Non-natural polynucleotides for modification of target nucleotide sequences

Abstract

The present invention relates to a non-natural polynucleotide capable of specifically binding to a sequence for modifying one or more nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell, wherein the non-natural polynucleotide comprises one or more mismatched nucleotides with respect to the sequence, has a cross-linked nucleic acid as described in at least one of (A) and (B) below, and additionally has the characteristics of (C) and (D). (A) One or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are cross-linked nucleic acids; (B) One or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are cross-linked nucleic acids; (C) A nucleotide at the 5' end is a cross-linked nucleic acid; and (D) The chain length is 22 to 95 nt.

Description

Technology Field

[0001] The present invention relates to a non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell. Background Technology

[0002] CRISPR-Cas is a genome editing technology that utilizes the acquired immune mechanisms of true bacteria or archaea and is used as a tool for genetic engineering. CRISPR-Cas9 (Patent Document 1), which uses a DNA sequence called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9, a DNA cutting enzyme of Streptococcus pyogenes, is characterized by inducing the cutting of a target two-stranded DNA sequence by having the target DNA sequence recognized as RNA, and is the most widely used genome editing technology due to its simplicity, speed, and high efficiency. On the other hand, CRISPR-Cas9 has problems with off-target effects, such as the guide RNA sequence misrecognizing the genome sequence and the introduction of unexpected mutations at locations other than the target DNA sequence resulting from the cutting of the two-stranded DNA.

[0003] Various solutions have been proposed regarding the off-target effects of CRISPR-Cas. For example, CRISPR-Cas3, which utilizes the fact that the recognition sequence of Cas3 derived from E. coli is 27 bases compared to the recognition sequence of Cas9 which is 20 bases, enabled the introduction of mutations with higher specificity (Patent Document 2). A method using a complex comprising a guide RNA capable of recognizing DNA sequences and a deaminase that performs nucleotide conversion, and a mutant Cas nuclease in which the cleavage activity of one of the two-stranded DNA strands is inactivated, achieved safer and more specific genome editing compared to CRISPR-Cas9 without inducing two-stranded DNA cleavage (Patent Document 3).

[0004] However, even if the problem of off-target effects could be improved, since CRISPR-Cas is a technology that introduces bacterial Cas nucleases or genes encoding Cas nucleases into cells, the problem of unexpected risks caused by the introduction of foreign genes still remains. As a genome editing technology that does not use Cas nuclease proteins, a genome editing technology using single-strand synthetic DNA containing a modifying nucleic acid is known.

[0005] As an example of a method for modifying a target nucleotide sequence in two-strand DNA using one-strand synthetic DNA in which a portion of the nucleotide sequence is substituted with a lock nucleotide (LNA), in cell-free experiments, a method is known that includes at least one mismatched nucleotide and at least two LNAs, and each LNA is an oligonucleotide positioned at a distance of at least one nucleotide from the at least one mismatched nucleotide (Patent Document 4).

[0006] As another example of a genome editing technique that does not use Cas nuclease proteins, it has been reported that a 1 to 3 base variation can be introduced into a target nucleotide sequence by introducing a single strand synthetic DNA, in which part of the nucleotide sequence is LNA, into mouse ES cells (Non-patent Literature 1). It is suggested that the technique of Non-patent Literature 1 can improve the problem of off-target effects compared to genome editing techniques that use Cas nuclease proteins. The authors of Non-patent Literature 1 confirmed, by decoding the base sequence, that the modification of the target nucleotide sequence was carried out as intended in 33 cells, and then decoded the base sequence of a region 335 bp surrounding the target nucleotide sequence and confirmed that no unintended modification was occurring at a point other than the target nucleotide sequence. From these results, it is explained that highly accurate genome editing could be achieved in mouse-derived ES cells in which the mismatch repair mechanism is functioning using the single-strand synthetic DNA used by the authors of Non-Patent Literature 1.

[0007] The authors of Non-Patent Literature 1 concluded that in order to alter the target nucleotide sequence by bypassing the intracellular mismatch repair mechanism, it is important that the mismatch nucleotide be LNA, and they have been conducting experiments with a focus on this. In fact, Non-Patent Literature 1 has conducted experiments using a total of more than 60 types of single-strand synthetic DNA, and 41 types of single-strand synthetic DNA had at least one mismatch nucleotide that was LNA.

[0008] Non-patent document 2 is a preliminary report by the same authors as Non-patent document 1, based on the discovery in Non-patent document 1 that if the mismatch nucleotide included in one-strand synthetic DNA is LNA, the mismatch repair mechanism within the cell can be bypassed. Non-patent document 2 focuses on elucidating the mechanism of how one-strand synthetic DNA in which the mismatch nucleotide is LNA carries out genome modification in mammalian cells. Prior art literature

[0009] Japanese Patent Publication No. 6343605, Japanese Patent Publication No. 6480647, Japanese Patent Publication No. 6206893, Japanese Patent Publication No. 5405121

[0010] 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, 4122-4127Thomas 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 The problem to be solved

[0011] There have been several reports regarding the alteration of target nucleotide sequences using single-strand synthetic DNA containing at least one LNA as a genome editing technique that does not use Cas nucleases, but they are not sufficient in terms of editing efficiency.

[0012] The objective of the present invention is to provide a new non-natural polynucleotide having structural features that enable improved editing efficiency in genome editing technology using non-natural polynucleotides for single-strand synthetic DNA in which part of the nucleotide sequence is a cross-linked nucleic acid. means of solving the problem

[0013] As a result of repeated research to solve the above problem, the inventors discovered that the editing efficiency (yield) of the target nucleotide sequence is improved by using a non-natural polynucleotide capable of specifically binding to the target nucleotide sequence for modifying one or more nucleotides included in the target nucleotide sequence in two-strand DNA within a cell, wherein the non-natural polynucleotide comprises one or more mismatched nucleotides with respect to the target nucleotide sequence, and nucleotides other than the mismatched nucleotides comprise nucleotides complementary to the target nucleotide sequence, and at least one nucleotide adjacent to the mismatched nucleotide or a nucleotide at the 5' end is a cross-linked nucleic acid, and is within a specific chain length range, thereby completing the present invention.

[0014] That is, the present invention relates to the following [1] to

[38] .

[0015] [1] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell, said target nucleotide sequence,

[0016] It comprises one or more mismatched nucleotides with respect to the above target nucleotide sequence, and

[0017] A non-natural polynucleotide having a cross-linked nucleic acid as shown in at least one of (A) and (B) below, and additionally having the features of (C) and (D).

[0018] (A) One or more nucleotides adjacent to the 5' upstream side of the mismatch nucleotide are cross-linked nucleic acids.

[0019] (B) One or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are cross-linked nucleic acids.

[0020] (C) The nucleotide at the 5' end is a cross-linked nucleic acid.

[0021] (D) The chain length is 22 to 95 nucleotides.

[0022] [2] The above-described non-natural polynucleotide having additional features of (E) below.

[0023] (E) The nucleotide at the 3' end is a cross-linked nucleic acid.

[0024] [3] The above-described non-natural polynucleotide having additional features of (F) below.

[0025] (F) One or more nucleotides adjacent to the nucleotide at the 5' end are cross-linked nucleic acids.

[0026] [4] A non-natural polynucleotide described in any one of [1] to [3] having additional characteristics of (G) below.

[0027] (G) One or more nucleotides adjacent to the nucleotide at the 3' end are cross-linked nucleic acids.

[0028] [5] A non-natural polynucleotide described in any one of [1] to [4] having additional features of (J) below.

[0029] (J) One or more phosphodiester bonds between nucleotides are substituted with phosphate group modification bonds.

[0030] [6] The non-natural polynucleotide described in [5] comprises at least one phosphate group selected from the group consisting of phosphorothioate bonds, methylphosphate bonds, boranophosphate bonds, and mesylphosphomidate bonds.

[0031] [7] A non-natural polynucleotide described in any one of [1] to [6] having additional features of (M) below.

[0032] (M) One or more nucleotides are cross-linked nucleic acids disposed between a nucleotide adjacent to the 5' upstream side of the mismatch nucleotide and a nucleotide at the 5' end, and disposed at a distance of at least 1 nucleotide from either the nucleotide adjacent to the 5' upstream side of the mismatch nucleotide or the nucleotide at the 5' end.

[0033] [8] A non-natural polynucleotide described in any one of [1] to [7] having additional characteristics of (N) below.

[0034] (N) One or more nucleotides are cross-linked nucleic acids disposed between a nucleotide adjacent to the 3' downstream side of the mismatch nucleotide and a nucleotide at the 3' end, and disposed at a distance of at least 1 nucleotide from either the nucleotide adjacent to the 3' downstream side of the mismatch nucleotide or the nucleotide at the 3' end.

[0035] [9] A non-natural polynucleotide described in any one of [1] to [8] having additional features of (O) and / or (X2) below.

[0036] (O) The pentose sugar in one or more nucleotides adjacent to the cross-linked nucleic acid at the 3' end is ribose.

[0037] (X2) The pentose sugar in one or more nucleotides adjacent to the cross-linked nucleic acid at the 5' end is ribose.

[0038]

[10] A non-natural polynucleotide described in any one of [1] to [9] having additional characteristics of (P) below.

[0039] (P) The nucleotide at the 3' end is phosphorylated.

[0040]

[11] A non-natural polynucleotide described in any one of [1] to

[10] , comprising at least one cross-linked nucleic acid selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA.

[0041]

[12] A non-natural polynucleotide described in any one of [1] to

[11] having additional features of (X1) below.

[0042] (X1) A nucleotide at the 3' end and / or one or more nucleotides adjacent to the nucleotide at the 3' end are nucleic acids in which the 2' site is modified.

[0043]

[13] The non-natural polynucleotide described in

[12] comprises at least one nucleic acid with the 2' site modified, selected from the group consisting of 2'-F, 2'-OMe, 2'-MOE, and 2'-O-(2-carbamoylethyl).

[0044]

[14] A non-natural polynucleotide described in any one of [1] to

[13] having additional features of (X4) below.

[0045] (X4) An adapter is attached to the 3' end.

[0046]

[15] The above adapter is a non-natural polynucleotide described in

[14] that has the function of inhibiting the repair of mismatch in host cells.

[0047]

[16] The non-natural polynucleotide described in

[14] or

[15] , wherein the adapter has the function of protecting the non-natural polynucleotide from nuclease digestion.

[0048]

[17] The above adapter is a non-natural polynucleotide described in any one of

[14] to

[16] that has the function of providing higher editing efficiency than the non-natural polynucleotide defined by SEQ ID No. 9 (designated as “GEO-8”).

[0049]

[18] The above adapter is a non-natural polynucleotide described in any one of

[14] to

[17] , which is a nucleotide containing a mismatch nucleotide.

[0050]

[19] The above adapter is a non-natural polynucleotide described in any one of

[14] to

[18] , which is a nucleotide forming a stem structure that may have a loop.

[0051]

[20] A non-natural polynucleotide described in any one of

[14] to

[17] , wherein the adapter is a compound other than a nucleotide that modifies the terminal (indicated as a “modifying compound”).

[0052]

[21] A non-natural polynucleotide described in

[20] having a molecular weight of 2,000 or less of the above-mentioned modified compound.

[0053]

[22] A non-natural polynucleotide described in any one of [1] to

[21] having additional features of (X5) below.

[0054] (X5) One or more nucleotides are inserted between the above mismatched nucleotide and the nucleotide at the 3' end.

[0055]

[23] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell, said target nucleotide sequence,

[0056] It comprises one or more mismatched nucleotides with respect to the above target nucleotide sequence, and

[0057] A non-natural polynucleotide having a cross-linked nucleic acid as shown in at least one of (A) and (B) below, having the feature of (D), and additionally having one or more features selected from the group consisting of (H2), (Y1) and (Y3).

[0058] (A) One or more nucleotides adjacent to the 5' upstream side of the mismatch nucleotide are cross-linked nucleic acids.

[0059] (B) One or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are cross-linked nucleic acids.

[0060] (D) The chain length is 22 to 95 nucleotides.

[0061] (H2) The phosphodiester bond portion between the 5' end nucleotide and one or more nucleotides adjacent to the 5' end nucleotide is substituted with a phosphate modification bond.

[0062] (Y1) The nucleotide at the 5' end is a mismatch nucleotide, and one or more nucleotides adjacent to the nucleotide at the 5' end are also mismatch nucleotides.

[0063] (Y3) An adapter is attached to the 5' end.

[0064]

[24] The above adapter is a non-natural polynucleotide described in

[23] having the function of inhibiting the repair of mismatch in host cells.

[0065]

[25] The non-natural polynucleotide described in

[23] or

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

[0066]

[26] The above adapter is a non-natural polynucleotide described in any one of

[23] to

[25] having the function of providing higher editing efficiency than the non-natural polynucleotide (GEO-8) defined by SEQ ID No. 9.

[0067]

[27] A non-natural polynucleotide described in any one of

[23] to

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

[0068]

[28] A non-natural polynucleotide described in any one of

[23] to

[27] having the following additional characteristics of (Y2).

[0069] (Y2) The pentose sugar in the 5' terminal nucleotide is ribose, and the pentose sugar in one or more nucleotides adjacent to the 5' terminal nucleotide is ribose.

[0070]

[29] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell, said target nucleotide sequence,

[0071] It comprises one or more mismatched nucleotides with respect to the above target nucleotide sequence, and

[0072] A non-natural polynucleotide having a cross-linked nucleic acid as shown in at least one of (A) and (B) below, and additionally having the features of (D) and (X7).

[0073] (A) One or more nucleotides adjacent to the 5' upstream side of the mismatch nucleotide are cross-linked nucleic acids.

[0074] (B) One or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are cross-linked nucleic acids.

[0075] (D) The chain length is 22 to 95 nucleotides.

[0076] (X7) One or more nucleotides placed between the mismatched nucleotide and the nucleotide at the 5' end are cross-linked nucleic acids.

[0077]

[30] The non-natural polynucleotide described in

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

[0078]

[31] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell, said target nucleotide sequence,

[0079] It comprises one or more mismatched nucleotides with respect to the above target nucleotide sequence, and

[0080] A non-natural polynucleotide having all the features of (C), (D), (I) and (X6) below.

[0081] (C) The nucleotide at the 5' end is a cross-linked nucleic acid.

[0082] (D) The chain length is 22 to 95 nucleotides.

[0083] (I) The phosphodiester bond portion between the 3' end nucleotide and one or more nucleotides adjacent to the 3' end nucleotide is substituted with a phosphorothioate bond.

[0084] (X6) The above mismatched nucleotide is a cross-linked nucleic acid.

[0085]

[32] The above-described non-natural polynucleotide having additional features of (E) below.

[0086] (E) The nucleotide at the 3' end is a cross-linked nucleic acid.

[0087]

[33] The above-mentioned non-natural polynucleotide having additional features of (P) as described in

[31] or

[32] .

[0088] (P) The nucleotide at the 3' end is phosphorylated.

[0089] 34] A kit for modifying a target nucleotide sequence containing a non-natural polynucleotide as described in any one of [1] to

[33] above.

[0090]

[35] A pharmaceutical composition containing a non-natural polynucleotide as described in any one of [1] to

[33] above.

[0091]

[36] A method for altering one or more nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell,

[0092] The process includes introducing a non-natural polynucleotide into a cell containing one or more mismatched nucleotides with respect to the above target nucleotide sequence, and

[0093] The modification of the target nucleotide sequence comprises at least one selected from the group consisting of deletion, insertion, and substitution of one or more nucleotides of the target nucleotide sequence, and

[0094] The above method, wherein the above non-natural polynucleotide is a non-natural polynucleotide described in any one of claims [1] to

[33] .

[0095]

[37] The method described in

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

[0096]

[38] The method described in

[37] wherein the eukaryotic cell is at least one selected from the group consisting of plant cells, insect cells and animal cells. Effects of the invention

[0097] According to the present invention, a non-natural polynucleotide with excellent editing efficiency of a target nucleotide sequence is provided. Brief explanation of the drawing

