Non-natural polynucleotides for modification of target nucleotide sequence

Abstract

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

Description

Non-naturally occurring polynucleotides for modifying target nucleotide sequences

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

[0002] CRISPR-Cas is a genome editing technology that utilizes the adaptive immune system of eubacteria and archaea and is used as a genetic engineering tool. CRISPR-Cas9 (Patent Document 1), which uses the DNA sequence CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the DNA cleavage enzyme Cas9 from Streptococcus pyogenes, is characterized by its ability to induce cleavage of target double-stranded DNA by RNA recognition of the target DNA sequence. Due to its simplicity, speed, and high efficiency, it is the most widely used genome editing technology. However, CRISPR-Cas9 has problems such as off-target effects, which are caused by misrecognition of genomic sequences by guide RNA sequences and the introduction of unexpected mutations in locations other than the target DNA sequence due to double-stranded DNA cleavage.

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

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

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

[0006] Another example of a genome editing technique that does not use Cas nuclease proteins has been reported: the introduction of single-stranded synthetic DNA containing LNA in part of the nucleotide sequence into mouse ES cells can introduce mutations of one to three bases into the target nucleotide sequence (Non-Patent Document 1). It has been suggested that this technique can improve the problem of off-target effects compared to genome editing techniques that use Cas nuclease proteins. The authors of Non-Patent Document 1 sequenced 335 bp surrounding the target nucleotide sequence for 33 cells in which the intended modification of the target nucleotide sequence was confirmed by sequencing, confirming that no unintended modifications had occurred outside the target nucleotide sequence. Based on these results, the authors explain that the single-stranded synthetic DNA used by the authors of Non-Patent Document 1 enabled highly accurate genome editing in mouse-derived ES cells with functional mismatch repair mechanisms. The authors of Non-Patent Document 1 concluded that it is important that the mismatch nucleotide is LNA in order to circumvent the intracellular mismatch repair mechanism and modify the target nucleotide sequence, and have conducted extensive experiments based on this conclusion. In fact, Non-Patent Document 1 conducted experiments using a total of more than 60 types of single-stranded synthetic DNAs, and 41 types of single-stranded synthetic DNAs had at least an LNA mismatch nucleotide.

[0007] Non-Patent Document 2 is a follow-up report by the same authors as Non-Patent Document 1, based on the discovery in Non-Patent Document 1 that when the mismatch nucleotide contained in single-stranded synthetic DNA is LNA, it is possible to avoid the mismatch repair mechanism in cells. Non-Patent Document 2 focuses on elucidating the mechanism by which single-stranded synthetic DNA containing an LNA mismatch nucleotide modifies the genome of mammalian cells.

[0008] Patent No. 6343605 Patent No. 6480647 Patent No. 6206893 Patent No. 5405121

[0009] 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

[0010] Several reports have been published on genome editing techniques that do not use Cas nucleases, such as modifying target nucleotide sequences using single-stranded synthetic DNA containing at least one LNA. However, the editing efficiency is not sufficient.

[0011] The present invention addresses the need to provide a novel non-natural polynucleotide that has structural features that enable improved editing efficiency in genome editing technology using non-natural polynucleotides involving single-stranded synthetic DNA in which part of the nucleotide sequence is a cross-linked nucleic acid.

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

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

[38] : [1] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence in intracellular double-stranded DNA, for modifying one or more nucleotides contained in the target nucleotide sequence, the non-natural polynucleotide containing one or more mismatch nucleotides with respect to the target nucleotide sequence, and having a bridged nucleic acid shown in at least one of the following (A) and (B), and further having the characteristics (C) and (D): (A) one or more nucleotides adjacent to the 5' upstream side of the mismatch nucleotide are bridged nucleic acids, (B) one or more nucleotides adjacent to the 3' downstream side of the mismatch nucleotide are bridged nucleic acids, (C) the 5'-terminal nucleotide is a bridged nucleic acid, and (D) the chain length is 22 to 95 nucleotides. [2] The non-natural polynucleotide according to [1] above, wherein the non-natural polynucleotide further has the following characteristic (E): (E) The 3'-terminal nucleotide is a bridged nucleic acid. [3] The non-natural polynucleotide according to any one of [1] to [2] above, wherein the non-natural polynucleotide further has the following characteristic (F): (F) One or more nucleotides adjacent to the 5'-terminal nucleotide are bridged nucleic acids. [4] The non-natural polynucleotide according to any one of [1] to [3] above, wherein the non-natural polynucleotide further has the following characteristic (G): (G) One or more nucleotides adjacent to the 3'-terminal nucleotide are bridged nucleic acids. [5] The non-natural polynucleotide according to any one of [1] to [4] above, wherein the non-natural polynucleotide further has the following characteristic (J): (J) One or more phosphodiester bond(s) between nucleotides are replaced with phosphate moiety-modified bond(s).

[0014] [6] The non-natural polynucleotide according to [5] above, wherein the phosphate moiety-modified bond comprises at least one bond selected from the group consisting of a phosphorothioate bond, a methylphosphate bond, a boranophosphate bond, and a mesyl phosphoramidate bond. [7] The non-natural polynucleotide according to any one of [1] to [6] above, wherein the non-natural polynucleotide further has the following characteristic (M): (M) one or more nucleotides located between the nucleotide adjacent to the 5' upstream side of the mismatch nucleotide and the 5'-terminal nucleotide, and located at a distance of at least one nucleotide from both the nucleotide adjacent to the 5' upstream side of the mismatch nucleotide and the 5'-terminal nucleotide, are crosslinked nucleic acids. [8] The non-natural polynucleotide according to any one of [1] to [7] above, wherein the non-natural polynucleotide further has the following characteristic (N): (N) One or more nucleotides positioned between the nucleotide adjacent to the 3'-downstream side of the mismatched nucleotide and the 3'-terminal nucleotide, and positioned at a distance of at least one nucleotide from both the nucleotide adjacent to the 3'-downstream side of the mismatched nucleotide and the 3'-terminal nucleotide, are bridged nucleic acids. [9] The non-natural polynucleotide according to any one of [1] to [8] above, wherein the non-natural polynucleotide further has the following characteristics (O) and / or (X2): (O) the pentose sugar in one or more nucleotides adjacent to the 3'-terminal bridged nucleic acid is ribose; (X2) the pentose sugar in one or more nucleotides adjacent to the 5'-terminal bridged nucleic acid is ribose.

[0015]

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

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

[10] above, wherein the crosslinked nucleic acid comprises at least one selected from the group consisting of LNA, AmNA, BNA NH, BNA N-Me, and ENA.

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

[11] above, wherein the non-natural polynucleotide further has the following characteristic (X1). (X1) The 3'-terminal nucleotide and / or one or more nucleotides adjacent to the 3'-terminal nucleotide are nucleic acids modified at the 2'-position.

[13] The non-natural polynucleotide according to

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

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

[13] above, wherein the non-natural polynucleotide further has the following feature (X4): (X4) An adapter is added to the 3'-terminus.

[15] The non-natural polynucleotide according to

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

[16] The non-natural polynucleotide according to

[14] or

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

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

[14] to

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

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

[14] to

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

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

[14] to

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

[0016]

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

[14] to

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

[21] The non-natural polynucleotide according to

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

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

[21] , wherein the non-natural polynucleotide further has the following characteristic (X5): (X5) one or more nucleotides are inserted between the mismatch nucleotide and the 3'-terminal nucleotide.

[23] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence, for modifying one or more nucleotides contained in the target nucleotide sequence in intracellular double-stranded DNA, the non-natural polynucleotide comprising one or more mismatch nucleotides with respect to the target nucleotide sequence, having at least one of the following crosslinked nucleic acids (A) and (B), having characteristic (D), and further having any one or more characteristics selected from the group consisting of (H2), (Y1), and (Y3). (A) one or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are bridged nucleic acids; (B) one or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are bridged nucleic acids; (D) the chain length is 22 to 95 nucleotides; (H2) the phosphodiester bond between the 5'-terminal nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide is replaced with a phosphate moiety-modified bond; (Y1) the 5'-terminal nucleotide is a mismatched nucleotide, and one or more nucleotides adjacent to the 5'-terminal nucleotide are mismatched nucleotides; (Y3) an adapter is attached to the 5'-terminal

[0017]

[24] The non-naturally occurring polynucleotide according to

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

[25] The non-naturally occurring polynucleotide according to

[23] or

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

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

[23] to

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

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

[23] to

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

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

[23] to

[27] above, wherein the non-naturally occurring polynucleotide further has the following characteristic (Y2): (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.

[29] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence, for modifying one or more nucleotides contained in the target nucleotide sequence in double-stranded DNA in a cell, the non-natural polynucleotide containing one or more mismatch nucleotides with respect to the target nucleotide sequence, having a bridged nucleic acid shown in at least one of the following (A) and (B), and further having the characteristics (D) and (X7): (A) one or more nucleotides adjacent to the 5'-upstream side of the mismatch nucleotide are bridged nucleic acids; (B) one or more nucleotides adjacent to the 3'-downstream side of the mismatch nucleotide are bridged nucleic acids; (D) the chain length is 22 to 95 nucleotides; (X7) one or more nucleotides located between the mismatch nucleotide and the 5'-terminal nucleotide are bridged nucleic acids.

[0018]

[30] The non-natural polynucleotide according to

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

[31] A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides contained in the target nucleotide sequence in intracellular double-stranded DNA, the non-natural polynucleotide comprising one or more mismatch nucleotides with respect to the target nucleotide sequence, and having all of the following characteristics (C), (D), (I), and (X6): (C) the 5'-terminal nucleotide is a bridged nucleic acid; (D) the chain length is 22 to 95 nucleotides; (I) the phosphodiester bond between the 3'-terminal nucleotide and one or more nucleotides adjacent to the 3'-terminal nucleotide is substituted with a phosphorothioate bond; (X6) the mismatch nucleotide is a bridged nucleic acid.

[32] The non-natural polynucleotide according to

[31] , wherein the non-natural polynucleotide further has the following characteristic (E): (E) The 3'-terminal nucleotide is a crosslinked nucleic acid.

[33] The non-natural polynucleotide according to

[31] or

[32] , wherein the non-natural polynucleotide further has the following characteristic (P): (P) The 3'-terminal nucleotide is phosphorylated.

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

[33] .

[35] A pharmaceutical composition comprising the non-natural polynucleotide according to any one of [1] to

[33] .

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

[33] .

[37] The method according to

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

[38] The method according to

[37] above, wherein the eukaryotic cell is at least one selected from the group consisting of a plant cell, an insect cell, and an animal cell.

