Non-naturally occurring polynucleotides for modifying target nucleotide sequences

Abstract

The present invention is a non-natural polynucleotide that can, to modify one or more nucleotides contained in a target nucleotide sequence in double-stranded DNA in a cell, specifically bind to the sequence, wherein the non-natural polynucleotide contains one or more mismatched nucleotides in the sequence, has a crosslinked nucleic acid represented by at least one of (A) and (B), and also has characteristics (C) and (D). (A) one or more adjacent nucleotides on the 5' upstream side of the mismatched nucleotide are crosslinked nucleic acids; (B) one or more adjacent nucleotides on the 3' downstream side of the mismatched nucleotide are crosslinked nucleic acids; (C) the 5'-terminal nucleotide is a crosslinked nucleic acid; and (D) the chain length is 22-95 nt.

Description

[Technical field]

[0001] The present invention relates to a non-natural polynucleotide capable of specifically binding to a target nucleotide sequence in intracellular double-stranded DNA for modifying one or more nucleotides contained in the target nucleotide sequence. [Background technology]

[0002] CRISPR-Cas is a genome editing technology that applies the adaptive immune mechanism of eubacteria and archaea, and is used as a tool for genetic engineering. CRISPR-Cas9 (Patent Document 1), which uses a DNA sequence CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequence and Cas9, a DNA cleavage enzyme of Streptococcus pyogenes, is characterized by inducing cleavage of the target double-stranded DNA by having RNA recognize the target DNA sequence, and is the most widespread 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 misrecognition of the genome sequence by the guide RNA sequence and the introduction of unexpected mutations in sites other than the target DNA sequence due to the cleavage of double-stranded DNA.

[0003] Various solutions have been proposed for the off-target effects of CRISPR-Cas. For example, CRISPR-Cas3, which utilizes the fact that the recognition sequence of Cas9 is 20 bases long while that of E. coli-derived Cas3 is 27 bases long, has enabled more specific mutation introduction (Patent Document 2). A method using a complex linking a guide RNA capable of recognizing DNA sequences with a deaminase that converts nucleic acid bases, and a mutant Cas nuclease that has inactivated the activity of cleaving one of the strands of double-stranded DNA, has realized safer and more specific genome editing than CRISPR-Cas9 without inducing double-stranded DNA cleavage (Patent Document 3).

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

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

[0006] As another example of genome editing technology that does not use Cas nuclease protein, it has been reported that a single-stranded synthetic DNA in which part of the nucleotide sequence is LNA can be introduced into mouse ES cells to introduce 1 to 3 base mutations 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 compared to genome editing technology that uses Cas nuclease protein. The authors of Non-Patent Document 1 decoded the base sequence of a 335 bp region surrounding 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 deciphering the base sequence, and confirmed that no unintended modification had occurred in any part other than the target nucleotide sequence. From this result, the authors explain that the single-stranded synthetic DNA used by the authors of Non-Patent Document 1 was able to achieve very accurate genome editing in mouse-derived ES cells in which the mismatch repair mechanism is functioning. The authors of Non-Patent Document 1 concluded that it is important that the mismatch nucleotide is LNA in order to avoid the intracellular mismatch repair mechanism and modify the target nucleotide sequence, and have conducted intensive experiments based on this conclusion. In fact, Non-Patent Document 1 conducted experiments using a total of more than 60 types of single-stranded synthetic DNAs, and 41 types of single-stranded synthetic DNAs had at least an LNA mismatch nucleotide.

[0007] Non-Patent Document 2 is a follow-up report by the same authors as Non-Patent Document 1, based on the discovery in Non-Patent Document 1 that the mismatch repair mechanism in cells can be avoided when the mismatch nucleotide contained in the single-stranded synthetic DNA is LNA. Non-Patent Document 2 focuses on elucidating the mechanism of how single-stranded synthetic DNA containing an LNA mismatch nucleotide modifies the genome of mammalian cells. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 6343605 [Patent Document 2] Patent No. 6480647 [Patent Document 3] Patent No. 6206893 [Patent Document 4] Patent No. 5405121 [Non-patent literature]

[0009] [Non-Patent Document 1] Thomas W. van Ravesteyn, Marleen Dekker, Alexander Fish, Titia K. Sixma, Astrid Wolters, Rob J. Dekker, and Hein PJ te Riele (2016) LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells, PNAS Vol.113, no.15, 4122-4127 [Non-Patent Document 2] Thomas W. van Ravesteyn, Marcos Arranz Dols, Wietske Pieters, Marleen Dekker, Hein te Riele (2020) Extensive trimming of short single-stranded DNA oligonucleotides during replication-coupled gene editing in mammalian cells. PLoS Genet 16(10): e1009041. https: / / doi.org / 10.1371 / journal.pgen.1009041 Summary of the Invention [Problem to be solved by the invention]

[0010] As a genome editing technique that does not use Cas nuclease, several reports have been published on modifying target nucleotide sequences using single-stranded synthetic DNA containing at least one LNA, but the editing efficiency is not sufficient.

[0011] The objective of the present invention is to provide a novel non-natural polynucleotide having structural features that enable improved editing efficiency in a genome editing technique using a non-natural polynucleotide related to a single-stranded synthetic DNA in which part of the nucleotide sequence is a cross-linked nucleic acid. [Means for solving the problem]

[0012] As a result of extensive research aimed at solving the above-mentioned problems, the inventors have discovered that the editing efficiency (yield) of a target nucleotide sequence can be improved by using a non-natural polynucleotide that is capable of specifically binding to a target nucleotide sequence in order to modify one or more nucleotides contained in the target nucleotide sequence in double-stranded DNA in a cell, the polynucleotide containing one or more mismatch nucleotides with respect to the target nucleotide sequence, wherein nucleotides other than the mismatch nucleotides contain nucleotides complementary to the target nucleotide sequence, and at least one nucleotide adjacent to the mismatch nucleotide or the 5'-terminal nucleotide is a cross-linked nucleic acid, and the polynucleotide is within a specific chain length range, thereby completing the present invention.

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

[38] . [1] A non-natural 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 mismatch 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 22 to 95 nucleotides. [2] The non-naturally occurring polynucleotide according to [1] above, further comprising the following characteristic (E): (E) The 3'-terminal nucleotide is a bridged nucleic acid. [3] The non-naturally occurring polynucleotide according to [1] or [2] above, 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 [1] to [3] above, further having 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 [1] to [4] above, further having the following characteristic (J): (J) one or more internucleotide phosphodiester bonds are replaced with phosphate-modified bonds

[0014] [6] The non-natural polynucleotide according to [5] above, wherein the phosphate moiety modification bond includes 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 [1] to [6] above, further having 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 [1] to [7] above, further having 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-naturally occurring polynucleotide according to any one of [1] to [8] above, further having 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.

[0015]

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

[11] The non-natural polynucleotide according to any one of [1] to

[10] above, 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-naturally occurring polynucleotide according to any one of [1] to

[11] above, further having 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

[12] above, 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-naturally occurring polynucleotide according to any one of [1] to

[13] above, further having the following characteristic (X4): (X4) An adapter is added to the 3' end

[15] The non-naturally occurring polynucleotide according to

[14] above, wherein the adaptor has a function of inhibiting mismatch repair in a host cell.

[16] The non-natural polynucleotide according to

[14] or

[15] above, 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

[14] to

[16] above, wherein the adapter has a function of imparting higher editing efficiency than the non-natural polynucleotide defined by SEQ ID NO: 9 (referred to as "GEO-8").

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

[14] to

[17] above, wherein the adaptor is a nucleotide containing a mismatched nucleotide.

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

[14] to

[18] above, wherein the adaptor is a nucleotide that forms a stem structure which may have a loop.

[0016]

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

[14] to

[17] above, wherein the adaptor is a compound (referred to as a "modifying compound") other than a nucleotide that modifies the end.

[21] The non-natural polynucleotide according to

[20] above, wherein the molecular weight of the modifying compound is 2,000 or less.

[22] The non-naturally occurring polynucleotide according to any one of [1] to

[21] above, further having the following characteristic (X5): (X5) one or more nucleotides are inserted between the mismatched nucleotide and the 3'-terminal nucleotide

[23] A non-natural 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 mismatch 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 22 to 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 adaptor is added to the 5' end

[0017]

[24] The non-naturally occurring polynucleotide according to

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

[25] The non-natural polynucleotide according to

[23] or

[24] above, 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

[23] to

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

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

[23] to

[26] above, wherein the adaptor is a modifying compound, and the molecular weight of the modifying compound is 2,000 or less.

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

[23] to

[27] above, further having 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-natural 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 mismatch 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 22 to 95 nucleotides. (X7) one or more nucleotides located between the mismatched nucleotide and the 5'-terminal nucleotide are bridged nucleic acids

[0018]

[30] The non-natural polynucleotide according to

[29] above, 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-natural 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 mismatch 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 22 to 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 according to

[31] above, further comprising the following characteristic (E): (E) The 3'-terminal nucleotide is a bridged nucleic acid.

[33] The non-naturally occurring polynucleotide according to

[31] or

[32] above, 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 [1] to

[33] above.

[35] A pharmaceutical composition comprising the non-natural 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 a 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-natural polynucleotide is the non-natural polynucleotide described in any one of [1] to

[33] above.

[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. Effect of the Invention

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

[0020] [Figure 1] FIG. 1 is a schematic diagram showing an overview of a method for modifying a base to be edited in a target nucleotide sequence using a non-natural polynucleotide of the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing an experimental system in which a mutation in an inactive luciferase gene in the genome of a 293-nLD1 cell is edited using a non-natural polynucleotide of the present invention, and the editing efficiency is detected as the luminescence of luciferase. [Figure 3A] 3A is a diagram showing the structures of the unnatural polynucleotides shown in Example 1-1 of the present invention, i.e., 12 types of unnatural polynucleotides, GEO-1 to GEO-9, GEO-242, GEO-11 and GEO-94. GEO-94 is a negative control having a base sequence without a mismatched nucleotide. In the figure, G indicates a mismatched nucleotide, and G with an asterisk (i.e., "G*") and L indicate that the pentose in the nucleotide has been replaced with LNA. [Figure 3B] FIG. 3B is a bar graph showing the editing efficiency (%) of the non-natural polynucleotides shown in Example 1-1 of the present invention, i.e., 12 types of non-natural polynucleotides, GEO-1 to GEO-9, GEO-242, GEO-11 and GEO-94. [Figure 4] 4 is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-2 of the present invention, in which G represents a mismatch nucleotide, and L represents that the pentose in the nucleotide has been replaced with LNA. [Diagram 5] 5 is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-3 of the present invention, in which G represents a mismatch nucleotide, and L represents that the pentose in the nucleotide has been replaced with LNA. [Figure 6]6 is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-4 of the present invention, in which G represents a mismatch nucleotide, and L represents that the pentose in the nucleotide has been replaced with LNA. [Figure 7] 7 is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-5 of the present invention, in which G represents a mismatch nucleotide, and L represents that the pentose in the nucleotide has been replaced with LNA. [Figure 8A] Figures 8A to 8C are diagrams showing the structure of a non-natural polynucleotide shown in Examples 1-6 of the present invention. Figure 8A is a diagram showing the structure of a non-natural polynucleotide in which the phosphodiester bond between the 5'-terminal LNA and the 1st to 5th nucleotides adjacent thereto is replaced with a phosphorothioate bond. In the figure, G represents a mismatch nucleotide, L represents that the pentose in the nucleotide has been replaced with LNA, and S represents that the phosphodiester bond has been replaced with a phosphorothioate bond. [Figure 8B] 8B is a diagram showing the structure of a non-natural polynucleotide in which the phosphodiester bond between the 3'-terminal nucleotide and the 1st to 7th nucleotides adjacent thereto is replaced with a phosphorothioate bond. In the figure, G indicates a mismatch nucleotide, L indicates that the pentose in the nucleotide is replaced with LNA, and S indicates that the phosphodiester bond is replaced with a phosphorothioate bond. [Figure 8C]8C is a diagram showing a non-natural polynucleotide configuration in which the phosphodiester bond between the 5'-terminal LNA and the four adjacent nucleotides is replaced with a phosphorothioate bond and the phosphodiester bond between the 3'-terminal nucleotide and the four adjacent nucleotides is replaced with a phosphorothioate bond; a non-natural polynucleotide configuration in which the phosphodiester bond between the LNA adjacent to the 5'-upstream side of the mismatch nucleotide G and the 2 or 4 nucleotides adjacent thereto is replaced with a phosphorothioate bond; and a non-natural polynucleotide configuration in which the nucleotide adjacent to the LNA adjacent to the 3'-downstream side of the mismatch nucleotide G and the phosphodiester bond between the 2 or 4 nucleotides adjacent to the adjacent nucleotide is replaced with a phosphorothioate bond. In the figure, G indicates a mismatch nucleotide, L indicates that the pentose in the nucleotide is replaced with LNA, and S indicates that the phosphodiester bond is replaced with a phosphorothioate bond. [Figure 9] 9 is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-7 of the present invention, in which G represents a mismatch nucleotide, L represents that the pentose in the nucleotide has been replaced with LNA, and S represents that the phosphodiester bond has been replaced with a phosphorothioate bond. [Figure 10A] 10A is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-8 of the present invention, in which G represents a mismatch nucleotide, and L represents that the pentose in the nucleotide has been replaced with LNA. [Figure 10B] 10B is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-8 of the present invention, in which G represents a mismatch nucleotide, and L represents that the pentose in the nucleotide has been replaced with LNA. [Figure 11]11 is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-9 of the present invention, in which G represents a mismatch nucleotide, L represents that the pentose in the nucleotide has been replaced with LNA, and R represents that the pentose in the nucleotide has been replaced with ribose. [Figure 12] 12 is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-10 of the present invention, in which G indicates a mismatch nucleotide, L indicates that the pentose in the nucleotide is replaced with LNA, P indicates that the 5'-terminal nucleotide and / or the 3'-terminal nucleotide is phosphorylated, and S indicates that the phosphodiester bond moiety is replaced with a phosphorothioate bond. [Figure 13] 13 is a diagram showing the structure of the non-natural polynucleotide shown in Example 1-11 of the present invention, in which G indicates a mismatch nucleotide, L indicates that the pentose in the nucleotide has been replaced with LNA, H indicates that the pentose in the nucleotide has been replaced with BNA-NH, M indicates that the pentose in the nucleotide has been replaced with BNA-N-Me, and E indicates that the pentose in the nucleotide has been replaced with ENA. [Figure 14] 1 is a diagram showing an outline of the structure of the non-natural polynucleotide (GEO-226) shown in Example 1-12 of the present invention, in which the arrow indicates the position of the mismatched nucleotide, L indicates that the pentose in the nucleotide is replaced with LNA, S indicates that the phosphodiester bond is replaced with a phosphorothioate bond, and P indicates that the 3'-end is phosphorylated. [Figure 15] FIG. 1 shows an outline of the structure of the non-natural polynucleotide (GEO-172) shown in Example 1-13 of the present invention. In the figure, the arrow indicates the position of the mismatched nucleotide, L indicates that the pentose in the nucleotide is replaced with LNA, S indicates that the phosphodiester bond is replaced with a phosphorothioate bond, R indicates that the nucleotide is replaced with RNA, and P indicates that the 3' end is phosphorylated. [Figure 16A]FIG. 1 shows an outline of the structure of the non-natural polynucleotides (GEO-602 to GEO-605) shown in Example 3-3 of the present invention. In the figure, G indicates a mismatch nucleotide, and L indicates that the pentose in the nucleotide is replaced with LNA. In the non-natural polynucleotides in the figure, the nucleotides L, G, and the black bars are DNA, and the nucleotides in the white bars are RNA. [Figure 16B] FIG. 1 shows an outline of the structure of the non-natural polynucleotides (GEO-606 and GEO-693 to GEO-695) shown in Example 3-3 of the present invention. In the figure, G indicates a mismatch nucleotide, and L indicates that the pentose in the nucleotide has been replaced with LNA. In the non-natural polynucleotides in the figure, the nucleotides at the positions of L, G, and black bars are DNA, and the nucleotides at the positions of white bars are RNA. [Figure 17A] FIG. 1 shows an outline of the structure of the non-natural polynucleotides (GEO-696, GEO-697, GEO-701, and GEO-702) shown in Example 3-3 of the present invention. In the figure, G indicates a mismatch nucleotide, L indicates that the pentose in the nucleotide has been replaced with LNA, and S indicates that the phosphodiester bond has been replaced with a phosphorothioate bond. In the non-natural polynucleotides in the figure, the nucleotides at the positions of L, G, and the black bands are DNA, and the nucleotides at the positions of the white bands are RNA. [Figure 17B] FIG. 1 shows an outline of the structure of the non-natural polynucleotides (GEO-703, GEO-822, and GEO-823) shown in Example 3-3 of the present invention. In the figure, G indicates a mismatch nucleotide, L indicates that the pentose in the nucleotide has been replaced with LNA, and P indicates that the 3' end has been phosphorylated. In the non-natural polynucleotides in the figure, the nucleotides at the positions of L, G, and black bars are DNA, and the nucleotides at the positions of white bars are RNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] <Summary of the Invention> In conventional techniques using single-stranded polynucleotides, the polynucleotide introduced into a cell specifically recognizes and binds to a target nucleotide sequence in the lagging strand of a replication fork during replication of double-stranded DNA, and acts as a primer for synthesis of Okazaki fragments by DNA polymerase. In this way, the polynucleotide is incorporated into the nascent DNA strand, thereby editing the genome sequence. Meanwhile, since the cells of living organisms are equipped with a mismatch repair mechanism that repairs mismatches that occur during DNA replication, mismatches caused by polynucleotides introduced from outside are also immediately repaired. One of the reasons why the editing efficiency of the modification of a target nucleotide sequence by a polynucleotide introduced from outside into a cell is lower than that of the modification of a target nucleotide sequence using a nuclease is thought to be the correction of editing by this mismatch repair mechanism.