[0098] 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. FIG. 2 is a schematic diagram showing an experimental system in which a mutation of the inactive luciferase gene in the genome of 293-nLD1 cells is edited using the non-natural polynucleotide of the present invention, and the editing efficiency is detected as the luminescence of the luciferase. FIG. 3a is a diagram showing the composition of 12 types of non-natural polynucleotides, namely GEO-1 to GEO-9, GEO-242, GEO-11, and GEO-94, which are non-natural polynucleotides shown in Example 1-1 of the present invention. GEO-94 is a negative control having a base sequence in which no mismatch nucleotides exist. In the figure, G represents a mismatch nucleotide, and G with an asterisk (i.e., "G*") and L indicate that the pentose sugar in the nucleotide is substituted with LNA. FIG. 3b is a bar graph showing the editing efficiency (%) of 12 types of non-natural polynucleotides, namely GEO-1 to GEO-9, GEO-242, GEO-11 and GEO-94, as shown in Example 1-1 of the present invention. FIG. 4 is a diagram showing the composition of a non-natural polynucleotide shown in Examples 1-2 of the present invention. In the diagram, G represents a mismatched nucleotide, and L represents that the pentose sugar in the nucleotide is substituted with LNA. FIG. 5 is a diagram showing the composition of a non-natural polynucleotide shown in Examples 1-3 of the present invention. In the diagram, G represents a mismatched nucleotide, and L represents that the pentose sugar in the nucleotide is substituted with LNA. FIG. 6 is a diagram showing the composition of a non-natural polynucleotide according to Examples 1-4 of the present invention. In the diagram, G represents a mismatched nucleotide, and L represents that the pentose sugar in the nucleotide is substituted with LNA. FIG. 7 is a diagram showing the composition of non-natural polynucleotides according to Examples 1-5 of the present invention. In the diagram, G represents a mismatched nucleotide, and L represents that the pentose sugar in the nucleotide is substituted with LNA. FIGS. 8a to 8c are drawings showing the composition of non-natural polynucleotides according to Examples 1-6 of the present invention. FIG. 8a is a drawing showing the composition of a non-natural polynucleotide in which the phosphodiester bond portion between the 5' end LNA and the adjacent 1 to 5 nucleotides is substituted with a phosphorothioate bond. In the drawings, G represents a mismatch nucleotide, L represents that the pentose sugar in the nucleotide is substituted with LNA, and S represents that the phosphodiester bond portion is substituted with a phosphorothioate bond. FIG. 8b is a diagram showing the composition of a non-natural polynucleotide in which the phosphodiester bond portion between a nucleotide at the 3' end and 1 to 7 adjacent nucleotides is substituted with a phosphorothioate bond. In the diagram, G represents a mismatch nucleotide, L represents that the pentose sugar in the nucleotide is substituted with LNA, and S represents that the phosphodiester bond portion is substituted with a phosphorothioate bond. FIG. 8c is a diagram showing each of the following: a configuration of a non-natural polynucleotide in which the phosphodiester bond portion between the 5' end LNA and the 4 nucleotides adjacent thereto is substituted with a phosphorothioate bond, and also the phosphodiester bond portion between the 3' end nucleotide and the 4 nucleotides adjacent thereto is substituted with a phosphorothioate bond; a configuration of a non-natural polynucleotide in which the phosphodiester bond portion between the LNA adjacent to the 5' upstream side of the mismatch nucleotide G and the additionally adjacent 2 or 4 nucleotides is substituted with a phosphorothioate bond; and a configuration of a non-natural polynucleotide in which the phosphodiester bond portion between the nucleotide adjacent to the LNA adjacent to the 3' downstream side of the mismatch nucleotide G and the 2 or 4 nucleotides adjacent to the adjacent nucleotide is substituted with a phosphorothioate bond. In the figure, G represents a mismatch nucleotide, L represents that the pentose sugar in the nucleotide is substituted with LNA, and S represents that the phosphodiester bond portion is substituted with a phosphorothioate bond. FIG. 9 is a diagram showing the composition of non-natural polynucleotides according to Examples 1-7 of the present invention. In the diagram, G represents a mismatch nucleotide, L represents that the pentose sugar in the nucleotide is substituted with LNA, and S represents that the phosphodiester bond portion is substituted with a phosphorothioate bond. FIG. 10a is a diagram showing the composition of a non-natural polynucleotide according to Examples 1-8 of the present invention. In the diagram, G represents a mismatched nucleotide, and L represents that the pentose sugar in the nucleotide is substituted with LNA. FIG. 10b is a diagram showing the composition of a non-natural polynucleotide according to Examples 1-8 of the present invention. In the diagram, G represents a mismatched nucleotide, and L represents that the pentose sugar in the nucleotide is substituted with LNA. FIG. 11 is a diagram showing the composition of a non-natural polynucleotide as shown in Examples 1-9 of the present invention. In the diagram, G represents a mismatched nucleotide, L represents that the pentose sugar in the nucleotide is substituted with LNA, and R represents that the pentose sugar in the nucleotide is substituted with ribose. FIG. 12 is a diagram showing the composition of a non-natural polynucleotide as shown in Examples 1-10 of the present invention. In the diagram, G represents a mismatched nucleotide, L represents that the pentose sugar in the nucleotide is substituted with LNA, P represents that the nucleotide at the 5' end and / or the nucleotide at the 3' end is phosphorylated, and S represents that the phosphodiester bond portion is substituted with a phosphorothioate bond. FIG. 13 is a diagram showing the composition of a non-natural polynucleotide as shown in Example 1-11 of the present invention. In the diagram, G represents a mismatched nucleotide, L represents that the pentose sugar in the nucleotide is substituted with LNA, H represents that the pentose sugar in the nucleotide is substituted with BNA-NH, M represents that the pentose sugar in the nucleotide is substituted with BNA-N-Me, and E represents that the pentose sugar in the nucleotide is substituted with ENA. FIG. 14 is a schematic diagram showing the composition of a non-natural polynucleotide (GEO-226) shown in Examples 1-12 of the present invention. In the figure, arrows indicate the positions of mismatched nucleotides, L indicates that the pentose sugar in the nucleotide is substituted with LNA, S indicates that the phosphodiester bond portion is substituted with a phosphorothioate bond, and P indicates that the 3' end is phosphorylated. FIG. 15 is a diagram showing a schematic representation of the composition of a non-natural polynucleotide (GEO-172) as shown in Examples 1-13 of the present invention. In the figure, an arrow indicates the position of a mismatched nucleotide, L indicates that the pentose sugar in the nucleotide is substituted with LNA, S indicates that the phosphodiester bond portion is substituted with a phosphorothioate bond, R indicates that the nucleotide is substituted with RNA, and P indicates that the 3' end is phosphorylated. FIG. 16a is a schematic diagram showing the composition of non-natural polynucleotides (GEO-602 to GEO-605) as shown in Example 3-3 of the present invention. In the figure, G represents a mismatched nucleotide, and L represents that the pentose sugar in the nucleotide is substituted with LNA. In the non-natural polynucleotides in the figure, L, G, and the nucleotide at the point of the black band are DNA, and the nucleotide at the point of the white band is RNA. FIG. 16b is a schematic diagram showing the composition of non-natural polynucleotides (GEO-606 and GEO-693 to GEO-695) shown in Example 3-3 of the present invention. In the figure, G represents a mismatched nucleotide, and L represents that the pentose sugar in the nucleotide is substituted with LNA. In the non-natural polynucleotides in the figure, L, G, and the nucleotide at the point of the black band are DNA, and the nucleotide at the point of the white band is RNA. FIG. 17a is a schematic diagram showing the composition 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 represents a mismatched nucleotide, L represents a nucleotide in which the pentose sugar is substituted with LNA, and S represents a nucleotide in which the phosphodiester bond portion is substituted with a phosphorothioate bond. In the non-natural polynucleotides in the figure, the nucleotides at the points of the black bands are DNA, and the nucleotides at the points of the white bands are RNA. FIG. 17b is a schematic diagram showing the composition of non-natural polynucleotides (GEO-703, GEO-822, and GEO-823) shown in Example 3-3 of the present invention. In the figure, G represents a mismatched nucleotide, L represents a nucleotide in which the pentose sugar is substituted with LNA, and P represents a nucleotide in which the 3' end is phosphorylated. In the non-natural polynucleotides in the figure, the nucleotides at the points of the black band are DNA, and the nucleotides at the points of the white band are RNA. Specific details for implementing the invention

[0099] <Overview of the Present Invention>

[0100] In conventional techniques using 1-strand polynucleotides, the polynucleotide introduced into the cell specifically recognizes and binds to the target nucleotide sequence in the lagging chain of the replication fork during 2-strand DNA replication, and acts as a primer for the synthesis of Okazaki fragments by DNA polymerase. In this way, the insertion of the polynucleotide into the newly formed DNA strand causes editing of the genome sequence. On the other hand, since the organism's cell is equipped with a mismatch repair mechanism that repairs mismatches that occur during DNA replication, the mismatch caused by the polynucleotide introduced from the outside is also immediately repaired. It is thought that the modification of editing by this mismatch repair mechanism is one of the reasons why the alteration of the target nucleotide sequence by the polynucleotide introduced into the cell from the outside is less efficient than the alteration of the target nucleotide sequence using nuclease.

[0101] Meanwhile, it is presumed that the non-natural polynucleotide of the present invention can significantly increase the editing efficiency of the target nucleotide sequence compared to conventional reports by promoting editing through the presence of cross-linked nucleic acid at a specific position within the non-natural polynucleotide and simultaneously reducing the influence of the mismatch repair mechanism.

[0102] In the present invention, in a non-natural polynucleotide, it is possible to further increase the efficiency of target nucleotide sequence modification by appropriately combining, for example, the number and position of mismatched nucleotides, the number and position of crosslinked nucleic acids, the chain length of the polynucleotide, the type of crosslinked nucleic acid, substitution of the phosphate portion of the phosphodiester bond between one or more nucleotides with a phosphate modification bond (e.g., a phosphorothioate bond), substitution of the pentose sugar in one or more nucleotides with ribose (which may be abbreviated as “RNA substitution” in this specification), phosphorylation of the 3’ end or 5’ end, or the addition of an adapter.

[0103] <Nucleotide>

[0104] In the present invention, "nucleotide" is a general term for a substance in which a phosphate group is attached to a nucleoside. A nucleoside is a molecule in which a purine base or a pyrimidine base is glycosidically bonded to the 1st position of a pentose sugar. A chain-type biopolymer in which a nucleotide is the unit is a polynucleotide (also called a "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 is similar to DNA in that it contains A, G, and T, but instead of T, it contains uridine monophosphate (UMP, hereinafter referred to as "U").

[0105] <2 Main Chain DNA>

[0106] In the present invention, "two-stranded DNA" refers to DNA in which one-stranded DNA having mutually complementary base sequences forms hydrogen bonds between bases in opposite directions to form a double screw. Examples of two-stranded DNA in the present invention are not particularly limited, but include genomic DNA, mitochondrial DNA, chloroplast DNA, etc.

[0107] In addition, in the present invention, the two-strand DNA containing the "target nucleotide sequence" may preferably be a gene related to a genetic disease, more preferably a gene related to a human genetic disease.

[0108] For example, the gene associated with adrenoleukodystrophy (ABCD1), the gene associated with heavy-chain acyl CoA dehydrogenase deficiency (ACADM), the gene associated with Wilson's disease (ATP7B), the gene associated with hereditary pulmonary hypertension (BMPR2), the gene associated with hereditary pulmonary hypertension (BMPR2), the gene associated with X-chain agammaglobulinemia (BTK), the gene associated with cystinuria (CTNS), the gene associated with Duchenne muscular dystrophy (DMD), the gene associated with hemophilia A (F8), the gene associated with hemophilia B (F9), the gene associated with tyrosinemia (FAH), the gene associated with hepatic glycogen storage disease type Ia (G6PC), the gene associated with familial frontotemporal degeneration (GRN), the gene associated with mucopolysaccharidosis type II (IDS), the gene associated with mucopolysaccharidosis type I (IDUA), the gene associated with primary immunodeficiency syndrome (IKBKB), and familial hypertrophic Examples include the gene associated with cardiomyopathy (MYH7), the gene associated with peroxisome dysplasia (PEX2), the gene associated with hepatic glycogen storage disease type IX (PHKA2), the gene associated with protein C deficiency (PROC), the gene associated with hepatic glycogen storage disease type V (PYGM), the gene associated with familial dilated cardiomyopathy (RBM20), the gene associated with α1-antitrypsin deficiency (SERPINA1), the gene associated with citrine deficiency (SLC25A13), the gene associated with cystinuria (SLC7A9), the gene associated with Niemann-Pick disease (SMPD1), and the gene associated with amyotrophic lateral sclerosis (SOD1). Furthermore, all of these diseases and genes are human diseases and genes.

[0109] <Complementary Nucleotides and Mismatched Nucleotides>

[0110] 2. In DNA strands, G and C, and A and T are typically 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."

[0111] In the present invention, "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, "a nucleotide that cannot form a Watson-Crick type hydrogen bond between two bases" is a nucleotide other than C for G, other than T for A, other than A for T, and other than G for C. "Unable to form a Watson-Crick type hydrogen bond between two bases because there are no corresponding nucleotides" means that for G, there are no corresponding nucleotides for A, T, G, and C; for A, there are no corresponding nucleotides for A, T, G, and C; for T, there are no corresponding nucleotides for A, T, G, and C; and for G, there are no corresponding nucleotides for A, T, G, and C.

[0112] In the non-natural polynucleotide of the present invention, there may be one or more "mismatch nucleotides" for modifying the base to be edited, and furthermore, there may be one or more "mismatch nucleotides" that do not participate in the modification. The latter "mismatch nucleotides" are mismatch nucleotides for the purpose of improving editing efficiency, and for example, include mismatch nucleotides inserted between the mismatch nucleotide for modification purposes and the nucleotide at the 3' end, or mismatch nucleotides among the nucleotides when the adapter is a nucleotide.

[0113] <Alteration of target nucleotide sequence>

[0114] In the present invention, "target nucleotide sequence" is a nucleotide sequence to which a non-natural polynucleotide to which the following description specifically binds. Here, "specifically binds" may include the following embodiments 1 and 2.

[0115] Aspect 1: In the case where the non-natural polynucleotide has only a mismatch with the base to be edited, it means binding to a sequence complementary to the non-natural polynucleotide, excluding the mismatch with the base to be edited.

[0116] Aspect 2: If the non-natural polynucleotide has a mismatch other than a mismatch with the base to be edited, it means that it binds to a sequence complementary to the non-natural polynucleotide, excluding the mismatch portions.

[0117] "Amendment" of a target nucleotide sequence refers to substituting a specific nucleotide (e.g., G) with any of three other types of nucleotides (A, T, or C) for one or more nucleotides of the target nucleotide sequence (hereinafter referred to as "substitution"), deleting one or more nucleotides of the target nucleotide sequence (hereinafter referred to as "deletion"), or inserting another nucleotide or nucleotide sequence between two specific nucleotides on the target nucleotide sequence (hereinafter referred to as "insertion"). "Amendment" of a target nucleotide sequence includes substitution, deletion, and insertion occurring individually, and combinations thereof.

[0118] <Non-natural polynucleotides>

[0119] In the present invention, "non-natural polynucleotide" refers to a polynucleotide that includes a nucleotide other than a naturally occurring nucleotide (A, T, G, C, or U) among the nucleotides constituting the polynucleotide (hereinafter referred to as "non-natural nucleotide"). The origin of the non-natural nucleotide is not particularly limited and includes those that are artificially synthesized and those extracted and purified from a substance containing non-natural nucleotides.

[0120] Non-natural nucleotides are not particularly limited but may include nucleic acids modified by a phosphate group, nucleic acids modified by a sugar group, nucleic acids modified by a base group, nucleic acids with an adapter added to the 3' end and / or 5' end, and nucleic acids in which one or more nucleotides are inserted between a mismatch nucleotide and a nucleotide at the 3' end. Nucleotides modified by a phosphate group are not particularly limited but may include nucleic acids in which the phosphodiester bond portion between one or more nucleotides is substituted with a phosphothioate bond. While a bond between ordinary nucleotides is called a phosphodiester bond, this sulfated bond is called a phosphothioate bond. In the present invention, a nucleotide having a phosphothioate bond is referred to as a "phosphothioated" or "S-modified" nucleotide.

[0121] Other examples of nucleic acids modified by a phosphate group include nucleic acids having methylphosphate bonds, nucleic acids having boranophosphate bonds in which one of the non-crosslinked oxygen atoms of the phosphate diester bond is substituted with a borano group (BH3), and nucleic acids having mesylphosphomidate bonds in which one of the non-crosslinked oxygen atoms of the phosphate diester bond is substituted with -NSO2CH3.

[0122] Examples of nucleic acids modified by sugar groups include, but are not particularly limited, nucleic acids modified at the 2' site, and cross-linked nucleic acids in which the 2' site and the 4' site are cross-linked. Examples of nucleic acids modified at the 2' site include, but are not particularly limited, 2'-F (F-ion), 2'-O-Methyl (2'-OMe), 2'-O-Methoxyethyl (2'-MOE), 2'-O-(2-carbamoylethyl).

[0123] Cross-linked nucleic acids are not particularly limited to 2'-O,4'-C-methylene-Bridged Nucleic Acid (2',4'-BNA), BNA NC Examples include , 2 or 3-cyclic cross-linked nucleic acids, and other cross-linked nucleic acids. 2',4'-BNA is also called Locked Nucleic Acid (LNA) and has the structure shown below.

[0124] [Chemical Formula 1]

[0125]

[0126] BNA / LNA flexible materials are not particularly limited to, but include ethylene-crosslinked BNA (ENA), amide-crosslinked 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 bound ethyl (cEt) LNA, 2'-O,4'-C bound methoxyethyl (cMOEt) LNA, carba (cLNA), BNACOC, spirocyclopropylene-crosslinked nucleic acid (scpBNA), heterocyclic-crosslinked BNA, urea-crosslinked BNA, sulfonamide-crosslinked BNA, 5'-methyl-substituted BNA, and guanidine-crosslinked nucleic acid (GuNA). Examples include 3,4-dehydro-2H-pyran nucleic acid (DpNA).

[0127] BNA NCExamples include BNA NH, BNA N-Me, and BNA N-Bn, although not specifically limited.

[0128] Examples of 2- or 3-circular cross-linked nucleic acids include, but are not limited to, TriNA, α-L-TriNA, F-bcDNA, tricycloDNA (tcDNA), F-tcDNA, 2-circular carbon-circular nucleotides, bicycloDNA (bcDNA), 2'-C-cross-linked 2-circular nucleotides (CBBN), etc.

[0129] Other cross-linked nucleic acids, though not specifically limited, include oxetane nucleotides, locked PMO derived from 2'-amino-LNA, cyclohexenyl nucleic acid (CeNA), aritriol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl RNA (p-RNA), 3'-deoxypyranosyl DNA (p-DNA), etc.

[0130] In the non-natural polynucleotide of the present invention, if the amount of cross-linked nucleic acid is excessive, the editing efficiency tends to decrease. For example, regarding the ratio of cross-linked nucleic acid in the non-natural polynucleotide of the present invention, if the total number of nucleotides constituting the non-natural polynucleotide is 100%, the ratio of cross-linked nucleic acid is preferably 2 to 30%, more preferably 2 to 20%, and even more preferably 2 to 15%.

[0131] In the non-natural polynucleotide of the present invention, it is preferable to add an adapter to the 3' end and / or 5' end so that higher editing efficiency is exhibited. An adapter in this specification may, for example, have a function that inhibits the repair of mismatch in host cells, have a function that protects the non-natural polynucleotide of the present invention from nuclease digestion, and have a function that provides higher editing efficiency than the non-natural polynucleotide (GEO-8) defined by SEQ ID No. 9 by adding it to the 3' end and / or 5' end. An adapter in the present invention has one or more of these functions, and more specific examples of such an adapter may include (1) a nucleotide as an adapter, or (2) a compound other than a nucleotide that modifies the end (referred to as "modifying compound" in this specification).

[0132] The presumed mechanism for inhibiting mismatch repair in host cells by adding an adapter to the 3' end and / or 5' end of the non-natural polynucleotide of the present invention is as follows. Mismatch repair is initiated when, in the case where one strand of DNA forming two strands has a 3' end structure, a mismatch repair enzyme in the cell recognizes the strand having the 3' end as a target for repair, and subsequently, the mismatch 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 mismatch nucleotide of the non-natural polynucleotide is repaired because the 3' end structure exists in the non-natural polynucleotide. When a nucleotide acting as an adapter is added to the 3' end of a non-natural polynucleotide, the 3' end of the non-natural polynucleotide is displaced without specifically binding to the target sequence, so it is possible to consider the possibility that it may be evaded from recognition by mismatch repair enzymes. Likewise, when a modifying compound is added to the 3' end of a non-natural polynucleotide, it is possible to consider the possibility that the 3' end of the non-natural polynucleotide may be evaded from recognition by mismatch repair enzymes. For this reason, it is presumed that the editing efficiency of a non-natural polynucleotide with a nucleotide acting as an adapter or a modifying compound added to the 3' end increases by inhibiting the host cell's mismatch repair.