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

[0020] FIG. 1 is a schematic diagram outlining a method for modifying a target 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 non-natural polynucleotide of the present invention is used to edit a mutation in an inactive luciferase gene in the genome of 293-nLD1 cells, and the editing efficiency is detected as luciferase luminescence. FIG. 3A is a diagram showing the structures of the 12 non-natural polynucleotides shown in Example 1-1 of the present invention, namely, GEO-1 to GEO-9, GEO-242, GEO-11, and GEO-94. GEO-94 is a negative control having a base sequence without a mismatched nucleotide. In the diagram, G indicates a mismatched nucleotide, and G with an asterisk (i.e., "G*") and L indicate that the pentose sugar in the nucleotide has been replaced with LNA. FIG. 3B is a bar graph showing the editing efficiency (%) of the 12 non-natural polynucleotides shown in Example 1-1 of the present invention, namely, GEO-1 to GEO-9, GEO-242, GEO-11, and GEO-94. FIG. 4 is a diagram showing the structure of the non-natural polynucleotides shown in Example 1-2 of the present invention. In the diagram, G represents a mismatched nucleotide, and L indicates that the pentose sugar in the nucleotide has been replaced with LNA. FIG. 5 is a diagram showing the structure of the non-natural polynucleotides shown in Example 1-3 of the present invention. In the diagram, G represents a mismatched nucleotide, and L indicates that the pentose sugar in the nucleotide has been replaced with LNA. FIG. 6 is a diagram showing the structure of the non-natural polynucleotides shown in Example 1-4 of the present invention. In the diagram, G represents a mismatched nucleotide, and L indicates that the pentose sugar in the nucleotide has been replaced with LNA. FIG. 7 is a diagram showing the structure of the non-natural polynucleotides shown in Example 1-5 of the present invention. In the diagram, G represents a mismatched nucleotide, and L indicates that the pentose sugar in the nucleotide has been replaced with LNA. 8A to 8C are diagrams showing the structure of the non-natural polynucleotide shown in Examples 1 to 6 of the present invention. Fig. 8A shows the structure of the non-natural polynucleotide in which the phosphodiester bond between the 5'-terminal LNA and the 1st to 5th nucleotides adjacent thereto is substituted with a phosphorothioate bond.In the figure, G indicates a mismatched nucleotide, L indicates that the pentose sugar in the nucleotide has been substituted with LNA, and S indicates that the phosphodiester bond has been substituted with a phosphorothioate bond. Figure 8B shows the structure of a non-natural polynucleotide in which the phosphodiester bond between the 3'-terminal nucleotide and the adjacent nucleotides 1 to 7 has been substituted with a phosphorothioate bond. In the figure, G indicates a mismatched nucleotide, L indicates that the pentose sugar in the nucleotide has been substituted with LNA, and S indicates that the phosphodiester bond has been substituted with a phosphorothioate bond. 8C shows the structure of a non-natural polynucleotide in which the phosphodiester bond between the 5'-terminal LNA and the four adjacent nucleotides is replaced with a phosphorothioate bond and the phosphodiester bond between the 3'-terminal nucleotide and the four adjacent nucleotides is replaced with a phosphorothioate bond; the structure of a non-natural polynucleotide in which the phosphodiester bond between the LNA adjacent to the 5'-upstream side of the mismatch nucleotide G and the two or four adjacent nucleotides thereto is replaced with a phosphorothioate bond; and the structure of a non-natural polynucleotide in which the phosphodiester bond between the nucleotide adjacent to the LNA adjacent to the 3'-downstream side of the mismatch nucleotide G and the two or four adjacent nucleotides thereto is replaced with a phosphorothioate bond. In the figure, G represents the mismatch nucleotide, L represents the substitution of the pentose sugar in the nucleotide with LNA, and S represents the substitution of the phosphodiester bond with a phosphorothioate bond. FIG. 9 shows the structure of the non-natural polynucleotide shown in Examples 1-7 of the present invention. In the figure, G indicates a mismatched nucleotide, L indicates that the pentose sugar in the nucleotide has been substituted with LNA, and S indicates that the phosphodiester bond has been substituted with a phosphorothioate bond. Figure 10A shows the structure of a non-natural polynucleotide shown in Examples 1-8 of the present invention. In the figure, G indicates a mismatched nucleotide, and L indicates that the pentose sugar in the nucleotide has been substituted with LNA. Figure 10B shows the structure of a non-natural polynucleotide shown in Examples 1-8 of the present invention.In the figure, G represents a mismatched nucleotide, and L indicates that the pentose sugar in the nucleotide has been replaced with LNA. FIG. 11 shows the structure of a non-natural polynucleotide shown in Examples 1-9 of the present invention. In the figure, G represents a mismatched nucleotide, L indicates that the pentose sugar in the nucleotide has been replaced with LNA, and R indicates that the pentose sugar in the nucleotide has been replaced with ribose. FIG. 12 shows the structure of a non-natural polynucleotide shown in Examples 1-10 of the present invention. In the figure, G represents a mismatched nucleotide, L indicates that the pentose sugar in the nucleotide has been replaced with LNA, P indicates that the 5'-terminal nucleotide and / or the 3'-terminal nucleotide has been phosphorylated, and S indicates that the phosphodiester bond moiety has been replaced with a phosphorothioate bond. FIG. 13 shows the structure of a non-natural polynucleotide shown in Examples 1-11 of the present invention. In the figure, G indicates a mismatched nucleotide, L indicates that the pentose sugar in the nucleotide has been replaced with LNA, H indicates that the pentose sugar in the nucleotide has been replaced with BNA-NH, M indicates that the pentose sugar in the nucleotide has been replaced with BNA-N-Me, and E indicates that the pentose sugar in the nucleotide has been replaced with ENA. This figure shows an outline of the structure of the unnatural polynucleotide (GEO-226) shown in Example 1-12 of the present invention. In the figure, the arrow indicates the position of the mismatched nucleotide, L indicates that the pentose sugar in the nucleotide has been replaced with LNA, S indicates that the phosphodiester bond has been replaced with a phosphorothioate bond, and P indicates that the 3' end has been phosphorylated. This figure shows an outline of the structure of the unnatural polynucleotide (GEO-172) shown in Example 1-13 of the present invention. In the figure, the arrow indicates the position of the mismatched nucleotide, L indicates that the pentose sugar in the nucleotide has been replaced with LNA, S indicates that the phosphodiester bond has been replaced with a phosphorothioate bond, R indicates that the nucleotide has been replaced with RNA, and P indicates that the 3' end has been phosphorylated. This figure shows an outline of the structure of the non-natural polynucleotides (GEO-602 to GEO-605) shown in Example 3-3 of the present invention.In the figure, G represents a mismatched nucleotide, and L represents that the pentose sugar in the nucleotide is replaced by LNA. In the unnatural polynucleotide in the figure, the nucleotides at the positions of L, G, and the black band are DNA, and the nucleotides at the positions of the white band are RNA. It is a diagram showing the schematic configuration of the unnatural 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 replaced by LNA. In the unnatural polynucleotide in the figure, the nucleotides at the positions of L, G, and the black band are DNA, and the nucleotides at the positions of the white band are RNA. It is a diagram showing the schematic configuration of the unnatural 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 that the pentose sugar in the nucleotide is replaced by LNA, and S represents that the phosphodiester bond portion is replaced by a phosphorothioate bond. In the unnatural polynucleotide in the figure, the nucleotides at the positions of L, G, and the black band are DNA, and the nucleotides at the positions of the white band are RNA. It is a diagram showing the schematic configuration of the unnatural 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 that the pentose sugar in the nucleotide is replaced by LNA, and P represents that the 3'-end is phosphorylated. In the unnatural polynucleotide in the figure, the nucleotides at the positions of L, G, and the black band are DNA, and the nucleotides at the positions of the white band are RNA.

[0021] <Summary of the Invention> In conventional techniques using single-stranded polynucleotides, the polynucleotide introduced into cells specifically recognizes and binds to a target nucleotide sequence in the lagging strand of a replication fork during double-stranded DNA replication, serving as a primer for Okazaki fragment synthesis by DNA polymerase. In this way, the polynucleotide is incorporated into the nascent DNA strand, resulting in genome sequence editing. Meanwhile, because living organisms possess a mismatch repair mechanism that repairs mismatches that occur during DNA replication, mismatches introduced by exogenously introduced polynucleotides are also immediately repaired. This mismatch repair mechanism is thought to be one of the reasons why modification of a target nucleotide sequence using exogenously introduced polynucleotides into cells has lower editing efficiency than modification of a target nucleotide sequence using nucleases.

[0022] On the other hand, it is presumed that the non-natural polynucleotide of the present invention promotes editing due to the presence of a cross-linked nucleic acid at a specific position in the non-natural polynucleotide, while at the same time reducing the effects of the mismatch repair mechanism, thereby significantly increasing the editing efficiency of the target nucleotide sequence compared to previous reports.

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

[0024] <Nucleotide> In the present invention, "nucleotide" is a general term for a substance in which a phosphate group is bound to a nucleoside. A nucleoside is a pentose sugar in which a purine or pyrimidine base is glycosidicly linked to its first position. A chain-like biopolymer made up of nucleotides 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"). Like DNA, RNA contains A, G, and T, but uridine monophosphate (UMP, hereinafter referred to as "U") instead of T.

[0025] <Double-stranded DNA> In the present invention, "double-stranded DNA" refers to DNA in which single-stranded DNAs having complementary base sequences form hydrogen bonds between bases in opposite directions to form a double helix. Examples of double-stranded DNA in the present invention include, but are not limited to, genomic DNA, mitochondrial DNA, and chloroplast DNA. Furthermore, in the present invention, double-stranded DNA containing a "target nucleotide sequence" preferably includes a gene involved in a genetic disease, more preferably a gene involved in a human genetic disease.

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

[0027] <Complementary nucleotides and mismatched nucleotides> Double-stranded DNA usually forms combinations of purine and pyrimidine bases, with G and C, and A and T bonding via hydrogen bonds. A nucleotide that pairs with a specific nucleotide is called a "complementary nucleotide."

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

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

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

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

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

[0033] Non-natural nucleotides include, but are not limited to, nucleic acids with modified phosphate moieties, nucleic acids with modified sugar moieties, nucleic acids with modified base moieties, nucleic acids with adapters attached to the 3'- and / or 5'-ends, and nucleic acids with one or more nucleotides inserted between the mismatch nucleotide and the 3'-terminal nucleotide. Nucleic acids with modified phosphate moieties include, but are not limited to, nucleic acids in which the phosphodiester bond between one or more nucleotides is replaced with a phosphorothioate bond. While a normal bond between nucleotides is called a phosphodiester bond, this sulfurized bond is called a phosphorothioate bond. In the present invention, a nucleotide having a phosphorothioate bond is called a "phosphorothioated" or "S-modified" nucleotide.

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

[0035] Nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids with modified 2' sites, cross-linked nucleic acids in which the 2' and 4' sites are cross-linked, etc. Nucleic acids with modified 2' sites include, but are not limited to, 2'-F (F-modified), 2'-O-Methyl (2'-OMe), 2'-O-Methoxyethyl (2'-MOE), 2'-O-(2-carbamoylethyl), etc.

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

[0037]

[0038] Examples of BNA / LNA analogs include, but are not limited to, ethylene-bridged BNA (ENA), amide-bridged BNA (AmNA), 2'-(alkylamino)-LNA, 2'-(acylamino)-LNA, 2'-N-substituted-2'-amino-LNA, α-LNA, α-L-LNA, β-D-LNA, 2'-amino-LNA, 2'-thio-LNA, xylo-LNA, 2'-O,4'-C-constrained ethyl (cEt)LNA, 2'-O,4'-C-constrained methoxyethyl (cMOEt)LNA, carba (cLNA), BNACOC, spirocyclopropylene-bridged nucleic acid (scpBNA), heterocyclic-bridged BNA, urea-bridged BNA, sulfonamide-bridged BNA, 5'-methyl-substituted BNA, guanidine-bridged nucleic acid (GuNA), and 3,4-dihydro-2H-pyran nucleic acid (DpNA).