[0022] On the other hand, in the case of the non-natural polynucleotide of the present invention, the presence of a cross-linked nucleic acid at a specific position in the non-natural polynucleotide promotes editing, and at the same time reduces the effects of the mismatch repair mechanism, presumably enabling the editing efficiency of the target nucleotide sequence to be significantly increased compared to previous reports.

[0023] In the present invention, in a non-natural polynucleotide, the efficiency of target nucleotide sequence modification can be further increased by appropriately combining, for example, the number and positions of mismatched nucleotides, the number and positions of crosslinked nucleic acids, the chain length of the polynucleotide, the type of crosslinked nucleic acid, replacement of a phosphodiester bond between one or more nucleotides with a phosphate-modified bond (e.g., phosphorothioate bond), replacement of a pentose sugar in one or more nucleotides with ribose (sometimes abbreviated as "RNA substitution" herein), phosphorylation of the 3' or 5' end, addition of an adapter, and the like.

[0024] <nucleotide> In the present invention, "nucleotide" is a general term for a substance in which a phosphate group is bound to a nucleoside. A nucleoside is a pentose in which a purine base or a pyrimidine base is bound to the first position via a glycosidic bond. A chain-like biopolymer in which nucleotides are the units is a polynucleotide (also called "nucleic acid"). There are two types of polynucleotides: deoxyribonucleotides (DNA) and ribonucleotides (RNA). DNA is composed of four types of nucleotides: adenosine monophosphate (AMP, hereinafter referred to as "A"), guanosine monophosphate (GMP, hereinafter referred to as "G"), cytidine monophosphate (CMP, hereinafter referred to as "C"), and thymidine monophosphate (dTMP, hereinafter referred to as "T"). RNA contains A, G, and T in common with DNA, but contains uridine monophosphate (UMP, hereinafter referred to as "U") instead of T.

[0025] <Double-stranded DNA> In the present invention, the term "double-stranded DNA" refers to a 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, but examples thereof include genomic DNA, mitochondrial DNA, and chloroplast DNA. In the present invention, the double-stranded DNA containing a "target nucleotide sequence" is preferably a gene involved in a genetic disease, more preferably a gene involved in a human genetic disease.

[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 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 glycogen storage disease type Ia (G6PC), genes related to familial frontotemporal lobar degeneration (GRN), genes related to mucopolysaccharidosis type II (IDS), mucopolysaccharidosis Examples of such diseases include a gene related to type I (IDUA), a gene related to primary immunodeficiency syndrome (IKBKB), a gene related to familial hypertrophic cardiomyopathy (MYH7), a gene related to peroxisome biogenesis disorder (PEX2), a gene related to hepatic glycogen storage disease type IX (PHKA2), a gene related to protein C deficiency (PROC), a gene related to hepatic glycogen storage disease type V (PYGM), a gene related to familial dilated cardiomyopathy (RBM20), a gene related to α1-antitrypsin deficiency (SERPINA1), a gene related to citrin deficiency (SLC25A13), a gene related to cystinuria (SLC7A9), a gene related to Niemann-Pick disease (SMPD1), and a gene related to amyotrophic lateral sclerosis (SOD1). Note that all of these diseases and genes are human diseases and genes.

[0027] <Complementary and mismatched nucleotides> In double-stranded DNA, G and C, and A and T are usually bonded by hydrogen bonds to form combinations of purine and pyrimidine bases. A nucleotide that pairs with a specific nucleotide is called a "complementary nucleotide."

[0028] In the present invention, the term "mismatch nucleotide" refers to a nucleotide that cannot form a Watson-Crick type hydrogen bond between two bases or a nucleotide that cannot form a Watson-Crick type hydrogen bond between two bases because there is no corresponding nucleotide. That is, the term "a 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. "A Watson-Crick type hydrogen bond between two bases cannot be formed because there is no corresponding nucleotide" refers to a nucleotide that does not have any of the corresponding nucleotides A, T, G, and C for G, a nucleotide that does not have any of the corresponding nucleotides A, T, G, and C for A, a nucleotide that does not have any of the corresponding nucleotides A, T, G, and C for T, and a nucleotide that does not have any of the corresponding nucleotides A, T, G, and C for G.

[0029] The non-natural polynucleotide of the present invention may have one or more "mismatch nucleotides" for modifying the base to be edited, and may also have one or more "mismatch nucleotides" that are not involved in the modification. The latter "mismatch nucleotide" is a mismatch nucleotide for the purpose of improving editing efficiency, and includes, for example, a mismatch nucleotide inserted between the mismatch nucleotide to be modified and the 3'-terminal nucleotide, and a mismatch nucleotide in the adaptor when the adaptor is a nucleotide.

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

[0031] "Modification" of a target nucleotide sequence refers to the substitution of a specific nucleotide (e.g., G) with any of the other three nucleotides (A, T, or C) (hereinafter referred to as "substitution") for one or more nucleotides in the target nucleotide sequence, the deletion of one or more nucleotides in the target nucleotide sequence (hereinafter referred to as "deletion"), or the insertion of another nucleotide or nucleotide sequence between two specific nucleotides in the target nucleotide sequence (hereinafter referred to as "insertion"). "Modification" of a target nucleotide sequence includes each of substitution, deletion, and insertion occurring alone, and combinations of these.

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

[0033] Examples of non-natural nucleotides include, but are not limited to, nucleic acids with modified phosphate moieties, nucleic acids with modified sugar moieties, nucleic acids with modified base moieties, nucleic acids with adapters added to the 3'-end and / or 5'-end, and nucleic acids with one or more nucleotides inserted between the mismatch nucleotide and the 3'-end nucleotide. Examples of nucleic acids with modified phosphate moieties include, but are not limited to, nucleic acids in which the phosphodiester bond between one or more nucleotides is replaced with a phosphorothioate bond. While a normal bond between 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 called a "phosphorothioated" or "S-modified" nucleotide.

[0034] Other examples of nucleic acids with modified phosphate moieties include nucleic acids having a methyl phosphate 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), and nucleic acids having a mesyl phosphoramidate bond in which one of the non-bridging oxygen atoms of the phosphodiester bond is replaced with -NSO2CH3.

[0035] Examples of nucleic acids whose sugar moieties are modified include, but are not limited to, nucleic acids whose 2' site is modified, crosslinked nucleic acids in which the 2' site and the 4' site are crosslinked, etc. Examples of nucleic acids whose 2' site is modified include, but are not limited to, 2'-F (F-modified), 2'-O-Methyl (2'-OMe), 2'-O-Methoxyethyl (2'-MOE), 2'-O-(2-carbamoylethyl), etc.

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

[0037] [ka]

[0038] Examples of BNA / LNA analogs include, but are not 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, but includes BNA NH, BNA N-Me, and BNA N-Bn.

[0040] Examples of bicyclic or tricyclic bridged nucleic acids include, but are not 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] Other cross-linked nucleic acids include, but are not limited to, oxetane nucleotides, locked PMOs derived from 2'-amino-LNA, cyclohexenyl nucleic acid (CeNA), alitriol 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 non-natural polynucleotide of the present invention, if the number of crosslinked nucleic acids is too large, the editing efficiency tends to decrease. For example, the ratio of crosslinked nucleic acids in the non-natural polynucleotide of the present invention is preferably 2 to 30%, more preferably 2 to 20%, and even more preferably 2 to 15%, when the total number of nucleotides constituting the non-natural polynucleotide is taken as 100%.

[0043] In the non-natural polynucleotide of the present invention, it is preferable to add an adapter to the 3'-end and / or 5'-end, since this shows higher editing efficiency. In the present specification, examples of the adapter include those having a function of inhibiting mismatch repair in host cells, those having a function of protecting the non-natural polynucleotide of the present invention from nuclease digestion, and those having a function of imparting a higher editing efficiency than the non-natural polynucleotide (GEO-8) defined by SEQ ID NO: 9 when added 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 adapters include (1) nucleotides as adapters and (2) compounds other than nucleotides that modify the ends (referred to as "modifying compounds" in the present specification).

[0044] The estimated mechanism by which mismatch repair in a host cell is inhibited by adding an adaptor to the 3'-end and / or 5'-end of the non-natural polynucleotide of the present invention is as follows. When one strand of a double-stranded DNA has a 3'-end structure, mismatch repair begins when a mismatch repair enzyme in the cell recognizes the strand having the 3'-end as a target for repair, and then the mismatched nucleotide on the strand having the 3'-end is removed and repaired. When the non-natural polynucleotide of the present invention is specifically bound to a target sequence, the non-natural polynucleotide has a 3'-end structure, so that the mismatched nucleotide of the non-natural polynucleotide is repaired. When a nucleotide is added as an adaptor to the 3'-end of the non-natural polynucleotide, the 3'-end of the non-natural polynucleotide does not specifically bind to the target sequence and dissociates, so that it may be avoided from being recognized by the mismatch repair enzyme. Similarly, when a modifying compound is added to the 3'-end of the non-natural polynucleotide, it may be possible that the 3'-end of the non-natural polynucleotide is also avoided from being recognized by the mismatch repair enzyme. For these reasons, it is presumed that the editing efficiency of a non-natural polynucleotide having an adaptor nucleotide or a modifying compound added to the 3' end is increased by inhibiting mismatch repair in the host cell.