[0133] The presumed mechanism for protecting the non-natural polynucleotide of the present invention from nuclease digestion by host cells by adding an adapter to the 3' end and / or 5' end of the non-natural polynucleotide of the present invention is as follows. A single strand of non-natural polynucleotide introduced into a host cell becomes subject to digestion by the host cell's nuclease. Although merely adding a nucleotide acting as an adapter to the 5' or 3' end of the non-natural polynucleotide cannot completely prevent the digestion of the non-natural polynucleotide by nucleases, it is presumed that the nucleotide acting as an adapter acts as a buffer, thereby inhibiting the digestion by exonucleases from proceeding to the mismatch nucleotide located in the center of the non-natural polynucleotide. Meanwhile, it is highly probable that digestion by exonucleases from the 3' end is inhibited by adding a modifying compound to the 3' end. For example, IdT is used to increase the nuclease resistance of antisense nucleic acids by adding it to the 3' end of the antisense nucleic acid.

[0134] As described above, since the function of inhibiting the repair of host cell mismatch and the function of protecting the non-natural polynucleotide of the present invention from nuclease digestion are based on different mechanisms, the non-natural polynucleotide of the present invention may possess both of these functions.

[0135] Examples of the structure of the nucleotide serving as the adapter include straight-chain nucleotides and nucleotides forming a stem structure that may have a loop. Furthermore, regarding the chain length of the nucleotide serving as the adapter, it is preferably 1 nt (nucleotide) or more, more preferably 3 nt or more, and even more preferably 6 or moreAnd, on the other hand, preferably 50 nt or less, more preferably 40 nt or less, and even more preferably 30 or less. Since the non-natural polynucleotide of the present invention does not include a nucleotide as an adapter, the chain length of the nucleotide as an adapter does not affect the chain length of the non-natural polynucleotide of the present invention.

[0136] Preferably, the nucleotide as an adapter is a nucleotide containing a mismatch nucleotide, and more preferably, the nucleotide sequence of the nucleotide as an adapter is a mismatch with respect to both the 5' upstream sequence adjacent to the target nucleotide sequence and the 3' downstream sequence adjacent to the target nucleotide sequence, but one or more may be complementary to the target nucleotide sequence.

[0137] The modifying compound as the adapter above is preferably a compound capable of binding to the 3' end and / or 5' end of the non-natural polynucleotide of the present invention.

[0138] 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, while 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.

[0139] Preferred specific examples of the modification compound include at least one selected from the group consisting of fluorescein (FAM), biotin, furomycin, cholesterol, digoxigenin (DIG), and Inverted dT.

[0140] The binding of the adapter to the non-natural polynucleotide of the present invention can be carried out by a known method.

[0141] These non-natural nucleotides with such formulas have a high affinity for natural DNA or RNA, so their application in nucleic acid pharmaceuticals is progressing.

[0142] <Basic Composition of Non-Natural Polynucleotides>

[0143] The non-natural polynucleotide of the present invention is a single-strand polynucleotide containing one or more mismatched nucleotides with respect to a target nucleotide sequence, and the nucleotides other than the mismatched nucleotides are polynucleotides that are complementary to the target nucleotide sequence. Accordingly, by introducing the non-natural polynucleotide of the present invention into a cell, it can specifically bind to the target nucleotide sequence. By doing so, one or more nucleotides in the target nucleotide sequence can be modified into a desired nucleotide. When there are multiple mismatched nucleotides, two or more mismatched nucleotides may be located adjacent to each other or located at separate positions. In the present invention, regarding the number of mismatched nucleotides, the term "plural" includes integers such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, and also includes a range in which these are the upper and lower limits. For example, 2 to 3, 2 to 4, 2 to 5, … 2 to 20, 3 to 4, 3 to 5, 3 to 6, … 3 to 20, 4 to 5, 4 to 6, 4 to 7, … 4 to 20, 5 to 6, 5 to 7, 5 to 8, … 5 to 20, … It is 18 ~ 19, 18 ~ 20, 19 ~ 20.

[0144] The basic composition of the non-natural polynucleotide of the present invention as described below may be, for example, those described in embodiments (i) to (vi).

[0145] (1) Mode (i)

[0146] One embodiment (embodiment (i)) of the non-natural polynucleotide of the present invention is,

[0147] (A) One or more nucleotides adjacent to the 5' upstream side of the mismatch nucleotide are cross-linked nucleic acids, and

[0148] (B) One or more nucleotides adjacent to the 3' downstream side of the mismatch nucleotide are cross-linked nucleic acids, and

[0149] (C) The nucleotide at the 5' end is a cross-linked nucleic acid, and

[0150] (D) The chain length is 22 to 95 nucleotides.

[0151] This configuration is such that it can produce the effects of the present invention.

[0152] The cross-linked nucleic acid included in the non-natural polynucleotide of embodiment (i) is not particularly limited, but at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA may be cited. In the present invention, LNA or BNA NH is preferred as the cross-linked nucleic acid of (A) and / or (B), and LNA is more preferred. In the present invention, LNA, AmNA, BNA NH, BNA N-Me, and ENA are preferred as the cross-linked nucleic acid of (C).

[0153] Here, the mismatch nucleotide may be a cross-linked nucleic acid or not a cross-linked nucleic acid, but in embodiment (i), it is more preferable that the mismatch nucleotide is not a cross-linked nucleic acid. Among the total of three nucleotides including the mismatch nucleotide and adjacent thereto, the number of cross-linked nucleic acids is preferably two. Also, regarding "multiple nucleotides are cross-linked nucleic acids" specified in (A) and (B) above, the term "multiple" is not particularly limited, but it is preferable to have two consecutive cross-linked nucleic acids rather than three or more consecutive ones, and one is more preferable.

[0154] (2) Mode (ii)

[0155] Another aspect of the non-natural polynucleotide of the present invention (Aspect (ii)) is,

[0156] (A) One or more nucleotides adjacent to the 5' upstream side of the mismatch nucleotide are cross-linked nucleic acids, and

[0157] (C) The nucleotide at the 5' end is a cross-linked nucleic acid, and

[0158] (D) The chain length is 22 to 95 nucleotides.

[0159] The configuration is as follows (provided that it does not have the features of (B) above). Even with such a configuration, the effects of the present invention can be achieved.

[0160] The cross-linked nucleic acid included in the non-natural polynucleotide of embodiment (ii) is not particularly limited, but at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA may be cited. In the present invention, LNA is preferred as the cross-linked nucleic acid included in the non-natural polynucleotide.

[0161] Here, the mismatch nucleotide may be a cross-linked nucleic acid or not a cross-linked nucleic acid, but in embodiment (ii), it is more preferable that the mismatch nucleotide is not a cross-linked nucleic acid. Among the total of three nucleotides including the mismatch nucleotide and adjacent thereto, the number of cross-linked nucleic acids is preferably two. Also, regarding "multiple nucleotides are cross-linked nucleic acids" specified in (A) above, the term "multiple" is not particularly limited, but it is preferable to have two consecutive cross-linked nucleic acids rather than three or more consecutive ones, and one is more preferable.

[0162] (3) Mode (iii)

[0163] An additional embodiment of the non-natural polynucleotide of the present invention (embodiment (iii)) is,

[0164] (B) One or more nucleotides adjacent to the 3' downstream side of the mismatch nucleotide are cross-linked nucleic acids, and

[0165] (C) The nucleotide at the 5' end is a cross-linked nucleic acid, and

[0166] (D) The chain length is 22 to 95 nucleotides.

[0167] The configuration is as follows (provided that it does not have the features of (A) above). Even with such a configuration, the effects of the present invention can be achieved.

[0168] The cross-linked nucleic acid included in the non-natural polynucleotide of embodiment (iii) is not particularly limited, but at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA may be cited. In the present invention, LNA is preferred as the cross-linked nucleic acid included in the non-natural polynucleotide.

[0169] Here, the mismatch nucleotide may be a cross-linked nucleic acid or not a cross-linked nucleic acid, but in embodiment (iii), it is more preferable that the mismatch nucleotide is not a cross-linked nucleic acid. Among the total of three nucleotides including the mismatch nucleotide and adjacent thereto, the number of cross-linked nucleic acids is preferably two. Also, regarding "multiple nucleotides are cross-linked nucleic acids" specified in (B) above, the term "multiple" is not particularly limited, but it is preferable to have two consecutive cross-linked nucleic acids rather than three or more consecutive ones, and one is more preferable.

[0170] [Chain length of non-natural polynucleotides]

[0171] As described above, the chain 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. It is desirable for the chain length of the non-natural polynucleotide to be within this numerical range because it allows for the modification of the target polynucleotide sequence with high editing efficiency.

[0172] [Relationship between mismatched nucleotides and adjacent cross-linked nucleic acids]

[0173] The non-natural polynucleotide of the present invention can exhibit sufficient editing efficiency for a target nucleotide sequence in any one of the above embodiments (i) to (iii), for example, even if the mismatch nucleotide is a cross-linked nucleic acid. In this case, it is more preferable if the cross-linked nucleic acid is an LNA.

[0174] In the present invention, at least one nucleotide adjacent to the mismatch nucleotide is a cross-linked nucleic acid. In the relationship between the mismatch nucleotide and the cross-linked nucleic acid adjacent thereto, the number of consecutive cross-linked nucleic acids is not particularly limited, but when there are multiple consecutive cross-linked nucleic acids, including the case where the mismatch nucleotide is a cross-linked nucleic acid, the number of consecutive cross-linked nucleic acids is preferably two.

[0175] [3' end cross-linked nucleic acid]

[0176] In any one of the above embodiments (i) to (iii), the non-natural polynucleotide of the present invention can further increase the editing efficiency of the target nucleotide sequence if, for example, (E) the nucleotide at the 3' end is a cross-linked nucleic acid.

[0177] [Nucleic acid with nucleotides adjacent to the 5' end that are cross-linked]

[0178] In any of the above embodiments (i) to (iii), or additionally in addition to (E), the non-natural polynucleotide of the present invention can further increase the editing efficiency of the target nucleotide sequence if, for example, one or more nucleotides adjacent to the nucleotide at the 5' end (F) are cross-linked nucleic acids. In this case, it is preferable that one nucleotide adjacent to the nucleotide at the 5' end is a cross-linked nucleic acid. Furthermore, it is more preferable that the cross-linked nucleic acid is an LNA. In this case, the number of cross-linked nucleic acids consecutive from the nucleotide at the 5' end is not particularly limited, but, for example, if the number of 5' ends from the mismatch nucleotide is 12 nt, it is preferable to have 2, and if the number of 5' ends is 25 nt, it is preferable to have 3.

[0179] [Nucleic acid with nucleotides adjacent to the 3' end that are cross-linked]

[0180] In any of the above embodiments (i) to (iii), or additionally in addition to (E) and / or (F), the non-natural polynucleotide of the present invention can further increase the editing efficiency of the target nucleotide sequence if, for example, one or more nucleotides adjacent to the nucleotide at the (G) 3' end are cross-linked nucleic acids. In this case, it is more preferable that the cross-linked nucleic acid is an LNA. In this case, the number of cross-linked nucleic acids consecutive from the nucleotide at the 3' end is not particularly limited, but, for example, when the number of 3' ends from the mismatch nucleotide is 27 nt, it is preferable to have 3 or fewer, more preferable to have 2 or fewer, and more preferable to have 1. Also, for example, when the number of 3' ends from the mismatch nucleotide is 12 nt, the number of cross-linked nucleic acids consecutive from the nucleotide at the 3' end is preferable to have 3 or fewer, more preferable to have 2 or fewer, and more preferable to have 1.

[0181] [Substitution into Phosphate Modification Combination]

[0182] The non-natural polynucleotide of the present invention can further enhance the editing efficiency of the present invention if, in any one of the embodiments (i) to (iii) above, or additionally in addition to (E), (F) and / or (G), for example, one or more phosphodiester bond portions between nucleotides are substituted with phosphate group modification bonds. Specific configurations are not particularly limited, but the following (J) may be cited.

[0183] (J) One or more phosphodiester bonds between nucleotides are substituted with phosphate group modification bonds.

[0184] In the present invention, a phosphate modification bond is a bond in which some atoms of a phosphate diester bond are replaced with other atoms or substituents, for example, sulfur atoms, boron atoms, nitrogen atoms, methyl groups, or ester groups. Specific examples of phosphate modification bonds include phosphorothioate bonds, methylphosphate bonds, boranophosphate bonds, and mesylphosphomidate bonds.

[0185] In the non-natural polynucleotide of the present invention, the number or location of phosphate group modification bonds is not particularly limited. In addition, in the non-natural polynucleotide of the present invention, the type of phosphate group modification bond may be one type, or two or more types may be mixed.

[0186] The number of phosphate group modification bonds may be 1, and the ratio of phosphate group modification bonds in phosphate diester bonds may be 60% or less, but from the perspective of editing efficiency, it is preferable that not all phosphate diester bonds are substituted.

[0187] As one embodiment of the above (J), the following may be cited.

[0188] (X3) The phosphodiester bond portion between the 3' end nucleotide and one or more nucleotides adjacent to the 3' end nucleotide is substituted with a phosphate modification bond.

[0189] In the above (J), from the perspective of editing efficiency, a phosphorothioate bond is preferred as the phosphate group modification bond. Examples of embodiments in which the phosphate group modification bond is a phosphorothioate bond include any of the following (H1), (I), (K) and (L) or combinations thereof.

[0190] (H1) The phosphodiester bond portion between the 5' end nucleotide and one or more nucleotides adjacent to the 5' end nucleotide is substituted with a phosphorothioate bond.

[0191] (I) The phosphodiester bond portion between the 3' end nucleotide and one or more nucleotides adjacent to the 3' end nucleotide is substituted with a phosphorothioate bond.

[0192] (K) The phosphodiester bond portion between the cross-linked nucleic acid adjacent to the 5' upstream side of the mismatched nucleotide and one or more nucleotides additionally adjacent to the cross-linked nucleic acid is substituted with a phosphorothioate bond.

[0193] (L) The phosphodiester bond portion between the cross-linked nucleic acid adjacent to the 3' downstream side of the mismatched nucleotide and one or more nucleotides additionally adjacent to the cross-linked nucleic acid is substituted with a phosphorothioate bond.

[0194] In the above features (K) and (L), the phosphodiester bond portion between the cross-linked nucleic acid and one nucleotide directly adjacent to the cross-linked nucleic acid may be substituted with a phosphorothioate bond, or may not be substituted with it.

[0195] The non-natural polynucleotide of the present invention may have both of the above features (H1) and (I), and in this case, the non-natural polynucleotide of the present invention preferably has the feature that "a phosphodiester bond portion between a 5' end nucleotide and one or more consecutive nucleotides adjacent to the 5' end nucleotide, and a phosphodiester bond portion between a 3' end nucleotide and one or more consecutive nucleotides adjacent to the 3' end nucleotide are substituted with phosphothioate bonds."

[0196] [The cross-linked nucleic acid adjacent to the 5' upstream side of the mismatch nucleotide and the cross-linked nucleic acid at the 5' end is the cross-linked nucleic acid]

[0197] In any of the above embodiments (i) to (iii), or additionally in addition to (E), (F), (G) and / or (J), for example (M), if one or more nucleotides are cross-linked nucleic acids disposed between a nucleotide adjacent to the 5' upstream side of the mismatch nucleotide and a nucleotide at the 5' end, and also disposed at a distance of at least one nucleotide from either the nucleotide adjacent to the 5' upstream side of the mismatch nucleotide or the nucleotide at the 5' end, the editing efficiency of the target nucleotide sequence can be further increased.

[0198] [The cross-linked nucleic acid adjacent to the 3' downstream side of the mismatch nucleotide and the cross-linked nucleic acid at the 3' end is the cross-linked nucleic acid]

[0199] In any of the above embodiments (i) to (iii), or additionally in addition to (E), (F), (G), (J) and / or (M), for example (N), if one or more nucleotides are cross-linked nucleic acids disposed between a nucleotide adjacent to the 3' downstream side of the mismatch nucleotide and a nucleotide at the 3' end, and are disposed at a distance of at least one nucleotide from either the nucleotide adjacent to the 3' downstream side of the mismatch nucleotide or the nucleotide at the 3' end, the editing efficiency of the target nucleotide sequence can be further increased.

[0200] [RNA Substitution]

[0201] The non-natural polynucleotide of the present invention is a one-chain polynucleotide, but deoxyribose in one or more nucleotides may be substituted with ribose.

[0202] Specifically, in any of the above embodiments (i) to (iii), or additionally in addition to (E), (F), (G), (J), (M) and / or (N), for example, if the pentose sugar in one or more nucleotides adjacent to the cross-linked nucleic acid at the (O) end is ribose, and / or if the pentose sugar in one or more nucleotides adjacent to the cross-linked nucleic acid at the (X2) end is ribose, the editing efficiency of the target nucleotide sequence can be further increased.

[0203] [3' terminal phosphorylation]

[0204] The non-natural polynucleotide of the present invention can further increase the editing efficiency of a target nucleotide sequence in any of the above embodiments (i) to (iii), or additionally in addition to (E), (F), (G), (J), (M), (N), (O) and / or (X2), for example, if the nucleotide at the 3' end of (P) is phosphorylated.

[0205] [Formula for the glycosylation of nucleic acids]

[0206] In any of the above embodiments (i) to (iii), or additionally in addition to (E), (F), (G), (J), (M), (N), (O), (X2) and / or (P), the non-natural polynucleotide of the present invention may further increase the editing efficiency of a target nucleotide sequence if, for example, one or more nucleotides adjacent to the 3' end nucleotide of (X1) are substituted with a nucleic acid having a modified 2' site.

[0207] Examples of nucleic acids with a modified 2' site include at least one selected from the group consisting of 2'-F, 2'-OMe, 2'-MOE, and 2'-O-(2-carbamoylethyl).

[0208] [Addition of adapter for 3' end]

[0209] The non-natural polynucleotide of the present invention can further increase the editing efficiency of a target nucleotide sequence if, in any of the above embodiments (i) to (iii), or additionally in addition to (E), (F), (G), (J), (M), (N), (O), (X2), (P) and / or (X1), an adapter is added to the (X4) 3' end.

[0210] [Insertion of a nucleotide between a mismatched nucleotide and a 3' terminal nucleotide]

[0211] In any of the above embodiments (i) to (iii), the non-natural polynucleotide of the present invention can further increase the editing efficiency of the target nucleotide sequence if, in addition to (E), (F), (G), (J), (M), (N), (O), (X2), (P), (X1) and / or (X4), (X5) one or more nucleotides are inserted between the mismatch nucleotide and the nucleotide at the 3' end. The "mismatch nucleotide" in (X5) is a mismatch nucleotide intended for modification. Also, in (X5), the inserted nucleotide is a mismatch nucleotide, but the mismatch nucleotide here is a mismatch nucleotide intended for improving editing efficiency and does not correspond to a mismatch nucleotide intended for modification. In addition, the inserted nucleotide may be a nucleic acid modified by the phosphate group described above, a nucleic acid modified by the sugar group described above, or a nucleic acid modified by the base group described above.