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

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

[0041] Other cross-linked nucleic acids include, but are not limited to, oxetane nucleotides, locked PMOs derived from 2'-amino-LNA, cyclohexenyl nucleic acid (CeNA), alitriol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), and 3'-deoxypyranosyl-DNA (p-DNA).

[0042] In the non-natural polynucleotide of the present invention, if the number of crosslinked nucleic acids is too large, the editing efficiency tends to decrease. For example, the proportion of crosslinked nucleic acids in the non-natural polynucleotide of the present invention is preferably 2 to 30%, more preferably 2 to 20%, and even more preferably 2 to 15%, when the total number of nucleotides constituting the non-natural polynucleotide is taken as 100%.

[0043] In the non-natural polynucleotide of the present invention, adding an adapter to the 3'-end and / or 5'-end is preferred because it exhibits higher editing efficiency. Examples of adapters used herein include those that inhibit mismatch repair in host cells, those that protect the non-natural polynucleotide of the present invention from nuclease digestion, and those that, when added to the 3'-end and / or 5'-end, confer higher editing efficiency than the non-natural polynucleotide (GEO-8) defined by SEQ ID NO: 9. The adapter of the present invention possesses one or more of these functions. Specific examples of such adapters include (1) nucleotides as adapters and (2) compounds other than nucleotides that modify the ends (referred to herein as "modifying compounds").

[0044] The predicted mechanism by which mismatch repair in host cells is inhibited by adding an adapter to the 3'-end and / or 5'-end of the non-natural polynucleotide of the present invention is as follows: When one strand of a double-stranded DNA has a 3'-end structure, mismatch repair begins when a mismatch repair enzyme in the cell recognizes the strand with the 3'-end as the target for repair, and then the mismatched nucleotide on the strand with the 3'-end is excised and repaired. When the non-natural polynucleotide of the present invention is specifically bound to a target sequence, the non-natural polynucleotide has a 3'-end structure, and the mismatched nucleotide in the non-natural polynucleotide is repaired. When a nucleotide is added as an adapter to the 3'-end of the non-natural polynucleotide, the 3'-end of the non-natural polynucleotide does not specifically bind to the target sequence but dissociates, potentially avoiding recognition by the mismatch repair enzyme. Similarly, when a modifying compound is added to the 3'-end of the non-natural polynucleotide, the 3'-end of the non-natural polynucleotide may also avoid recognition by the mismatch repair enzyme. For these reasons, it is presumed that the editing efficiency of non-natural polynucleotides to which adapter nucleotides or modifying compounds have been added at the 3' end is increased by inhibiting mismatch repair in host cells.

[0045] The mechanism by which the addition of an adapter to the 3' and / or 5' end of a non-natural polynucleotide of the present invention protects the non-natural polynucleotide of the present invention from digestion by nucleases in host cells is presumed to be as follows: Single-stranded non-natural polynucleotides introduced into host cells are subject to digestion by nucleases in host cells. While the addition of an adapter nucleotide to the 5' or 3' end of a non-natural polynucleotide alone cannot completely prevent nuclease digestion of the non-natural polynucleotide, it is presumed that the adapter nucleotide acts as a buffer, inhibiting exonuclease digestion from progressing to the mismatched nucleotide located in the center of the non-natural polynucleotide. On the other hand, the addition of a modifying compound to the 3' end is likely to inhibit exonuclease digestion from the 3' end. For example, IdT has been used to increase the nuclease resistance of antisense nucleic acids by adding it to the 3' end of the antisense nucleic acid.

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

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

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

[0049] These unnatural nucleotides with such modifications have a high affinity for natural DNA and RNA, and are therefore being applied to nucleic acid pharmaceuticals and other applications.

[0050] <Basic Structure of Non-Natural Polynucleotides> The non-natural polynucleotides of the present invention are single-stranded polynucleotides that contain one or more mismatched nucleotides relative to a target nucleotide sequence, and the nucleotides other than the mismatched nucleotides are nucleotides complementary to the target nucleotide sequence. Therefore, by introducing the non-natural polynucleotides of the present invention into cells, they can specifically bind to a target nucleotide sequence. This allows one or more nucleotides in the target nucleotide sequence to be modified to desired nucleotides. When there are multiple mismatched nucleotides, the two or more mismatched nucleotides may be adjacent to each other or may be spaced apart. In the present invention, the term "multiple" with respect to the number of mismatched nucleotides refers to an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, including ranges with these 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, ... 18 to 19, 18 to 20, 19 to 20.

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

[0052] (1) Aspect (i) One aspect (aspect (i)) of the non-natural polynucleotide of the present invention is configured as follows: (A) one or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are bridged nucleic acids, (B) one or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are bridged nucleic acids, (C) the 5'-terminal nucleotide is a bridged nucleic acid, and (D) the chain length is 22 to 95 nucleotides. Such a configuration can exert the effects of the present invention.

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

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

[0055] (2) Aspect (ii) Another aspect (aspect (ii)) of the non-natural polynucleotide of the present invention is configured in such a manner that (A) one or more nucleotides adjacent to the 5'-upstream side of the mismatched nucleotide are bridged nucleic acids, (C) the 5'-terminal nucleotide is a bridged nucleic acid, and (D) the chain length is 22 to 95 nucleotides (however, the polynucleotide does not have the characteristic (B) above). Even with such a configuration, the effects of the present invention can be achieved.

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

[0057] Here, the mismatched nucleotide may or may not be a bridged nucleic acid, but in aspect (ii), it is more preferable that the mismatched nucleotide is not a bridged nucleic acid. Of the total three nucleotides adjacent to the mismatched nucleotide, including the mismatched nucleotide, the number of bridged nucleic acids is preferably two. Furthermore, the term "multiple" in "multiple nucleotides are bridged nucleic acids" as defined in (A) above is not particularly limited, but two consecutive bridged nucleic acids are preferred over three or more consecutive bridged nucleic acids, with one being more preferred.

[0058] (3) Aspect (iii) A further aspect (aspect (iii)) of the non-natural polynucleotide of the present invention is one in which: (B) one or more nucleotides adjacent to the 3'-downstream side of the mismatched nucleotide are bridged nucleic acids; (C) the 5'-terminal nucleotide is a bridged nucleic acid; and (D) the chain length is 22 to 95 nucleotides (however, the polynucleotide does not have the characteristic of (A) above). Even with such a configuration, the effects of the present invention can be achieved.

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

[0060] Here, the mismatched nucleotide may or may not be a bridged nucleic acid, but in aspect (iii), it is more preferable that the mismatched nucleotide is not a bridged nucleic acid. Of the total three nucleotides adjacent to the mismatched nucleotide, including the mismatched nucleotide, the number of bridged nucleic acids is preferably two. Furthermore, the term "multiple" in "multiple nucleotides are bridged nucleic acids" as defined in (B) above is not particularly limited, but two consecutive bridged nucleic acids are preferred over three or more consecutive bridged nucleic acids, with one being more preferred.

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

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

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

[0064] [3'-Terminal is a Crosslinked Nucleic Acid] In any of the above aspects (i) to (iii), when the non-natural polynucleotide of the present invention has (E) a crosslinked nucleic acid at the 3'-terminus, the editing efficiency of the target nucleotide sequence can be further increased.

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

[0066] [The nucleotide adjacent to the 3'-terminus is a bridged nucleic acid] In any of the above aspects (i) to (iii), or in addition to the above aspects (E) and / or (F), the non-natural polynucleotide of the present invention can further enhance the editing efficiency of a target nucleotide sequence by, for example, (G) when one or more nucleotides adjacent to the 3'-terminus nucleotide are bridged nucleic acids. In this case, it is more preferable that the bridged nucleic acid is an LNA. In this case, the number of consecutive bridged nucleic acids at the 3'-terminus is not particularly limited, but, for example, when the number of nucleotides from the mismatched nucleotide to the 3'-terminus is 27 nt, the number is preferably three or less, more preferably two or less, and even more preferably one. Furthermore, for example, when the number of nucleotides from the mismatched nucleotide to the 3'-terminus is 12 nt, the number of consecutive bridged nucleic acids at the 3'-terminus is preferably three or less, more preferably two or less, and even more preferably one.

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

[0068] (J) One or more internucleotide phosphodiester bonds are substituted with phosphate-modified bonds. In the present invention, a phosphate-modified bond refers to a bond in which some atoms in a phosphodiester bond are substituted with another atom or substituent, such as a sulfur atom, a boron atom, a nitrogen atom, a methyl group, or an ester group. Specific examples of phosphate-modified bonds include phosphorothioate bonds, methylphosphate bonds, boranophosphate bonds, and mesylphosphoramidate bonds. In the non-natural polynucleotide of the present invention, the number and position of phosphate-modified bonds are not particularly limited. Furthermore, the non-natural polynucleotide of the present invention may contain one type of phosphate-modified bond, or two or more types may be present in combination. The number of phosphate-modified bonds may be one, and the proportion of phosphate-modified bonds in the phosphodiester bonds may be 60% or less. However, from the viewpoint of editing efficiency, it is preferable that not all phosphodiester bonds are substituted.

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

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

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

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

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

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

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

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

[0077] [Modifications of the Sugar Moiety of Nucleic Acid] In any of the above aspects (i) to (iii), or in addition to the above aspects (E), (F), (G), (J), (M), (N), (O), (X2), and / or (P), the non-natural polynucleotide of the present invention can further enhance the editing efficiency of the target nucleotide sequence by modifying the sugar moiety of the nucleic acid, for example, by substituting (X1) the 3'-terminal nucleotide and / or one or more nucleotides adjacent to the 3'-terminal nucleotide with a nucleic acid modified at the 2' position. Examples of nucleic acids modified at the 2' position include at least one selected from the group consisting of 2'-F, 2'-OMe, 2'-MOE, and 2'-O-(2-carbamoylethyl).

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

[0079] [Insertion of a Nucleotide Between the Mismatched Nucleotide and the 3'-Terminal Nucleotide] In any of the above aspects (i) to (iii), or in addition to the above aspects (E), (F), (G), (J), (M), (N), (O), (X2), (P), (X1), and / or (X4), the non-natural polynucleotide of the present invention can further improve the editing efficiency of a target nucleotide sequence by (X5) inserting one or more nucleotides between the mismatched nucleotide and the 3'-terminus nucleotide. The "mismatched nucleotide" in (X5) refers to a mismatched nucleotide intended for modification. Furthermore, although the inserted nucleotide in (X5) is a mismatched nucleotide, this mismatched nucleotide is intended for the purpose of improving editing efficiency and does not fall under the category of a mismatched nucleotide intended for modification. Furthermore, the inserted nucleotide may be the above-mentioned nucleic acid with a modified phosphate moiety, the above-mentioned nucleic acid with a modified sugar moiety, or the above-mentioned nucleic acid with a modified base moiety. In (X5), the number of inserted nucleotides may be one, and when there are multiple nucleotides, it is preferably two or more, and on the other hand, it is preferably six or less, more preferably three or less.