[0045] The estimated mechanism by which the non-natural polynucleotide of the present invention is protected from nuclease digestion in host cells by adding an adaptor 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 subject to digestion by nuclease in the host cell. Although the digestion of the non-natural polynucleotide by nuclease cannot be completely prevented by simply adding a nucleotide as an adaptor to the 5'-end or 3'-end of the non-natural polynucleotide, it is estimated that the nucleotide as an adaptor acts as a buffer to inhibit the digestion by exonuclease from proceeding to the mismatched nucleotide located in the center of the non-natural polynucleotide. On the other hand, the addition of a modifying compound to the 3'-end is highly likely to inhibit digestion by exonuclease from the 3'-end. For example, IdT is used to increase the nuclease resistance of an antisense nucleic acid by adding it to the 3'-end of the antisense nucleic acid.

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

[0047] Examples of the structure of the nucleotide as the adaptor include linear nucleotides and nucleotides that form a stem structure which may have a loop. The chain length of the nucleotide as the adaptor is preferably 1 nt (nucleotide) or more, more preferably 3 nt or more, and even more preferably 6 or more, and is preferably 50 nt or less, more preferably 40 nt or less, and even more preferably 30 nt or less. The non-natural polynucleotide of the present invention does not contain a nucleotide as an adaptor, and therefore the chain length of the nucleotide as the adaptor does not affect the chain length of the non-natural polynucleotide of the present invention. The nucleotides serving as adapters are preferably nucleotides containing mismatched nucleotides, and it is more preferable that the nucleotide sequences of the nucleotides serving as adapters are all mismatched with respect to the 5' upstream sequence adjacent to the target nucleotide sequence or the 3' downstream sequence adjacent to the target nucleotide sequence, although one or more may be complementary to the target nucleotide sequence.

[0048] The modifying compound serving as the adaptor is preferably a compound capable of binding to the 3'-terminus and / or 5'-terminus of the non-natural polynucleotide of the present invention. The modifying compound is preferably a compound of a certain size. For example, the molecular weight of the modifying compound is preferably 50 or more, more preferably 100 or more, and is preferably 2,000 or less, more preferably 1,500 or less, even more preferably 1,000 or less, even more preferably 800 or less, and even more preferably 500 or less. Preferred specific examples of the modifying compound include at least one selected from the group consisting of fluorescein (FAM), biotin, puromycin, cholesterol, digoxigenin (DIG) and inverted dT. The binding of an adaptor to the non-natural polynucleotide of the present invention can be carried out by known methods.

[0049] These unnatural nucleotides with such modifications have a high affinity for natural DNA and RNA, and therefore their application in nucleic acid drugs and other fields is being promoted.

[0050] <Basic structure of non-natural polynucleotides> The non-natural polynucleotide of the present invention is a single-stranded polynucleotide, which contains one or more mismatched nucleotides with respect to a target nucleotide sequence, and the nucleotides other than the mismatched nucleotides are nucleotides complementary to the target nucleotide sequence. Therefore, by introducing the non-natural polynucleotide of the present invention into a cell, it can specifically bind to the target nucleotide sequence. As a result, one or more nucleotides in the target nucleotide sequence can be modified to desired nucleotides. When the mismatched nucleotides are multiple, the two or more mismatched nucleotides may be adjacent to each other or may be located at different positions. In the present invention, the term "multiple" 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 also includes ranges with these as upper and lower limits. For example, 2~3, 2~4, 2~5, ···2~20, 3~4, 3~5, 3~6, ​​···3~20, 4~5, 4~6, 4~7, ···4~20, 5~6, 5~7, 5~8, ···5~20, ···18~19, 18~20, 19~20.

[0051] Such basic structures of the non-natural polynucleotide of the present invention include, for example, those described in the following embodiments (i) to (vi).

[0052] (1) Aspect (i) One embodiment (embodiment (i)) of the non-naturally occurring polynucleotide of the present invention is (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; and (D) The length is 22 to 95 nucleotides. This configuration can achieve the effects of the present invention.

[0053] The bridged nucleic acid contained in the non-natural polynucleotide of aspect (i) is not particularly limited, and may be at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA. In the present invention, the bridged nucleic acid of (A) and / or (B) is preferably LNA or BNA NH, more preferably LNA. In the present invention, the bridged nucleic acid of (C) is preferably LNA, AmNA, BNA NH, BNA N-Me, or ENA.

[0054] Here, the mismatched nucleotide may or may not be a bridged nucleic acid, but in embodiment (i), it is more preferable that the mismatched nucleotide is not a bridged nucleic acid. Of the total of three nucleotides adjacent to the mismatched nucleotide, the number of bridged nucleic acids is preferably two. In addition, the term "multiple" in "multiple nucleotides are bridged nucleic acids" as defined in (A) and (B) above is not particularly limited, but it is preferable that the number of consecutive bridged nucleic acids is two rather than three or more, and more preferably one.

[0055] (2) Aspect (ii) Another embodiment (embodiment (ii)) of the non-naturally occurring polynucleotide of the present invention is (A) one or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are bridged nucleic acids; (C) the 5'-terminal nucleotide is a bridged nucleic acid; and (D) The length is 22 to 95 nucleotides. (However, it does not have the feature (B) above.) Even with this configuration, the effects of the present invention can be achieved.

[0056] The bridged nucleic acid contained in the non-natural polynucleotide of embodiment (ii) is not particularly limited, and may be at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA. In the present invention, the bridged nucleic acid contained in the non-natural polynucleotide is preferably LNA.

[0057] Here, the mismatched nucleotide may or may not be a bridged nucleic acid, but in embodiment (ii), it is more preferable that the mismatched nucleotide is not a bridged nucleic acid. Of the total of three nucleotides adjacent to the mismatched nucleotide, the number of bridged nucleic acids is preferably two. In addition, the term "multiple" in "multiple nucleotides are bridged nucleic acids" as defined in (A) above is not particularly limited, but it is preferable that the number of consecutive bridged nucleic acids is two rather than three or more, and more preferably one.

[0058] (3) Aspect (iii) A further aspect (aspect (iii)) of the non-naturally occurring polynucleotide of the present invention is (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; and (D) The length is 22 to 95 nucleotides. (However, it does not have the feature (A) above.) Even with such a configuration, the effects of the present invention can be achieved.

[0059] The bridged nucleic acid contained in the non-natural polynucleotide of embodiment (iii) is not particularly limited, and may be at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA. In the present invention, the bridged nucleic acid contained in the non-natural polynucleotide is preferably LNA.

[0060] Here, the mismatched nucleotide may or may not be a bridged nucleic acid, but in embodiment (iii), it is more preferable that the mismatched nucleotide is not a bridged nucleic acid. Of the total of three nucleotides adjacent to the mismatched nucleotide, the number of bridged nucleic acids is preferably two. In addition, the term "multiple" in "multiple nucleotides are bridged nucleic acids" as defined in (B) above is not particularly limited, but it is preferable that the number of consecutive bridged nucleic acids is two rather than three or more, and more preferably one.

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

[0062] [Relationship between mismatched nucleotides and adjacent cross-linked nucleic acids] In any one of the above aspects (i) to (iii), the non-natural polynucleotide of the present invention can exhibit sufficient editing efficiency for a target nucleotide sequence even if the mismatched nucleotide is, for example, a bridged nucleic acid. In this case, it is more preferable that the bridged nucleic acid is an LNA.

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

[0064] [3'-end cross-linked nucleic acid] In any one of the above aspects (i) to (iii), when the 3'-terminal nucleotide (E) is a crosslinked nucleic acid, the non-natural polynucleotide of the present invention can further increase the editing efficiency of the target nucleotide sequence.

[0065] [Nucleic acid with bridged nucleotide adjacent to the 5' end] In any of the above aspects (i) to (iii), or in addition to the above aspect (E), the non-natural polynucleotide of the present invention can further improve the editing efficiency of the target nucleotide sequence when, for example, (F) one or more nucleotides adjacent to the 5'-terminal nucleotide are bridged nucleic acids. In this case, it is preferable that one nucleotide adjacent to the 5'-terminal nucleotide is a bridged nucleic acid. Furthermore, it is more preferable that the bridged nucleic acid is an LNA. In this case, the number of consecutive bridged nucleic acids at the 5'-terminal nucleotide is not particularly limited, but for example, when the number of 5'-terminal nucleotides from the mismatch nucleotide is 12 nt, it is preferably 2, and when the number of 5'-terminal nucleotides is 25 nt, it is preferably 3.

[0066] [Nucleic acid with bridged nucleotide adjacent to the 3' end] In the non-natural polynucleotide of the present invention, 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 (G) are bridged nucleic acids, the editing efficiency of the target nucleotide sequence can be further improved. In this case, it is more preferable that the bridged nucleic acid is an LNA. In this case, the number of bridged nucleic acids contiguous at the 3'-terminal nucleotide is not particularly limited, but for example, when the number of 3'-terminal nucleotides from the mismatched nucleotide is 27 nt, the number is preferably 3 or less, more preferably 2 or less, and even more preferably 1. Furthermore, for example, when the number of 3'-terminal nucleotides from the mismatched nucleotide is 12 nt, the number of bridged nucleic acids contiguous at the 3'-terminal nucleotide is preferably 3 or less, more preferably 2 or less, and even more preferably 1.

[0067] [Substitution of modified bonds at the phosphate moiety] In the non-natural polynucleotide of the present invention, in any one of the above aspects (i) to (iii), or in addition to the above (E), (F) and / or (G), for example, when one or more internucleotide phosphodiester bonds are replaced with phosphate-modified bonds, the editing efficiency of the present invention can be further improved. Specific examples of the configuration include, but are not limited to, the following (J).

[0068] (J) One or more internucleotide phosphodiester bonds are replaced with phosphate-modified linkages. In the present invention, the phosphate moiety modified bond is a bond in which some atoms of a phosphodiester bond are replaced with other atoms or substituents, such as sulfur atoms, boron atoms, nitrogen atoms, methyl groups, and ester groups. Specific examples of the phosphate moiety modified bond include phosphorothioate bonds, methyl phosphate bonds, boranophosphate bonds, and mesyl phosphoramidate bonds. In the non-natural polynucleotide of the present invention, the number and positions of the phosphate moiety modification bonds are not particularly limited. Furthermore, the non-natural polynucleotide of the present invention may have one type of phosphate moiety modification bond or two or more types of phosphate moiety modification bonds. The number of phosphate moiety modified bonds may be 1, or the ratio of phosphate moiety modified bonds in the phosphodiester bonds may be 60% or less. However, from the viewpoint of editing efficiency, it is preferable that not all of the phosphodiester bonds are substituted.

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

[0070] In the above (J), from the viewpoint of editing efficiency, the phosphate moiety modification bond is preferably a phosphorothioate bond. Examples of the phosphate moiety modification bond being a phosphorothioate bond include any one or combination of the following (H1), (I), (K) and (L). (H1) The phosphodiester bond between the 5'-terminal nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide is replaced with a phosphorothioate bond. (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. (K) The phosphodiester bond between the bridged nucleic acid adjacent to the 5' upstream side of the mismatched nucleotide and one or more nucleotides further adjacent to the bridged nucleic acid is substituted with a phosphorothioate bond. (L) The phosphodiester bond between the bridged nucleic acid adjacent to the 3' downstream side of the mismatched nucleotide and one or more nucleotides further adjacent to the bridged nucleic acid is substituted with a phosphorothioate bond.

[0071] In the above features (K) and (L), the phosphodiester bond between the crosslinked nucleic acid and one nucleotide immediately adjacent to the crosslinked nucleic acid may or may not be replaced by a phosphorothioate bond.

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

[0073] [The bridged nucleic acid is between the bridged nucleic acid adjacent to the 5' upstream side of the mismatched nucleotide and the bridged nucleic acid at the 5' end] In any of the above aspects (i) to (iii), or in addition to the above aspects (E), (F), (G) and / or (J), the non-natural polynucleotide of the present invention can further improve the editing efficiency of a target nucleotide sequence, for example, when (M) one or more nucleotides located between the nucleotide adjacent to the 5' upstream side of the mismatch nucleotide and the 5'-terminal nucleotide and spaced at a distance of at least one nucleotide from both the nucleotide adjacent to the 5' upstream side of the mismatch nucleotide and the 5'-terminal nucleotide are crosslinked nucleic acids.

[0074] [The bridged nucleic acid is between the bridged nucleic acid adjacent to the 3' downstream side of the mismatched nucleotide and the bridged nucleic acid at the 3' end] In the non-natural polynucleotide of the present invention, in any of the above aspects (i) to (iii), or in addition to the above (E), (F), (G), (J) and / or (M), for example, when (N) one or more nucleotides located between the nucleotide adjacent to the 3' downstream side of the mismatch nucleotide and the 3'-terminal nucleotide and spaced at a distance of at least one nucleotide from both the nucleotide adjacent to the 3' downstream side of the mismatch nucleotide and the 3'-terminal nucleotide are crosslinked nucleic acids, the editing efficiency of the target nucleotide sequence can be further improved.

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

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

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

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

[0079] [Nucleotide insertion between the mismatched nucleotide and the 3'-terminal nucleotide] In the non-natural polynucleotide of the present invention, in any of the above aspects (i) to (iii), or in addition to the above (E), (F), (G), (J), (M), (N), (O), (X2), (P), (X1) and / or (X4), when one or more nucleotides are inserted between the mismatch nucleotide and the 3'-terminal nucleotide (X5), the editing efficiency of the target nucleotide sequence can be further improved. The "mismatch nucleotide" in (X5) refers to a mismatch nucleotide for the purpose of modification. Furthermore, the inserted nucleotide in (X5) is a mismatch nucleotide, but the mismatch nucleotide here is a mismatch nucleotide for the purpose of improving the editing efficiency, and does not correspond to a mismatch nucleotide for the purpose of modification. Furthermore, the inserted nucleotide may be the above-mentioned nucleic acid with a modified phosphate moiety, the above-mentioned nucleic acid with a modified sugar moiety, or the above-mentioned nucleic acid with a modified base moiety. In (X5), the number of inserted nucleotides may be 1, and when multiple nucleotides are inserted, it is preferably 2 or more, while it is preferably 6 or less, more preferably 3 or less.