[0212] (X5) In this case, the number of inserted nucleotides may be 1, and if there are multiple nucleotides, it is preferably 2 or more, while preferably 6 or less, more preferably 3 or less.

[0213] (4) Mode (iv)

[0214] An additional embodiment of the non-natural polynucleotide of the present invention (embodiment (iv)) is,

[0215] (A) One or more nucleotides adjacent to the 5' upstream side of the mismatch nucleotide are cross-linked nucleic acids, and / or (B) One or more nucleotides adjacent to the 3' downstream side of the mismatch nucleotide are cross-linked nucleic acids, and

[0216] (D) The chain length is 22 to 95 nucleotides, and additionally

[0217] (H2) The phosphodiester bond portion between a nucleotide at the 5' end and one or more nucleotides adjacent to the 5' end nucleotide is substituted by a phosphate modification bond, (Y1) the nucleotide at the 5' end is a mismatch nucleotide, and one or more nucleotides adjacent to the 5' end nucleotide are also mismatch nucleotides, and (Y3) an adapter is attached to the 5' end. One or more selected from the group consisting of these.

[0218] This configuration is such that it can produce the effects of the present invention.

[0219] (A), (B), and (D) in mode (iv) are identical to mode (i).

[0220] Here, the mismatch nucleotide may be a cross-linked nucleic acid or not a cross-linked nucleic acid, but in embodiment (iv), it is more preferable that the mismatch nucleotide is not a cross-linked nucleic acid.

[0221] [Substitution into Phosphate Modification Combination]

[0222] In the above embodiment (iv), the non-natural polynucleotide of the present invention is preferably such that, for the sake of editing efficiency, the phosphodiester bond portion between the (H2) 5' end nucleotide and one or more nucleotides adjacent to the 5' end nucleotide is substituted with a phosphate group modification bond.

[0223] In the present invention, a phosphate modification bond is a bond in which some atoms of a phosphate diester bond are replaced with other atoms or substituents, for example, sulfur atoms, boron atoms, nitrogen atoms, methyl groups, or ester groups. Specific examples of phosphate modification bonds include phosphorothioate bonds, methylphosphate bonds, boranophosphate bonds, and mesylphosphomidate bonds.

[0224] In the non-natural polynucleotide of the present invention, the number or location of phosphate group modification bonds is not particularly limited. In addition, in the non-natural polynucleotide of the present invention, the type of phosphate group modification bond may be one type, or two or more types may be mixed.

[0225] The number of phosphate group modification bonds may be 1, and the ratio of phosphate group modification bonds in phosphate diester bonds may be 60% or less, but from the perspective of editing efficiency, it is preferable that not all phosphate diester bonds are substituted.

[0226] [Introduction of a mismatched nucleotide to the 5' end]

[0227] In the non-natural polynucleotide of the present invention, in the above embodiment (iv), from the perspective of editing efficiency, it is preferable that the nucleotide at the 5' end of (Y1) is a mismatch nucleotide, and that one or more nucleotides adjacent to the nucleotide at the 5' end are also mismatch nucleotides. The mismatch nucleotides in (Y1) are "mismatch nucleotides for the purpose of improving editing efficiency" and do not correspond to "mismatch nucleotides for altering the target nucleotide sequence." When there are multiple mismatch nucleotides in (Y1), the mismatch nucleotides may be adjacent to each other or not adjacent to each other. Furthermore, the number is preferably 2 or more, while 10 or fewer is preferable, and 7 or fewer is more preferable.

[0228] [Addition of adapter for 5' end]

[0229] In the above embodiment (iv), the non-natural polynucleotide of the present invention preferably has an adapter attached to the (Y3) 5' end for editing efficiency. The details of the adapter are as described above.

[0230] [RNA Substitution]

[0231] The non-natural polynucleotide of the present invention is a one-chain polynucleotide, but deoxyribose in one or more nucleotides may be substituted with ribose.

[0232] Specifically, in embodiment (iv), for example, if the pentose sugar in the nucleotide at the 5' end of (Y2) is ribose and the pentose sugar in one or more nucleotides adjacent to the nucleotide at the 5' end is ribose, the editing efficiency of the target nucleotide sequence can be further increased.

[0233] (5) Mode (v)

[0234] An additional embodiment (embodiment (v)) of the non-natural polynucleotide of the present invention is,

[0235] (A) One or more nucleotides adjacent to the 5' upstream side of the mismatch nucleotide are cross-linked nucleic acids, and / or (B) One or more nucleotides adjacent to the 3' downstream side of the mismatch nucleotide are cross-linked nucleic acids, and

[0236] (D) The chain length is 22 to 95 nucleotides, and

[0237] (X7) One or more nucleotides placed between the mismatched nucleotide and the nucleotide at the 5' end are cross-linked nucleic acids.

[0238] This configuration is such that it can produce the effects of the present invention.

[0239] (A), (B), and (D) in mode (v) are identical to mode (i).

[0240] Here, the mismatch nucleotide may be a cross-linked nucleic acid or not a cross-linked nucleic acid, but in embodiment (v), it is more preferable that the mismatch nucleotide is not a cross-linked nucleic acid.

[0241] In the above embodiment (v), the non-natural polynucleotide of the present invention is, for the sake of editing efficiency, (X7) one or more nucleotides disposed between the mismatch nucleotide and the nucleotide at the 5' end are cross-linked nucleic acids. The cross-linked nucleic acid specified in (X7) is not particularly limited, but at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA may be cited, and LNA is preferred.

[0242] (6) Mode (vi)

[0243] An additional embodiment of the non-natural polynucleotide of the present invention (embodiment (vi)) is,

[0244] (C) The nucleotide at the 5' end is a cross-linked nucleic acid, and

[0245] (D) The chain length is 22 to 95 nucleotides, and

[0246] (I) The phosphodiester bond portion between a 3'-terminal nucleotide and one or more nucleotides adjacent to the 3'-terminal nucleotide is substituted with a phosphorothioate bond, and

[0247] (X6) The above mismatched nucleotide is a cross-linked nucleic acid.

[0248] This configuration is such that it can produce the effects of the present invention.

[0249] (C), (D) and (I) in mode (vi) are identical to mode (i).

[0250] In the non-natural polynucleotide of the present invention, in aspect (vi), from the perspective of editing efficiency, (X6) the mismatch nucleotide is a cross-linked nucleic acid. The cross-linked nucleic acid specified in (X6) is not particularly limited, but at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA may be cited, and LNA is preferred.

[0251] [3' end cross-linked nucleic acid]

[0252] In the above embodiment (vi), the non-natural polynucleotide of the present invention can further increase the editing efficiency of the target nucleotide sequence if, for example, the nucleotide at the 3' end (E) is a cross-linked nucleic acid. The cross-linked nucleic acid specified in (E) is not particularly limited, but at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA may be cited, and LNA is preferred.

[0253] [3' terminal phosphorylation]

[0254] The non-natural polynucleotide of the present invention can further increase the editing efficiency of a target nucleotide sequence if, in the above embodiment (vi) or additionally in addition to (E), for example, the nucleotide at the (P) 3' end is phosphorylated.

[0255] <Editing Efficiency>

[0256] The efficiency of modifying a target nucleotide sequence by the non-natural polynucleotide of the present invention (referred to as "editing efficiency" in this specification) can be expressed relatively with respect to the editing efficiency of the non-natural polynucleotide represented by SEQ No. 9, GEO-8 of the example described below, as 1.0, and may be, for example, in the range of 0.4 or higher, preferably 0.5 or higher, and more preferably 0.6 or higher.

[0257] <Kit>

[0258] In the present invention, "kit" refers to a genetic engineering tool used for modifying a target nucleotide sequence containing a non-natural polynucleotide. The kit of the present invention may include, to the extent that it does not impede the effects of the present invention, a buffer, a stabilizer, a preservative, other reagents, an instruction manual describing a protocol for modifying a target nucleotide sequence using a non-natural polynucleotide.

[0259] <Pharmaceutical Composition>

[0260] By using a pharmaceutical composition containing a non-natural polynucleotide of the present invention (hereinafter referred to as the “pharmaceutical composition” of the present invention), a pharmaceutical composition capable of treating a target disease can be provided by modifying a gene having a mutation that does not function normally into a gene that functions normally through the action of the non-natural polynucleotide.

[0261] When using the pharmaceutical composition of the present invention, it may be administered, for example, orally, intravenously, intramuscularly, via the oral mucosa, rectum, vaginally, transdermally, via the nasal cavity, or via inhalation. Alternatively, an ex vivo treatment method may be used in which the pharmaceutical composition of the present invention is applied to cells extracted from a patient, the cells are cultured after treatment as needed, and then returned to the patient's body. The non-natural polynucleotide of the pharmaceutical composition of the present invention may be formulated alone or in combination with other components, but may also be provided in the form of a formulation by incorporating a pharmaceutically acceptable carrier or formulation additive therein. Pharmaceutically acceptable carriers or additives are not particularly limited but may include lipid nanoparticles, excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, solubilizers, solubilization aids, isotonic agents, pH adjusters, stabilizers, etc.

[0262] The administration of the pharmaceutical composition of the present invention depends on the severity and responsiveness of the condition being treated and is continued for several days to several months, or until a cure is achieved, or until a reduction in the condition is achieved. A person skilled in the art can determine the optimal dose, method of administration, and frequency of repetition.

[0263] <Diseases Targeted by the Pharmaceutical Composition>

[0264] Diseases to which the pharmaceutical composition of the present invention is applied include, for example, diseases caused by a one-base substitution in the human genome, such as adrenoleukodystrophy, heavy-chain acyl CoA dehydrogenase deficiency, Wilson's disease, hereditary pulmonary hypertension, X-chain agammaglobulinemia, cystinuria, Duchenne muscular dystrophy, hemophilia A, hemophilia B, tyrosinemia, hepatic glycogen storage disease type Ia, familial frontotemporal degeneration, mucopolysaccharidosis type II, mucopolysaccharidosis type I, primary immunodeficiency syndrome, familial hypertrophic cardiomyopathy, peroxisome dysplasia, hepatic glycogen storage disease type IX, protein C deficiency, hepatic glycogen storage disease type V, familial dilated cardiomyopathy, α1-antitrypsin deficiency, citrine deficiency, cystinuria, Niemann-Pick disease, and amyotrophic lateral sclerosis. It is possible.

[0265] <Preparation of Non-Natural Polynucleotides>

[0266] The non-natural polynucleotide in the present invention can be easily prepared by methods known in the field of the present invention.

[0267] <Introduction of Non-Natural Polynucleotides into Cells>

[0268] In the present invention, "introducing a non-natural polynucleotide into a cell" can be carried out according to known methods depending on the cell to be introduced. Known methods for introducing polynucleotides into cells (transfection methods) are broadly classified into two types: viral vector systems and non-viral vector systems. Viral vector systems are methods for introducing genes into a cell by utilizing the cell entry mechanism inherent to viruses, and are not particularly limited but use adenoviruses, retroviruses, lentiviruses, etc. as vectors. Non-viral vector systems are not particularly limited but include the lipofection method, electroporation method, microinjection method, particle gun method, etc. The lipofection method is a method that utilizes the phenomenon in which a positively charged cationic liposome binds to a negatively charged polynucleotide to form a complex, thereby introducing the polynucleotide from the cell surface into the cell through the phenomenon of endocytosis. The electroporation method is a method of introducing polynucleotides through small, empty pores on the cell surface by directly applying high-voltage pulses to the cell using a dedicated machine. The microinjection method is a method of introducing a substance into a glass needle with a tip diameter of about 1 μm by placing the substance to be introduced directly into the cell. The particle gun method is a method of coating the surface of gold particles with DNA by co-precipitating gold particles and DNA, and then ejecting the gold particles toward target cells using helium gas pressure or the like. The present invention is a method of modifying the target nucleotide sequence by introducing only non-natural polynucleotides without introducing Cas nuclease into the cell, and from the perspective of safety, a non-viral vector-based method is more preferable. Examples of the method of the present invention include a method performed in the human body or a method including a process performed in the human body.

[0269] The non-natural polynucleotide introduced into the cell in this manner (also referred to as "editing nucleic acid" in this specification) selectively binds to a target nucleotide sequence in genomic DNA, as schematically illustrated in FIG. 1. It is preferable that the non-natural polynucleotide of the present invention be designed such that it is mismatched with the base to be edited in the target nucleotide sequence of two-stranded DNA in the cell, such as genomic DNA, and that all base sequences other than the base to be edited are base sequences complementary to the target nucleotide sequence. However, to the extent that it does not contradict the purpose of the present invention, mismatched nucleotides other than the base to be edited may be present.

[0270] For example, if the base to be edited in the target nucleotide sequence is T and it is to be changed 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 to change the base of the base to be edited to a desired base based on common technical knowledge.

[0271] <Method for modifying one or more nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell>

[0272] The "method for modifying one or more nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell" of the present invention comprises a process of introducing the non-natural polynucleotide of the present invention described above into a cell. Here, the modification of the target nucleotide sequence comprises at least one selected from the group consisting of deletion, insertion, and substitution of one or more nucleotides of the target nucleotide sequence. Preferably, the non-natural polynucleotide described in embodiments (i) to (vi) above may be used as the modification method of the present invention.

[0273] <Method for Confirming Alteration of Target Nucleotide Sequence>

[0274] Methods for confirming changes in the target nucleotide sequence in the present invention include methods for measuring the activity of a gene containing the target nucleotide sequence, and methods for directly measuring the target nucleotide sequence using a next-generation sequencer (NGS) and digital PCR.

[0275] <Cell>

[0276] The method of modifying a target nucleotide sequence using a non-natural polynucleotide according to the present invention can be applied to all cells having two-stranded DNA. Furthermore, as long as the DNA replication mechanism within the cell can be utilized, the method is not limited to two-stranded DNA inherent in the cell, but may also modify virus-derived one-stranded DNA from a virus-infected cell as the target nucleotide sequence. In short, the term "cell" in the present invention includes prokaryotic cells and eukaryotic cells.

[0277] Prokaryotic cell

[0278] In the present invention, "prokaryotic cell" is not particularly limited, but may include bacterial cells and archaea.

[0279] Eukaryotic cells

[0280] In the present invention, "eukaryotic cell" is not particularly limited but may include animal cells, insect cells, plant cells, algae cells, and fungal cells.

[0281] Animal Cells

[0282] In the present invention, "animal cells" are not particularly limited but include cells of vertebrates, cells of invertebrates, cells derived from animal tissues, germ cells, somatic cells, and stem cells. Germ cells include oocytes and sperm cells. Somatic cells are not particularly limited but include fibroblasts, hematopoietic cells, neurons, muscle cells, osteocytes, liver cells, pancreatic cells, brain cells, kidney cells, etc. Stem cells are not particularly limited but include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).

[0283] <Mammalian Cells>

[0284] In the present invention, "mammal" refers to a group of vertebrates belonging to the mammal class, and humans are also included in mammals. "Mammalian cell" refers to a cell that constitutes a mammal or a cell derived from a mammal. Examples of mammals other than humans are not particularly limited but include deer, sea lions, hamsters, dogs, rats, wolves, whales, zebras, donkeys, weasels, bats, dolphins, anteaters, seals, cattle, wild boars, horses, squirrels, bears, cats, moles, monkeys, raccoons, kangaroos, pigs, foxes, sheep, etc.

[0285] Regarding human cells, as described above, cells derived from tissues, germ cells, somatic cells, and stem cells are included. In the embodiments of this specification, alterations to target nucleotide sequences were confirmed using HEK293 cells, but they may also be confirmed using other cells, such as HeLa cells.

[0286] <Insect Cells>

[0287] In the present invention, "insect" is a general term for arthropods classified as the class Insecta.

[0288] Insects used are not particularly limited, but include silkworm larvae, fruit flies, crickets, etc. In the present invention, "insect cells" are not particularly limited, but may include cells constituting the body tissues of insects and cells derived from insect tissues.

[0289] Plant Cells

[0290] In the present invention, the term "plant" is not specifically limited but may include seed plants, ferns, bryophytes, 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 are not specifically limited but may include morning glory, dandelion, azalea, rhododendron, eggplant, rapeseed, pea, etc. Monocotyledons are not specifically limited but may include rice, lily, tulip, Japanese pampas grass, corn, etc. Gymnosperms are not specifically limited but may include pine, cedar, ginkgo, cypress, etc. Ferns are not specifically limited but may include bracken, ostrich fern, kelp leaf grass, horsetail, etc. Bryophytes are not specifically limited but may include umbrella moss, horn moss, pine moss, water moss, etc. Algae include multicellular algae and unicellular algae. Examples of multicellular algae include, but are not specifically limited, kelp, wakame, green laver, and sea mustard. Examples of unicellular algae include, but are not specifically limited, Chlorella, Euglena, Spirulina, Chlamydomonas, Cocomixa, Botryococcus, Closterium, and diatoms.

[0291] Examples

[0292] The present invention will be described in detail below based on specific embodiments, but the present invention is not limited to these.

[0293] A. Genome editing experiment using 293-nLD1 cells (1)

[0294] 1. Preparation of the variant NanoLuc (registered trademark) plasmid

[0295] DNA in the region of base numbers 847 to 1,380 was synthesized based on the wild-type base sequence of the luciferase gene NanoLuc (GenBank JQ513379) (Gene Universal Inc.), XhoI and ApaI linkers were combined, and pcDNA TM It was inserted into the corresponding restriction enzyme site of the 5 / FRT / TO plasmid (Thermo Fisher Scientific Inc.) (pcDNA5-nLW1). Here, the wild-type nucleotide sequence of the NanoLuc gene is denoted as Sequence No. 1.

[0296] Meanwhile, a variant base sequence was synthesized in which cytosine (C) at base number 922 of the NanoLuc gene was changed to thymine (T), and pcDNA was prepared in the same manner as above. TM A variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD1). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation.