[0080] (4) Aspect (iv) A further aspect of the non-natural polynucleotide of the present invention (Aspect (iv)) is one in which: (A) one or more nucleotides adjacent to the 5'-terminal upstream of the mismatched nucleotide are bridged nucleic acids, and / or (B) one or more nucleotides adjacent to the 3'-terminal downstream of the mismatched nucleotide are bridged nucleic acids; (D) the chain length is 22 to 95 nucleotides; and (H2) the phosphodiester bond between the 5'-terminal nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide is replaced with a phosphate-modified bond; (Y1) the 5'-terminal nucleotide is a mismatched nucleotide, and one or more nucleotides adjacent to the 5'-terminal nucleotide are also mismatched nucleotides; and (Y3) an adaptor is attached to the 5'-terminal. This aspect can achieve the effects of the present invention. (A), (B), and (D) in Aspect (iv) are the same as Aspect (i).

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

[0082] [Substitution with a Phosphate Moiety-Modified Bond] In the non-natural polynucleotide of the present invention, in aspect (iv) above, from the viewpoint of editing efficiency, (H2) the phosphodiester bond between the 5'-terminal nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide is preferably substituted with a phosphate moiety-modified bond.

[0083] In the present invention, a phosphate-modified bond refers to a bond in which some atoms in a phosphodiester bond are substituted with another atom or substituent, such as a sulfur atom, a boron atom, a nitrogen atom, a methyl group, or an ester group. Specific examples of phosphate-modified bonds include phosphorothioate bonds, methylphosphate bonds, boranophosphate bonds, and mesylphosphoramidate bonds. In the non-natural polynucleotide of the present invention, the number and position of phosphate-modified bonds are not particularly limited. Furthermore, the non-natural polynucleotide of the present invention may contain one type of phosphate-modified bond, or two or more types may be present in combination. The number of phosphate-modified bonds may be one, and the proportion of phosphate-modified bonds in the phosphodiester bonds may be 60% or less. However, from the viewpoint of editing efficiency, it is preferable that not all phosphodiester bonds are substituted.

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

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

[0086] [RNA replacement] The non-natural polynucleotide of the present invention is a single-stranded polynucleotide, and deoxyribose in one or more nucleotides may be replaced with ribose. Specifically, in embodiment (iv), for example, if the pentose in the 5'-terminal nucleotide (Y2) is ribose and the pentose in one or more nucleotides adjacent to the 5'-terminal nucleotide is also ribose, the editing efficiency of the target nucleotide sequence can be further improved.

[0087] (5) Aspect (v) A further aspect (aspect (v)) of the non-natural polynucleotide of the present invention is configured as follows: (A) one or more nucleotides adjacent to the 5' upstream side of the mismatched nucleotide are bridged nucleic acids, and / or (B) one or more nucleotides adjacent to the 3' downstream side of the mismatched nucleotide are bridged nucleic acids; (D) the chain length is 22 to 95 nucleotides; and (X7) one or more nucleotides located between the mismatched nucleotide and the 5'-terminal nucleotide are bridged nucleic acids. This configuration can exert the effects of the present invention. (A), (B), and (D) in aspect (v) are the same as in aspect (i).

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

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

[0090] (6) Aspect (vi) A further aspect (aspect (vi)) of the non-natural polynucleotide of the present invention is configured as follows: (C) the 5'-terminal nucleotide is a bridged nucleic acid; (D) the chain length is 22 to 95 nucleotides; (I) the phosphodiester bond between the 3'-terminal nucleotide and one or more nucleotides adjacent to the 3'-terminal nucleotide is substituted with a phosphorothioate bond; and (X6) the mismatch nucleotide is a bridged nucleic acid. This configuration can exhibit the effects of the present invention. (C), (D), and (I) in aspect (vi) are the same as aspect (i).

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

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

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

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

[0095] <Kit> In the present invention, a "kit" refers to a genetic engineering tool used to modify a target nucleotide sequence containing a non-natural polynucleotide. The kit of the present invention may also contain buffers, stabilizers, preservatives, other reagents, instructions describing a protocol for modifying a target nucleotide sequence with a non-natural polynucleotide, and the like, provided that the effects of the present invention are not impaired.

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

[0097] The pharmaceutical compositions of the present invention can be administered, for example, orally, intravenously, intramuscularly, via the oral mucosa, rectally, vaginally, transdermally, nasally, or via inhalation. Alternatively, ex vivo therapy can be used in which the pharmaceutical compositions of the present invention are applied to cells extracted from a patient, and the treated cells are optionally cultured and then returned to the patient. The non-natural polynucleotides of the pharmaceutical compositions of the present invention may be formulated alone or in combination with other components, or may be formulated with a pharmaceutically acceptable carrier or formulation additive. Pharmaceutically acceptable carriers or additives include, but are not limited to, lipid nanoparticles, excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, solubilizers, solubilizers, isotonicity agents, pH adjusters, stabilizers, etc.

[0098] Administration of the pharmaceutical compositions of the present invention depends on the severity and responsiveness of the condition being treated, and may last from several days to several months, or until a cure is effected or a diminution of the condition is achieved. Optimal dosages, administration methods, and recurrence frequencies can be determined by those skilled in the art.

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

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

[0101] <Introduction of Non-Natural Polynucleotides into Cells> In the present invention, "introducing a non-natural polynucleotide into a cell" can be carried out according to known methods depending on the cell to be introduced. Known methods for introducing polynucleotides into cells (transfection methods) can be broadly divided into two types: viral vector systems and non-viral vector systems. Viral vector systems are methods for introducing genes into cells using the cell entry mechanism inherent in viruses, and include, but are not limited to, methods using adenoviruses, retroviruses, lentiviruses, etc. as vectors. Non-viral vector systems include, but are not limited to, lipofection, electroporation, microinjection, particle gun technology, etc. Lipofection is a method that utilizes the phenomenon in which a negatively charged polynucleotide is bound to a positively charged cationic liposome to form a complex, and the polynucleotide is taken up into the cell from the cell surface by endocytosis. Electroporation is a method in which a high-voltage pulse is applied directly to cells using a dedicated machine, allowing the polynucleotide to be taken up through small pores on the cell surface. The microinjection method involves placing a substance to be delivered into a glass needle with a tip diameter of approximately 1 μm and directly introducing it into cells. The particle gun method involves co-precipitating gold particles with DNA to coat the surface of the gold particles, and then ejecting the gold particles toward target cells using helium gas pressure or similar. The present invention is a method for modifying a target nucleotide sequence by introducing only a non-natural polynucleotide without introducing a Cas nuclease into cells; from the perspective of safety, non-viral vector-based methods are more preferred. Examples of the methods of the present invention include methods performed inside the human body and methods that include steps performed inside the human body.

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

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

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

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

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

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

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

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

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

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

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

[0113] <Plant Cells> In the present invention, "plants" include, but are not limited to, seed plants, ferns, mosses, algae, and the like. "Plant cells" refer to cells that constitute plants or cells derived from plants. Seed plants include angiosperms and gymnosperms. Angiosperms include dicotyledons and monocotyledons. Dicotyledons include, but are not limited to, morning glory, dandelion, azalea, azalea, eggplant, rapeseed, and pea. Monocotyledons include, but are not limited to, rice, lily, tulip, miscanthus, and corn. Gymnosperms include, but are not limited to, pine, cedar, ginkgo, and cypress. Ferns include, but are not limited to, bracken, fern, Japanese oak, and horsetail. Bryophytes include, but are not limited to, liverwort, hornwort, Polytrichum sphagnum, and Sphagnum. Algae include multicellular algae and unicellular algae. Examples of multicellular algae include, but are not limited to, kelp, wakame, Ulva, Spirogyra, etc. Examples of unicellular algae include, but are not limited to, Chlorella, Euglena, Spirulina, Chlamydomonas, Coccomyxa, Botryococcus, Mikania, and Diatom.

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

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

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

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

[0118] 3. Introduction of non-natural polynucleotides into 293-nLD1 cells. 293-nLD1 cells were cultured at 1x10 4 Cells were placed in a 96-well plate (Nunc® MicroWell TM The cells were seeded onto a 96-well Nunclon Delta-Treated Flat-Bottom Microplate (Thermo Fisher Scientific Inc.) and cultured in DMEM + 10% FBS medium at 37°C in a 5% CO environment. After 24 hours, 0.05 or 0.1 μg of non-natural polynucleotide was transfected using Lipofectamine 3000 according to the standard protocol.

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

[0120] Counting of viable cells Viable cell counts were measured using the CellTiter-Blue® Cell Viability Assay (Promega Corporation) according to the standard protocol. CellTiter-Blue fluorescence was measured using an EnSpire multimode plate reader (PerkinElmer Co., Ltd.). The viable cell count of edited cells was 5 x 10 3 From 2 x 10 5 A calibration curve of CellTiter-Blue measurement values ​​and cell numbers was created using a two-fold dilution series of 293-nLD1 cells between 0.01 and 0.1, and the number of viable cells was calculated based on this calibration curve.

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

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

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

[0124] Example 1-1: Modification of a Target Nucleotide Sequence with a Non-Natural Polynucleotide An experiment was conducted to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells. As described above, the codon (CAA) encoding glutamine 22 (Gln-22) in the luciferase gene introduced into 293-nLD1 cells was point mutated to a stop codon (TAA), resulting in no luciferase activity. Substituting a T for a C in the stop codon (TAA) in the inactive luciferase gene restored luciferase activity in 293-nLD1 cells, allowing modification of the target nucleotide sequence to be detected by luciferase-induced luminescence (Figure 2).

[0125] In this Example 1-1, 11 types of unnatural polynucleotides, GEO-1 to GEO-9, GEO-242, and GEO-11, as well as the unnatural polynucleotide GEO-94, which serves as a negative control and cannot repair inactivating mutations due to the absence of mismatched nucleotides, were transfected into 293-nLD1 cells (Figure 3A).

[0126] In GEO-1 to GEO-9, GEO-242, and GEO-11, the nucleotide at the position corresponding to the T in the stop codon TAA in the target polynucleotide sequence is G, i.e., a mismatch, and the nucleotides other than the mismatch nucleotide are nucleotides complementary to the target polynucleotide sequence. GEO-1 to GEO-4 are 21 nt long, and GEO-5 to GEO-9, GEO-242, GEO-11, and GEO-94 are 25 nt long. In GEO-1 and GEO-5, the mismatch nucleotide G is LNA. In GEO-2 and GEO-6, one nucleotide 5' upstream and one nucleotide 3' downstream adjacent to the mismatch nucleotide G are LNA. In GEO-3 and GEO-7, the mismatch nucleotide G and the 5' terminal nucleotide are LNA. In GEO-4 and GEO-8, one nucleotide 5' upstream and one nucleotide 3' downstream adjacent to the mismatch nucleotide G are LNA, and the 5' terminal nucleotide is LNA. GEO-11 has an LNA at the 5' end. GEO-242 has an LNA at the 3' end. GEO-9 has no LNA.

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

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

[0129]

[0130] In Table 1, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotides substituted with bridged nucleic acids are shown in capital letters. All of the bridged nucleic acids in Table 1 were LNAs.