[0080] (4) Aspect (iv) A further aspect (aspect (iv)) of the non-naturally occurring polynucleotide of the present invention is (A) one or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are bridged nucleic acids, and / or (B) one or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are bridged nucleic acids, (D) the length is 22 to 95 nucleotides, and (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 mismatch nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide are mismatch nucleotides; and (Y3) an adaptor is added to the 5'-terminal. This configuration can achieve the effects of the present invention. In the embodiment (iv), (A), (B) and (D) are the same as those in the embodiment (i).

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

[0082] [Substitution of modified bonds at the phosphate moiety] In the non-natural polynucleotide of the present invention, in view of editing efficiency, (H2) the phosphodiester bond between the 5'-terminal nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide is preferably replaced with a phosphate moiety-modified bond.

[0083] In the present invention, the phosphate moiety modified bond is a bond in which some atoms of a phosphodiester bond are replaced with other atoms or substituents, such as sulfur atoms, boron atoms, nitrogen atoms, methyl groups, and ester groups. Specific examples of the phosphate moiety modified bond include phosphorothioate bonds, methyl phosphate bonds, boranophosphate bonds, and mesyl phosphoramidate bonds. In the non-natural polynucleotide of the present invention, the number and positions of the phosphate moiety modification bonds are not particularly limited. Furthermore, the non-natural polynucleotide of the present invention may have one type of phosphate moiety modification bond or two or more types of phosphate moiety modification bonds. The number of phosphate moiety modified bonds may be 1, or the ratio of phosphate moiety modified bonds in the phosphodiester bonds may be 60% or less. However, from the viewpoint of editing efficiency, it is preferable that not all of the phosphodiester bonds are substituted.

[0084] [Introduction of mismatch nucleotides at the 5' end] In the non-natural polynucleotide of the present invention, in view of editing efficiency, it is preferable that the 5'-terminal nucleotide of (Y1) is a mismatched nucleotide, and one or more nucleotides adjacent to the 5'-terminal nucleotide are mismatched nucleotides. The mismatched nucleotide in (Y1) is a "mismatched nucleotide for the purpose of improving editing efficiency" and does not fall under the category of "mismatched nucleotide for modifying the target nucleotide sequence." When there are multiple mismatched nucleotides in (Y1), the mismatched nucleotides may or may not be adjacent to each other. The number is preferably 2 or more, and more preferably 10 or less, and more preferably 7 or less.

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

[0086] [RNA replacement] The non-natural polynucleotide of the present invention is a single-stranded polynucleotide, and deoxyribose in one or more nucleotides may be replaced with ribose. Specifically, in aspect (iv), for example, if (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, the editing efficiency of the target nucleotide sequence can be further improved.

[0087] (5) Mode (v) A further embodiment (embodiment (v)) of the non-naturally occurring polynucleotide of the present invention is (A) one or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are bridged nucleic acids, and / or (B) one or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are bridged nucleic acids, (D) The length of the strand is 22 to 95 nucleotides. (X7) one or more nucleotides located between the mismatched nucleotide and the 5'-terminal nucleotide are bridged nucleic acids This configuration can achieve the effects of the present invention. In the embodiment (v), (A), (B) and (D) are the same as those in the embodiment (i).

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

[0089] In the non-natural polynucleotide of the present invention, in view of editing efficiency in the aspect (v), (X7) one or more nucleotides located between the mismatch nucleotide and the 5'-terminal nucleotide are bridged nucleic acids. The bridged nucleic acid defined in (X7) is not particularly limited, but may be at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me and ENA, with LNA being preferred.

[0090] (6) Mode (vi) A further aspect (aspect (vi)) of the non-naturally occurring polynucleotide of the present invention is (C) the 5'-terminal nucleotide is a bridged nucleic acid; (D) The length of the strand is 22 to 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. This configuration can achieve the effects of the present invention. (C), (D) and (I) in embodiment (vi) are the same as those in embodiment (i).

[0091] In the non-natural polynucleotide of the present invention, in view of editing efficiency, (X6) the mismatched nucleotide is a bridged nucleic acid. The bridged nucleic acid defined in (X6) is not particularly limited, but may be at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me and ENA, and LNA is preferred.

[0092] [3'-end cross-linked nucleic acid] In the non-natural polynucleotide of the present invention, in the aspect (vi), for example, when the 3'-terminal nucleotide (E) is a bridged nucleic acid, the editing efficiency of the target nucleotide sequence can be further improved. The bridged nucleic acid defined in (E) is not particularly limited, but includes at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA, and LNA is preferred.

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

[0094] <Editing efficiency> The efficiency of modifying a target nucleotide sequence using the non-natural polynucleotide of the present invention (referred to as "editing efficiency" in this specification) is, when expressed relative to the editing efficiency of 1.0 when using GEO-8 in the Examples described below, which is the non-natural polynucleotide shown in SEQ ID NO: 9, can be, for example, in the range of 0.4 or more, preferably 0.5 or more, and more preferably 0.6 or more.

[0095] <Kit> In the present invention, a "kit" refers to a genetic engineering tool used to modify a target nucleotide sequence containing a non-natural polynucleotide. The kit of the present invention may contain 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, within the scope of not impairing the effects of the present invention.

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

[0097] When the pharmaceutical composition of the present invention is used, it can be administered, for example, orally, intravenously, in muscle, oral mucosa, rectally, vaginally, transdermally, via the nasal cavity, or via inhalation. Alternatively, an ex vivo therapy can be used in which the pharmaceutical composition of the present invention is used on cells extracted from a patient, and the treated cells are cultured as necessary and 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 formulation by adding a pharma- ceutically acceptable carrier or formulation additive thereto. Examples of pharma-ceutically acceptable carriers or additives include, but are not limited to, lipid nanoparticles, excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, solubilizers, solubilization aids, isotonicity agents, pH adjusters, stabilizers, and the like.

[0098] Administration of the pharmaceutical compositions of the present invention will continue for several days to several months, depending on the severity and responsiveness of the condition being treated, or until a cure is effected or a diminution of the condition is achieved. Optimal dosages, dosing methods, and repetition frequencies can be determined by those skilled in the art.

[0099] <Diseases for which the pharmaceutical composition is intended> Examples of diseases that the pharmaceutical composition of the present invention can treat include diseases caused by the substitution of a single base in the human genome, such as adrenoleukodystrophy, medium-chain acyl-CoA dehydrogenase deficiency, Wilson's disease, hereditary pulmonary hypertension, X-linked agammaglobulinemia, cystinuria, Duchenne muscular dystrophy, hemophilia A, hemophilia B, tyrosinemia, hepatic glycogen storage disease type Ia, familial frontotemporal lobar degeneration, mucopolysaccharidosis type II, mucopolysaccharidosis type I, primary immunodeficiency syndrome, familial hypertrophic cardiomyopathy, peroxisome biogenesis disorder, hepatic glycogen storage disease type IX, protein C deficiency, hepatic glycogen storage disease type V, familial dilated cardiomyopathy, α1-antitrypsin deficiency, citrin deficiency, cystinuria, Niemann-Pick disease, and amyotrophic lateral sclerosis.

[0100] <Preparation of non-natural polynucleotides> The non-naturally occurring polynucleotide of the present invention can be easily prepared by methods known in the field of the present invention.

[0101] <Introduction of non-natural polynucleotides into cells> In the present invention, "introducing a non-natural polynucleotide into a cell" can be carried out according to a known method depending on the cell to be introduced. Known methods for introducing a polynucleotide into a cell (transfection method) are broadly divided into two types: a viral vector system and a non-viral vector system. The viral vector system is a method for introducing a gene into a cell by utilizing the cell entry mechanism that a virus originally has, and is a method using, but not limited to, adenovirus, retrovirus, lentivirus, etc. as a vector. Examples of non-viral vector systems include, but are not limited to, a lipofection method, an electroporation method, a microinjection method, a particle gun method, etc. The lipofection method is a method that utilizes the phenomenon in which a positively charged cationic liposome binds around a negatively charged polynucleotide to form a complex, and the polynucleotide is taken up into the cell from the cell surface by the endocytosis phenomenon. The electroporation method is a method in which a high voltage pulse is directly applied to the cell using a dedicated machine, and the polynucleotide is taken up through small holes on the cell surface. The microinjection method is a method in which a substance to be introduced is placed in a glass needle with a tip diameter of about 1 μm and directly introduced into a cell. The particle gun method is a method in which the surface of gold particles is coated with DNA by co-precipitating gold particles and DNA, and the gold particles are ejected toward a target cell by 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 Cas nuclease into a cell, and a non-viral vector-based method is more preferable from the viewpoint of safety. Examples of the method of the present invention include a method performed inside the human body and a method including a step performed inside the human body.

[0102] The non-natural polynucleotide (also referred to as "edited nucleic acid" in this specification) introduced into the cell in this manner selectively binds to the target nucleotide sequence in genomic DNA, as shown diagrammatically in Figure 1. The non-natural polynucleotide of the present invention is preferably designed so that it is mismatched with the base to be edited in the target nucleotide sequence in double-stranded DNA in a cell, such as genomic DNA, and all base sequences other than the base to be edited are complementary to the target nucleotide sequence. However, there may be mismatched nucleotides other than the base to be edited, as long as it does not contradict the object of the present invention.

[0103] For example, when the base to be edited in the target nucleotide sequence is T and it is desired to change it to C, the mismatch nucleotide in the non-natural polynucleotide of the present invention is set to G. A person skilled in the art can set the mismatch nucleotide in the non-natural polynucleotide based on common technical knowledge in order to change the base of the base to be edited to a desired base.

[0104] <Method for modifying one or more nucleotides contained in a target nucleotide sequence in a double-stranded DNA in a cell> The "method for modifying one or more nucleotides contained in a target nucleotide sequence in a double-stranded DNA in a cell" of the present invention comprises the step of introducing the above-mentioned non-natural polynucleotide of the present invention into a cell. Here, the modification of the target nucleotide sequence includes at least one selected from the group consisting of deletion, insertion, and substitution of one or more nucleotides in the target nucleotide sequence. As the non-natural 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> Methods for confirming modification of a target nucleotide sequence in the present invention include a method for measuring the activity of a gene containing the target nucleotide sequence, and a method for directly measuring the target nucleotide sequence using a next-generation sequencer (NGS) and digital PCR.

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

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

[0108] <Eukaryotic cells> In the present invention, the term "eukaryotic cells" is not particularly limited, but includes animal cells, insect cells, plant cells, algae cells, and fungal cells.

[0109] <Animal cells> In the present invention, the term "animal cells" includes, but is not limited to, vertebrate cells, invertebrate cells, cells derived from animal tissues, germ cells, somatic cells, and stem cells. Germ cells include oocytes and sperm cells. Somatic cells include, but are not limited to, fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells, brain cells, kidney cells, and the like. Stem cells include, but are not limited to, induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).

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

[0111] As described above, human cells include tissue-derived cells, germ cells, somatic cells, and stem cells. In the examples of the present specification, the modification of the target nucleotide sequence was confirmed using HEK293 cells, but it can also be confirmed using other cells, such as HeLa cells.

[0112] <Insect cells> In the present invention, "insects" is a general term for arthropods classified in the class Insecta. Insects that can be used include, but are not limited to, silkworm larvae, fruit flies, crickets, etc. In the present invention, "insect cells" include, but are not limited to, cells that constitute the body tissue of insects and cells derived from insect tissue.

[0113] <Plant cells> In the present invention, the term "plant" includes, but is not limited to, seed plants, ferns, mosses, algae, etc. The term "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 dicotyledons and monocotyledons. Dicotyledons include, but are not limited to, morning glory, dandelion, azalea, azalea, eggplant, rapeseed, pea, etc. Monocotyledons include, but are not limited to, rice, lily, tulip, Japanese silver grass, corn, etc. Gymnosperms include, but are not limited to, pine, cedar, ginkgo, cypress, etc. Ferns include, but are not limited to, bracken, fern, Japanese oak, horsetail, etc. Bryophytes include, but are not limited to, Marchantia polymorpha, Hornwort, Polytrichum japonicum, Sphagnum, etc. Algae include multicellular algae and unicellular algae. Examples of multicellular algae include, but are not limited to, kelp, wakame, Ulva, Spirogyra, etc. Examples of unicellular algae include, but are not limited to, Chlorella, Euglena, Spirulina, Chlamydomonas, Coccomyxa, Botryococcus, Mikania, Diatom, etc. EXAMPLES

[0114] The present invention will be described in detail below with reference to specific examples, but the present invention is not limited to these.

[0115] A. Genome editing experiment using 293-nLD1 cells (1) 1. Construction of mutant NanoLuc® plasmids Based on the wild-type base sequence of the luciferase gene NanoLuc (GenBank JQ513379), DNA from base number 847 to 1,380 was synthesized (Gene Universal Inc.), and XhoI and ApaI linkers were ligated to create pcDNA TM The wild-type nucleotide sequence of the NanoLuc gene was shown in SEQ ID NO:1. On the other hand, a mutant base sequence was synthesized in which the cytosine (C) at base number 922 of the NanoLuc gene was changed to thymine (T), and the mutant was transformed into pcDNA TM A mutant was created by incorporating the luciferase gene into the 5 / FRT / TO plasmid (pcDNA5-nLD1). The luciferase gene in this mutant is an inactive luciferase gene that does not show luciferase activity due to the point mutation described above.