[0297] 2. Preparation of a stable transformed cell line containing the mutated NanoLuc gene

[0298] Flp-In, a human-derived cell line TM -293 cell line (Thermo Fisher Scientific Inc.) at 1 × 10⁻⁶ 6Cells were seeded into 6 cm dishes 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 a 5% CO2 environment. After 24 hours, pcDNA5-nLD1 or pcDNA5-nLW1 plasmid (1 μg) and pOG44 plasmid (3 μg, Thermo Fisher Scientific Inc.) were transfected using Lipofectamine 3000 (Thermo Fisher Scientific Inc.) according to standard protocol. After 48 hours, TrypLE TM Express Enzyme (Thermo Fisher Scientific Inc.) was added, and the cells were incubated at 37°C for 3 minutes. The exfoliated cells were recovered, suspended in DMEM + 10% FBS medium containing 50 μg / ml of hygromycin (Thermo Fisher Scientific Inc.), and cultured in two 10 cm dishes. Subsequently, culture was continued while changing the medium at 3-day intervals. After approximately 20 days of culture, the formation of colonies of sufficient number and size was confirmed, and the entire cell population was tested using TrypLE TM It was detached from the petri dish using Express Enzyme and recovered.

[0299] Cells in which the variant NanoLuc was inserted into the genome were named 293-nLD1 cells. The variant NanoLuc gene in 293-nLD1 cells is an inactive luciferase gene, as described above. These 293-nLD1 cells were used in subsequent genome editing experiments.

[0300] Meanwhile, cells into which wild-type NanoLuc was inserted were named 293-nLW1 and used as a positive control for subsequent genome editing experiments.

[0301] 3. Introduction of non-natural polynucleotides into 293-nLD1 cells

[0302] 293-nLD1 cells 1 × 10 4 96-well plate (Nunc (registered trademark) MicroWell) for cell formation TM 96, Nunclon Delta-Treated, Flat-Bottom Microplate, Thermo Fisher Scientific Inc.) was seeded and cultured in DMEM + 10% FBS medium at 37°C under a 5% CO2 environment. After 24 hours, 0.05 or 0.1 μg of non-natural polynucleotides were transfected with Lipofectamine 3000 according to a standard protocol.

[0303] 4. Measurement of Editing Efficiency

[0304] · NanoLuc Luciferase Assay

[0305] After continuing incubation for 72 hours from transfection, NanoLuc Luciferase activity was measured. NanoLuc Luciferase activity was measured according to a standard protocol using the Nano-Glo (registered trademark) Luciferase Assay System (Promega Corporation). An EnSpire multimode plate reader (PerkinElmer Co., Ltd.) was used to measure the luminescence of luciferase.

[0306] · Count of live cells

[0307] The number of live cells was measured according to a standard protocol using the CellTiter-Blue (registered trademark) Cell Viability Assay (Promega Corporation). An EnSpire multimode plate reader (PerkinElmer Co., Ltd.) was used to measure the fluorescence values ​​of CellTiter-Blue.

[0308] The number of live cells in the edited cells is 5 × 10 3 Up to 2 × 10 5 A calibration curve for the measured values ​​of CellTiter-Blue and the number of cells was constructed using a 2x dilution series of 293-nLD1 cells, and the number of live cells was calculated based on this calibration curve.

[0309] · Calculation of editing efficiency

[0310] As a result of measuring NanoLuc Luciferase activity in 293-nLW1 cells into which wild-type luciferase was inserted into the genome, the measured value was approximately 20,000 counts / cell. Therefore, when the mutation in the inactive luciferase gene is repaired and wild-type NanoLuc is synthesized, luciferase activity of 20,000 counts per cell is detected. Based on this value, the editing efficiency (%) is,

[0311] NanoLuc Luciferase Activity ÷ 20,000 ÷ Number of Cells × 100

[0312] It was calculated using the formula.

[0313] 5. Measurement of Editing Efficiency by Next Generation Sequencing (NGS)

[0314] Genomic DNA was prepared from 293-nLD1 cells transfected with non-natural polynucleotides using NucleoSpin Tissue (Takara Inc.) according to a standard protocol. For the prepared genomic DNA, a region of approximately 200 bases upstream to downstream of the bases to be edited was amplified by PCR to create a library, and amplicon sequencing was performed on this library 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 number of edited sequences to the number of wild-type sequences.

[0315] 6. Synthesis of Non-Natural Polynucleotides

[0316] All non-natural polynucleotides containing nucleic acid modifications were synthesized by Gene Design (Japan) and purified using a simple column or HPLC.

[0317] Example 1-1 Modification of target nucleotide sequence by non-natural polynucleotides

[0318] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD1 cells.

[0319] As previously mentioned, the codon (CAA) encoding glutamine (Gln-22) at position 22 of the luciferase gene introduced into 293-nLD1 cells is point-mutated into a stop codon (TAA) and does not exhibit luciferase activity as is. If the T of the stop codon (TAA) in the inactive form of the luciferase gene can be replaced with C, the luciferase activity of the 293-nLD1 cells is restored, and changes in the target nucleotide sequence can be detected by luminescence produced by luciferase (Fig. 2).

[0320] In Example 1-1, 11 types of non-natural polynucleotides, including GEO-1 to GEO-9, GEO-242, and GEO-11, and as a negative control, non-natural polynucleotide GEO-94, which cannot repair inactive mutations because no mismatch nucleotides are present, were each introduced into 293-nLD1 cells by the transfection method (Fig. 3a).

[0321] For 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. For GEO-1 to GEO-4, it is 21 nt, and for GEO-5 to GEO-9, GEO-242, GEO-11, and GEO-94, it is 25 nt. For GEO-1 and GEO-5, the mismatch nucleotide G is an LNA. For GEO-2 and GEO-6, one nucleotide on the 5' upstream and 3' downstream sides adjacent to the mismatch nucleotide G is an LNA. In GEO-3 and GEO-7, the mismatch nucleotide G and the nucleotide at the 5' end are LNAs. In GEO-4 and GEO-8, one nucleotide on the 5' upstream and 3' downstream sides adjacent to the mismatch nucleotide G is an LNA, and the nucleotide at the 5' end is also an LNA. In GEO-11, the nucleotide at the 5' end is an LNA. In GEO-242, the nucleotide at the 3' end is an LNA. GEO-9 does not have an LNA.

[0322] In GEO-94, the nucleotide at the position corresponding to the base to be edited is A, that is, complementary. Also, in GEO-94, one nucleotide on the 5' upstream and 3' downstream sides adjacent to this nucleotide A is LNA, and the nucleotide at the 5' end is also LNA.

[0323] The nucleotide sequences, chain lengths, and editing efficiencies (%) of each of the above-mentioned edited nucleic acids GEO-1 to GEO-9, GEO-242, GEO-11, and negative control (GEO-94) are as shown in Table 1. In addition, the editing efficiencies (%) of each edited nucleic acid are also shown as a bar graph (Fig. 3b).

[0324] [Table 1]

[0325]

[0326] In Table 1, guanosine monophosphate, the mismatch nucleotide, was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 1 were LNA.

[0327] As shown in Table 1, the absolute value of the editing efficiency was highest for GEO-8 at 0.25%, and second highest for GEO-7 at 0.05%. Additionally, the editing efficiency of the negative control, GEO-94, was approximately 0%. If the editing efficiency of GEO-8 is set to 1.0, the relative editing efficiencies of GEO-1 through GEO-6, GEO-9, GEO-242, and GEO-11 were all less than 0.2. Furthermore, GEO-1, GEO-2, GEO-3, GEO-5, GEO-6, and GEO-7 are identical to the composition disclosed in Non-Patent Literature 1. Thus, GEO-8, the non-natural polynucleotide of the present invention, exhibited an editing efficiency more than five times higher than that of the previously reported GEO-7.

[0328] In the subsequent Examples 1-2 to 1-13, the editing efficiency of GEO-8 was used as a standard, but if the relative editing efficiency with the editing efficiency of GEO-8 set to 1.0 is 0.4 or higher, it indicates a value sufficiently higher than the editing efficiency reported in the past.

[0329] Example 1-2 Chain length of non-natural polynucleotide

[0330] GEO-4 and GEO-8 of Example 1-1 showed a significant difference in editing efficiency due to a difference of 4 nt, even though the arrangement of LNAs was the same.

[0331] In Example 1-2, to investigate the preferred chain length of the non-natural polynucleotide, the same experiment as in Example 1-1 was conducted using nine types of non-natural polynucleotides with different chain lengths and identical GEO-8 and LNA batches (Fig. 4). The nine types of non-natural polynucleotides used in Example 1-2 are 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).

[0332] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 2.

[0333] [Table 2]

[0334]

[0335] In Table 2, guanosine monophosphate, the mismatch nucleotide, was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 2 were LNA.

[0336] As shown in Table 2, the relative editing efficiency of edited nucleic acids with chain lengths of 25 nt or more and 95 nt or less, including GEO-8 used as a control, was 0.4 or higher, and that of GEO-174 with 45 nt showed the highest relative editing efficiency at 3.74. Although GEO-281 and GEO-282, which have longer chain lengths, showed a tendency for their relative editing efficiency to decrease, it was found that they still showed high values ​​of 0.58 or higher.

[0337] Examples 1-3 Modification of the 3' end of non-natural polynucleotides with LNA

[0338] In Examples 1-3, the editing efficiency of a configuration in which a pentose sugar in a 3'-terminal nucleotide is substituted with LNA was investigated.

[0339] Specifically, editing efficiency was measured in the same manner as in Example 1-1 using each of GEO-16, in which the 3' terminal nucleotide of GEO-6 is LNA; and GEO-17, in which the 3' terminal nucleotide of GEO-8 is additionally LNA (Fig. 5).

[0340] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 3.

[0341] [Table 3]

[0342]

[0343] In Table 3, guanosine monophosphate, the mismatch nucleotide, was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 3 were LNA.

[0344] As shown in Table 3, the relative editing efficiency of GEO-16 was 0.16, and that of GEO-17 was 2.00. From these results, it was found that the configuration (GEO-16) in which one nucleotide on the 5' upstream and 3' downstream side adjacent to the mismatch nucleotide is LNA and the 3' end is LNA has low relative editing efficiency. In the configuration in which one nucleotide on the 5' upstream and 3' downstream side adjacent to the mismatch nucleotide is LNA and the 5' end is LNA (hereinafter referred to as the "configuration of GEO-8"), it was found that the relative editing efficiency improves if the 3' end is additionally LNA. On the other hand, it was found that even when the 3' end was LNA, when the 5' end was an unmodified configuration (GEO-16), the editing efficiency was the same as that of GEO-6 and did not exhibit sufficient editing efficiency. In this regard, it was shown that in order to exhibit sufficient editing efficiency, it is important that the nucleotide at the 5' end be a cross-linked nucleic acid such as LNA.

[0345] Examples 1-4 Modification of the LNA of one or more nucleotides adjacent to the LNA of the 5' end of a non-natural polynucleotide

[0346] In the present Example 1-4, the editing efficiency of a configuration in which one or more nucleotides adjacent to the 5' end are additionally substituted with LNA was investigated for the configuration of GEO-8 of Example 1-1 and the configuration of GEO-17 of Example 1-3.

[0347] Specifically, editing efficiency was measured in the same manner as in Example 1-1 using each of GEO-18, in which 1 nucleotide adjacent to the 5' end is additionally LNA in GEO-8; GEO-19, in which 2 consecutive nucleotides adjacent to the 5' end are additionally LNA in GEO-8; and GEO-68, in which 1 nucleotide adjacent to the 5' end is additionally LNA in GEO-17 (Fig. 6).

[0348] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 4.

[0349] [Table 4]

[0350]

[0351] In Table 4, guanosine monophosphate, the mismatch nucleotide, was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 4 were LNA.

[0352] As shown in Table 4, the relative editing efficiency of GEO-18 was 2.05, and that of GEO-19 was 0.66. From these results, it was found that in the configuration of GEO-8, sufficient editing efficiency is exhibited even if one or more nucleotides adjacent to the 5' end are additionally substituted with LNA. In addition, the relative editing efficiency of GEO-17 was 2.00, and that of GEO-68 was 1.64. From these results, it was found that in a configuration where one nucleotide on the 5' upstream and 3' downstream sides adjacent to the mismatch nucleotide is LNA, and the 5' end is LNA, and additionally the 3' end is LNA (hereinafter referred to as the "configuration of GEO-17"), sufficient editing efficiency is exhibited even if one nucleotide adjacent to the 5' end is additionally substituted with LNA.

[0353] In the configuration of GEO-8, the relative editing efficiency of GEO-19, in which two nucleotides adjacent to the 5' end were additionally substituted with LNA, was 0.66, which was slightly lower than the relative editing efficiency of the configuration of GEO-8. Meanwhile, in the following Examples 1-8, when the 5' upstream side of the mismatch nucleotide was extended, the relative editing efficiency of GEO-313, in which one nucleotide adjacent to the 5' end was additionally substituted with LNA in the configuration of GEO-8, was 5.23, and the relative editing efficiency of GEO-315, in which two nucleotides adjacent to the 5' end were additionally substituted with LNA in the configuration of GEO-8, was 6.59. It has been suggested that in the composition of GEO-8, even when multiple nucleotides adjacent to the 5' end are additionally substituted with LNA, editing efficiency may increase if other compositions are changed, such as by changing the chain length of the non-natural polynucleotide.

[0354] Examples 1-5 Modification of the LNA of one or more nucleotides adjacent to the LNA of the 3' end of a non-natural polynucleotide

[0355] In Examples 1-5, the editing efficiency of a configuration in which one or more nucleotides adjacent to the 3' end nucleotide are additionally substituted with LNA was investigated for the configuration of GEO-17 of Example 1-3.

[0356] Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using each of GEO-69, in which, in addition to GEO-17, one nucleotide adjacent to the 3' end nucleotide is LNA; and GEO-71, in which, in addition to GEO-17, two consecutive nucleotides adjacent to the 3' end nucleotide are LNA (Fig. 7).

[0357] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 5.

[0358] [Table 5]

[0359]

[0360] In Table 5, guanosine monophosphate, the mismatch nucleotide, was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 5 were LNA.

[0361] 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, regarding the configuration of GEO-17, if one nucleotide adjacent to the 3' end nucleotide is additionally substituted with LNA, it exhibits an editing efficiency similar to that of GEO-8, and if two nucleotides adjacent to the 3' end nucleotide are substituted with LNA, it exhibits an editing efficiency about half that of GEO-8.

[0362] Meanwhile, in Examples 1-8 described below, when the 3' downstream side of the mismatch nucleotide was extended, the relative editing efficiency of GEO-319, in which one nucleotide adjacent to the 3' end was additionally substituted with LNA in the configuration of GEO-17, was 2.18, and the relative editing efficiency of GEO-321, in which two nucleotides adjacent to the 3' end were additionally substituted with LNA in the configuration of GEO-17, was 1.40. It was suggested that even when multiple nucleotides adjacent to the 3' end are additionally substituted with LNA in the configuration of GEO-17, the editing efficiency may increase if other configurations are changed, such as by changing the chain length of the non-natural polynucleotide.

[0363] Example 1-6 Substitution of a phosphodiester bond portion between one or more nucleotides in a non-natural polynucleotide with a phosphorothioate bond (1)

[0364] In Examples 1-6, the editing efficiency of a configuration in which the phosphodiester bond portion between one or more nucleotides of the GEO-8 of Example 1-1 is additionally substituted with a phosphorothioate bond was investigated.

[0365] Specifically, GEO-158, GEO-159, GEO-160, GEO-29, and GEO-161 (Fig. 8a), wherein in GEO-8, the phosphodiester bond portion between the 5' end nucleotide and the consecutive 1 to 5 nucleotides adjacent to the 5' end nucleotide is further substituted with a phosphorothioate bond; GEO-162, GEO-163, GEO-164, GEO-30, GEO-165, GEO-272, and GEO-196 (Fig. 8b), wherein in GEO-8, the phosphodiester bond portion between the 3' end nucleotide and the consecutive 1 to 7 nucleotides adjacent to the 3' end nucleotide is further substituted with a phosphorothioate bond; GEO-31 (Fig. 8c), wherein in GEO-8, the phosphodiester bond portion between a 5'-terminal nucleotide and a consecutive 4 nucleotides adjacent to the 5'-terminal nucleotide is further substituted with a phosphorothioate bond, and the phosphodiester bond portion between a 3'-terminal nucleotide and a consecutive 4 nucleotides adjacent to the 3'-terminal nucleotide is also substituted with a phosphorothioate bond; GEO-126, GEO-127 (Fig. 8c), wherein in GEO-8, the phosphodiester bond portion between a 5'-upstream LNA adjacent to a mismatch nucleotide G and a consecutive 2 or 4 nucleotides adjacent to the LNA is further substituted with a phosphorothioate bond; In addition, regarding GEO-8, the phosphodiester bond portion between the nucleotide adjacent to the LNA on the 3' downstream side adjacent to the mismatch nucleotide G and the consecutive 2 or 4 nucleotides adjacent to the adjacent nucleotide was substituted with a phosphorothioate bond, respectively, GEO-128 and GEO-129 (Fig. 8c), and the editing efficiency was measured in the same manner as in Example 1-1.

[0366] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 6.

[0367] [Table 6]

[0368]

[0369] In Table 6, guanosine monophosphate, the mismatched nucleotide, was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 6 were LNA. At points where the phosphodiester bond between two or more nucleotides was substituted with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0370] As shown in Table 6, the relative editing efficiency of GEO-158 was 2.40, that of GEO-159 was 3.19, that of GEO-160 was 3.02, that of GEO-29 was 2.86, and that of GEO-161 was 2.27. From these results, it was shown that in the composition of GEO-8, the editing efficiency is improved when the phosphodiester bond portion between the 5' end nucleotide and the 1 to 5 nucleotides adjacent to the 5' end nucleotide is replaced with a phosphorothioate bond.

[0371] In addition, the relative editing efficiency of GEO-162 was 3.64, that of GEO-163 was 2.04, that of GEO-164 was 3.08, that of GEO-30 was 4.17, that of GEO-165 was 4.24, that of GEO-272 was 3.40, and that of GEO-196 was 3.97. From these results, it was shown that in the composition of GEO-8, the editing efficiency is improved when the phosphodiester bond portion between the 3' end nucleotide and the 1 to 7 nucleotides adjacent to the 3' end nucleotide is replaced with a phosphorothioate bond.

[0372] In addition, the relative editing efficiency of GEO-31 was 5.68. From this result, it was shown that in the composition of GEO-8, the editing efficiency is improved when the phosphodiester bond portions between the 5' end nucleotide and the 4 consecutive nucleotides adjacent to the 5' end nucleotide, and between the 3' end nucleotide and the 4 consecutive nucleotides adjacent to the 3' end nucleotide, are replaced with phosphorothioate bonds.

[0373] Also, 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 composition of GEO-8, if the phosphodiester bond portion between the LNA adjacent to the 5' upstream side of the mismatch nucleotide and the consecutive 2 or 4 nucleotides additionally adjacent to the LNA is substituted with a phosphorothioate bond, the editing efficiency is of the same degree as that of GEO-8.