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

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

[0133] Example 1-2 Chain Length of Non-Natural Polynucleotides GEO-4 and GEO-8 in Example 1-1 showed a large difference in editing efficiency with a difference of 4 nt, despite the same LNA configuration. In this Example 1-2, to investigate the preferred chain length of non-natural polynucleotides, an experiment similar to that in Example 1-1 was performed using nine non-natural polynucleotides with the same LNA configuration as GEO-8 but different chain lengths (Figure 4). The nine non-natural polynucleotides used in this Example 1-2 were GEO-23 (29 nt), GEO-79 (31 nt), GEO-24 (35 nt), GEO-25 (41 nt), GEO-174 (45 nt), GEO-175 (49 nt), GEO-176 (53 nt), GEO-281 (75 nt), and GEO-282 (95 nt). The base sequence, chain length and relative editing efficiency of each of the above edited nucleic acids are as shown in Table 2.

[0134]

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

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

[0137] Example 1-3: LNA Modification of the 3' End of a Non-Natural Polynucleotide In this Example 1-3, the editing efficiency of a construct in which the pentose sugar in the 3'-terminal nucleotide was replaced with LNA was examined. Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-16, in which the 3'-terminal nucleotide of GEO-6 is LNA, and GEO-17, in which the 3'-terminal nucleotide of GEO-8 is further LNA (Figure 5). The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are shown in Table 3.

[0138]

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

[0140] As shown in Table 3, the relative editing efficiency of GEO-16 was 0.16, and that of GEO-17 was 2.00. These results indicated that the configuration in which one nucleotide adjacent to the mismatched nucleotide on the 5' upstream side and one nucleotide adjacent to the 3' downstream side were LNA and the 3' terminus was LNA (GEO-16) had a low relative editing efficiency. It was also shown that the relative editing efficiency was improved when the 3' terminus was LNA in the configuration in which one nucleotide adjacent to the mismatched nucleotide on the 5' upstream side and one nucleotide adjacent to the 3' downstream side were LNA. On the other hand, when the 5' terminus was unmodified (GEO-16), even though the 3' terminus was LNA, the editing efficiency was comparable to that of GEO-6, indicating insufficient editing efficiency. This indicates that it is important for the 5' terminus nucleotide to be a bridged nucleic acid such as LNA in order to achieve sufficient editing efficiency.

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

[0142]

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

[0144] As shown in Table 4, the relative editing efficiency of GEO-18 was 2.05, and that of GEO-19 was 0.66. These results indicated that sufficient editing efficiency was observed even in the GEO-8 configuration, where one or more nucleotides adjacent to the 5' end were further replaced with LNA. Furthermore, the relative editing efficiency of GEO-17 was 2.00, and that of GEO-68 was 1.64. These results indicated that the GEO-17 configuration, where one nucleotide 5' upstream and one nucleotide 3' downstream adjacent to the mismatched nucleotide were LNA, the 5' end was LNA, and the 3' end was LNA (hereinafter referred to as the "GEO-17 configuration"), also showed sufficient editing efficiency even in the configuration where one nucleotide adjacent to the 5' end was further replaced with LNA.

[0145] The relative editing efficiency of GEO-19, in which two nucleotides adjacent to the 5' end of the GEO-8 structure were further replaced with LNA, was 0.66, a slightly lower relative editing efficiency than that of the GEO-8 structure. On the other hand, in Examples 1-8 described below, when the 5' upstream side of the mismatched nucleotide was extended, the relative editing efficiency of GEO-313, in which one nucleotide adjacent to the 5' end of the GEO-8 structure was further replaced with LNA, was 5.23, and the relative editing efficiency of GEO-315, in which two nucleotides adjacent to the 5' end of the GEO-8 structure were further replaced with LNA, was 6.59. These results suggest that even when multiple nucleotides adjacent to the 5' end of the GEO-8 structure are further replaced with LNA, the editing efficiency may be increased if other structural changes, such as a change in the chain length of the non-natural polynucleotide, are made.

[0146] Example 1-5: LNA Modification of One or More Nucleotides Adjacent to the LNA at the 3' End of a Non-Natural Polynucleotide In this Example 1-5, the editing efficiency of a configuration in which one or more nucleotides adjacent to the 3'-terminal nucleotide of the GEO-17 configuration of Example 1-3 were further replaced with LNA was examined. Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-69, in which one nucleotide adjacent to the 3'-terminal nucleotide of GEO-17 is further LNA, and GEO-71, in which two consecutive nucleotides adjacent to the 3'-terminal nucleotide of GEO-17 are further LNA (Figure 7). The base sequence, chain length, and relative editing efficiency of each of the above-mentioned edited nucleic acids are as shown in Table 5.

[0147]

[0148] In Table 5, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted by the bridged nucleic acid is shown in capital letters. All of the bridged nucleic acids in Table 5 were LNAs.

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

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

[0151] Example 1-6: Substitution of phosphodiester bonds between one or more nucleotides contained in a non-natural polynucleotide with phosphorothioate bonds (1) In this Example 1-6, the editing efficiency of a configuration in which the phosphodiester bonds between one or more nucleotides in the GEO-8 configuration of Example 1-1 were further substituted with phosphorothioate bonds was examined.

[0152] Specifically, GEO-158, GEO-159, GEO-160, GEO-29, and GEO-161 (FIG. 8A) are GEO-8 in which the phosphodiester bond between the 5'-terminal nucleotide and 1 to 5 consecutive nucleotides adjacent to the 5'-terminal nucleotide has been replaced with a phosphorothioate bond; GEO-162, GEO-163, GEO-164, GEO-30, GEO-165, GEO-272, and GEO-196 (FIG. 8B) are GEO-8 in which the phosphodiester bond between the 3'-terminal nucleotide and 1 to 7 consecutive nucleotides adjacent to the 3'-terminal nucleotide has been replaced with a phosphorothioate bond; and GEO-8 in which the phosphodiester bond between the 5'-terminal nucleotide and 4 consecutive nucleotides adjacent to the 5'-terminal nucleotide has been replaced with a phosphorothioate bond. The editing efficiency was measured in the same manner as in Example 1-1 using GEO-31 ( FIG. 8C ), in which the phosphodiester bond between the 3'-terminal nucleotide and the four consecutive nucleotides adjacent to the 3'-terminal nucleotide was replaced with a phosphorothioate bond; GEO-126 and GEO-127 ( FIG. 8C ), in which the phosphodiester bond between the 5'-upstream LNA adjacent to the mismatch nucleotide G and the two or four consecutive nucleotides adjacent to the LNA was replaced with a phosphorothioate bond in GEO-8; and GEO-128 and GEO-129 ( FIG. 8C ), in which the phosphodiester bond between the nucleotide adjacent to the 3'-downstream LNA adjacent to the mismatch nucleotide G and the two or four consecutive nucleotides adjacent to the adjacent nucleotide in GEO-8 was replaced with a phosphorothioate bond. The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are shown in Table 6.

[0153]

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

[0155] As shown in Table 6, the relative editing efficiency of GEO-158 was 2.40, that of GEO-159 was 3.19, that of GEO-160 was 3.02, that of GEO-29 was 2.86, and that of GEO-161 was 2.27. These results demonstrated that in the GEO-8 configuration, the editing efficiency was improved when the phosphodiester bond between the 5'-terminal nucleotide and the 1st to 5th nucleotides adjacent to the 5'-terminal nucleotide was replaced with a phosphorothioate bond.

[0156] Furthermore, the relative editing efficiency of GEO-162 was 3.64, that of GEO-163 was 2.04, that of GEO-164 was 3.08, that of GEO-30 was 4.17, that of GEO-165 was 4.24, that of GEO-272 was 3.40, and that of GEO-196 was 3.97. These results indicate that in the GEO-8 configuration, the editing efficiency is improved when the phosphodiester bond between the 3'-terminal nucleotide and nucleotides 1 to 7 adjacent to the 3'-terminal nucleotide is replaced with a phosphorothioate bond.

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

[0158] Furthermore, as shown in Table 6, the relative editing efficiency of GEO-126 was 0.99, and that of GEO-127 was 0.70. These results demonstrate that in the GEO-8 configuration, when the phosphodiester bond between the LNA adjacent to the 5' upstream side of the mismatched nucleotide and the two or four consecutive nucleotides further adjacent to the LNA is replaced with a phosphorothioate bond, the editing efficiency is comparable to that of GEO-8.

[0159] Furthermore, Table 6 shows that the relative editing efficiency of GEO-128 was 0.55, and that of GEO-129 was 2.15. These results indicate that in the GEO-8 configuration, when the phosphodiester bond between two adjacent nucleotides 3' downstream of the LNA adjacent to the mismatched nucleotide was replaced with a phosphorothioate bond, the editing efficiency was half that of GEO-8, and when the phosphodiester bond between four adjacent nucleotides 3' downstream of the LNA adjacent to the mismatched nucleotide was replaced with a phosphorothioate bond, the editing efficiency was improved. Note that, as shown in Table 6 and Figure 8C, in GEO-128 and GEO-129, the phosphodiester bond between the LNA adjacent to the 3' downstream of the mismatched nucleotide and the nucleotide directly adjacent to the LNA was not replaced with a phosphorothioate bond.

[0160] Example 1-7: Substitution of Phosphorothioate Linkages for One or More Internucleotide Phosphodiester Linkages in a Non-Natural Polynucleotide (2) In this Example 1-7, the editing efficiency of a configuration in which the phosphodiester linkages between one or more nucleotides in the GEO-17 configuration of Example 1-3 were further substituted with phosphorothioate linkages was examined. Specifically, GEO-155, GEO-167, GEO-168, and GEO-169, which are GEO-17 variants in which the phosphodiester linkages between the 3'-terminal nucleotide and the 1 to 4 consecutive nucleotides adjacent to the 3'-terminal nucleotide were further substituted with phosphorothioate linkages, were used to measure the editing efficiency in the same manner as in Example 1-1 (Figure 9). The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are listed in Table 7.

[0161]

[0162] In Table 7, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted with a bridged nucleic acid is indicated in uppercase. All bridged nucleic acids in Table 7 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides.

[0163] As shown in Table 7, the relative editing efficiency of GEO-155 was 3.63, that of GEO-167 was 6.05, that of GEO-168 was 5.81, and that of GEO-169 was 2.90. These results demonstrated that in the GEO-17 configuration, the editing efficiency was improved when the phosphodiester bond between the 3'-terminal nucleotide and the 1st to 4th nucleotides adjacent to the 3'-terminal nucleotide was replaced with a phosphorothioate bond.

[0164] Example 1-8: Extension of chain length 5' upstream and 3' downstream of mismatched nucleotide and position of LNA In this Example 1-8, when a non-naturally occurring polynucleotide contained one mismatched nucleotide, the effect on editing efficiency of changing the chain length of the 5' upstream region and the chain length of the 3' downstream region of the mismatched nucleotide was investigated. Furthermore, the editing efficiency was investigated when the number and position of LNAs near the 5' end and the number and position of LNAs near the 3' end were appropriately changed.

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

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

[0167]

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

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

[0170] Comparison of GEO-8 and GEO-311 showed that extending the length of the 5' upstream region of the mismatched nucleotide increased the editing efficiency. Comparison of GEO-313 and GEO-18 in Example 1-4, and GEO-315 and GEO-19 in Example 1-4, showed that extending the length of the 5' upstream region of the mismatched nucleotide increased the editing efficiency, even when multiple nucleotides adjacent to the 5'-terminal nucleotide were LNA.