[0116] 2. Generation of stable transformed cell lines carrying the mutant NanoLuc gene Flp-In, a human-derived cell line TM -293 cell line (Thermo Fisher Scientific Inc.) at 1x10 6 The cells were seeded on a 6 cm dish 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 under 5% CO2. After 24 hours, the cells were transfected with pcDNA5-nLD1 or pcDNA5-nLW1 plasmid (1 μg) and pOG44 plasmid (3 μg, Thermo Fisher Scientific Inc.) using Lipofectamine 3000 (Thermo Fisher Scientific Inc.) according to the standard protocol. After 48 hours, the cells were transfected with TrypLE TM Express Enzyme (Thermo Fisher Scientific Inc.) was added and incubated at 37°C for 3 minutes. The detached cells were collected and suspended in DMEM + 10% FBS medium containing 50μg / ml of hygromycin (Thermo Fisher Scientific Inc.), and cultured in two 10cm dishes. Culture was then continued with the same medium replaced every 3 days. After approximately 20 days of culture, the formation of sufficient numbers and sizes of colonies was confirmed, and all cells were incubated with TrypLE. TMThe cells were detached from the petri dish using Express Enzyme and collected.

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

[0118] 3. Introduction of non-natural polynucleotides into 293-nLD1 cells 1x10 293-nLD1 cells 4 A 96-well plate (Nunc® MicroWell) was used to culture the cells. TM The cells were seeded onto a 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 polynucleotide was transfected using Lipofectamine 3000 according to the standard protocol.

[0119] 4. Measuring editing efficiency NanoLuc Luciferase Assay After 72 hours of culture from the transfection, NanoLuc Luciferase activity was measured. NanoLuc Luciferase activity was measured using the Nano-Glo® Luciferase Assay System (Promega Corporation) according to the standard protocol. Luciferase luminescence was measured using an EnSpire multimode plate reader (PerkinElmer Co., Ltd.).

[0120] Counting viable cells The number of viable cells was measured using CellTiter-Blue® Cell Viability Assay (Promega Corporation) according to the standard protocol. The fluorescence value of CellTiter-Blue was measured using an EnSpire multimode plate reader (PerkinElmer Co., Ltd.). The number of viable edited cells was 5 x 10 3 From 2 x 10 5 A standard curve of CellTiter-Blue measurement values ​​and cell numbers was created using a two-fold dilution series of 293-nLD1 cell numbers between 100 and 1000 μg / mL, and the number of live cells was calculated based on this standard curve.

[0121] Editing efficiency calculation When the NanoLuc Luciferase activity of 293-nLW1 cells with wild-type luciferase integrated into the genome was measured, 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, a 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 x100 The calculation was done using the formula:

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

[0123] 6. Synthesis of non-natural polynucleotides All non-natural polynucleotides, including those with modified nucleic acids, were synthesized by Gene Design Co., Ltd. (Japan) and purified using a simple column or HPLC.

[0124] Example 1-1 Modification of target nucleotide sequence by non-natural polynucleotide Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells. As mentioned above, the codon (CAA) encoding the 22nd glutamine (Gln-22) in the luciferase gene introduced into 293-nLD1 cells is mutated to a stop codon (TAA), and the gene does not show luciferase activity in its current state. If the T in the stop codon (TAA) in the inactive luciferase gene can be replaced with a C, the luciferase activity of the 293-nLD1 cells is restored, and the modification of the target nucleotide sequence can be detected by the luminescence produced by luciferase (Figure 2).

[0125] In this Example 1-1, 11 types of non-natural polynucleotides, GEO-1 to GEO-9, GEO-242, and GEO-11, as well as the non-natural polynucleotide GEO-94, which cannot repair inactivating mutations because it does not contain 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 stop codon TAA in the target polynucleotide sequence is G, i.e., a mismatch, and the nucleotides other than the mismatch nucleotide are nucleotides 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 mismatch nucleotide G is LNA. In GEO-2 and GEO-6, one nucleotide on the 5' upstream side and one nucleotide on the 3' downstream side adjacent to the mismatch nucleotide G are LNA. In GEO-3 and GEO-7, the mismatch nucleotide G and the 5' terminal nucleotide are LNA. In GEO-4 and GEO-8, one nucleotide on the 5' upstream side and one nucleotide on the 3' downstream side adjacent to the mismatch nucleotide G are LNA, and the 5' terminal nucleotide is LNA. GEO-11 has an LNA nucleotide at the 5' end. GEO-242 has an LNA nucleotide at the 3' end. GEO-9 has no LNA.

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

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

[0129] [Table 1]

[0130] In Table 1, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted by the bridged nucleic acid is shown in uppercase. All bridged nucleic acids in Table 1 were LNA.

[0131] As shown in Table 1, the absolute value of the editing efficiency was the highest for GEO-8 at 0.25%, and the second highest for GEO-7 at 0.05%. Furthermore, the editing efficiency of the negative control GEO-94 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 have the same configuration as disclosed in Non-Patent Document 1. Thus, GEO-8, which is a non-natural polynucleotide of the present invention, showed an editing efficiency 5 times or more higher than that of the previously reported GEO-7.

[0132] In the following Examples 1-2 to 1-13, the editing efficiency of GEO-8 was used as the standard, and a relative editing efficiency of 0.4 or higher, with the editing efficiency of GEO-8 taken as 1.0, indicates a value sufficiently higher than previously reported editing efficiencies.

[0133] Example 1-2 Chain length of non-natural polynucleotide Although GEO-4 and GEO-8 in Example 1-1 had the same LNA arrangement, a difference of 4 nt resulted in a large difference in editing efficiency. In this Example 1-2, in order to investigate the preferred chain length of the non-natural polynucleotide, an experiment similar to that in Example 1-1 was carried out using nine types of non-natural polynucleotides having the same LNA arrangement as GEO-8 but different chain lengths (FIG. 4). The nine types of non-natural polynucleotides used in this 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 sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 2.

[0134] [Table 2]

[0135] In Table 2, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted by the bridged nucleic acid is shown in uppercase. All bridged nucleic acids in Table 2 were LNA.

[0136] As shown in Table 2, the relative editing efficiency of edited nucleic acids with a chain length of 25 nt to 95 nt, including the control GEO-8, was 0.4 or more, and that of GEO-174 with a chain length of 45 nt was 3.74, the highest relative editing efficiency. GEO-281 and GEO-282, which have longer chain lengths, showed a tendency to have a lower relative editing efficiency, but still showed a high value of 0.58 or more.

[0137] Example 1-3 LNA modification of the 3' end of a non-natural polynucleotide In this Example 1-3, the editing efficiency of a construct in which the pentose sugar in the 3'-terminal nucleotide 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 3'-terminal nucleotide is LNA in GEO-6; and GEO-17, in which the 3'-terminal nucleotide is further LNA in GEO-8 (Figure 5). The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 3.

[0138] [Table 3]

[0139] In Table 3, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted by the bridged nucleic acid is shown in uppercase. All bridged nucleic acids in Table 3 were LNA.

[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 this result, it was found that 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 LNA and the 3' end is LNA (GEO-16) has a low relative editing efficiency. It was shown that 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 LNA and the 5' end is LNA (hereinafter referred to as the "GEO-8 configuration"), the relative editing efficiency is improved when the 3' end is also LNA. On the other hand, it was found that even if the 3' end is LNA, when the 5' end is unmodified (GEO-16), the editing efficiency was about the same as that of GEO-6, and sufficient editing efficiency was not shown. From this, it was shown that it is important that the 5' end nucleotide is a bridged nucleic acid such as LNA in order to exert sufficient editing efficiency.

[0141] Examples 1-4 LNA modification of one or more nucleotides adjacent to the LNA at the 5' end of a non-natural polynucleotide In this Example 1-4, the editing efficiency of a configuration in which one or more nucleotides adjacent to the 5' end are further replaced with LNA was examined in relation to the GEO-8 configuration in Example 1-1 and the GEO-17 configuration in Example 1-3. Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-18, in which one nucleotide adjacent to the 5' end of GEO-8 is further LNA; GEO-19, in which two consecutive nucleotides adjacent to the 5' end of GEO-8 are further LNA; and GEO-68, in which one nucleotide adjacent to the 5' end of GEO-17 is further LNA (Figure 6). The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 4.

[0142] [Table 4]

[0143] In Table 4, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted by the bridged nucleic acid is shown in uppercase. All bridged 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 this result, it was found that sufficient editing efficiency was exhibited even in the configuration of GEO-8 in which one or more nucleotides adjacent to the 5' end were further replaced with LNA. Furthermore, the relative editing efficiency of GEO-17 was 2.00, and that of GEO-68 was 1.64. From this result, it was found that sufficient editing efficiency was exhibited even in the configuration in which one nucleotide adjacent to the 5' end was replaced with LNA ...

[0145] The relative editing efficiency of GEO-19, in which two nucleotides adjacent to the 5' end in the GEO-8 configuration were further replaced with LNA, was 0.66, which was slightly lower than that of the GEO-8 configuration. On the other hand, in Example 1-8 described later, when the 5' upstream side of the mismatched nucleotide was extended, the relative editing efficiency of GEO-313, in which one nucleotide adjacent to the 5' end in the GEO-8 configuration 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 in the GEO-8 configuration were further replaced with LNA, was 6.59. It was suggested that even when multiple nucleotides adjacent to the 5' end in the GEO-8 configuration were further replaced with LNA, the editing efficiency may be increased if other configurations, such as the chain length of the non-natural polynucleotide, are changed.

[0146] Examples 1-5 LNA modification of one or more nucleotides adjacent to the LNA at the 3' end of a non-natural polynucleotide In this Example 1-5, the editing efficiency of a configuration in which one or more nucleotides adjacent to the 3'-terminal nucleotide were further replaced with LNA in comparison with the GEO-17 configuration in Example 1-3 was examined. Specifically, the editing efficiency was measured in a manner similar to that of Example 1-1 using GEO-69, in which one nucleotide adjacent to the 3'-terminal nucleotide of GEO-17 is LNA; and GEO-71, in which two consecutive nucleotides adjacent to the 3'-terminal nucleotide of GEO-17 are LNA (Figure 7). The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 5.

[0147] [Table 5]

[0148] In Table 5, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted by the bridged nucleic acid is shown in uppercase. All bridged 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, when one nucleotide adjacent to the 3'-terminal nucleotide in the GEO-17 configuration was further replaced with LNA, the editing efficiency was comparable to that of GEO-8, and when two nucleotides adjacent to the 3'-terminal nucleotide were replaced with LNA, the editing efficiency was about half that of GEO-8.

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

[0151] Example 1-6 Replacement of one or more internucleotide phosphodiester bonds contained in a non-natural polynucleotide with phosphorothioate bonds (1) In this Example 1-6, the editing efficiency of a configuration in which one or more internucleotide phosphodiester bonds were further replaced with phosphorothioate bonds in addition to the configuration of GEO-8 in Example 1-1 was examined.

[0152] Specifically, GEO-158, GEO-159, GEO-160, GEO-29 and GEO-161 (FIG. 8A) are GEO-8 in which the phosphodiester bond between the 5'-terminal nucleotide and 1 to 5 consecutive nucleotides adjacent to the 5'-terminal nucleotide is replaced with a phosphorothioate bond; GEO-162, GEO-163, GEO-164, GEO-30, GEO-165, GEO-272 and GEO-196 (FIG. 8B) are GEO-8 in which the phosphodiester bond between the 3'-terminal nucleotide and 1 to 7 consecutive nucleotides adjacent to the 3'-terminal nucleotide is replaced with a phosphorothioate bond; and The editing efficiency was measured in the same manner as in Example 1-1 using each of GEO-31 (Figure 8C), in which the phosphodiester bond between the 3'-terminal nucleotide and the four consecutive nucleotides adjacent to the 3'-terminal nucleotide was replaced with a phosphorothioate bond; GEO-126 and GEO-127 (Figure 8C), in which the phosphodiester bond between the 5'-upstream LNA adjacent to the mismatch nucleotide G and the two or four consecutive nucleotides adjacent to the LNA in GEO-8 was replaced with a phosphorothioate bond; and GEO-128 and GEO-129 (Figure 8C), in which the phosphodiester bond between the nucleotide adjacent to the 3'-downstream LNA adjacent to the mismatch nucleotide G and the two or four consecutive nucleotides adjacent to the adjacent nucleotide in GEO-8 was replaced with a phosphorothioate bond. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 6.

[0153] [Table 6]

[0154] In Table 6, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide replaced by the bridged nucleic acid is shown in uppercase. All bridged nucleic acids in Table 6 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced by a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[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. These results showed that in the configuration of GEO-8, the editing efficiency was improved when the phosphodiester bond between the 5'-terminal nucleotide and the 1st to 5th nucleotides adjacent to the 5'-terminal nucleotide was replaced with a phosphorothioate bond.

[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. These results showed that in the configuration of GEO-8, the editing efficiency was improved when the phosphodiester bond moiety between the 3'-terminal nucleotide and the 1st to 7th nucleotides adjacent to the 3'-terminal nucleotide was replaced with a phosphorothioate bond.

[0157] Furthermore, the relative editing efficiency of GEO-31 was 5.68. This result showed that in the configuration of GEO-8, the editing efficiency was improved when the phosphodiester bond moiety between the 5'-terminal nucleotide and the four consecutive nucleotides adjacent to the 5'-terminal nucleotide and between the 3'-terminal nucleotide and the four consecutive nucleotides adjacent to the 3'-terminal nucleotide were replaced with phosphorothioate bonds.