[0374] Also, 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 composition of GEO-8, if the phosphodiester bond between two consecutive nucleotides adjacent to the 3' downstream side is substituted with a phosphorothioate bond by adding an LNA adjacent to the 3' downstream side of the mismatch nucleotide, it exhibits half the editing efficiency of GEO-8, and if the phosphodiester bond between four consecutive nucleotides adjacent to the 3' downstream side is substituted with a phosphorothioate bond by adding an LNA adjacent to the 3' downstream side of the mismatch nucleotide, the editing efficiency is improved. In addition, as shown in Table 6 and Figure 8c, in GEO-128 and GEO-129, the phosphodiester bond portion between the LNA adjacent to the 3' downstream side of the mismatch nucleotide and the nucleotide directly adjacent to the LNA is not substituted with a phosphorothioate bond.

[0375] Example 1-7 Substitution of a phosphodiester bond portion between one or more nucleotides in a non-natural polynucleotide with a phosphorothioate bond (2)

[0376] In Examples 1-7, the editing efficiency of a configuration in which one or more phosphodiester bond portions between nucleotides were additionally substituted with phosphothioate bonds was investigated for the configuration of GEO-17 of Example 1-3.

[0377] Specifically, GEO-155, GEO-167, GEO-168, and GEO-169 were used in which the phosphodiester bond portion between the 3' end nucleotide and the 1 to 4 consecutive nucleotides adjacent to the 3' end nucleotide was additionally substituted with a phosphorothioate bond in GEO-17, and editing efficiency was measured in the same manner as in Example 1-1 (Fig. 9).

[0378] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 7.

[0379] [Table 7]

[0380]

[0381] In Table 7, guanosine monophosphate, the mismatched nucleotide, was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 7 were LNA. At points where the phosphodiester bond between two or more nucleotides was substituted with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0382] As shown in Table 7, the relative editing efficiency of GEO-155 was 3.63, that of GEO-167 was 6.05, that of GEO-168 was 5.81, and that of GEO-169 was 2.90. From these results, it was shown that in the composition of GEO-17, the editing efficiency is improved when the phosphodiester bond portion between the 3' end nucleotide and the 1 to 4 nucleotides adjacent to the 3' end nucleotide is replaced with a phosphorothioate bond.

[0383] Examples 1-8 Chain length elongation at the 5' upstream and 3' downstream sides of mismatched nucleotides and the position of LNA

[0384] In Examples 1-8, the effect on editing efficiency was investigated when the chain length of the 5' upstream region and the chain length of the 3' downstream region of the mismatched nucleotide were changed in the case where there is one mismatched nucleotide included in the non-natural polynucleotide. Furthermore, the editing efficiency was investigated when the number and location of LNAs near the 5' end and the number and location of LNAs near the 3' end were appropriately changed.

[0385] Specifically, GEO-25, with a chain length of 41 nt, a 5' upstream region of mismatch nucleotide G of 20 nt and a 3' downstream region of 20 nt, one nucleotide on the 5' upstream and 3' downstream sides adjacent to the mismatch nucleotide G of LNA, and a nucleotide at the 5' end of LNA; GEO-311, with a chain length of 38 nt, a 5' upstream region of mismatch nucleotide G of 25 nt and a 3' downstream region of 12 nt, one nucleotide on the 5' upstream and 3' downstream sides adjacent to the mismatch nucleotide G of LNA, and a nucleotide at the 5' end of LNA; GEO-312, wherein in GEO-311, the 12th nucleotide towards the 5' upstream side, counting from the nucleotide 5' upstream adjacent to the mismatch nucleotide G, is an LNA; GEO-313, wherein in GEO-311, the 1 nucleotide adjacent to the 5' end LNA is an LNA; GEO-314, wherein in GEO-311, the 1 nucleotide adjacent to the 5' end LNA is an LNA, and the 12th nucleotide towards the 5' upstream side, counting from the nucleotide 5' upstream adjacent to the mismatch nucleotide G, is an LNA; GEO-315, wherein in GEO-311, the 2 consecutive nucleotides adjacent to the 5' end LNA are LNAs; And, using each of GEO-316, in which the two consecutive nucleotides adjacent to the LNA at the 5' end are LNA, and the 12th nucleotide towards the 5' upstream side, counting from the nucleotide adjacent to the mismatch nucleotide G, is LNA, the editing efficiency was measured in the same manner as in Example 1-1 (Fig. 10a).

[0386] More specifically, GEO-317, wherein the chain length is 40 nt, the 5' upstream region of the mismatch nucleotide G is 12 nt and the 3' downstream region is 27 nt, the 1 nucleotide on the 5' upstream and 3' downstream side adjacent to the mismatch nucleotide G is LNA, and the nucleotides at the 5' end and 3' end are LNA; GEO-318, wherein in GEO-317, the 12th nucleotide towards the 3' downstream side, counting from the nucleotide on the 3' downstream side adjacent to the mismatch nucleotide G, is LNA; and GEO-319, wherein in GEO-317, the 1 nucleotide adjacent to the 3' end LNA is LNA; Editing efficiency was measured in the same manner as in Example 1-1 using each of GEO-320, in which the 1 nucleotide adjacent to the 3' end LNA in GEO-317 is LNA and the 12th nucleotide towards the 3' downstream side, counted from the nucleotide adjacent to the mismatch nucleotide G in GEO-317; GEO-321, in which the 2 consecutive nucleotides adjacent to the 3' end LNA in GEO-317 are LNA; and GEO-322, in which the 2 consecutive nucleotides adjacent to the 3' end LNA in GEO-317 are LNA and the 12th nucleotide towards the 3' downstream side, counted from the nucleotide adjacent to the mismatch nucleotide G in GEO-317 (Fig. 10b).

[0387] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 8.

[0388] [Table 8]

[0389]

[0390] In Table 8, the mismatched nucleotide guanosine monophosphate was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 8 were LNA.

[0391] 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.

[0392] From a comparison of GEO-8 and GEO-311, it was found that the editing efficiency increases when the chain length of the 5' upstream region of the mismatch nucleotide is extended. When comparing GEO-313 with GEO-18 of Examples 1-4, and GEO-315 with GEO-19 of Examples 1-4, it was found that the editing efficiency increases when the chain length of the 5' upstream region of the mismatch nucleotide is extended, even when multiple nucleotides adjacent to the 5' terminal nucleotide are LNAs.

[0393] When comparing GEO-311 and GEO-312, GEO-313 and GEO-314, and GEO-315 and GEO-316, it was found that the editing efficiency increased further when the 12th nucleotide towards the 5' upstream side, starting from the nucleotide 5' upstream adjacent to the mismatch nucleotide, was LNA.

[0394] Compared to GEO-311, GEO-312, GEO-313, GEO-314, GEO-315, and GEO-316, which extended the chain length of the 5' upstream region of the mismatch nucleotide, GEO-317, GEO-318, GEO-319, GEO-320, GEO-321, and GEO-322, which extended the chain length of the 3' downstream region of the mismatch nucleotide, showed relatively low editing efficiency, suggesting that extending the chain length of the 5' upstream region of the mismatch nucleotide is effective.

[0395] As a result of comparing GEO-317 and GEO-318, and GEO-319 and GEO-320, there was no significant difference in editing efficiency, suggesting that when the chain length of the 3' downstream region of the mismatch nucleotide is extended, the effect on editing efficiency is small even if the 12th nucleotide towards the 3' downstream side, starting from the nucleotide adjacent to the mismatch nucleotide G, is substituted with LNA.

[0396] Examples 1-9 RNA Substitution of Nucleotides Near the 3' End

[0397] In Examples 1-9, the editing efficiency of a configuration in which one or more nucleotides adjacent to the 3' end nucleotide are additionally RNA substituted with respect to the configuration of GEO-17 of Example 1-3 was investigated.

[0398] Specifically, in GEO-17, editing efficiency was measured in the same manner as in Example 1-1 using each of GEO-150, in which 1 nucleotide adjacent to the 3' end nucleotide was RNA substituted; GEO-73, in which 2 consecutive nucleotides adjacent to the 3' end nucleotide were RNA substituted; GEO-151, in which 3 consecutive nucleotides adjacent to the 3' end nucleotide were RNA substituted; and GEO-74, in which 4 consecutive nucleotides adjacent to the 3' end nucleotide were RNA substituted (Fig. 11).

[0399] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 9.

[0400] [Table 9]

[0401]

[0402] In Table 9, the mismatched nucleotide guanosine monophosphate was underlined, and the nucleotide substituted with cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 9 were LNA. For the RNA-substituted nucleotides, an arrow was placed next to the base symbol of the corresponding nucleotide.

[0403] As shown in Table 9, the relative editing efficiency of GEO-150 was 1.65, that of GEO-73 was 4.20, that of GEO-151 was 0.78, and that of GEO-74 was 1.12. From these results, it was found that in the composition of GEO-17, if 1 or 2 nucleotides adjacent to the 3' end nucleotide are RNA substituted, the editing efficiency is improved compared to GEO-8, and if 3 or 4 nucleotides are RNA substituted, the editing efficiency is the same as that of GEO-8.

[0404] Examples 1-10 Phosphorylation of nucleotides at the 3' and / or 5' ends

[0405] In the present Example 1-10, the editing efficiency of a configuration in which the nucleotides at the 3' end and / or 5' end were additionally phosphorylated was investigated for the configuration of GEO-8 of Example 1-1 or GEO-17 of Example 1-3.

[0406] Specifically, editing efficiency was measured in the same manner as in Example 1-1 using each of GEO-26, in which the nucleotide at the 5' end of GEO-8 is phosphorylated; GEO-27, in which the nucleotide at the 3' end is phosphorylated; GEO-28, in which both the nucleotides at the 5' end and the 3' end are phosphorylated; GEO-223, in which the nucleotide at the 3' end of GEO-17 is phosphorylated; and GEO-224, in which the phosphodiester bond portion between the nucleotide at the 3' end and the adjacent nucleotide is substituted with a phosphorothioate bond and the nucleotide at the 3' end is phosphorylated (Fig. 12).

[0407] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 10.

[0408] [Table 10]

[0409]

[0410] In Table 10, the mismatched nucleotide guanosine monophosphate was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 10 were LNA. At points 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. Additionally, to indicate that the 5' end and / or 3' end was phosphorylated, "p" was attached to the 5' end and / or 3' end.

[0411] As shown in Table 10, the relative editing efficiency of GEO-26 was 1.14, that of GEO-27 was 2.23, and that of GEO-28 was 2.91; the relative editing efficiency of GEO-223 was 2.70, and that of GEO-224 was 2.29. From these results, it was shown that in the configuration of GEO-8, phosphorylating only the 5' end did not affect editing efficiency, but phosphorylating the 3' end improved editing efficiency. Furthermore, when both the 5' and 3' ends were phosphorylated, a higher editing efficiency was observed than when only the 3' end was phosphorylated. Similarly, in the configuration of GEO-17, it was shown that phosphorylating the 3' end improved editing efficiency. In this case, even if the phosphodiester bond portion between the 3' terminal nucleotide and the adjacent nucleotide is replaced with a phosphorothioate bond, no increase in editing efficiency was confirmed.

[0412] Examples 1-11 Types of cross-linked nucleic acids

[0413] In the present Example 1-11, the editing efficiency of a configuration in which the 5' end LNA of the GEO-8 of Example 1-1 was substituted with another cross-linked nucleic acid was investigated.

[0414] Specifically, in GEO-8, editing efficiency was measured in the same manner as in Example 1-1 using each of GEO-41, in which the 5' end LNA was substituted with BNA-NH; GEO-43, in which the 5' end LNA was substituted with BNA-N-Me; and GEO-57, in which the 5' end LNA was substituted with ENA (Fig. 13).

[0415] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 11.

[0416] [Table 11]

[0417]

[0418] In Table 11, guanosine monophosphate, which is a mismatch nucleotide, is underlined, and the nucleotide substituted with a cross-linked nucleic acid is indicated in uppercase. The types of cross-linked nucleic acids among each edited nucleic acid in Table 11 are as described above.

[0419] As shown in Table 11, the relative editing efficiency of GEO-41 was 1.38, that of GEO-43 was 1.51, and that of GEO-57 was 1.57. From these results, it was shown that in the composition of GEO-8, the 5' end can be substituted with other cross-linked nucleic acids such as BNA-NH, BNA-N-Me, and ENA, in addition to LNA, and that the editing efficiency may actually increase upon substitution.

[0420] Examples 1-12 Combinations of nucleic acid formulas (1)

[0421] In the present Example 1-12, the editing efficiency of a configuration in which the substitution of the phosphodiester bond portion to a phosphorothioate bond and phosphorylation of the 3' end were additionally combined with the configuration of GEO-8 of Example 1-1 was investigated.

[0422] Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-226, in which the phosphodiester bond portion between consecutive 5 nucleotides adjacent to the 3' end nucleotide in GEO-8 is substituted with a phosphorothioate bond and the 3' end is phosphorylated (Fig. 14).

[0423] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 12.

[0424] [Table 12]

[0425]

[0426] In Table 12, guanosine monophosphate, the mismatched nucleotide, was underlined, and the nucleotide substituted with the cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 12 were LNA. At points 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. Additionally, "p" was attached to the 3' end to indicate that the 3' end was phosphorylated.

[0427] As shown in Table 12, the relative editing efficiency of GEO-226 was 8.19. From this result, it was shown that in the composition of GEO-8, the editing efficiency increases significantly when the substitution of the phosphodiester bond portion with a phosphorothioate bond and the phosphorylation of the 3' end are additionally combined.

[0428] Examples 1-13 Combinations of nucleic acid formulas (2)

[0429] In the present Example 1-13, the editing efficiency of a configuration in which the phosphodiester binding portion of the GEO-23 of Example 1-3 was further combined with substitution of the phosphodiester binding portion with a phosphorothioate binding, RNA substitution, and phosphorylation of the 3' end was investigated.

[0430] Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-172, in which the phosphodiester bond portion between four consecutive nucleotides adjacent to the 5' end nucleotide in GEO-23 was substituted with a phosphorothioate bond, two consecutive nucleotides adjacent to the 3' end nucleotide were RNA substituted, and the 3' end was phosphorylated (Fig. 15).

[0431] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 13.

[0432] [Table 13]

[0433]

[0434] In Table 13, guanosine monophosphate, the mismatched nucleotide, was underlined, and the nucleotide substituted with cross-linked nucleic acid was indicated in uppercase. All cross-linked nucleic acids in Table 13 were LNA. At points 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. For RNA-substituted nucleotides, an arrow was attached to the base symbol of the corresponding nucleotide. Additionally, "p" was attached to the 3' end to indicate that the 3' end was phosphorylated.

[0435] As shown in Table 13, the relative editing efficiency of GEO-172 was 8.48. From this result, it was shown that in the composition of GEO-17, the editing efficiency increased significantly when the substitution of the phosphodiester binding portion with a phosphorothioate binding portion, RNA substitution, and phosphorylation of the 3' end were additionally combined.

[0436] Discussion

[0437] Examples 1-1 to 1-13 of the present invention above are compared with disclosures in patent and non-patent literature.

[0438] (1) From Example 1-1, it was found that GEO-8, a non-natural polynucleotide of the present invention, exhibited a higher editing efficiency of more than 5 times compared to GEO-7 (i.e., the chain length, the position of the mismatch nucleotide, and the position of the LNA are the same as GEO-8), which is the polynucleotide with the highest editing efficiency among the compositions disclosed in Non-Patent Literature 1. When comparing GEO-8 and GEO-7, GEO-8 of the present invention differs in that the mismatch nucleotide itself is not an LNA, and both the nucleotide on the 5' side and the nucleotide on the 3' side adjacent to the mismatch nucleotide are substituted with an LNA.

[0439] Regarding this point, Patent Document 4 states that while an oligonucleotide containing an LNA adjacent to a mismatch nucleotide did not show improvement, an oligonucleotide having an LNA spaced at an additional interval of 1 nucleotide from the mismatch showed an average increase of 5 times, so it can be said that Patent Document 4 actively rejects configurations containing an LNA adjacent to a mismatch nucleotide.

[0440] Non-patent literature 1 focuses on avoiding mismatch repair, and Non-patent literatures 1 and 2 focus on elucidating the mechanism of how one-strand synthetic DNA, in which the mismatch nucleotide is LNA, carries out genome modification in mammalian cells. Among the total of 60 or more types of one-strand synthetic DNA attempted in Non-patent literature 1, there is only one type in which both the 5' side nucleotide and the 3' side nucleotide adjacent to the mismatch nucleotide are substituted with LNA, and among the total of 50 or more types of one-strand DNA attempted in Non-patent literature 2, all of them have the mismatch nucleotide itself as LNA, and no attempt has been made in which both the 5' side nucleotide and the 3' side nucleotide adjacent to the mismatch nucleotide are substituted with LNA. In addition, the review of Non-Patent Literature 1 emphasizes that making the mismatch nucleotide itself an LNA is important for avoiding mismatch repair, but it does not mention that both the 5' side nucleotide and the 3' side nucleotide adjacent to the mismatch nucleotide are substituted with an LNA.

[0441] Therefore, the descriptions in Patent Document 4 and Non-Patent Documents 1 and 2 can be considered a hindering factor in the motivation to adopt the feature (A) and feature (B) of the present invention, which is the feature in which both the 5' side nucleotide and the 3' side nucleotide adjacent to the mismatch nucleotide are substituted with LNA in order to improve editing efficiency. Furthermore, the experimental results shown in Example 1-1 of this specification are the exact opposite of the indications in Patent Document 4, and in terms of the effects of the invention, the effects of the present invention can be considered heterogeneous effects that cannot be predicted from Patent Document 4 or Non-Patent Documents 1 and 2.

[0442] Among a total of more than 110 types of single-strand synthetic DNA attempted in non-patent literature 1 and 2, there was only one type in each literature of single-strand synthetic DNA in which the nucleotide at the 5' end is LNA and the nucleotide at the 3' end is not LNA. Although single-strand synthetic DNA in which two nucleotides—the nucleotide at the 5' end and the mismatch nucleotide—are LNA showed approximately twice the editing efficiency compared to single-strand synthetic DNA in which only the mismatch nucleotide is LNA, no further investigation was conducted on single-strand synthetic DNA in which the 5' end is LNA.

[0443] Non-patent document 2 concludes that the 5' side region of the mismatch nucleotide is degraded by endonucleases rather than exonucleases, and therefore suggests that the effect of protecting the 5' end from exonucleases by modifying the 5' end with LNA is limited. In fact, since the editing efficiency is only doubled by the LNA modification of the 5' end, it is derived that the LNA modification of the 5' end is not that important. Therefore, a combination of LNA modification of the 5' end and the substitution of both the 5' side nucleotide and the 3' side nucleotide adjacent to the mismatch nucleotide with LNA has not been attempted.