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

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

[0173] Comparing GEO-317 with GEO-318, and GEO-319 with 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, replacing the 12th nucleotide 3' downstream from the nucleotide 3' downstream adjacent to the mismatch nucleotide G with LNA has little effect on editing efficiency.

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

[0175]

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

[0177] As shown in Table 9, the relative editing efficiency of GEO-150 was 1.65, that of GEO-73 was 4.20, that of GEO-151 was 0.78, and that of GEO-74 was 1.12. These results showed that in the GEO-17 configuration, when one or two nucleotides adjacent to the 3'-terminal nucleotide were substituted with RNA, the editing efficiency was improved compared to GEO-8, and when three or four nucleotides were substituted with RNA, the editing efficiency was comparable to that of GEO-8.

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

[0179]

[0180] In Table 10, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted with the bridged nucleic acid is indicated in uppercase. All bridged nucleic acids in Table 10 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. Furthermore, to indicate that the 5'-end and / or 3'-end were phosphorylated, a "p" was added to the 5'-end and / or 3'-end.

[0181] As shown in Table 10, the relative editing efficiencies of GEO-26, GEO-27, and GEO-28 were 1.14, 2.23, and 2.91, respectively. The relative editing efficiencies of GEO-223 and GEO-224 were 2.70 and 2.29, respectively. These results indicate that in the GEO-8 construct, phosphorylation of only the 5' end did not affect editing efficiency, but phosphorylation of the 3' end improved editing efficiency. Furthermore, phosphorylation of both the 5' and 3' ends showed higher editing efficiency than phosphorylation of only the 3' end. Similarly, phosphorylation of the 3' end also improved editing efficiency in the GEO-17 construct. In this case, replacing the phosphodiester bond between the 3'-terminal nucleotide and the adjacent nucleotide with a phosphorothioate bond did not result in an increase in editing efficiency.

[0182] Example 1-11: Types of Bridged Nucleic Acids In this Example 1-11, the editing efficiency of the GEO-8 structure of Example 1-1, in which the LNA at the 5' end was replaced with another bridged nucleic acid, was examined. Specifically, the editing efficiency was measured in the same manner as in Example 1-1 using GEO-41, in which the LNA at the 5' end of GEO-8 was replaced with BNA-NH; GEO-43, in which the LNA at the 5' end was replaced with BNA-N-Me; and GEO-57, in which the LNA at the 5' end was replaced with ENA (Figure 13). The base sequence, chain length, and relative editing efficiency of each of the above editing nucleic acids are shown in Table 11.

[0183]

[0184] In Table 11, the mismatched nucleotide, guanosine monophosphate, is underlined, and the nucleotide substituted with the bridged nucleic acid is indicated in uppercase. The type of bridged nucleic acid in each edited nucleic acid in Table 11 is as described above.

[0185] As shown in Table 11, the relative editing efficiency of GEO-41 was 1.38, that of GEO-43 was 1.51, and that of GEO-57 was 1.57. These results indicate that in the structure of GEO-8, the 5' end may be replaced with not only LNA but also other bridged nucleic acids such as BNA-NH, BNA-N-Me, and ENA, and that such replacement may actually increase the editing efficiency.

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

[0187]

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

[0189] As shown in Table 12, the relative editing efficiency of GEO-226 was 8.19. This result indicated that the editing efficiency was significantly increased by further combining the substitution of phosphorothioate bonds for phosphodiester bonds and phosphorylation of the 3'-end in the GEO-8 construct.

[0190] Example 1-13 Combination of Nucleic Acid Modifications (2) In this Example 1-13, the editing efficiency of a configuration in which the GEO-23 configuration of Example 1-3 was further combined with the substitution of phosphorothioate linkages for phosphodiester bonds, RNA substitution, and phosphorylation of the 3' end was examined. Specifically, the editing efficiency was measured using GEO-172, which was a GEO-23 construct in which the phosphodiester linkages between four consecutive nucleotides adjacent to the 5'-terminal nucleotide were substituted with phosphorothioate linkages, two consecutive nucleotides adjacent to the 3'-terminal nucleotide were substituted with RNA, and the 3' end was phosphorylated (Figure 15). The base sequence, chain length, and relative editing efficiency of each of the above edited nucleic acids are listed in Table 13.

[0191]

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

[0193] As shown in Table 13, the relative editing efficiency of GEO-172 was 8.48. This result indicated that the editing efficiency was significantly increased by further combining the substitution of phosphorothioate bonds for phosphodiester bonds, RNA substitution, and 3'-end phosphorylation in the GEO-17 construct.

[0194] Discussion The above Examples 1-1 to 1-13 of the present invention are compared with the disclosures in patent documents and non-patent documents. (1) Example 1-1 shows that GEO-8, a non-natural polynucleotide of the present invention, exhibits an editing efficiency that is at least five times higher than that of GEO-7, which corresponds to the polynucleotide with the highest editing efficiency among the configurations disclosed in Non-Patent Document 1 (i.e., the chain length, mismatch nucleotide position, and LNA position are identical to those of GEO-8). Comparing GEO-8 and GEO-7, GEO-8 of the present invention differs in that the mismatch nucleotide itself is not LNA, but both the 5'-side nucleotide and the 3'-side nucleotide adjacent to the mismatch nucleotide are substituted with LNA.

[0195] In this regard, Patent Document 4 states that oligonucleotides containing an LNA adjacent to the mismatched nucleotide did not show any improvement, but oligonucleotides having an LNA spaced one nucleotide further from the mismatch showed an average increase of 5-fold, and it can be said that Patent Document 4 actively excludes configurations containing an LNA adjacent to the mismatched nucleotide.

[0196] Non-Patent Document 1 focuses on avoiding mismatch repair, and Non-Patent Documents 1 and 2 further focus on elucidating the mechanism by which single-stranded synthetic DNAs containing LNA mismatch nucleotides modify the genome of mammalian cells. Of the more than 60 types of single-stranded synthetic DNAs tested in Non-Patent Document 1, only one had both the 5'- and 3'-nucleotides adjacent to the mismatch nucleotide substituted with LNA, while all of the more than 50 types of single-stranded DNAs tested in Non-Patent Document 2 had the mismatch nucleotide itself as LNA, and no single-stranded DNAs in which both the 5'- and 3'-nucleotides adjacent to the mismatch nucleotide were substituted with LNA were tested. Furthermore, the discussion in Non-Patent Document 1 emphasizes the importance of using LNA for the mismatch nucleotide itself in avoiding mismatch repair, but does not mention DNAs in which both the 5'- and 3'-nucleotides adjacent to the mismatch nucleotide are substituted with LNA.

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

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

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

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

[0201] (2) Examples 1-2 demonstrated that a certain level of editing efficiency was observed when the chain length of the non-natural polynucleotide was between 25 and 95 nt. Regarding chain length, many previous studies have suggested that a chain length of approximately 25 bases is preferable and that longer chain lengths result in lower effectiveness. However, the present invention differs from existing research in that it is highly effective even when the chain length is longer. Specifically, Non-Patent Document 1 investigated the optimal length of an edited nucleic acid using a polynucleotide in which only the mismatch nucleotides were LNAs, and found that a length of 25 bases was optimal, with the results showing that editing efficiency decreased with lengths longer than 25 bases. Furthermore, Patent Document 4 used a polynucleotide with a length of 24 bases. On the other hand, it was discovered that non-natural polynucleotides of the present invention exhibit even higher editing efficiency with lengths longer than 25 bases.

[0202] (3) Examples 1-3 demonstrated that for a gene in which both the 5'- and 3'-terminal nucleotides adjacent to a mismatched nucleotide are substituted with LNA, the characteristic (C) of the 5'-terminal nucleotide being a bridged nucleic acid is important for the gene to be effective. Furthermore, comparison with GEO-8, GEO-16, and GEO-17 revealed that when both the 5'- and 3'-terminal nucleotides are bridged nucleic acids, the editing efficiency increased two-fold. Furthermore, it was found that the effect of the 3'-terminal nucleotide being an LNA is more pronounced when the 5'-terminal nucleotide is an LNA. (4) Examples 1-4 and 1-5 demonstrated sufficient editing efficiency even when one or more nucleotides adjacent to the 5'- or 3'-terminus are substituted with LNA, and that the editing efficiency may be improved depending on the position and number of nucleotides substituted with LNA. Such an effect was not described or suggested in any of the above patent documents or non-patent documents.

[0203] (5) Examples 1-6 and 1-7 demonstrated that in a sample in which both the 5'- and 3'-nucleotides adjacent to the mismatched nucleotide are substituted with LNA, the editing efficiency is improved when the phosphodiester bond between the 5'- or 3'-terminal nucleotide and the 1st to 7th nucleotides adjacent thereto is substituted with a phosphorothioate bond. Since the editing efficiency does not decrease even when the number of phosphorothioate bonds is increased by one on both the 5'- and 3'-terminal sides, it is likely that sufficient editing efficiency is exhibited even when the number of phosphodiester bonds is 8 or more. This effect was not described or suggested in any of the above-mentioned patent documents or non-patent documents. Furthermore, the present inventors have made a completely new discovery in this specification that sulfonation from both ends particularly strongly improves the editing effect when the 5'- or 3'-end is LNA.

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

[0205] (7) Examples 1-9, 1-10, 1-12, and 1-13 demonstrated that the editing efficiency can be significantly increased by combining the substitution of crosslinked nucleic acids with modifications such as substitution of phosphodiester bonds with phosphorothioate bonds, RNA substitution, and 3'-end or 5'-end phosphorylation. Such an effect was not described or suggested in any of the above patent documents or non-patent documents.

[0206] (8) Examples 1-11 demonstrated that substitution of the 5'-end with a bridged nucleic acid not only with LNA but also with other bridged nucleic acids such as BNA-NH, BNA-N-Me, and ENA can improve editing efficiency. Such an effect was not described or suggested in any of the above patent documents or non-patent documents.

[0207] B. Genome editing experiments using 293-nLD2 cells, 293-nLD3 cells, 293-nLD4-1 cells, 293-nLD4-2 cells, 293-nLD4-3 cells, 293-nLD5 cells, 293-nLD6 cells, 293-nLD7 cells, 293-nLD8 cells, or 293-nLD9 cells 1. Preparation of various mutant NanoLuc (registered trademark) plasmids (1) Mutant base sequences were synthesized in which the guanine (G) at base number 894 of the nDL2 NanoLuc gene was changed to adenine (A) and the cytosine (C) at base number 895 was changed to thymine (T), and the mutant base sequences were transformed into pcDNA 1616 in the same manner as above. TM We created a mutant (pcDNA5-nLD2) containing the luciferase gene integrated into the 5 / FRT / TO plasmid. The luciferase gene contained in this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the above mutation.

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

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

[0210] (4) A mutant base sequence was synthesized in which the cytosine (C) at base number 922 of the nDL4-2 NanoLuc gene was changed to thymine (T) and the guanine (G) at base number 937 was changed to thymine (T), and the mutant base sequence was then transformed into pcDNA in the same manner as above.TM We created a mutant (pcDNA5-nLD4-2) by incorporating the luciferase gene into the 5 / FRT / TO plasmid. The luciferase gene in this mutant is an inactive luciferase gene that does not exhibit luciferase activity due to the point mutation described above.