[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, it was found that in the configuration of GEO-8, when the phosphodiester bond 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, 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, it was shown that in the configuration of GEO-8, when the phosphodiester bond between two consecutive nucleotides 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, the editing efficiency was half that of GEO-8, and when the phosphodiester bond between four consecutive nucleotides 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, the editing efficiency was improved. Note that, as shown in Table 6 and FIG. 8C, in GEO-128 and GEO-129, the phosphodiester bond 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 Replacement of one or more internucleotide phosphodiester bonds contained in a non-natural polynucleotide with phosphorothioate bonds (2) In this Example 1-7, the editing efficiency of a configuration in which one or more internucleotide phosphodiester bonds are further replaced with phosphorothioate bonds in addition to the configuration of GEO-17 in Example 1-3 was examined. Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-155, GEO-167, GEO-168, and GEO-169, which are GEO-17 in which the 3'-terminal nucleotide and the phosphate diester bond between 1 to 4 consecutive nucleotides adjacent to the 3'-terminal nucleotide have been replaced with phosphorothioate bonds (Figure 9). The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 7.

[0161] [Table 7]

[0162] In Table 7, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide replaced by the bridged nucleic acid is shown in uppercase. All bridged nucleic acids in Table 7 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced by a phosphorothioate bond, 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. These results showed that in the configuration of GEO-17, the editing efficiency was improved when the phosphodiester bond between the 3'-terminal nucleotide and 1 to 4 nucleotides adjacent to the 3'-terminal nucleotide was replaced with a phosphorothioate bond.

[0164] Example 1-8 Extension of chain length on the 5' upstream and 3' downstream sides of the mismatched nucleotide and the position of LNA In this Example 1-8, when the non-natural polynucleotide contains one mismatched nucleotide, the influence of changing the length of the 5' upstream region and the length of the 3' downstream region of the mismatched nucleotide on the editing efficiency was examined. Furthermore, the editing efficiency was examined when the number and position of LNA near the 5' end and the number and position of LNA near the 3' end were appropriately changed.

[0165] Specifically, GEO-25 has a chain length of 41 nt, a 5' upstream region of the mismatch nucleotide G of 20 nt and a 3' downstream region of 20 nt, one nucleotide adjacent to the mismatch nucleotide G on the 5' upstream side and the 3' downstream side is LNA, and the 5' terminal nucleotide is LNA; GEO-311 has a chain length of 38 nt, a 5' upstream region of the mismatch nucleotide G of 25 nt and a 3' downstream region of 12 nt, one nucleotide adjacent to the mismatch nucleotide G on the 5' upstream side and the 3' downstream side is LNA, and the 5' terminal nucleotide is LNA; GEO-312 in which the 12th nucleotide counting from the 5' upstream nucleotide adjacent to the mismatch nucleotide G toward the 5' upstream side in GEO-311 is LNA; GEO-313 in which the nucleotide adjacent to the 5' terminal LNA in GEO-311 is LNA; The editing efficiency was measured in the same manner as in Example 1-1 using GEO-314, in which the 12th nucleotide counting from the upstream nucleotide toward the 5' upstream side is LNA; GEO-315, in which two consecutive nucleotides adjacent to the LNA at the 5' end of GEO-311 are LNA; and GEO-316, in which two consecutive nucleotides adjacent to the LNA at the 5' end of GEO-311 are LNA and the 12th nucleotide counting from the 5' upstream nucleotide adjacent to the mismatch nucleotide G is LNA (Figure 10A).

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

[0167] [Table 8]

[0168] In Table 8, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted by the bridged nucleic acid is shown in uppercase. All bridged 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] Comparison of GEO-8 and GEO-311 showed that the editing efficiency increased when the chain length of the 5' upstream region of the mismatched nucleotide was extended. Comparison of GEO-313 and GEO-18 in Example 1-4, and GEO-315 and GEO-19 in Example 1-4 showed that even when multiple nucleotides adjacent to the 5'-terminal nucleotide were LNA, the editing efficiency increased when the chain length of the 5' upstream region of the mismatched nucleotide was extended.

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

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

[0173] Comparing GEO-317 and GEO-318, and GEO-319 and GEO-320, there was no significant difference in the editing efficiency, suggesting that when the length of the 3' downstream region of the mismatch nucleotide is extended, replacing the 12th nucleotide 3' downstream from the nucleotide 3' downstream adjacent to the mismatch nucleotide G with LNA has little effect on the editing efficiency.

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

[0175] [Table 9]

[0176] In Table 9, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide replaced by the bridged nucleic acid is shown in capital letters. All bridged nucleic acids in Table 9 were LNA. The RNA-substituted nucleotides are indicated by an arrow at the base symbol of the corresponding nucleotide.

[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. These results show that in the GEO-17 configuration, when 1 or 2 nucleotides adjacent to the 3'-terminal nucleotide were substituted with RNA, the editing efficiency was improved compared to GEO-8, and when 3 or 4 nucleotides were substituted with RNA, the editing efficiency was comparable to that of GEO-8.

[0178] Examples 1-10 Phosphorylation of 3'- and / or 5'-Terminal Nucleotides In this Example 1-10, the editing efficiency of a configuration in which the 3'-end and / or 5'-end nucleotides were phosphorylated in addition to the configuration of GEO-8 in Example 1-1 or GEO-17 in Example 1-3 was examined. Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-8, GEO-26 in which the 5'-terminal nucleotide is phosphorylated; GEO-27 in which the 3'-terminal nucleotide is phosphorylated, and GEO-28 in which both the 5'-terminal and 3'-terminal nucleotides are phosphorylated; GEO-17, GEO-223 in which the 3'-terminal nucleotide is phosphorylated; and GEO-17, GEO-224 in which the phosphodiester bond between the 3'-terminal nucleotide and the adjacent nucleotide is replaced with a phosphorothioate bond and the 3'-terminal nucleotide is phosphorylated (Figure 12). The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 10.

[0179] [Table 10]

[0180] In Table 10, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide replaced by the bridged nucleic acid is shown in capital letters. All bridged nucleic acids in Table 10 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced by a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. Furthermore, to indicate that the 5'-end and / or 3'-end were phosphorylated, a "p" was added 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, and that of GEO-223 was 2.70 and that of GEO-224 was 2.29. From these results, it was shown that in the configuration of GEO-8, phosphorylation of only the 5' end did not affect the editing efficiency, but phosphorylation of the 3' end improved the editing efficiency. In addition, when both the 5' end and the 3' end were phosphorylated, a higher editing efficiency was shown than when only the 3' end was phosphorylated. Similarly, in the configuration of GEO-17, it was shown that phosphorylation of the 3' end improved the editing efficiency. In this case, no increase in editing efficiency was observed even when the phosphodiester bond between the nucleotide at the 3' end and the nucleotide adjacent to it was replaced with a phosphorothioate bond.

[0182] Examples 1-11 Types of cross-linked nucleic acids In this Example 1-11, the editing efficiency of a configuration in which the LNA at the 5' end in the configuration of GEO-8 in Example 1-1 was replaced with another crosslinked nucleic acid was examined. Specifically, 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 of GEO-8 was replaced with BNA-NH; 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 sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 11.

[0183] [Table 11]

[0184] In Table 11, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide replaced with the bridged nucleic acid is shown in capital letters. The type of bridged nucleic acid in each edited nucleic acid in Table 11 is 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. This result indicates that in the structure of GEO-8, the 5' end may be replaced with other bridged nucleic acids such as BNA-NH, BNA-N-Me, and ENA, in addition to LNA, and that the editing efficiency may be increased by replacing it.

[0186] Examples 1-12 Combination of nucleic acid modifications (1) In this Example 1-12, the editing efficiency of a configuration in which the phosphodiester bond moiety in the GEO-8 configuration of Example 1-1 was further combined with substitution of a phosphorothioate bond and phosphorylation of the 3′ end was examined. Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-226, in which the phosphodiester bond between the five consecutive nucleotides adjacent to the 3'-terminal nucleotide in GEO-8 was replaced with a phosphorothioate bond and the 3'-terminal was phosphorylated (Figure 14). The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 12.

[0187] [Table 12]

[0188] In Table 12, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide replaced by the bridged nucleic acid is shown in uppercase. All bridged nucleic acids in Table 12 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced by a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. Furthermore, to indicate that the 3' end was phosphorylated, a "p" was added to the 3' end.

[0189] As shown in Table 12, the relative editing efficiency of GEO-226 was 8.19. This result indicated that the further combination of replacing the phosphodiester bond with a phosphorothioate bond and phosphorylating the 3' end in the GEO-8 configuration significantly increased the editing efficiency.

[0190] Examples 1-13 Combination of nucleic acid modifications (2) In this Example 1-13, the editing efficiency of a configuration in which the phosphodiester bond moiety is replaced with a phosphorothioate bond, RNA replacement, and phosphorylation of the 3' end are further combined in the GEO-23 configuration of Example 1-3 was examined. Specifically, in GEO-23, the phosphodiester bond between the four consecutive nucleotides adjacent to the 5'-terminal nucleotide was replaced with a phosphorothioate bond, and two consecutive nucleotides adjacent to the 3'-terminal nucleotide were replaced with RNA, and the 3'-terminal was phosphorylated. The editing efficiency was measured using GEO-172 in the same manner as in Example 1-1 (Figure 15). The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 13.

[0191] [Table 13]

[0192] In Table 13, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted with the bridged nucleic acid is shown in capital letters. All bridged nucleic acids in Table 13 were LNA. Where a phosphodiester bond between two or more nucleotides was substituted with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. For the RNA-substituted nucleotide, an arrow was added to the base symbol of the corresponding nucleotide. Furthermore, to indicate that the 3' end was phosphorylated, a "p" was added to the 3' end.

[0193] As shown in Table 13, the relative editing efficiency of GEO-172 was 8.48. This result indicated that the further combination of replacing the phosphodiester bond with a phosphorothioate bond, RNA replacement, and 3'-end phosphorylation in the GEO-17 configuration significantly increased the editing efficiency.

[0194] Consideration The above-mentioned Examples 1-1 to 1-13 of the present invention will be compared with the disclosures in patent documents and non-patent documents. (1) Example 1-1 shows that GEO-8, a non-natural polynucleotide of the present invention, exhibited an editing efficiency 5 times or more higher than that of GEO-7 (i.e., the chain length, the position of the mismatched nucleotide, and the position of the 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. GEO-8 is different from GEO-7 in that the mismatched nucleotide itself is not LNA, and both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide are substituted with LNA.

[0195] In this regard, Patent Document 4 describes that oligonucleotides containing an LNA adjacent to the mismatched nucleotide showed no improvement, whereas oligonucleotides having an LNA spaced one nucleotide further from the mismatch showed an average increase of 5-fold, and it can be said that Patent Document 4 actively excludes configurations that include an LNA adjacent to the mismatched nucleotide.

[0196] Non-Patent Document 1 focuses on avoiding mismatch repair, and Non-Patent Documents 1 and 2 focus on elucidating the mechanism of how single-stranded synthetic DNA with a mismatched nucleotide of LNA modifies the genome of mammalian cells. Of the total of 60 or more types of single-stranded synthetic DNAs tested in Non-Patent Document 1, only one type has both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide substituted with LNA, and all of the total of 50 or more types of single-stranded DNAs tested in Non-Patent Document 2 have the mismatched nucleotide itself as LNA, and none have been tested in which both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide have been substituted with LNA. In addition, the discussion in Non-Patent Document 1 emphasizes that it is important to make the mismatched nucleotide itself LNA in avoiding mismatch repair, and does not mention those in which both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide have been substituted with LNA.

[0197] Therefore, it can be said that the descriptions in Patent Document 4 and Non-Patent Documents 1 and 2 are obstacles to the adoption of the feature in which both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatched nucleotide are replaced with LNA to improve editing efficiency, i.e., the motivation to adopt feature (A) and feature (B) of the present invention. Furthermore, the experimental results shown in Example 1-1 of this specification are the exact opposite of the suggestion of Patent Document 4, and it can be said that the effect of the present invention is a heterogeneous effect that cannot be predicted from Patent Document 4 and Non-Patent Documents 1 and 2.

[0198] Of the total of 110 or more types of single-stranded synthetic DNAs tested in Non-Patent Documents 1 and 2, only one type of single-stranded synthetic DNA in which the 5'-terminal nucleotide was LNA and the 3'-terminal was not LNA was found in each document. Although single-stranded synthetic DNA in which the 5'-terminal nucleotide and both the mismatch nucleotides were LNA showed approximately twice the editing efficiency compared to single-stranded synthetic DNA in which only the mismatch nucleotide was LNA, no further investigation was conducted on single-stranded synthetic DNA in which the 5'-terminal was LNA.

[0199] Non-Patent Document 2 concludes that the region on the 5' side of the mismatched nucleotide is degraded by endonucleases, not exonucleases, and therefore suggests that the effect of LNA modification of the 5' end to protect it from exonucleases is limited.In fact, the LNA modification of the 5' end only increases the editing efficiency by two times, which leads to the conclusion that the LNA modification of the 5' end is not very important.Therefore, no attempt has been made to combine the 5' end modification with the 5' end modification in which both the 5' end nucleotide and the 3' end nucleotide adjacent to the mismatched nucleotide are replaced with LNA.

[0200] Therefore, it can be said that the descriptions in Non-Patent Documents 1 and 2 are an obstacle to attempts to substitute the 5' end of a non-natural polynucleotide with LNA in order to improve editing efficiency.