[0444] Therefore, the descriptions in non-patent literatures 1 and 2 can be considered an inhibitory factor to attempts to substitute the 5' end of a non-natural polynucleotide with LNA in order to improve editing efficiency.

[0445] (2) From Examples 1-2, it was found that if the chain length of the non-natural polynucleotide is 25 nt or more and 95 nt or less, an editing efficiency of a certain degree or higher is confirmed.

[0446] Regarding chain length, there have been many studies in the past stating that a length of about 25 bases is preferable and that the effect decreases as the chain length increases, whereas the present invention differs from existing research in that the effect is high even with a long chain length. Specifically, Non-Patent Document 1 examines the optimal length of an edited nucleic acid in a polynucleotide in which only mismatched nucleotides are used as LNAs, and as a result, it is shown that a length of 25 bases is optimal and that the editing efficiency decreases when it is longer than 25 bases. In addition, Patent Document 4 uses a polynucleotide of 24 bases in length.

[0447] Meanwhile, in the non-natural polynucleotides of the present invention, it has been found that editing efficiency is further increased when they are longer than 25 bases.

[0448] (3) From Examples 1-3, it was found that for the effect of having both the 5' side nucleotide and the 3' side nucleotide adjacent to the mismatch nucleotide substituted with LNA to be effective, the characteristic (C) that the nucleotide at the 5' end is a cross-linked nucleic acid is important. In addition, from comparison with GEO-8, GEO-16, and GEO-17, it was found that when both the 5' end and the 3' end nucleotides are cross-linked nucleic acids, the editing efficiency increased twofold. In addition, it was found that the effect of the nucleotide at the 3' end being an LNA is strongly manifested when the nucleotide at the 5' end is an LNA.

[0449] (4) For Examples 1-4 and 1-5, sufficient editing efficiency was shown even when one or more nucleotides adjacent to the 5' end or 3' end were substituted with LNA, and in some cases, editing efficiency was improved depending on the position and number of nucleotides substituted with LNA. Such effects were not described or suggested in any of the above patent or non-patent literature.

[0450] (5) From Examples 1-6 and 1-7, it was shown that in cases where both the 5' side nucleotide and the 3' side nucleotide adjacent to the mismatch nucleotide are substituted with LNA, the phosphodiester bond portion between the nucleotide at the 5' end or 3' end and the adjacent nucleotide 1 to 7 is substituted with a phosphothioate bond, thereby improving editing efficiency. Since editing efficiency does not decrease even when the number of phosphothioate bonds is increased by one on both the 5' end and 3' end sides, it is derived that sufficient editing efficiency is likely to be achieved even if the number of phosphodiester bonds is 8 or more. Such an effect was not described or suggested in any of the aforementioned patent or non-patent literature. In addition, when the 5' end or the 3' end is LNA, the S-transformation from both ends particularly strongly enhances the editing effect, which is a completely new discovery disclosed by the inventors in this specification.

[0451] (6) From Examples 1-8, it was shown that the editing efficiency increases when the chain length of the 5' upstream and 3' downstream regions of the mismatched nucleotides is extended, and also that the editing efficiency increases when several nucleotides in the extended regions are replaced with LNA. These effects were not described or suggested in any of the aforementioned patent or non-patent literature.

[0452] (7) From Examples 1-9, 1-10, 1-12 and 1-13, in addition to substituting with cross-linked nucleic acids, the editing efficiency was significantly increased by combining the formulas of substitution of the phosphodiester binding portion to a phosphorothioate binding portion, RNA substitution, 3' end phosphorylation, or 5' end phosphorylation. This effect was not described or suggested in any of the above patent or non-patent literatures.

[0453] (8) From Examples 1-11, regarding the substitution of the 5' end with a cross-linked nucleic acid, it was shown that the editing efficiency was improved when the substitution was made with other cross-linked nucleic acids such as BNA-NH, BNA-N-Me, and ENA, in addition to LNA. This effect was not described or suggested in any of the above patent or non-patent literatures.

[0454] 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

[0455] 1. Preparation of various variant NanoLuc (registered trademark) plasmids

[0456] (1) nDL2

[0457] Synthesized a variant base sequence in which guanine (G) at base number 894 of the NanoLuc gene was changed to adenine (A) and cytosine (C) at base number 895 was changed to thymine (T), and pcDNA was prepared in the same manner as above. TM A variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD2). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the mutation.

[0458] (2) nDL3

[0459] Synthesized a variant nucleotide sequence in which thymine (T) at base number 956 of the NanoLuc gene was changed to adenine (A), thymine (T) at base number 957 to guanine (G), and cytosine (C) at base number 958 to thymine (T), and pcDNA in the same manner as above TM A variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD3). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the mutation.

[0460] (3) nDL4-1

[0461] Synthesized a variant base sequence in which cytosine (C) at base number 922 of the NanoLuc gene was changed to thymine (T) and guanine (G) at base number 931 was changed to thymine (T), and pcDNA was prepared in the same manner as above. TM A variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD4-1). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the mutation.

[0462] (4) nDL4-2

[0463] Synthesized a variant nucleotide sequence in which cytosine (C) at base number 922 of the NanoLuc gene was changed to thymine (T) and guanine (G) at base number 937 was changed to thymine (T), and pcDNA was prepared in the same manner as above. TM A variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD4-2). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation.

[0464] (5) nDL4-3

[0465] Synthesized a variant nucleotide sequence in which guanine (G) at base number 937 of the NanoLuc gene was changed to thymine (T) and cytosine (C) at base number 958 was changed to thymine (T), and pcDNA was prepared in the same manner as above. TM A variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD4-3). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation.

[0466] (6) nDL5

[0467] Synthesized a variant nucleotide sequence in which guanine (G) at base number 976 of the NanoLuc gene is deleted, and pcDNA in the same manner as above. TMA variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD5). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation.

[0468] (7) nDL6

[0469] A variant nucleotide sequence was synthesized in which adenine (A) at base number 911 and cytosine (C) at base number 912 of the NanoLuc gene were deleted, and pcDNA was prepared in the same manner as above. TM A variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD6). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation.

[0470] (8) nDL7

[0471] Synthesized a variant nucleotide sequence in which thymine (T) is inserted between thymine (T) at base number 930 and guanine (G) at base number 931 of the NanoLuc gene, and pcDNA in the same manner as above TM A variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD7). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation.

[0472] (9) nDL8

[0473] Synthesize a variant nucleotide sequence in which thymine (T) and adenine (A) are 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 pcDNA in the same manner as above TMA variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD8). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation.

[0474] (10) nDL9

[0475] A variant nucleotide sequence was synthesized by deleting thymine (T) at base number 981, cytosine (C) at base number 982, cytosine (C) at base number 983, and guanine (G) at base number 984 of the NanoLuc gene, and pcDNA was prepared in the same manner as above. TM A variant inserted into the 5 / FRT / TO plasmid was prepared (pcDNA5-nLD9). The luciferase gene in this variant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation.

[0476] 2. Preparation of stable transformed cell lines containing various mutated NanoLuc genes

[0477] Flp-In, a human-derived cell line TM -293 cell line (Thermo Fisher Scientific Inc.) at 1 × 10⁻⁶ 6Cells were seeded into 6 cm dishes 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 a 5% CO2 environment. After 24 hours, pcDNA5-nLD2, pcDNA5-nLD3, pcDNA5-nLD4-1, pcDNA5-nLD4-2, pcDNA5-nLD4-3, pcDNA5-nLD5, pcDNA5-nLD6, pcDNA5-nLD7, pcDNA5-nLD8, or pcDNA5-nLD9 plasmids (1 μg) and pOG44 plasmid (3 μg, Thermo Fisher Scientific Inc.) were transfected using Lipofectamine 3000 (Thermo Fisher Scientific Inc.) according to standard protocol. After 48 hours, TrypLE TM Express Enzyme (Thermo Fisher Scientific Inc.) was added, and the cells were incubated at 37°C for 3 minutes. The exfoliated cells were recovered, suspended in DMEM + 10% FBS medium containing 50 μg / ml of hygromycin (Thermo Fisher Scientific Inc.), and cultured in two 10 cm dishes. Subsequently, culture was continued while changing the medium at 3-day intervals. After approximately 20 days of culture, the formation of colonies of sufficient number and size was confirmed, and the entire cell population was tested using TrypLE TM It was detached from the petri dish using Express Enzyme and recovered.

[0478] Each cell line obtained using the above various plasmids was named as follows.

[0479] Cells into which pcDNA5-nLD2 was inserted into the genome were named 293-nLD2 cells. Cells into which pcDNA5-nLD3 was inserted into the genome were named 293-nLD3 cells. Cells into which pcDNA5-nLD4-1 was inserted into the genome were named 293-nLD4-1 cells. Cells into which pcDNA5-nLD4-2 was inserted into the genome were named 293-nLD4-2 cells. Cells into which pcDNA5-nLD4-3 was inserted into the genome were named 293-nLD4-3 cells. Cells into which pcDNA5-nLD5 was inserted into the genome were named 293-nLD5 cells. Cells into which pcDNA5-nLD6 was inserted into the genome were named 293-nLD6 cells. Cells into which pcDNA5-nLD7 was inserted into the genome were named 293-nLD7 cells. Cells into which pcDNA5-nLD8 was inserted into the genome were named 293-nLD8 cells. Cells into which pcDNA5-nLD9 was inserted into the genome were named 293-nLD9 cells.

[0480] The variant NanoLuc gene possessed by each of the above cells is an inactive luciferase gene, as described above. Each of the above cells was used in subsequent genome editing experiments.

[0481] 3. Introduction of non-natural polynucleotides into 293-nLD2 ~ 9 cells

[0482] 293-nLD2 cells 1 × 10 4 96-well plate (Nunc (registered trademark) MicroWell) for cell formation TM96, Nunclon Delta-Treated, Flat-Bottom Microplate, Thermo Fisher Scientific Inc.) was sown and cultured in DMEM + 10% FBS medium at 37°C under a 5% CO2 environment. After 24 hours, non-natural polynucleotides at concentrations of 0.0125 μg, 0.025 μg, 0.050 μg, 0.100 μg, and 0.200 μg were transfected with Lipofectamine 3000 according to a standard protocol.

[0483] 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 way as 293-nLD2 cells.

[0484] 4. Measurement of Editing Efficiency

[0485] · NanoLuc Luciferase Assay

[0486] After continuing incubation for 72 hours from transfection, NanoLuc Luciferase activity was measured. NanoLuc Luciferase activity was measured according to a standard protocol using the Nano-Glo (registered trademark) Luciferase Assay System (Promega Corporation). An EnSpire multimode plate reader (PerkinElmer Co., Ltd.) was used to measure the luminescence of luciferase.

[0487] · Count of live cells

[0488] The number of live cells was measured according to a standard protocol using the CellTiter-Blue (registered trademark) Cell Viability Assay (Promega Corporation). An EnSpire multimode plate reader (PerkinElmer Co., Ltd.) was used to measure the fluorescence values ​​of CellTiter-Blue.

[0489] The number of live cells in the edited cells is 5 × 10 3 Up to 2 × 10 5 A calibration curve for the measured values ​​of CellTiter-Blue and the number of cells was constructed using a 2x dilution series of 293-nLD2 cells, and the number of live cells was calculated based on this calibration curve.

[0490] The number of live cells was calculated for 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 using the same method as for 293-nLD2 cells.

[0491] · Calculation of editing efficiency

[0492] As a result of measuring NanoLuc Luciferase activity in 293-nLW1 cells into which wild-type luciferase was inserted into the genome, the measured value was approximately 20,000 counts / cell. Therefore, when the mutation in the inactive luciferase gene is repaired and wild-type NanoLuc is synthesized, luciferase activity of 20,000 counts per cell is detected. Based on this value, the editing efficiency (%) is,

[0493] NanoLuc Luciferase Activity ÷ 20,000 ÷ Number of Cells × 100

[0494] It was calculated using the formula.

[0495] 5. Evaluation of editing efficiency of non-natural polynucleotides by AUC log

[0496] The editing efficiency of each non-natural polynucleotide was evaluated as follows.

[0497] NanoLuc Luciferase activity was measured at 5 concentrations of non-natural polynucleotides (0.0125 μg, 0.025 μg, 0.050 μg, 0.100 μg, 0.200 μg), and a dose-response curve was constructed with the axis representing the concentration of non-natural polynucleotides (X-axis) as the logarithm. From the constructed dose-response curve, the value of the area under the curve (hereinafter referred to as "AUC") with respect to the X-axis (hereinafter referred to as "AUC log") was calculated. A Prism (GraphPad) was used to calculate AUC log.

[0498] 6. Synthesis of Non-Natural Polynucleotides

[0499] All non-natural polynucleotides containing nucleic acid modifications were synthesized by Gene Design (Japan) and purified using a simple column or HPLC.

[0500] Example 2-1 Examination of 2 base substitutions (1)

[0501] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD2 cells.

[0502] 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.

[0503] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 14.

[0504] [Table 14]

[0505]

[0506] In Table 14, mismatched nucleotides were underlined, and nucleotides replaced with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 14 were LNAs. At points where the phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0507] The structure of D2-2 is identical to the structure reported in Non-Patent Literature 1. The relative editing efficiency was calculated based on the AUC value of D2-2 as a reference (1.00) and is shown in Table 14. In addition, 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.

[0508] As shown in Table 14, the relative editing efficiency of D2-1 was 0.13, that of D2-3 was 4.94, that of D2-4 was 3.13, that of D2-5 was 1.71, and that of D2-6 was 1.64. From these results, it was found that the non-natural polynucleotide of the present invention can modify two nucleotides included in the target nucleotide sequence.

[0509] Example 2-2 3. Review of Base Substitution

[0510] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD3 cells.

[0511] 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.

[0512] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 15.

[0513] [Table 15]

[0514]

[0515] In Table 15, mismatched nucleotides were underlined, and nucleotides replaced with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 15 were LNAs. At points where the phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0516] All structures shown in Table 15 are 3-base substitutions, and there have been no previous reports of such examples.

[0517] The relative editing efficiency was calculated based on the AUC value of D3-2 as the reference (1.00) and is shown in Table 15. In addition, 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 included in the target nucleotide sequence.

[0518] Example 2-3 Review of 2 base substitutions (2)

[0519] Experiments were conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD4-1 cells, 293-nLD4-2 cells, or 293-nLD4-3 cells.

[0520] In experiments 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.

[0521] In experiments using 293-nLD4-2 cells, each of the three types of non-natural polynucleotides, D4-2-1 to D4-2-4, was introduced into 293-nLD4-2 cells by transfection.

[0522] In experiments using 293-nLD4-3 cells, each of the three types of non-natural polynucleotides, D4-3-1 to D4-3-4, was introduced into 293-nLD4-3 cells by transfection.

[0523] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 16.

[0524] [Table 16]

[0525]

[0526] In Table 16, mismatched nucleotides were underlined, and nucleotides replaced with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 16 were LNAs. At points where the phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0527] All structures shown in Table 16 are 2-base substitutions located at separate positions, and there have been no previous reports of such examples.

[0528] In D4-1-1 to D4-1-4, there is an 8-base gap between the two mismatched nucleotides for the target nucleotide sequence. The relative editing efficiency was calculated based on the AUC value of D4-1-2 as the reference (1.00) and is shown in Table 16. Additionally, 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.

[0529] For D4-2-1 to D4-2-4, there is a 14-base gap between the two mismatched nucleotides for the target nucleotide sequence. The relative editing efficiency was calculated based on the AUC value of D4-2-2 as the reference (1.00) and is shown in Table 16. Additionally, 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.

[0530] For D4-3-1 to D4-3-4, there is a 20-base gap between the two mismatched nucleotides for the target nucleotide sequence. The relative editing efficiency was calculated based on the AUC value of D4-3-2 as the reference (1.00) and is shown in Table 16. Additionally, 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.

[0531] From these results, it was found that when there are multiple changes to nucleotides included in the target nucleotide sequence, the sites of change do not need to be adjacent, and multiple nucleotides included in the target nucleotide sequence can be changed simultaneously even if they are separated by at least 20 bases.

[0532] Example 2-4 1. Review of base insertion

[0533] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD5 cells.

[0534] In this example, four types of non-natural polynucleotides, D5-2, D5-4, D5-5, and D5-6, and non-natural polynucleotide D5-1 as a control, were each introduced into 293-nLD5 cells by transfection.

[0535] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 17.

[0536] [Table 17]

[0537]

[0538] In Table 17, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 17 were LNAs. At points where the phosphodiester bond between two or more nucleotides was substituted with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. Additionally, the nucleotides within [ ] represent nucleotides intended to insert the nucleotide complementary to the nucleotide within [ ] into the target nucleotide sequence.

[0539] The structure of D5-2 is identical to the structure reported in Non-Patent Literature 1. The relative editing efficiency was calculated based on the AUC value of D5-2 as a reference (1.00) and is shown in Table 17. In addition, 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.

[0540] As shown in Table 17, the relative editing efficiency of D5-1 of the control was 0.33, that of D5-4 was 10.44, that of D5-5 was 16.19, and that of D5-6 was 2.11. From these results, it was found that the non-natural polynucleotide of the present invention can insert one desired base into a deletion site included in the target nucleotide sequence.

[0541] Example 2-5 2 Review of base insertion

[0542] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD6 cells.

[0543] 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.

[0544] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 18.

[0545] [Table 18]

[0546]

[0547] In Table 18, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 18 were LNAs. At points where phosphodiester bonds between two or more nucleotides were substituted with phosphothioate bonds, an asterisk (*) was inserted between the corresponding two or more nucleotides. Additionally, the nucleotides within [ ] represent nucleotides intended for inserting the nucleotide complementary to the nucleotide within [ ] into the target nucleotide sequence.

[0548] All structures shown in Table 18 are 2-base insertions, and there have been no previous reports of such examples.

[0549] The relative editing efficiency was calculated based on the AUC value of D6-2 as the reference (1.00) and is shown in Table 18. Additionally, the AUC when TE was used as a negative control was 158, and the AUC of D3-2 was 4924. As shown in Table 18, the relative editing efficiency of D6-1 was 0.60, that of D6-3 was 12.62, that of D6-4 was 17.70, that of D6-5 was 2.29, that of D6-6 was 7.52, that of D6-7 was 40.12, that of D6-8 was 24.11, that of D6-9 was 7.66, and that of D6-10 was 17.37. From this result, it was found that according to the non-natural polynucleotide of the present invention, a desired base can be inserted even if there are two deletion sites included in the target nucleotide sequence.