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

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

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

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

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

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

[0217] 2. Creation of stable transformed cell lines carrying various mutant NanoLuc genes. TM -293 cell line (Thermo Fisher Scientific Inc.) at 1x10 6Cells were seeded onto 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 in a 5% CO2 environment. After 24 hours, the cells were transfected with pcDNA5-nLD2, pcDNA5-nLD3, pcDNA5-nLD4-1, pcDNA5-nLD4-2, pcDNA5-nLD4-3, pcDNA5-nLD5, pcDNA5-nLD6, pcDNA5-nLD7, pcDNA5-nLD8, or pcDNA5-nLD9 plasmid (1 μg) and pOG44 plasmid (3 μg, Thermo Fisher Scientific Inc.) using Lipofectamine 3000 (Thermo Fisher Scientific Inc.) according to the standard protocol. After 48 hours, the cells were transfected with TrypLE. TM Express Enzyme (Thermo Fisher Scientific Inc.) was added and incubated at 37°C for 3 minutes. The detached cells were collected and suspended in DMEM + 10% FBS medium containing 50 μg / ml hygromycin (Thermo Fisher Scientific Inc.). The cells were then divided into two 10 cm dishes and cultured. Culture was continued with the same medium every three days. After approximately 20 days of culture, the formation of sufficient colonies and colonies of sufficient size was confirmed, and all cells were treated with TrypLE. TM The cells were detached from the petri dish using Express Enzyme and collected.

[0218] The cell lines obtained using the various plasmids listed above were named as follows: Cells with pcDNA5-nLD2 integrated into the genome were named 293-nLD2 cells. Cells with pcDNA5-nLD3 integrated into the genome were named 293-nLD3 cells. Cells with pcDNA5-nLD4-1 integrated into the genome were named 293-nLD4-1 cells. Cells with pcDNA5-nLD4-2 integrated into the genome were named 293-nLD4-2 cells. Cells with pcDNA5-nLD4-3 integrated into the genome were named 293-nLD4-3 cells. Cells with pcDNA5-nLD5 integrated into the genome were named 293-nLD5 cells. Cells with pcDNA5-nLD6 integrated into the genome were named 293-nLD6 cells. Cells with pcDNA5-nLD7 integrated into the genome were named 293-nLD7 cells. The cells with pcDNA5-nLD8 integrated into the genome were named 293-nLD8 cells. The cells with pcDNA5-nLD9 integrated into the genome were named 293-nLD9 cells. The mutant NanoLuc gene contained in each of these cells was an inactive luciferase gene, as described above. These cells were used in subsequent genome editing experiments.

[0219] 3. Introduction of non-natural polynucleotides into 293-nLD2-9 cells. 293-nLD2 cells were cultured at 1x10 4 Cells were placed in a 96-well plate (Nunc® MicroWell TMCells were seeded onto 96 Nunclon Delta-Treated Flat-Bottom Microplates (Thermo Fisher Scientific Inc.) and cultured in DMEM + 10% FBS medium at 37°C in a 5% CO environment. After 24 hours, the cells were transfected with 0.0125 μg, 0.025 μg, 0.050 μg, 0.100 μg, or 0.200 μg of unnatural polynucleotides using Lipofectamine 3000 according to standard protocols. 293-nLD3, 293-nLD4-1, 293-nLD4-2, 293-nLD4-3, 293-nLD5, 293-nLD6, 293-nLD7, 293-nLD8, and 293-nLD9 cells were transfected in the same manner as for 293-nLD2 cells.

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

[0221] Counting of viable cells Viable cell counts were measured using the CellTiter-Blue® Cell Viability Assay (Promega Corporation) according to the standard protocol. The fluorescence value of CellTiter-Blue was measured using an EnSpire multimode plate reader (PerkinElmer Co., Ltd.). The viable cell count of edited cells was 5 x 10 3 From 2 x 10 5A standard curve of CellTiter-Blue measurement values ​​and cell counts was created using a two-fold dilution series of 293-nLD2 cells, and the viable cell count was calculated based on this standard curve. The viable cell counts of 293-nLD3, 293-nLD4-1, 293-nLD4-2, 293-nLD4-3, 293-nLD5, 293-nLD6, 293-nLD7, 293-nLD8, and 293-nLD9 cells were calculated in the same manner as for 293-nLD2.

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

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

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

[0225] Example 2-1 Examination of Two-Base Substitution (1) An experiment was carried out to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD2 cells.

[0226] In this example, five types of unnatural polynucleotides, D2-1 to D2-6, were transfected into 293-nLD2 cells. The base sequence, chain length, and relative editing efficiency of each of the edited nucleic acids are shown in Table 14.

[0227]

[0228] In Table 14, mismatched nucleotides are underlined, and nucleotides replaced with bridged nucleic acids are indicated in capital letters. All bridged nucleic acids in Table 14 were LNA. Where a phosphodiester bond between two or more nucleotides was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding two or more nucleotides. The structure of D2-2 is the same as that reported in Non-Patent Document 1. The relative editing efficiency was calculated using the AUC value of D2-2 as the reference (1.00), and is shown in Table 14. When Tris-EDTA (Tris-HCl 10 mM, EDTA 1 mM, pH 7.4, hereinafter referred to as "TE") was used as a negative control, the AUC was 123, and the AUC of D2-2 was 64,642. As shown in Table 14, the relative editing efficiency of D2-1 was 0.13, that of D2-3 was 4.94, that of D2-4 was 3.13, that of D2-5 was 1.71, and that of D2-6 was 1.64. These results demonstrate that the non-natural polynucleotides of the present invention can modify two nucleotides contained in a target nucleotide sequence.

[0229] Example 2-2: Examination of Three-Base Substitution An experiment was carried out to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD3 cells.

[0230] In this example, four types of unnatural polynucleotides, D3-1 to D3-5, were transfected into 293-nLD3 cells. The base sequence, chain length, and relative editing efficiency of each of the edited nucleic acids are shown in Table 15.

[0231]

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

[0233] All of the structures shown in Table 15 contain three base substitutions, a finding that has not been previously reported. The relative editing efficiencies were calculated using the AUC value of D3-2 as the reference (1.00), and are shown in Table 15. When TE was used as a negative control, the AUC was 119, while the AUC of D3-2 was 14,436. As shown in Table 15, the relative editing efficiencies of D3-1 were 0.23, D3-3 3.44, D3-4 2.47, and D3-5 7.89. These results demonstrate that the non-natural polynucleotides of the present invention can modify three nucleotides in a target nucleotide sequence.

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

[0235] In experiments using 293-nLD4-1 cells, three types of unnatural polynucleotides, D4-1-1 to D4-1-4, were transfected into 293-nLD4-1 cells. In experiments using 293-nLD4-2 cells, three types of unnatural polynucleotides, D4-2-1 to D4-2-4, were transfected into 293-nLD4-2 cells. In experiments using 293-nLD4-3 cells, three types of unnatural polynucleotides, D4-3-1 to D4-3-4, were transfected into 293-nLD4-3 cells. The base sequence, chain length, and relative editing efficiency of each of the above editing nucleic acids are shown in Table 16.

[0236]

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

[0238] The structures shown in Table 16 all involve two base substitutions at distant positions, a phenomenon not previously reported. In D4-1-1 to D4-1-4, the two mismatched nucleotides in the target nucleotide sequence are spaced 8 bases apart. The relative editing efficiencies were calculated using the AUC value of D4-1-2 as the reference (1.00) and are shown in Table 16. When TE was used as the negative control, the AUC was 47.16, and the AUC of D4-1-2 was 14,904. As shown in Table 16, the relative editing efficiencies of D4-1-1 were 0.67, D4-1-3 were 3.19, and D4-1-4 were 9.45. In D4-2-1 to D4-2-4, the two mismatched nucleotides in the target nucleotide sequence are spaced 14 bases apart. The relative editing efficiencies were calculated using the AUC value of D4-2-2 as the reference (1.00) and are shown in Table 16. When TE was used as a negative control, the AUC was 47.16, and the AUC for D4-2-2 was 63,770. As shown in Table 16, the relative editing efficiency for D4-2-1 was 0.04, for D4-2-3 2.83, and for D4-2-4 3.87. In D4-3-1 to D4-3-4, the two mismatched nucleotides in the target nucleotide sequence were spaced 20 bases apart. The relative editing efficiency was calculated using the AUC value for D4-3-2 as the reference (1.00) and is shown in Table 16. When TE was used as a negative control, the AUC was 47.16, and the AUC for D4-3-2 was 104,690. As shown in Table 16, the relative editing efficiency for D4-3-1 was 0.00, for D4-3-3 1.76, and for D4-3-4 4.70.

[0239] These results show that when there are multiple nucleotide modifications in a target nucleotide sequence, the modification sites do not need to be adjacent, and multiple nucleotides in the target nucleotide sequence can be modified simultaneously even if they are at least 20 bases apart.

[0240] Example 2-4: Study of Single Base Insertion An experiment was carried out to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD5 cells.

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

[0242]

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

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

[0245] As shown in Table 17, the relative editing efficiency of the control D5-1 was 0.33, that of D5-4 was 10.44, that of D5-5 was 16.19, and that of D5-6 was 2.11. These results demonstrate that the non-natural polynucleotide of the present invention enables insertion of a single desired base into a deletion site contained in a target nucleotide sequence.

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

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

[0248]

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

[0250] All of the structures shown in Table 18 involve two-base insertions, a phenomenon not previously reported. The relative editing efficiencies were calculated using the AUC value of D6-2 as the reference (1.00), and are shown in Table 18. When TE was used as a negative control, the AUC was 158, and the AUC of D3-2 was 4924. As shown in Table 18, the relative editing efficiencies of D6-1 were 0.60, D6-3 12.62, D6-4 17.70, D6-5 2.29, D6-6 7.52, D6-7 40.12, D6-8 24.11, D6-9 7.66, and D6-10 17.37. These results demonstrate that the non-natural polynucleotides of the present invention can insert desired bases even when the target nucleotide sequence contains two deletion sites.

[0251] Example 2-6: Study of Single-Base Deletion An experiment was carried out to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD7 cells.

[0252] In this example, three types of unnatural polynucleotides, D7-1 to D7-4, were transfected into 293-nLD7 cells. The base sequence, chain length, and relative editing efficiency of each of the edited nucleic acids are shown in Table 19.

[0253]

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

[0255] All of the structures shown in Table 19 have a single-base deletion, and no such examples have been reported previously. The relative editing efficiencies were calculated using the AUC value of D7-2 as the reference (1.00), and are shown in Table 19. When TE was used as a negative control, the AUC was 9964, and the AUC of D7-2 was 12715. As shown in Table 19, the relative editing efficiencies of D7-1 were 0.72, D7-3 were 2.10, and D7-4 were 11.31. These results demonstrate that the non-natural polynucleotides of the present invention can remove desired bases from target nucleotide sequences.

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

[0257] In this example, three types of unnatural polynucleotides, D8-1 to D8-4, were transfected into 293-nLD8 cells. The base sequence, chain length, and relative editing efficiency of each of the edited nucleic acids are shown in Table 20.