[0201] (2) Example 1-2 demonstrated that a certain level of editing efficiency was observed when the chain length of the non-natural polynucleotide was 25 nt or more and 95 nt or less. Regarding the chain length, many literatures have said that a chain length of about 25 bases is good, and that the longer the chain length, the lower the effect, whereas the present invention is different from existing research in that the effect is high even if the chain length is long. Specifically, Non-Patent Document 1 examines the optimal length of an edited nucleic acid using a polynucleotide in which only the mismatch nucleotide is LNA, and as a result, it is shown that 25 bases is optimal, and editing efficiency decreases when the length is longer than 25 bases. In addition, Patent Document 4 uses a polynucleotide with a length of 24 bases. On the other hand, it has been discovered that in the case of the non-natural polynucleotide of the present invention, the editing efficiency is further increased when the polynucleotide is longer than 25 bases.

[0202] (3) From Examples 1-3, it was shown that in order for a compound in which both the 5'-side nucleotide and the 3'-side nucleotide adjacent to a mismatched nucleotide are replaced with LNA to exert an effect, the feature (C) that the 5'-terminal nucleotide is a bridged nucleic acid is important. Furthermore, a comparison with GEO-8, GEO-16, and GEO-17 showed that when both the 5'-terminal and 3'-terminal nucleotides are bridged nucleic acids, the editing efficiency increased by two times. It was also found that the effect of the 3'-terminal nucleotide being LNA is strongly exhibited when the 5'-terminal nucleotide is LNA. (4) In Examples 1-4 and 1-5, sufficient editing efficiency was observed even when one or more nucleotides adjacent to the 5'-end or 3'-end were replaced with LNA, and it was shown that the editing efficiency could be improved depending on the position and number of nucleotides replaced with LNA. Such an effect was not described or suggested in any of the above patent documents or non-patent documents.

[0203] (5) From Examples 1-6 and 1-7, it was shown that in those in which both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatch nucleotide are substituted with LNA, the editing efficiency is improved when the phosphodiester bond between the 5'-end or 3'-end nucleotide and the 1-7 nucleotides adjacent thereto are substituted with phosphorothioate bonds. Since the editing efficiency does not decrease even if the number of phosphorothioate bonds is increased by one each at both the 5'-end and 3'-end, it is likely that sufficient editing efficiency is exhibited even if 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. In addition, when the 5'-end or 3'-end is LNA, S-conjugation from both ends particularly strongly improves the editing effect, which is a completely new discovery revealed by the present inventors in this specification.

[0204] (6) Examples 1-8 show that extending the length of the 5' upstream region and 3' downstream region of the mismatched nucleotide increases the editing efficiency, and further shows that substituting some nucleotides in the extended region with LNA increases the editing efficiency. Such an effect was not described or suggested in any of the above patent documents or non-patent documents.

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

[0206] (8) Examples 1-11 show that the effect of improving the editing efficiency can be achieved by substituting a bridged nucleic acid at the 5' end with not only LNA but also other bridged nucleic acids such as BNA-NH, BNA-N-Me, ENA, etc. 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® plasmids (1) nDL2 A mutant base sequence was synthesized in which the guanine (G) at base number 894 of the NanoLuc gene was changed to adenine (A) and the cytosine (C) at base number 895 was changed to thymine (T), and the mutant base sequence was synthesized using pcDNA TM A mutant was created by incorporating the luciferase gene into the 5 / FRT / TO plasmid (pcDNA5-nLD2). The luciferase gene contained in 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 was synthesized in which the thymine (T) at base number 956 of the NanoLuc gene was changed to adenine (A), the thymine (T) at base number 957 to guanine (G), and the cytosine (C) at base number 958 to thymine (T), and the mutant base sequence was synthesized in the same manner as above. TM A mutant was created by incorporating the luciferase gene into the 5 / FRT / TO plasmid (pcDNA5-nLD3). The luciferase gene contained in 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 was synthesized in which the cytosine (C) at base number 922 of the NanoLuc gene was changed to thymine (T) and the guanine (G) at base number 931 was changed to thymine (T), and the mutant base sequence was then transformed into pcDNA in the same manner as above. TM We created a mutant that incorporates the luciferase gene into the 5 / FRT / TO plasmid (pcDNA5-nLD4-1). The luciferase gene contained in 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 was synthesized in which the cytosine (C) at base number 922 of the NanoLuc gene was changed to thymine (T) and the guanine (G) at base number 937 was changed to thymine (T), and the mutant base sequence was then transformed into pcDNA in the same manner as above.TM A mutant was created by incorporating the luciferase gene into the 5 / FRT / TO plasmid (pcDNA5-nLD4-2). The luciferase gene contained in this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation described above.

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

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

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

[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 the mutant base sequence was transformed into pcDNATM A mutant was created by incorporating the luciferase gene into the 5 / FRT / TO plasmid (pcDNA5-nLD7). The luciferase gene contained in this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation described above.

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

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

[0217] 2. Creation of stable transformed cell lines carrying various mutant NanoLuc genes Flp-In, a human-derived cell line TM -293 cell line (Thermo Fisher Scientific Inc.) at 1x10 6The cells were seeded on a 6 cm dish 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 under 5% CO2. 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 standard protocol. After 48 hours, TrypLE TM Express Enzyme (Thermo Fisher Scientific Inc.) was added and incubated at 37°C for 3 minutes. The detached cells were collected and suspended in DMEM + 10% FBS medium containing 50μg / ml of hygromycin (Thermo Fisher Scientific Inc.), and cultured in two 10cm dishes. Culture was then continued with the same medium replaced every 3 days. After approximately 20 days of culture, the formation of sufficient numbers and sizes of colonies was confirmed, and all cells were incubated with TrypLE. TM The cells were detached from the petri dish using Express Enzyme and collected.

[0218] The cell lines obtained using the above-mentioned various plasmids were 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, and the cells in which pcDNA5-nLD9 was integrated into the genome were named 293-nLD9 cells. The mutant NanoLuc gene in each of the cells was an inactive luciferase gene as described above. Each of the cells was used in the subsequent genome editing experiment.

[0219] 3. Introduction of non-natural polynucleotides into 293-nLD2-9 cells 1x10 293-nLD2 cells 4 A 96-well plate (Nunc® MicroWell) was used to culture the cells. TM The cells were seeded onto a 96, Nunclon Delta-Treated, Flat-Bottom Microplate (Thermo Fisher Scientific Inc.) and cultured in DMEM + 10% FBS medium at 37°C under a 5% CO2 environment. After 24 hours, non-natural polynucleotides at concentrations of 0.0125μg, 0.025μg, 0.050μg, 0.100μg, and 0.200μg were transfected using Lipofectamine 3000 according to the standard protocol. 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 293-nLD2 cells.

[0220] 4. Measuring editing efficiency NanoLuc Luciferase Assay After 72 hours of culture from the transfection, NanoLuc Luciferase activity was measured. NanoLuc Luciferase activity was measured using the Nano-Glo® Luciferase Assay System (Promega Corporation) according to the standard protocol. Luciferase luminescence was measured using an EnSpire multimode plate reader (PerkinElmer Co., Ltd.).

[0221] Counting viable cells The number of viable cells was measured using CellTiter-Blue (registered trademark) Cell Viability Assay (Promega Corporation) according to the standard protocol. The fluorescence value of CellTiter-Blue was measured using an EnSpire multimode plate reader (PerkinElmer Co., Ltd.). The number of viable edited cells was 5 x 10 3 From 2 x 10 5 A standard curve of CellTiter-Blue measurement values ​​and cell numbers was created using a two-fold dilution series of 293-nLD2 cell numbers between 100 and 1000 μg / mL, and the number of live cells was calculated based on this standard curve. The viable cell numbers of 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 calculated in the same manner as for 293-nLD2 cells.

[0222] Editing efficiency calculation When the NanoLuc Luciferase activity of 293-nLW1 cells with wild-type luciferase integrated into the genome was measured, 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, a 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 x100 The calculation was done using the formula:

[0223] 5. Evaluation of editing efficiency of non-natural polynucleotides by AUC log The editing efficiency of each non-natural polynucleotide was evaluated as follows. NanoLuc Luciferase activity was measured at five concentrations of non-natural polynucleotide (0.0125μg, 0.025μg, 0.050μg, 0.100μg, 0.200μg), and a dose-response curve was prepared with the axis (X-axis) showing the concentration of non-natural polynucleotide as a logarithm. From the prepared dose-response curve, the area under the curve (hereinafter referred to as "AUC") was calculated with the X-axis as a logarithm (hereinafter referred to as "AUC log"). Prism (GraphPad) was used to calculate AUC log.

[0224] 6. Synthesis of non-natural polynucleotides All non-natural polynucleotides, including those with modified nucleic acids, were synthesized by Gene Design Co., Ltd. (Japan) and purified using a simple column or HPLC.

[0225] Example 2-1 Examination of two base substitutions (1) Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD2 cells.

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

[0227] [Table 14]

[0228] In Table 14, mismatched nucleotides are underlined, and nucleotides replaced with bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 14 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. The structure of D2-2 is similar to that reported in Non-Patent Document 1. The relative editing efficiency was calculated with the AUC value of D2-2 as the standard (1.00), and is shown in Table 14. When Tris-EDTA (Tris-HCl 10 mM, EDTA 1 mM, pH 7.4, hereinafter referred to as "TE") was used as a negative control, the AUC 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. These results demonstrated that the non-natural polynucleotide of the present invention can modify two nucleotides contained in a target nucleotide sequence.

[0229] Example 2-2 Examination of 3-base substitutions Experiments were performed to measure the editing efficiency of a 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 transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 15.

[0231] [Table 15]

[0232] In Table 15, mismatched nucleotides are underlined, and nucleotides replaced with bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 15 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0233] All of the structures shown in Table 15 have three base substitutions, and no such examples have been reported previously. The relative editing efficiency was calculated based on the AUC value of D3-2 as the standard (1.00), and is shown in Table 15. When TE was used as a negative control, the AUC 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 these results, it was found that the non-natural polynucleotide of the present invention can modify three nucleotides contained in the target nucleotide sequence.

[0234] Example 2-3 Examination of 2-base substitution (2) Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD4-1, 293-nLD4-2, or 293-nLD4-3 cells.

[0235] In an experiment using 293-nLD4-1 cells, each of the three types of non-natural polynucleotides D4-1-1 to D4-1-4 was introduced into 293-nLD4-1 cells by transfection. In an experiment using 293-nLD4-2 cells, each of the three types of non-natural polynucleotides D4-2-1 to D4-2-4 was introduced into the 293-nLD4-2 cells by transfection. In an experiment using 293-nLD4-3 cells, each of the three types of non-natural polynucleotides D4-3-1 to D4-3-4 was introduced into the 293-nLD4-3 cells by transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 16.

[0236] [Table 16]

[0237] In Table 16, mismatched nucleotides are underlined, and nucleotides replaced with bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 16 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0238] The structures shown in Table 16 all have two base substitutions at distant positions, and no such examples have been reported previously. In D4-1-1 to D4-1-4, the two mismatched nucleotides for the target nucleotide sequence are spaced apart by 8 bases. The relative editing efficiency was calculated based on the AUC value of D4-1-2 as the standard (1.00), and is 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 for the target nucleotide sequence are spaced 14 bases apart. The relative editing efficiency was calculated based on the AUC value of D4-2-2 as the standard (1.00) and shown in Table 16. When TE was used as a negative control, the AUC was 47.16, and the AUC of D4-2-2 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 in the target nucleotide sequence are spaced 20 bases apart. The relative editing efficiency was calculated based on the AUC value of D4-3-2 as the standard (1.00) and shown in Table 16. When TE was used as a negative control, the AUC was 47.16, and the AUC of D4-3-2 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] These results indicate that when there are multiple nucleotide modifications in a target nucleotide sequence, the modification sites do not need to be adjacent, and multiple nucleotides in the target nucleotide sequence can be modified simultaneously even if they are at least 20 bases apart.

[0240] Example 2-4: Examination of 1 base insertion Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD5 cells.

[0241] In this example, four types of non-natural polynucleotides, D5-2, D5-4, D5-5 and D5-6, and the non-natural polynucleotide D5-1 as a control, were each introduced into 293-nLD5 cells by transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 17.

[0242] [Table 17]

[0243] In Table 17, mismatched nucleotides are underlined, and nucleotides replaced with bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 17 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. Furthermore, the nucleotides in [ ] indicate nucleotides for inserting a nucleotide complementary to the nucleotide in [ ] into the target nucleotide sequence.

[0244] The structure of D5-2 is similar to that reported in Non-Patent Document 1. The relative editing efficiency was calculated with the AUC value of D5-2 as the standard (1.00), and is shown in Table 17. When Tris-EDTA (Tris-HCl 10 mM, EDTA 1 mM, pH 7.4, hereinafter referred to as "TE") was used as a negative control, the AUC 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. These results demonstrated that the non-natural polynucleotide of the present invention allows insertion of one desired base into a deletion site contained in a target nucleotide sequence.

[0246] Example 2-5: Examination of 2-base insertion Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD6 cells.

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

[0248] [Table 18]

[0249] In Table 18, mismatched nucleotides are underlined, and nucleotides replaced with bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 18 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. Furthermore, the nucleotides in [ ] indicate nucleotides for inserting a nucleotide complementary to the nucleotide in [ ] into the target nucleotide sequence.

[0250] All of the structures shown in Table 18 are two-base insertions, and no such examples have been reported previously. The relative editing efficiency was calculated based on the AUC value of D6-2 as the standard (1.00), and is shown in Table 18. When TE was used as a negative control, the AUC 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 these results, it was found that the non-natural polynucleotide of the present invention can insert a desired base even if the target nucleotide sequence contains two deletion sites.

[0251] Example 2-6 Examination of one base deletion Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural 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 transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 19.