[0550] Examples 2-6 1. Examination of base deletions

[0551] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD7 cells.

[0552] 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.

[0553] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 19.

[0554] [Table 19]

[0555]

[0556] In Table 19, mismatched nucleotides were underlined, and nucleotides replaced with cross-linked nucleic acids were indicated in uppercase. In Table 19, the mismatched nucleotide x indicates that one nucleotide complementary to the nucleotide present in the target nucleotide sequence is missing in the edited nucleic acid. All cross-linked nucleic acids in Table 19 were LNAs. At points 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.

[0557] All structures shown in Table 19 are single-base deletions, and such examples have not been reported in the past. Relative editing efficiency was calculated based on the AUC value of D7-2 (1.00) and is shown in Table 19. In addition, 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.

[0558] Example 2-7 2 Examination of base deletion

[0559] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD8 cells.

[0560] 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.

[0561] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 20.

[0562] [Table 20]

[0563]

[0564] In Table 20, mismatched nucleotides were underlined, and nucleotides replaced with cross-linked nucleic acids were indicated in uppercase. In Table 20, mismatched nucleotides xx indicate that two nucleotides complementary to the nucleotides present in the target nucleotide sequence are missing in the edited nucleic acid. All cross-linked nucleic acids in Table 20 were LNAs. At points 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.

[0565] All structures shown in Table 20 are 2-base deletions, and such examples have not been reported in the past. The relative editing efficiency was calculated based on the AUC value of D7-2 (1.00) and is shown in Table 19. In addition, the AUC when TE was used as a negative control 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.

[0566] Examples 2-8 4. Review of base insertion

[0567] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD9 cells.

[0568] 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.

[0569] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 21.

[0570] [Table 21]

[0571]

[0572] In Table 21, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 21 were LNAs. At points where phosphodiester bonds between two or more nucleotides were substituted with phosphothioate bonds, an asterisk (*) was inserted between the corresponding two or more nucleotides. Additionally, the nucleotides within [ ] represent nucleotides intended for inserting the nucleotide complementary to the nucleotide within [ ] into the target nucleotide sequence.

[0573] The structure of D9-2 is identical to the structure reported in Non-Patent Literature 1. Relative editing efficiency was calculated based on the AUC value of D9-2 as the reference (1.00) and is shown in Table 21. Additionally, 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 this result, it was found that according to the non-natural polynucleotide of the present invention, a desired base can be inserted even if there are 4 deletion sites included in the target nucleotide sequence.

[0574] From Examples 2-1 to 2-8 above, it was found that by using the non-natural polynucleotide of the present invention, it is possible to substitute two or more bases on a target polynucleotide, insert one or more bases into a target polynucleotide, and remove one or more bases from a target polynucleotide.

[0575] Genome editing experiment using C. 293-nLD1 cells (2)

[0576] Using the 293-nLD1 cells prepared in the above “A. Genome editing experiment using 293-nLD1 cells (1)”, the following genome editing experiment was performed.

[0577] 1. Introduction of non-natural polynucleotides into 293-nLD1 cells

[0578] 293-nLD1 cells 1 × 10 4 96-well plate (Nunc (registered trademark) MicroWell) for cell formation TM 96, Nunclon Delta-Treated, Flat-Bottom Microplate, Thermo Fisher Scientific Inc.) was seeded and cultured in DMEM + 10% FBS medium at 37°C under a 5% CO2 environment. After 24 hours, 0.05 or 0.1 μg of non-natural polynucleotides were transfected with Lipofectamine 3000 according to a standard protocol.

[0579] 2. Measurement of Editing Efficiency and Evaluation of Editing Efficiency of Non-Natural Polynucleotides by AUC log

[0580] Measurement of editing efficiency and evaluation of editing efficiency were carried out using the method described in the above “B. Genome editing experiment using 293-nLD2 cells, 293-nLD3 cells, 293-nLD4-1 cells, 293-nLD4-2 cells, 293-nLD4-3 cells, 293-nLD5 cells, 293-nLD6 cells, 293-nLD7 cells, 293-nLD8 cells or 293-nLD9 cells”.

[0581] 3. Synthesis of Non-Natural Polynucleotides

[0582] All non-natural polynucleotides containing nucleic acid modifications were synthesized by Gene Design (Japan) and purified using a simple column or HPLC.

[0583] Example 3-1 Modification of target nucleotide sequence by non-natural polynucleotides having various characteristics

[0584] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD1 cells.

[0585] In this example, each of the non-natural polynucleotides shown in Table 31 was introduced into 293-nLD1 cells by transfection.

[0586] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 31.

[0587] [Table 31-1]

[0588]

[0589] [Table 31-2]

[0590]

[0591] In Table 31, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 31 were LNAs. For nucleotides substituted with RNA, an arrow was placed next to the base symbol of the corresponding nucleotide. An asterisk (*) was inserted between the corresponding nucleotides at the point where the phosphodiester bond between nucleotides was replaced by a phosphorothioate bond. For non-natural polynucleotides with phosphorylated 5' and / or 3' ends, "p" was placed at the 5' and / or 3' ends.

[0592] In addition, G(F) and T(F) in GEO-204 each indicate that they are modified by 2'-Fluoro, A(MT) in GEO-213 indicates that it is modified by 2'-O-Methyl, and t*(MP)a in GEO-651 indicates that the bond between t and a is a methylphosphate bond.

[0593] As shown in Table 31, it was found that the non-natural polynucleotide of the present invention has an editing efficiency more than 4 times higher than that of the standard GEO-8.

[0594] Example 3-2 Modification of a target nucleotide sequence by a non-natural polynucleotide accompanied by a mismatch nucleotide at the 3' end

[0595] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD1 cells.

[0596] In this example, each of the non-natural polynucleotides shown in Table 32 was introduced into 293-nLD1 cells by transfection.

[0597] The base sequences, chain lengths, and relative editing efficiencies of each of the above-mentioned edited nucleic acids are as described in Table 32. In addition, in Table 32, the chain lengths of the edited nucleic acid body and the adapter are listed separately, but each sequence defined by a sequence number is the combined total of the edited nucleic acid body and the adapter.

[0598] [Table 32-1-1]

[0599]

[0600] [Table 32-1-2]

[0601]

[0602] [Table 32-2-1]

[0603]

[0604] [Table 32-2-2]

[0605]

[0606] [Table 32-3]

[0607]

[0608] [Table 32-4]

[0609]

[0610] [Table 32-5]

[0611]

[0612] [Table 32-6]

[0613]

[0614] In Table 32, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 32 were LNAs. For RNA-substituted nucleotides, an arrow was placed next to the base symbol of the corresponding nucleotide. An asterisk (*) was inserted between the corresponding nucleotides at the point where the phosphodiester bond between nucleotides was replaced by a phosphorothioate bond. For non-natural polynucleotides with phosphorylated 5' and / or 3' ends, "p" was placed at the 5' and / or 3' ends. Also, [t], [tt] and [ttt] represent 1, 2, and 3 thymidine monophosphates inserted, respectively, and [a], [aa] and [aaa] represent 1, 2, and 3 adenosine monophosphates inserted, respectively.

[0615] As shown in Table 32, it was found that the non-natural polynucleotide of the present invention had a higher editing efficiency than the standard GEO-8.

[0616] Example 3-3 Modification of a target nucleotide sequence by a non-natural polynucleotide accompanied by an adapter at the 3' end

[0617] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD1 cells.

[0618] In this example, each of the non-natural polynucleotides shown in Table 33 was introduced into 293-nLD1 cells by transfection.

[0619] The base sequences, chain lengths, and relative editing efficiencies of each of the above-mentioned edited nucleic acids are as described in Table 33. In addition, in Table 33, the chain lengths of the edited nucleic acid body and the adapter are listed separately, but each sequence defined by a sequence number is the combined total of the edited nucleic acid body and the adapter.

[0620] [Table 33-1]

[0621]

[0622] [Table 33-2]

[0623]

[0624] In Table 33, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 33 were LNAs. For RNA-substituted nucleotides, an arrow was placed next to the base symbol of the corresponding nucleotide. An asterisk (*) was inserted between the corresponding nucleotides at the point where the phosphodiester bond between nucleotides was replaced by a phosphorothioate bond. For non-natural polynucleotides with phosphorylated 5' and / or 3' ends, "p" was placed at the 5' and / or 3' ends.

[0625] In addition, among each non-natural polynucleotide shown in Table 33, the nucleotide in the second row is an adapter, and each adapter forms a stem structure that may have a loop. Figures 16a, 16b, 17a, and 17b show schematic diagrams of the stem structures of each non-natural polynucleotide.

[0626] As shown in Table 33, it was found that the non-natural polynucleotide of the present invention has an editing efficiency equivalent to or higher than that of the standard GEO-8.

[0627] Modification of the target nucleotide sequence by the non-natural polynucleotide of Example 3-4 Aspect (vi)

[0628] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD1 cells.

[0629] In this example, each of the non-natural polynucleotides shown in Table 34 was introduced into 293-nLD1 cells by transfection.

[0630] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 34.

[0631] [Table 34]

[0632]

[0633] In Table 34, mismatched nucleotides were underlined, and nucleotides substituted with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Table 34 were LNAs. At points where the phosphodiester bond between nucleotides was replaced by a phosphorothioate bond, an asterisk (*) was inserted between the corresponding nucleotides. For non-natural polynucleotides with phosphorylated 5' and / or 3' ends, "p" was attached to the 5' and / or 3' ends.

[0634] As shown in Table 34, it was found that the non-natural polynucleotide of the present invention had a higher editing efficiency than the standard GEO-8.

[0635] Examples 3-5 Modification of target nucleotide sequence by non-natural polynucleotides where the 5' terminal nucleotide is not an LNA

[0636] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD1 cells.

[0637] In this example, each of the non-natural polynucleotides shown in Table 41 was introduced into 293-nLD1 cells by transfection.

[0638] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Table 41.

[0639] [Table 41-1]

[0640]

[0641] [Table 41-2]

[0642]

[0643] In Table 41, mismatched nucleotides were underlined, and nucleotides replaced with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids here were LNA. For nucleotides replaced with RNA, an arrow was placed next to the base symbol of the corresponding nucleotide. At points where the phosphodiester bond between nucleotides was replaced by a phosphorothioate bond, an asterisk (*) was inserted between the corresponding nucleotides.

[0644] The cross-linked nucleic acids in GEO-827 and GEO-831 were all LNAs. Also, the mismatch nucleotides at or near the 5' end in GEO-649, GEO-650, GEO-653, GEO-654, GEO-656, and GEO-657 are mismatch nucleotides for the purpose of improving editing efficiency, and are not mismatch nucleotides for the purpose of modification.

[0645] As shown in Table 41, it was found that the non-natural polynucleotide of the present invention had a higher editing efficiency than the standard GEO-8.

[0646] Examples 3-6 Modification of target nucleotide sequences by non-natural polynucleotides accompanied by modifying compounds at the 5' or 3' ends

[0647] An experiment was conducted to measure the editing efficiency of target polynucleotide sequences by introducing non-natural polynucleotides into 293-nLD1 cells.

[0648] In this example, each of the non-natural polynucleotides shown in Tables 42 and 43 was introduced into 293-nLD1 cells by transfection.

[0649] The base sequences, chain lengths, and relative editing efficiencies of each of the above edited nucleic acids are as described in Tables 42 and 43.

[0650] [Table 42]

[0651]

[0652] [Table 43]

[0653]

[0654] In Tables 42–43, mismatched nucleotides were underlined, and nucleotides replaced with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Tables 42–43 were LNAs. At points where the phosphodiester bond between nucleotides was replaced by a phosphorothioate bond, an asterisk (*) was inserted between the corresponding nucleotides.

[0655] Also, in Tables 42 to 43, Biotin is biotin as a modifying compound, FAM is fluorescein (FAM) as a modifying compound, Pur is puromycin as a modifying compound, Chol is cholesterol as a modifying compound, DIG is digoxigenin as a modifying compound, and IdT is Inverted dT as a modifying compound. Here, the molecular weight of Biotin 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. In addition, the mismatch nucleotides at the 5' end or near it in GEO-649, GEO-650, GEO-653, GEO-654, GEO-656, and GEO-657 are mismatch nucleotides for the purpose of improving editing efficiency, and are not mismatch nucleotides for the purpose of modification.

[0656] As shown in Tables 42 to 43, it was found that the non-natural polynucleotide of the present invention has a higher editing efficiency than the standard GEO-8.

[0657] Example 3-7 Review of the introduction of phosphate modification bonds to the 5' side or 3' side

[0658] An experiment was conducted to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide with a phosphate group modification linkage on the 5' or 3' side from a mismatch nucleotide intended for modification into 293-nLD1 cells.

[0659] In this example, each of the non-natural polynucleotides shown in Tables 44 to 45 was introduced into 293-nLD1 cells by the transfection method.

[0660] The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are as described in Tables 44 to 45.

[0661] [Table 44]

[0662]

[0663] [Table 45]

[0664]

[0665] In Tables 44–45, mismatched nucleotides were underlined, and nucleotides replaced with cross-linked nucleic acids were indicated in uppercase. All cross-linked nucleic acids in Tables 44–45 were LNAs. At points where the phosphodiester bond between nucleotides was replaced by a phosphorothioate bond, an asterisk (*) was inserted between the corresponding nucleotides.

[0666] As shown in Tables 44 to 45, it was found that the non-natural polynucleotide of the present invention has a higher editing efficiency than the standard GEO-8.

[0667] Industrial applicability

[0668] The method using the non-natural polynucleotide of the present invention enables the modification of a target nucleotide sequence by introducing the polynucleotide alone, without introducing a foreign nuclease or a gene encoding a foreign nuclease into the cell, and has industrial applicability because it can be applied to highly safe genome editing technology.

Claims

Claim 1 A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying 1 to 4 nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell, comprising 1 to 4 mismatch nucleotides with respect to the target nucleotide sequence, having a cross-linked nucleic acid as shown in (A) and (B) below, and additionally having the characteristics of (C) and (D) (provided that a polynucleotide in which cross-linked nucleic acids and non-cross-linked nucleic acids exist alternately throughout the entire region of the polynucleotide). (A) One nucleotide adjacent to the 5' upstream side of the mismatch nucleotide is a cross-linked nucleic acid. (B) One nucleotide adjacent to the 3' downstream side of the mismatch nucleotide is a cross-linked nucleic acid. (C) A nucleotide at the 5' end is a cross-linked nucleic acid. (D) Chain length 24 ~ It is 95 nucleotides. Claim 2 In claim 1, the non-natural polynucleotide further comprises the following characteristic (E): (E) The nucleotide at the 3' end is a cross-linked nucleic acid. Claim 3 In claim 1, the non-natural polynucleotide further comprises the following characteristic (F): (F) One or more nucleotides adjacent to the nucleotide at the 5' end are cross-linked nucleic acids. Claim 4 In claim 2, the above-mentioned non-natural polynucleotide further comprises the following characteristic (G): (G) One or more nucleotides adjacent to the nucleotide at the 3' end are cross-linked nucleic acids. Claim 5 In claim 1, the non-natural polynucleotide further comprises the following feature (J): (J) One or more phosphodiester bond portions between nucleotides are substituted with phosphate group modification bonds. Claim 6 In claim 1, the non-natural polynucleotide further comprises the following characteristic (M): (M) One or more nucleotides are cross-linked nucleic acids disposed between a nucleotide adjacent to the 5' upstream side of the mismatch nucleotide and a nucleotide at the 5' end, and disposed at a distance of at least one nucleotide from either the nucleotide adjacent to the 5' upstream side of the mismatch nucleotide or the nucleotide at the 5' end. Claim 7 In claim 2, the non-natural polynucleotide further comprises the following characteristic (N): (N) One or more nucleotides are cross-linked nucleic acids disposed between a nucleotide adjacent to the 3' downstream side of the mismatch nucleotide and a nucleotide at the 3' end, and disposed at a distance of at least one nucleotide from either the nucleotide adjacent to the 3' downstream side of the mismatch nucleotide or the nucleotide at the 3' end. Claim 8 In claim 2, the non-natural polynucleotide further comprises the following features (O) and / or (X2): (O) A pentose sugar in one or more nucleotides adjacent to the cross-linked nucleic acid at the 3' end is ribose; (X2) A pentose sugar in one or more nucleotides adjacent to the cross-linked nucleic acid at the 5' end is ribose. Claim 9 In claim 1, the above-mentioned non-natural polynucleotide further comprises the following characteristic (P): (P) The nucleotide at the 3' end is phosphorylated. Claim 10 In claim 1, the non-natural polynucleotide comprising at least one cross-linked nucleic acid selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA. Claim 11 In claim 2, the non-natural polynucleotide further comprises the following feature (X1): (X1) a nucleotide at the 3' end and / or one or more nucleotides adjacent to the nucleotide at the 3' end are nucleic acids in which the 2' site is modified. Claim 12 In claim 1, the non-natural polynucleotide further comprises the following features (X4). (X4) An adapter is attached to the 3' end. Claim 13 In claim 12, the adapter is a non-natural polynucleotide having the function of inhibiting the repair of mismatch in host cells. Claim 14 In claim 12, the adapter has the function of protecting the non-natural polynucleotide from nuclease digestion. Claim 15 In claim 12, the adapter is a non-natural polynucleotide having the function of providing higher editing efficiency than a non-natural polynucleotide (designated as "GEO-8") consisting of the base sequence defined as SEQ ID No.

9. Claim 16 In claim 12, the adapter is a non-natural polynucleotide that is a nucleotide containing a mismatch nucleotide. Claim 17 In claim 12, the adapter is a non-natural polynucleotide that forms a stem structure having a loop. Claim 18 In claim 12, the adapter is a non-natural polynucleotide that is a compound other than a nucleotide modifying the terminal (indicated as "modifying compound"). Claim 19 In claim 1, the non-natural polynucleotide further comprises the following characteristic (X5): (X5) One or more nucleotides are inserted between the mismatch nucleotide and the nucleotide at the 3' end (provided that insertion between the mismatch nucleotide and one nucleotide adjacent to the 3' downstream side is excluded.) Claim 20 A method for modifying 1 to 4 nucleotides included in a target nucleotide sequence in two-stranded DNA within a cell, comprising the step of introducing into the cell a non-natural polynucleotide comprising 1 to 4 mismatched nucleotides with respect to the target nucleotide sequence, wherein the modification of the target nucleotide sequence comprises at least one selected from the group consisting of deletion, insertion, and substitution of 1 to 4 nucleotides of the target nucleotide sequence, and wherein the non-natural polynucleotide is a non-natural polynucleotide described in any one of claims 1 to 19 (provided, cases of introduction into human germ cells and cases of being performed in the human body are excluded). Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete

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