. 37. The method of claim 36, wherein the cell is a prokaryotic or eukaryotic cell.

38. 38. The method of claim 37, wherein the eukaryotic cell is at least one selected from the group consisting of a plant cell, an insect cell, and an animal cell.

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