[0253] [Table 19]

[0254] In Table 19, the mismatched nucleotides are underlined, and the nucleotides replaced by the bridged nucleic acid are shown in capital letters. The mismatched nucleotide x in Table 19 means that one nucleotide complementary to a nucleotide present in the target nucleotide sequence is missing in the edited nucleic acid. All bridged nucleic acids in Table 19 were LNA. In the places where the phosphodiester bond between two or more nucleotides was replaced by a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0255] All of the structures shown in Table 19 have one base deleted, and no such examples have been reported in the past. The relative editing efficiency was calculated based on the AUC value of D7-2 as the standard (1.00), and is 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 these results, it was found that the non-natural polynucleotide of the present invention can remove a desired base from a target nucleotide sequence.

[0256] Example 2-7 Examination of 2-base deletion Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a 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 transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 20.

[0258] [Table 20]

[0259] In Table 20, the mismatched nucleotides are underlined, and the nucleotides replaced by the bridged nucleic acid are shown in capital letters. The mismatched nucleotide xx in Table 20 means that two nucleotides complementary to the nucleotides present in the target nucleotide sequence are missing in the edited nucleic acid. All bridged nucleic acids in Table 20 were LNA. In the places where the phosphodiester bond between two or more nucleotides was replaced by a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0260] All of the structures shown in Table 20 are two-base deletions, and no such examples have been reported in the past. The relative editing efficiency was calculated based on the AUC value of D7-2 as the standard (1.00), and is 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 these results, it was found that the non-natural polynucleotide of the present invention can remove multiple desired bases from a target nucleotide sequence.

[0261] Example 2-8 Examination of 4-base insertion Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD9 cells.

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

[0263] [Table 21]

[0264] In Table 21, mismatched nucleotides are underlined, and nucleotides replaced with bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 21 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. Furthermore, the nucleotides in [ ] indicate nucleotides for inserting a nucleotide complementary to the nucleotide in [ ] into the target nucleotide sequence.

[0265] The structure of D9-2 is the same as that reported in Non-Patent Document 1. The relative editing efficiency was calculated based on the AUC value of D9-2 as the standard (1.00), and is shown in Table 21. When Tris-EDTA (Tris-HCl 10 mM, EDTA 1 mM, pH 7.4, hereinafter referred to as "TE") was used as a negative control, the AUC 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 these results, it was found that the non-natural polynucleotide of the present invention can insert a desired base even if the target nucleotide sequence contains four deletion sites.

[0266] From the above Examples 2-1 to 2-8, it was found that by using the non-natural polynucleotide of the present invention, it is possible to replace two or more bases on a target polynucleotide, to insert one or more bases into a target polynucleotide, and to remove one or more bases from a target polynucleotide.

[0267] C. Genome editing experiment using 293-nLD1 cells (2) The following genome editing experiment was carried out using the 293-nLD1 cells prepared in the above section “A. Genome editing experiment using 293-nLD1 cells (1)”.

[0268] 1. Introduction of non-natural polynucleotides into 293-nLD1 cells 1x10 293-nLD1 cells 4 A 96-well plate (Nunc® MicroWell) was used to culture the cells. TM The cells were seeded onto a 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 polynucleotide was transfected using Lipofectamine 3000 according to the standard protocol.

[0269] 2. Measurement of editing efficiency and evaluation of editing efficiency of non-natural polynucleotides by AUC log The measurement and evaluation of editing efficiency were performed as described above in "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."

[0270] 3. Synthesis of non-natural polynucleotides All non-natural polynucleotides, including those with modified nucleic acids, were synthesized by Gene Design Co., Ltd. (Japan) and purified using a simple column or HPLC.

[0271] Example 3-1 Modification of target nucleotide sequence by non-natural polynucleotides having various characteristics Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells.

[0272] In this example, each of the non-natural polynucleotides shown in Table 31 was introduced into 293-nLD1 cells by transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 31.

[0273] [Table 31-1]

[0274] [Table 31-2]

[0275] In Table 31, mismatched nucleotides are underlined, and nucleotides replaced by bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 31 were LNA. For RNA-substituted nucleotides, an arrow is attached to the base symbol of the corresponding nucleotide. Where the phosphodiester bond between nucleotides has been replaced by a phosphorothioate bond, an asterisk (*) is inserted between the corresponding nucleotides. For non-natural polynucleotides with phosphorylated 5'- and / or 3'-ends, "p" is attached to the 5'- and / or 3'-ends. Furthermore, G(F) and T(F) in GEO-204 indicate that they are 2'-Fluoro modified, A(MT) in GEO-213 indicates that they are 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, the non-natural polynucleotides of the present invention were found to have editing efficiency four or more times higher than that of the benchmark GEO-8.

[0277] Example 3-2 Modification of target nucleotide sequence by non-natural polynucleotide with mismatch nucleotide at the 3' end Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a 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 transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 32. In Table 32, the chain length of the edited nucleic acid body and the chain length of the adapter are listed separately, but each sequence defined as a SEQ ID NO is the combined length of the edited nucleic acid 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 are underlined, and nucleotides replaced by bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 32 were LNA. For RNA-substituted nucleotides, an arrow is attached to the base symbol of the corresponding nucleotide. Where the phosphodiester bond between nucleotides is replaced by a phosphorothioate bond, an asterisk (*) is inserted between the corresponding nucleotides. For non-natural polynucleotides phosphorylated at the 5'-end and / or 3'-end, "p" is attached to the 5'-end and / or 3'-end. Furthermore, [t], [tt], and [ttt] indicate that one, two, and three thymidine monophosphates have been inserted, respectively, and [a], [aa], and [aaa] indicate that one, two, and three adenosine monophosphates have been inserted, respectively.

[0288] As shown in Table 32, the non-naturally occurring polynucleotides of the present invention were found to have higher editing efficiency than the benchmark GEO-8.

[0289] Example 3-3 Modification of target nucleotide sequence using non-natural polynucleotide with an adaptor at the 3' end Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells.

[0290] In this example, each of the non-natural polynucleotides shown in Table 33 was introduced into 293-nLD1 cells by transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 33. Note that in Table 33, the chain length of the edited nucleic acid body and the chain length of the adapter are listed separately, but each sequence defined as a sequence number is the combined length of the edited nucleic acid body and the adapter.

[0291] [Table 33-1]

[0292] [Table 33-2]

[0293] In Table 33, mismatched nucleotides are underlined, and nucleotides replaced by bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 33 were LNA. For RNA-substituted nucleotides, an arrow is attached to the base symbol of the corresponding nucleotide. Where the phosphodiester bond between nucleotides is replaced by a phosphorothioate bond, an asterisk (*) is inserted between the corresponding nucleotides. For non-natural polynucleotides with phosphorylated 5'-end and / or 3'-end, "p" is attached to the 5'-end and / or 3'-end. Furthermore, in each of the non-natural polynucleotides shown in Table 33, the nucleotides in the second line are adaptors, and each adaptor forms a stem structure that may have a loop. Figures 16A, 16B, 17A, and 17B show schematic diagrams of the stem structure of each of the non-natural polynucleotides.

[0294] As shown in Table 33, the non-naturally occurring polynucleotides of the present invention were found to have editing efficiency equivalent to or higher than that of the benchmark GEO-8.

[0295] Example 3-4 Modification of target nucleotide sequence by non-natural polynucleotide of embodiment (vi) Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells.

[0296] In this example, each of the non-natural polynucleotides shown in Table 34 was introduced into 293-nLD1 cells by transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 34.

[0297] [Table 34]

[0298] In Table 34, mismatched nucleotides are underlined, and nucleotides replaced by bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Table 34 were LNA. Where internucleotide phosphodiester bonds were replaced by phosphorothioate bonds, an asterisk (*) was inserted between the corresponding nucleotides. Non-natural polynucleotides phosphorylated at the 5'-end and / or 3'-end were marked with "p" at the 5'-end and / or 3'-end.

[0299] As shown in Table 34, the non-naturally occurring polynucleotides of the present invention were found to have higher editing efficiency than the benchmark GEO-8.

[0300] Example 3-5 Modification of target nucleotide sequence using non-natural polynucleotide having a 5'-terminal nucleotide other than LNA Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells.

[0301] In this example, each of the non-natural polynucleotides shown in Table 41 was introduced into 293-nLD1 cells by transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 41.

[0302] [Table 41-1]

[0303] [Table 41-2]

[0304] In Table 41, the mismatched nucleotides are underlined, and the nucleotides replaced by the bridged nucleic acid are shown in capital letters. All bridged nucleic acids here were LNA. The RNA-substituted nucleotides are indicated by an arrow at the base symbol of the corresponding nucleotide. Where the phosphodiester bond between nucleotides has been replaced by a phosphorothioate bond, an asterisk (*) is inserted between the corresponding nucleotides. The bridged nucleic acid in GEO-827 and GEO-831 was LNA. Furthermore, the mismatched nucleotide at or near the 5' end in GEO-649, GEO-650, GEO-653, GEO-654, GEO-656, and GEO-657 is a mismatched nucleotide for the purpose of improving editing efficiency, and is not a mismatched nucleotide for the purpose of modification.

[0305] As shown in Table 41, the non-naturally occurring polynucleotides of the present invention were found to have higher editing efficiency than the benchmark GEO-8.

[0306] Examples 3-6 Modification of target nucleotide sequence by non-natural polynucleotides with modifying compounds at the 5' or 3' end Experiments were performed to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells.

[0307] In this example, each of the non-natural polynucleotides shown in Table 42 and Table 43 was introduced into 293-nLD1 cells by transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Tables 42 and 43.

[0308] [Table 42]

[0309] [Table 43]

[0310] In Tables 42 to 43, mismatched nucleotides are underlined, and nucleotides replaced with bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Tables 42 to 43 were LNA. Where the internucleotide phosphodiester bond was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding nucleotides. In Tables 42 and 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 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 or near the 5' end in GEO-649, GEO-650, GEO-653, GEO-654, GEO-656 and GEO-657 are mismatched nucleotides for the purpose of improving editing efficiency and are not mismatched nucleotides for the purpose of modification.

[0311] As shown in Tables 42 to 43, the non-natural polynucleotides of the present invention were found to have higher editing efficiency than the standard GEO-8.

[0312] Example 3-7: Examination of introduction of phosphate modification bond to the 5' or 3' side An experiment was carried out to measure the editing efficiency of a target polynucleotide sequence by introducing into 293-nLD1 cells a non-natural polynucleotide having a phosphate-modified bond on the 5' or 3' side of the mismatched nucleotide to be modified.

[0313] In this example, each of the non-natural polynucleotides shown in Tables 44 and 45 was introduced into 293-nLD1 cells by transfection. The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Tables 44 to 45.

[0314] [Table 44]

[0315] [Table 45]

[0316] In Tables 44 to 45, mismatched nucleotides are underlined, and nucleotides replaced with bridged nucleic acids are shown in capital letters. All bridged nucleic acids in Tables 44 to 45 were LNA. Where the internucleotide phosphodiester bond was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding nucleotides.

[0317] As shown in Tables 44 to 45, the non-natural polynucleotides of the present invention were found to have higher editing efficiency than the standard GEO-8. [Industrial Applicability]

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

Claims

1. A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying 1 to 4 nucleotides contained in the target nucleotide sequence in a double-stranded DNA in a cell, comprising: containing 1 to 4 mismatch 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) (excluding polynucleotides in which crosslinked nucleic acids and non-crosslinked nucleic acids exist alternately throughout the entire region of the polynucleotide). (A) one nucleotide adjacent to the 5' upstream side of the mismatched nucleotide is a bridged nucleic acid; (B) one nucleotide adjacent to the 3' downstream side of the mismatched nucleotide is a bridged nucleic acid; (C) The 5'-terminal nucleotide is a bridged nucleic acid. (D) The length is 24 to 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, 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 claim 2, 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 claim 1, 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 claim 1, 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 claim 2, 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-naturally occurring polynucleotide according to claim 2, further comprising the following characteristics (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 of claim 1, further comprising the following characteristic (P): (P) The 3'-terminal nucleotide is phosphorylated

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

12. The non-naturally occurring polynucleotide according to claim 2, 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-naturally occurring polynucleotide according to claim 1, 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 , wherein the adaptor has a function of protecting the non-natural polynucleotide from nuclease digestion.

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

18. The non-natural polynucleotide according to claim 14, wherein the adaptor is a nucleotide containing a mismatched nucleotide.

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

20. The non-natural polynucleotide according to claim 14, 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-naturally occurring polynucleotide according to claim 1, further comprising the following characteristic (X5): (X5) one or more nucleotides are inserted between the mismatched nucleotide and the 3'-terminal nucleotide (excluding the insertion between the mismatched nucleotide and one nucleotide adjacent to it on the 3' downstream side).

23. A kit for modifying a target nucleotide sequence, comprising the non-natural polynucleotide according to any one of claims 1 to 22.

24. A pharmaceutical composition comprising the non-natural polynucleotide according to any one of claims 1 to 22.

25. A method for modifying 1 to 4 nucleotides contained in a target nucleotide sequence in a double-stranded DNA in a cell, comprising: introducing into a cell a non-naturally occurring polynucleotide containing 1 to 4 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 1 to 4 nucleotides of the target nucleotide sequence; The method, wherein the non-naturally occurring polynucleotide is the non-naturally occurring polynucleotide according to any one of claims 1 to 22 (excluding cases where the polynucleotide is introduced into human germ cells or is carried out inside the human body).

26. The method of claim 25, wherein the cell is a prokaryotic or eukaryotic cell.

27. 27. The method of claim 26, wherein the eukaryotic cell is at least one selected from the group consisting of a plant cell, an insect cell, and an animal cell.

28. An ex vivo therapeutic composition comprising the non-natural polynucleotide according to any one of claims 1 to 22.

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