[0258]

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

[0260] All of the structures shown in Table 20 have two-base deletions, and no such examples have been reported previously. The relative editing efficiencies were calculated using the AUC value of D7-2 as the reference (1.00), and are shown in Table 19. When TE was used as a negative control, the AUC was 1243, and the AUC of D8-2 was 1303. As shown in Table 20, the relative editing efficiencies of D8-1 were 1.03, D8-3 were 4.84, and D8-4 were 22.95. These results demonstrate that the non-natural polynucleotides of the present invention can remove multiple desired bases from a target nucleotide sequence.

[0261] Example 2-8: Examination of 4-base insertion An experiment was carried out to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD9 cells.

[0262] In this example, four types of unnatural polynucleotides, D9-1, D9-2, D9-3, D9-5, and D9-6, were transfected into 293-nLD9 cells. The base sequence, chain length, and relative editing efficiency of each of the edited nucleic acids are shown in Table 21.

[0263]

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

[0265] The structure of D9-2 is similar to that reported in Non-Patent Document 1. The relative editing efficiency was calculated using the AUC value of D9-2 as the reference (1.00), and is shown in Table 21. When Tris-EDTA (Tris-HCl 10 mM, EDTA 1 mM, pH 7.4, hereinafter referred to as "TE") was used as a negative control, the AUC was 383.8, and the AUC of D9-2 was 18,543. 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. These results demonstrate that the non-natural polynucleotides of the present invention can insert desired bases even when the target nucleotide sequence contains four deletion sites.

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

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

[0268] 1. Introduction of non-natural polynucleotides into 293-nLD1 cells. 293-nLD1 cells were cultured at 1x10 4Cells were placed in a 96-well plate (Nunc® MicroWell TM The cells were seeded onto a 96-well Nunclon Delta-Treated Flat-Bottom Microplate (Thermo Fisher Scientific Inc.) and cultured in DMEM + 10% FBS medium at 37°C in a 5% CO environment. After 24 hours, 0.05 or 0.1 μg of non-natural polynucleotide was transfected using Lipofectamine 3000 according to the standard protocol.

[0269] 2. Measurement of editing efficiency and evaluation of editing efficiency of non-natural polynucleotides by AUC log Measurement and evaluation of editing efficiency were performed using the method described above in "B. Genome editing experiments using 293-nLD2 cells, 293-nLD3 cells, 293-nLD4-1 cells, 293-nLD4-2 cells, 293-nLD4-3 cells, 293-nLD5 cells, 293-nLD6 cells, 293-nLD7 cells, 293-nLD8 cells, or 293-nLD9 cells."

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

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

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

[0273]

[0274]

[0275] In Table 31, mismatched nucleotides are underlined, and nucleotides replaced by bridged nucleic acids are indicated in capital letters. All bridged nucleic acids in Table 31 were LNA. RNA-substituted nucleotides are indicated by an arrow next to the corresponding nucleotide base symbol. Where an internucleotide phosphodiester bond was replaced with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding nucleotides. Non-natural polynucleotides phosphorylated at the 5' and / or 3' ends were marked with a "p" at the 5' and / or 3' end. Furthermore, G(F) and T(F) in GEO-204 indicate 2'-fluoro-modification, A(MT) in GEO-213 indicates 2'-O-methyl modification, and t*(MP)a in GEO-651 indicates that the bond between t and a is a methylphosphate bond.

[0276] As shown in Table 31, the non-natural polynucleotides of the present invention were found to have an editing efficiency four times or more higher than that of the standard GEO-8.

[0277] Example 3-2 Modification of target nucleotide sequence by non-natural polynucleotide with mismatch nucleotide at the 3' end An experiment was carried out to measure the editing efficiency of the target polynucleotide sequence by introducing non-natural polynucleotide into 293-nLD1 cells.

[0278] In this example, each of the non-natural polynucleotides shown in Table 32 was introduced into 293-nLD1 cells by transfection. The base sequence, chain length, and relative editing efficiency of each of the edited nucleic acids are as shown in Table 32. Note that in Table 32, the chain length of the edited nucleic acid body and the chain length of the adapter are listed separately, but each sequence specified as a SEQ ID NO: is the combined length of the edited nucleic acid body and the adapter.

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287] In Table 32, mismatched nucleotides are underlined, and nucleotides substituted with bridged nucleic acids are indicated in capital letters. All bridged nucleic acids in Table 32 were LNA. RNA-substituted nucleotides are indicated by an arrow next to the base symbol of the corresponding nucleotide. Where an internucleotide phosphodiester bond has been replaced with a phosphorothioate bond, an asterisk (*) is inserted between the corresponding nucleotides. Non-natural polynucleotides phosphorylated at the 5' and / or 3' ends are marked with a "p" at the 5' and / or 3' end. Furthermore, [t], [tt], and [ttt] indicate the insertion of one, two, and three thymidine monophosphates, respectively; [a], [aa], and [aaa] indicate the insertion of one, two, and three adenosine monophosphates, respectively.

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

[0289] Example 3-3 Modification of target nucleotide sequence by non-natural polynucleotide with an adapter at the 3' end An experiment was carried out to measure the editing efficiency of the target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells.

[0290] In this example, each of the non-natural polynucleotides shown in Table 33 was introduced into 293-nLD1 cells by transfection. The base sequence, chain length, and relative editing efficiency of each of the edited nucleic acids are as shown in Table 33. Note that in Table 33, the chain length of the edited nucleic acid body and the chain length of the adapter are listed separately, but each sequence specified as a SEQ ID NO: is the combined length of the edited nucleic acid body and the adapter.

[0291]

[0292]

[0293] In Table 33, mismatched nucleotides are underlined, and nucleotides replaced by bridged nucleic acids are indicated in capital letters. All bridged nucleic acids in Table 33 were LNA. RNA-substituted nucleotides are indicated by an arrow next to the corresponding nucleotide base symbol. Where an internucleotide phosphodiester bond has been replaced with a phosphorothioate bond, an asterisk (*) is inserted between the corresponding nucleotides. Non-natural polynucleotides phosphorylated at the 5' and / or 3' ends are marked with a "p" at the 5' and / or 3' end. Furthermore, for each non-natural polynucleotide listed in Table 33, the nucleotide in the second line is an adapter, and each adapter forms a stem structure that may have a loop. Schematic diagrams of the stem structure of each non-natural polynucleotide are shown in Figures 16A, 16B, 17A, and 17B.

[0294] As shown in Table 33, the non-natural polynucleotides of the present invention were found to have editing efficiencies equal to or higher than that of the standard GEO-8.

[0295] Example 3-4 Modification of target nucleotide sequence with non-natural polynucleotide of embodiment (vi) An experiment was carried out to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells.

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

[0297]

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

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

[0300] Example 3-5 Modification of target nucleotide sequence by non-natural polynucleotide whose 5'-terminal nucleotide is not LNA An experiment was carried out to measure the editing efficiency of the target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells.

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

[0302]

[0303]

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

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

[0306] Example 3-6 Modification of target nucleotide sequence by non-natural polynucleotide with a modifying compound at the 5' or 3' end An experiment was conducted to measure the editing efficiency of a target polynucleotide sequence by introducing a non-natural polynucleotide into 293-nLD1 cells.

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

[0308]

[0309]

[0310] In Tables 42 and 43, mismatched nucleotides are underlined, and nucleotides substituted with crosslinked nucleic acids are indicated in uppercase. All crosslinked nucleic acids in Tables 42 and 43 were LNA. Where an internucleotide phosphodiester bond was substituted with a phosphorothioate bond, an asterisk (*) was inserted between the corresponding nucleotides. In Tables 42 and 43, Biotin represents biotin as a modifying compound, FAM represents fluorescein (FAM) as a modifying compound, Pur represents puromycin as a modifying compound, Chol represents cholesterol as a modifying compound, DIG represents digoxigenin as a modifying compound, and IdT represents 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 dioxygenin (DIG) is 414.31, and the molecular weight of inverted dT (IdT) is 304.2. Furthermore, the mismatch nucleotide at or near the 5' end in GEO-649, GEO-650, GEO-653, GEO-654, GEO-656, and GEO-657 is a mismatch nucleotide for the purpose of improving editing efficiency, and is not a mismatch nucleotide for the purpose of modification.

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

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

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

[0314]

[0315]

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

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

[0318] The method of the present invention using non-natural polynucleotides enables modification of target nucleotide sequences by simply introducing polynucleotides into cells, without introducing exogenous nucleases or genes encoding exogenous nucleases, and can be applied to extremely safe genome editing technology, making it industrially applicable.

Claims

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

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

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

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

5. The non-natural polynucleotide according to any one of claims 1 to 4, further comprising the following characteristic (J): (J) one or more internucleotide phosphodiester bonds are replaced with phosphate-modified bonds; 6. The non-natural polynucleotide according to claim 5, wherein the phosphate moiety modified bond comprises at least one bond selected from the group consisting of phosphorothioate bond, methyl phosphate bond, boranophosphate bond and mesyl phosphoramidate bond.

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

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

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

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

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

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

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

14. The non-natural polynucleotide according to any one of claims 1 to 13, further comprising the following feature (X4): (X4) an adaptor is added to the 3' end 15. The non-naturally occurring polynucleotide according to claim 14, wherein the adaptor has the function of inhibiting mismatch repair in a host cell.

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

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

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

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

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

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

22. The non-natural polynucleotide according to any one of claims 1 to 21, further comprising the following feature (X5): (X5) one or more nucleotides are inserted between the mismatched nucleotide and the 3'-terminal nucleotide 23. A non-natural polynucleotide capable of specifically binding to a target nucleotide sequence for modifying one or more nucleotides contained in the target nucleotide sequence in a double-stranded DNA in a cell, the non-natural polynucleotide containing one or more mismatch nucleotides with respect to the target nucleotide sequence, having at least one of the following (A) and (B) crosslinked nucleic acids, having the characteristic (D), and further having any one or more characteristics selected from the group consisting of (H2), (Y1) and (Y3): (A) one or more nucleotides adjacent to the 5' upstream side of the mismatch nucleotide are crosslinked nucleic acids, (B) one or more nucleotides adjacent to the 3' downstream side of the mismatch nucleotide are crosslinked nucleic acids, (D) having a chain length of 22 to 95 nucleotides, (H2) a phosphodiester bond between the 5'-terminal nucleotide and one or more nucleotides adjacent to the 5'-terminal nucleotide is replaced with a phosphate moiety modified bond, (Y1) the 5'-terminal nucleotide is a mismatch nucleotide, and one or more nucleotides adjacent to the 5'-terminal nucleotide are mismatch nucleotides, (Y3) an adaptor is added to the 5'-terminal.

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

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

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

9.

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

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

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

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

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

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

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

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

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

36. A method for modifying one or more nucleotides contained in a target nucleotide sequence in a double-stranded DNA in a cell, comprising the step of introducing into a cell a non-natural polynucleotide containing one or more mismatch nucleotides with respect to the target nucleotide sequence, wherein the modification of the target nucleotide sequence comprises at least one or more selected from the group consisting of deletion, insertion and substitution of one or more nucleotides of the target nucleotide sequence, and wherein the non-natural polynucleotide is a non-natural polynucleotide described in any one of claims 1 to 33.

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

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

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